CN101998989A - Identification of TRPML3 (MCOLN3) as a salty taste receptor and use in assays for identifying taste (salty) modulators and/or therapeutics that modulate sodium transport, absorption or excretion and/or aldosterone and/or vasopressin production or release - Google Patents

Abstract

The present invention relates to high-throughput mammalian and medium-throughput oocyte-based electrophysiological assays for identifying human TRPML3 modulators, preferably TRPML3 enhancers. Compounds that modulate TRPML3 function in the assay are expected to affect salty taste in humans. The inventive electrophysiological assays, such as the two-electrode voltage-clamp technique, facilitate the identification of compounds which specifically modulate human TRPML3. The assays of the invention provide a robust screen useful to detect compounds that facilitate (enhance) or inhibit TRPML3 function. Compounds that enhance or block TRPML3 channel activity should thereby modulate salty taste. In addition, these compounds may be used to regulate sodium excretion, urinary output and other biological functions relating to sodium levels. This invention relates to the elucidation that TRPML3 is involved in salty taste perception in primates including humans and likely other mammals (given the significance of sodium and other ions to physiological functions and conditions this phenotype is likely strongly conserved in different animals). The TRPML3 gene also modulates one or more of sodium metabolism, sodium excretion, blood pressure, fluid retention, cardiac function and urinary functions such as urine production and excretion. The inventors have identified TRPML3 as encoding a salty taste receptor in primates and humans (and likely other mammals) based on gene expression assays which have determined that TRPML3 is expressed specifically in taste bud cells and not in lingual epithelial cells, similar assays that have determined that TRPML3 is specifically expressed or enriched in the top half of taste bud cells in a subset of taste cells which do not express TRPM5 or PKD2L1, prior reports that document the expression of TRPML3 in other sensory organs such as the ear (therefore further substantiating the role of TRPML3 as a 'professional' sensory gene).

Description

TRPML3 (MCOLN3) is as the evaluation of saline taste acceptor and in the purposes of the mensuration of the sense of taste (saline taste) conditioning agent of generation that is used for identifying regulation and control sodium transport, absorption or drainage and/or aldosterone and/or vasopressing or release and/or therapeutical agent

Relevant interim and non-provisional application

The application relates to by relating to of submitting to before this transferee Senomyx Inc and is used to the provisional application identifying primate sense of taste specific gene and be used in particular for identifying the new principle of primate saline taste acceptor gene.These provisional application: No. the 60/929th, 017, the U. S. application sequence of submitting on June 8th, 2007; No. the 60/929th, 007, the U. S. application sequence of submitting on June 8th, 2007; No. the 60/947th, 052, the U. S. application sequence of submitting on June 29th, 2007; No. the 60/935th, 297, the U. S. application sequence of submitting on August 3rd, 2007; No. the 60/987th, 611, the U. S. application sequence of submitting on November 13rd, 2007; No. the 60/988th, 938, the U. S. application sequence of submitting on November 19th, 2007; No. the 60/991st, 274, the U. S. application sequence of submitting on November 30th, 2007; No. the 60/991st, 289, the U. S. application sequence of submitting on November 30th, 2007; No. the 60/992nd, 502, the U. S. application sequence of submitting on December 5th, 2007; No. the 60/992nd, 517, the U. S. application sequence of submitting on December 5th, 2007; No. the 61/017th, 244, the U. S. application sequence of submitting on December 28th, 2007; No. the 61/021st, 437, the U. S. application sequence of submitting on January 16th, 2008; No. the 61/043rd, 257, the U. S. application sequence of submitting on April 8th, 2008; With No. the 61/053rd, 310, the U. S. application sequence of submitting on May 10th, 2008.In addition, the application relates to the right of priority of No. the 11/808th, 356, U.S.'s sequence also submitting on June 8th, 1 and the attorney docket of submitting on the same day 67824.703201.All above-mentioned interim and non-provisional applications integral body are by reference incorporated this paper into.

These applications comprise and relate to the various sense of taste specific genes disclosure of (comprising TRPML3 or MCOLN3), when this gene when being known alternatively.This gene is emphasized to be the gene by inventor's functionalization in electrophysiological detection.This gene is turned to the candidate gene of coding saline taste acceptor by inventor's theory, because it is predicted the special sodium-ion channels of the sense of taste of the saline taste sense of taste of its coding and regulating primate (for example people), rodent and other animals.As described herein, use the cell (Mammals and Amphibians cell) of the TRPML3 polypeptide of expressing wild-type or sudden change and external and body interior (animal) mensuration of rodent zooblast to confirm that TRPML3 or MCOLN3 participate in the saline taste impression, and the incoherent other biological function of the influence and the sense of taste, (sudden change as rodent TRPML3 proves such as the sense of hearing and balance, the hair cell of inner ear has been killed in this sudden change, hearing had undesirable action (deafness), cause impaired balance, and the destruction that causes the normal pegmentation calmness, the loss of the melanocyte that this obviously causes owing to being suddenlyd change by this identical TRPML3).

The inventor obtains, open and the previous unknown result of this paper also shows, because this gene is at sodium transport, possible effect in drainage and the absorption, its effect in sodium impression and the tissue (comprising suprarenal gland and hypophysis) of expressing based on known this gene specific wherein, this gene shows that it regulates and control with aldosterone and/or vasopressing or participate in regulating sodium transport, metabolism, drain and other sodium and the relevant function that relates to aldosterone and/or vasopressing of other ions of possibility, because these hormones are at the different mammiferous sodium transport that comprises people and rodent, play a significant role in metabolism and the drainage.

Invention field

The TRPML subfamily member specific ion passage polypeptide (TRPML3 or MCOLN3) that the present invention relates to transient receptor potential cationic channel (transient receptorpotential cation) superfamily participates in the discovery that the sense of taste (saline taste) is experienced (for example sodium taste perception).

More specifically, consider and keep sodium and the importance of potassium ion level in the different concns threshold value, and consider the material impact of the different organic healthy important physical processes of these ion pairs, the present invention relates to following discovery: specific ion passage polypeptide (TRPML3 or MCOLN3) participates in the sense of taste (saline taste) impression of primate (people and non-human primate), rodent and other Mammalss and other vertebratess of possibility (bird, Reptilia, Amphibians).

In addition, the present invention relates to following discovery: TRPML3 or MCOLN3 polypeptide or its functional variant when separately or with other sense of taste specific polypeptides (, or comprising for example ionic channel of TRPML1, TRPML2, NKAIN3 or NALCN) for example such as other accessory molecules of GPCR when combination is expressed in response in saline taste stimulate and potentially the Taste Receptors of other sense of taste trigger molecules work.

In addition, the present invention relates to following discovery: because effect and the wherein tissue of known its specifically expressing (comprise suprarenal gland and hypophysis) of this gene in sodium impression, this genes encoding separately or the polypeptide that plays a role in the level of regulation and control aldosterone and vasopressing and release and physiologically active that therefore regulation and control are relevant by the sodium of aldosterone and/or vasopressing adjusting together with other accessory molecules.

In addition, the present invention relates to following discovery: because TRPML3 participates in the sodium impression and further expresses at the parathyroid gland organ based on it, the TRPML3 conditioning agent can be used to treatment and relates to parathyroid disease, these diseases include but not limited to that calcium homeostasis, hyperparathyroidism, hypoparathyroidism, hypercalcemia, capsule osteitis, Albright's syndrome to type, Jansen metaphysis cartilage generate, the Blomstrand cartilage generates and the osteoporosis of different reasons, and described reason comprises that for example the age is relevant, menopause or medicine, chemotherapy or radiotherapy-induced.

The invention still further relates to the application of the animal model (Varitint waddler mouse) of the mutant form that contains TRPML3 or MCOLN3 gene, wherein TRPML3 saline taste gustatory cell is excised from taste bud specifically, and wherein the saline taste sense of taste greatly weakened in case study TRPML3 to the influence of the saline taste sense of taste and this ionic channel to other influences of sodium transport, metabolism and excretory, because this gene may have identical influence other animals that comprise the people.

The invention still further relates to and use mutant TRPML3 gene (A419P TRPML3) to come special excision to comprise the cell type of gustatory cell or melanocyte and the mouse model system that generation lacks these different cell colonys.

The invention still further relates to and use mutant TRPML3 ionic channel polypeptide (A419P TRPML3) to kill particular cell types such as saline taste gustatory cell or melanocyte as toxin.

The invention still further relates to this gene and corresponding polypeptide or its and comprise that the variant of the mutant (it is active or keep ionic channel to be in the mutant of open or off-position that for example (strengthen or weaken) regulated and control in design) of allelic variant, mosaic, fragment and transformation is being used for identifying that TRPML3 regulates the application of the mensuration of compound (TRPML3 agonist, antagonist, toughener, co-inhibitor).These compounds can be used as sense of taste conditioning agent and regulation and control or treatment potentially and relate to unusual vasopressing release, the generation of aldosterone, the function of melanocyte or the therapeutical agent of sodium transport, absorption and/or the excretory physiological function and the patient's condition.For example these compounds can be used as saline taste sense of taste co-inhibitor, toughener or inhibitor, or be used for the treatment of hypertension, urinary function, cardiovascular disorder, be used for the treatment of the relevant patient's condition of melanocyte, such as pigmentation disorder, melanoma with such as the mucus related conditions of the glutinous lipidosis of IV type.

Based on aforementioned, the present invention relates to the high flux screening mensuration (HTS mensuration) based on mammalian cell that is used to identify the TRPML3 conditioning agent particularly.In exemplary, what the present invention was taught in the toughener that is used for identifying the TRPML3 function or co-inhibitor uses the cell of the active variant (A419P-TRPML3) of expressing TRPML3 based on the mensuration of cell.The compound anticipated impact people of regulation and control TRPML3 function and the saline taste sense of taste in other Mammalss in based on the mensuration of cell.The mensuration of describing among the present invention can be with the high-throughput mode operation in standard 96 or 384 well culture plates.

In a more particular embodiment, the present invention identifies and provides in function (electric physiology), the body and the immunohistochemistry data, and these data produce evidence to show the polypeptide that TRPML3 (MCOLN3) coding works as primate (for example people) saline taste acceptor.

In relevant embodiment, the invention provides and use the TRPML3 polypeptide to can be used to enrichment, evaluation or separate the saline taste acceptor and the sign of other TRPML3 express cells with nucleotide sequence and specific probe conduct thereof.

In relevant embodiment, the invention provides and use the TRPML3 polypeptide can or express the sign that the relevant specified disease TRPML3 relevant with the patient's condition suddenlys change with abnormal T RPML3 function as can be used to identify with nucleotide sequence and specific probe thereof, the described disease and the patient's condition are such as relating to unusual sodium impression, transhipment, drain and absorbing, the disease of melanocyte function (cancer, pigmentation disorder etc.) and the disease that relates to the generation of unusual aldosterone or vasopressing or discharge are such as cardiovascular and urinary disorders.

Another concrete aspect, the invention provides the cell that uses TRPML3 (MCOLN3) and express TRPML3 or TRPML3 transgenic animal model and identify and cause or regulation and control (prevent or strengthens) comprise the interior mensuration of external and body of agonist, antagonist or toughener compound of the saline taste sense of taste of people's primate.These measure to use express cell or the animal of TRPML3 (wild-type or its variant), or use and express with such as other auxiliary polypeptide of other sense of taste specific polypeptides (such as NALCN or NKAIN3, GPCR) or such as the TRPML3 ionic channel of the relevant ions passage of TRPML1 and/or TRPML2 or the cell of variant (for example having enhanced activity or genetic modification for being fixed in the functional variant that open to the outside world or " closing " are orientated).

In yet another aspect, the invention provides transgenic animal (preferably rodent) and use its confirm TRPML3 the mammiferous saline taste sense of taste and relating to sodium and other physiological functions of other ions (such as potassium, calcium, lithium) and ion (sodium) metabolism, blood pressure, fluid retention and drainage, urinary function and heart function in effect.

In yet another aspect, the invention provides in the mensuration of for example nervous physiology behavior or electrophysiological detection in the external and bodies of the cell that uses TRPML3 and express TRPML3 or transgenic animal and measure, so that evaluation alleviates the therapeutic compound of the disease that relates to or caused by this ionic channel polypeptide expression defective and the patient's condition and/or its influence to the specific cells that comprises excision.These patient's condition comprise and relate to TRPML3 overexpression, the low patient's condition of sudden change of expressing and influencing the TRPML3 polypeptide of its ability that works as the special sodium channel of the sense of taste in the Mammals (comprising for example people and non-human primate) as an example.Other patient's condition comprise Addison's disease and relate to unusual aldosterone or the disease of vasopressing generation or release as an example, such as hypertension, ypotension, fluid retention and impaired uropoiesis or heart function, such as arrhythmia, heart attack and apoplexy.

In another embodiment, the present invention relates to identify that independent TRPML3 is used in regulation and control or the mensuration of the compound of the TRPML3 function expressed with another sense of taste specific gene (such as NALCN or NKAIN3 or TRPML1 or TRPML2) and use compounds identified to regulate and control saline taste impression in people and other Mammalss or the vertebrates.In another embodiment, the present invention relates to identify that independent TRPML3 is used in regulation and control or the mensuration of the compound of the TRPML3 function expressed with another sense of taste specific gene (such as NALCN or NKAIN3 or TRPML1 or TRPML2) and use compounds identified to regulate and control saline taste impression in people and other Mammalss or the vertebrates.

The present invention further provides based on the TRPML3 that uses method provided herein to identify express or do not express separate, the ad hoc approach of purifying and mark saline taste and other TRPML3 express cell types and gustatory cell pedigree, described cell lineage comprises sense of taste stem cell and other prematurities and sophisticated gustatory cell pedigree, comprises the cell that is divided into taste bud cell, gustatory cell neurone, sense of taste immunocyte etc.These separate and purification process comprises positive and negative cells separation method.Gustatory cell pedigree that for example needs or type can by for example positive cell system of selection by using fluorescence-activated cell sorter (FACS), for example the electric physiological vision of the pearl by using antibody sandwich identify needs cell for example the magnetic bead cell of single transfectional cell select to separate.Alternatively, the gustatory cell pedigree that needs or type can by the negative cells purifying and separation method reclaims or purifying, wherein, for example contact with the cytotoxic antibody of the gene that is specific to target gene or on unwanted gustatory cell type to be removed, expresses from the gustatory cell that contains needs of tongue, oral cavity or gi tract and relevant organ and the blended cell suspension of unwanted cells by for example making by removing cell type enrichment or purifying from the blended cell colony of one or several unwanted cells pedigree with needs.

The invention still further relates to and use Varitint waddler mouse to detect of the influence of TRPML3 function melanocyte, hypophysis, suprarenal gland, the sense of taste, uropoiesis or gustatory cell.

The invention still further relates to and use Varitint waddler mouse to detect of the influence of TRPML3 function melanocyte, hypophysis, suprarenal gland, the sense of taste, uropoiesis or gustatory cell.

In addition, the present invention relates to following discovery: owing to TRPML3 participation sodium impression and further based on its expression at the parathyroid gland organ, the TRPML3 conditioning agent can be used to treatment and relates to parathyroid disease, described disease includes but not limited to that calcium homeostasis, hyperthyroidism, hypothyroidism, hypercalcemia, capsule osteitis, false hypothyroidism, Jansen metaphysis cartilage generate, the Blomstrand cartilage generates and the osteoporosis of different reasons, and described reason comprises that for example the age is relevant, menopause or medicine, chemotherapy or radiotherapy-induced.

The invention still further relates to and use Varitint waddler mouse in mensuration, to detect specifically expressing in the saline taste gustatory cell and the gene of specifically expressing (because wherein having excised the saline taste gustatory cell) in Varitint waddler mouse not, the function of the adjustable TRPML3 of this gene, or work or regulate and control growth differentiation or the apoptosis of saline taste sense of taste signal from the transmission of TRPML3 neuralward fiber and/or control saline taste gustatory cell as the saline taste acceptor.These gene tests are measured to comprise and are used gene chip or microarray technology to come comparison gene of expressing and the gene of expressing in the saline taste gustatory cell in Varitint waddler mouse.

The present invention further provides based on the TRPML3 that uses method provided herein to identify express or do not express separate, purifying and mark saline taste and other TRPML3 express cell types and cytophyletic ad hoc approach, described cell lineage comprises sense of taste stem cell and other prematurities and sophisticated gustatory cell pedigree, comprises the cell that is divided into taste bud cell, gustatory cell neurone, sense of taste immunocyte etc.These separate and purification process comprises positive and negative cells separation method.Gustatory cell pedigree that for example needs or type can be by for example positive cell systems of selection by using fluorescence-activated cell sorter (FACS), for example the magnetic bead cell of the cell (such as single cells transfected) of the electric physiological vision evaluation needs of the pearl by using antibody sandwich is selected to separate.Alternatively, the gustatory cell pedigree that needs or type can by the negative cells purifying and separation method reclaims or purifying, wherein, for example contact with the cytotoxic antibody of the gene that is specific to target gene or on unwanted gustatory cell type to be removed, expresses from the gustatory cell that contains needs of tongue, oral cavity or gi tract and relevant organ and the blended cell suspension of unwanted cells by for example making by removing cell type enrichment or purifying from the blended cell colony of one or several unwanted cells pedigree with needs.The invention still further relates to and use Varitint waddler mouse to detect of the influence of TRPML3 function melanocyte, hypophysis, suprarenal gland, the sense of taste, uropoiesis or gustatory cell.

The invention still further relates to and use Varitint waddler mouse to detect of the influence of TRPML3 function melanocyte, hypophysis, suprarenal gland, the sense of taste, uropoiesis or gustatory cell.

The invention still further relates to and use Varitint waddler mouse in mensuration, to detect specifically expressing in the saline taste gustatory cell and the gene of specifically expressing (because wherein having excised the saline taste gustatory cell) in Varitint waddler mouse not, the function of the adjustable TRPML3 of this gene, or work or regulate and control growth differentiation or the apoptosis of saline taste sense of taste signal from the transmission of TRPML3 neuralward fiber and/or control saline taste gustatory cell as the saline taste acceptor.These gene tests are measured to comprise and are used gene chip or microarray technology to come comparison gene of expressing and the gene of expressing in the saline taste gustatory cell in Varitint waddler mouse.

The invention still further relates to and use the sign be specific to TRPML3 and/or relevant auxiliary polypeptide for example to participate in the zone of the specific sense of taste (saline taste) and non-sense of taste specific function in antibody or oligonucleotide allocation tongue and the oral cavity, localization package is contained in the specific sense of taste specific gene of expression on specific (saline taste) taste perception cell in gi tract and the relevant organ (such as enteric epithelium or urethra) and therefore participates in one or more cell of gustatory cell specific function disclosed herein, and/or in gustatory cell differentiation research, use this gene and its special sign, for example be used to identify the compound of inducing gustatory cell differentiation or dedifferentiation, for example adult or embryonic stem cell and other multipotencys or immature cell type are divided into gustatory cell pedigree and the gustatory cell type that needs.

Background of invention

Various ionic channels had been studied, so that illustrate its potential participation saline taste sense of taste and regulate sodium transport, metabolism and drainage.Especially, studied the ionic channel member of ENaC/ degenerin family epithelium sodium channel (ENaC) in great detail, it comprises that mammiferous sour is subjected to mechanical sensitivity degenerin passage and the molluscan FMRF-amidated peptide gated channel (Kellenger of ionic channel (ASIC), nematode, S. and Schild, L. (2002) Physiol.Rev.82:735-767).What ENaC mediation comprised guanamprazine-sensitivity in the multiple tissue of kidney, colon and lung passes through the antiepithelial top of high resistant film Na ⁺Transhipment.

Known ENaC is the different trimerization passage that comprises α, β and γ subunit or δ, β and γ subunit.Suppose this different trimerization passage participant's saline taste impression.Previous this transferee has developed and has used the ENaC sequence to identify the mensuration of the compound of regulation and control δ β γ and α β γ people ENaC, whether will regulate and control people's saline taste impression potentially so that check these compounds.These compounds also can be used for treating the people's pathology that relates to unusual ENaC function potentially.

Do not resemble other Mammalss, when using with the concentration of regulating and control the ENaC function specifically, guanamprazine only reduces the intensity of the sodium-chlor sense of taste slightly, promptly reduces about 15-20% (Halpern, B.P. (1998) Neuroscience and Behavioral Reviews.23:5-47).The experiment that the inventor carries out shows that guanamprazine or more effective guanamprazine derivative phenamil do not cause the remarkably influenced to people's salt intensity of impression when the horizontal checkout of the IC50 value (being equal to 10 times to guanamprazine and 100 times of IC50 values that the benzyl guanamprazine are higher than δ β γ ENaC) that is higher than α β γ ENaC with 300 times (to guanamprazines) and 3000 times (to benzyl guanamprazine (benzamil)).Based on more than, draw the theory of other (ENaC gene) participant's saline tastes impression.

In addition, report that recently Taste Receptors can express at non-oral cavity tissue, for example at Digestive tract with express such as other organs of kidney potentially, this shows that it has the relevant activity of the non-sense of taste, such as digesting in food impression and regulation and control etc.Especially, it is reported that sweet taste, delicate flavour and bitter taste Taste Receptors expressing in removing extrabuccal cell, such as gastrointestinal cell (referring to for example Sternini etc., Amer J Physiol.Gastrointestinal and Liver Physiology, 292:G457-G461,2007; Mace, O.J. etc., J.Physiology.10.1113/jphysiol.2007.130906.On May 10th, 2007 online delivering).Also according to the different (Margolskee etc. of study group, Bezencon etc., Rozengurt etc., with (2007) (the same) such as Sternini), bitter taste and delicate flavour Taste Receptors and in GI specialized cells, express (referring to for example Margolskee etc., Genes Brain Behavior 2007 (March 21 delivered on the net) such as other sense of taste signal transduction molecules of TRPM5 and gusducin; Rozengurt etc., Amer.J.Physiol.Gastroent.Liver Physiol.291 (2): G171-7 (2006); Bezencon etc., Chem Senses32 (1): 41-47 (2007)).Margolskee etc. (the same) are report further, and the forfeiture of T1R3 or gusducin causes the change of insulin metabolism and such as the release of the peptide of being satiated with food of GLP-1 (glucagon-like peptide 1) in the rodent.

Based on these observations, express in tissue such as suprarenal gland that the saline taste acceptor may play a significant role in control sodium ion homeostasis and the hypophysis other Taste Receptors.Because Taste Receptors is expressed in such as Alimentary non-gustatory cell, and may in the organ of urinary system, express, it may participate in not directly function such as the digestive function (such as stomach motion, absorption, food detection, metabolism and oral cavity or gastral immunomodulatory) relevant with the sense of taste, and also can influence with sodium and absorb, drain the function relevant, such as blood pressure and fluid retention with transhipment.Therefore identification of taste cell-specific gene and these genes of evaluation are expressed other non-taste functions that should help better to understand these Taste Receptors in which specific cells, and also help to use these cell of these genes, gene product and expression in the mensuration that is used for identifying the novel treatment of for example treating digestive ailment, the immunomodulatory of described digestive ailment autoimmunization, inflammation and cancer, metabolism, diabetes, eating disorder, obesity, gustatory cell turnover, hypertension, fluid retention and Digestive tract.In the particular instance of sodium (saline taste) Taste Receptors, illustrate that the specific identity that saline taste is experienced important function of gene should help to understand other sodium correlation functions of these gene pairss and to the effect of participation sodium transport, metabolism and the excretory polypeptide (such as vasopressing or aldosterone) of uropoiesis and cardiovascular function key.

Mention as mentioned, the present invention is specifically related to TRPML3 and participates in Mammals and the discovery of the ionic channel polypeptide of other vertebrate sodium (saline taste) taste perceptions potentially, consider sodium and other ions (such as potassium, calcium, lithium) importance to many physiological functions, this discovery will hint that this gene may guard in different vertebratess.Before this paper contriver's specific discovery, it is the ionic channel (further may in relating to sodium transport and excretory relevant physiological function, play a role) that TRPML3 (MCOLN3) coding participates in primate and other mammiferous saline taste impressions, previous this gene of report is responsible for being called the phenotype of the mouse mutant of varitint-waddler, this mutant shows the early onset hearing disability, the vestibular defective, (DiPalma etc., Mutationsin Mcoln3 associated withdeafness and pigmentation defects in varitint-waddler (Va) mice. (sudden change of the Mcoln3 relevant with the pigmentation defective with deafness in varitint-waddler (Va) mouse) Proc.Natl.Acad.Sci USA 99:14994-14999 cause death pigment anomaly and perinatal period; 2002).According to further, MCOLN3 or TRPML3 express (in the ear) in hair cell and stereocilium plasma membrane.Especially, reported that the sudden change that causes ala 419 in the 5th membrane spaning domain to be replaced to pro in this peptide causes highly active MCOLN3, this causes expressing death (so deafness of Va mouse) (Grimm C etc., the Proc Natl.Acad.Sci.USA 104:19583-8 of the cell (being the hair cell of ear) of this molecule; 2007).

Although in mouse, the sudden change of A419P-TRPML 3 causes varitint-waddler phenotype (Kim etc., J.Biol.Chem., on December 14th, 2007 of severe form; 282 (50): 36138-42.2007 delivered on the net October 25; Nagata etc., Proc.Natl.Acad.Sci, USA, on January 8th, 2008; 105 (1): 353-8.2007 delivered on the net December 27; Grimm etc., Proc.Natl.Acad.Sci, USA, on December 4th, 2007,104 (49): 19583-8.2007 delivered on the net November 28), it is characterized by pigmentation defective, hearing disability and embryonic death, (TRPML3 (I362T/A419P) causes the varitint-waddler phenotype of slight form in another TRPML3 sudden change.

Sudden change is that what how to cause each phenotype is unknown.It is reported that first channel characteristic of TRPML3 is as strong inside rectification cationic channel, be subjected to the new adjusting (Kim etc. 2007 (the same)) of the outer Na+ of born of the same parents.Born of the same parents' outside surface that they further are reported in preincubate TRPML3 in the substratum that does not contain Na+ is that passage activation is required, but the slow inactivation of passage subsequently.Therefore the A419P sudden change schedules the open state of not regulated with passage lock.Investigators such as Kim further observe the similar function of A419G sudden change and obtain, and itself and the similar expection of A419P remove to stablize the 5th membrane spaning domain of α spiral of TRPML3.On the contrary, Kim etc. observe the passage that the I362T sudden change causes non-activity, but the channel characteristic of TRPML3 (I362T/A419P) and TRPML3 (A419P) are similar.Yet they report that the surface expression of TRPML3 (I362T/A419P) and current density are lower than TRPML3 (A419P), and it is reported that the A419P sudden change has influenced the glycosylation of passage, and cause a large amount of necrocytosiss.It is reported that their discovery confirmed that further the varitint-waddler phenotype is because the function of the TRPML3 (A419P) that TRPML3 (I362T/A419P) mutant alleviates obtains to cause, causes slighter phenotype.

In addition, it is reported that some sudden changes of relevant member TRPML1 (glutinous fat 1/MCOLN1) of TRPML3 gene family cause the IV type to stick lipidosis, serious heredity nerve degenerative diseases (Xu etc., on November 13rd, 2007, Proc Acad Sci USA, 104 (46): 18321-62007 delivered on the net November 7).This disease is the particular form of glutinous lipidosis, is the autosomal recessive lysosome storage disorder that is characterized as serious neurological, achlorhydria and impaired vision (such as the corneal opacity and stravismus).This disease is (Venkatanulum etc., 23 days 281 (25) June of J Biol.Chem.2006: 17517-27 delivered on the net on April 10th, 2006) that the sudden change owing to the relevant cationic channel TRPML1 of the 2nd group of transient receptor potential (TRP) takes place.

It is reported the member of the TRPML3 gene family connection that is relative to each other.For example same (the same) such as reference Venkatanulum shows that these (TRPML1,2 and 3) albumen tend to multimerization, and instructs its ubcellular to distribute and the mechanism of regulating and control these albumen transportations is limited.They also declare TRPML interact form with and heteromultimeric.In addition, Venkatanulum etc. claims that also the existence of TRPML1 or TRPML2 influences the spatial distribution of TRPML3 specifically.Although they declare that TRPML1 and TRPML2 are lysosomal proteins with polymer, TRPML3 is positioned at endoplasmic reticulum with polymer.In addition, they declare that TRPML3 is positioned lysosome with TRPML1 or TRPML2 coexpression the time, TRPML3 location of mistake similarly when the lysosome target of TRPML1 and TRPML2 is destroyed.On the contrary, they state that TRPML3 does not cause TRPML1 or the TRPML2 delay in endoplasmic reticulum.Venkatanulum etc. also show the grade of the ubcellular distribution that has control TRPML, so that TRPML1 and TRPML2 indicate the location of TRPML 3 rather than vice versa.

Also reported MCOLN3 or TRPML3 in document and the public gene database at the suprarenal gland strongly expressed, known suprarenal gland plays a significant role in regulating the metabolism of body sodium.Further, the adrenal destruction that is characterized as that it is reported human autoimmune disease (Ai Dishengshi), and an indication symptom of this disease is that intensive salt is craved for.

Further, it is reported TRPML3 at the hypophysis strongly expressed, and in melanocyte, express.As noted above, varitint sudden change and the death that causes the hearing cell cause the death of melanocyte.

Yet known to the inventor, previous nobody shows the Taste Receptors polypeptide that in different Mammalss or other vertebratess TRPML3 or genes involved TRPML1 or TRPML2 participate in the saline taste sense of taste or coding impression and stimulate in response to saline taste.

Invention summary and purpose

Therefore, the purpose of this invention is to provide and determine for the first time following discovery and support data: TRPML3 different Mammalss with bring into play active function in other vertebrate sense of taste (the particularly saline taste sense of taste) potentially, and based on this this ionic channel independent or with other ion channel genes (such as TRPML1, TRPML2 or NALCN, NKAIN3) or other accessory proteins can further absorb together such as sodium, transport cell relevant and physiologically active and discharge such as urine with excretory sodium, blood pressure regulation with similarly play a significant role in relevant attached influence and the activity.

Equally, the purpose of this invention is to provide express TRPML3 or its variant and randomly be suitable for use in the mensuration of identifying the TRPML3 conditioning agent or be suitable for studying TRPML3 to the saline taste sense of taste and relate to sodium transport, metabolism and other physiological processs of excretory influence other ionic channels or such as conversion or the cells transfected or the transgenic animal of the accessory protein of the special GPCR of the sense of taste.

Equally, the purpose of this invention is to provide and comprise test cell, preferably recombinant mammalian cells or Amphibians ovocyte or express the saline taste gustatory cell of endogenous TRPML3 or the mensuration system of other TRPML3 express cells (for example hypophysis or suprarenal gland) of expressive function TRPML3 or variant, fragment or functional equivalent, and based on mammalian cell with infer the mensuration (preferably high throughput assay) of conditioning agent based on the TRPML3 of Amphibians ovocyte.

More specifically, the purpose of this invention is to provide and express the human cell line who can be used on screening TRPML3 conditioning agent, for example HEK293T cell, Chinese hamster ovary celI and Amphibians ovocyte based on the functional TRPML3 in the mensuration of cell or variant, fragment or functional equivalent.

Equally, the mammalian cell and the Amphibians ovocyte that the purpose of this invention is to provide expressive function TRPML3 or variant, fragment or functional equivalent are used for identifying functionally the activity of TRPML3 and identify the compound (this paper is called saline taste sense of taste conditioning agent) that strengthens or prevent the saline taste impression.These compounds can be used as the saline taste sense of taste that becomes to assign to strengthen, regulate and control, suppress or prevent of food, medicine and beverage.

Disclosed as the provisional application of the claimed benefit of priority of the application and application for a patent for invention formerly; the inventor uses the new principle of identifying primate sense of taste specific gene to identify that originally this gene is coding sense of taste specific gene (be disclosed in provisional application in detail, incorporate this paper by reference into).This gene of these application code displaying membrane-spanning proteins is expressed in the taste bud top of conducting sodium, taste perception cell.Infer that based on this TRPML3 may participate in the saline taste impression function relevant with other sodium.

Data disclosed herein provided below, comprise that (the varitint-waddler mouse of mutant form TRPML3 gene is expressed in use to external and intravital function (electric physiology) data, it causes forfeiture of deafness, pigment and impaired balance), immunohistochemistry data and other information, confirm the supposition that the inventor is initial, and provide convictive this genes encoding that experimental results show that to allow the saline taste acceptor of experiencing gustatory cell detection sodium-chlor (salt) of the taste bud of tongue.

In addition, based on the fact that this gene is also expressed at suprarenal gland and hypophysis, its expection participates in together with other polypeptide such as vasopressing and aldosterone or regulates in the different cells of body, tissue and the organ sodium transport, metabolism and excretory and generally regulate.

The evidence that this paper that provides convictive evidence to prove the ionic channel that the TRPML3 genes encoding works as the saline taste acceptor jointly in different animals comprises comprises following:

(1) MCOLN3 or TRPML3 specifically expressing in primate and people's sense of taste cell at least, and do not express at the tongue epithelial cell, and at the taste bud top, do not express TRPM5 (being that it is not sweet taste, bitter taste or delicate flavour), do not express PKD2L1 (being that it is not a tart flavour) and find taste perception cell subsets specifically expressing towards taste pore.Therefore, the MCOLN3 positive cell comprises the gustatory cell of the different subgroups that are different from the known gustatory cell that participates in other (non-saline taste) sense of taste forms of detection.

(2) MCOLN3 or TRPML3 also express in the recipient cell of other organs (for example ear).Therefore it is the receptor gene of ' specialty '.

(3) MCOLN3 or TRPML3 are at the suprarenal gland strongly expressed.These bodies of gland are brought into play important effect in regulating the metabolism of body sodium.Therefore MCOLN3 may (based on these and other data of inventor's acquisition) be to regulate the metabolic key molecule of sodium, and can regulate or participate in suprarenal gland generation aldosterone.

(4) human autoimmune disease (Ai Dishengshi) is characterized as adrenal destruction.An indication symptom of this disease is that salt is craved for.The latter may be at the existence of the autoantibody of MCOLN3 or destroy MCOLN3 in the taste bud or the result of this transgenation of TRPML3 function.

(5) also high expression level in participating in the hypophysis that vasopressing discharges of MCOLN3 or TRPML3, this then influence urine generation and renal function.Based on this and other data relevant with TRPML3 that the inventor obtains, this polypeptide may be regulated the release of vasopressing, therefore regulates the sodium excretion in the urine.

(6) MCOLN3 or TRPML3 conduct sodium in electrophysiologic study, and show the correct biochemical character (promptly detecting K+, Li+ and guanamprazine insensitivity) that is predicted as primate saline taste acceptor.

(7) in varitint mouse (have TRPML3 sudden change), use the nervous physiology experiments experiment (the neural record) of sodium to show that the Varitint mouse is to replying of sodium impaired (do not show that intensive saline taste reply).These mouse have been excised the gustatory cell (saline taste gustatory cell) of expressing TRPML3 in the taste bud, and this has confirmed that these specific cells are the prerequisites that detect saline taste.

(8) use the mammalian cell of the TRPML3 polypeptide (sudden change causes ionic channel to be fixed in the open to the outside world orientation) of expressing sudden change and the mensuration based on cell of Amphibians ovocyte to identify the TRPML3 toughener and the co-inhibitor that in taste test, should strengthen or prevent the saline taste sense of taste.

Therefore, based on preamble, the objective of the invention is definite MCOLN3 or TRPML3 identity as people's saline taste acceptor, and cell or animal based on this this gene that used transfection or variant (for example functional mosaic, have enhanced activity, be fixed in open orientation or help the mutant of the change of its proteic other needs of TRPML3 that are used to measure) are designed screening assay, purpose is to identify agonist, antagonist or the toughener (conditioning agent) of this molecular function, and it will regulate and control the saline taste sense of taste and other TRPML3 functions.

Equally, the purpose of this invention is to provide isolating gustatory cell, adrenal cells, pituicyte or uropoiesis device cell or comprise and express to participate in the saline taste impression, the sodium metabolism, the gustatory cell of the TRPML3 that aldosterone generation and/or vasopressing discharge, adrenal cells, the cell sample of the enrichment of pituicyte or uropoiesis device cell, wherein said isolating gustatory cell, adrenal cells, pituicyte, or the gustatory cell sample of uropoiesis device cell or enrichment is expressed TRPML3 gene or its variant, its coding regulation and control saline taste sense of taste, sodium transport, at least a sodium channel that metabolism or drainage and/or aldosterone or vasopressing discharge or produce.Preferably, this cell will be people, non-human primate or rodent source.

Equally, specific purpose of the present invention is to identify to be used for the treatment of to relate to that aldosterone discharges or the TRPML3 conditioning agent of the disease that produces.Can use the disease of TRPML3 modulators for treatment and the patient's condition to comprise can be by the disease of excitement or antagonism aldosterone so excitement or antagonism sodium transport and excretory compounds for treating, described compound comprises oedema as an example, blood pressure (high or low blood pressure), liver cirrhosis, the primary hyperaldosteronemia, renal tubal dysfunction, diabetes (I or II type) and relative pathological symptom, comprise circulatory problems, oedema, the illness in eye relevant with Cycle Difference, high cortisone mass formed by blood stasis, the atherosclerosis or the obesity of abdominal obesity for example, and hepatopathy, sexual dysfunction (sex), cerebrovascular disease, vascular disease, retinopathy, neuropathy, Insulinopathy, endothelial function disturbance, pressure function of receptors obstacle, migraine, hectic fever and premenstrual tension and other cardiovascular patient's condition are such as atherosclerosis, in heart failure, congestive heart failure, vascular disease, apoplexy, myocardial infarction, endothelial function disturbance, ventricular hypertrophy, renal tubal dysfunction, target organ damage, thrombosis, arrhythmia, plaque rupture and aneurysma.

Equally, specific purpose of the present invention is to identify to be used for the treatment of to relate to that vasopressing discharges or the TRPML3 conditioning agent of the disease that produces.Can use the disease of TRPML3 modulators for treatment and the patient's condition to comprise can be by the disease of the compounds for treating of excitement or antagonizing vessel vassopressin, described compound similarly comprises diabetes, obesity, ephrosis as an example, such as cystic kidney disease, acquired cystic kidney disease, such as the eye circular correlation illness of myopia; Feel sick, vomiting, sexual dysfunction (sex), oedema, hypertension, congestive heart failure (scope from the II class of New York heart association to ruddy pulmonary edema), periodically spontaneous oedema, nephrotic syndrome, because the liver cirrhosis or the ascites of other reasons, the cerebral edema of a variety of causes, and dilutional hyponatremia and the metabolism that is referred to as inappropriate ADH secretion syndrome changes and wherein need the other diseases or the patient's condition of vasorelaxation and/or anti-oxytocic activity in the treatment.

Equally more specifically, the isolating Taste Receptors that the purpose of this invention is to provide regulation and control saline taste impression, it comprises mosaic or the fragment of TRPML3 that TRPML3 polypeptide or its variant (such as transforming the variant that has the enhanced ion channel activity or remain secured to open orientation as) or regulation and control comprise people's the mammiferous saline taste sense of taste.This Taste Receptors can be monomer or polymer (different aggressiveness or same aggressiveness), and can comprise other sense of taste specific polypeptides, for example such as other ionic channel polypeptide of TRPML2, TRPML1, NKAIN3 or NALCN.

Equally more specifically, the purpose of this invention is to provide by genetic modification is that expression and/or the further genetic modification that has knocked out endogenous TRPML3 knocked in it directly to homologue or variant (for example transforming the variant that strengthens ion channel activity or passage is fixed in the open to the outside world position as) transgenic nonhuman animal.These animals that comprise expressing human or other primate TRPML3 genes or variant can be used to identify the compound of regulation and control (strengthen or prevent) people and other Mammals saline taste sense of taste.

More specifically, the purpose of this invention is to provide by genetic modification is the transgenic nonhuman animal of expressing heterologous TRPML3 polypeptide (for example people TPML3 or variant).

Equally more specifically, the purpose of this invention is to provide the transgenic animal of use expressing TRPML3 or mutant forms (such as the sudden change that causes the Varitint-waddler phenotype) at antagonist, agonist or the toughener of identifying TRPML3 and research TRPML3 to the measuring method in the screening that comprises the saline taste sense of taste and sodium transport, metabolism and the influence of the different physiologically actives of excretory.

Equally more specifically, the purpose of this invention is to provide the method for using corresponding transgenic animal, wherein the TRPML3 gene " is knocked out " so that illustrate TRPML3 to the sense of taste with to heart or urinary function, particularly the influence that aldosterone generation, sodium metabolism, saline taste impression or vasopressing are discharged.Expect that these animals can comprise the patient's condition relevant with sodium transport and metabolism, such as hypertension, ypotension, fluid retention, heart attack and apoplexy.Therefore the present invention also comprises and uses these animals as disease model be used to assess the potential therapeutical agent that is used for the treatment of or prevents this type of patient's condition.

Equally more specifically, the objective of the invention is to use the TRPML3 polypeptide (comprising the mutant form that produces the Varitint-Waddler phenotype) of mutant form, so that kill or excise specific cells, comprise saline taste gustatory cell, melanocyte, hypophysis or adrenal cells.

Equally more specifically, the objective of the invention is to use the mutant form (comprising the mutant form that produces the Varitint-Waddler phenotype) of the gene of coding TRPML3 polypeptide, so that produce and wherein to have excised the transgenic animal of specific cells, and use these transgenic animal to test potential therapeutical agent and as disease model.

Equally more specifically, the objective of the invention is to use the TRPML3 polypeptide (comprising the mutant form that produces the Varitint-Waddler phenotype) of mutant form as the toxin that kills specific cells.

Equally more specifically, the purpose of this invention is to provide to use regulation and control or treat melanoma, adrenal carcinoma, hypophysis cancer etc. and relate to the patient's condition of melanocyte, such as pigmentation disorder or hypophysis or the relevant sufferer of suprarenal gland with other in conjunction with the molecule (for example molecule of excitement or antagonism or this polypeptide of specific combination) of TRPML3.

Equally more specifically, the reconstitution cell that the purpose of this invention is to provide the saline taste acceptor of expressing the TRPML3 comprise encoding function sodium-ion channel polypeptide or its variant.

Equally more specifically, the purpose of this invention is to provide the mensuration that is used to identify excitement, antagonism or strengthens the active compound of TRPML3, it comprises that the reorganization of expressing TRPML3 or the endogenous sense of taste or other cells contact with TRPML3 toughener, agonist or the antagonist of supposition and determines that it is to the active influence of TRPML3.Preferably, these mensuration will be electrophysiological detections, and for example patch clamp or two electrode voltages pincers are measured, its can be automatization and generally will use Mammals or Amphibians cell.

Equally more specifically, the purpose of this invention is to provide by ionic current and measure the method for identifying the TRPML3 conditioning agent.

Equally more specifically, the purpose of this invention is to provide the method for identifying TRPML3 conditioning agent (toughener, co-inhibitor) by automatic electric physiology (patch clamp) mensuration (being that IonWorks measures system).

Equally more specifically, the purpose of this invention is to provide the method for identifying TRPML3 conditioning agent (toughener, co-inhibitor) by the electrophysiological detection that uses the frog ovocyte.

Equally more specifically, the purpose of this invention is to provide the method for identifying TRPML3 conditioning agent (toughener, co-inhibitor) by the electrophysiological detection that uses mammalian cell.

Equally more specifically, this type of TRPML3 that the purpose of this invention is to provide agonist, antagonist or toughener compound that wherein assessment is identified in taste test measures.

Equally more specifically, this type of TRPML3 that the purpose of this invention is to provide wherein the influence that in the animal agonist of Test Identification, antagonist or toughener compound produce aldosterone measures.

Equally more specifically, this type of TRPML3 that the purpose of this invention is to provide wherein the influence that in the animal agonist of Test Identification, antagonist or toughener compound discharge vasopressing measures.

Equally more specifically, the purpose of this invention is to provide wherein in the animal agonist of Test Identification, antagonist or toughener compound measures this type of TRPML3 of at least a of heart or urinary function and the influence that more specifically blood pressure, fluid retention, sodium metabolism or urine produced.

Equally more specifically, agonist, antagonist or the toughener compound that the purpose of this invention is to provide evaluation is used for the treatment of and relates to the disease that aldosterone produces or the application of the patient's condition, and it comprises the compound of the regulation and control TRPML3 that uses significant quantity.

Equally more specifically, agonist, antagonist or the toughener compound that the purpose of this invention is to provide evaluation is used for the treatment of and relates to the disease that vasopressing discharges or the application of the patient's condition, and it comprises the compound of the regulation and control TRPML3 that uses significant quantity.

Equally more specifically, agonist, antagonist or the toughener compound that the purpose of this invention is to provide evaluation be used to regulate and control to have in requisition for curee's the application of heart function (for example blood pressure, arrhythmia or apoplexy or fluid retention), it comprises the compound of the regulation and control TRPML3 that uses significant quantity.

Equally more specifically, the gustatory cell sample that the purpose of this invention is to provide isolating gustatory cell, adrenal cells, pituicyte, melanocyte or uropoiesis device cell or enrichment, the cell sample of wherein said separation or enrichment comprise the cell of expressing TRPML3 ionic channel polypeptide.

Equally more specifically, the purpose of this invention is to provide the Taste Receptors of isolating regulation and control saline taste impression, it comprises TRPML3 polypeptide or its variant of the regulation and control Mammals saline taste sense of taste.

Equally more specifically, the purpose of this invention is to provide by genetic modification is to knock out or damage the transgenic nonhuman animal that endogenous TRPML3 expresses, and condition is that described transgenic animal are not the Varitint mouse.

Equally more specifically, the purpose of this invention is to provide by genetic modification is the transgenic nonhuman animal of expressing heterologous TRPML3 polypeptide, and condition is that described transgenic animal are not the Varitint mouse.

Equally more specifically, the purpose of this invention is to provide a kind of method of in the screening of the compound of identifying the regulation and control saline taste sense of taste, using transgenic animal.

Equally more specifically, the purpose of this invention is to provide the method for a kind of antagonist that uses transgenic animal to identify TRPML3, agonist or toughener, wherein further randomly assessment in the test of people's sense of taste of institute's compounds identified.

Equally more specifically, the purpose of this invention is to provide the use transgenic animal and illustrate the method for TRPML3 the influence of aldosterone generation, sodium metabolism, saline taste impression or vasopressing release.

Equally more specifically, the purpose of this invention is to provide use to express coding to the transgenic animal (inhuman) of the TRPML3 gene of the deleterious ionic channel of cell of expressing ionic channel with assessment relate to that unusual aldosterone produces, vasopressing discharges, the method for the treatment plan of the disease of sodium metabolism and/or melanocyte loss or the patient's condition.

The invention still further relates to Varitint waddler mouse and detect of the application of TRPML3 function the influence of melanocyte, hypophysis, suprarenal gland, the sense of taste, uropoiesis or gustatory cell.

The invention still further relates to Varitint waddler mouse and in Varitint waddler mouse, do not express application in the mensuration of gene of (because wherein having excised the saline taste gustatory cell) detecting specifically expressing in the saline taste gustatory cell, the function of the adjustable TRPML3 of this gene perhaps works as the saline taste acceptor or regulates and control growth differentiation or the apoptosis of saline taste sense of taste signal from the transmission of TRPML3 neuralward fiber and/or control saline taste gustatory cell.These gene tests are measured to comprise and are used gene chip or microarray technology to come comparison gene of expressing and the gene of expressing in the saline taste gustatory cell in Varitint waddler mouse.

The present invention also provides the method for treatment parathyroid gland relative disease, described disease such as calcium homeostasis, hypercalcemia, osteitis, hypoparathyroidism, hyperparathyroidism, Engel-Recklinghausen disease, Albright's syndrome to type, Jansen metaphysis cartilage generate, the Blomstrand cartilage generates and the osteoporosis of different reasons, described reason such as disease, age, menopause, chemotherapy, radiotherapy, medicine and similar reason.

Equally more specifically, the purpose of this invention is to provide the reconstitution cell of expressing the saline taste acceptor that comprises TRPML3 or its variant, for example, yeast, Amphibians, insect, bacterium, Reptilia, bird or mammalian cell, preferably mammalian cell or frog ovocyte, but such as CHO-K1, HEK-293, COS, CHO, the bhk cell of described TRPML3 polypeptide of transient expression or the described TRPML3 polypeptide of stably express.

Equally more specifically, the purpose of this invention is to provide a kind ofly in conjunction with the method for identifying the saline taste sense of taste conditioning agent of inferring in measuring, it comprises: the TRPML3 polypeptide is provided or expresses the cell of TRPML3 and make described polypeptide or cell contacts with the TRPML3 adjusting compound of supposition and identify potential TRPML3 conditioning agent based on the particular combination of itself and TRPML3 polypeptide.

Equally more specifically, the purpose of this invention is to provide a kind of regulation and control have in requisition for curee's blood pressure or the method for fluid retention, it comprises the compound of the regulation and control TRPML3 that uses significant quantity.

Equally more specifically, the purpose of this invention is to provide a kind of regulation and control have in requisition for curee's urine produce and/or the excretory method, it comprises the compound of the regulation and control TRPML3 that uses significant quantity.

Equally more specifically, the purpose of this invention is to provide a kind of treatment have in requisition for curee's Addison's disease or the method for the glutinous lipidosis (mocolipidosis) of IV type, it comprises the compound of the regulation and control TRPML3 that uses significant quantity.

Equally more specifically, the purpose of this invention is to provide specific codon optimized and the sequence of TRPML3 sudden change and the mensuration of using these sequences.

Equally more specifically, agonist, antagonist or the toughener compound that the purpose of this invention is to provide evaluation like this be used to regulate and control to have in requisition for curee's urine generation and/or the application of drainage or oedema, it comprises the compound of the regulation and control TRPML3 that uses significant quantity.

Equally more specifically, the agonist, antagonist or the toughener compound that the purpose of this invention is to provide this type of evaluation that can comprise polypeptide, antibody, small molecules, siRNA, sense-rna, ribozyme etc.

More specifically, the mensuration (preferably high or middle isoflux) that the purpose of this invention is to provide based on Mammals and ovocyte is used for analyzing and screening saline taste acceptor (TRPML3), and this mensuration randomly can comprise the compound that adds regulation and control TRPML3 function.These class methods can be used to characterize functionally the activity of TRPML3, and identify required specific motif or the residue of salt impression in the different TRPML3 ionic channels, and identify the compound (this paper is called saline taste sense of taste conditioning agent) that strengthens or prevent the saline taste impression.

Equally, the purpose of this invention is to provide by using the TRPML3 conditioning agent and treat or prevent to relate to the novel method of sodium transport and the metabolic patient's condition (such as hypertension, ypotension, fluid retention, heart attack and apoplexy) and the patient's condition mentioned above.

Aspect specific, the TRPML3 that the invention provides a kind of mammalian cell that utilizes expressive function TRPML3 sodium channel or ovocyte and supposition regulates the method that compound is identified the conditioning agent of TRPML3, and it comprises: (i) measure the influence of described compound to the sodium transport by the TRPML3 passage; And (ii) identify based on its enhancing or inhibition influence whether described compound is the TRPML3 conditioning agent to sodium transport.The present invention also comprises the (iii) affirmation compounds identified regulation and control saline taste sense of taste in people or Mammals taste test.In one embodiment, described TRMPL3 is mammiferous TRMPL3.In another embodiment, TRMPL3 is the TRMPL3 of people, non-human primate, rodent (mouse or rat), ox, pig, horse or sheep.

In further embodiment, the compound of Test Identification is to sodium extraction or urinary function or cardiovascular or relate in the body of other functions of TRPML3 and influencing in people or Mammals.In one embodiment, described TRMPL3 is mammiferous TRMPL3.In another embodiment, TRMPL3 is the TRMPL3 of people, non-human primate, rodent, ox, pig, horse or sheep.

In one aspect of the invention, mammalian cell is selected from the group of being made up of HEK293, HEK293T, Swiss3T3, CHO, BHK, NIH3T3 and COS cell.Aspect second, ovocyte is Mammals, Amphibians, fowl or Reptilia ovocyte.Further, the Amphibians ovocyte is Xenopus laevis (Xenopus) ovocyte.In another aspect of this invention, other sense of taste gene, preferably ionic channels of cell expressing.

In the embodiment that the present invention is correlated with, these mensuration are used for identifier TRPML3 toughener or inhibitor, wherein ovocyte with contact with inhibitor or the activator of people TRPML3 before the people TRPML3 toughener of inferring contacts.In other embodiments, this mensuration comprises that also use is not by the negative control of the ovocyte of microinjection people TRPML3 cRNA.In another aspect of this invention, the conditioning agent of supposition is used with the concentration of scope from about 1nM to about 1mM.In another aspect of this invention, people TRPML3 toughener shows at least 20% reinforcing coefficient.Further, people TRPML3 toughener shows at least 50% reinforcing coefficient.Further, people TRPML3 toughener shows at least 100% reinforcing coefficient.

Equally more generally, the purpose of this invention is to provide the method or the principle of gene that a kind of identification code participates in the polypeptide of primate (people or inhuman) saline taste impression, it comprises:

(i) identify a cover gene, it is included in fungi shape and randomly expression and gene that the gene of not expressing and/or the expression level in gustatory cell are significantly higher than the expression level in the tongue cell in profile shape, the lobate or palate gustatory cell in the tongue cell;

(ii) in the genes identified, randomly identify a cover gene in (i), it is not expressed in the gustatory cell of the sign (T1R or T2R, TRPM5 and PKD2L1/PKD1L3) of expressing delicate flavour, sweet taste, bitter taste or tart flavour Taste Receptors or these cells;

(iii) randomly identify be included in step (i) or step (ii) after the subgroup of the sense of taste specific gene of genes identified in belonging to, it is not expressed in lower part of taste bud at upper part specifically expressing of taste bud, perhaps its upper part of taste bud than lower part enrichment of taste bud (at least 1.2-1.5 doubly more the highland express); And

(iv) expression basis functionally (ii) or also definite which the described gene of one or more genes of (iii) identifying responds ionic channel as sodium or sodium responds acceptor or transhipment works, so identify that this gene is as the supposition gene of regulating and control the saline taste sense of taste.

(preferably, the genes identified of functionalization is at upper part enrichment of taste bud 1.2-1.5 gene doubly at least with respect to lower part of taste bud).

In these methods, step (i) preferably includes uses laser capture micro-dissection (LCD) to come to dissect and the purifying taste tissue from non-taste tissue, and/or step (i) comprises the gene of RNA amplification from gustatory cell and tongue cell, the gene chip screening that contains the sample that is specific to the specific mammalian genes that obtains the sense of taste and ligule tissue of the gene of amplification.

Further, in these methods, step (i) preferably includes the high-throughput PCR of use at the primer of people or genomic each ionic channel of non-human primate, and step (ii) preferably is specific to the in situ hybridization of the antisense RNA probes of genes identified in the step (i) by use so that determine expression level in gustatory cell and tongue cell, or is specific to immunocytochemistry by the antibody of the mark of step (i) genes identified encoded protein by use and detects and finish.

Equally more generally, the purpose of this invention is to provide the method for gene that a kind of identification code participates in the polypeptide of primate (people or inhuman) saline taste impression, it comprises:

(i) identify a cover gene, it is included in the gene that the gene of expressing in fungi shape, profile shape, the lobate or palate gustatory cell and not expressing and/or the expression level in described gustatory cell are significantly higher than the expression level in the tongue cell in the tongue cell;

(ii) in the genes identified, identify a cover gene in (i), it is not expressed in the gustatory cell of the sign (T1R or T2RTRPM5 or PKD2L1/PKD1L3) of expressing delicate flavour, sweet taste, bitter taste or tart flavour Taste Receptors or these cells;

(iii) at (i) or (ii) in the genes identified, identify randomly whether this gene is expressed and do not expressed in lower part of taste bud in upper part of taste bud specifically, perhaps with respect to its upper part enrichment of expression (doubly more highland expression of 1.2-1.5 at least) at taste bud in taste bud lower part; And

(iv) determine that (ii) which described gene works as sodium response ionic channel or sodium response acceptor or transhipment, therefore identifies the supposition gene of this gene as the regulation and control saline taste sense of taste in the primary neuronal of genes identified expressing one or more bases.

(same, the sense of taste specific gene of the selection of functionalization will preferably by enrichment, promptly the high 1.2-1.5 of lower part times of the expression contrast taste bud of upper part of taste bud.)

Equally more specifically, the purpose of this invention is to provide and a kind ofly identify that potential terrain planted agent is used to regulate and control the mensuration of the compound of people's saline taste sense of taste, it comprises following:

(i) cell of gene of expressing the independent TRPML3 ionic channel of coding or having NALCN, NKAIN3, TRPML1 or TRPML2 or directly having a polypeptide of at least 90% sequence identity to the gene of homologue or coding and its encoded polypeptides is contacted with antagonist, agonist or the toughener compound of at least a supposition;

(ii) under the situation of the agonist, antagonist or the toughener that have and do not exist described supposition, measure sodium conduction, receptor active or sodium transport; With

(iii) whether regulate and control the sodium conduction and identify that with other transport properties consistent this compound is a potential saline taste sense of taste toughener with people's saline taste acceptor based on compound.

Equally more specifically, the purpose of this invention is to provide the method that probe that a kind of use is specific to TRPML3 gene or gene product comes in gustatory cell sample evaluation and/or separation and/or enrichment saline taste specific cell (preferably primate alt expression of receptor cell).

Equally more specifically, the purpose of this invention is to provide the application that comes the gustatory cell hypotype or the gustatory cell pedigree of purifying or enrichment needs with TRPML3, it comprises the magnetic bead that uses fluorescence-activated cell sorter (FACS) or applying marking.

Equally more specifically, the purpose of this invention is to provide the application that comes the gustatory cell hypotype or the gustatory cell pedigree of purifying or enrichment needs with TRPML3, gustatory cell hypotype that wherein needs or gustatory cell pedigree are separated by the method that comprises negative cells selection technology, purifying, enrichment or mark, expression or expression based at least one other gustatory cell specific gene lack, and described negative cells selects technology for example by using the cytotoxic antibody that kills at least one non-target cell type or pedigree specifically to eliminate at least one non-target gustatory cell hypotype or pedigree.

Equally more specifically, the purpose of this invention is to provide use separately or with the TRPML3 of other sense of taste specific genes (for example TRPML1, TRPML2, NALCN and/or NKAIN3) as a token of identify, the method for separation or enrichment saline taste recipient cell.

Based on preamble, can understand the result that the present invention partly is new departure of identification of taste specific gene.These genes are to use two kinds of different technology (gene chip and polymerase chain reaction (PCR) screening) to identify, so that identify new saline taste receptor target gene.At first, the Affymetrix gene chip that use contains nearly all known macaque gene is determined which gene specifically expressing in the fungi shape nipple gustatory cell of the primate profile shape gustatory cell of tongue rear face and tongue front, and is not expressing by the isolating tongue epithelial cell of laser capture micro-dissection.Second, use PCR to determine which ionic channel (passage of in people/macaque genome, making a catalogue from us) specifically expressing in macaque fungi shape and/or profile shape (CV) nipple gustatory cell, and by the isolating tongue epithelial cell expression of laser capture micro-dissection.In addition, in these genes, taste bud upper part specifically expressing or enrichment (with 1.2-1.5 at least doubly more the highland express) subgroup be accredited as particularly preferred functionalization candidate gene.By the specific expressed use of the sense of taste of any method genes identified independently Histological method's (such as in situ hybridization or immunohistochemistry) confirm express in gustatory cell so that determine which gene.Use double-tagging Histological method, determine which new sense of taste specific gene in sweet taste, bitter taste and the umami cells of expressing sense of taste specific ion passage TRPM5, express in the tart flavour cell of sense of taste specific ion passage PKD2L1/PKD1L3 or do not express in unique cell type of TRPM5 or PKD2L1/PKD1L3 and express.The sense of taste specific gene (preferably ionic channel) that can conduct sodium or be activated by sodium and in TRPM5-and PKD2L1/PKD1L3-negative cells colony, express be possible be used for screening effort candidate gene in case the gene of identification code Mammals saline taste acceptor and wherein these saline taste acceptor genes and be used for designing and be used for the high throughput assay of identifier's saline taste sense of taste toughener such as the particular cell types of expressing at oral cavity and urethra.Use these general methods, TRPML3 is accredited as potential saline taste acceptor.

Use these principles and identify and influence salt experience (with may be affected thus such as sodium absorption, transhipment and excretory other biological active and such as the influence of fluid retention and blood pressure regulation) new sense of taste specific gene generally can influence the impression of other sense of taste forms and taste alternatively.Although the inventor identifies TRPML3 as the saline taste acceptor, and have convictive performance data support, expection TRPML3 participates in all non-sense of taste biological functions as discussed above.Therefore, this gene is a target useful in the therapeutic screening assay, for example identify to be used for the treatment of the treatment of diseases agent relevant with TRPML3, described disease such as Addison's disease, such as mucus (mocoid) illness, uropoiesis illness and the cardiovascular disease of the glutinous lipidosis of IV type with sodium transport, metabolism and drainage and vasopressing or aldosterone discharges or generation is relevant pathology.

The sense of taste specific gene that the present invention generally also relates to invention and its specific probe comprise positive and negative cells separation method separate and purification process in application.For example, gustatory cell pedigree that needs or type can be separated by the positive cell system of selection, for example by using fluorescence-activated cell sorting (FACS), magnetic bead cell to select, for example pass through cell, for example single cells transfected such as the electric physiology method visual identification needs of the pearl of using antibody sandwich.Alternatively, the gustatory cell pedigree that needs or type can by the negative cells purifying and separation method reclaims or purifying, the cell type that wherein needs comes enrichment or purifying by removing one or more unwanted cells pedigrees from the blended cell colony, for example by the sense of taste (saline taste) cell that contains needs and the blended cell suspension (for example from tongue, oral cavity or gi tract and relevant organ) of unwanted cells are contacted with the cytotoxic antibody that is specific to the target gene of expressing on unwanted gustatory cell type to be removed.

The present invention generally also relates to the application of the sense of taste specific gene of invention, it participates in the specific sense of taste and non-sense of taste exceptional function, be included in express specific sense of taste specific gene and therefore participate in gustatory cell specific function disclosed herein one or more gi tract and the cell drawing of the specific region of relevant organ (such as enteric epithelium or urethra), and/or in gustatory cell differentiation research, use this gene and its special sign, for example be used to identify the compound of inducing gustatory cell differentiation or dedifferentiation, for example adult or embryonic stem cell and other multipotencys or immature cell type are divided into gustatory cell pedigree and the gustatory cell type that needs.

Equally more specifically, as hereinafter describing in detail, the present invention provides the principle and the standard of candidate's saline taste sense of taste gene (preferably ionic channel) more widely, and it is:

A) at primate (macaque) gustatory cell specifically expressing in fungi shape and/or profile shape nipple deutero-gustatory cell and lobate and the palate gustatory cell particularly, and do not express at the tongue epithelial cell, perhaps the expression level in gustatory cell is higher than the tongue cell.

B) by the expression in the gustatory cell of Histological method.Particularly, in unique gustatory cell type of not expressing sweet taste, bitter taste and umami cells sign TRPM5 or tart flavour cell sign PKD2L1/PKD1L3, express.This uniqueness cell type may be corresponding to the gustatory cell pedigree of uniqueness, for example special salt impression or fatty recipient cell.

C) in heterologous expression system (such as xenopus leavis oocytes and mammalian cell) or primary neuronal (such as dorsal root ganglion neurons) as the sodium channel with essentially consist sexual function (be part passage colony open and when static, pass through sodium) or the functional expression of sodium activated receptor.

D) randomly, in the top of taste bud cell particular expression or enrichment, preferably taste bud upper part contrast lower part doubly higher expression of 1.2-1.5 at least.

The gene that satisfies these standards further carries out high flux screening and makes great efforts so that identify the compound that strengthens the impression of people's salt.These methods with express mutant TRPML3 gene and produce external functional examination in the mouse of Varitint-waddler phenotype and the coupling of nervous physiology data and disclosed the saline taste acceptor that TRPML3 is primate (people) and non-human primate, other animals of most probable, described other animals comprise for example other Mammalss, such as dog, cat, horse, ox, pig, sheep and other vertebratess.

More specifically, as hereinafter describing in detail, the invention provides the principle and the standard of candidate's saline taste sense of taste gene (preferably ionic channel), it is:

A) at primate (macaque) gustatory cell specifically expressing in fungi shape and/or profile shape nipple deutero-gustatory cell and lobate and the palate gustatory cell particularly, and do not express at the tongue epithelial cell, perhaps the expression level in gustatory cell is higher than the tongue cell.

B) by the expression in the gustatory cell of Histological method.Particularly, in unique gustatory cell type of not expressing sweet taste, bitter taste and umami cells sign TRPM5 or tart flavour cell sign PKD2L1/PKD1L3, express.This uniqueness cell type may be special salt recipient cell.

C) in heterologous expression system (such as xenopus leavis oocytes and mammalian cell) or primary neuronal (such as dorsal root ganglion neurons) as the sodium channel with essentially consist sexual function (be part passage colony open and when static, pass through sodium) or the functional expression of sodium activated receptor.

Summary of the invention

Use the new principle (it is required protection in the related application of submitting on the same day with the application) of disclosed identification of taste specific gene in the early stage temporary patent application incorporate this paper by reference into; the inventor has identified the sense of taste specific polypeptides that works as primate (people) saline taste receptor polypeptides; it participates in relating to other physiological functions of sodium transport, absorption and excretory probably, such as uropoiesis and heart function.

Especially, the inventor has identified the gene of glutinous fat 3 (MCOLN3) of optional called after or TRPML3, the membrane-spanning proteins that express on its taste bud top that is coded in the taste perception cell of conduction sodium.Various evidences illustrate that convictively this polypeptide is as primate saline taste receptor polypeptides.

Particularly, this gene itself and/or represent the saline taste acceptor together such as TRPML1, TRPML2, NALCN or NKAIN3 with other sense of taste specific polypeptides or ionic channel (relevant family member), it allows the gustatory cell of experiencing of the taste bud of tongue to detect sodium-chlor (salt).In addition, because this gene, expects reasonably that it is played a positive role at suprarenal gland and hypophysis high expression level in the sodium metabolism of regulating body.Below the evidence that shows this gene behaviour saline taste acceptor comprises at least:

(1) new principle of the gustatory receptor genes of use to identify inferring, the inventor determines that MCOLN3 is not expressing TRPM5 (being that it is not sweet taste, bitter taste or delicate flavour), do not expressing PKD2L1 (being that it is not a tart flavour) and towards the taste bud top specifically expressing of the taste perception cell subsets of taste pore.

(2) known MCOLN3 also expresses in the recipient cell such as other organs of ear.Therefore, it is the recipient cell of ' specialty '.

(3) further known MCOLN3 is at the suprarenal gland strongly expressed.These bodies of gland are brought into play important effect in the sodium metabolism of regulating body.Therefore, MCOLN3 regulates the metabolic key molecule of sodium, and can regulate suprarenal gland generation aldosterone.

(4) relevant with preamble, also the feature of known person autoimmune disease (Ai Dishengshi) is adrenal destruction.An indication symptom of this disease is that salt is craved for.The latter may result from existence at the autoantibody of MCOLN3 or destroy the sudden change of this gene of MCOLN3 function in the taste bud.

(5) also known MCOLN3 or TRPML3 high expression level in the hypophysis that produce to participate in the vasopressing that urine produces, this has further confirmed the possible effect in sodium excretion and urinary function of this gene.

(6) confirm MCOLN3 conduction sodium and show biochemical character prediction and consistent with people's saline taste acceptor (detecting K+, Li+ and guanamprazine insensitivity) in electrophysiologic study as the data that are included in the EXPERIMENTAL EXAMPLE hereinafter.

(7) in varitint-waddler mouse (having the TRPML3 sudden change), use the nervous physiology experiment (neural record) of sodium to show that the Varitint mouse is to the response of sodium impaired (not showing the intensive saline taste replys).In addition, confirmed these same mouse cut express the gustatory cell (saline taste gustatory cell) of TRPML3 or MCOLN3, the gustatory cell subgroup of the uniqueness of the taste bud cell of this further definite expression TRPML3 has function, and promptly they are prerequisites of saline taste impression.

(8) use the mammalian cell of expression mutation T RPML3 polypeptide (sudden change causes ionic channel to be fixed in the open to the outside world orientation) and the mensuration based on cell of Amphibians ovocyte to identify the TRPML3 toughener and the co-inhibitor that in the saline taste test, should strengthen or prevent the saline taste sense of taste.

This discovery is extremely important, because identification of M COLN3 or TRPML3 are used to identify the purpose of agonist, antagonist or the toughener (conditioning agent) of this molecular function for the saline taste acceptor (and inferring it may is other Mammalss or vertebrate saline taste acceptor) of people and other primates and rodent allows design to use the screening assay of the cell of this gene transfection.These compounds can be used as sense of taste conditioning agent, and also can be used as and be used for the treatment of and regulate and control heart and uropoiesis correlation function and the patient's condition such as high or low blood pressure, apoplexy, heart attack, arrhythmia, fluid retention, unusual sodium and other ionic metabolism and excretory therapeutical agent.

As mentioned, this gene originally be to use be intended to identify the full genome strategy of the gene of people's taste bud top specifically expressing find (referring to provisional application, it incorporates this paper by reference into, and it identifies the sense of taste specific gene of TRPML3 as rodent and primate).The inventor is early stage also to determine that from previous experiment the taste bud top contains the known gustatory receptor genes of overexpression and other sense of taste specific genes cell of (comprising similarly the sodium-ion channel TRPML3 in the enrichment of taste bud upper part).On the contrary, the precursor cell of experiencing the taste bud cell that is positioned at taste bud top is contained in the bottom of taste bud.This database allows the inventor to identify the gene of many taste buds top (impression) cell specific expression, comprises TRPML3.

The inventor points out that further the previous report of one of these genes (MCOLN3 or TRPML3) is responsible for being called the phenotype of the mouse mutant of varitint-waddler when the sense of taste specific gene of look back identifying, this mouse shows as the early onset hearing loss, the vestibular defective, (Dipalma etc., Mutations in Mcoln3 associated withdeafness and pigmentation defects in variant-waddler (Va) mice. (the Mcoln3 sudden change relevant with the pigmentation defective with the deafness of variant-waddler (Va) mouse) Proc.Natl.Acad.Sci USA 99:14994-14999 cause death pigment anomaly and perinatal period; 2002).As pointing out in the background of invention, MCOLN3 or TRPML3 express in the plasma membrane of hair cell and stereocilium (in the ear), and the sudden change special event that the ala 419 that causes the 5th membrane spaning domain becomes the replacement of pro causes highly active MCOLN3, it causes the necrocytosis of expressing this molecule, hair cell (therefore causing the deafness of Va mouse) (Grimm C etc., ProcNatl.Acad.Sci.USA 104:19583-8 such as ear; 2007).

Illustrate TRPML3 as primate and may other mammiferous saline taste acceptors based on the inventor, predict that further this mouse also will show saline taste parageusia (because unusual MCOLN3 molecule and may damage the influence of saline taste impression in tongue taste bud cell).In fact, this point that the inventor is verified.The inventor uses the mouse (varitint mouse) of expressing this mutation T RPML3 gene to carry out nervous physiology research (describing in the EXPERIMENTAL EXAMPLE hereinafter), and confirm as wish and expection that these mouse show impaired to replying of stimulating of saline taste (as stimulating the nerve record result of these mouse to be proved wherein normally will observe the concentration that the positive neurons record replys with saline taste).Use the neural record of CT, show that Varitint waddler mouse shows the sodium-responsible defective of chlorination.Particularly, the benzyl guanamprazine of Varitintwaddler mouse-insensitive CT nerve has weakened replying greatly of sodium-chlor.

The taste bud that the inventor also has molecule and immunohistochemistry data to disclose these same mouse has excised the taste bud cell of expressing TRPML3.This has confirmed the inventor's supposition: this unique gustatory cell subgroup participates in detecting saline taste and is the prerequisite of saline taste impression.

Therefore, the inventor has the body internal evidence to it is confirmed that as drawing a conclusion: the existence of functional TRPML3 ionic channel in specific gustatory cell (" specialty " saline taste gustatory cell) is the prerequisite of rodent and other Mammalss of possibility (comprising the most special people and other primates) saline taste impression.

In addition, because the inventor uses public database to determine MCOLN3 strongly expressed in suprarenal gland and hypophysis, this has further supported the inventor's discovery and has shown this gene and detection specifically or this gene of target and/or regulate and control other application of the compound of its function.The fact that this gene is expressed in suprarenal gland and hypophysis is crucial observation, because suprarenal gland is represented one of main sodium metabolism setter of body.The salt level of these body of gland monitoring of blood, justacrine keeps sodium and discharges blood pressure and the water salt equilibrated aldosterone (mineralocorticoid) that potassium is regulated body by helping kidney.When the generation decline of aldosterone was too low, kidney can not be regulated the salt solution balance, causes Q volume of blood and blood pressure drops.

Hypophysis also produces vasopressing (AVP), and it is the hormone that participates in sodium level in the urine and play a role in by urine excretion at sodium.Especially, AVP most important effect is to regulate the body retained water; When body dewatered, it was released, and caused that kidney keeps water, so concentrated urine and reduce the urine amount.It also improves blood pressure by the vasoconstriction of inducing appropriateness.

In addition, AVP has increased in the nephron of kidney distal convoluted tubule and has collected the permeability of tubule to water, therefore allows heavily absorbing and the drainage-antidiuresis of the concentrated urine of small volume more of water.This is to insert the epithelial top of uriniferous tubules/collection tube film by other aquaporin (aquaporin-2) to take place.This aquaporin permission water is by the nephron (at distal convoluted tubule and conduction tubule) and enter cell, and this has increased from the re-absorbed water yield of filtrate.

AVP has also increased the permeability of the marrow sample part of collection tube to urea, and the urea that allows to increase heavily absorbs into matter between the marrow sample, has reduced the concentration gradient that produces by removing the water in the cortex collection tube.In addition, the effect of another kidney of AVP is its heavily absorption that thickens sodium in the ring that stimulates gland of Henle.Therefore, find that based on the inventor TRPML3 participates in saline taste and detects, surprisingly TRPML3 does not express in producing two bodies of gland that very obviously participate in sodium transport and excretory polypeptide, and this gene also relates to other processes of sodium transport, absorption and excretory in regulation and control except participating in the saline taste impression, particularly plays a positive role in the process by vasopressing or aldosterone adjusting.

Therefore, based on these observations with illustrate this gene as the saline taste acceptor, the further identification of M COLN3 of the inventor is crucial salt/sodium monitoring molecule, the generation of aldosterone and/or regulate hypophysis and discharge vasopressing in the control suprarenal gland.

In tongue, be accredited as the molecule that detects the Taste Receptors that saline taste stimulates as expection, MCOLN3 or TRPML3 are responsible for detecting the saline taste recipient cell Expression of Subsets of the saline taste sense of taste by being positioned at the taste bud top.Therefore, the invention provides strong evidence and prove that MCOLN3 or TRPML3 detect and regulate the vital role in the salt in various tissues.

Although reported before that MCOLN3 was the sodium conduction pathway, have many sodium-ion channel polypeptide, and this passage before be not considered to participate in the saline taste impression or regulates sodium metabolism, drainage, transports or relate to the sodium correlated process of vasopressing and aldosterone.Therefore the present invention has constituted new unexpected discovery, because it provides the new application (saline taste acceptor) of known (MCOLN3).In hypophysis, the inventor is based on its discovery and be contained in this paper and confirm that TRPML3 further contemplates that as the data of the effect of saline taste acceptor MCOLN3 further may participate in regulating the release of vasopressing.As mention, be the crucial regulon that urine produces by its vasopressing that influences to kidney.Importantly, known vasopressing is subjected to the NaCl concentration adjustment from postpituitary release.This albumen is at the hypophysis high expression level.Therefore, based on its expression at hypophysis, by it vasopressing is discharged possible influence, TRPML3 may regulate the NaCl metabolism of body by its influence to fluid retention, NaCl impression and concentration and blood pressure.

Therefore, in one embodiment, the present invention has identified that MCOLN3 or TRPML3 are as people's saline taste acceptor, and provide the screening assay of the cell that uses this gene transfection based on this, be used to identify agonist, antagonist or the toughener (conditioning agent) of this molecular function, it will regulate and control saline taste sense of taste TRPML3 function relevant with other sense of taste and non-sense of taste correlation function, such as the function of the pathology patient's condition relevant with abnormal T RPML3 expression that relates to sodium excretion in the different tissues, metabolism and transhipment and identify such as this paper.

More specifically, in another embodiment, the invention provides the gustatory cell of separation and purifying, adrenal cells, pituicyte or uropoiesis device cell or comprise and express to participate in the saline taste impression, the sodium metabolism, aldosterone produces, and/or the gustatory cell of the TRPML3 of vasopressing release, adrenal cells, the gustatory cell sample of the enrichment of pituicyte or uropoiesis device cell, wherein said isolating gustatory cell, adrenal cells, the gustatory cell sample of pituicyte or uropoiesis device cell or enrichment is expressed the coding regulation and control saline taste sense of taste, the sodium metabolism, TRPML3 gene or its variant of at least a sodium channel that aldosterone generation and vasopressing discharge.Preferably, described cell will be people, non-human primate or rodent source.

In another embodiment, the present invention also provides the isolating Taste Receptors of regulation and control saline taste impression, and it comprises the mammiferous saline taste sense of taste that the TRPML3 polypeptide that is used in the mensuration that is used for identifying the TRPML3 conditioning agent or its variant and/or the regulation and control of described Taste Receptors polypeptide comprise the people.This Taste Receptors can be monomer or polymer (with aggressiveness or different aggressiveness), and can comprise other sense of taste specific polypeptides, and other ionic channel polypeptide for example are such as NKAIN3 or NALCN or such as the relevant ions passage of TRPML1 or TRPML2.This TRPML3 polypeptide or nucleotide sequence can be Mammals or other source of species.As mention, consider the sodium metabolism and drain, directly in homologue may and be drained in different Mammalss and saline taste impression and salt (sodium) metabolism that may other vertebratess (such as Reptilia, Amphibians and fowl), the playing a role of this gene and various species thereof organic homeostasis and healthy importance.

The TRPML3 gene can be a wild-type according to the present invention, can is that introducing influence (strengthening or inhibition) ion channel function and/or non diffusible ion passage are in the sudden change of opening or closing the needs of orientation by genetic modification perhaps.It also can be modified by the preferred codon of host.This type of sudden change is illustrated in this paper, and those skilled in the art can use methods known in the art to design other sudden changes.Therefore, should be understood that the TRPML3 polypeptide can be modified with respect to natural TRPML3 polypeptide herein, and can have 80,85,90,95,96,97,98,99 or higher sequence identity with natural TRPML3 polypeptide or function fragment.In addition, this TRPML3 polypeptide can comprise chimeric ionic channel, and promptly one or more structural domains of wherein endogenous TRPML3 ionic channel or zone are by the ionic channel (TRPML1 or TRPML2) of relevant (for example directly to homologue) ionic channel, same TRPML family or for example the counter structure territory or the zone of another ionic channel of another sodium channel (such as NALCN or NKAIN3) replace.

These mosaics can make up based on known TRPML3 albumen topological framework.This topological framework is illustrated in following.

In this diagram, membrane spaning domain classifies 1 to 6 as.Amino (N) and carboxyl (C) are in cell.In addition, between extracellular TM1 and TM2, there is big born of the same parents' outer shroud.There is the mosaic of function (still in response to sodium) for example to make up across the born of the same parents outer region of TM1 and TM2 or by corresponding TM replacement TM by born of the same parents outer region replacement potentially with another ionic channel with another TRPML3 or another ionic channel polypeptide.Mosaic also can prepare between people and mouse TRPML3, and wherein the big born of the same parents' outer shroud between TM1 and the TM2 is exchanged.

Residue around porose area and the TM5 also can for example be modified by the corresponding residue of other ionic channels potentially.

Based on comparison and comparison (Figure 35) from the protein sequence of people (NM_018298) and mouse (NM_134160) TRPML3 sequence, wherein the people is expressed as Hs, and mouse is expressed as Mm, can find that these proteic 91% are identical, and the 96%th, similar.This has confirmed the inventor's supposition (reasonably): consider the important physiological function of its regulation and control, TRPML3 is different Mammalss and potentially may be very conservative in other vertebratess, and can be built with the mosaic and the mutant of function.In fact, this application contains the sequence of bird and fish TRPML3 gene in the sequence table before the claim.Use these and other TRPML3 genes can identify that as probe other are directly to homologue.

Comparison among the figure (Figure 35) has shown that similarly 6 membrane spaning domains of the TRPML3 polypeptide of people and mouse TRPML3 are underlined TM1 to TM6.Porose area between TM5 and the TM6 is expressed as ' porose area '.Diagram predicts that amino and C-terminal are positioned at cell as mentioned.When making up mutant, the residue of needs maintenance porose area is complete or modify considerably less residue, if there be the corresponding residue of these modifications corresponding to the residue that is present in other TRPML3 polypeptide porose areas or TRPML1 or TRPML2 porose area.

A419P sudden change locking TRPML3 this paper discussion and that be found in the varitint-waddler mouse is in open conformation, and it is positioned at TM5, highlights in Figure 35.

As hereinafter embodiment discussion and demonstration, this sudden change is used in the mensuration of TRPML3 conditioning agent (co-inhibitor), can be used on especially in the FLIPR mensuration.Another sudden change V412P partly activates TRPML3, represents with red-violet colour.This sudden change has strengthened the activity of TRPML3, and can be used to screen toughener in FLIPR measures.Other sudden changes also can produce around TM5 and porose area potentially so that change the activity of TRPML3 ionic channel and produce the active channel that can be used for high flux screening.

The present invention is general use comprise the TRPML3 polypeptide that uses wild-type or sudden change or wherein codon according to wherein expressing the mensuration that the host cell (for example people's cell) that takes place has carried out the TRPML3 polypeptide optimized.Yet, in some instances, need determine that the institute of TRPML3 is influential in the body by eliminating the TRPML3 polypeptide expression.Therefore, in another embodiment, the invention provides a kind of transgenic nonhuman animal, it is to have knocked out the expression of endogenous TRPML3 or expressed non-functional TRPML3 polypeptide by genetic modification.

In another embodiment, the present invention also provides a kind of transgenic nonhuman animal, it is expressing heterologous TRPML3 polypeptide or sudden change or chimeric TRPML3 polypeptide by genetic modification, so that can screen the conditioning agent that this animal is identified described allos TRPML3 polypeptide, the TRPML3 of people or other primate TRPML3 or domestic animal (dog, cat etc.) for example.

In another embodiment, the present invention also provides and has used the measuring method (so that screen conditioning agent that this animal identify sudden change or allos TRPML3 polypeptide) of transgenic animal in the screening of antagonist, agonist or the toughener of identifying TRPML3 of expressing sudden change or allos TRPML3.

In relevant embodiment, the present invention has also produced and has contained the transgenic animal of finding in the Varitint-mouse that TRPML3 suddenlys change or other function equivalents suddenly change, so that produce the transgenic animal of the cell (such as saline taste gustatory cell, pituicyte, adrenal cells, melanocyte or uropoiesis device system cells) of having removed particular type expression TRPML3, and the influence of studying these cell ablations in these animals, or use the disease model of these animals as the patient's condition that relates to these excision cells.

In another embodiment, the present invention also provides the wherein TRPML3 gene quilt method of the transgenic animal of " excision " of basis of using, so that illustrate TRPML3 to the sense of taste with to heart or urinary function, the particularly influence that aldosterone generation, sodium metabolism, saline taste impression or vasopressing are discharged.

And, because knocking out, TRPML3 should produce the patient's condition that relates to unusual sodium transport, absorption and drainage and relevant uropoiesis or cardiovascular effect or unusual, these transgenic animal also can be used as the model of these patient's condition potentially, and are used to test potential therapeutical agent and treatment plan.

In a more particular embodiment, the present invention also provides and has used Varitint waddler mouse to study saline taste sense of taste behavior when lacking the TRPML3 gustatory cell.

In a more particular embodiment, the invention still further relates to use Varitint waddler mouse and detect of the influence of TRPML3 function melanocyte, hypophysis, suprarenal gland, the sense of taste, uropoiesis or gustatory cell.

In a more particular embodiment, the invention still further relates to and detecting in saline taste gustatory cell specifically expressing and in Varitint waddler mouse, do not expressing in the mensuration of gene of (because wherein having excised the saline taste gustatory cell) and use Varitint waddler mouse, the function of the adjustable TRPML3 of described gene, or work or regulate and control growth differentiation or the apoptosis of saline taste sense of taste signal from the transmission of TRPML3 neuralward fiber and/or control saline taste gustatory cell as the saline taste acceptor.These gene tests are measured to comprise and are used gene chip or microarray technology to come comparison gene of expressing and the gene of expressing in the saline taste gustatory cell in Varitint waddler mouse.

In a more particular embodiment, the present invention also provides the method for treatment parathyroid gland relative disease, such as the osteoporosis of calcium homeostasis, hypercalcemia, osteitis, hypoparathyroidism, hyperparathyroidism, Engel-Recklinghausen disease, Albright's syndrome to type, the generation of Jansen metaphysis cartilage, the generation of Blomstrand cartilage and different reasons, described reason such as disease, age, menopause, chemotherapy, radiation therapy, medicine and similar reason.

In another embodiment, the present invention also provides the reconstitution cell of expressing the saline taste acceptor, and it comprises TRPML3 or its variant of coding sodium-ion channel polypeptide.

In another embodiment, the present invention also provides and has used A419P TRPML3 polypeptide to kill the specific cell type of expressing TRPML3, for example saline taste gustatory cell, pituicyte, adrenal cells, melanocyte and/or urinary system cell as toxin.

In another embodiment, the present invention also provides sodium transport, metabolism or the excretory of the tagged molecule research body of specific combination TRPML3 to use.

In another embodiment, the present invention also provides the application to specific site (for example saline taste gustatory cell, adrenal cells, melanocyte, pituicyte etc.) of the molecule guiding therapeutical agent of specific combination TRPML3 or diagnostic reagent.

Equally more specifically, in another embodiment, the invention provides the mensuration that is used to identify excitement, antagonism or strengthens the active compound of TRPML3, it comprises that the reorganization of expressing TRPML3 or the endogenous sense of taste or other cells contact with TRPML3 toughener, agonist or the antagonist of supposition and determines that it is to the active influence of TRPML3.Preferably, these mensuration will be electrophysiological detections, and for example, patch clamp or two electrode voltages pincers are measured.

Equally more specifically, in another embodiment, the invention provides and be used for measuring the method for identifying the TRPML3 conditioning agent by ionic current.

Equally more specifically, in another embodiment, the invention provides agonist, antagonist or toughener compound this type of the evaluated TRPML3 in taste test that wherein identifies and measure.

Equally more specifically, in another embodiment, the invention provides agonist, antagonist or the toughener compound wherein identified influence this type of tested TRPML3 in animal that aldosterone produces is measured.

Equally more specifically, in another embodiment, the invention provides agonist, antagonist or the toughener compound wherein identified influence this type of tested TRPML3 in animal that vasopressing discharges is measured.

Equally more specifically, in another embodiment, the invention provides wherein agonist, antagonist or the toughener compound identified measures heart or urinary function and at least a influence this type of tested TRPML3 in animal of more specifically blood pressure, fluid retention, sodium metabolism or urine being produced.

Equally more specifically, in another embodiment, agonist, antagonist or the toughener compound that the invention provides evaluation is used for the treatment of and relates to the disease that aldosterone produces or the application of the patient's condition, and it comprises the compound of using significant quantity regulation and control TRPML3.

Equally more specifically, in another embodiment, agonist, antagonist or the toughener compound that the invention provides evaluation is used for the treatment of and relates to the disease that vasopressing discharges or the application of the patient's condition, and it comprises the compound of using significant quantity regulation and control TRPML3.As mention, these patient's condition comprise the disease and the patient's condition of the TRPML3 modulators for treatment that can use excitement or antagonizing vessel vassopressin as an example, such as diabetes, obesity, such as the ephrosis of cystic kidney disease, acquired cystic kidney disease, such as the eye circular correlation illness of myopia; Feel sick, vomiting, sexual dysfunction (sex), oedema, hypertension, congestive heart failure (scope from the II class of New York heart association to ruddy pulmonary edema), periodically spontaneous oedema, nephrotic syndrome, the ascites that causes by liver cirrhosis or other reasons, the cerebral edema of a variety of causes, and dilutional hyponatremia and the metabolism that is referred to as inappropriate ADH secretion syndrome changes and wherein need the other diseases or the patient's condition of vasorelaxation and/or anti-oxytocic activity in the treatment.

Equally more specifically, in another embodiment, agonist, antagonist or the toughener compound that the invention provides evaluation is used for the treatment of and relates to the disease that aldosterone produces or the application of the patient's condition, and it comprises uses the compound that therefore significant quantity regulation and control TRPML3 regulates and control aldosterone.Can use the disease and the patient's condition of excitement or antagonism aldosterone so excitement or antagonism sodium transport and excretory TRPML3 modulators for treatment to comprise for example oedema, blood pressure (high or low blood pressure), liver cirrhosis, the primary hyperaldosteronemia, renal tubal dysfunction, diabetes (I or II type) and relative pathological symptom, comprise circulatory problems, oedema, the illness in eye relevant with Cycle Difference, high cortisone mass formed by blood stasis, the atherosclerosis or the obesity of abdominal obesity for example, and hepatopathy, sexual dysfunction (sex), cerebrovascular disease, vascular disease, retinopathy, neuropathy, Insulinopathy, endothelial function disturbance, pressure function of receptors obstacle, migraine, hectic fever and premenstrual tension and other cardiovascular patient's condition are such as atherosclerosis, in heart failure, congestive heart failure, vascular disease, apoplexy, myocardial infarction, endothelial function disturbance, ventricular hypertrophy, renal tubal dysfunction, target organ damage, thrombosis, arrhythmia, plaque rupture and aneurysma.

Equally more specifically, in another embodiment, agonist, antagonist or the toughener compound that the invention provides evaluation is used for the treatment of the application of the disease that relates to TRPML3 or the patient's condition (such as Addison's disease or the glutinous lipidosis of IV type).

Equally more specifically, in another embodiment, agonist, antagonist or the toughener compound that the invention provides evaluation be used to regulate and control to have in requisition for curee's the application of heart function (for example blood pressure, arrhythmia or apoplexy or fluid retention), it comprises the compound of using significant quantity regulation and control TRPML3.

Equally more specifically, in another embodiment, agonist, antagonist or the toughener compound that the invention provides this type of evaluation be used to regulate and control to have in requisition for curee's urine produce and/or excretory is used, it comprises the compound of using significant quantity regulation and control TRPML3.

Equally more specifically, another embodiment provides hair or the growth of skin or the painted application that the TRPML3 conditioning agent of toughener, agonist or antagonist for example is used for the treatment of the patient's condition (such as melanoma and pigmentation disorder) that relates to melanocyte and promotes for example to decolour owing to disease, aging, UV radiation, chemotherapy or hormone imbalances.

Equally more specifically, another embodiment provides the TRPML3 conditioning agent of toughener, agonist or antagonist for example to be used for the treatment of the application of the patient's condition (such as hypophysis cancer or diabetes or disease of pituitary gland) that relates to pituicyte.

Equally more specifically, another embodiment provides the TRPML3 conditioning agent of toughener, agonist or antagonist for example to be used for the treatment of the application of the patient's condition (such as adrenal carcinoma or other suprarenal gland patient's condition) that relates to adrenal cells.

Equally more specifically, another embodiment provides the TRPML3 conditioning agent of toughener, agonist or antagonist for example to be used for the treatment of the application of the patient's condition (such as relevant malignant tumour other taste bud related conditions of taste bud) that relates to taste bud.

Equally more specifically, in another embodiment, the agonist, antagonist or the toughener compound that the invention provides this type of evaluation are used for the sense of taste and therapeutic application, and it can comprise polypeptide, antibody, small molecules, siRNA, sense-rna, ribozyme etc.

TRPML3 can be partly by the sodium of specifically expressing in gustatory cell or the hypothesis of other ionic channels and transhipment and GPCR mediation based on people's saline taste sense of taste as the discovery part of saline taste acceptor.The active compound of these target genes of regulation and control that uses these gene products and derivative thereof to identify can be used as the conditioning agent of the food, beverage and the medicine people saline taste sense of taste that are used for people's consumption potentially.This compounds and derivative thereof also can be used to treat the disease that relates to unusual ion channel function potentially.The cell compounds identified of further using according to the present invention genes identified and expressing this gene is used in the therapeutical agent screening assay of discussing as this paper of the potential therapeutical agent that is used for identifying other gustatory cell correlation functions of regulation and control and phenotype.

Based on its selective expression in being found in the profile shape nipple gustatory cell that tongue forward primate fungi shape nipple gustatory cell neutralization is found in back of tongue, the gene chip microarray that uses the Taste Receptors cell with compare by the isolating non-sense of taste tongue epithelial cell of laser capture micro-dissection (LCM), the inventor thinks that this gene is important.This scheme has also been identified two at other sense of taste of upper part enrichment of taste bud special ionic channel NKAIN3 and NALCN.Because the saline taste impression is the most general in the tongue front portion, prediction saline taste acceptor gene is contained in this cover genes identified.(described according to whole specification sheets, the inventor has identified " people or Mammals saline taste acceptor " rather than " this people or Mammals saline taste acceptor ", because can expect regulating the saline taste sense of taste in people or other Mammalss and there are some redundancies in the metabolic gene of sodium.

Originally this gene is accredited as infers the sense of taste specific ion passage polypeptide that participates in the Mammals saline taste sense of taste.This scheme comprises step (i) evaluation one cover gene, it is included in the macaque gustatory cell (fungi shape and profile shape nipple gustatory cell) and expresses, but gene of not expressing in the tongue epithelial cell and/or the expression level in gustatory cell are significantly higher than the gene of the expression level in the tongue cell; Identified gene subgroup in the cover gene of (ii) identifying in (i), it is based on and shows that it may be the Standard Selection of saline taste acceptor candidate (i.e. the ionic channel of Tui Ceing) and/or coding Multidomain transmembrane protein.Check that then these genes determine that these genes are expressed and still do not express in expressing the gustatory cell of delicate flavour, sweet taste or bitter taste Taste Receptors (TIR or T2R) or tart flavour Taste Receptors (PKD2L1/PKD1L3); (iii) express according to the one or more genes in the subgroup of (ii) identifying functionally and determine in these genes which works as sodium response ionic channel or sodium response acceptor or transhipment, therefore identify the supposition gene of this gene as the regulation and control saline taste sense of taste.The taste tissue that is used for this method is generally from people, primate or rodent source.In a preferred embodiment of present method, step gene (iii) works as sodium response ionic channel, more preferably when these genetic expressions, and the part of passage colony opening and when tranquillization, pass through sodium.

In preferred embodiments, step (i) comprises that use laser capture micro-dissection (LCM) dissects and the purifying taste tissue from non-taste tissue.In a pattern of the present embodiment, step (i) comprises from gustatory cell and tongue cell carries out the RNA amplification to gene, the gene of amplification screens with containing the gene chip that is specific to the specific mammiferous gene samples that obtains the sense of taste and ligule tissue, and preferably gene chip comprises that one overlaps the people's gene of note.In an alternative mode of the present embodiment, step (i) comprises the high-throughput PCR of use at the primer of each ionic channel of mammalian genes group.

In a further preferred embodiment, the in situ hybridization that is specific to the antisense RNA probes of a cover gene of identifying in the step (i) by use is carried out step (ii) so that determine expression level in gustatory cell and tongue cell.In optional preferred embodiment, be specific in the step (i) immunochemistry of the traget antibody of genes identified encoded protein by use and detect and carry out step (ii).

Be used for another embodiment of method of gene that identification code relates to the polypeptide of Mammals saline taste impression, method of the present invention comprises step (i) evaluation one cover macaque gene, it is included in the gustatory cell and expresses, but gene of not expressing in the tongue cell and/or the expression level in gustatory cell are significantly higher than the gene of the expression level in macaque tongue cell; Identified gene subgroup in the cover gene of (ii) in (i), identifying, it is not expressed in the gustatory cell of expressing delicate flavour, sweet taste or bitter taste Taste Receptors (TIR or T2R) or tart flavour Taste Receptors (PKD2L1/PKD1L3); Determine in the primary neuronal of one or more genes in expressing that (iii) therefore which identify the gene of this gene as the regulation and control saline taste sense of taste of inferring as sodium response ionic channel or sodium response acceptor or transhipment in the described gene according to the subgroup of (ii) identifying.In a pattern of the present embodiment, step (iii) comprises this gene of neurone and specific combination and suppresses its active antibody and contacts.

In another common-mode, the invention provides the mensuration that is used to identify the compound of using in the potential body with regulation and control people saline taste sense of taste.The method comprising the steps of (i) expresses coding ionic channel, acceptor or transhipment, be accredited as the cell of gene that the gene of the gene that influences the saline taste sense of taste of supposition or coding and its encoded polypeptides have a polypeptide of at least 90% sequence identity according to any method above contacts with the toughener compound of at least one supposition; (ii) when existing and do not exist, the toughener of described supposition measures sodium conduction, receptor active or sodium transport; (iii) identify that based on its activity or sodium transport that whether strengthens sodium conduction, described acceptor this compound is as potential saline taste sense of taste toughener.In various embodiments, this genes encoding ionic channel, perhaps this genes encoding GPCR.Preferably, this gene is a people's gene.More preferably, this method also comprises this compound or derivatives thereof effect in human or animal's taste test of test.Preferably, the compound promoted sodium ion of selection is transported in the taste bud cell.The gene of inferring that influences the saline taste sense of taste can be at Amphibians ovocyte or mammalian cell, and preferably xenopus leavis oocytes or be selected from the mammalian cell of the group of being made up of HEK293, HEK293T, Swiss3T3, CHO, BHK, NIH3T3, monkey L cell, African green monkey kidney cell, Ltk-cell and COS cell is expressed.Preferably, the expression of gene that influences the saline taste sense of taste of supposition is under the control that can regulate promotor.The gene of inferring that influences the saline taste sense of taste can stably or instantaneously be expressed.In preference pattern, the gene that influences the saline taste sense of taste is TRPML3.

In preference pattern, step mensuration (ii) is to use the electrophysiological detection of sodium sensitive dye, preferred dyestuff comprises the membrane potential dyestuff that is selected from by the following group of forming: MolecularDevices membrane potential test kit (catalog number (Cat.No.) R8034), Di-4-ANEPPS (4-(2-(6-(dibutylamino)-2-naphthalene-yl) vinyl)-1-(3-sulfopropyl) hydroxide pyridine, inner salt, DiSBACC4 (2) (two-(1,2-two barbituric acids)-three acetylene oxprenolols), Cc-2-DMPE (Pacific Ocean indigo plant 1,2-two mnyristoyls-sn-glycerol-3 phosphorylethanolamine, triethylammonium salts) and SBFI-AM (1,3-benzene dicarboxylic acid (benzenedicrboxylicacid), 4,4-[1,4,10-trioxa-7,13-diazacyclo pentadecane (cylopentadecane)-7, two (the 5-methoxyl groups-6 of 13-two bases, 1,2-cumarone two bases) } two-four { (acetyl oxygen) methyl } ester (MolecularProbes), more preferably, this sodium sensitive dye is tetraacethyl sodium green (Molecular Probes) or Na-sensitive dye test kit (Molecular Devices).In another preference pattern, step mensuration (ii) is that the two electrode voltages pincers in the xenopus leavis oocytes are measured, and perhaps this mensuration is the patch clamp mensuration in the mammalian cell.Preferably, this mensuration is measured by ionic current and is measured activity, comprises using atomic absorption spectrum to detect ionic current.

Alternatively, this mensuration can be used fluorescent plate reader (FLIPR) or voltage imaging plate reader (VIPR), and it is used for strengthening sodium or the liquid-absorbent that depends on ionic channel.In the preferred embodiment of present method, the activity of the gene of inferring that influences the saline taste sense of taste clamps electric physiology ground by two electrode voltages and measures in the frog ovocyte, perhaps preferably using in mammalian cell can be the automated imaging Instrument measuring of fluorescent plate reader (FLIPR) or voltage imaging plate reader (VIPR) or patch clamp.

In another pattern, the invention provides the mensuration that is used to identify the compound of using in the potential body with regulation and control people sweet taste, bitter taste, delicate flavour or tart flavour sense of taste.The cell of expression genes identified according to the present invention that the method comprising the steps of (i) contacts with the toughener or the co-inhibitor compound of at least one supposition; (ii) when existing and do not exist, the toughener of described supposition or co-inhibitor measure sodium conduction, receptor active or sense of taste gene product function; (iii) whether regulate and control sodium conduction, the activity of described acceptor or the function of sense of taste gene product and identify that this compound is as potential sweet taste, bitter taste or delicate flavour sense of taste toughener or co-inhibitor based on it.

In another pattern, the invention provides the mensuration that is used to identify the compound of using in the potential body that has as potential therapeutical agent.The cell of expression genes identified according to the present invention that the method comprising the steps of (i) contacts with the toughener or the co-inhibitor compound of at least one supposition; (ii) when existing and do not exist, the toughener of described supposition or co-inhibitor measure sodium conduction, receptor active or sense of taste gene product function; (iii) whether regulate and control the activity of sodium conduction, described acceptor or the function of sense of taste gene product identifies that this compound is as the gustatory cell correlation function or the immunomodulatory of phenotype such as digestion illness or disease, gustatory cell or taste bud turnover or regeneration, oral cavity or Digestive tract or the potential therapeutical agent of treatment metabolism disorder (such as diabetes, obesity, eating disorder etc.) that can be used to directly do not relate to the sense of taste based on it.

Accompanying drawing describes in detail

It is the RT-PCR data of sense of taste specific gene that Fig. 1 contains announcement TRPML3.In this experiment, use people (left side) by laser capture micro-dissection collection and monkey (right side) taste bud (sense of taste) and tongue epithelial cell (tongue) sample to carry out RT-PCR.Can find that 3 of TRPML express in gustatory cell, similar to known sense of taste specific gene T1R2 with TRPM5.The house-keeping gene beta-actin is all expressed in the sense of taste and tongue cell, and the RNA that shows two samples is high-quality.'+' shows and carried out reverse transcription, and '-' shows and do not carry out reverse transcription (negative control).Only carry out reverse transcription and just can observe band.All bands are cloned and the identity of the gene for confirmation that checks order.

Fig. 2 contains and shows that TRPML3 forms the electric Physiological Experiment of sodium channel.The cell of expressing human TRPML3 is carried out full cell patch clamp electric physiology.These result of experiment disclose TRPML3 and produce the sodium leakage electric current, and it is being removed sodium and is being prevented when positively charged ion NMDG replaces with big not seeing through.Trace demonstration-the 60mV at top keeps the electric current under the current potential among Fig. 2.The intermediary trace is presented at when having sodium (NaCl) and not having sodium (NMDG-Cl) the current-voltage trace from-100mV to+60mV among the figure.Figure bottom among Fig. 2 is presented at sodium and has (dark blue colo(u)r streak; Rhombus) and not there is (purple colo(u)r streak; Square) time i-v curve.TRPML3 shows inside rectification (comparing electric current higher under the negative voltage with positive voltage) as can be seen.

Fig. 3 contains other electric Physiological Experiment results that show that people TRPML3 channel characteristic is consistent with the psychophysics of people's saline taste sense of taste.The last figure of Fig. 3 contains sodium conduction (the dark blue colo(u)r streak that shows TRPML3; Rhombus) is not subjected to 30uM guanamprazine (purple colo(u)r streak; Square) current-voltage curve of preventing.People's saline taste sense of taste and TRPML3 are not subjected to preventing of guanamprazine.Figure below of same figure contains and shows that TRPML3 can see through salt cation sodium (dark blue colo(u)r streak equably; Rhombus) and lithium (purple colo(u)r streak; Square) current-voltage curve.Consider that known sodium and lithium are the salty equably facts (because two kinds of positively charged ions permeate people TRPML3 passage equably) to the people, this result is that people's saline taste acceptor is consistent with TRPL3.

Fig. 4 contains the experiment that shows that near the top diaphragm area of TRPML3 albumen taste pore expressed.The TRPML3 antibody labeling extends to the gustatory cell process (left figure) of taste pore as can be seen.Illustrated clearly that towards the amplification of the top taste bud structural domain of saliva TRPML3 albumen expresses (3 right figure in the taste pore zone; Taste pore is represented with blue arrow).This observation is that people's saline taste acceptor is consistent with TRPML3 also, experiences sodium in the saliva because this position is ideally suited TRPML3.Similar to TRPML3, other known Taste Receptors (sweet taste, bitter taste, delicate flavour and tart flavour) are also to it tastes the taste pore polarization of its flavor unit that needs in the saliva therein.

Fig. 5 contains the data of the immunochemistry double-label experiment that shows that TRPML3 albumen is not expressed in the TRPM5 cell.This figure shows in the monkey CV nipple with TRPM5 (green; Left figure) and TRPML3 (redness; Middle figure) the immunohistochemistry result of double-tagging.The cell of expressing TRPM5 and TRPML3 as can be seen among this figure is different (right side is the figure that merges).These data show that TRPML3 does not express in TRPM5 cell (comprising sweet taste, bitter taste and umami cells), but express in professional saline taste gustatory cell specially.

Fig. 6 contains the data of another immunochemistry double-label experiment.The data announcement TRPML3 albumen that is contained among Fig. 6 is not expressed in the PKD2L1 cell.This figure contains in monkey CV nipple with PKD2L1 (green; Left figure) and TRPML3 (redness; Middle figure) the immunohistochemistry result of double-tagging.The cell of as can be seen from the figure expressing PKD2L1 and TRPML3 is different (right side is the figure that merges).These data show that TRPML3 does not express in PKD2L1 cell (comprising the tart flavour cell), but express in the saline taste gustatory cell of specialty.

Fig. 7 has illustrated the example of the I/V curve of the ovocyte that personnel selection TRPML3 cRNA injects.

Fig. 8 has illustrated that screening contains the active examples for compounds of adjustable TRPML3 in the people TRPML3 cRNA ovocyte.

Fig. 9 has illustrated the example of the I/V curve that uses TRPML3 co-inhibitor gadolinium.

Figure 10 uses two electrode voltages to clamp in the ovocyte expression system to check the experiment flow figure of compound to the active influence of people TRPML3.

Figure 11 has illustrated that the expression of the active sodium channel of composition has strengthened the substrate fluorescence in the cell that is loaded with certain films current potential dyestuff.

Figure 12 illustrates and uses the enhancing that gadolinium has reduced substrate fluorescence in the cell of expressing A419P-TRPML3.

Figure 13 illustrates and uses gadolinium has reduced substrate fluorescence in the cell of expressing A419P-TRPML3 in the mode that depends on dosage enhancing.

Figure 14 illustrates the titration of TRPML3 plasmid.

Figure 15 illustrates that the influence of gadolinium is specific to TRPML3.

Figure 16 illustrates that the baculovirus transduction HEK293 cell with coding A419P-TRPML3 has doubled the mensuration window.

Figure 17 is to use the example of the garbled data of the cell acquisition of expressing A419P-TRPML3.

10,000 general introductions that compound screens on a small scale that Figure 18 is to use the cell of expression A419P-TRPML3 to carry out.

Figure 19 shows the wild-type (non-codon optimized) of people TRPML3 and the comparison of codon optimized dna sequence dna, and these dna sequence dnas are 76.4% identical.Wild-type (non-codon optimized), codon optimized and A419P TRPML3 express in ovocyte, and measure sodium current.

Figure 20: people's wild-type is (non-codon optimized; With blue diamond indicia WT), the functional expression of codon optimized (with pink colour square marks WT-CO) and A419P (with yellow warning triangle mutant) TRPML3 cRNA.The I/V curve of the inside rectification of being represented by more weak electric current under electric current stronger under the hyperpolarization current potential (more negative potential) and the depolarize current potential (more positive potential) shows the functional expression of TRPML3 ionic channel.Should note comparing with no codon optimized wild-type TRPML3, codon optimized TRPML3 and A419P TRPML3 observe the electric current of increase.

Figure 21: screen the ovocyte of injecting with codon optimized people TRPML3 cRNA so that identify the example of the compound (TRPML3 toughener) of activation TRPML3.In a plurality of ovocytes, the TRPML3 toughener with the TRPML3 increased activity 169+/-26% (the representative trace at top), and the not injection ovocyte that does not have TRPML3 to express not have to influence (the representative trace of bottom).Only add damping fluid to the not influence of TRPML3 electric current, and in case use the second time, the influence of TRPML3 toughener is reproducible.

Figure 22: the TRPML3 toughener is to the example of TRPML3 I/V curve influence.Stimulate (red-violet colour square marks toughener) with the ovocyte unprocessed (blue warning triangle contrast) of codon optimized people TRPML3 cRNA injection or with the TRPML3 toughener, from-90mV extremely+measure electric current under the voltage of 30mV.Should notice that TRPML 3 tougheners activate TRPML3 electric current (inwardly electric current is bigger compared with the control for toughener) under negative voltage, cause the increase of I/V rate of curve.Should also be noted that zero current drifts about to the right, hint is the conduction of enhanced sodium when toughener exists.

Figure 23: the example of TRPML3 toughener influence when sodium exists and do not exist outside born of the same parents.The ovocyte of expressing codon optimized people TRPML3 cRNA with NMDG (not having sodium), TRPML3 toughener add sodium, only damping fluid or TRPML3 toughener add NMDG (not having sodium) stimulates.Should notice that the TRPML3 toughener has strengthened the activity of TRPML3 when sodium exists, but when sodium does not exist, not influence.These data declarations TRPML3 toughener has been opened the TRPML3 passage, and has increased sodium ion inflow ovocyte.

Figure 24: the expression level of WT TRPML3 depends on mammiferous cell type.A. express the current/voltage analysis (I/V figure) of the cell of A419P mutant TRPML3 passage in WT and the HEK293 cell.A419P mutant TRPML3 passage is expressed big inside rectified current (pink colour), yet only observes little WT TRPML3 electric current (blueness).B.WT has similar functional character with A419P mutant TRPML3 passage in Chinese hamster ovary celI.

Figure 25: TRPML3 is used for the application of Chinese hamster ovary celI toughener and co-inhibitor screening.The WT people TRPML3 passage of transient expression is used for identifying the passage toughener in the A.CHO cell.I/V figure demonstration is compared (blueness with independent damping fluid; Contrast), use toughener to cause the enhancing (pink colour) of inside electric current under the negative potential.The mutant A419P TRPML3 passage of stably express is used for identifying the passage co-inhibitor in the B.CHO cell.Compare (blueness with independent damping fluid; Contrast), add weaken (pink colour) that 1mM GdCl3 (Gadolinium trichloride) causes inside electric current.

Figure 26: codon optimized WT TRPML3 is used to screen the application of compound that strengthens the TRPML3 function.A. the transient expression (light blue) of non-codon optimized WT TRPML3 causes electric current very little in the HEK293 cell.On the contrary, the WT TRPML3 (mazarine that son optimizes that accesses to your password; Cod Opt WT) causes having the electric current (pink colour) of similar average intensity to A419P mutant passage.B. the codon optimized WT TRPML3 (blueness) that uses the baculovirus transduction to send causes the mean current similar to A419P TRPML3 (pink colour).C. be used for identifying the toughener of TRPML3 function with the cell of codon optimized WT TRPML3 baculovirus transduction.Compare (blueness with independent damping fluid; Contrast), add the increase (pink colour) that the toughener compound causes inside electric current.

The functional surface that Figure 27: WT and A419P TRPML3 co expression have increased in the HEK293 cell is expressed.The electric current transfection that A.A419P TRPML3 cDNA (0.5ug) causes produces the electric current with the inside rectification of characteristic in the HEK293 cell.The non-codon optimized TRPML3 of B.WT (1.5ug) expresses in the HEK293 cell, and does not produce electric current.It is the big inside electric current of twice when only expressing A419P cDNA that C.A419P (0.5ug) causes with WT (1.5ug) TRPML3 cDNA co expression in the HEK293 cell.D. the I/V of the mean current that WT (blueness), A419P (pink colour) and WT and A419P coexpression (yellow) TRPML3cDNA cause in the HEK293 cell schemes.

Figure 28 contains the example of TRPML3 function in the IonWorks PPC diaphragm plate.A has the view in all 384 holes that A419 TRPML3 can induce clone's PPC diaphragm plate, and it shows the result of scanning before compound.Yellow shows wherein-hole (in control experiment, not having the hole to be marked as yellow) of electric current≤0nA under the 120mV with parent CHO-K1 cell.Blueness show even sealed wherein too low (＜10mOhm) so that can not measure the hole of electric current reliably.Can in 94% hole, measure A419P TRPML3 electric current.B, from the adding 4mM GdCl3 of the diaphragm plate that shows among the A or the outer damping fluid of born of the same parents (simulation is added) before and mean current ± SEM afterwards.GdCl3 is added on the post 1-38, and the outer damping fluid of born of the same parents is added on the post 39-48.For relatively, comprised from the independent experiment of using parent CHO-K1 cell.The stability of TRPML3 electric current showed that this mensuration should detect the compound that strengthens or prevent the TRPML3 electric current after simulation was added.

Figure 29 contains the example (last figure) of the IonWorks scanning of using the induced CHO-K1 clone of expressing A419P TRPML3.The more still less inside rectification of electric current indication TRPML3 under multiple current and the depolarize current potential (more positive potentials) under the hyperpolarization current potential (more negative potentials).Add GdCl3 and prevented the TRPML3 electric current.Red line is illustrated in the scanning in sodium (NaCl) solution.Blue line is illustrated in the scanning in the 4mM GdCl3 solution.Middle figure is from parent CHO-K1 cell, as negative control.Positive current under the negative potential is because the excessive down leakage difference of correcting electric current of negative potential subtracts.Figure below shows the voltage instruction scheme of record current of being used for.Be used for calculating leakage current (electric current of flowing through by sealing leak) from the grade of 0mV to 10mV, from total current, deduct the electric current that leakage current obtains flowing out film.The result is from the single hole in the PPC diaphragm plate, and representative reaches the mean current of 64 cells.

Figure 30 contains be used for checking the experiment flow figure of compound to TRPML3 (hTRPML3) activity influence in IonWorks measures.

Figure 31 is presented at that the TRPML3 cell is excised from taste bud specifically in the Varitint waddler mouse.Taste bud (TB) and tongue epithelial cell (LE) by isolating Varitint waddler of laser capture micro-dissection (Va) or wild-type (WT) mouse are carried out terminal point RT-PCR experiment.TRPML3 only expresses in the taste bud of WT mouse and lacks in the taste bud of Va mouse, and every other sense of taste gene (T1R2, GPR113, TRPM5) and house-keeping gene (beta-actin, GAPDH) are expressed in TB and LE equably.'+' shows and carried out reverse transcription, and '-' shows and do not carry out reverse transcription.Have only the ThermoScript II of use just can observe the PCR band, hint PCR product is from mRNA rather than genomic dna.

Figure 32 is also by using PCR in real time to be presented at that the TRPML3 cell is excised from taste bud specifically in the Varitint waddler mouse.Taste bud (TB) by isolating Varitint waddler of laser capture micro-dissection (Va) or wild-type (WT) mouse is carried out the real-time quantitative RT-PCR experiment.TRPML3 only expresses in the taste bud of WT mouse and lack (using two different primers that the Mcoln3_1 of mark is obtained similar result with Mcoln3_2) in the taste bud of Va mouse, and every other sense of taste gene (Tas1r2, Tas1r3, PKD211, TRPM5, Plcb2, Tas2r108 and Tas2r116) and house-keeping gene (contrast) are expressed in the taste bud of Va and WT mouse.

Figure 33 contains and show that sweet taste, delicate flavour, bitter taste and tart flavour cell are complete experiments in the taste bud of Varitint-waddler mouse.From wild-type (row of going up of figure) and Varitintwaddler (Va; The following row of figure) in situ hybridization of the profile shape nipple of mouse back of tongue.PKD1L3 (left side; Tart flavour), PKD2L1 (centre; Tart flavour) and TRPM5 (right side; Sweet taste, bitter taste, delicate flavour and GPR113) gustatory cell is present in wild-type and the Va mouse with similar level.

Figure 34 contains the neural record experiment of CT that proof Varitint waddler mouse lacks the saline taste sense of taste.From wild-type (left side) or Varitint waddler (Va; The right side) the neural record of the CT of mouse.Add 5uM benzyl guanamprazine with 0.1M NaCl or 0.1M NaCl and stimulate the forward tongue so that suppress the guanamprazine sensitive ingredients of CT neural response.In the middle of NaCl stimulates, with the low salts solution flushing tongue that contains 10mM KCl.Should notice that the insensitive component of benzyl guanamprazine of CT neural response eliminated (red arrow) in a large number in the Va mouse, hint excision TRPML3 gustatory cell has significantly damaged the saline taste impression.In addition, (red circle) replied the instant phase place of NaCl and greatly weakened in the Va mouse.Scale shows that time frame (x-axle) and CT that salt is used reply intensity (y-axle; Arbitrary unit).

Figure 35 contains the sequence alignment of people and mouse TRPML3 gene and polypeptide.Identified membrane spaning domain, born of the same parents' outer shroud and porose area, and the residue (419) that causes Varitint waddler mouse.

Detailed Description Of The Invention

The present invention relates to identify to regulate mammal and the gene experienced of other vertebrates (for example fowl, reptile and amphibian) saline taste potentially. This gene TRPML3 or MCOLN3 be specifically expressing in the gustatory that stimulates in response to saline taste, and expresses in hypophysis, adrenal gland and melanocyte. This gene code ion channel polypeptide, its separately or potentially with other accessory molecules or ion channel (such as TRPML1, TRPML2, NALCN or NKAIN3) detect together the saline taste stimulation, and may regulate sodium transport, metabolism and drainage and/or further can affect the sodium correlated process that relates to aldosterone and/or vasopressing based on the following fact: this ion channel is mainly expressed in the adrenal gland that produces aldosterone (a kind of remarkable participation affect the hormone of the sodium correlated process of uropoiesis and cardiovascular system and other organs) and basically by secreting vasopressing (at sodium transport, bring into play extremely important effect and affect blood pressure in metabolism and the drainage, urine is discharged and fluid retention etc.) another kind of hormone) the hypophysis expression. Therefore, this ion channel polypeptide may play a significant role in sodium transport, metabolism and the drainage of different cells and organ, and participates in the saline taste impression.

The glutinous fat 3 (MCOLN3) of this gene or TRPML3 (using this paper and disclosed new principle evaluation in incorporating by reference other provisional application of this paper into) are coded in the membrane-spanning proteins of the taste bud top expression of the taste perception cell that conducts sodium. It is believed that this gene code allows the saline taste acceptor of experiencing gustatory detection sodium chloride (salt) of the taste bud of tongue. In addition, because this gene is expressed in adrenal gland and hypophysis, it may participate in regulating the sodium metabolism of body. The inventor obtains evidence and shows that this gene is that people's saline taste is acceptor as follows with reporting this gene-correlation:

(1) MCOLN3 is not expressing TRPM5 (being that it is not sweet taste, bitter taste or delicate flavour) and is expressing towards the taste bud top of the taste perception cell subsets of taste pore.

(2) MCOLN3 also expresses in the permissive cell such as other organs of ear. Therefore it is the receptor gene of ' specialty '.

(3) MCOLN3 is at the adrenal gland strongly expressed. These bodies of gland are the very important effect of performance in the sodium metabolism of regulating body. Therefore MCOLN3 may be the key molecule of regulating the sodium metabolism, and may regulate adrenal gland generation aldosterone.

(4) feature of a kind of human autoimmune disease (Ai Dishengshi) is adrenal destruction. An indication symptom of this disease is that salt is craved for. The latter may result from the existence for the autoantibody of MCOLN3, or destroys the sudden change of this gene of MCOLN3 function in the taste bud.

(5) MCOLN3 high expressed in the hypophysis that the vasopressing that participates in the generation of adjusting urine discharges. This has further supported this ion channel in the sodium metabolism of body and the importance in the drainage.

(6) MCOLN3 conduction sodium in electrophysiologic study, and show the correct biochemical character (detecting K+, Li+ and amiloride sensitiveness) that is predicted as the saline taste acceptor.

(7) in varitint mouse (having the TRPML3 sudden change), use the nervous physiology experiment (neural recording) of sodium to show that the Varitint mouse is to the response defective (not showing strong saline taste replys) of sodium.

(8) use the mammalian cell of the TRPML3 polypeptide (sudden change causes ion channel to be fixed in the open to the outside world orientation) of expressing sudden change and the mensuration based on cell of amphibian egg mother cell to identify TRPML3 reinforcing agent and the co-inhibitor that should in the saline taste test, strengthen or prevent the saline taste sense of taste.

MCOLN3 is the design of the Screening test of the discovery of the people's saline taste acceptor cell, the activator for the identification of this molecular function, antagonist or reinforcing agent (conditioning agent) purpose that have realized using this gene transfection.

This gene is to use the gene and the screening that are intended to identify specifically expressing in gustatory to identify at the full genome screening strategy of its subgroup of people's taste bud top enrichment by the inventor at first. From their previous experiment, the inventor infers that the taste bud top contains the cell of the known gustatory receptor genes of overexpression. On the contrary, the precursor of experiencing taste buds cell that is positioned at the taste bud top section is contained in the taste bud bottom. This database allows the inventor to identify the gene of many taste buds top (impression) cell specific expression. One of these genes MCOLN3 before had been described as being responsible for being called the phenotype of the mouse mutant of varitint-waddler, it shows as the early onset hearing disability, the vestibular defective, (Dipalma etc., Mutations in MCOLN3associated with deafness and pigmentation defects invaritint-waddler (Va) mice. (the MCOLN3 sudden change relevant with the pigment defective with the deafness of varitint-waddler (Va) mouse) Proc.Natl.Acad.Sci USA 99:14994-14999 cause death pigment anomaly and perinatal period; 2002). MCOLN3 expresses in the plasma membrane of hair cell and stereocilium (in the ear). This sudden change causes the ala 419 of the 5th membrane spaning domain to become the replacement of pro. The result is highly active MCOLN3, and it causes the cell death of expressing this molecule, such as hair cell (therefore causing the deafness of Va mouse) (Grimm C etc., the Proc Natl.Acad.Sci.USA 104:19583-8 of ear; 2007). Therefore, the inventor expects that this mouse also will show saline taste parageusia (because unusual MCOLN3 molecule and may affect in the saline taste taste buds cell of tongue thereof). In fact, as be contained in shown in the nervous physiology experiment and data of this paper, when the saline taste with the concentration that normally should cause saline taste impression stimulates, these mouse do not stimulate (the neural recording result confirmation of the Varitint mouse of hereinafter discussing that responds consumingly to saline taste, it is active and because the forfeiture of hair cell in the inner ear causes that the TRPML3 of deaf and equilibrium problem suddenlys change and also destroyed the saline taste sense of taste that this confirms that further the TRPML3 of normal function or MCOLN3 ion channel are the prerequisites that saline taste is experienced to destroy TRPML3).

Equally, because public database shows MCOLN3 strongly expressed in adrenal gland and hypophysis, and because adrenal gland represents one of main sodium metabolism adjuster of body, TRPML3 may significantly participate in the sodium metabolism. (the salt level justacrine of adrenal gland monitoring of blood is by helping kidney to keep sodium and discharging potassium and regulate the aldosterone (mineralocorticoid) of body blood pressure and water salt balance. In addition, when the generation of aldosterone descend too low the time, kidney can not be regulated the salt solution balance, causes blood volume and blood pressure drops. )

Therefore, it is predicted that also MCOLN3 controls salt crucial in the adrenal gland of aldosterone generation/sodium monitoring molecule. On the contrary, the MCOLN3 in the tongue is by the taste perception lymphocyte subgroups that is positioned at the taste bud top, and is responsible for detecting saline taste. By any mode, MCOLN3 has crucial effect in the salt that detects various tissues. Therefore the present invention consists of the important discovery with important application.

In addition, because TRPML3 or MCOLN3 great expression in hypophysis also, the inventor predicts that also MCOLN3 participates in regulating vasopressing and discharges. By its key adjusting that vasopressing is the urine generation that affects to kidney. Importantly, known vasopressing is subjected to the adjusting of NaCl concentration from postpituitary release. Otherwise, by its impact to fluid retention, NaCl impression and concentration and blood pressure, the key of seemingly body NaCl metabolism of MCOLN3 is regulated son.

Therefore the present invention identifies and provides function (electric physiology) and immunohistochemistry data and animal data (nervous physiology research), it shows the polypeptide that TRPML3 (MCOLN3) coding works as primate (for example people) saline taste acceptor, and plays a significant role in the sodium impression of system and metabolism.

Based on this, the present invention mainly concentrates on exploitation and measures for the identification of the reliable and effective of compound of regulation and control TRPML3 polypeptide (prevent, strengthen, activate), these compounds should be regulated and control the saline taste sense of taste and the regulation and control biological function relevant with TRPML3, sodium transport, absorption and drainage such as cell, tissue and organ, and impact (excitement or antagonism) aldosterone or relevant activity and the patient's condition of vasopressing, release or the generation of wherein regulating and control aldosterone or vasopressing are the bases for the treatment of. As known in the art, the compound of excitement or antagonizing vessel pitressin and aldosterone can be advantageously applied in the treatment, particularly uropoiesis and the cardiovascular patient's condition, and the patient's condition that relates to oedema or unusual circulation. For example, these compounds are used for the treatment of hypertension, oedema, congestive heart failure, diabetes and symptom thereof, and many other patient's condition.

In addition, because TRPML3 affects melanocyte and hair cell, the compound of regulation and control TRPML3 can be used for promoting propagation and the differentiation of melanocyte, can be used for treating the pigmentation disorder, can prevent or recover gray hair or skin to its normal coloring (for example owing to disease, age, hormone function obstacle, UV radiation or chemotherapy lose painted), and can promote hair follicle growth and propagation. The TRPML3 conditioning agent also can be used for treating melanoma, because it optionally kills the MC of expressing TRPML3 potentially.

Based on preamble, the invention provides the mensuration system of the test cell (preferably based on mammalian cell and egg mother cell) that comprises expressive function TRPML3, it can comprise the wild type TRPML3 of the species of any needs, the TRPML3 of the sudden change that wherein suddenlys change natural generation or introduce by design, for example in order to modify the function (enhancer or inhibitor) of TRPML3 or keep it to be in fixing perforate orientation in order to help its Screening test that is used for conditioning agent, perhaps it can comprise the domain of TRPML3 wherein or the outer part of born of the same parents and another TRPML3 ion channel or another ion channel (such as TRPML1, TRPML2, NALCN or NKAIN3) or the domain of another TRP ion channel or chimeric TRPML3 ion channel or the function fragment of the outer part exchange of born of the same parents. The exemplary sudden change of TRPML3 is disclosed in this paper, and can be by those skilled in the art with information disclosed herein with well known to a person skilled in the art method design. Equally, as described herein, provide coding TRPML3, the nucleotide sequence of (being to be preferred in the cell that will measure therein of codon) has superiority to contain the preferred codon of host. For example, the inventor provides hereinafter by the molecular TRPML3 gene of the preferred password of people, in order to strengthen the activity of people's (for example HEK-293 cell) TRPML3 ion channel, is used for according to preferred mensuration of the present invention.

Preferably, the invention provides based on mammalian cell and be used for analyzing and the mensuration of the preferably high or isoflux of screening saline taste acceptor (TRPML3) based on egg mother cell. More specifically, the invention provides and express the amphibian egg mother cell based on the TRPML3 of the mensuration of cell can be used on for screening TRPML3 conditioning agent. The present invention also provides the amphibian egg mother cell of expressive function TRPML3, is used for identifying functionally the TRPML3 activity, and can be used to identify the compound (this paper is called saline taste sense of taste conditioning agent) that strengthens or prevent the saline taste impression. These compounds can be used as the saline taste sense of taste that becomes to assign to strengthen, regulate and control, suppress or prevent of food, medicine and beverage. These compounds also have potential therapeutic and use, for example regulate blood pressure, cardiac function, renal function particularly urine generation and drainage, treatment Addison's disease, IV type stick the physiological effect of lipidosis and aldosterone and/or vasopressing and wherein treat the disease that needs to use aldosterone or vasopressing activator or antagonist.

Therefore the present invention identifies and provides function (electric physiology), molecule and immunohistochemistry data, and it shows the polypeptide that TRPML3 (MCOLN3) coding works as primate (for example people) saline taste acceptor.

Further the invention provides these sense of taste specific genes as the application of the sign of the cell that can be used to enrichment, evaluation or separation expression saline taste acceptor.

The present invention also provides use TRPML3 (MCOLN3) and has expressed the cell of TRPML3 or the TRPML3 transgenic animal model is identified and caused or regulation and control (prevent or strengthen) comprise the external and in vivoassay of activator, antagonist or reinforcing agent compound of the saline taste sense of taste of people's primate. These measure to use the cell of single expression TRPML3 or express the TRPML3 ion channel and such as the cell of other sense of taste specific polypeptides of NALCN or NKAIN3.

In addition, the invention provides transgenic animals (preferably rodent) and being used for confirm TRPML3 the mammiferous saline taste sense of taste and relating to sodium and other physiological functions (such as sodium metabolism, blood pressure, fluid retention and drainage, urinary function and cardiac function) of other ions in effect.

The present invention also provide the cell that uses TRPML3 and express TRPML3 or transgenic animals in mensuration (preferably electrophysiological detection) in order to identify the external and in vivoassay of the therapeutic compound that alleviates the disease that relates to this expression of polypeptides defective (comprising overexpression, the low sudden change of expressing and affecting the TRPML3 polypeptide of its ability that works as the sense of taste special sodium channel in the mammal that comprises for example people and non-human primate) and the patient's condition. These patient's condition comprise Addison's disease and relate to or be subjected to unusual aldosterone generation or vasopressing to discharge the other diseases that affects as an example, such as hypertension, low blood pressure, fluid retention and impaired uropoiesis or cardiac function (such as cardiac arrhythmia, heart attack and apoplexy).

This gene is to identify by the method for using the new sense of taste specific gene of following evaluation at first:

1) Laser capture microdissection (LCM) and RNA amplification: in the laser capture microdissection, very thin laser beam is used for dissecting and the gustatory of purifying from histotomy. The method has been separated the tongue epithelial cell that lacks pollution and the gustatory of connective tissue, and allows the technical staff that highly enriched gustatory colony is carried out molecular biology experiment. Abreast, separate the tongue epithelial cell by LCM, and as the negative control that lacks gustatory. It is favourable that LCM dissects the artificial or enzymatic of papilla gustatosia, because these primitive technologies produce the heterogeneous mixture of the sense of taste and tongue cell, wherein gustatory comprises the collection material of 1-20%. RNA amplification reaches 1,000 with agonic mode total RNA that increases from the gustatory that separates by LCM and tongue cell, 000 times, carry out molecular biology research (genetic chip or PCR) in order to produce enough genetic stocks. We have found that 5,000 gustatories enough are used for using the gene chip experiment of macaque taste tissue, enough are used for using the PCR experiment of macaque taste tissue more than 10,000 gustatories.

2) Genetic chip: genetic chip contains the gene of nearly all note on the little chip. Which specific gene the RNA that separates from the sense of taste and tongue cell and increase and gene chip hybridization can be used to determine in gustatory and not at the tongue cells, and the expression of which specific gene in gustatory is higher than the tongue cell. Use Affymetrix macaque genome array to use paired macaque fungi shape (FG) and profile shape (CV) sense of taste and tongue sample to carry out gene chip experiment, use GeneSpring GX v7.3 software (Agilent Technologies) to analyze. 5000 fungi shapes separate separately by LCM with the tongue cell with the CV sense of taste, and total RNA of each sample of purifying. Then cloning RNA and and gene chip hybridization. Use two different algorithms to carry out data analysis: to consider on the genetic chip Affymetrix Microarray Suite 5 (MAS5) of fully coupling and mismatch probe and only considered to mate fully on the genetic chip the reliable multi-chip algorithm (RMA) of probe. Identified the sense of taste specific gene of coding transmembrane protein in this analysis.

3) PCR: use the primer that is specific to people/macaque genome intermediate ion passage in 96 orifice plates, to carry out high flux PCR, the RNA of the people that amplification separates by LCM/macaque sense of taste and tongue cell. Detect the product of suitable size in gustatory rather than the tongue cell and the PCR product is carried out dna sequencing (gene identity for confirmation) and show that interested ion channel is sense of taste specific gene. Before the high flux PCR of the primer of the ion channel of in using for the macaque genome, identifying, at first to reaching 4 known sense of taste specific genes and 2 house-keeping genes carry out quality control PCR reaction, in order to guarantee that the sense of taste and tongue RNA are high-quality. 4 sense of taste specific genes that can be detected are gα protein gusducin (GNAT3), sweet receptor component T1R2, ion channel TRPM5 and enzyme phospholipase C hypotype β 2 (PLC β 2); Two house-keeping genes that detect are beta-actin and GAPDH. Sense of taste gene by gustatory rather than tongue cell specific expression add gustatory and tongue cell all express all over house-keeping gene, show high-quality RNA material.

Analyze the PCR product at Ago-Gel and determine suitably whether the band of size is present in gustatory rather than the tongue cell. Gene with this expression pattern is the sense of taste specific gene of inferring. Clone all sense of taste specific genes and order-checking and confirm the identity of gene.

4) In situ hybridization: the antisense RNA probes and the histotomy hybridization that contains gustatory that are specific to individual gene (identifying by genetic chip or PCR), so that whether determine the mRNA transcript of gene of interest expresses in gustatory, particularly in tart flavour, sweet taste, bitter taste and/or umami cells, express, or in the cell type of the uniqueness that may participate in the saline taste detection, express. In double labelling in situ hybridization, produce two different rna probes and come two different genes of mark, two different sense of taste specific genes particularly identifying by genetic chip and/or PCR method. Alternatively, can produce whether a probe comes the mark individual gene expresses in order to determine this gene in gustatory. To double labelling research, first gene is used in the FITC probe mark that produces a kind of color under the fluorescence microscope, and second gene is used in foxalin (DIG) probe mark that produces different colours under the fluorescence microscope. It is to express in identical or different cell types that the stack of probe 1 and probe 2 has disclosed gene. For example, if the ion channel of the uniqueness of identifying by genetic chip or PCR method is located altogether with the cell of expressing TRPM5, this unique ion channel is at the cells of being responsible for sweet taste, bitter taste and/or the delicate flavour sense of taste. On the contrary, if the ion channel of the uniqueness of identifying by genetic chip or PCR method is not located altogether with the cell of expressing TRPM5, this unique ion channel is expressed in the different cell types that may be responsible for the saline taste sense of taste (or another kind of sense of taste form), and ion channel that should uniqueness may be participated in sodium directly and detects.

5) Immunohistochemistry: the antibody that is specific to single albumen (its gene is identified by genetic chip or PCR) is applied to contain the histotomy of gustatory so that whether determine interested albumen expresses in gustatory, particularly expresses in tart flavour, sweet taste, bitter taste and/or umami cells or expresses in unique cell type that may participate in the saline taste detection. In the double labelling immunohistochemistry, coming mark two different albumen, particularly its gene with two different antibody is two different sense of taste differential proteins identifying by genetic chip and/or PCR method. Whether alternatively, can come the single albumen of mark with an antibody probe expresses in gustatory in order to determine this albumen. To double labelling research, first albumen is with the antibody labeling of the very dilute concentration that can only detect with the Sensitive Detection method that is called tyrasamine amplification of signal (TSA). Second albumen also detects with non-TSA method with another antibody labeling then. First antibody of dilution can not be detected by the non-TSA method of standard; Therefore from two different antibodies of same species (for example rabbit polyclonal antibody) will be not can cross reaction, therefore can be used in the double-label experiment. It is to express in identical or different cell types that the stack of albumen probe 1 and albumen probe 2 has disclosed albumen. For example, if the ion channel of the uniqueness of identifying by genetic chip or PCR method is located altogether with the cell of expressing TRPM5, this unique ion channel is at the cells of being responsible for sweet taste, bitter taste and/or the delicate flavour sense of taste. On the contrary, if the ion channel of the uniqueness of identifying by genetic chip or PCR method is not located altogether with the cell of expressing TRPM5, this unique ion channel is expressed in the different cell types that may be responsible for the saline taste sense of taste (or another kind of sense of taste form), and ion channel that should uniqueness may be participated in sodium directly and detects.

Further, identify that this ion channel gene comprises also that as the ion channel that participates in potentially the saline taste impression following principle selects potential saline taste acceptor or ion channel candidate.

At first, use LCM as indicated above from macaque (machin), to separate taste bud. The macaque gene is identical with the average 90-95% of people's gene, and macaque is the biological fabulous experimental model of people that research comprises the sense of taste. Therefore the sense of taste gene of identifying in the macaque will be similar to its people's straight homologues height, and the similar function of observing among the executor. Use LCM, very thin laser beam is used for dissecting and the purifying gustatory from histotomy. The method has been separated the tongue epithelial cell that lacks pollution and the gustatory of connective tissue, and allows highly enriched gustatory colony is carried out molecular biology experiment. Abreast, separate the tongue epithelial cell by LCM, and as the negative control that lacks gustatory. It is favourable that LCM dissects the artificial and enzymatic of papilla gustatosia, because these original technology tend to produce the heterogeneous mixture of gustatory and tongue cell, wherein gustatory only comprises the collection material of about 1-20%.

The second, the RNA that uses genetic chip/microarray analysis to separate with non-gustatory from the sense of taste. Genetic chip contains the gene of nearly all note on the little chip. Can be used to determine which specific gene at gustatory and not at the tongue cells from RNA and the gene chip hybridization of gustatory and tongue cell separation, and the expression of which specific gene in gustatory is higher than the tongue cell. In order to identify that its probe set does not have the gene of function at genetic chip, genetic chip carries out 21 non-taste tissue of macaque. The probe set that does not produce the gene that is higher than the background level data comprises not with the probe set of the effective hybridization of gene target and not by the probe set of 21 rhesus monkey tissue figure representatives. These genes of the gene that representative is not comprised by method for gene chip are higher than gene by the tongue cell of LCM separation by PCR and/or histologic analysis in order to identify at the gene (gene of the transmembrane protein of particularly encoding) of gustatory rather than tongue cells and the expression in gustatory separately.

The 3rd, the sense of taste specific gene of identifying by genetic chip and/or PCR checks by histology with double labelling method. Use in situ hybridization, be specific to the antisense probe and the histotomy hybridization that contains gustatory of individual gene, so that whether determine the mRNA transcript of gene of interest expresses in gustatory, in sweet taste, bitter taste, tart flavour and/or delicate flavour gustatory, express especially, or in unique cell type that may participate in saline taste or other sense of taste forms (for example fat taste detection), express. Use is specific to the immunohistochemistry antibody of single albumen (its gene is identified by genetic chip), whether these antibody are applied to contain the histotomy of gustatory in order to determine interested albumen and express in gustatory, particularly in sweet taste, bitter taste, tart flavour and/or umami cells, express, or in the particular cell types that may participate in the detection of saline taste or fat taste, express. The gene of expressing in the gustatory of expressing TRPM5 (sign of sweet taste, bitter taste and umami cells) will be encoded and may be regulated and control the albumen of sweet taste, bitter taste and/or the delicate flavour sense of taste. The gene of expressing in the gustatory of expressing PKD2L1 or PKD1L3 (sign of tart flavour cell) will be encoded and may be regulated and control the albumen of the tart flavour sense of taste. Do not express the gene of expressing in the gustatory of PKD2L1 or PKD1L3 yet and will be coded in the albumen of expressing in can the particular cell types in response to saline taste or adipocyte neither expressing TRPM5. Therefore, the gene of expressing in specific gustatory type can be in response to saline taste acceptor or fat taste receptors, and can regulate and control saline taste or fat taste detects.

The 4th, use similar LCM program and genetic chip or PCR expression, test to identify that where overlapping gene specific ground expresses in the first half rather than the latter half of taste bud, perhaps where overlap gene in the first half enrichment, namely with respect to the cell of the latter half in the cell that comprises taste bud the first half at least 1.2-1.5 doubly more the highland express. (consider its orientation on taste bud, these genes are preferred candidate of people's sense of taste acceptor).

The 5th, the sense of taste specific gene of expressing in unique cell type produces sodium response acceptor or sodium-conducting ion passage by with comprising that electrophysiological functional examination analyzes in order to determine the gene outcome of expressing in the allos system such as HEK293 cell, Chinese hamster ovary celI or xenopus leavis oocytes. Saline taste acceptor target should be in response to the sodium ion (20-140mM sodium) of the concentration relevant with people's sense of taste.

The 6th, for the effect of last confirmation gene as the saline taste acceptor, the gene that satisfies standard proposed above further carries out high flux screening and identifies reinforcing agent and co-inhibitor, and these compounds of test in the saline taste test are in order to determine whether the enhancer or inhibitor saline taste is experienced for it. Abreast, generation lacks the knock-out mice of gene of interest (or expressing as variant form that this paper Varitint mouse is used), and carries out physiology (neural recording) and behavior (the 2-bottle is preferably tested and the sense of taste is measured test) and test and determine whether defective or lack saline taste and experience of this animal.

Therefore, this TRPML3 gene is accredited as the polypeptide ion channel that coding participates in the sodium taste perception and may participate in sodium impression and metabolism, and it is more widely based on following standard: 1) in genetic chip and/or PCR experiment specifically in gustatory expression or the expression in gustatory be higher than gene (these are defined as sense of taste specific gene) in the tongue cell; 2) do not correspond to the gene of expressing in unique cell type of sweet taste, bitter taste, tart flavour and/or umami cells in histology; 3) in electric physiology or functional experiment, produce the gene outcome that sodium responds acceptor or sodium channel; With 4) reinforcing agent or co-inhibitor and/or the knock-out mice of the gene outcome of regulation and control saline taste impression or express non-activity or the variant form defective of interested ion channel gene or lack saline taste and reply.

Use these principles, method and scheme, it is special that many primate genes are accredited as gustatory at first. These genes that comprise TRPML3 are contained in the early stage provisional application, and it incorporates this paper by reference into. Consider to identify comprehensive and correct method of these genes, the inventor predicts that this big cover gene is included in the gene of specifically expressing in the primate gustatory.

From this big group of gene, identified the sense of taste special, in the little subgroup of the sense of taste specific gene of the first half specifically expressing of taste bud or enrichment and coding sodium-ion channel. Three ion channel specific genes have been identified especially, i.e. TRPML3, NKAIN3 and NALCN. In these 3 genes, confirmed that the TRPML3 coding participates in the saline taste impression and be the required ion channel of saline taste impression.

Particularly, be contained in the data that obtain in the hereinafter function of embodiment (electric physiology) and immunohistochemistry data and Varitint mouse show MCOLN3 (TRPML3) as rodent, people and other primates and may other mammals in the saline taste acceptor work, and may in relating to other physiological functions of sodium metabolism, absorption and drainage (such as relating to that aldosterone produces and the physiological function of vasopressing release), play a role. This standard of the test of the ion channel gene of support selecting is further in general introduction and the table 1 hereinafter.

Table 1

The gene name

TB is to the ratio of LE

The top is to the ratio of substrate TB

Report the list of references of this gene code sodium channel

NALCN(akaVGCNL1)	11.2	7.2	Cell.2007 April 20; 129 (2): 371-83The neuronal channel NALCNcontributes resting sodiumpermeability and is required fornormal respiratory rhythm. (the neuron passage helps the permeability of tranquillization sodium, and is that normal respiratory rhythm is required)Lu B， Su Y， Das S， Liu J， Xia J， Ren D. NALCN is described as the sodium leakage passage, consistent with the saline taste acceptor of prediction.
				TRPML3(akaMCOLN3)	10.2	1.6	J Biol Chem.On October 25th, 2007; [being published in before the galley on the net] Gain-of-function mutation in TRPML3causes the mouse varitint-waddlerphenotype. (the acquisition function mutation of TRPML3 causes the phenotype of varitint-waddler mouse).Kim HJ， Li Q， Tjon-Kon-Sang S， So I， Kiselyov K， Muallem S. TRPML3 is described first as the passage that can see through sodium, consistent with the saline taste acceptor.
NKAIN3(akaFAM77D)	3.3	1.5	Hum Mol Genet.On October 15th, 2007; 16 (20): 3394-410. is published in A novel on the 2nd family of transmembraneproteins interacting with July in 2007 (beta) subunits of the Na on the net, K-ATPase. (with
							Na, the interactional transmembrane protein new family of the β subunit of K-ATP enzyme)Gorokhova S， Bibert S， Geering K， Heintz N. the fruit bat homologue of NKAIN3 has been described as amiloride-insensitive sodium channel, consistent with the saline taste acceptor.

Therefore, based on preamble, the present invention relates generally to for the identification of the method for sense of taste gene (comprise participate in the saline taste impression or such as the gene of other taste perception of fat taste impression) and its for the identification of people's saline taste sense of taste reinforcing agent and other sense of taste conditioning agent compounds with for the identification of the application in the Screening test of the potential therapeutic agent of other gustatory correlation functions of regulation and control and phenotype (comprise directly and the sense of taste transduce relevant disease and the patient's condition, namely relate in the different tissues unusually disease and the patient's condition of sodium transport, metabolism and drainage and impression).

The compound of regulation and control TRPML3 has potential application in the impression of regulation and control people saline taste. Acceptable derivates test in people's sense of taste test of end user volunteer on the compound of for example in electrophysiological detection, identifying and the biology thereof, its impact to the impression of people's saline taste for confirmation. In addition, the compound that is accredited as potential therapeutic agent will be evaluated in suitable external and body inner model, depend on the essence that expection is used. The compound that for example is accredited as the potential therapeutic agent of diabetes can (such as NOD mouse model or BioBreeding rat model) assessment in known diabetes animal model. Similarly, the compound that is accredited as the potential therapeutic agent of IBD or Crohn disease can be tested in the rodent models of IBD or Crohn disease.

Be used for that identification of taste (for example saline taste sense of taste) is regulated or the mensuration based on cell for the treatment of compound will preferably comprise the high flux screening platform of the compound of the gene activity that makes up to identify that regulation and control (enhancing) participation saline taste is experienced with the cell of expressing gene disclosed herein or its. In addition, these sequences can be modified in order to introduce silent mutation or have the sudden change (such as the definite sudden change that affects ion (sodium) and flow into) of function effect. Mention as mentioned, this mensuration will be preferably included in the electrophysiological detection that carries out in the amphibian egg mother cell or use fluorescence ion-sensitive dyestuff or film potential dyestuff (for example sodium sensitive dye) uses the mensuration of expressing according to the mammalian cell of ion channel of the present invention. Preferably, the compound of regulating and control this type of ion channel is by the Screening and Identification of using the electrophysiological detection (for example patch-clamp or two electrode voltages pincers) that the egg mother cell of expressing the ion channel that this paper identifies carries out.

Alternatively, the regulation and control supposition participates in the compound of the ion channel of the saline taste sense of taste and can measure to detect by ion current, and for example the radio-labeled ion current is measured or the ion current of atomic absorption spectrum coupling is measured. As mentioned open, these compounds have potential application in regulation and control people saline taste impression or for the other biological process of regulating and control to relate to unusual or normal ion channel function.

Use in the cell of the needs in usually being used in the screening of regulating compound for the identification of ion channel or GPCR the nucleotide sequence of the sudden change that (preferably egg mother cell or such as people's cell or other people or the mammalian cell of HEK-293 cell) express based on the mensuration of cell. These cells can further be transformed in order to express other sequences, and other sense of taste GPCR for example is namely such as the T1R that describes in other patent applications of this assignee Senomyx or T2R and suitable G albumen. The egg mother cell system has superiority because its allow multiple mRNA species direct injection, provide high protein to express, and be the adverse effect of ion channel overexpression in holding. Yet shortcoming is to use the electric physiology screening of amphibian egg mother cell to be not suitable for a large amount of compound of high flux screening, and is not mammlian system. As point out that the present invention comprises the mensuration of using mammalian cell, preferably high throughput assay. These high flux screenings measure generally will use transfection be inoculated into the hole or culture plate in mammalian cell, wherein when test compounds exists, allow to carry out functional expression, and use film potential fluorescence or ion (sodium) fluorescent dye come detection of active.

These screening techniques are used for identifying the TRPML3 conditioning agent with nucleic acid provided herein and protein sequence, such as the potential therapeutic agent of the activator of people's saline taste sense of taste or other sense of taste forms, inhibitor, stimulon, reinforcing agent etc. and other gustatory functions of target or phenotype. This type of conditioning agent can affect the saline taste sense of taste or other sense of taste forms or gustatory correlation function and phenotype, for example by regulatory transcription, translation, mRNA or protein stability; By changing the interaction of ion channel and plasma membrane or other molecules; Or by affecting the activity of ionophorous protein.

Use high flux screening (HTS) to come screening compounds, so as to identify can in conjunction with and/or regulation and control taste receptors or sense of taste ion channel polypeptide or transhipment is sub or those compounds of its fragment activity. Among the present invention, albumen is expressed with recombinating in the cell of for example people's cell or frog egg mother cell, any measuring method by use ion channel, acceptor or transhipment subfunction is measured active regulation and control, such as the change of sodium or lithium level in measurement film potential or the measurement born of the same parents. The method of measuring ion (for example cation) channel function for example comprises, Imaging-PAM and the ion current of patch clamp technique, two electrode voltages pincers, the full cell currents of measurement and use ion-sensitive fluorescent dye are measured, and for example radiolabeled ion current is measured or ion current is measured.

The gene reinforcing agent of identifying according to the present invention can be used for many different purposes. For example, it can comprise the saline taste sense of taste of regulating and control food, beverage, soup, medicine and other people consumable products as flavor enhancement. In addition, the invention provides be used to the kit that carries out mensuration disclosed herein.

In fact, as be shown in the examples, this type of mensuration based on the TRPML3 cell has been identified TRPML3 co-inhibitor and reinforcing agent, and it will testedly in taste test determine that it is to the impact of saline taste impression.

The present invention also provides the application of TRPML3 as the sign of the cell that can be used to enrichment, evaluation or separation expression saline taste acceptor.

The present invention also provides use TRPML3 (MCOLN3) and has expressed the cell of TRPML3 or the TRPML3 transgenic animal model is identified and caused or regulation and control (prevent or strengthen) comprise the external and in vivoassay of activator, antagonist or reinforcing agent compound of the saline taste sense of taste of people's primate. These measure the cell that uses single expression TRPML3 or the cell of expressing TRPML3 ion channel and other sense of taste specific polypeptides (such as NALCN or NKAIN3) or relevant TRPML member (such as TRPML1 or TRPML2).

Further, the invention provides transgenic animals (preferably rodent) and being used for confirm TRPML3 the mammiferous saline taste sense of taste and relating to sodium and other physiological functions (such as sodium metabolism, blood pressure, fluid retention and drainage, urinary function and cardiac function) of other ions in the application of effect.

In addition, the invention provides the cell that uses TRPML3 and express TRPML3 or transgenic animals in mensuration (preferably electrophysiological detection) in order to identify the external and in vivoassay of the therapeutic compound that alleviates the disease that relates to this expression of polypeptides defective (overexpression, the low sudden change of expressing and affecting the TRPML3 polypeptide of its ability that in comprising for example people's and non-human primate mammal, works as the special sodium channel of the sense of taste) and the patient's condition. These patient's condition comprise Addison's disease and relate to the disease that unusual aldosterone produces or vasopressing discharges as an example, such as hypertension, low blood pressure, fluid retention and impaired uropoiesis or cardiac function, such as cardiac arrhythmia, heart attack and apoplexy.

Definition

" the saline taste acceptor of supposition or saline taste sense of taste ion channel gene " refers to express in gustatory and not at tongue cells or the obvious more weak gene of the expression in the tongue cell, preferably do not express in the gustatory of expressing T1R, T2R, TRPM5 or PKD2L1/PKD1L3 gene in addition. Preferably, these genes are in the cell the first half contrast the latter half specifically expressing that comprises taste bud or enrichment (at least doubly more highland expression of 1.2-1.5). This comprises chemoreception or gustatory, particularly macaque and other mammiferous gustatories of possibility. As point out and in preferred aspects of the invention, TRPML3 has been accredited as this type of saline taste acceptor (sodium-ion channel) polypeptide.

" TRPML3 " or " MCOLN3 " refers to participate in rodent, people and non-human primate and may other mammals and gene or its variant of the saline taste impression of vertebrate (comprising bird, reptile and amphibian). The application comprises just the TRPML3 gene of the people before claim, mouse, ox, mouse, zebra fish, chicken and other mammalian species and the exemplary sequence of polypeptide. The peptide sequence that has relatively disclosed the TRPML3 polypeptide of these sequences is very similar in different plant species, and namely mouse and people TRPML3 have 96% sequence homogeneity and 91% sequence similarity. Therefore to those of ordinary skill in the art, in other mammalian genes or genome and polypeptide libraries, identify that the TRPML3 gene should be relative simple. And, consider the sodium metabolism to the key effect of cell viability and organism general health, other vertebrates may be expressed TRPML3. According to this, the invention is intended to comprise widely the TRPML3 gene that contains different plant species (but most preferably people and other mammals) and the saline taste acceptor of functional variety (such as chimera). The TRPML3 gene of this paper and polypeptide also are included in TRPML3 mutator and the fragment of one or more site mutations particularly, described sudden change is for example in order to modify (strengthening or reduce the TRPML3 activity), ion channel is more suitable for for such as (for example TRPML3 of different plant species or non-TRPML3 ion channel are such as for example TRPML1 so that this ion channel is fixed in open to the outside world or " closing " position or the domain by producing one of them TRPML3 polypeptide or born of the same parents' outer shroud or its part and another ion channel by modified polypeptide, TRPML2, NKAIN3 or NALCN) exchange fragment or chimeric mensuration in. Term TRPML3 polypeptide and nucleotide sequence also specifically comprise with polypeptide disclosed herein and have at least 80% sequence homogeneity, more preferably have at least 90% sequence homogeneity or the TRPML3 ion channel polypeptide of at least 95%, 96%, 97%, 98% or 99% sequence homogeneity more preferably with natural TRPML3 polypeptide (such as the TRPML3 polypeptide of natural human, non-human primate, rodent (rat, mouse etc.), dog, cat, horse, ox, sheep etc.). The TRPML3 nucleotide sequence also comprises all nucleic acid coding sequences, such as cDNA, genome sequence, cRNA, mRNA and strand, two strands and three chain nucleic acid sequence and complementary strand thereof. Among the people, exist in the sequence table before being contained in claim of 3 principal modes of TRPML3 mRNA, its sequence at taste bud, hypophysis and adrenal gland.

The TRPML3 sequence also comprises specifically with coding and participates in the nucleotide sequence of coding TRPML3 of sodium-ion channel of saline taste impression and/or sodium transport, metabolism or drainage or the sequence of its complementary strand hybridization. Be suitable for identifying that the exemplary hybridization patient's condition of other TRPML3 straight homologuess and related gene is known in the art, and be defined in the application hereinafter.

" gustatory " refers to express the cell of directly or indirectly regulating or regulating and control specific sense of taste form such as sweet taste, tart flavour, delicate flavour, saline taste, bitter taste, fat, metal or other taste perceptions or general taste perception such as at least one acceptor, transhipment or the ion channel of the duration of sense of taste intensity or sense of taste response when ripe. This is particularly including the gene of expressing in chemical sensation or gustatory (particularly macaque and other mammal gustatories of possibility). Gustatory is expressed the albumen (chromosome reading frame 15) of mRNA and/or gene C 6orf15-be also referred to as STG. This gene is described as sense of taste specific gene (M.Neira etc., Mammalian Genome 12:60-66,2001), and in the macaque sense of taste specific gene of this paper report. In addition, ripe taste receptor cells is generally with mRNA and/or the albumen of express alpha ENaC. We have data (this paper does not show) announcement α ENaC expressing in sweet taste, bitter taste, delicate flavour, tart flavour and the most probable saline taste gustatory at least. Further, ripe taste receptor cells is with mRNA and/or the albumen of General Expression Cyfra21-1. This albumen is only expressed in the gustatory of maturation, is not found in substrate or stem cell (L.Wong etc., Chemical Senses 19 (3): 251-264,1994). In addition, but based on its characteristic morphology those of ordinary skill in the art identification of taste cell. Especially, the gustatory of ripe taste receptors is the spindle bodily form of elongation. Ripe taste receptor cells also has the cell tip (top film) that penetrates taste pore, therefore approaches or is exposed to saliva. On the contrary, immature gustatory (for example basal cell or stem cell) is circular, and is not exposed to taste pore and saliva. Be different from ripe gustatory, basal cell and stem cell also tend to the base portion location towards taste bud.

" chemoreception cell " is the cell that participates in the impression that stimulates such as the chemical stimulus of flavor unit with such as other chemoreceptions of odorant. The chemoreception cell of this paper is particularly including taste receptor cells and be contained in the cell of expressing one or more taste receptors in alimentary canal or urethra or other organs when ripe. For example, known stomach and intestine chemoreception cellular expression T1R or T2R, and these cells may participate in food impression, metabolism, digestion, diabetes, food absorption, stomach motion etc. In addition, the cell of finding in the urethra may be expressed the saline taste acceptor, and participates in sodium transport, drainage and relative function, such as blood pressure and fluid retention. Further, the chemoreception cell of expressing taste receptors in the digestive system also can be expressed Chromogranin A, this albumen is the sign (C.Sternini of secretory granules, " Taste Receptors in thegastrointestinal tract.IV.Functional Implications of BitterTaste Receptors in Gastrointestinal Chemosensing ". (function of bitter taste taste receptors hint in the taste receptors .IV. stomach and intestine chemoreception in the intestines and stomach) American Journal of Physiology, Gastrointestinal and LiverPhysiology. "; 292:G457-G461,2007).

" the gustatory related gene " of this paper refers to that gustatory is expressed rather than the gene of the participation sense of taste of tongue cellular expression or the relevant gustatory function of the non-sense of taste or phenotype. The gene of specifically expressing in particularly from the chemoreception of macaque or gustatory of reporting in this early stage provisional application of quoting particularly including this paper and this paper. Gustatory comprise in the oral cavity (such as tongue) of expressing taste receptors cell and at the gustatory in other zones of body (such as digestive system and urethra) of expressing taste receptors. This genoid comprises the gene in the table that is contained in the application of incorporating by reference this paper into. These genes comprise the gene that participates in the sense of taste and non-sense of taste correlation function, such as gustatory turnover, affect disease, oral cavity and/or the digestive system in digestive system or oral cavity the immunity accent, relate to the digestion and metabolism function of gustatory, such as diabetes, obesity, blood pressure, fluid retention etc. When referring to the specific sense of taste specific gene that this paper identifies, these genes comprise corresponding to the gene that is contained in this paper and straight homologues thereof and comprise the chimera of its allelic variant and the nucleotide sequence of variant. Especially, this type of variant comprise coding with by at least 80% identical (sequence of at least 90% or 95% identical polypeptide more preferably of the polypeptide corresponding to the gene code of quoting gene or its straight homologues (particularly people and non-human primate straight homologues). In addition, this gene be included under the stringent hybridization condition with corresponding to the nucleotide sequence corresponding to the nucleic acid array hybridizing of a kind of gene order of the gene in the early stage temporary patent application.

" cationic channel " is to regulate the multiple protein that anionic current is crossed cell membrane. The ability of certain cationic passage transhipment certain cationic generally changes with the specificity of the special modality of cationic chemical valence and certain cationic.

" with poly-passage " refers to the cationic channel that is made up of identical α subunit, and " different poly-passage " refers to the cationic channel that is made up of two or more dissimilar α subunits. Can comprise auxiliary β subunit with poly-with different poly-passage.

The polypeptide monomer of the auxiliary subunit of the cationic channel that " β subunit " is made up of the α subunit; And independent β subunit can not form passage (referring to for example, United States Patent (USP) the 5th, 776, No. 734). Known β subunit for example increases the number of passage by helping the α subunit to arrive cell surface, changes the sensitiveness that activates dynamics and change the native ligand the joint passage. The β subunit can be in porose area outside, and associates with the α subunit that comprises porose area. They also can facilitate the collar extension of porose area.

Term " really " or " wild type " or " natural " nucleotide sequence refer to be contained in wild-type nucleic acid sequence and general other nucleotide sequences of being familiar with of splice variant, allelic variant and other variants and this area of this paper sequence table.

Term " really " or " wild type " or " natural " polypeptide refer to by the application and incorporate by reference the polypeptide of disclosed gene and nucleic acid sequence encoding in the early stage temporary patent application of this paper into.

Term " the enhancing receptor nucleic acids sequence of modification " or " nucleotide sequence of optimization " refer to contain the nucleotide sequence of the sudden change of gene activity in one or more sudden changes, particularly impact (suppress or strengthen) recombinant host cell (the most especially egg mother cell or people's cell, such as the HEK-293 cell). Especially, these sudden changes comprise the sudden change that affects gate by the ion channel that contains sudden change subunit sequence that obtains. This ion channel can comprise this type of sudden change in one or several of three subunits that consist of the specific ion passage. The nucleotide sequence of modifying for example can at impact (infringement) gate control function or lack to contain to replace in the subunit of surface expression and suddenlys change. The present invention comprises other mutator sequences, i.e. splice variant, the variant, the chimera of this sequence and the application of similar sequence that contain disappearance or increase. Further, the present invention can use and can be modified in order to introduce the sequence of the preferred codon of host cell (the particularly preferred codon of amphibian or human host cell).

The nucleic acid of term acceptor or ionophorous protein or its fragment or encode specific taste receptors or ion channel or transhipment or its fragment refers to nucleic acid and polypeptide polymorphie variant according to the present invention, allele, homologue between mutant and species, its: (1) has with the amino acid sequence of the amino acid sequence of wild-type nucleic acid coding or sense of taste albumen and for example is higher than about 60% amino acid sequence identity by the albumen of the gene nucleic acid sequential coding that is contained in this paper table 1 and fragment thereof and its conservative variant of modifying, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or the amino acid sequence of homoamino acid sequence homogeneity more, preferably at least about 25,50,100,200,500,1000 or the zone of amino acids more; (3) by under stringent hybridization condition specifically with corresponding to the nucleotide sequence of the antisense strand hybridization of the nucleotide sequence of the gene of one of described gene of coding coding and the polypeptide of the conservative variant coding of modifying thereof; (4) have to have with for example nucleic acid disclosed herein and be higher than about 60% sequence homogeneity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% or the nucleotide sequence of higher nucleotide sequence homology, preferably at least about 25,50,100,200,500,1000 or the zone of polynucleotides more.

The saline taste of inferring or other sense of taste specific genes or polynucleotides or peptide sequence are generally from mammal, and it includes but not limited to primate (for example people); Rodent (for example rat, mouse, hamster); Ox, pig, horse, sheep or any other mammal. Yet, as it may have in other (nonmammalian) vertebrates of identity function and expresses therein to mention the TRPML3 ion channel. Nucleic acid of the present invention and albumen comprise naturally occurring or recombinant molecule. These genes generally have coding the albumen of ion channel activity, and namely it can see through sodium or lithium. This comprises primate TRPML3 gene and people thereof and other mammiferous straight homologuess and keeps the functional fragment of TRPML3 and variant especially, and namely performance is similar at monitoring sodium, lithium conduction and in to the electrophysiological detection of replying (lacking) of amiloride and in other functional examinations that are fit to.

Mean the impact of measuring increase or reducing the compound that indirectly or directly is in the parameter under sense of taste gene (the preferably saline taste gene of this paper evaluation) impact, for example function, physics, phenotype and chemical affect by " determining functional impact " or " determining the impact to cell ". This type of function effect includes but not limited to ion current, film potential, current strength and valtage-gated change, and other biological learns impact, such as change and the similar impact of the gene expression of any marker gene. Ion current can comprise any ion by passage, and for example sodium or lithium and analog thereof are such as radio isotope. This type of function effect can be measured by any method known to those skilled in the art, for example uses the patch-clamp of voltage sensitive dye or by measuring the change such as the parameter of spectral signature (for example fluorescence, absorbance, refractive index), fluid dynamic (for example shape), chromatogram or dissolubility property. The electrophysiological detection that is fit to of use expressing the cell of TRPML3 is illustrated in hereinafter the EXPERIMENTAL EXAMPLE.

The polynucleotides that this gustatory is expressed and " inhibitor ", " activator " and " conditioning agent " of peptide sequence are used to refer to activation, inhibition or the regulatory molecule of the external and in vivoassay evaluation of using these polynucleotides and peptide sequence. Inhibitor be for example in conjunction with, partially or completely suppress active, reduce, stop, delay to activate, inactivation, desensitization or the compound reducing the active of these sense of taste differential proteins or express, for example antagonist. " activator " is the compound that strengthens, opens, activates, promotes, strengthens activation, sensitization, antagonism or upregulated protein activity. Inhibitor, activator or conditioning agent generally also comprise the genetic modification form of this gustatory differential protein, for example have the form that changes activity, and natural existence and synthetic part, antagonist, activator, peptide, cyclic peptide, nucleic acid, antibody, antisense molecule, siRNA, ribozyme, little organic molecule and similar molecule. This type of mensuration of inhibitor and activator for example comprises expresses this gustatory differential protein in external, cell, cell extract or cell membrane, use the conditioning agent compound inferred and definite function effect to activity then, as indicated above.

The sample or measure that comprises the albumen of the gene code of being identified by this paper that processes with potential activator, inhibitor or conditioning agent relatively checks with the control sample that does not have inhibitor, activator or conditioning agent and to activate or degree that migration is regulated and control. The relative protein active value of control sample (not processing with inhibitor) designated 100%. When the activity value with respect to contrast be about 80%, preferably 50%, more preferably during 25-0%, realize the inhibition of ion channel. When the activity value with respect to contrast (not processing with activator) is 110%, more preferably 150%, more preferably 200-500% (namely with respect to 2 to 5 times higher of contrasts), 1000-3000% or when higher more preferably, realize the activation of ion channel.

As used herein, term " test compounds " or " drug candidate " or " conditioning agent " or phraseological equivalent are described the natural existence of ability of its regulation and control creeping chill to be tested or any molecule of synthetic compound, (for example length is from about 5 to about 25 amino acid for preferably little molecule or protein, oligopeptides, preferably length is from about 10 to 20 or 12 to 18 amino acid, and preferably length is 12,15 or 18 amino acid), little organic molecule, polysaccharide, lipid, aliphatic acid, polynucleotides, siRNA, oligonucleotides, ribozyme etc. Test compounds can be the form with the test compounds library, such as the multifarious combination of enough scopes or random library are provided. Test compounds randomly links to each other with fusion partner, for example target compound, rescue compound, dimeric compounds, stable compound, addressable compound and other funtion parts. By convention, be tested and appraised the test compounds (being called " candidate compound ") with some desirable characteristics or activity (for example suppressing active) and produce the new chemical entities with useful properties, produce the variant of lead compound and assess characteristic and the activity of those variant compounds. High flux screening (HTS) method is adopted in this alanysis usually.

" little organic molecule " refers to that molecular weight is greater than about 50 dalton and less than about 2500 daltonian natural existence or synthetic organic molecules, preferably less than about 2000 dalton, preferably between about 100 between about 1000 dalton, more preferably between about 200 to about 500 dalton.

" biological sample " comprises such as the histotomy of biopsy and postmortem sample and the freezing microtome section of getting for the histology purpose. This type of sample comprises blood, sputum, tissue, the cultured cell such as primary culture, explant and transformant, ight soil, urine etc. Biological sample is generally available from the eucaryon organism, and mammal most preferably is such as primate (for example chimpanzee or people); Ox; Dog; Cat; Rodent, for example cavy, mouse, rat; Rabbit; Or bird; Reptile; Or fish.

Term " identical " or " homogeneity " percentage refer in the context of two or more nucleic acid or peptide sequence that two or more sequences or subsequence are identical have the amino acid residue of particular percentile or nucleotides identical (namely when with maximum correspondence when relatively window or appointed area compare and compare, about 60% homogeneity that (for example is contained in gene or the sequence of this paper table 1) in the specific region, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher homogeneity), measured such as the BLAST or BLAST 2.0 sequence comparison algorithms that use default parameters hereinafter described, or by manpower comparing to and visual inspection (referring to for example NCBI website or similar website). Can say that then this type of sequence is " substantially identical ". This definition also refers to or can be applicable to the complementary series (compliment) of cycle tests. This definition also comprises the sequence that has disappearance and/or add, and the sequence with replacement. As mentioned below, optimization algorithm can be considered breach and similar parameters. Preferably, homogeneity is present in length at least about the zone of 25 amino acid or nucleotides, and more preferably being present in length is the zone of 50-100 amino acid or nucleotides.

To sequence relatively, a general reference sequences that sequence compares with it as cycle tests. When using sequence comparison algorithm, test and reference sequences are transfused to computer, specify the subsequence coordinate, if necessary, and specified sequence algorithm routine parameter. Preferably, the default program parameter can be used, perhaps optional parameters can be specified. Calculate cycle tests with respect to the sequence homogeneity percentage of reference sequences based on the program parameter sequence comparison algorithm then.

As used herein, " comparison window " comprises the reference of fragment that is selected from by any of many adjoining positions of the following group that forms: from 20 to 600, common about 50 to about 200, more generally about 100 to about 150, wherein after two sequences were compared by the best, sequence can compare with the reference sequences of similar number adjoining position. It is well known in the art being used for sequence alignment method relatively. Can carry out the best comparison for sequence relatively, for example by Smith ﹠ Waterman, local homology's algorithm of Adv.Appl.Math.2:482 (1981), by Needleman﹠ Wunsch, the sequence analysis algorithm of J.Mol.Biol.48:443 (1970), by Pearson ﹠ Lipman, the similarity searching method of Proc.Na t ' l.Acad.Sci.USA 85:2444 (1988), (WisconsinGenetics software kit (Genetics Computer Group is implemented in computerization by these algorithms, 575 Science Dr., Madison, Wis. the GAP in, BESTFIT, FASTA and TFASTA), or by manpower comparing to and visual inspection (referring to such as Current Protocols in Molecular Biology (molecular biology fresh approach) (Ausubel etc., editor, nineteen ninety-five supplementary issue)).

The preferred embodiment that is fit to the algorithm of definite sequence homogeneity and sequence similarity percentage is BLAST and BLAST 2.0 algorithms, it is described in respectively Altschul etc., Nucl.AcidsRes.25:3389-3402 (1977) and Altschul etc., J.Mol.Biol.215:403-410 (1990). Determine the sequence homogeneity percentage of nucleic acid of the present invention and protein with BLAST and BLAST 2.0 with parameter described herein. The software that is used for execution BLAST analysis can openly obtain by NCBI. This algorithm relates to that at first to be tested and appraised length in the inquiry sequence be that the short character of W is identified high sub-sequence to (HSP), these characters when with database sequence in the character of same length mate when comparing or satisfy some on the occasion of threshold value score T. T refer to adjacent character score threshold value (Altschul etc., above). These initial adjacent characters hit the longer HSP that finds to contain these characters as the seed of initial search. Character hits at the both direction along each sequence and extends until the score of accumulation comparison increases. Nucleotide sequence operation parameter M (reward score of a pair of coupling residue, always＞0) and N (penalty values of not mating residue, always＜0) are calculated the accumulation score. To amino acid sequence, calculate the accumulation score with score matrix. When: accumulation comparison score has reduced quantity X from its maximum acquisition value; Since the accumulation of one or more negative score residue comparisons, accumulation score vanishing or following; When perhaps reaching arbitrary sequence terminal, the extension that each direction character hits suspends. BLAST algorithm parameter W, T and X determine sensitivity and the speed of comparison. The default parameters of BLASTN program (for nucleotide sequence) is: character length (W) is 11, desired value (E) is 10, M=5, N=-4 and two chains of comparison. To amino acid sequence, the default parameters of BLASTP program is: character length is 3, desired value (E) is 10, and the BLOSUM62 score matrix is (referring to Henikoff ﹠ Henikoff, Proc.Natl.Acad.Sci., USA 89:10915 (1989)) in, comparison (B) is 50, desired value (E) is 10, M-5, N=-4 and two chains of comparison.

" nucleic acid " refers to the deoxyribonucleotide of strand or double chain form or ribonucleotide and polymer or its complementary strand. This term comprises the framework residue that contains known nucleotide analog or modification or the nucleic acid of bonding, it is for that synthesize, naturally occurring and the non-natural existence, it has the binding characteristic similar to reference nucleic acid, and with the mode metabolism similar to reference nucleotide. The example of this type of analog includes but not limited to thiophosphate, phosphoramidate, methyl phosphorodithioate, chirality-methyl phosphorodithioate, 2-O-methyl ribonucleotides, peptide-nucleic acid (PNA).

Except as otherwise noted, the specific nucleotide sequence sequence that also comprises implicitly its conservative variant (for example degenerate codon replacement) of modifying and complementary series and clearly indicate. Particularly, degenerate codon replaces and can realize (Batzer etc., Nucleic Acid Res.19:5081 (1991) with the sequence of mixing the replacement of base and/or deoxyinosine residue by the 3rd position that produces wherein one or more selections (or owning) codon; Ohtsuka etc., J.Biol.Chem.260:2605-2608 (1985); Rossolini etc., Mol.Cell.Probes 8:91-98 (1994)). The commutative use of term nucleic acid and gene, cDNA, mRNA, oligonucleotides and polynucleotides.

Specific nucleic acid sequence also comprises " splice variant " implicitly. Similarly, the specific protein by nucleic acid coding comprises any albumen of being encoded by the splice variant of this nucleic acid implicitly. Show that such as its title " splice variant " is the product of gene alternative splicing. After transcribing, initial transcribed nucleic acid thing can be by montage so that the different polypeptide of difference (replacing) nucleic acid montage product coding. The mechanism that produces splice variant is different, but comprises the alternately montage of extron. The alternately polypeptide that passes through read-through transcription from identical nucleic acid also is contained in this definition. The spawn (recombinant forms that comprises the montage product) of montage reaction is included in this definition. The example of potassium channel splice variant is discussed among J.Biol.Chem.273 (52): the 35095-35101 (1998) at Leicher etc.

Term " polypeptide ", " peptide " and " protein " refer to the polymer of amino acid residue in the commutative use of this paper. This term application is corresponding natural polymers that have amino acid whose artificial chemistry analogies in wherein one or more amino acid residues, and there is amino acid whose polymer in natural amino acid whose polymer and the non-natural of existing.

Term " amino acid " refers to natural existence and synthetic amino acid, and amino acid analogue and amino acid analog thing to work with naturally occurring amino acid similarity mode. Naturally occurring amino acid is the amino acid by genetic code coding, and afterwards adorned amino acid, for example hydroxyproline, Gla and O-phosphoserine. Amino acid analogue refers to have the compound with the identical basic chemical structure of naturally occurring amino acid, the carbon of namely being combined with hydrogen, carboxylic group, amino group and R group, for example homoserine, nor-leucine, Methionine sulfoxide, methionine methyl sulfonium. This type of analog has the R group (for example nor-leucine) of modification or the peptide backbone of modifying, but keeps the basic chemical structure identical with naturally occurring amino acid. The amino acid analog thing refers to have the structure that is different from the general chemical constitution of amino acid but the compound that works in the mode with naturally occurring amino acid similarity.

Herein, the one-letter symbol of its known trigram symbol or IUPAC-IUB biochemical nomenclature commission recommendation all can refer to amino acid. Equally, its single-letter code of usually accepting can refer to nucleotides.

" the conservative variant of modifying " is applied to amino acid and nucleotide sequence. About specific nucleotide sequence, the conservative variant of modifying refer to the to encode nucleic acid of identical or basic identical amino acid sequence, or the do not encode nucleic acid of amino acid sequence of basic identical sequence of this nucleic acid wherein. Because the degeneracy of genetic code, the identical any specific albumen of nucleic acid coding on many functions. For example, codon GCA, GCC, GCG and GCU coded amino acid alanine all. Therefore codon is appointed as each position of alanine therein, and this codon can be changed into the described any corresponding codon that does not change coded polypeptide. This type of nucleic acid is changed into " the reticent change ", and it is a kind of change of conservative modification. Each nucleotide sequence of coded polypeptide has also been described each possible silence change of this nucleic acid herein. The technical staff will understand, and each codon of nucleic acid (except the AUG of unique password that is generally methionine and the TGG of unique password that is generally tryptophan) can be modified in order to produce molecule identical on the function. Correspondingly, each of the nucleic acid of coded polypeptide reticent change to imply in each be not in relation in the described sequence of actual probes sequence about expression product.

About amino acid sequence, single replacement, disappearance or the interpolation that it will be understood by those skilled in the art that change in the coded sequence, increases or lack single amino acids or the amino acid whose nucleic acid of little percentage, peptide, polypeptide or protein sequence is wherein to change to cause with chemically similar 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor amino acid whose " variant of conservative modification ". Provide that similar amino acid whose conservative replacement table is well known in the art on the function. This type of conservative variant of modifying be additional to and do not get rid of polymorphie variant of the present invention, plant between homologue and allele.

Below 8 groups every group contain the mutually conservative amino acid that replaces: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (Q); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); With 8) cysteine (C), methionine (M) (referring to for example Creighton, Proteins (1984)).

Macromolecular structure such as polypeptide structure can be described from the various levels of organizing. The general discussion of this tissue is referring to such as Alberts etc., Molecular Biology of the Cell (molecular biology of the cell) (third edition, 1994) and Cantor and Schimmel, biophysical chemistry part I:The conformation of Biological Macromolecules (conformation of large biological molecule) (1980). " primary structure " refers to the amino acid sequence of particular peptide. " secondary structure " refers to the three-dimensional structure of polypeptide local order. These structures are commonly referred to domain, for example membrane spaning domain, pore structure territory and kytoplasm stern construction territory. Domain is the polypeptide portion that forms the polypeptide compacted unit, is generally 15 to 350 amino acid longs. The exemplary configurations territory comprises ectodomain, membrane spaning domain and cytoplasmic structure territory. Typical domain is made of the part of lower tissue, such as. β .-folding and. the stretching of α .-spiral. " tertiary structure " refers to the complete three-dimensional structure of polypeptide monomer. " quaternary structure " refers to the three-dimensional structure that formed by the non-covalent association of three grades of units independently. The anisotropy term is also referred to as energy terms.

" mark " or " but test section " is to pass through the composition that spectrum, photochemistry, biochemistry, immunochemistry, chemistry or other physical methods detect. For example useful mark comprise 32P, fluorescent dye, electron dense agent, enzyme (for example, usually being used among the ELISA), biotin, digoxin or haptens and can be for example by radioactive label being mixed become detectable protein or be used for detecting specifically protein with the antibody of reactive polypeptide of peptide.

When for example referring to that cell or nucleic acid, albumen or carrier use, term " restructuring " shows that this cell, nucleic acid, albumen or carrier are modified by the change of introducing heterologous nucleic acids or albumen or natural acid or albumen, and perhaps this cell is from the cell of modification like this. Therefore, for example, recombinant cell is expressed the gene of not finding or is expressed the otherwise natural gene of unconventionality expression (reduce and express or do not express) in natural (non-restructuring) form of cell.

When referring to that nucleic acid moiety uses, term " allos " shows that this nucleic acid comprises two or more subsequences of not finding with mutually the same relation at nature. For example, this nucleic acid generally is recombinantly produced, have two or more from uncorrelated gene and be arranged as the sequence that produces new functional nucleic acid, for example from the promoter in a source with from the code area in another source. Similarly, heterologous protein shows that this albumen comprises two or more subsequences (for example fusion) of not finding with mutually the same relation at nature.

Phrase " stringent hybridization condition " refer to probe under this condition will with generally be in the nucleic acid complex mixture its target subsequence hybridization and not with the condition of other sequence hybridizations. Stringent condition is sequence-dependent, and is different under different situations. Longer sequence is specific hybridization under higher temperature. The extensive guidance of nucleic acid hybridization is found in Tijssen, Techniques inBiochemistry and Molecular Biology-Hybridization with NucleicProbes (hybridization of biochemistry and Protocols in Molecular Biology-nucleic acid probe), " Overviewof principles of hybridization and the strategy of nucleic acidassays " (" general introduction of Hybridization principle and nucleic acid determination strategy ") (1993). Stringent condition is generally elected as under specific ion intensity pH than low about 5-10 ℃ of the thermal melting point (Tm) of particular sequence. Tm is 50% temperature (under specific ion intensity, pH and nucleic acid concentration) (because target sequence is excessive existence, under the Tm, being occupied at 50% of equalization point probe) in equalization point and target sequence hybridization with the probe of target complementation. The stringent condition also destabilizing agent of available interpolation such as formamide is realized. To selective or specific hybrid, positive signal is at least twice of background, preferably 10 of background hybridization times. Exemplary stringent hybridization condition can be as follows: 50% formamide, 5X SSC and 1%SDS, under 42 ℃, hatch, perhaps 5X SSC, 1%SDS, under 65 ℃, hatch, with 0.2X SSC and 0.1%SDS 65 ℃ of lower washings.

If the polypeptide of its coding is substantially identical, the nucleic acid of not hybridizing each other under the stringent condition so still is substantially identical. For example when the maximum codon degeneracy generation nucleic acid that uses genetic code to allow copied, this thing happens. In this case, this nucleic acid is generally hybridized under the strict hybridization conditions of appropriateness. Exemplary " hybridization conditions that appropriateness is strict " is included in the buffer solution of 40% formamide, 1M NaCl, 1%SDS 37 ℃ of lower hybridization, washing under 45 ℃ in 1X SSC. Positive hybridization is at least twice of background. Those of ordinary skills will readily appreciate that the condition that similar stringency can be provided with optional hybridization and wash conditions. Other criterions of be used for determining Crossbreeding parameters are provided in many lists of references, CurrentProtocols in Molecular Biology (molecular biology fresh approach) for example, the editors such as Ausubel.

To PCR, about 36 ℃ temperature generally is used for low stringency amplification, can change between about 32 ℃ and 48 ℃ although depend on the primer length annealing temperature. To high stringency pcr amplification, generally be about 62 ℃ temperature, although depend on primer length and specificity, high stringency annealing temperature can from about 50 ℃ to about 65 ℃ scope. The general cycling condition of high and low stringency amplification comprises 90 ℃-95 ℃ 30 seconds-2 minutes sex change phases, continues 30 seconds-2 minutes annealing phase and about 72 ℃ of extended peroids of 1-2 minute. Scheme and criterion low and high stringency amplified reaction are provided in such as (1990) PCR Protocols such as Innis, AGuide to Methods and Applications (PCR scheme, the guidance of methods and applications), Academic Press, Inc.N.Y.).

" antibody " refers to comprise from the polypeptide of specific bond with the framework region of the immunoglobulin gene of identifying antigen or its fragment. The immunoglobulin gene of identification comprises κ, λ, α, γ, δ, ζ and μ constant region gene, and countless immune globulin variable region gene. Light chain is categorized as κ or λ. Heavy chain is categorized as γ, μ, α, δ or ζ, and it has defined immunoglobulin class then, is respectively IgG, IgM, IgA, IgD and IgE. The antigen binding domain of general antibody is most critical to specificity and the affinity of combination.

As used herein, term antibody comprises that also the modification by complete antibody produces or use again synthetic antibody fragment (such as scFv), chimera, humanized antibody or use the antibody fragment that phage display library identifies (referring to such as McCafferty etc., Nature 348:552-554 (1990)) of recombinant DNA method. To the preparation of the antibody of for example restructuring, monoclonal or polyclonal antibody, can use many technology known in the art (referring to for example Kohler ﹠Milstein, Nature 256:495-497 (1975); Kozbor etc., Immunology Today4:72 (1983); Cole etc., 77-96 page or leaf, Monoclonal Antibodies andCancer Therapy (monoclonal antibody and treatment of cancer), Alan R.Liss, Inc. (1985); Coligan, Current Protocols in Immunology (immunology fresh approach) (1991); Harlow ﹠ Lane, Antibodies, A Laboratory Manual (antibody laboratory manual) (1988) and Harlow ﹠ Lane, Using Antibodies, ALaboratory Manual (using the antibody laboratory manual) (1999); And Goding, Monoclonal Antibodies:Principles and Practice (monoclonal antibody: principle and time) (second edition 1986)).

When finger protein or peptide, phrase " special (or selective) in conjunction with " antibody or " special (or selective) and ... immune response " refer to usually determine the association reaction that albumen exists in the heterogeneous population of albumen and other biological goods. Therefore, specifying under the immunoassays condition, specific antibodies is with at least twice of background and being combined with specific protein above 10 to 100 times of background more generally. Need to be for its antibody that specificity of specific protein is selected with the specific bond of antibody under this condition. For example, the polyclonal antibody of albumen, polymorphie variant, allele, straight homologues and the conservative variant of modifying or splice variant or the preparation of its part can be selected in order to only obtain with this albumen not those polyclonal antibodies with other albumen Immunel responses. This selection can realize by deduction and the antibody of other molecule cross reactions. Useful panimmunity mensuration form is selected specifically and the immunoreactive antibody of specific protein. For example, solid phase ELISA immunoassays generally be used for selecting specifically with the antibody of protein immunization reaction (referring to, for example, Harlow ﹠ Lane, Antibodies, A Laboratory Manual (antibody laboratory manual) (1988) has described can be used to determine specific immunoreactive immunoassays form and condition).

" treatment effective dose " means and produces it and use the agent of impact herein. Accurate dosage will depend on therapeutic purposes, and can by those skilled in the art with known technology determine (referring to, for example, Lieberman, Pharmaceutical Dosage Forms (pharmaceutical dosage forms) (1-3 volume, 1992); Lloyd, The Art, Science and Technologyof Pharmaceutical compounding (art of medical compounds, Science and Technology) (1999); And Pickar, Dosage Calculations (Rapid Dose Calculation) (1999)).

The sense of taste (saline taste) gene that this paper identifies recombinant expressed

For obtaining the high level expression of clone gene (such as the cDNA of this gene of coding), the technical staff generally with this gene subclone in expression vector, described expression vector contains and instructs the strong promoter transcribe, transcribes/translation termination, if and to the nucleic acid of encoding proteins, be used for the ribosome bind site of translation initiation. The eucaryon and the prokaryotic promoter that are fit to are well known in the art, and are described in such as Sambrook etc. and Ausubel etc., above. For example, the bacterial expression system that is used for expression sense of taste differential protein can be available from for example Escherichia coli (E.coli), bacillus (Bacillus sp.) and salmonella (Salmonella) (Palva etc., Gene22:229-235 (1983); Mosbach etc., Nature 302:543-545 (1983). The kit that is used for this type of expression system can commercially obtain. The eukaryotic expression system that is used for mammalian cell, yeast and insect journal is well known in the art, and also can commercially obtain. For example, the retroviruse expression system can be used in the invention. As mentioned below, preferably at the people's cells such as the HEK-293 cell, it is widely used in high flux screening to the gene that affects the saline taste sense of taste of this supposition.

Be used for instructing the selection of the promoter that heterologous nucleic acids expresses to depend on specific application. Promoter be preferably located under its natural setting distance with transcription initiation site identical with the distance allos transcription initiation site. Yet, as known in the art, can hold some changes of this distance and not lose the function of promoter.

Except promoter, expression vector generally contains and comprises nucleic acid is expressed required every other element in host cell transcript unit or expression cassette. Therefore general expression cassette comprises operationally promoter and required signal, ribosome bind site and the translation termination signal of the effective poly-adenosine of transcript that the nucleotide sequence of the gene of identifying with coding links to each other. Other elements of expression cassette can comprise enhancer, if use genomic DNA as structural gene, comprise the introne with function donor splicing site and acceptor site.

Except promoter sequence, the transcription termination region that expression cassette also should comprise the structural gene downstream provides effective termination. The terminator can be available from the gene identical with promoter sequence, perhaps can be available from different genes.

The particular expression carrier that is used for hereditary information is transferred in the cell is not especially crucial. Can use any common vector of expressing at eucaryon or prokaryotic. The bacterial expression vector of standard comprises plasmid (such as plasmid, pSKF, the pET23D based on pBR322) and amalgamation and expression system (such as MBP, GST and LacZ). Epitope tag also can be added to the separation method of providing convenience in the recombinant protein, for example c-myc. Sequence label can be included in the expression cassette for the nucleic acid rescue. Can be included in the carrier sign as carrier transduction such as the sign of fluorescin, green or red fluorescent protein, β-gal, CAT and analog.

The expression vector that contains from eucaryon virus regulating element generally is used in the carrier for expression of eukaryon, for example SV40 carrier, papillomatosis poisonous carrier, retrovirus vector and from the carrier of dust bar virus. Other exemplary eukaryotic vectors comprise pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculoviral pDSVE and allow albumen in CMV promoter, SV40 early promoter, SV40 late promoter, metallothionein promoter, MuMTV promoter, rous sarcoma virus promoter, polyhedrin promoter or show other carriers of expressing under effective other promoters guidances of eukaryotic expression. Particularly preferred expression system is the BacMam expression system, and it uses the carrier based on baculoviral to come express polypeptide in mammalian cell, and this paper is at the TRPML3 of HEK-293 cells polypeptide.

Also can regulate with inducible promoters from the eukaryotic vector expressing protein. Use inducible promoters, add this promoter by the response element with these materials, expression depends on the concentration such as the derivant of tetracycline or moulting hormone. Only when derivant existed, high level expression was generally available from inducible promoters; The substrate expression is minimum.

The carrier that uses among the present invention can comprise can regulate promoter, for example, tet-regulating system and RU-486 system (referring to for example Gossen Bujard, Proc.Nat ' l Acad.SciUSA 89:5547 (1992); Oligino etc., Gene Ther.5:491-496 (1998); Wang etc., Gene Ther.4:432-441 (1997); Neering etc., Blood88:1147-1155 (1996); With Rendahl etc., Nat.Biotechnol.16:757-761 (1998)). These give the expression of little molecular Control candidate's target nucleic acid. This beneficial features can be used to determine that desired phenotype is that cDNA rather than somatic mutation by transfection causes.

The sign of the gene magnification of providing is provided some expression systems, such as thymidine kinase and dihyrofolate reductase. Alternatively, the high-yield expression system that does not relate to gene magnification also is fit to, and such as use baculovirus vector in insect cell, uses gene order or other strong bacilliform virus promoters under the polyhedrin promoter instructs.

The element that generally comprises in expression vector also is included in the replicon that works in the particular host cell. In Escherichia coli, this carrier can comprise the gene of the antibiotic resistance of encoding in case allow to contain recombinant plasmid bacterium selection and at the restriction site of the uniqueness of plasmid nonessential region in order to allow the insertion of eucaryon sequence. The certain antibiotics resistant gene of selecting is not crucial, and any of many resistant genes known in the art is suitable. Preferably select the protokaryon sequence so that it does not disturb DNA copying in eukaryotic, if need.

The transfection method of standard can be used to produce bacterium, mammal, yeast or the insect cell line of expressing the sense of taste differential protein that needs in a large number, the Application standard technology is carried out purifying (referring to such as Colley etc., J.Biol.Chem.264:17619-17622 (1989) then; Guideto Protein Purification (protein purification guidance) is in Methods in Enzymology the 182nd volume (Deutscher edits, 1990)). The conversion of eucaryon and prokaryotic according to standard technique carry out (referring to for example Morrison, J.Bact.132:349-351 (1977); Clark-Curtiss ﹠ Curtiss, Methods in Enzymology 101:347-362 (Wu etc., editor, 1983). Can use any known procedure of the extraneous nucleotide sequence being introduced host cell. These programs comprise use calcium phosphate transfection, cohesion amine, protoplast fusion, electroporation, biological projectile, liposome, microinjection, blood plasma carrier, viral vectors and be used for will the clone genomic DNA, cDNA, synthetic DNA or other exogenic heredity materials introduce any of other known methods of host cell (referring to such as Sambrook etc., above). Unique necessity be: the specific genetic modification program of use can be successfully introduced at least one gene and can be expressed in the host cell of this gene.

After expression vector was introduced cell, transfectional cell was cultivated under the condition of supporting gene expression. In some instances, can use the standard technique of hereinafter identifying from culture, to reclaim this type of polypeptide.

The mensuration that is used for the gustatory specific gene product conditioning agent of supposition

The regulation and control of the gustatory differential protein of inferring can be evaluated with multiple external and in vivoassay, comprise the model based on cell as indicated above. This type of mensuration can be used to test inhibitor and the activator of this albumen or its fragment, and therefore tests its inhibitor and activator. This type of conditioning agent is used for drug therapy potentially, or regulate and control the saline taste sense of taste or other sense of taste forms or the whole sense of taste as flavor enhancement, or be used for one or several relevant function or the phenotype of gustatory of gustatory specific gene that regulation and control relate to the evaluation of this paper report as potential therapeutic agent.

With the mensuration of the cell of expressing restructuring or naturally occurring this sense of taste differential protein can with in external, the body as described herein and the various mensuration that exsomatize carry out. Can to regulate and control its active molecule in order identifying, to measure to detect various candidate modulator to preferably in the impact of the activity of cells.

Especially, the channel activity of ionophorous protein can be measured in order to measure the change of ion current with various mensuration, comprise patch clamp technique, measure full cell currents, radiolabeled ion current is measured or with the flow measurement of atomic absorption spectrum coupling and use voltage sensitive dye or lithium or sodium sensitive dye fluoremetry (referring to such as Vestergarrd-Bogind etc., J.Membrane Biol.88:67-75 (1988); Daniel etc., J.Pharmacol.Meth.25:185-193 (1991); Hoevinsky etc., J.Membrane Biol.137:59-70 (1994)). For example, the nucleic acid of encoding proteins or its homologue can be injected into xenopus leavis oocytes or be transfected in the mammalian cell, preferably such as people's cell of HEK-293 cell or Chinese hamster ovary celI. Can evaluate channel activity by the change (being the change of film potential) of measuring the film polarization then.

The method for optimizing that obtains electrophysiologicalmeasurements measurements is by measuring electric current with patch clamp technique, for example " cell adheres to " pattern, " interior-outer " pattern and " full cell " pattern (referring to, for example, Ackerman etc., New Engl.J.Med.336:1575-1595,1997). But full cell currents Application standard method determines, such as by Hamil etc., and the method that Pflugers.Archiv.391:185 (1981) describes.

Also evaluate channel activity by the change of measuring ion concentration (being sodium or lithium) in the born of the same parents easily. These class methods are illustrated in this paper. For example, can be by the picked-up of evaluation radiosodium or by measuring the sodium flow with the fluorescent dye that is fit to. In general microfluorometric determination test, be loaded in the kytoplasm of the cell of expressing gustatory specific ion passage with regard to the dyestuff that experiences the fluorescence change in case combine single sodium ion. In case be exposed to activator, by when sodium in conjunction with the time fluorescence that takes place change the increase that has reflected kytoplasm sodium.

Except these method for optimizing, the activity of this gustatory specific polypeptides also can with multiple other external evaluations with in vivoassay in order to determine function, chemistry and physical influence, for example be measured itself and the combination that comprises other molecules of peptide, little organic molecule and lipid; Measure protein and/or rna level, perhaps measure other aspects of this polypeptide, for example transcriptional level or affect the physiological change of gustatory differential protein activity. When using intact cell or animal to determine that function as a result, the technical staff also can measure multiple impact, such as the change of Growth of Cells pH changes or born of the same parents in second messenger (such as IP3, cGMP or cAMP) or phospholipase C signal transduction path component or regulate the change of son. This type of mensuration can be used to activator and the inhibitor of tested K CNB albumen. The conditioning agent of identifying like this is used for for example many diagnosis and treatment is used.

External test

Evaluation has the mensuration of the compound of regulation activity and preferably carries out external to this gene. The mensuration of this paper is preferably used according to full-length proteins of the present invention or its variant. This albumen optionally merges to form chimera with heterologous protein. In the mensuration of this paper example, the cell that preferably is used in the expression full-length polypeptide in the high throughput assay is used for identifying the compound of controlling gene function. Alternatively, the restructuring of purifying or naturally occurring albumen can be used in the in-vitro method of the present invention. Except purifying protein or its fragment, the part that restructuring or naturally occurring gustatory albumen can be cell lysate or cell membrane. As mentioned below, can be solid-state or soluble in conjunction with measuring. Preferably, this albumen, its fragment or film covalently or non-covalently are combined with solid support. Usually, external test of the present invention is non-competing or competition ligand binding or ligand affinity mensuration (having the outer part of known born of the same parents, such as menthol). These external tests comprise the change of spectrum (for example fluorescence, absorbance, refractive index), fluid dynamic (for example shape), chromatogram or the dissolubility property of measuring albumen.

Preferably, carry out high flux in conjunction with mensuration, wherein albumen contacts with potential conditioning agent, and hatches the time of appropriate amount. As mentioned below, can use multiple conditioning agent, comprise little organic molecule, peptide, antibody and ligand analogs. Many measure can be used to identify the combination of conditioning agent, and the protein-protein that comprises mark is in conjunction with mensuration, mobility shift assay change, immunoassays, such as enzymatic determination and the similar mensuration of phosphorylation assay. In some instances, the combination of candidate modulator is wherein measured the interference of using the known ligand combination by determining in conjunction with measuring with competition when potential conditioning agent exists. In this type of was measured, known ligand is combination at first, added then the compound (reinforcing agent of namely inferring) that needs. After specific protein is washed, determine the interference of potential conditioning agent or known ligand combination. Common potential conditioning agent or known ligand are labeled.

In addition, high flux functional genome measures also to can be used to be tested and appraised and destroys sense of taste specific polypeptides and identify the creeping chill conditioning agent with the compound of the protein interaction of other albumen of its combination. This type of mensuration can for example use clone or blastema to detect change, the change of cellular calcium or the change of membrance current that cell surface marker is expressed. Cell generally contacts with cDNA or random peptide library (nucleic acid coding). This cDNA library can comprise the cDNA of justice, antisense, total length and brachymemma. This peptide library is by nucleic acid coding. Use then mensuration monitoring cDNA as indicated above or peptide library to the impact of cell phenotype. The impact of cDNA or peptide for example can be used, and the regulatable expression of nucleic acid (such as the expression from the tetracycline promoter) is proved and distinguishes with somatic mutation. The cDNA of encoded peptide and nucleic acid can use technology well known by persons skilled in the art (for example using sequence label) and be saved.

Albumen according to the present invention with the protein-interacting of being encoded by cDNA can separate with yeast two-hybrid system, Mammalian two-hybrid system or phage display screening etc. The target of identifying like this can be further as the bait of these mensuration identify can with interactional other components of specific ion passage, acceptor or transport protein, its member also is that the target of drug development is (referring to such as Fields etc., Nature 340:245 (1989); Vasavada etc., Proc.Nat ' lAcad.Sci.USA 88:10686 (1991); Fearon etc., Proc.Nat ' l Acad.Sci.USA 89:7958 (1992); Dang etc., Mol.Cell.Biol.11:954 (1991); Chien etc., Proc.Nat ' l Acad.Sci.USA 9578 (1991); With United States Patent (USP) the 5th, 283,173,5,667,973,5,468,614,5,525,490 and 5,637, No. 463).

In vivoassay based on cell

In preferred embodiments, wild type and mutant gustatory differential protein be at cells, and measure its function (for example physics and chemistry) or phenotypic alternation in order to identify its function of regulation and control or recover the conditioning agent of mutant gene (gene that for example has impaired gate control function) function. The cell of expressing protein also can be used in conjunction with in measuring. As described hereinly measure any suitable function effect. For example, lithium or the change of sodium level and the combination of part all are to be fit to use the mensuration of identifying potential conditioning agent based on the system of cell in the change of film potential, the born of the same parents. Be used for the recombinant cell lines that this type of comprises blastema and transform expressing protein as based on the cell that is fit to of the mensuration of cell. Therefore this gustatory differential protein can be naturally occurring or restructuring. As indicated above, have the fragment of these albumen of ion channel activity or chimera and also can be used in the mensuration based on cell. For example, the membrane spaning domain according to ion channel of the present invention can merge with the cytoplasmic structure territory of heterologous protein (preferably allos ionophorous protein). This chimeric protein will have ion channel activity, and can be used in the mensuration based on cell of the present invention. In another embodiment, the domain of gustatory differential protein (the outer or cytoplasmic structure territory such as born of the same parents) is used in the mensuration based on cell of the present invention.

In another embodiment, the cell polypeptide level of particular target sense of taste polypeptide can be determined by the level of measuring albumen or mRNA. Use the immunoassays (such as Western blotting, ELISA and similar mensuration) of the antibody of selective binding polypeptide or its fragment to measure the protein level relevant with the ion channel activation. To measuring mRNA, it is preferred for example using the hybridization assays of the amplification of PCR, LCR or for example northern hybridization, RNA enzyme protection, dot blot. As described herein, use the detection agent (for example antibody and the analog of nucleic acid, radioactivity or the enzyme labeling of fluorescence or radioactivity mark) of direct or indirect mark to detect the level of albumen or mRNA.

Alternatively, but the operation report genic system is measured protein expression. This system can use the target gene promoters that operationally links to each other with reporter gene (such as chloramphenicol acetyltransferase, Fluc, bacteriofluorescein enzyme, beta galactosidase and alkaline phosphatase) to design. In addition, interested albumen can be used as by linking to each other with second reporter gene (such as redness or green fluorescent protein) indirect reporter gene (referring to, for example, Mistili ﹠ Spector, Nature Biotechnology 15:961-964 (1997)). Cell is advanced in the general transfection of reporter gene construct. After potential conditioning agent processing, according to standard technique measurement report well known by persons skilled in the art genetic transcription, translation or active amount.

In another embodiment, can measure the function effect relevant with signal transduction. The ion channel that activates or suppress will change the characteristic of target enzyme, second messenger, passage and other effect proteins potentially. This example comprises the activation of phospholipase C and other signal systems. Also the downstream result be can check, DG and IP3 produced such as phospholipase C.

The mensuration of ion channel activity comprises with the dyestuff loading cell of ion or voltage-sensitive reports activity, and for example sodium flows into or the release of the interior sodium of born of the same parents by observing. Mensuration that be used for to determine this type of receptor active also can be evaluated test compounds with activator and the antagonist of these known acceptors as feminine gender or positive control activity. In for the identification of the mensuration of regulating compound (for example activator, antagonist), the change of kytoplasm intermediate ion level or membrane voltage will be monitored with ion-sensitive or membrane voltage fluorescence indicator respectively. Adoptable ion-sensitive indicator and voltage probe comprise those that are disclosed in Molecular Probes 1997 catalogues. Also can use radiolabeled ion current measure or with the flow measurement of atomic absorption spectrum coupling.

The nucleic acid that separates coding TRPML3 albumen

The present invention partly depends on the routine techniques that genetic recombination is learned the field. The basic textbook of the conventional method that uses among open the present invention comprises Sambrook and Russell, MolecularCloning, A Laboratory Manual (molecular cloning laboratory manual) (third edition, 2001); Kriegler, Gene Transfer and Expression:A Laboratory Manual (transgenosis and expression laboratory manual) (1990); With Current Protocols inMolecular Biology (molecular biology fresh approach) (Ausubel etc., editor, 1994)).

Coding TRPML3 albumen, polymorphie variant, straight homologues and allelic nucleic acid can separate by the screening library under stringent hybridization condition with oligonucleotides with the TRPML3 nucleic acid probe. Alternatively, upward with for antiserum or the antibody purification homology of TRPML3 or the preparation of its part express by detecting immunity, expression library can be used to clone TRPML3 albumen, polymorphie variant, straight homologues and allele.

In order to prepare cDNA library, the technical staff should select to be rich in the source of TRPML3 RNA. Use then reverse transcriptase that mRNA is become cDNA, be connected in the recombinant vector, and be transfected in the recombinant host for breeding, screening and clone. For the preparation of with the screening cDNA library method be known (referring to, for example, Gubler ﹠ Hoffman, Gene 25:263-269 (1983); Sambrook etc., above; Ausubel etc., above).

To genomic library, from tissue extraction DNA, mechanical shearing or enzymic digestion produce the fragment of about 12-20kb. This fragment is separated by gradient centrifugation and the fragment that does not need size then, and is building up in the bacteriophage λ carrier. These carriers and bacteriophage are in vitro package. By such as Benton ﹠ Davis, the described patch hybridization analysis of Science 196:180-182 (1977) recombinant phage. Such as Grunstein etc., Proc.Natl.Acad.Sci.USA., the general description of 72:3961-3965 (1975) is carried out colony hybridization.

Alternatively, use T7 RNA polymer from the linearizing DNA in-vitro transcription of suitable Restriction Enzyme cRNA, can produce from TRPML3 DNA plasmid the TRPML3 cRNA of the TRPML3 that encodes, the cRNA microinjection that obtains is advanced in the suitable cell, for example egg mother cell, preferably frog egg mother cell.

The optional method that separates TRPML3 nucleic acid and straight homologues thereof, allele, mutant, polymorphie variant and the conservative variant of modifying is used in combination synthetic Oligonucleolide primers and cloning RNA or dna profiling (referring to United States Patent (USP) the 4th, 683,195 and 4,683, No. 202; PCRProtocols:A Guide to Methods and Applications (PCR scheme: the methods and applications guide) (Innis etc., editor, 1990)). Can be used to directly from mRNA, from cDNA, from the nucleotide sequence of genomic library or cDNA library amplification TRPML3 such as the method for polymerase chain reaction (PCR) and ligase chain reaction (LCR). Degenerate oligonucleotide can be designed to the sequence provided herein TRPML3 homologue that increases. The restriction endonuclease site can add in the primer. Polymerase chain reaction or other amplification in vitro method also can be used for the nucleotide sequence, preparation nucleic acid of clones coding albumen to be expressed for example in case use its as probe for detection of the existence of the mRNA of coding TRPML3 in the physiologically sample, be used for nucleic acid sequencing or be used for other purposes. Gene by PCR reaction amplification can and be cloned into the suitable carrier from the Ago-Gel purifying.

The gene expression of TRPML3 also can be by technical Analysis known in the art; for example reverse transcription with amplification mRNA, separate total RNA or poly A+RNA, northern hybridization, dot blot, in situ hybridization, RNA enzyme protection, high density polynucleotides array technique, for example similar techniques.

The nucleic acid of coding TRPML3 albumen can use with the high density oligonucleotide array technology (for example GeneChip) to identify TRPML3 albumen, straight homologues, allele, conservative variant and the polymorphie variant of modifying among the present invention. In the example that the homologue of evaluation is relevant with the regulation and control of t cell activation and migration therein, it can be used as diagnostic tool with GeneChip and come disease in the detection of biological sample, referring to, for example, Gunthand etc., AIDSRes.Hum.Retroviruses 14:869-876 (1998); Kozal etc., Nat.Med.2:753-759 (1996); Matson etc., Anal.Biochem.224:110-106 (1995); Lockhart etc., Nat.Biotechnol.14:1675-1680 (1996); Gingeras etc., Genome Res.8:435-448 (1998); With Hacia etc., Nucleic Acids Res.26:3865-3866 (1998).

As point out, identify that the preferred mensuration of the compound of regulation and control (namely strengthen, suppress or prevent) TRPML3 comprises the electrophysiological detection that electric current change in the cell of the TRPML3 that contacts with the TRPML3 conditioning agent (reinforcing agent or inhibitor) of at least one supposition is expressed in monitoring. These mensuration can be used any cell of expressive function TRPML3. In preferred embodiments, this cell will comprise egg mother cell, and preferably frog egg mother cell, mammalian cell, yeast cells or insect cell perhaps are suitable for another expression system of expressive function TRPML3 ion channel. Preferably, this expression system will show strongly and the rapidly expression of TRPML3 sodium channel, and will not express any or considerably less endogenous ion channel ideally, therefore help to identify the compound of special regulation and control TRPML3 sodium channel function. Therefore, unwanted background is replied and is minimized or eliminates. In addition, need such as the strong cell of egg mother cell, because this can be used in again according in the mensuration of the present invention cell. Reported before that egg mother cell was rapid and expressed consumingly other functional ion passages (Pascal, CRC Crit.Rev.Biotech.22 (4): 317-87 (1987); Wagner etc., Cell Physiol.Biochem.10:1-12 (2000); Canessa etc., Nature 367:463-467 (1994)).

Particularly preferred electrophysiological detection is the mensuration of measuring the appropriate flux of TRPML3 channel function in the frog egg mother cell by two electrode voltage tongs technologies. This strongly, expression system provides only can reaching millions of ion channels after about 18-24 hour and has expressed in oocyte membrane fast. In addition, because egg mother cell relatively big (diameter 1mm compares relatively large with most of mammals), it is processed easily and operates.

Based on these advantages, single egg mother cell can be used to obtain electrophysiological recording a plurality of and that repeat. The general interior source channels of also expressing seldom of egg mother cell. Therefore egg mother cell allows the direct measurement target compound of repetition to the impact of TRPML3 sodium channel function.

In preferred two electrode voltages pincers according to the present invention are measured, with microinjection the frog egg mother cell of TRPML3 cRNA transfer to the glass scintillation bottle, and hatch under proper condition in order to help the TRPML3 protein expression.

Obtained the TRPML3 sodium-ion channel express after (generally after the cRNA microinjection about 24 hours), use suitable two electrode voltage measurement mechanisms (for example parallel egg mother cell voltage clamp of OpusXpress 6000A system (MDS AnalyticalTechnologies)) to measure the TRPML3 function according to two electrode voltage tongs technologies. Two electrode voltage tongs technologies are measured and are passed macroscopical electric current of complete oocyte membrane by the TRPML3 sodium-ion channel. The electricity consumption pressure sensitivity is subjected to electrode and current-sense electrode puncture egg mother cell; Use voltage to experience electrode and will stride voltage or the potential difference of oocyte membrane and be fixed in particular value, and use the current-sense electrode measurement to keep this voltage required electric current of striding oocyte membrane or ion-flow rate. The OpusXpress system be can the commercial two electrode voltage measurement mechanisms that obtain an example, it is automanual, and comprises the work station that allows to record simultaneously the electrophysiological recording that produces from 8 egg mother cells. This system also provides automatic egg mother cell to pierce through and has sent target compound by the computer-controlled compound of sending to the fluid actuated bar of 96-hole compound plate. This system can be described as medium or appropriate flux system best, can reach 100 kinds of compounds because it allows to assess weekly. Certainly, can screen more compound by other voltage measuring apparatus that add as described.

In this mensuration system, the TRPML3 reinforcing agent will cause passing in the oocyte membrane enhancing of the electric current of TRPML3 passage. This value is calculated by normalized form. This type of mensuration also can comprise suitable negative control, and example is the TRPML3 inhibitor as is known. Therefore, this compound works to confirm egg mother cell expressive function TRPML3 as internal reference, and allow the TRPML3 reinforcing agent (if target compound is the TRPML3 reinforcing agent, it will cause passing in the oocyte membrane enhancing of the electric current of TRPML3 passage) of screening supposition after using compound.

Ideally, calculate the enhancing coefficient % of each reinforcing agent. For example, 100% reinforcing agent has increased TRPML3 activity 100% with respect to substrate control value (not having compound).

Also confirm that with negative control the egg mother cell of not injecting with TRPML3 cRNA does not show identical impact ideally.

Ideally, the compound that also strengthens valve to showing strong % carries out more complicated analysis (for example, current/voltage (I/V) curve, competitive assay and dose response curve) and determines that compound shows the concentration of half maximum activity (EC50 value). These experiments will confirm further that the impact of this compound is that TRPML3-is special.

These mensuration will provide the evaluation to TRPML3 conditioning agent (preferably TRPML3 reinforcing agent), and it can be used as the additive of food, beverage, medicine and similar substance in order to regulate and control the relative saline taste sense of taste. Ideally, the TRPML3 reinforcing agent will show at least 20% enhancing coefficient, and more preferably at least 50% and even at least 100% enhancing coefficient more preferably.

Test can be any little organic molecule or biological entities as the compound of TRPML3 protein modulators, such as protein (for example antibody or peptide), sugar, nucleic acid (for example ASON or ribozyme) or lipid. Alternatively, conditioning agent can be the hereditary change form of TRPML3 albumen. Test compounds generally will be little organic molecule, peptide, lipid and lipid analogs. Preferably, test compounds is safe to people's consumption.

In essence, any compound can be used as conditioning agent potential in the mensuration of the present invention or part, although normally used most compounds is dissolvable in water in water or organic (particularly based on DMSO) solution. This mensuration is designed to screen big chemical library by the automatic assay step with for mensuration provides the compound from any convenient source, both move (for example, surveying the form be fixed on the microtiter plate with microtitration in robot) abreast. Should understand, chemical compound lot supplier is arranged, comprise ChemDiv (San Diego, Calif.), Sigma-Aldrich (St.Louis, Mo.), Fluka Chemika-Biochemica-Analytika (BuchsSwitzerland) and similar supplier.

In preferred embodiments, appropriateness or high-throughput screening method relate to provides little organic molecule or the peptide library that contains a large amount of potential TRPML3 conditioning agents (potential activator or inhibitor compound). Screen in mensuration this type of " chemical library " as described herein identifies that those demonstrate the library member of the feature activity that needs (specific chemical species or subclass) then. The compound of identifying like this can be used as conventional " lead compound ", perhaps itself can be used as potential or actual product. As point out that preferred egg mother cell two voltage clamp electrode systems (single assembly) allow to detect weekly about 60 compounds.

The combinatorial chemistry library is the set of the multiple compounds that produces by chemical synthesis or biosynthesis, by combination number of chemical " construction unit " (such as reagent). For example, linear combination chemistry library (such as polypeptide libraries) is to form by one group of chemical constitution unit (amino acid) of combination in every kind of specific compound length possible mode (being amino acid whose number in the polypeptide compound). This combined hybrid by the chemical constitution unit can be synthesized countless compounds.

The preparation in combinatorial chemistry library and screening are well known to a person skilled in the art. This type of combinatorial chemistry library includes but not limited to that peptide library is (referring to for example United States Patent (USP) the 5th, 010, No. 175, Furka, Int J.Pept.Prot.Res.37:487-493 (1991) and Houghton etc., Nature 354:84-88 (1991)). Also can use other chemical substances that produce the Chemical Diversity library. This type of chemical substance includes but not limited to: the class peptide (for example, PCT announces WO No. 91/19735), encoded peptide (for example, PCT announces WO No. 93/20242), biological oligomer (for example at random, PCT announces WO No. 92/00091), the benzene diazepine (for example, United States Patent (USP) the 5th, 288, No. 514), such as glycolylurea, various body (diversomer) (Hobbs etc. of benzene diazepine and dipeptides, Proc.Nat.Acad.Sci.USA90:6909-6913 (1993)), ethene polypeptide (Hagihara etc., J.Amer.Chem.Soc.114:6568 (1992)), non-peptide simulating peptide (Hirschmann etc. with glucose support, J.Amer.Chem.Soc.114:9217-9218 (1992)), the similar organic synthesis thing (Chen etc. of little library of compounds, J.Amer.Chem.Soc.116:2661 (1994)), oligomerization carbamate (Cho etc., Science 261:1303 (1993)), and/or peptidyl phosphate (Campbell etc., J.Org.Chem.59:658 (1994)), nucleic acid library is (referring to Ausubel, Berger and Sambrook, all see above), the peptide nucleic acid library (referring to, for example, United States Patent (USP) the 5th, 539, No. 083), antibody library (referring to, for example, Vaughn etc., Nature Biotechnology, 14 (3): 309-314 (1996) and PCT/US96/10287), the carbohydrate library (referring to, for example, Liang etc., Science, 274:1520-1522 (1996) and United States Patent (USP) the 5th, 593, No. 853), little organic molecule library (referring to, for example, the benzene diazepine, Baum C﹠EN, January 18, the 33rd page (1993); Isoprenoid, United States Patent (USP) the 5th, 569, No. 588; Thiazolidone and a Buprofezin (metathiazanone), United States Patent (USP) the 5th, 549, No. 974; Pyrrolidines, United States Patent (USP) the 5th, 525,735 and 5,519, No. 134; Morpholinium compound, United States Patent (USP) the 5th, 506, No. 337; The benzene diazepine, United States Patent (USP) the 5th, 288, No. 514, and similar substance).

For the preparation of the device of combinatorial libraries can commercially obtain (referring to, for example 357MPS, 390MPS, Advanced Chem Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition, multiple combination library itself can commercial obtain (referring to, for example, ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc., St.Louis, Mo., ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).

For example use high flux screening (HTS) screening compounds identify can in conjunction with and/or regulation and control taste receptors or sense of taste ion channel polypeptide or transhipment is sub or those compounds of its fragment activity. Among the present invention, albumen is recombinant expressed in cell (for example mammalian cell or frog egg mother cell), and measures active regulation and control by any measurement (such as the change of sodium or lithium level in measurement film potential or the measurement born of the same parents) of using ion channel, acceptor or transhipment subfunction. The mensuration for example method of cationic ion channel function for example comprises, patch clamp technique, two electrode voltages pincers, the Imaging-PAM of measuring full cell currents and use ion-sensitive fluorescent dye and ion current mensuration (for example radiolabeled ion current mensuration or ion current are measured).

The F﹠B group that contains the TRPML3 adjusting compound that uses disclosed mensuration evaluation Compound

Use to identify that TRPML3 regulates compound that the mensuration of compound identifies potentially as composition or the spices that can digest composition (being F﹠B) and Orally administered medicine. The compound of regulation and control or the impression of enhancing saline taste can be used as the spices of food or beverage alone or in combination. In a preferred application, this conditioning agent will be impregnated in has food or the beverage that falls low-level sodium, and the saline taste of the product that obtains will be similar to high sodium product. The example of this type of F﹠B comprises snack products such as potato block, crispbread, soup, the meat products that dips in material, soft drink, packing, pretzel and other.

Saline taste flavor potentiator or the co-inhibitor identified according to the present invention can be mixed in the various F﹠B. Other examples of F﹠B comprise multiple F﹠B, for example, such as the beverage of fruit drink, sports drink, vegetable juice, fermentation lactic acid beverage, soda, coffee, cocoa, black tea, oolong tea, green tea, pure mellow wine, alcohol and powder drink; Such as candy, chewing gum, piece candy, chewing gum, soda pop sugar and chocolate confectionery; The food that bakes such as cookies, biscuit and bread; Such as sour milk and ice-cream dessert; Snack such as potato block and crispbread; Stew, curried food, soup, condiment, dip in material, noodle soup, soup-stock, miso, fast food meat soup, dip, meat soup, jam, sprinkling water material, Japanese flapjack, miso shiru, pickles, rice dumpling water material, ordinary food and drink water material, such as half prepared food or prepared food or its refrigeration and the frozen food of wheat, buckwheat and Chinese style noodles; Fast food food such as the instant noodles; With such as the powdery condiments that mixes and the condiments of mayonnaise.

In the F﹠B that the saline taste flavor potentiator mixes with it, be particularly conducive to improve body be nutrition and nourish beverage, comprise the drinks of nutritional supplement beverage, such as the snack products of potato block and local flavor crispbread, such as the fragrant processed food of flavor, stew and soup and the similar food of curry. The fragrant processed food form of flavor above comprises prepared food and half prepared food and foods packed in carton containers, its refrigeration or frozen food.

The saline taste flavor potentiator of the present invention that mixes or the amount of co-inhibitor can change according to the form of saline taste flavor potentiator or co-inhibitor and the food or the beverage that mix with it, but preferable range with respect to food or beverage 0.000001% to 1.0wt%, more preferably 0.0001% to 0.1wt%, and more preferably 0.00001% to 0.01wt%. Saline taste flavor potentiator of the present invention or co-inhibitor can mix by any of known method.

Alternatively, prevent or the compound that suppresses saline taste impression can be used as natural composition or the spices that contains the food of high salt concentration, in order to prevent or cover its saline taste. These materials comprise sports drink and wherein have electrolytical other compositions that comprise in a large number sodium, for example be used for medicine or displacement purpose after the sick or strenuous exercise of loss electrolyte balance.

Can comprise that the composition that is used for ingesting of regulating compound according to TRPML3 of the present invention will comprise the composition of ingesting for people, animal (domestic, zoo animal, pet), and will comprise food, beverage, medicament, nutriment (neuticeutical) and cosmetics.

The amount that this type of TRPML3 regulates compound will be the amount that produces the impression of desirability saline taste. Certainly, it is safe being used in that this type of compound in using will be defined as the people is consumed, and can accept in the test of people's sense of taste.

Preferred embodiment-use two electrode voltages pincers the electrophysiological recording of measuring is measured egg mother cell In the TRPML3 electric current

Use the electrophysiological detection for the identification of the TRPML3 conditioning agent of the amphibian egg mother cell of expressive function people TRPML3

The egg mother cell expression system has inherent advantages (expression, strong, low endogenous ion channel is expressed), and this makes it be used to check that compound is to the impact of the sodium transport by the TRPML3 passage. These compounds are for the candidate who strengthens the saline taste impression. Early stage egg mother cell expression system be used for the rapid and strong expression ion channel of functional study (Dascal, CRC Crit.Rev.Biochem. (1987) 22 (4): 317-387; Wagner etc., CellularPhysiology and Biochemistry (2000) 10:1-12; Canessa etc., Nature (1994) 367:463-467). Therefore, select this system to be used for using the two electrode voltages pincers of method as described below and material to measure.

The egg mother cell expression system comprises following steps and method, and it comprises screening TRPML3 reinforcing agent altogether: the frog operation is separated with egg mother cell, the microinjection of the preparation of cRNA, egg mother cell and use two electrode voltages to clamp electrophysiological recording and measure the TRPML3 electric current in egg mother cell. Below with reference to document description be used for the frog operation and separate (Marcus-Sekura etc., Methods in Enzymology (1987) 152:284-288 with egg mother cell; Goldin, Methods in Enzymology (1992) 207:266-279), cRNA prepares (Swanson etc., Methods in Enzymology (1992) 207:310-319; Goldin etc., Methods in Enzymology (1992) 207:279-297), Oocyte micro-injection (Matten etc., Methods in Enzymology (1995) 254:458-466; Hitchcock etc., Methods in Enzymology (1987) 152:276-284) and two electrode voltages pincers electrophysiological recording (Stuhmer, Methods in Enzymology (1992) 207:319-339; Wagner etc., Cellular Physiology and Biochemistry (2000) 10:1-12) general practice. Every kind of these methods (because it relates to screening TRPML3 reinforcing agent) is described in further detail in hereinafter.

The frog operation is separated with egg mother cell

Length more than or equal to the female Africa xenopus South Africa pawl frog of 9cm available from NASCO (FortAtkinson, Wis.). Frog is 0.15%MS-222 (tricaine or the ethyl-3-amino benzoic Acid methane sulfonate in the distilled water; Sigma) anaesthetize in and place on ice. Use aseptic surgical technique and tools, pass outer skin and the continuous 1-2cm otch of inner peritoneal layer generation at belly. The ovary leaf that cuts (containing 1000-2000 egg mother cell) places the not medium of calcic (82.5mM NaCl, 2mM KCl, 1mM MgCl2,5mM HEPES make pH 7.5 with NaOH) of OR-2, and use successively 2mg/ml IA Collagenase Type (Sigma) (preparation before using) digestion 45 minutes, on swaying platform, digested in room temperature in 15 minutes with the digestion of 1mg/ml IA Collagenase Type then. After the enzymic digestion, at the time point of most of egg mother cells from the release of ovary leaf, egg mother cell does not wash in having the OR-2 of clostridiopetidase A, and transfers to and contain Barth ' s salt solution (88mM NaCl, 2mM KCl, the 0.82mM MgSO that is supplemented with the 2.5mM Sodium Pyruvate₄、0.33mM Ca(NO ₃) ₂、0.41mM CaCl ₂、2.4mM NaHCO ₃With 5mM HEPES pH 7.5; Specialty Media) in the culture dish. Selection contains corresponding to the clear animal pole of the dark side of ovum that contains the melanin pigment particle with corresponding to maturity period V or the VI egg mother cell (1mm diameter) of the plant pole of the bright side of ovum that contains livetin and is used for microinjection. Use the C6 pin to sew up frog with 3-0 black establishment suture (Harvard Apparatus), and after 2-3 month convalescence, be reused for the separation of egg mother cell.

The cRNA preparation

Specification (Ambion) according to manufacturer produces TRPML3 cRNA from the people TRPML3 DNA plasmid that is described in WO 02/087306A2 with mMESSAGE mMACHINE kit, at external use T7 RNA polymerase from transcribing cRNA with the linearizing DNA of Restriction Enzyme. Check that by sex change agarose gel electrophoresis and the spectrophotometric absorbance reading at 260nm and 280nm place the quality of cRNA is to guarantee to produce the undegradable cRNA of total length.

Microinjection

Injection needle is enclosed within on the Model P-97 Flaming/Brown micropipettor (Sutter Instrument Co.) that uses borosilicate glass capillary tube (World PrecisionInstruments Inc.), rear end filling mineral oil (Sigma) uses Nanoliter 2000 injectors (WorldPrecision Instruments) front end with Micro4 MicroSyringe pump controller to fill TRPML3 cRNA then. With the 10-25nl Oocyte micro-injection that contains 10-25ng people TRPML3 cRNA in animal pole. After the microinjection, egg mother cell is transferred in the glass scintillation bottle that contains the Barth ' s solution that is supplemented with the 2.5mM Sodium Pyruvate, and under ordinary atmospheric conditions in 18-19 ℃ of overnight incubation. During this period, this egg mother cell is translated as protein with the TRPML3 cRNA of injection.

Use the TRPML3 electric current in the two electrode voltages pincers measurement egg mother cell

Microinjection 10-25ng people TRPML3 cRNA 24 to 48 hours afterwards uses the TRPML3 function of two electrode voltage tongs technologies in the parallel egg mother cell voltage clamp of OpusXpress 6000A system (MDSAnalytical Technologies) measurement egg mother cell. Two electrode voltage tongs technologies are to measure the electric physiology method (Stuhmer, Methods in Enzymology (1992) 207:319-339) that flows through macroscopical electric current of whole oocyte membrane by the albumen passage. The electricity consumption pressure sensitivity is subjected to electrode and current-sense electrode to pierce through egg mother cell; Use voltage to experience electrode and make voltage or the potential difference of striding oocyte membrane be fixed in particular value, and measure the required electric current of striding oocyte membrane or the ion-flow rate of this voltage of maintenance with the current-sense electrode. The OpusXpress system is the semi-automatic two electrode voltages pincers work station that allows to record simultaneously 8 egg mother cells. Piercing through of egg mother cell is automatically, and sending of compound is to be undertaken by computer-controlled fluid actuated bar from 96 hole compound plates.

Egg mother cell places the OpusXpress system, and dipping bath (96mMNaCl, 2.5mM KCl, 1mM CaCl2,1mM MgCl2 and 5mM HEPES make pH 7.5 with NaOH) in ND-96 solution. The electricity consumption pressure sensitivity is subjected to pierce through egg mother cell with the current-sense electrode, be enclosed within on the ModelP-97 Flaming/Brown micropipettor (Sutter Instrument Co.) that uses borosilicate glass capillary tube (World Precision Instruments Inc.), the 3M KCl that contains the silver chlorate lead is filled in the rear end. Experience electrode for voltage, electrode shows the impedance of 2-10Mohm; For the current-sense electrode, electrode shows the impedance of 0.5-2Mohm2-10Mohm. After piercing through, the voltage of egg mother cell is fixed in-60mV, and initial experimental record. Obtain data at the 50Hz place, and use 4 hole Bessel filters to hang down by filtering at the 5Hz place.

Illustrate that the inspection compound that carries out is shown in Figure 10 to the schema of the experimental sequence of the influence of TRPML3 function, comprise the ovocyte that the screening of maintenance current potential, I/V curve, NDMG competition test, dose response curve and the test of 60mV are not injected.

Preferred embodiment-use two electrode voltages pincers the electrophysiological recording and codon optimized of measuring TRPML3 sequence measuring ovocyte in the TRPML3 electric current

Codon comprises 3 Nucleotide of specific amino acids in the coding protein sequence.Because there are 20 amino acid whose codon nucleotide triplets of 61 different codings, most of amino acid can be by more than codon coding.Use in the specific species speed that codon codon optimized of each amino acid preference can be by increasing translation and accuracy and do not change protein sequence and improve proteic functional expression.

Be created in dna level and non-codon optimized TRPML3 has the codon optimized form (Figure 19) of 76.4% homologous people TRPML3 gene.Codon is optimised for the best translation that reaches the human sequence.By the L-Ala 419 in the 5th membrane spaning domain is sported proline(Pro), we have also produced the activity form (A419P TRPML3) of TRPML3.This sudden change cause the TRPML3 passage be in open conformation (confirmation) (Xu etc., PNAS 104 (46): 18321-18326,2007; Grimm etc., PNAS 104 (49): 19583-19588,2007; Nagata etc., PNAS 105 (1): 353-358,2008; Kim etc., J.Biol.Chem.282 (50): 36138-36142,2007); Therefore, A419P TRPML3 is used in particular for identifying the TRPML3 co-inhibitor.

Wild-type (non-codon optimized), codon optimized and A419P TRPML3 express in ovocyte, and measure sodium current.Figure 20 illustrates that wild-type TRPML3 produces low-level sodium current, and codon optimized wild-type TRPML3 produces the sodium current of medium level, and A419PTRPML3 produces high-caliber sodium current.Therefore, codon optimized wild-type TRPML3 and A419P TRPML3 help to screen the compound of regulation and control TRPML3 function.

Codon optimized wild-type TRPML3 can be used to screening open (enhancing) compound of TRPML3 function as previously described.Hereinafter to use these sequences electricity physiology ovocytes to measure how to be used for identifying be how the TRPML3 toughener and the codon optimized TRPML3 of wild-type of candidate's saline taste sense of taste toughener helps to identify the TRPML3 toughener in the ovocyte electrophysiological detection to the data declaration among the embodiment.

Preferred embodiment--the TRPML3 mammalian cell electrophysiological detection of measuring

Can use different mammalian cells to carry out this mensuration.In preferred exemplary, the mammalian cell of immortalization or tissue culture cells (such as human embryonic kidney cell's (HEK293 cell) or Chinese hamster ovary cell (Chinese hamster ovary celI)) are used for checking the influence of compound to the sodium transport of the people TRPML3 cationic channel by heterogenous expression.These compounds are candidates of regulation and control saline taste impression.The expression of ionic channel in mammalian tissues culturing cell system is widely used in the rapid and intensive expression of the ionic channel that is used for functional study.The mammalian cell of use cultivating comprises as the advantage of expression system: be used for interested cDNA is introduced the multiple complete method of cell, comprise the ability that produces stable cell lines, relatively easily carry out patch clamp experiments, compare high-caliber electric current with electric current from the ionic channel of endogenous expression; Directly measure the ability of ion channel function.

Carry out following steps that the technician was familiar with and the method that electrophysiological recording comprises electric physiology, tissue culture and biology field from mammalian tissues culturing cell system: cells in culture is kept, the preparation of cDNA and purifying, by transfection and/or virus transduction with cDNA introduce cell, stable clone is selected and the electric physiology of patch clamp.Below with reference to document description electrophysiological (Sackman B. and E.Neher (editor) .1995.Single-channel recording (single channel recording), the second edition generally put into practice of patch clamp of mammalian cell; Hille is channels of excitable membranes (the easily ionic channel of excitation membrane) B.2001.Ion, the third edition).

Use the TRPML3 in the full cell voltage pincers electrophysiological recording measurement mammalian cell Stream

Full cell voltage tongs technology is to measure the electric physiology method that flows through macroscopical electric current of whole plasma membrane by the albumen passage.The live body mammalian cell places the special Microscopy Room that contains the outer solution of born of the same parents.Instructed and be in vision control by micromanipulator, the minor diameter glass pipette that is full of with the conductivity salts solution at first uses the soft negative pressure that causes high anti-gigaOhm to seal to be attached to cytolemma.Use causes the further suction of full cell patch pincers configuration to destroy the interior patch clamp of suction pipe.Full cell patch pincers configuration allows all ionophorous proteins or the macroscopical electric current in the multiple measurement film.Use patch clamp amplifier and the combination of full cell patch pincers configuration to allow operator's control to stride the voltage or the potential difference of intact cell film, and cell interior and outside ionic composition.Therefore this technology provide extremely sensitive and flexibly platform be used for the characteristic that ionic channel is studied on biophysics ground, include, but is not limited to voltage-dependent, activation and deactivation kinetics and to the permeability of different ions and the screening platform of ionic channel co-inhibitor, toughener and conditioning agent.Utilize computer-controlled patch clamp amplifier proportioning valve controller to allow voltage schemes to automatically perform, and allow the outer solution of born of the same parents to exchange rapidly.Therefore individual cells can stand multiple voltage scheme and compound interpolation.

Before any electrophysiological detection, must obtain TRPML3 cDNA and effectively be delivered to the mammalian tissues culturing cell.This can 3 kinds of modes realize at least: 1) use the method transient transfection TRPML3 cDNA based on lipid, 2) transduction of the virus infection of use such as baculovirus, adenovirus and slow virus, 3) by TRPML3 being stablized the clone that TRPML3 is expressed in adding karyomit(e) stably express cDNA and selection.The mammiferous electric physiology scheme that is used for screening the toughener of TRPML3 electric current and co-inhibitor is with previous to describe the scheme that is used for xenopus leavis oocytes similar.Briefly, these schemes comprise the NMDG competition and the dose response of current/voltage analysis, candidate TRPML3 co-inhibitor and toughener.In addition, full cell patch pincers electrophysiological technique can overcome some limitation of the two electrode voltages pincers generation of ovocyte.For example, the mammalian cell of smaller szie allows the more detailed biological and physical analysis (such as the influence of compound to activation and deactivation kinetics) of quick process.The ability of solution also allows to measure any change of the passage permeability that the interpolation by compound causes in the control cell born of the same parents.At last, cell attachment, interior-ability outer and outer-outer membrane configuration allow to measure the ability of single channel current, and this allows to identify in detail the mechanism of action of any toughener or co-inhibitor.

Chinese hamster ovary celI is used for functional expression and the screening saline taste sense of taste conditioning agent of wild-type TRPML3 ApplicationPrevious report uses patch clamp to measure to show in the HEK293 cell wild-type (WT) TRPML3 seldom or do not have function (Xu etc., a PNAS104 (46): 18321-18326,2007; Grimm etc., PNAS 104 (49): 19583-19588,2007; Nagata etc., PNAS 105 (1): 353-358,2008; Kim etc., J.Biol.Chem.282 (50): 36138-36142,2007).On the contrary, it is believed that A419P mutant TRPML3 is not regulated in the HEK293 cell, cause intensive electric current when transient expression.Among Figure 24 A, the HEK293 cell is with WT and A419P mutant TRPML 3 transient transfections, and its electric current is measured by a series of voltage steps from-100 to+60mV, so that generation current-voltage correlation figure (I/V figure).Compare with WT, the expression of A419P mutant TRPML3 passage causes big, inside rectified current.We have described the application (Figure 24 B) such as the optional cell system of Chinese hamster ovary celI of the WT TRPML3 that allows to increase function among the embodiment hereinafter.In addition, we show that average macroscopical electric current is identical to WT with A419P TRPML3 passage with inside rectification.Therefore, allow to provide the more effective functional expression of WT TRPML3 in the mammlian system of saline taste sense of taste regulator screening platform such as the application of the specific cells of Chinese hamster ovary celI system.Among Figure 25, we have illustrated the practice of using the WT that expresses and A419P mutant TRPML3 passage to test and study potential saline taste sense of taste toughener and co-inhibitor in Chinese hamster ovary celI.In full cellular electrophysiologicalsensor is measured, the TRPML3 toughener will be observed to the enhancing when cell fixation inside electric current during in negative potential.The I/V of Figure 25 A analyzes and shows that the combined thing of WTTRPML3 strengthens.In same mensuration, the reduction of inside electric current when the co-inhibitor of TRPML3 causes being in negative potential.Among Figure 25 B, constructive expression's the combined thing Gadolinium trichloride of A419P mutant TRPML3 passage (inhibitor of many ionic channels) is prevented in Chinese hamster ovary celI.

Codon optimized cDNA is used for mammalian cell effective expression TRPML3 and screening The application of saline taste sense of taste conditioning agent

Codon comprises 3 Nucleotide of specific amino acids in the coding protein sequence.Because there are 20 amino acid whose codon nucleotide triplets of 61 different codings, most of amino acid can be by more than codon coding.Use in the specific species speed that codon codon optimized of each amino acid preference can be by increasing translation and accuracy and do not change protein sequence and improve proteic functional expression.As before pointing out, be created in dna level and non-codon optimized TRPML3 has the codon optimized form (Figure 19) of 76.4% homologous people TRPML3 gene.Codon is optimised for the best translation that reaches the human sequence.Before we showed not expressive function passage (Figure 24 A) in the HEK293 cell effectively of WT TRPML3 passage.We illustrate that the TRPML3 that the son that accesses to your password is optimized has greatly overcome observed expression problem in the HEK293 cell in Figure 26.When in the HEK293 cell, expressing codon optimized WT TRPML3 (Figure 26 B), observe the intensive electric current that has similar characteristics with A419P TRPML3 mutant passage by transient transfection or the transduction of use baculovirus.Therefore the TRPML3 that the son that accesses to your password is optimized has allowed to provide the improved functional expression of WTTRPML3 ionic channel in the HEK293 cell of saline taste sense of taste regulator screening platform.Among Figure 26 C, we have illustrated that WT TRPML3 passage that the son that accesses to your password is optimized tests and study the practice of potential saline taste sense of taste conditioning agent.In this experiment, codon optimized WT TRPML3 cDNA is delivered to mammalian cell via the baculovirus transduction.The be activated compound of TRPML3 of the inside electric current of codon optimized WT TRPML3 mediation strengthens (Figure 26 C).

The TRPML3 heteromultimers is used to express and screen the application of saline taste sense of taste conditioning agent

TRPML3 ionic channel subunit is the member of the bigger 6TMD ionic channel family of ionic channel subunit.Similar to other 6TMD ionic channels, it is believed that need reach 4 TRPML subunits produces single ionic channel (Hille is the Channels of excitablemembranes ionic channel of excitation membrane (easily) B.2001.Ion, the third edition; Venkatachalam etc., J BiolChem.2006 June 23; 281 (25): 17517-27).Functional passage can be fully by same subunit form (with poly-) or with closely-related subunit associate (different poly-).The general feature of different poly-passage is that it has usually with it and compares intermediary biophysics function with poly-counterpart, has therefore strengthened passage potential functional diversity.Change that also can be by plasma membrane transportation and posttranslational modification (such as phosphorylation, ubiquitinization and glycosylation) come regulation activity by the different compositions of subunit.The research of different poly-passage can be by sending a plurality of passage subunit cDNA and realize via injecting cRNA in cotransfection in the mammalian cell or the xenopus leavis oocytes altogether in the mammalian cell.In addition, multimerization also can be by realizing covalently bound the transduction to the stable cell lines that produces a plurality of cDNA of expression together or with the virus of a plurality of viruses of sending a plurality of passage subunit cDNA of passage subunit cDNA.

In the practice, the multimerization of WT and A419P TRPML3 passage subunit can be used to strengthen surface-active level (Figure 27).As showing, passage expressive function passage (Figure 27 A) in the HEK293 cell of forming by A419P TRPML3 subunit exclusively.In same cell, when non-codon optimized WT TRPML3 subunit expression, even when using the cDNA of 3 times of amounts, do not observe electric current (Figure 27 B).On the contrary, A419P TRPML3 cDNA and WT TRPML3cDNA co expression in the HEK293 cell causes synergistic effect, causes the bigger electric current (Figure 27 C-D) than two simple addition predictions of independent passage colony.These data show that the passage of being made up of WT and A419P mutant subunit is present on the film, and can be used for TRPML3 toughener and co-inhibitor mensuration.As shown in this embodiment, use limited amount A419P cDNA contrast WT should increase the ratio of WT TRPML3 subunit in the passage tetramer, may give passage intermediary biophysics function.Therefore may may open very much (Po) (Xu etc. owing to shown poly-together A419P TRPML3 passage, PNAS 104 (46): 18321-18326,2007), use different poly-A419P/WT TRPML3 passage may be more suitable for being used to screen the TRPML3 toughener.

The preferred function of measuring embodiment-use membrane potential dyestuff monitoring variant TRPML3

Developed the specific mensuration based on cell that is used to find the TRPML3 conditioning agent, it can finally be used in regulates and control the saline taste impression in the F﹠B.In the HEK293 cell of the gene transient transfection of using coding A419P-TRPML3 or transduction, use specific membrane potential dyestuff (FMPs; Molecular Devices) function of monitoring A419P-TRPML3.

In one embodiment of the invention, express Mammals or frog ovocyte the membrane potential fluorescence dye or the preload of sodium fluorescence dye of TRPML3 or variant, fragment or functional equivalent.This cell contacts with the adjusting compound of TRPML3 supposition when having sodium or lithium then.The change of the change of cation mediated cell fluorescence when lacking conditioning agent relatively comes to determine the regulation and control degree of TRPML3 when the conditioning agent of inferring exists.

Alternatively, mammalian cell can be with functional TRPML3 splice variant and fragment transfection.Cell inoculation is in the hole of porous plate then, and hatches and be enough to reach the time of joining at least about 70%.Load cell with membrane potential dyestuff dyestuff then, and contact with sodium with the regulating compound of at least one supposition.Any change by conditioning agent/membrane potential dye fluorescence that TRPML3 interaction causes uses fluorescent plate reader or voltage strength plate reader to monitor.The saline taste sense of taste conditioning agent of Tui Ceing can be identified by the change of fluorescence then.

Preferred embodiment-IonWorks TRPML3 the patch clamp of measuring is measured

The automatic patch clamp of IonWorks system is used for checking the influence of compound to the sodium transport by people TRPML3 cationic channel.These compounds are candidates of regulation and control saline taste impression.The IonWorks system is widely used in high-flux electric physiology.Use its 384-well format, the IonWorks system can check thousands of kinds of compounds every day, is the most high-throughout in any automatic electric physiological system.The advantage of IonWorks instrument is that it can be with the mode standard operation corresponding to individual cells of each hole wherein, perhaps provide colony's patch clamp (PPC) mode operation of the mean current of 64 cells, therefore increased overall success ratio and reduced the otherness of Kong Yukong with each hole wherein.Other advantages of IonWorks system are that it uses mammalian cell, and it provides the direct measurement of ion channel function by the record ion(ic)current.

Use the IonWorks patch clamp to measure the TRPML3 electric current of measuring in the CHO-K1 cell

Can measure the multi-form of people TRPML3, include but not limited to the gain-of-function mutation bodily form formula that wild-type form and coding A419P replace.Can in the CHO-K1 cell, express TRPML3:(i by any of 3 kinds of methods) transient transfection, (ii) BacMam transduction or (iii) stable transfection, and use the perforation patch clamp technique to go up the function of measuring TRPML3 at IonWorks Quattro instrument (MDSAnalytical Technologies).Use Detachin cellular segregation solution (Genlantis) dissociated cell, centrifugal, and externally resuspended in the record buffer liquid (150mMNaCl, 2mM KCl, 1.5mM CaCl2,1mM MgCl2,10mM HEPES make pH 7.4 with NaOH).Dissociated cell is added in the PPC plate of 384-hole, and wherein each hole substrate has 64 holes, and the diameter in each hole is 1-2 μ m.A cell drops on the hole, and uses negative pressure to form high mega-Ohm sealing between cell and hole.After sealing forms, use the perforation patch clamp technique to obtain electricity and enter cell interior.In this technology, the amphotericin microbiotic of hole (form) be applied to the hole below, and in the plasma membrane section that separates, exposes, form aperture, cause that electricity enters cell below the PPC plate.In case enter cell, initial experimental record.Two class electrodes, the independent recording electrode in the common ground electrode under the flat board and each hole of immersion allows control voltage (current potential of cross-cell membrane) and permission record to stride the flow of the ion(ic)current of intact cell film.IonWorks Quattro system is the semi-automatic patch clamp workstation that allows record 384 holes.Cell is scattered in diaphragm plate, sealing forms and electricity to enter be automatically, and sending by computer-controlled fluid actuated bar of compound carried out from 384 hole compound flat boards.

The IonWorks screening assay that the compound that is used for identifying regulation and control (activate or prevent) TRPML3 function has been described is below described.In order to make dull and stereotyped hole that data are provided, must satisfy two standards: (i) at first, the hole must have most of holes openeds (average impedance in 64 holes is necessary＞1mOhm also＜10mOhm) and (ii) after cell adds, most of holes must have with diaphragm plate form high mOhm sealing cell (average impedance in 64 holes is necessary＞10mOhm).If satisfied this two standards, instrument is collected the data that scan behind preceding scanning of compound and the compound.In the illustrated embodiment (Figure 28), the impedance in 94% hole is greater than 10mOhm.In each scanning, when changing, measures voltage electric current (referring to the embodiment voltage instruction trace of Figure 29).

Because the TRPML3 channel table reveals inside rectification, record will show under inside electric current big under the hyperpolarization current potential and the depolarize current potential little of extrinsic current (Figure 29).Compound will with between-1uM and-concentration between the 100uM uses.If compound works as the TRPML3 toughener, the electric current that passes the TRPML3 passage so in the cytolemma strengthens.If compound works as the TRPML3 co-inhibitor, pass the current decay of TRPML3 passage so in the cytolemma.The TRPML3 electric current can be checked under two different voltages: for example ,-120mV and-40mV.By influence compound that rectification that voltage relies on strengthens TRPML3 preferentially rectification therein strong-detected under the 40mV, and prevent the compound of TRPML3 will be preferentially therein the TRPML3 electric current bigger-detected under the 120mV.For the influence of quantification compound to the TRPML3 function, we use following formula: [(A-Ao)/(B-Bo)] x100.B and Bo are the electric currents of measuring before the compound adding, and A and Ao are the electric currents of measuring after adding compound.A and B be test voltage (120mV or-electric current under 40mV), and Ao or Bo are the electric currents under the 0mV.This value causes being used for measuring the active regulation and control coefficient % of compound in our mensuration.For example, if regulation and control coefficient % equals 200%, compare with the control value that lacks compound so, this compound has doubled the activity of TRPML3.If regulation and control coefficient % equals 50%, this compound has reduced the substrate control value (lacking compound) of TRPML3 activity to 1/2 so.

In parental cell, carry out the negative control experiment so that explanation uses the observed influence of compound to be owing to flow through the electric current of TRPML3 passage in the cell of expressing TRPML3, rather than owing to flow through the electric current of the passage of endogenous expression in the cytolemma.The compound of special regulation and control TRPML3 should not influence the electric current in the contrast CHO-K1 cell, and should show the regulation and control coefficient % near 100%.

Carry out more complicated analysis to showing big regulation and control coefficient % and not influencing the compound that contrasts the CHO-K1 cell.Mensuration comprises current/voltage (I/V) curve, GdCl3 competitive assay (GdCl3 is the co-inhibitor of TRPML3) and dose response curve.To the I/V curve, when existing or lacking compound, from 120mV to+60mV measures electric current with the voltage progression of the increase of 10mV, so that the regulation and control amplitude of research compound.The electric current regulation and control amplitude of I/V slope of a curve indication compound of interest.Strong toughener increases the I/V slope of a curve, the opening of the TRPML3 ionic channel that indication increases.Strong co-inhibitor reduces the I/V slope of a curve, the closing of the TRPML3 ionic channel that indication increases.The I/V curve that the contrast I/V curve that carries out when having compound should carry out when lacking compound is identical and overlapping.

Carry out the influence that the GdCl3 competitive assay illustrates compound and depend on TRPML3.At first, use compound, use the GdCl3 (or some other TRPML3 co-inhibitor) of Sa then so that determine regulation and control coefficient %.For making toughener directly act on the TRPML3 passage, the electric current that the toughener of using by oneself adds the cell that GdCl3 handles should be similar to the electric current that the cell observation only handled with GdCl3 arrives.This experiment shows that compound does not have enhancement when passage is prevented, so the function of the necessary direct regulation and control TRPML3 passage of this compound.

Carry out dose response curve and determine that compound shows the concentration of half maximum activity (toughener is EC50, and co-inhibitor is IC50).EC50 or IC50 value are more little, and compound is strong more as the activity of TRPML3 conditioning agent.The compound that increases concentration by continuous application ((1nM), develops into high dosage (1mM)) and carries out dose response curve from low dosage.Coefficient % is regulated and control in calculating as indicated above, and maps to determine the EC50 or the IC50 value of compound with logarithmic scale as the function of compound concentration.

Illustrate check compound to the flow chart description of the experimental sequence of the influence of TRPML3 function in Figure 30, be included in 120mV and-screening down of maintenance current potential, I/V curve, GdCl3 competition test, dose response curve and the negative control of 40mV test.

Animal model

The also potential active conditioning agent of screening-gene that is used for of animal model.The transgenic animal technology causes gene overexpression, and siRNA and gene knockout technology cause with suitable gene targeting vector homologous recombination after the genetic expression that lacks or reduce.Identical technology also can be applicable to preparation and knocks out cell.When needs, the tissue specific expression of target gene or to knock out may be necessary.The transgenic animal that produce by this class methods as with the animal model of gene target associated responses.This type of animal of for example expressing according to gene of the present invention can be used to produce super gustation person (supertaster) phenotype, such as being used to screen chemistry and biotoxin, rancid/rotten/F﹠B that pollutes, perhaps be used to screen the therapeutic compound of sense of taste differentiation of stem cells.

Can produce and knock out cell and transgenic mice by marker gene or other heterologous genes are inserted the genomic native gene of mouse site via homologous recombination.This type of mouse also can be by replacing native gene or by for example producing by being exposed to known mutagenic compound sudden change native gene with the mutant form of target gene.

DNA construct is introduced in the nuclear of embryonic stem cell.The cell that contains the genetic damage of new transformation is injected into the embryo of the host mouse of being implanted the recipient female mouse again.Among these embryos some develop into has the gomphosis mouse of part from the sexual cell of mutant cells system.Therefore, by the breeding gomphosis mouse, may obtain to contain the genetic damage of introducing new mouse system (referring to, for example, Capecchi etc., Science 244:1288 (1989)).According to Hogan etc., Manipulating the Mouse Embryo:A Laboratory Manual (mice embryonic operation experiments handbook) (1988) and Teratocarcinomas and Embryonic StemCells:A Practical Approach (teratoma and embryonic stem cell hands-on approach) (Robertson, editor, 1987), can derive chimeric target mouse.

Preferred animal model is measured embodiment-Varitint-Waddler mice study

The mouse model that is called Varitint waddler mouse or the application of similar transgenic animal are also contained in the present invention, wherein TRPML3 saline taste gustatory cell is excised from taste bud specifically, and wherein saline taste is greatly weakened so that study TRPML3 in the body to the saline taste sense of taste with relate to the influence of metabolic other functions of sodium, and the application of the sudden change of this gene in producing the animal that particular cell types (such as saline taste gustatory cell, melanocyte, pituicyte and adrenal cells) reduces.

The TRPML3 ionic channel of Varitint waddler mouse has A419P sudden change (the Di Palma etc. that function obtains, PNAS 99 (23): 14994-14999,2002), it is from 1942 spontaneous mutation (Cloudman etc., J.Heredity 36:258-263,1945).It is because two obvious phenotypes that these mouse are called as Varitint waddler: piebald hair color (the variable tone of fur) and vestibular system defective (turn-take behavior with wave as duck).The A419P TRPML3 of enhanced activity has changed the ionic equilibrium of expressing the cell (comprising the melanocyte of skin and the hair cell of inner ear and vestibular system) of TRPML3, and cause the death (Xu etc. of these cell colonys, PNAS 104 (46): 18321-18326,2007; Grimm etc., PNAS 104 (49): 19583-19588,2007; Nagata etc., PNAS 105 (1): 353-358,2008; Kim etc., J.Biol.Chem.282 (50): 36138-36142,2007).Necrocytosis may be attributable to sodium and/or calcium ion enters tenuigenin uncontrollably.Therefore, Varitint waddler mouse is the model of wherein expressing the cell ablation that the cell of A419P TRPML3 dies off.

Because specifically expressing in the gustatory cell of TRPML3 on tongue experimentizes to determine whether the gustatory cell of expression TRPML3 is cut in Varitint waddler mouse.Use terminal point PCR (Figure 31) or real-time quantitative PCR (Figure 32) in gustatory cell, to detect less than TRPML3 from the purifying of Varitint waddler mouse.On the contrary, the expression of gene that participates in sweet taste, bitter taste, delicate flavour and the tart flavour sense of taste is similar (Figure 31-32) in Varitint waddler and wild-type control mice.Figure 33 using-system hybridization in situ technique explanation TRPM5 (sweet taste, bitter taste, delicate flavour, GPR113) and PKD2L1/PKD1L3 (tart flavour) gustatory cell are complete in the Varitintwaddler mouse.Therefore, it is unaffected at Varitint waddler mouse model not express the gustatory cell colony of TRPML3 (comprising sweet taste, bitter taste, delicate flavour, GPR113 and tart flavour).Therefore, Varitint waddler mouse contains the taste bud that lacks the TRPML3 gustatory cell, and Varitint waddler mouse can be used to study the saline taste sense of taste when lacking this cell colony.

In addition, the neural record of electric physiology CT is the biological improving method of the sense of taste of research rodent system, and has been used to illustrate influence (Damak etc., the Science.2003301 (5634): 850-3 of genetic mutation to the physiologic response of multiple taste stimulation; Lyall etc., J Physiol.2004558 (Pt 1): 147-59; Zhao etc., Cell.2003 115 (3): 255-66; Mueller etc., Nature.2005434 (7030): 225-9).The CT nerve comprises the anterior tongue of taste bud at fungi shape and some lobate papilla gustatosia moderate stimulation; Therefore the activity of CT nerve has been represented in response to flavor unit and has been applied to measuring of tongue forward Taste Receptors cell function.The program of using those skilled in the art to be familiar with is as mentioned before carried out CT neural recording method (Lyall etc., J Physiol.2004558 (Pt 1): 147-59; Treesukosol etc., Am J Physiol Regul IntegrComp Physiol.2007 May, 292 (5): R1799-809; Dahl etc., BrainResearch.1997 756:22-34).

Use the neural record of CT, show that Varitint waddler mouse shows the defective to the sodium-chlor response.Particularly, Varitint waddler mouse has the insensitive CT nerve of benzamil-that extremely weakens reply (Figure 34) to sodium-chlor.Because TRPML3 is not prevented by guanamprazine or guanamprazine analogue benzamil, the insensitive CT of benzamil-replys mainly owing to TRPML3.Initial (phase type) that the CT nerve is replied and lasting (catatonic type) component are weakened in Varitint waddler mouse.These data show that elimination TRPML3 gustatory cell has significantly reduced mouse and experienced the ability of saline taste, and point out TRPML3 gustatory cell central role as professional saline taste gustatory cell.

These results show that further Varitint waddler mouse has wherein the TRPML3 gustatory cell by the taste bud of special excision, and these mouse can be used in the wherein special sense of taste research that influences the saline taste sense of taste.

These results show that also Varitint waddler mouse shows the insensitive CT nerve of benzamil-to the sodium-responsible defective of chlorination, and Varitint waddler mouse shows the defective of CT nerve to chlorination sodium-responsible initial (phase type) and lasting (catatonic type) component.

Importantly, these results show that can use the expression of A419P TRPML3 also produces the mouse model system that lacks different cell colonys so that excise cell type specifically.

These animals also can be used to study A419P TRPML3 kills particular cell types as toxin influence.

Candidate modulator

Test is as the sense of taste associated protein of inferring or relate to other non-sense of taste correlation functions of gustatory cell and the compound of the conditioning agent of phenotype can be any little organic molecule or biological entities, such as the nucleic acid or the lipid of protein, sugar, for example antisense oligonucleotide or the ribozyme of for example antibody or peptide.Alternatively, conditioning agent can be proteic hereditary change form.Test compounds generally will be little organic molecule, peptide, lipid and lipid analogs.In one embodiment, this compound is natural existence or synthetic menthol analogue.

Any in essence compound can be used as potential conditioning agent or the part in the present invention's mensuration, although normally used most compounds is dissolvable in water water or organic (particularly based on DMSO) solution.This mensuration is designed to screen big chemical library by the automatic assay step with for mensuration provides the compound from any convenient source, and it generally carries out (for example carrying out with the microtitration form) abreast on microtiter plate in robot measures.Should understand, compound has many suppliers, comprise Sigma (St.Louis, Mo.), Aldrich (St.Louis, Mo.), Sigma-Aldrich (St.Louis, Mo.), Fluka Chemika-BiochemicaAnalytika (Buchs Switzerland) and similar supplier.

In a preferred embodiment, high-throughput screening method relates to the little organic molecule that combination is provided or contains the peptide library of a large amount of potential therapeutic compounds (potential conditioning agent or ligand compound).In one or more mensuration as described herein, screen active those libraries of feature member (specific chemical species or subclass) that this type of " combinatorial chemistry library " or " ligand library " identify that demonstration needs then.Compounds identified can be used as conventional " lead compound " like this, and perhaps they itself can be used as potential or actual therapeutical agent.

The combinatorial chemistry library is the set of the multiple compound that produces by chemosynthesis or biosynthesizing, by combination many chemistry " structural unit " (such as reagent).For example, the linear combination chemistry library such as polypeptide libraries is to make up one group of chemical structure unit (amino acid) by every kind of possible mode with specific compound length (being amino acid whose number in the polypeptide compound) to form.Mixing by unitary this type of combination of chemical structure can be synthesized countless compounds.

The preparation in combinatorial chemistry library and screening are well known to a person skilled in the art.This type of combinatorial chemistry library include but not limited to peptide library (referring to, for example, United States Patent (USP) the 5th, 010, No. 175, Furka, Int.J.Pept.Prot.Res.37:487-493 (1991) and Houghton etc., Nature 354:84-88 (1991)).Also can use other chemistry that produce the Chemical Diversity library.This type of chemistry includes but not limited to: the class peptide (for example, PCT announces WO No. 91/19735), the peptide of coding (for example, PCT announces WO 93/20242), biological at random oligomer (for example, PCT announces WO No. 92/00091), the benzene diazepine (for example, United States Patent (USP) the 5th, 288, No. 514), such as glycolylurea, various body (Hobbs etc. of benzene diazepine and dipeptides, Proc.Nat.Acad.Sci.USA 90:6909-6913 (1993)), vinyl polypeptide (Hagihara etc., J.Amer.Chem.Soc.114:6568 (1992)), have the non-peptide simulating peptide of glucose support (Hirschmann etc., J.Amer.Chem.Soc.114:9217-9218 (1992)), the similar organic synthesis thing (Chen etc. of little library of compounds, J.Amer.Chem.Soc.116:2661 (1994)), oligomerization carbamate (Cho etc., Science 261:1303 (1993)), and/or peptidyl phosphoric acid ester (Campbell etc., J.Org.Chem.59:658 (1994)), nucleic acid library is (referring to Ausubel, Berger and Sambrook, all see above), the peptide nucleic acid(PNA) library (referring to, for example, United States Patent (USP) the 5th, 539, No. 083), antibody library (referring to, for example, Vaughn etc., Nature Biotechnology, 14 (3): 309-314 (1996) and PCT/US96/10287), the carbohydrate library (referring to, for example, Liang etc., Science, 274:1520-1522 (1996) and United States Patent (USP) the 5th, 593, No. 853), little organic molecule library (referring to, for example, benzene diazepine, Baum C﹠amp; EN, January 18, the 33rd page (1993); Isoprenoid, United States Patent (USP) the 5th, 569, No. 588; A thiazolidone and a Buprofezin, United States Patent (USP) the 5th, 549, No. 974; Tetramethyleneimine, United States Patent (USP) the 5th, 525,735 and 5,519, No. 134; Morpholinium compound, United States Patent (USP) the 5th, 506, No. 337; The benzene diazepine, United States Patent (USP) the 5th, 288, No. 514, and similar substance).

The device that is used to prepare combinatorial library can commercially obtain (referring to, for example 357MPS, 390MPS, Advanced Chem Tech, Louisville Ky., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, Foster City, Calif., 9050 Plus, Millipore, Bedford, Mass.).In addition, multiple combinatorial library itself can commercial obtain (referring to, for example, ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc., St.Louis, Mo., ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).C. solid-state and solvable high throughput assay

The proteic cell or tissue of the target sense of taste that can use target sense of taste differential protein or express natural existence disclosed herein or reorganization carries out other solubilities and measures.Alternatively, can high throughput format carry out the external test based on solid phase, wherein albumen or its fragment (such as the cytoplasmic structure territory) are attached to the solid phase substrate.Any be suitable for high flux screening of mensuration described herein, for example part combination, calcium current amount, membrane potential change etc.

In solvable or solid-state high throughput assay of the present invention, may in one day, screen several thousand kinds of different conditioning agents or part.This method is used in external test albumen, or is used to comprise proteic based on cell or based on the mensuration of film.Especially, every hole of microtiter plate can be used to move the single mensuration at the potential conditioning agent of selecting, and perhaps if observe the influence of concentration or incubation time, single conditioning agent can be tested in every 5-10 hole.Therefore, single standard microtiter plate can be measured about 100 kinds (for example 96 kinds) conditioning agents.If use 1536 hole flat boards, so single flat board can easily be measured the about 1500 kinds of different compounds of about 100-.May measure many flat boards every day; May use integration system of the present invention to measure screening and reach about 6,000,20,000,50,000 or surpass 100,000 kinds of different compounds.

To solid state reaction, protein of interest or its fragment (for example ectodomain) or comprise protein of interest or its fragment can directly or indirectly combine with solids fraction by bonding (for example passing through label) covalently or non-covalently as the cell of a fusion rotein part or film.This label can be any of various ingredients.Usually, the molecule of combination tag (label wedding agent) is fixed in solid support, and the interaction of interested tag molecule by label and label wedding agent is attached on the solid support.

Based on the clear known interaction of molecules of describing in the document, can use many labels and label wedding agent.For example, when label had natural wedding agent (for example, vitamin H, albumin A or Protein G), it can use with suitable label wedding agent (the Fc district of avidin, Streptavidin, neutravidin, immunoglobulin (Ig) etc.).The antibody that has such as the molecule of the natural wedding agent of vitamin H also is the suitable label wedding agent of general available; Referring to SIGMA Immunochemicals 1998 catalogue SIGMA, St.Louis Mo.).

Similarly, any haptens or antigen compound can be used in combination with suitable antibody so that form label/label wedding agent right.Thousands of species specific antibodies can commercially obtain, and many other antibody are described in the document.For example, in a kind of common configuration, label is one anti-, and the label wedding agent that to be identification one anti-is two anti-.Except antibody-AI, receptor-ligand binding also is that suitable label and label wedding agent is right.For example, the agonist of cell-membrane receptor and antagonist are (for example, cell receptor-ligand interaction is such as transfer, c-kit, virus receptor part, cytokine receptor, Chemokine Receptors, interleukin-2-receptor, immunoglobulin receptor and antibody, cadherin family, integrin family, selectin family and similar acceptor; Referring to, for example, Pigott ﹠amp; Power, The Adhesion Molecule FactsBook I (the true handbook I of adhesion molecule) (1993).Similarly, toxin and venom, virus epitopes, hormone (for example, opiate, steroid etc.), (for example it mediates the influence of various little parts to intracellular receptor, comprises steroid, Triiodothyronine, retinoid and vitamins D; Peptide), medicine, lectin, sugar, nucleic acid (linear and ring-type polymer configuration), oligosaccharides, protein, phosphatide and antibody can both interact with various cell receptors.

Synthetic polymer such as urethane, polyester, polycarbonate, polyureas, polymeric amide, polymine, poly arylidene thio-ester, polysiloxane, polyimide and poly-acetic ester also can form suitable label or label wedding agent.It is apparent to those skilled in the art that many other label/label wedding agents are to also being used in the mensuration described herein system in case looked back present disclosure.

Joint commonly used such as peptide, polyethers and analogue also can be used as label, and comprises such as the polyglycine polypeptide of sequence sequence between about 5 to 200 amino acid.This type of flexible joint is that those of ordinary skill in the art are known.For example, poly-(ethylene glycol) joint can be available from Shearwater Polymers, Inc.Huntsville, Ala.These structures randomly have amide linkage, sulfydryl bonding or exclusive-OR function bonding.

Any that uses present available several different methods is fixed in the solid phase substrate with the label wedding agent.The solid phase substrate generally is exposed to by all or a part with substrate chemical group is fixed in the chemical reagent of a part of reacted surface of label wedding agent by derivatize or functionalization.For example, being suitable for being attached to more, the group of long-chain part will comprise amine, hydroxyl, thiol group and carboxylic group.Aminoalkyl group silane and hydroxyalkyl silane can be used to the functionalization kinds of surface, such as glass surface.The structure of this type of solid phase biological polymer array is clear to be described in the document.Referring to, for example, Merrifield, J.Am.Chem.Soc.85:2149-2154 (1963) (having described for example solid phase synthesis of peptide); Geysen etc., J.Immunol.Meth.102:259-274 (1987) (described on the pin solid components synthetic); Frank ﹠amp; Doring, Tetrahedron 44:6031-6040 (1988) (described on the Mierocrystalline cellulose dish various peptide sequences synthetic); Fodor etc., Science, 251:767-777 (1991); Sheldon etc., Clinical Chemistry 39 (4): 718-719 (1993); With Kozal etc., NatureMedicine 2 (7): 753-759 (1996) (all having described the biopolymer array that is fixed in the solid phase substrate).Be used for the method non-chemically that the label wedding agent is fixed in substrate is comprised the method that other are commonly used, such as heat, by UV radiation crosslinking and similar approach.

Above described the present invention, embodiment provided below further illustrates certain preferred embodiments of the present invention.These embodiment are only for illustrative purposes provides, and should not be construed as restriction the present invention.

The application in practice of invention

The active compound of sense of taste specific gene that regulation and control (preferably strengthen) are identified according to the present invention is in regulation and control people's saline taste sense of taste and other sense of taste forms or totally have important application in the sense of taste potentially.In addition, these compounds are used to relate to the therapeutic application of other gustatory cell correlation functions and phenotype potentially, such as adjusting and the similar functions and the phenotype of immunity in gustatory cell turnover, digestive ailment, digestive function, metabolic adjusting, oral cavity and/or the Digestive tract.

The compound that activates the sense of taste ionic channel of papilla gustatosia on the tongue can be used to by promoting Na ⁺Transhipment is advanced the taste bud cell and is strengthened the saline taste impression.This has tangible consumer applications in the sense of taste of improving the less salt F﹠B and palatability.

In addition, the gene of this paper can be used as evaluation with gene product, separates or the sign of specific gustatory cell type of enrichment or pedigree.

Further, be specific to the gustatory cell of transcription factor that the gene of the gustatory cell that this paper identifies and gene product can be used to identify that regulation and control gustatory cell apoptosis, regulation and control control Taste Receptors express, regulation and control autocrine/paracrine regulation and control growth, prolong the taste bud life-span, the compound that produces super gustation person animal phenotype is used in such as the screening that is used for bio-terrorism or be used in the animal that the screening induced dry-cell activated and be divided into the compound of gustatory cell in vivo.

The cell of this gene, gene product and this gene of expression can be used to screen influences the transportation that Taste Receptors comes and goes top film/taste pore district, so that strengthen or suppress the overall or special sense of taste, regulate gustatory cell action potential discharge frequency/membrane potential and control the intensity of overall or the special sense of taste, regulate neurotransmitter and be discharged into esodic nerve and control the intensity of overall or the special sense of taste and the Taste Receptors function of autocrine/paracrine regulation and control.

Further, the cell of this gene, gene product and this gene of expression can be used to identify such as regeneration gustatory cell in suffering from cancer, chemotherapy radiation, the damage that influences the sense of taste or surgical operation, drug-induced dysgeusia, ageusic older individuals or patient and is used to alleviate the compound of taste bud forfeiture.

Further, the cell of this gene and gene product and this gene of expression can be used to screen the compound of bacterium, virus and other immunogenic neutralization/eliminations in the detoxifcation that influences toxic substance in oral hygiene, halitosis, the oral cavity and saliva/mouth or the digestive tube.

Preferably, the TRPML3 conditioning agent can be used as odor additive so that initiation or regulation and control (strengthening or inhibition) saline taste impression and treatment relate to sodium metabolism, absorption and the excretory patient's condition and physiological function.Especially, the TRPML3 conditioning agent can be added in food, beverage and medicament and other consuming product, so that regulation and control or cover its saline taste.In addition, the TRPML3 conditioning agent can be used to treat heart relevant with regulating and control TRPML3 and urinary function with toughener, produces and vasopressing release such as blood pressure, fluid retention, urine generation, apoplexy, heart attack, arrhythmia, aldosterone.

In addition, transgenosis TRPML3 animal (for example knocking out and knock in animal) has application in practice in research sodium metabolism and other activity in to physiological process and the influence such as the disease of Addison's disease, and the compound that is used to identify regulation and control TRPML3.

The following examples that relate to TRPML3 provide the evidence of confirming to show TRPML3 coding saline taste receptor polypeptides and have used above-described material and method is carried out.Propose these embodiment so that for those of ordinary skills provide complete disclosure and describe how to produce and use the present invention, and be not intended to limit and be considered to scope of the present invention.

Embodiment

Embodiment 1:

Present embodiment relates to experiment and the molecular biology data that are contained in Fig. 1, and it shows that TRPML3 is a sense of taste specific gene.People (left side) who collects by the laser capture micro-dissection and monkey (right side) taste bud (sense of taste) and tongue epithelial cell (tongue) carry out RT-PCR.Fig. 1 shows that TRPML3 only expresses in gustatory cell, similar with known sense of taste specific gene T1R2 and TRPM5.This figure has shown that also the house-keeping gene beta-actin expresses in the sense of taste and tongue cell, show that the RNA from two samples is high-quality.'+' shows and carried out reverse transcription, and ' ' show and do not carry out reverse transcription (negative control).Only have reverse transcription and observe band.All bands are cloned and are checked order and confirm the identity of gene.

Embodiment 2:

Present embodiment contains the electrophysiological detection that is contained in Fig. 2, and it has disclosed TRPML 3 and has formed the sodium channel.The full cell patch that carries out the cell of expressing human TRPML3 as described therein clamps electric physiology.TRPML3 is created in the sodium leakage electric current of removing sodium and being prevented when replacing with big not penetrating positively charged ion NMDG as can be seen.Top trace among the same figure shown be in-60mV keeps the electric current under the current potential.Current-voltage trace from-100mV to+60mV when the middle part trace of Fig. 2 is presented at sodium and has (NaCl) and do not have (NMDG-Cl).Figure below is presented at sodium and has (dark blue colo(u)r streak among the figure; Rhombus) and not there is (purple colo(u)r streak; Square) time i-v curve.TRPML3 shows inside rectification (comparing electric current stronger under the negative voltage with positive voltage) as can be seen.

Embodiment 3:

Present embodiment relates to the electrophysiological detection that the result is contained in Fig. 3.These results that end user TRPML3 channel characteristic obtains are consistent with people's saline taste sense of taste psychophysics.Last figure among the figure contains and shows TRPML3 sodium conduction (dark blue colo(u)r streak; Rhombus) not by 30uM guanamprazine (purple colo(u)r streak; Square) current-voltage curve of preventing.People's saline taste sense of taste and TRPML3 are not prevented by guanamprazine.Figure below of same figure contains and shows that TRPML3 can see through saline taste positively charged ion sodium (dark blue colo(u)r streak equably; Rhombus) and lithium (purple colo(u)r streak; Square).This is consistent with TRPML3 coding people saline taste acceptor, because sodium and lithium are impartial saline tastes to the people, and two positively charged ions all see through people TRPML3 passage.

Embodiment 4:

Present embodiment relates to the immunohistochemistry labelling experiment that is contained among Fig. 4.Wherein TRPML3 albumen is expressed in the film district, top near taste pore as can be seen.Especially, the TRPML3 antibody labeling extends to the gustatory cell process (left figure) of taste pore as can be seen.In addition, clearly demonstrate TRPML3 albumen towards the amplification of the top taste bud structural domain of saliva and expressed (3 right figure in the taste pore district; Taste pore is represented with blue arrow).This position is ideally suited TRPML3 and experiences sodium in the saliva.Similar with TRPML3, other Taste Receptors (sweet taste, bitter taste, delicate flavour and tart flavour) are also to the taste pore polarization, and wherein its sampling saliva is used for its required flavor unit.

Embodiment 5:

Present embodiment relates to the immuno-chemical marker experiment that is contained in Fig. 5.These results show that TRPML3 albumen do not express in the TRPM5 cell.Particularly, use TRPM5 (green; Left figure) and TRPML3 (redness; Middle figure) in monkey CV nipple, carries out the double-tagging immunohistochemistry.The cell of expressing TRPM5 and TRPML3 as can be seen from this figure is different (figures of the right for merging).These data show that TRPML3 does not express in TRPM5 cell (comprising sweet taste, bitter taste and umami cells), and only express in the saline taste gustatory cell of specialty.

Embodiment 6:

Present embodiment relates to the immunohistochemistry labelling experiment that is contained in Fig. 6.These results show that TRPML3 albumen do not express in the PKD2L1 cell.Use PKD2L1 (green; Left figure) and TRPML3 (redness; Middle figure) in monkey CV nipple, carries out the double-tagging immunohistochemistry.The cell of as can be seen from the figure expressing PKD2L1 and TRPML3 is different (figures of the right for merging).These data show that TRPML3 does not express in PKD2L1 cell (comprising the tart flavour cell), and express in the saline taste gustatory cell of specialty.

Embodiment 7:

I/V curve in the ovocyte of personnel selection TRPML3 cRNA injection

Following examples have illustrated the ovocyte screening assay of the compound of identifying regulation and control (activate or prevent) TRPML3 function.The ovocyte of expression TRPML3 is replaced by the sodium of I/V tracing analysis and use NMDG and is identified.Referring to Fig. 7.With the increment measurement electric current of the voltage range from 100mV to+60mV with 10mV.Because the TRPML3 channel table reveals inside rectification, have ovocyte little under big inside electric current and the depolarize current potential under the hyperpolarization current potential and express the TRPML3 passage to extrinsic current.NMDG is the big positively charged ion that can not see through TRPML3.Therefore, the electric current representative that is suppressed by NMDG depends on the sodium current of TRPML3.Use NMDG inhibition electric current to be used as another internal reference and confirm this ovocyte expressive function TRPML3 albumen.

The inside rectification I/V curve of being represented by more weak electric current under electric current stronger under the hyperpolarization current potential (more negative current potential) and the depolarize current potential (current potential of corrigendum) shows ovocyte expression TRPML3 ionic channel.Use NMDG displacement sodium to prevent the TRPML3 electric current.The I/V curve of sodium and NMDG subtracts each other and has produced the special sodium current of TRPML3.I/V curve in rhombus () expression sodium (NaCl) solution; Square (■) expression does not contain the I/V curve in the NMDG solution of sodium; Triangle (▲) is represented subtracting each other of NaCl and NMDG I/V curve, and represents the special sodium current of TRPML3-.

Embodiment 8

The active compound of adjustable TRPML3 in the ovocyte of screening personnel selection TRPML3 cRNA injection

Concentration between-1uM and-compound between the 100uM is applied to show the ovocyte of TRPML3 function.Referring to Fig. 2.If this compound works as the TRPML3 toughener, then the electric current by the TRPML3 passage strengthens (becoming more negative) in the oocyte membrane.If this compound works as the TRPML3 co-inhibitor, the then current decay (cannot not becoming negatively) by the TRPML3 passage in the oocyte membrane.For of the influence of quantification compound, use following equation to the TRPML3 function:

[(A-Ao)/(B-Bo)]x-100

A is the electric current after the compound treatment, and Ao is the electric current before the compound treatment, and B is the electric current after NMDG handles, and Bo is the electric current before NMDG handles.This value causes being used for the active regulation and control coefficient % of compound in the measuration.For example, if regulation and control coefficient % equals 100%, this compound has increased the substrate control value (lacking compound) of TRPML3 activity 100% so.If regulation and control coefficient % equals-100%, this compound has reduced the substrate control value (lacking compound) of TRPML3 activity 100% so.In the OpusXpress system, calculate the regulation and control coefficient % of each ovocyte separately, determine the average and standard deviation of each compound then.

In the ovocyte of not injecting TRPML3 cRNA, carry out the negative control experiment, influence with explanation observed compound in the ovocyte of expressing TRPML3 is owing to the electric current that flows through the TRPML3 passage, rather than owing to flows through the electric current of the passage of endogenous expression in the oocyte membrane.The compound of special regulation and control TRPML3 should not influence the electric current in the ovocyte of not injecting, but should show the regulation and control coefficient % near zero.

As shown in Figure 8, calculate the regulation and control coefficient % of the compound of each screening.The amplitude that this value changes corresponding to the electric current that caused by compound multiply by-100% divided by the amplitude that the electric current that is caused by NMDG changes.In the present embodiment, voltage is fixed in 3 kinds of compounds of step sizing (100uM) in 4 of 60mV (may maximum 8) ovocytes in the OpusXpress system.All 4 ovocytes are expressed TRPML3, as NMDG the retarding effect of the ovocyte electric current of measurement are proved.All 3 compounds are not significantly regulated and control the function of TRPML3, are similar because add before the compound with afterwards electric current.

Embodiment 9:

Use the I/V curve of TRPML3 co-inhibitor gadolinium

The compound that shows big regulation and control coefficient % and do not influence the ovocyte of not injecting TRPML3 cRNA is carried out more complicated analysis.Mensuration comprises current/voltage (I/V) curve, NMDG competitive assay and dose response curve.To the I/V curve, when existing and lacking compound, with the increment measurement electric current of the voltage range from 100mV to+60mV, so that the regulation and control amplitude of research compound with 10mV.The electric current regulation and control amplitude of I/V slope of a curve indication compound of interest.Strong toughener increases the I/V slope of a curve, the opening of the TRPML3 ionic channel that indication increases.Strong co-inhibitor reduces the I/V slope of a curve, the closing of the TRPML3 ionic channel that indication increases.Fig. 9 illustrates how gadolinium is prevented TRPML3 and reduced the I/V slope of a curve.In not injecting the ovocyte of TRPML3cRNA, the I/V curve that carries out when compound exists should be identical and overlapping with the I/V curve when lacking compound.I/V curve in the little square explanation sodium solution, and the I/V curve in large square explanation 300uM gadolinium (GdCl3) solution.Should notice that gadolinium prevents the TRPML3 electric current and reduce the I/V slope of a curve.

Embodiment 10:

The NMDG competitive assay

Carry out the NMDG competitive assay so that the influence of explanation compound depends on TRPML3.At first, use NMDG so that explanation TRPML3 expresses in ovocyte.Then, use compound so that determine regulation and control coefficient %.Last common application NMDG and compound.For making toughener directly act on the TRPML3 passage, common application NMDG additional compound should show the NMDG-type and reply, and means that electric current is suppressed, and does not strengthen.This experiment shows when lack sodium, and when replacing with impermeable positively charged ion NMDG, this compound can not have reinforcing effect; Therefore this compound must direct regulation and control TRPML3 sodium channel function.

Embodiment 11:

Dose response curve

Carry out dose response curve and show the concentration of half maximum activity (is EC50 to toughener, and be IC50 to co-inhibitor) so that determine compound.EC50 or IC50 value are more little, and this compound is strong more as the activity of TRPML3 conditioning agent.((1nM) begin to be developed to high dosage (1mM)) and carry out dose response curve by using the compound that increases concentration successively from low dosage.Calculating as indicated above regulation and control coefficient %, and on logarithmic scale with the function construction of compound concentration so that determine the EC50 or the IC50 value of compound.

Embodiment 12:

The expression of the activated sodium channel of composition has strengthened the substrate fluorescence with certain films current potential dyestuff loaded cells

The HEK293 cell is with RFP, α ENaC or A419P-TRPML3 transient transfection, and is used for the membrane potential dyestuff (R-8034 of HBSS; Molecular Devices) under room temperature, loaded 30 minutes.Go up monitoring membrane potential (fluorescent signal) in FLIPR system (Molecular Devices).Referring to Figure 11.The result show when with RFP transfectional cell (RFP is a control vector) when comparing, express the cell demonstration enhanced substrate fluorescence of A419P-TRPML and α ENaC.These results mean that the composition activity of A419P-TRPML3 and α ENaC causes more depolarize of cytolemma, and we can measure the activity of A419P-TRPML3 in this FLIPR measures.

Embodiment 13:

The application gadolinium has reduced the enhancing of substrate fluorescence in the cell of expressing A419P-TRPML3

The HEK293 cell is with RFP or A419P-TRPML3 transient transfection, and is used for the membrane potential dyestuff (R-8034 of HBSS; Molecular DeviceS) under room temperature, loaded 30 minutes.Referring to Figure 12.Go up monitoring membrane potential (fluorescent signal) in FLIPR system (Molecular Devices).The result show add gadolinium (big, short arrow) significantly reduced with the RFP transfectional cell in the approaching substrate fluorescence of value of (long arrow and trace 2) acquisition.These results show that the composition of A419P-TRPML3 activity active and the TRPML3 conditioning agent can both detect in this mensuration.

Embodiment 14:

The application gadolinium has reduced the enhancing of substrate fluorescence in the cell of expressing A419P-TRPML3 in the mode that depends on dosage

The HEK293 cell is with RFP or A419P-TRPML3 transient transfection, and is used for the membrane potential dyestuff (R-8034 of HBSS; Molecular Devices) under room temperature, loaded 30 minutes.The gadolinium of HBSS, NMDG and increase concentration is added in the cell, and goes up the change of monitoring the membrane potential (fluorescent signal) that causes in FLIPR system (MolecularDevices).Referring to Figure 13.Result's demonstration is compared with observed influence in the RFP transfectional cell, and the gadolinium that increases concentration has significantly reduced substrate fluorescence to a greater degree.

Embodiment 15:

The titration of TRPML3 plasmid

The HEK293 cell is with A419P-TRPML3 plasmid (from 0.02ug to the 2ug) transient transfection of RFP (0ug) or increasing amount, and is used for the membrane potential dyestuff (R-8034 of HBSS; Molecular Devices) under room temperature, loaded 30 minutes.Referring to Fig. 4.HBSS (contrast) and 4mM gadolinium are added in the cell, and go up the change of monitoring the membrane potential (fluorescent signal) that causes in FLIPR system (Molecular Devices).The result shows the size that has strengthened the gadolinium influence to 0.5g of measuring that increases the TRPML3 plasmid.

Embodiment 16:

The influence of gadolinium is specific to TRPML3

The HEK293 cell is with RFP, A419P-TRPML3 or α ENaC plasmid transient transfection, and is used for the membrane potential dyestuff (R-8034 of HBSS; Molecular Devices) under room temperature, loaded 30 minutes.HBSS (contrast), 3mM gadolinium and 30uM guanamprazine are added in the cell, and go up the change of monitoring the membrane potential (fluorescent signal) that causes in FLIPR system (Molecular Devices).Referring to Figure 15.The result shows that gadolinium preferentially reduces the substrate fluorescence counting in the A419P-TRPML3 transfectional cell, and guanamprazine preferentially reduces the substrate fluorescence counting in the α ENaC-transfectional cell.

Embodiment 17:

Baculovirus transduction HEK293 cell with coding A419P-TRPML3 has doubled the mensuration window

The HEK293 cell is transduceed with the baculovirus of the modification that allows A419P-TRPML3 to express in mammalian cell (BacMaM).The cell of 24 hours postoperative infections is used for the membrane potential dyestuff (R-8034 of HBSS; Molecular Devices) under room temperature, loaded 30 minutes.HBSS (contrast), 2mM and 3mM gadolinium are added in the cell, and go up the change of monitoring the membrane potential (fluorescent signal) that causes in FLIPR system (MolecularDevices).Referring to Figure 16.

Embodiment 17:

The screening embodiment of the data of the cell acquisition of expressing A419P-TRPML3

The HEK293 cell is transduceed with the baculovirus of the modification that allows A419P-TRPML3 to express in mammalian cell (BacMaM).The cell of 24 hours postoperative infections is used for the membrane potential dyestuff (R-8034 of HBSS; Molecular Devices) under room temperature, loaded 30 minutes.Referring to Figure 17.320 kinds of different compounds (red point), HBSS (stain) and gadolinium (Bluepoint) are added in the cell of 384 hole compound flat boards, and go up the change of monitoring the membrane potential (fluorescent signal) that causes in FLIPR system (Molecular Devices).In this experiment, identified and obviously prevented two of A419P-TRMPL3 mainly to hit.

Embodiment 18:

General introduction with the little screening of 10,000 compounds of the cell of expressing A419P-TRPML3

The HEK293 cell is transduceed with the baculovirus of the modification that allows A419P-TRPML3 to express in mammalian cell (BacMaM).The cell of 24 hours postoperative infections is used for the membrane potential dyestuff (R-8034 of HBSS; Molecular Devices) under room temperature, loaded 30 minutes, and handle as shown in figure 17.Referring to Figure 18.Screen 10,000 kinds of compounds, identified and severally mainly hit, comprise that 52 co-inhibitors hit with 113 tougheners to hit.

Embodiment 11-18 is presented under these experiment conditions, and with respect to using control vector (RFP) cells transfected, the cell of expressing A419P-TRPML3 has shown the remarkable enhancing of substrate fluorescence.TRP passage co-inhibitor gadolinium is to depend on the enhancing that dosage and ad hoc fashion have reversed fluorescence.α ENaC co-inhibitor guanamprazine is not to being influenced by the enhanced substrate fluorescence of expressing the A419P-TRPML3 initiation.Now screen thousands of kinds of compounds in this mensuration, and identified several hitting.These hit and will further assess by electric physiology and taste test.

Embodiment 19:

The codon optimized TRPML3 gene and the structure of mutant

As shown in figure 19, make up codon optimized TRPML3 gene.Codon comprises 3 Nucleotide of coding specific amino acids in the protein sequence.Because there are 20 amino acid whose codon nucleotide triplets of 61 different codings, most of amino acid can be by more than codon coding.Use in the specific species speed that codon codon optimized of each amino acid preference can be by increasing translation and accuracy and do not change protein sequence and improve proteic functional expression.

The inventor uses has the codon optimized form (Figure 19) of 76.4% homologous people TRPML3 gene at dna level and non-codon optimized TRPML3.Codon is optimised for the best translation that reaches the human sequence.By the L-Ala 419 in the 5th membrane spaning domain is sported proline(Pro), also produced the activity form (A419P TRPML3) of TRPML3.This sudden change cause the TRPML3 passage be in open conformation (confirmation) (Xu etc., PNAS 104 (46): 18321-18326,2007; Grimm etc., PNAS 104 (49): 19583-19588,2007; Nagata etc., PNAS 105 (1): 353-358,2008; Kim etc., J.Biol.Chem.282 (50): 36138-36142,2007); Therefore, A419P TRPML3 is used in particular for identifying the TRPML3 co-inhibitor.

Embodiment 20:

As be contained in shown in the experiment of Figure 20, the expression of wild-type TRPML3 produces low sodium current level, and codon optimized wild-type TRPML3 produces medium sodium current level, and A419PTRPML3 produces high sodium current level.Therefore, codon optimized wild-type TRPML3 and A419PTRPML3 help to screen the compound of regulation and control TRPML3 function.

Embodiment 21:

The ovocyte that screening is injected with codon optimized people TRPML3 cRNA

Figure 21 contains and relates to the experiment of screening with the ovocyte of codon optimized people TRPML3 cRNA injection, so that identify the compound (TRPML3 toughener) that activates TRPML3.The presentation of results that wherein comprises when ovocyte be fixed in-during 60mV, identified the toughener that activates TRPML3.

In multiple ovocyte, the TRPML3 toughener has strengthened active 169+/-26% (the representative trace in top) of TRPML3, and to do not influence (the representative trace in bottom) with the not injection ovocyte that does not have TRPML3 to express.Only add damping fluid to the not influence of TRPML3 electric current, and in case use the second time, the influence of TRPML3 toughener is reproducible.

Embodiment 22:

The TRPML3 toughener is to the influence of TRPML3 I/V curve

Figure 22 contains the experiment of explanation TRPML3 toughener to the embodiment of TRPML3 I/V curve influence.This figure is presented at the toughener that negative voltage is identical with front embodiment down in the I/V tracing analysis and activates TRPML3.Stimulate (toughener of red-violet colour square marks) with the ovocyte unprocessed (contrast of blue warning triangle) of codon optimized people TRPML3cRNA injection or with the TRPML3 toughener, and from-90mV extremely+measure electric current under the voltage of 30mV.Should notice that the TRPML3 toughener activates TRPML3 electric current (use compared with the control the inside electric current of toughener bigger) under negative voltage, cause the increase of I/V rate of curve.Should also be noted that zero current drifts about to the right, hint is the conduction of enhanced sodium when toughener exists.

Embodiment 23:

The experiment of TRPML3 toughener influence when Figure 23 has contained example sodium exists and do not exist outside born of the same parents.The ovocyte of expressing codon optimized people TRPML3cRNA with NMDG (not having sodium), TRPML3 toughener add sodium, only damping fluid or TRPML3 toughener add NMDG (not having sodium) stimulates.Should notice that the TRPML3 toughener has strengthened the activity of TRPML3 when sodium exists, but when lacking sodium, not influence.The open TRPML3 passage of these data declarations TRPML3 toughener, and increase the flow that sodium ion flows to ovocyte.Figure 23 illustrates that this identical toughener does not activate TRPML3 during sodium outside lacking born of the same parents.Because the open TRPML3 of this compound also increases the sodium flow that flows into cell, it is candidate's a saline taste sense of taste toughener.Jointly, these data declaration electricity physiology ovocytes measure how to be used for identifying to be the TRPML3 toughener of candidate's saline taste sense of taste toughener, and how the codon optimized TRPML3 of wild-type helps to identify the TRPML3 toughener in the ovocyte electrophysiological detection.

Embodiment 24:

The expression level of WT TRPML3 in the different mammalian cell types

As be contained in shown in the experiment among Figure 24, the expression level of WT TRPML3 depends on the type of mammalian cell.The figure A of this figure is the current/voltage analysis (I/V figure) of expressing the HEK293 cell of WT and A419P mutant TRPML3 passage.It shows the big inside rectified current (pink colour) of A419P mutant TRPML3 passage expression, and only observes little WT TRPML3 electric current (blueness).B.WT has similar functional character with A419P mutant TRPML3 passage in Chinese hamster ovary celI.

Embodiment 25:

TRPML3 is used for the application of Chinese hamster ovary celI toughener and co-inhibitor screening

Experiment among Figure 25 relates to the application that TRPML3 is used for Chinese hamster ovary celI toughener and co-inhibitor screening.Figure A shows WT people's TRPML3 passage transient expression in Chinese hamster ovary celI, and it is used for identifying the passage toughener.I/V figure shows and independent damping fluid (blueness; Contrast) compares, use toughener to cause the increase (pink colour) of inside electric current under the negative potential.The mutant A419P TRPML3 passage that figure B is presented at stably express in the Chinese hamster ovary celI is used for identifying the passage co-inhibitor.With independent damping fluid (blueness; Contrast) compares, add weaken (pink colour) that 1mM GdCl3 causes inside electric current.

Embodiment 26:

The screening assay of the TRPML3 that the son that accesses to your password in mammalian cell is optimized

The experiment of Figure 26 contains uses the identical codon optimized WT TRPML3 that is contained in Figure 19 to be used to screen the experiment of the compound that strengthens the TRPML3 function in mammalian cell.Figure A shows that the transient expression of non-codon optimized WT TRPML3 (light blue) causes in the HEK293 cell electric current seldom.On the contrary, the WT TRPML3 (mazarine that son optimizes that accesses to your password; CodOpt WT) causes electric current (pink colour) to the similar average amplitude of A419P mutant passage.Figure B shows that the codon optimized WT TRPML3 (blueness) that uses the baculovirus transduction to send causes the mean current similar to A419P TRPML3 (pink colour).Figure C shows that the cell with codon optimized WT TRPML3 baculovirus transduction is used for identifying the toughener of TRPML3 function.With independent damping fluid (blueness; Contrast) compares, add the increase (pink colour) that the toughener compound causes inside electric current.

Embodiment 27:

The co expression of WT and A419P TRPML3

The experiment of Figure 27 relates to the experiment of WT and A419P TRPML3 co expression.The result of this figure shows that co expression has increased the functional surface expression in the HEK293 cell.The electric current of A419P TRPML3 cDNA (0.5ug) initiation of HEK293 cell is advanced in figure A demonstration transfection, produces the electric current with the inside rectification of characteristic.Figure B shows that the non-codon optimized TRPML3 of WT (1.5ug) expresses in the HEK293 cell, and does not produce electric current.Figure C shows that A419P (0.5ug) and WT (1.5ug) TRPML3 cDNA co expression in the HEK293 cell cause big inside electric current, and its twice for the electric current when single expression A419P cDNA is big.Figure D contains the mean current that the TRPML3 cDNA of WT (blueness), A419P (pink colour) and WT and A419P co expression (yellow) causes in the HEK293 cell I/V schemes.

Embodiment 28

The experiment of Figure 28 is the embodiment that measures the TRPML3 function in the experiment of IonWorks PPC diaphragm plate.Figure A contains and has the view from all 384 holes of PPC diaphragm plate that A419 TRPML3 can induce the clone, and it has shown the result of scanning before the compound.Yellow shows wherein-120mV under the hole (in the control experiment of using parent CHO-K1 cell, not having the hole to be marked as yellow) of electric current≤0nA.Blueness show wherein even sealed impedance too low (＜10mOhm) so that can not measure the hole of electric current reliably.Can in 94% hole, measure A419P TRPML3 electric current.

Figure B contain interpolation shown in A from the 4mM GdCl3 of diaphragm plate or the outer damping fluid of born of the same parents (simulation is added) before and mean current ± SEM afterwards.GdCl3 is added to post 1-38, and the outer damping fluid of born of the same parents is added to post 39-48.For relatively, comprise data from the independent experiment of using parent CHO-K1 cell.The stability of TRPML3 electric current showed that this mensuration should detect the compound that strengthens or prevent the TRPML3 electric current after simulation was added.

Embodiment 29

Figure 29 contains the embodiment (last figure) of the IonWorks scanning of using the induced CHO-K1 clone of expressing A419P TRPML3.The inside rectification of TRPML3 is represented by more weak electric current under electric current stronger under the hyperpolarization current potential (more negative current potential) and the depolarize current potential (current potential of corrigendum).Add GdCl3 and prevented the TRPML3 electric current.Red line is illustrated in the scanning in sodium (NaCl) solution.Blue line is illustrated in the scanning in the 4mM GdCl3 solution.Middle figure is from the parent CHO-K1 cell as negative control.Positive current under the negative potential is because the excessive down leakage difference of correcting electric current of negative potential subtracts.Figure below shows the voltage instruction scheme of record current of being used for.Be used for calculating leakage current (electric current that flows to by sealing leak) from the grade of 0mV to 10mV, deduct the electric current that leakage current obtains flowing through film from total current.

The result is from the single hole in the PPC diaphragm plate, and represents the mean current of 64 cells of Ke Da.Figure 30 contains be used for checking the experiment flow figure of compound to the active influence of people TRPML3 (hTRPML3) in IonWorks measures.

Embodiment 30

As be contained in shown in the PCR in real time experimental result of Figure 31, the TRPML3 gustatory cell is excised from taste bud specifically in the Varitint waddler mouse.Shown experiment by the terminal point RT-PCR on the isolating Varitint waddler of laser capture micro-dissection (Va) or wild-type (WT) mouse taste bud (TB) and the tongue epithelial cell (LE).

The result shows that TRPML3 only expresses in the taste bud of WT mouse, and lacks in the taste bud of Va mouse, and every other sense of taste gene (T1R2, Gpr113, TRPM5) and house-keeping gene (beta-actin, GAPDH) expression equably in TB and LE.'+' shows and carried out reverse transcription, and ' ' show and do not carry out reverse transcription.Have only the ThermoScript II of use just can observe the PCR band, show the PCR product derived from mRNA, rather than genomic dna.

Embodiment 31

Present embodiment relates to the experiment that is summarized in Figure 32, and it contains and shows that the TRPML3 cell is by specifically from the PCR in real time experiment of the taste bud excision of Varitint waddler mouse.Real-time quantitative RT-PCR experiment by isolating Varitint waddler of laser capture micro-dissection (Va) or wild-type (WT) mouse taste bud.TRPML3 only expresses in the taste bud of WT mouse, and in the taste bud of Va mouse, lack (using the Mcoln3_1 of two different primer cover marks to obtain similar result) with Mcoln3_2, and every other sense of taste gene (Tas1r2, Tas1r3, PKD211, TRPM5, Plcb2, Tas2r108 and Tas2r116) and house-keeping gene (contrast) are expressed in the taste bud of Va and WT mouse.

Embodiment 32

Present embodiment relates to the experiment among Figure 33, and it shows that sweet taste, bitter taste, delicate flavour and tart flavour gustatory cell are kept perfectly in Varitint waddler mouse.From wild-type (row of going up of figure) and Varitint waddler (Va; The following row of figure) in situ hybridization of mouse back of tongue profile shape nipple.PKD1L3 (left side; Tart flavour), PKD2L1 (centre; Tart flavour) and TRPM5 (right side; Sweet taste, bitter taste and delicate flavour) gustatory cell is present in wild-type and the Va mouse with similar level.

Embodiment 33

Present embodiment relates to the experiment of using the neural record of CT in wild-type that stimulates with the saline taste that is contained in Figure 34 and Varitint waddler mouse, its saline taste that shows the Varitintwaddler mouse is experienced defective, and wild-type mice detects saline taste under the same conditions.Wild-type (left side) or Varitint waddler (Va; The right side) the neural record of the CT of mouse.Anterior tongue adds 5uM benzamil with 0.1M NaCl or 0.1M NaCl to stimulate and suppresses the guanamprazine sensitive ingredients that the CT nerve is replied.In the middle of NaCl stimulated, tongue washed with the low salts solution that contains 10mM KCl.It should be noted that the insensitive component of benzamil-greatly elimination (red arrow) of quilt in the Va mouse that the CT nerve is replied, hint excision TRPML3 gustatory cell has significantly damaged the saline taste impression.In addition, the instant phase place of NaCl is replied the Va mouse greatly weakened (red circle).Scale shows that time frame (x-axle) and CT that salt is used reply intensity (y-axle; Arbitrary unit).

Figure 35 contains the comparison from the protein sequence of people (NM_018298) and mouse (NM_134160) TRPML3 sequence, and wherein the people is expressed as Hs, and mouse is expressed as Mm.People and murine protein sequence are 91% identical similar with 96%.6 membrane spaning domains of the TRPML3 polypeptide of people and mouse TRPML3 are underlined TM1 to TM6.Porose area between TM5 and the TM6 is expressed as ' porose area '.Prediction amino and C-terminal are positioned at cell interior.This paper discussion and the A419P sudden change locking TRPML3 that is found in the varitint-waddler mouse are in open conformation, and it is positioned at TM5, and highlights with redness.Another sudden change V412P partly activates TRPML3, and represents with red-violet colour.

Although the present invention describes by embodiment and preferred embodiment, should understand word used herein is descriptive words, rather than restricted word.Can change within the scope of the appended claims, and not deviate from scope and spirit of the present invention with its wider aspect.Although the present invention describes with reference to specific method, material and embodiment at this paper, should understand and the invention is not restricted to disclosed details.The present invention extends to all equivalent constructions, methods and applications within the scope of the appended claims.

Mention as preamble, gustatory cell specific gene of identifying according to the present invention and corresponding gene product and express for example endogenous gustatory cell of cell or the chemoreception cell of this gene and comprise these sense of taste specific genes and directly to homologue, allelic variant, have with the variant of its at least 90% sequence identity and/or the hybridization conditions of above negating under can be used on identification of taste with the reconstitution cell of the gene of its specific hybridization and regulate in the mensuration of compound, and be used in the therapeutic screening assay.

For example these cell of these sense of taste specific genes, polypeptide and expression can be used to screen the compound that is used for the treatment of the Digestive tract disorder.These disorders comprise the patient's condition, the autoimmunization that influences Digestive tract and the inflammatory diseases such as dyspeptic influence digestion as an example, such as ulcerative colitis, inflammatory bowel syndrome, Crow grace syndrome, celiac disease, with precancer that influences Digestive tract and cancer, such as the cancer that influences sialisterium, taste bud, stomach, pancreas, gall-bladder, oesophagus, little or large intestine, anus or colon.

These sense of taste specific genes also can be used in the screening assay of identifying the compound that influences the gustatory cell turnover.Known gustatory cell turnover is (every approximately several Zhou Yici) rapidly.In addition, the many patient's condition and the aged ability that can influence the gustatory cell growth negatively that comprise chemotherapy or radiation therapy.The result is the taste perception that weakens, and it can cause the reduction of cancer patients or life of elderly person quality.This is obvious especially in the patient who suffers from head and neck cancer, the esophageal carcinoma, cancer of the stomach, lung cancer or carcinoma of the pancreas.Additionally, this can evolve with another patient's condition (emaciation or wasting syndrome), and it makes up and reduces the feed desire.Lacking suitable nutrition is that the cancer patients falls ill and dead serious reason.This sense of taste specific gene contains the gene of expressing in stem cell, show that these genes are to be gustatory cell precursor and the sign of evolution for the stem cell of gustatory cell.These genes or the cell of expressing these genes can be used to identify and quicken the signal that gustatory cell is grown.May comprise the growth of these signal mediation gustatory celies of the cytokine-like acceptor that is present on the gustatory cell, and can be used in the screening of identifying the compound of inducing gustatory cell differentiation or propagation.Therefore part can separate from saliva potentially, and soluble saliva influences the ability of taste function.For example, the patient who suffers from the Sjogren syndrome autoimmune disease of sialisterium (attack) shows the taste function of change.This gene and will help to understand these differentiation mechanism by using the gene array to study the expression of this gene in sialisterium.

Also can use this gustatory cell specific gene and corresponding gene product and the cell of expressing these genes, so that identify the potential therapeutical agent that is used to regulate and control the oral immunisation system.The oral cavity is by the normal microflora group feature the same with digestive tube.The change of normal microflora can produce such as gingivitis, halitosis, stomach trouble and can cause decaying tooth or the patient's condition of other infection of loss of tooth.The gustatory cell specific gene that this paper identifies comprises many immunity system genes.These genes and corresponding polypeptide or the cell of expressing this gene can be used to identify the immunizator inner equilibrium that is used for keeping the oral cavity, prevent the pathogenic bacterium hypertrophy and be used for identifying the therapeutical agent of oral cavity at the cell type that keeps suitable oral immunisation performance keying action.

In addition, this gustatory cell specific gene and corresponding gene product or the cell of expressing this gene be used in be used for identifying the treatment diabetes, such as the screening assay of the compound of the feed disorder of obesity.

The compound of inventor's genes identified product and enhancing or suppressor gene product can influence: the detoxifcation of toxic substance in oral hygiene, halitosis, the apocleisis, voracity and other metabolism disorders.The expression of Taste Receptors may be represented in the digestive process at different local food and the dissimilar complex systems of detecting in the Digestive tract.Therefore, the existence of " impression " food or particular type (such as carbohydrate, fat, delicate flavour food, salt), should trigger the various signals that to regulate the molecule generation that participates in the digestion adjusting, such as entering GIP (depending on the pancreotropic hormone polypeptide of glucose) and the GLP-1 (glucagon-like peptide 1) that endocrine cell produces in the intestines.Taste Receptors on possible these cells is regulated the generation of other molecular signals in other cells of Digestive tract when triggering.These phenomenons can be studied by determining the different acceptor of which cell expressing, the molecule that uses the gene array to study these cells generations when activating then.

More specifically, the present invention identifies and provides functional (electric physiology) and immunohistochemistry data, and it shows the polypeptide that TRPML3 (MCOLN3) coding works as primate (for example people) saline taste acceptor.

More specifically, the present invention also provides the application of these sense of taste specific genes as the sign of the cell that can be used to enrichment, evaluation or separation expression saline taste acceptor.

More specifically, the present invention also provides use TRPML3 (MCOLN3) and has expressed the cell of TRPML3 or the TRPML3 transgenic animal model is identified and caused or regulation and control (prevent or strengthens) comprise mensuration in the external and body of agonist, antagonist or the toughener compound of the saline taste sense of taste in people's the primate.These measure cell that uses single expression TRPML3 or the cell of expressing TRPML3 ionic channel and other sense of taste specific polypeptides (such as NALCN or NKAIN3).

More specifically, the present invention transgenic animal (preferably rodent) also are provided and use these transgenic animal confirm TRPML3 the Mammals saline taste sense of taste and relating to sodium and other physiological functions of other ionic (such as sodium metabolism, blood pressure, fluid retention and drainage, urinary function and heart function) in effect.

More specifically, the present invention also provides the cell that uses TRPML3 and express TRPML3 or transgenic animal to alleviate mensuration in the external and body that relates to the disease that influence this polypeptide expression defective that work in special sodium channel as the sense of taste in its Mammals that is comprising for example people and non-human primate and the rodent (comprising overexpression, the low expression and the sudden change of TRPML3 polypeptide) and the patient's condition so that identify in measuring (preferably electrophysiological detection).These patient's condition comprise Addison's disease and relate to the disease that unusual aldosterone produces or vasopressing discharges as an example, such as hypertension, ypotension, fluid retention and impaired uropoiesis or heart function, such as arrhythmia, heart attack and apoplexy.In addition, the patient's condition that can use TRPML3 to regulate compounds for treating comprises melanoma and relates to other patient's condition of melanocyte (melanocyes), such as the pigmentation disorder.

Therefore, in sum, the present invention relates to identification of M COLN3 and be coding people saline taste acceptor, it allows design to use the cell of this gene transfection to be used to identify the screening assay of agonist, antagonist or the toughener (adjusting compound) that influence this molecular function.These compounds can be used as sense of taste conditioning agent and are used as the metabolism of regulation and control sodium, absorption and excretory therapeutical agent.In order to further describe the present invention and exemplary, following TRPML3 nucleic acid and peptide sequence are hereinafter provided from different Mammalss (comprising the people).Yet, as mentioned before, gustatory cell specific gene of identifying according to the present invention and corresponding gene product and express for example endogenous gustatory cell of cell or the chemoreception cell of this gene and comprise these sense of taste specific genes and directly to homologue, allelic variant, mosaic and have at least 80% with it, 90%, 95%, 96%, 97%, 98%, 99% or the fragment of the genetic modification of higher sequence identity and variant and/or under the hybridization conditions of above negating the reconstitution cell of the gene of special and its hybridization can be used on identification of taste and regulate in the mensuration of compound, and be used in the therapeutic screening assay.

Reference

All reference integral body of quoting among the application are incorporated this paper by reference into.

Sequence table

(sequence of example T RPML3, TRPML2, TRPML1 polypeptide and nucleotide sequence and NKAIN3 and NALCN)

Homo sapiens (homo sapiens) TRPML3 (NM_018298) (SEQ ID NO:1)

ATGGCAGATCCTGAGGTAGTTGTGAGTAGCTGCAGCTCTCATGAAGAGGAAAATCGCTGCAATTTTAACCAGCAAACATCTCCATCTGAGGAGCTTCTATTAGAAGACCAGATGAGGCGAAAACTCAAATTTTTTTTCATGAATCCCTGTGAGAAGTTCTGGGCTCGAGGTAGAAAACCATGGAAACTTGCCATACAAATTCTAAAAATTGCAATGGTGACTATCCAGCTGGTCTTATTTGGGCTAAGTAACCAGATGGTGGTAGCTTTCAAGGAAGAGAATACTATAGCATTCAAACACCTTTTCCTAAAAGGATATATGGACCGAATGGATGACACATATGCAGTGTACACACAAAGTGACGTGTATGATCAGTTAATCTTCGCAGTAAACCAGTACTTGCAGCTATACAATGTCTCCGTTGGGAATCATGCTTATGAGAACAAAGGTACCAAGCAATCTGCTATGGCAATCTGTCAGCACTTCTACAAGCGAGGAAACATCTACCCTGGAAATGATACCTTTGACATCGATCCAGAAATTGAAACTGAGTGTTTCTTTGTGGAGCCAGATGAACCTTTTCACATTGGGACACCAGCAGAAAATAAACTGAACTTAACACTGGACTTCCACAGACTCCTAACAGTGGAGCTTCAGTTTAAACTGAAAGCCATTAATCTGCAGACAGTTCGTCATCAAGAACTCCCTGACTGTTATGACTTTACTCTGACTATAACATTTGACAACAAGGCCCATAGTGGAAGAATTAAAATAAGTTTAGATAATGACATTTCCATCAGAGAATGTAAAGACTGGCATGTATCTGGATCAATTCAGAAGAACACTCATTACATGATGATCTTTGATGCCTTTGTCATTCTGACTTGCTTGGTTTCATTAATCCTCTGCATTAGATCTGTGATTAGAGGACTTCAGCTTCAGCAGGAGTTTGTCAATTTTTTCCTCCTCCATTATAAGAAGGAAGTTTCTGTTTCTGATCAAATGGAATTTGTCAATGGATGGTACATTATGATTATTATTAGTGACATATTGACAATCATTGGATCAATTCTAAAAATGGAAATCCAAGCTAAGAGTCTAACTAGTTATGATGTCTGTAGCATACTTCTTGGGACTTCTACCATGCTCGTGTGGCTTGGAGTCATCCGATACCTCGGTTTCTTTGCAAAGTACAACCTCCTCATTTTGACCCTTCAGGCAGCGCTGCCCAATGTCATCAGGTTCTGCTGCTGTGCAGCTATGATTTACTTAGGTTACTGCTTCTGTGGATGGATCGTGCTGGGGCCTTACCATGACAAGTTTCGTTCTCTGAACATGGTCTCTGAGTGCCTTTTCTCTCTGATAAATGGAGATGATATGTTTGCCACGTTTGCAAAAATGCAGCAAAAAAGTTACTTAGTCTGGCTGTTTAGTAGAATTTACCTCTACTCATTCATCAGCCTCTTTATATATATGATTTTAAGTCTTTTCATTGCACTGATCACTGATACATACGAAACAATTAAGCAATACCAACAAGATGGCTTCCCAGAGACTGAACTTCGTACATTTATATCAGAATGCAAAGATCTACCCAACTCTGGAAAATACAGATTAGAAGATGACCCTCCAGTATCTTTATTCTGCTGTTGTAAAAAG(SEQ?ID?NO：1)

Homo sapiens TRPML3 (NM_018298) (SEQ ID NO:2)

MADPEVVVSSCSSHEEENRCNFNQQTSPSEELLLEDQMRRKLKFFFMNPCEKFWARGRKPWKLAIQILKIAMVTIQLVLFGLSNQMVVAFKEENTIAFKHLFLKGYMDRMDDTYAVYTQSDVYDQLIFAVNQYLQLYNVSVGNHAYENKGTKQSAMAICQHFYKRGNIYPGNDTFDIDPEIETECFFVEPDEPFHIGTPAENKLNLTLDFHRLLTVELQFKLKAINLQTVRHQELPDCYDFTLTITFDNKAHSGRIKISLDNDISIRECKDWHVSGSIQKNTHYMMIFDAFVILTCLVSLILCIRSVIRGLQLQQEFVNFFLLHYKKEVSVSDQMEFVNGWYIMIIISDILTIIGSILKMEIQAKSLTSYDVCSILLGTSTMLVWLGVIRYLGFFAKYNLLILTLQAALPNVIRFCCCAAMIYLGYCFCGWIVLGPYHDKFRSLNMVSECLFSLINGDDMFATFAKMQQKSYLVWLFSRIYLYSFISLFIYMILSLFIALITDTYETIKQYQQDGFPETELRTFISECKDLPNSGKYRLEDDPPVSLFCCCKK(SEQ?ID?NO：2)

Homo sapiens TRPML3A419P (SEQ ID NO:3)

ATGGCAGATCCTGAGGTAGTTGTGAGTAGCTGCAGCTCTCATGAAGAGGAAAATCGCTGCAATTTTAACCAGCAAACATCTCCATCTGAGGAGCTTCTATTAGAAGACCAGATGAGGCGAAAACTCAAATTTTTTTTCATGAATCCCTGTGAGAAGTTCTGGGCTCGAGGTAGAAAACCATGGAAACTTGCCATACAAATTCTAAAAATTGCAATGGTGACTATCCAGCTGGTCTTATTTGGGCTAAGTAACCAGATGGTGGTAGCTTTCAAGGAAGAGAATACTATAGCATTCAAACACCTTTTCCTAAAAGGATATATGGACCGAATGGATGACACATATGCAGTGTACACACAAAGTGACGTGTATGATCAGTTAATCTTCGCAGTAAACCAGTACTTGCAGCTATACAATGTCTCCGTTGGGAATCATGCTTATGAGAACAAAGGTACCAAGCAATCTGCTATGGCAATCTGTCAGCACTTCTACAAGCGAGGAAACATCTACCCTGGAAATGATACCTTTGACATCGATCCAGAAATTGAAACTGAGTGTTTCTTTGTGGAGCCAGATGAACCTTTTCACATTGGGACACCAGCAGAAAATAAACTGAACTTAACACTGGACTTCCACAGACTCCTAACAGTGGAGCTTCAGTTTAAACTGAAAGCCATTAATCTGCAGACAGTTCGTCATCAAGAACTCCCTGACTGTTATGACTTTACTCTGACTATAACATTTGACAACAAGGCCCATAGTGGAAGAATTAAAATAAGTTTAGATAATGACATTTCCATCAGAGAATGTAAAGACTGGCATGTATCTGGATCAATTCAGAAGAACACTCATTACATGATGATCTTTGATGCCTTTGTCATTCTGACTTGCTTGGTTTCATTAATCCTCTGCATTAGATCTGTGATTAGAGGACTTCAGCTTCAGCAGGAGTTTGTCAATTTTTTCCTCCTCCATTATAAGAAGGAAGTTTCTGTTTCTGATCAAATGGAATTTGTCAATGGATGGTACATTATGATTATTATTAGTGACATATTGACAATCATTGGATCAATTCTAAAAATGGAAATCCAAGCTAAGAGTCTAACTAGTTATGATGTCTGTAGCATACTTCTTGGGACTTCTACCATGCTCGTGTGGCTTGGAGTCATCCGATACCTCGGTTTCTTTGCAAAGTACAACCTCCTCATTTTGACCCTTCAGGCAGCGCTGCCCAATGTCATCAGGTTCTGCTGCTGTCCAGCTATGATTTACTTAGGTTACTGCTTCTGTGGATGGATCGTGCTGGGGCCTTACCATGACAAGTTTCGTTCTCTGAACATGGTCTCTGAGTGCCTTTTCTCTCTGATAAATGGAGATGATATGTTTGCCACGTTTGCAAAAATGCAGCAAAAAAGTTACTTAGTCTGGCTGTTTAGTAGAATTTACCTCTACTCATTCATCAGCCTCTTTATATATATGATTTTAAGTCTTTTCATTGCACTGATCACTGATACATACGAAACAATTAAGCAATACCAACAAGATGGCTTCCCAGAGACTGAACTTCGTACATTTATATCAGAATGCAAAGATCTACCCAACTCTGGAAAATACAGATTAGAAGATGACCCTCCAGTATCTTTATTCTGCTGTTGTAAAAAG(SEQ?ID?NO：3)

Homo sapiens TRPML3 A419P (SEQ ID NO:4)

MADPEVVVSSCSSHEEENRCNFNQQTSPSEELLLEDQMRRKLKFFFMNPCEKFWARGRKPWKLAIQILKIAMVTIQLVLFGLSNQMVVAFKEENTIAFKHLFLKGYMDRMDDTYAVYTQSDVYDQLIFAVNQYLQLYNVSVGNHAYENKGTKQSAMAICQHFYKRGNIYPGNDTFDIDPEIETECFFVEPDEPFHIGTPAENKLNLTLDFHRLLTVELQFKLKAINLQTVRHQELPDCYDFTLTITFDNKAHSGRIKISLDNDISIRECKDWHVSGSIQKNTHYMMIFDAFVILTCLVSLILCIRSVIRGLQLQQEFVNFFLLHYKKEVSVSDQMEFVNGWYIMIIISDILTIIGSILKMEIQAKSLTSYDVCSILLGTSTMLVWLGVIRYLGFFAKYNLLILTLQAALPNVIRFCCCPAMIYLGYCFCGWIVLGPYHDKFRSLNMVSECLFSLINGDDMFATFAKMQQKSYLVWLFSRIYLYSFISLFIYMILSLFIALITDTYETIKQYQQDGFPETELRTFISECKDLPNSGKYRLEDDPPVSLFCCCKK(SEQ?ID?NO：4)

Homo sapiens TRPML1 (NM_020533) (SEQ ID NO:5)

ATGACAGCCCCGGCGGGTCCGCGCGGCTCAGAGACCGAGCGGCTTCTGACCCCCAACCCCGGGTATGGGACCCAGGCGGGGCCTTCACCGGCCCCTCCGACACCCCCAGAAGAGGAAGACCTTCGCCGTCGTCTCAAATACTTTTTCATGAGTCCCTGCGACAAGTTTCGAGCCAAGGGCCGCAAGCCCTGCAAGCTGATGCTGCAAGTGGTCAAGATCCTGGTGGTCACGGTGCAGCTCATCCTGTTTGGGCTCAGTAATCAGCTGGCTGTGACATTCCGGGAAGAGAACACCATCGCCTTCCGACACCTCTTCCTGCTGGGCTACTCGGACGGAGCGGATGACACCTTCGCAGCCTACACGCGGGAGCAGCTGTACCAGGCCATCTTCCATGCTGTGGACCAGTACCTGGCGTTGCCTGACGTGTCACTGGGCCGGTATGCGTATGTCCGTGGTGGGGGTGACCCTTGGACCAATGGCTCAGGGCTTGCTCTCTGCCAGCGGTACTACCACCGAGGCCACGTGGACCCGGCCAACGACACATTTGACATTGATCCGATGGTGGTTACTGACTGCATCCAGGTGGATCCCCCCGAGCGGCCCCCTCCGCCCCCCAGCGACGATCTCACCCTCTTGGAAAGCAGCTCCAGTTACAAGAACCTCACGCTCAAATTCCACAAGCTGGTCAATGTCACCATCCACTTCCGGCTGAAGACCATTAACCTCCAGAGCCTCATCAATAATGAGATCCCGGACTGCTATACCTTCAGCGTCCTGATCACGTTTGACAACAAAGCACACAGTGGGCGGATCCCCATCAGCCTGGAGACCCAGGCCCACATCCAGGAGTGTAAGCACCCCAGTGTCTTCCAGCACGGAGACAACAGCTTCCGGCTCCTGTTTGACGTGGTGGTCATCCTCACCTGCTCCCTGTCCTTCCTCCTCTGCGCCCGCTCACTCCTTCGAGGCTTCCTGCTGCAGAACGAGTTTGTGGGGTTCATGTGGCGGCAGCGGGGACGGGTCATCAGCCTGTGGGAGCGGCTGGAATTTGTCAATGGCTGGTACATCCTGCTCGTCACCAGCGATGTGCTCACCATCTCGGGCACCATCATGAAGATCGGCATCGAGGCCAAGAACTTGGCGAGCTACGACGTCTGCAGCATCCTCCTGGGCACCTCGACGCTGCTGGTGTGGGTGGGCGTGATCCGCTACCTGACCTTCTTCCACAACTACAATATCCTCATCGCCACACTGCGGGTGGCCCTGCCCAGCGTCATGCGCTTCTGCTGCTGCGTGGCTGTCATCTACCTGGGCTACTGCTTCTGTGGCTGGATCGTGCTGGGGCCCTATCATGTGAAGTTCCGCTCACTCTCCATGGTGTCTGAGTGCCTGTTCTCGCTCATCAATGGGGACGACATGTTTGTGACGTTCGCCGCCATGCAGGCGCAGCAGGGCCGCAGCAGCCTGGTGTGGCTCTTCTCCCAGCTCTACCTTTACTCCTTCATCAGCCTCTTCATCTACATGGTGCTCAGCCTCTTCATCGCGCTCATCACCGGCGCCTACGACACCATCAAGCATCCCGGCGGCGCAGGCGCAGAGGAGAGCGAGCTGCAGGCCTACATCGCACAGTGCCAGGACAGCCCCACCTCCGGCAAGTTCCGCCGCGGGAGCGGCTCGGCCTGCAGCCTTCTCTGCTGCTGCGGAAGGGACCCCTCGGAGGAGCATTCGCTGCTGGTGAAT(SEQ?ID?NO：5)

Homo sapiens TRPML1 (NM_020533) (SEQ ID NO:6)

MTAPAGPRGSETERLLTPNPGYGTQAGPSPAPPTPPEEEDLRRRLKYFFMSPCDKFRAKGRKPCKLMLQVVKILVVTVQLILFGLSNQLAVTFREENTIAFRHLFLLGYSDGADDTFAAYTREQLYQAIFHAVDQYLALPDVSLGRYAYVRGGGDPWTNGSGLALCQRYYHRGHVDPANDTFDIDPMVVTDCIQVDPPERPPPPPSDDLTLLESSSSYKNLTLKFHKLVNVTIHFRLKTINLQSLINNEIPDCYTFSVLITFDNKAHSGRIPISLETQAHIQECKHPSVFQHGDNSFRLLFDVVVILTCSLSFLLCARSLLRGFLLQNEFVGFMWRQRGRVISLWERLEFVNGWYILLVTSDVLTISGTIMKIGIEAKNLASYDVCSILLGTSTLLVWVGVIRYLTFFHNYNILIATLRVALPSVMRFCCCVAVIYLGYCFCGWIVLGPYHVKFRSLSMVSECLFSLINGDDMFVTFAAMQAQQGRSSLVWLFSQLYLYSFISLFIYMVLSLFIALITGAYDTIKHPGGAGAEESELQAYIAQCQDSPTSGKFRRGSGSACSLLCCCGRDPSEEHSLLVN(SEQID?NO：6)

Homo sapiens TRPML2 (NM_153259) (SEQ ID NO:7)

ATGGCCCGGCAGCCTTATCGTTTTCCCCAGGCAAGGATTCCGGAGAGAGGATCAGGTGTTTTCAGGTTAACCGTCAGAAACGCAATGGCACATCGTGATTCTGAGATGAAAGAAGAATGTCTAAGGGAAGACCTGAAGTTTTACTTCATGAGCCCTTGTGAAAAATACCGAGCCAGACGCCAGATTCCGTGGAAACTGGGTTTGCAGATTTTGAAGATAGTCATGGTCACCACACAGCTTGTTCGTTTTGGTTTAAGTAACCAGCTGGTGGTTGCTTTCAAAGAAGATAACACTGTTGCTTTTAAGCACTTGTTTTTGAAAGGATATTCTGGTACAGATGAAGATGACTACAGCTGCAGTGTATATACTCAAGAGGATGCCTATGAGAGCATCTTTTTTGCTATTAATCAGTATCATCAGCTAAAGGACATTACCCTGGGGACCCTTGGTTATGGAGAAAATGAAGACAATAGAATTGGCTTAAAAGTCTGTAAGCAGCATTACAAGAAAGGGACCATGTTTCCTTCTAATGAGACACTGAATATTGACAACGACGTTGAGCTCGATTGTGTTCAATTAGACCTTCAGGACCTCTCCAAGAAGCCTCCGGACTGGAAGAACTCATCATTCTTCAGACTGGAATTTTATCGGCTCTTACAGGTTGAAATCTCCTTTCATCTTAAAGGCATTGACCTACAGACAATTCATTCCCGTGAGTTACCAGACTGTTATGTCTTTCAGAATACGATTATCTTTGACAATAAAGCTCACAGTGGCAAAATCAAAATCTATTTTGACAGTGATGCCAAAATTGAAGAATGTAAAGACTTGAACATATTTGGATCTACTCAGAAAAATGCTCAGTATGTCCTGGTGTTTGATGCATTTGTCATTGTGATTTGCTTGGCATCTCTTATTCTGTGTACAAGATCCATTGTTCTTGCTCTAAGGTTACGGAAGAGATTTCTAAATTTCTTCCTGGAGAAGTACAAGCGGCCTGTGTGTGACACCGACCAGTGGGAGTTCATCAACGGCTGGTATGTCCTGGTGATTATCAGCGACCTAATGACAATCATTGGCTCCATATTAAAAATGGAAATCAAAGCAAAGAATCTCACAAACTATGATCTCTGCAGCATTTTTCTTGGAACCTCTACGCTCTTGGTTTGGGTTGGAGTCATCAGATACCTGGGTTATTTCCAGGCATATAATGTGCTGATTTTAACAATGCAGGCCTCACTGCCAAAAGTTCTTCGGTTTTGTGCTTGTGCTGGTATGATTTATCTGGGTTACACATTCTGTGGCTGGATTGTCTTAGGACCATACCATGACAAGTTTGAAAATCTGAACACAGTTGCTGAGTGTCTGTTTTCTCTGGTCAACGGTGATGACATGTTTGCAACCTTTGCCCAAATCCAGCAGAAGAGCATCTTGGTGTGGCTGTTCAGTCGTCTGTATTTATATTCCTTCATCAGCCTTTTTATATATATGATTCTCAGTCTTTTTATTGCACTTATTACAGATTCTTATGACACCATTAAGAAATTCCAACAGAATGGGTTTCCTGAAACGGATTTGCAGGAATTCCTGAAGGAATGCAGTAGCAAAGAAGAGTATCAGAAAGAGTCCTCAGCCTTCCTGTCCTGCATCTGCTGTCGGAGGAGGAAAAGAAGTGATGATCACTTGATACCTATTAGC(SEQ?ID?NO：7)

Homo sapiens TRPML2 (NM_153259) (SEQ ID NO:8)

MARQPYRFPQARIPERGSGVFRLTVRNAMAHRDSEMKEECLREDLKFYFMSPCEKYRARRQIPWKLGLQILKIVMVTTQLVRFGLSNQLVVAFKEDNTVAFKHLFLKGYSGTDEDDYSCSVYTQEDAYESIFFAINQYHQLKDITLGTLGYGENEDNRIGLKVCKQHYKKGTMFPSNETLNIDNDVELDCVQLDLQDLSKKPPDWKNSSFFRLEFYRLLQVEISFHLKGIDLQTIHSRELPDCYVFQNTIIFDNKAHSGKIKIYFDSDAKIEECKDLNIFGSTQKNAQYVLVFDAFVIVICLASLILCTRSIVLALRLRKRFLNFFLEKYKRPVCDTDQWEFINGWYVLVIISDLMTIIGSILKMEIKAKNLTNYDLCSIFLGTSTLLVWVGVIRYLGYFQAYNVLILTMQASLPKVLRFCACAGMIYLGYTFCGWIVLGPYHDKFENLNTVAECLFSLVNGDDMFATFAQIQQKSILVWLFSRLYLYSFISLFIYMILSLFIALITDSYDTIKKFQQNGFPETDLQEFLKECSSKEEYQKESSAFLSCICCRRRKRSDDHLIPIS(SEQ?ID?NO：8)

House mouse TRPML3 (NM_134160) (SEQ ID NO:9)

ATGGCAAATCCCGAGGTGCTGGTTAGCAGCTGCAGAGCTCGCCAAGATGAAAGCCCCTGCACTTTCCACCCGAGCTCGTCCCCGTCAGAGCAGCTTCTCTTAGAAGACCAGATGAGGCGGAAACTCAAGTTCTTTTTTATGAATCCTTGTGAGAAGTTCTGGGCTCGGGGTAGGAAGCCATGGAAACTTGCCATACAGATTCTGAAAATCGCGATGGTGACTATCCAGCTGGTTCTGTTTGGACTAAGTAACCAGATGGTAGTAGCTTTCAAAGAGGAGAACACTATAGCCTTCAAACACCTCTTCCTAAAGGGCTACATGGATCGAATGGACGACACCTATGCAGTGTACACTCAGAGTGAAGTGTATGACCAGATCATCTTTGCAGTGACCCAGTACTTGCAGCTTCAGAACATCTCCGTGGGCAATCACGCTTATGAGAACAAGGGGACTAAGCAGTCGGCGATGGCAATCTGTCAGCACTTCTACAGGCAAGGAACCATCTGCCCCGGGAACGACACCTTTGACATCGATCCAGAAGTTGAAACAGAATGTTTCCTTGTAGAGCCAGATGAAGCTTCCCACCTTGGAACGCCTGGAGAAAATAAACTCAACCTGAGCCTGGACTTCCACAGACTTCTGACGGTGGAGCTCCAGTTTAAGCTCAAAGCCATCAATCTGCAGACAGTTCGACACCAGGAGCTTCCTGACTGTTACGACTTTACGCTGACTATAACATTCGACAACAAGGCTCACAGTGGAAGAATCAAAATAAGCTTAGACAACGACATTTCTATCAAAGAATGCAAAGACTGGCATGTGTCTGGATCAATTCAGAAGAACACACACTACATGATGATCTTTGATGCCTTTGTCATTCTGACCTGCTTGGCCTCACTGGTGCTGTGTGCCAGGTCTGTGATTAGGGGTCTTCAGCTTCAGCAGGAGTTTGTCAACTTCTTCCTTCTTCACTACAAGAAGGAAGTTTCGGCCTCTGATCAGATGGAGTTCATCAACGGGTGGTACATTATGATCATCATTAGTGACATATTGACAATCGTTGGATCAGTTCTGAAAATGGAAATCCAAGCCAAGAGTCTCACAAGCTATGATGTCTGCAGCATACTTCTCGGGACGTCAACTATGCTCGTGTGGCTTGGAGTTATCCGATACCTGGGTTTCTTTGCGAAGTACAATCTCCTTATTCTGACCCTCCAGGCAGCGCTGCCCAACGTCATGAGGTTCTGTTGCTGCGCTGCTATGATCTATCTAGGCTATTGCTTTTGCGGATGGATTGTGCTGGGCCCTTACCATGAGAAGTTCCGTTCCCTGAACAGGGTCTCCGAGTGCCTGTTCTCGCTGATAAACGGAGACGATATGTTTTCCACATTTGCGAAAATGCAGCAGAAGAGTTACCTGGTGTGGCTGTTCAGCCGAGTCTACCTGTACTCGTTCATCAGCCTCTTCATTTACATGATTCTGAGCCTTTTCATCGCGCTCATCACAGACACATACGAAACAATTAAGCACTACCAGCAAGATGGCTTCCCAGAGACGGAACTTCGAAAGTTTATAGCGGAATGCAAAGACCTCCCCAACTCCGGAAAATACAGATTAGAAGATGACCCTCCGGGTTCTTTACTCTGCTGCTGCAAAAAG(SEQ?ID?NO：9)

House mouse (mus musculus) TRPML3 (NM_134160) (SEQ ID NO:10)

MANPEVLVSSCRARQDESPCTFHPSSSPSEQLLLEDQMRRKLKFFFMNPCEKFWARGRKPWKLAIQILKIAMVTIQLVLFGLSNQMVVAFKEENTIAFKHLFLKGYMDRMDDTYAVYTQSEVYDQIIFAVTQYLQLQNISVGNHAYENKGTKQSAMAICQHFYRQGTICPGNDTFDIDPEVETECFLVEPDEASHLGTPGENKLNLSLDFHRLLTVELQFKLKAINLQTVRHQELPDCYDFTLTITFDNKAHSGRIKISLDNDISIKECKDWHVSGSIQKNTHYMMIFDAFVILTCLASLVLCARSVIRGLQLQQEFVNFFLLHYKKEVSASDQMEFINGWYIMIIISDILTIVGSVLKMEIQAKSLTSYDVCSILLGTSTMLVWLGVIRYLGFFAKYNLLILTLQAALPNVMRFCCCAAMIYLGYCFCGWIVLGPYHEKFRSLNRVSECLFSLINGDDMFSTFAKMQQKSYLVWLFSRVYLYSFISLFIYMILSLFIALITDTYETIKHYQQDGFPETELRKFIAECKDLPNSGKYRLEDDPPGSLLCCCKK(SEQ?ID?NO：10)

Jungle fowl (gallus gallus) TRPML3 (XM_426647) (SEQ ID NO:11)

ATGATCACCCGTGGTTTCCGTTCAGGCTTTTACGGCTGTAGCAATCGATATAGTGGTGCTAAGTGCCAGTTAGTTGCTCAGACGTTTGTCATTTCAGCAATGGAGACTCCTGAAGTGGCTGTAAGCAGCTGCAGTGCTCGGGATGACGAAGGGCTCTGCAGCTACGGACAACACCTATTGCTGCCACAAGAGCTGGTGGCGGAAGACCAGCTGAGGAGGAAGCTGAAGTTCTTCTTCATGAACCCATGTGAAAAATTCTGGGCTCGGGGCAGAAAACCTTGGAAACTTGGGATTCAGCTGCTCAAAATAGCAATGGTTACCATTCAGCTGGTGCTTTTTGGATTGAGCAATCAAATGGTGGTTGCTTTCAAAGAAGAGAACACTATTGCATTCAAACATCTCTTCTTGAAAGGGTACATGGACAGAATGGATGATACCTATGCGGTATACACACAGACAGATGTCTATGACCAAATATTCTTTGCCATCAATCAGTACTTACAGTTGCCCAACATTTCTGTTGGAAACCATGCTTATGAGAAGAAAGGAGCAGAAGAGACAGCTCTGGCTGTATGTCAACAGTTCTACAAGCAAGGAACCATCTGTCCTGGAAATGACACCTTTGATATAGACCCAGAGATTGTGACTGACTGCTTGTACATTGAGCCGATGATGTCTTTAGACAACAGAACAGTGGGAAAGCACAATTTGAATTTCACTCTGGATTTCCACAGGCTCGTGGCAGTGCAACTCATGTTCAATCTGAAGGCAATCAACCTCCAGACCGTCCGTCACCACGAGCTCCCTGACTGTTACGATTTCACCCTGACGATAGTGTTTGATAATAAAGCCCACAGTGGAAGAATCAAAATCAGTCTAGACAACGACATAGAGATCAGGGAATGTAAAGACTGGCACGTTTCTGGATCAATACAGAAGAATACGCATTACATGATGATCTTCGATGCTTTTGTCATACTGATCTGTCTGAGCTCATTGATCCTTTGCACTCGATCAGTAGTCAAAGGAATTCGGCTCCAAAGAGAATTTGTAAGTTTTTTCCTATATTATTACAAGAAAGAGGTATCTTACAATGATCAGATGGAATTTGTCAATGGCTGGTATATCCTCATTATGGTTAGTGATGTCCTCACTATCGTTGGATCAACTCTCAAAATGGAGATACAGGCCAAGAGTCTGACAAGTTACGACGTCTGTAGCATACTCTTAGGAACATCCACTATGCTGGTGTGGCTTGGAGTCATTCGCTACCTCGGTTTCTTTCAGAAGTATAATCTTCTCATTCTAACGCTGCGAGCAGCACTACCCAACGTCATGAGGTTCTGCTGTTGTGCTGCTATGATCTATCTAGGTTATTGTTTCTGCGGATGGATTGTACTGGGGCCATACCACGTGAAGTTCCGTTCTCTGAATGTGGTTTCTGAATGCCTCTTTTCATTGATAAATGGAGATGACATGTTTGCCACTTTTGCAAAAATGCAGCAGAAAAGTTACTTGGTTTGGTTATTCAGTAGAATCTACCTCTACTCCTTCATCAGCCTGTTCATCTACATGGTGCTAAGTCTCTTCATTGCACTCATTACAGATACATATGAAACTATCAAGCACTACCAACAAGATGGCTTTCCAGAGACAGAACTTCAGAGATTTATATCACAGTGCAAAGACTTACCAAACTCTGGAAGGTACAGATTAGAAGAGGAAGGTTCTGTATCTCTCTTCTGTTGTTGCAGTGGTCCTAGTGAACATATC(SEQ?ID?NO：11)

Jungle fowl TRPLM3 (XM_426647) (SEQ ID NO:12)

MITRGFRSGFYGCSNRYSGAKCQLVAQTFVISAMETPEVAVSSCSARDDEGLCSYGQHLLLPQELVAEDQLRRKLKFFFMNPCEKFWARGRKPWKLGIQLLKIAMVTIQLVLFGLSNQMVVAFKEENTIAFKHLFLKGYMDRMDDTYAVYTQTDVYDQIFFAINQYLQLPNISVGNHAYEKKGAEETALAVCQQFYKQGTICPGNDTFDIDPEIVTDCLYIEPMMSLDNRTVGKHNLNFTLDFHRLVAVQLMFNLKAINLQTVRHHELPDCYDFTLTIVFDNKAHSGRIKISLDNDIEIRECKDWHVSGSIQKNTHYMMIFDAFVILICLSSLILCTRSVVKGIRLQREFVSFFLYYYKKEVSYNDQMEFVNGWYILIMVSDVLTIVGSTLKMEIQAKSLTSYDVCSILLGTSTMLVWLGVIRYLGFFQKYNLLILTLRAALPNVMRFCCCAAMIYLGYCFCGWIVLGPYHVKFRSLNVVSECLFSLINGDDMFATFAKMQQKSYLVWLFSRIYLYSFISLFIYMVLSLFIALITDTYETIKHYQQDGFPETELQRFISQCKDLPNSGRYRLEEEGSVSLFCCCSGPSEHI(SEQ?ID?NO：12)

Domesticated dog (canis familiaris) TRPML3 (XM_547306) (SEQ ID NO:13)

ATGACCCCTTTTGGCAGCTTGGCTTCTGCAAAGGCTTCAAACTCAAGAGCTGCCTGGAAGATTGTGGTGGATTCATTCAGCTCGCAGCACACGCCCCAGGTGGGCACTGGTGGTCCCCAGGAATCAGACAAGGCCCTTACCTTCGGAGAGTTAACGTTCTCCCACTCATCTCCATTCTTCATCTGCGCCAGCCCTTCTCTCCCACCGTTGCACAGCAGTGGGCTAGACGATGAGCCATACTGCTGGACAGGTTTTCACTGCATCAAGTACCTCGCGGGCCCAGCGAGTGTCCCAAACTCCCTTGAAAGAGGGAGTAAGATATTGGTTTCCCAAGCCTCCTTTCCCATCCGGACCTCCCCTTACCTGACACTGCTAAGCCGAGGGGAAAAGAAGCCTCTCTGCAGTTCCGTGGAGAAAAGGCCTTTGGGGGTTTTGGAGATGGGAAGTCTGACTCTCCTCTCGGAAGAGCTCAAACGGCAGCTCCCCGGCACGCTGTTGCTAGGACAACCTCCGTTGCTAAGGGAAAGAGGCGGCTCCTCTGCTGAGATTGACAAGACGCCGCTACCAATAGGGCGTCTACTCTCCGGCCGCCTCTTCAAGGCCGCACTTGTGATTGGCTGCTGTCGTGCTGACGTCACGCAACTCGAACCGCCGAGAGATCCTGGGTTTTCGCCCGCTCGGCAGGAGGTGTGCGGTTTGGGTTCCCGCGTGGAGGGTGCTGCTGGCTCGAATGTAAACAATCTTTTTTTTTTTCTCCCCCTAGAGATGGCAAATCCTACGGTTGTTATAAGTAGCTGCAGCTCTCATGAAGAGGAAAATCGTTGCACTTTTAGCCAGCACACATCGCCCTCTGAGGAGCTTCTGTTAGAAGACCAGATAAGGCGAAAACTCAAATTTTTTTTCATGAATCCTTGTGAAAAGTTCTGGGCTCGAGGTAGAAAACCATGGAAGCTTGCCATACAAATTCTAAAAATTGCAATGGTGACTATCCAGCTGGTCTTTTTTGGGCTAAGTAACCAGATGGTTGTAGCTTTCAAGGAAGAAAACACTATAGCATTCAAACACCTCTTCTTAAAAGGATATATGGACCGAATGGATGACACATATGCAGTGTACACACAACGTGATGTATATGATCAGATCATCTTTGCAGTGAACCAGTACTTGCTTCTACGCAATACCTCGGTTGGGAATCATGCTTATGAGAACAAGGGGACGGAACAGTCTGCTATGGCAATCTGTCAGCACTTCTACAAGCAGGGAAACATCTGTCCTGGAAATGATACCTTCGACATTGATCCAGAGATTGAAACTGAGTGTTTCTCTGTAGAGCCAGCTGAGCCTTTCCACGTCGGAACACTGGAAGAAAATAAACTCAACTTAACGCTGGACTTTCACAGACTCCTCACGGTGGACCTGCAGTTTAAGCTGAAGGCCATTAATCTGCAGACCATTCGGCATCACGAGCTCCCTGACTGTTATGACTTTACTCTCACTATAACATTTGACAATAAGGCCCATAGTGGAAGAATTAAGATAAGTTTAGATAATGATATTTCCATCAGAGAATGTAAAGACTGGCATGTATCGGGATCAATTCAGAAGAACACTCACTACATGATGATCTTTGATGCCTTTGTTATTCTGACATGCTTGGCTTCACTAACCCTGTGCCTTCGATCTGTAATTAGAGGACTTCAGCTTCAACAGGAATTTGTCAATTTTTTCCTCCTCCATTATAAGAAGGAAGTTTCTGTTTCTGATCGAATGGAATTTGTCAATGGATGGTACATTATGATTATTATTAGTGACATGTTGACAATTATTGGATCAATTCTGAAAATGGAAATTCAAGCTAAGAGTCTAACAAGTTATGATGTTTGTAGCATACTTCTTGGGACTTCCACCATGCTTGTGTGGCTTGGAGTTATTCGATACCTCGGCTTCTTTCAGAAGTACAATCTCCTTATTCTGACCCTGCAGGCAGCACTGCCCAGTGTCATCAGGTTCTGTTGCTGTGCCGCTATGATTTATTTAGGCTATTGCTTCTGTGGATGGATTGTGCTGGGGCCGTACCATGATAAGTTCCGTTCTCTGAACATGGTCTCTGAGTGCCTTTTCTCTCTGATAAATGGAGATGATATGTTTGCCACATTTGCAAAAATGCAACAAAAAAGTTACTTGGTCTGGCTGTTTAGCAGAATTTATCTCTACTCATTCATCAGCCTCTTTATATATATGATTTTAAGTCTTTTCATCGCACTGATCACTGATACGTATGAAACAATTAAGCATTACCAACAAGATGGCTTTCCAGAGACTGAACTTCGTACATTTATATCAGAGTGCAAAGATCTACCCAATTCTGGAAAATACAGATTAGAAGATGACACTCCAATATCTATATTCTGCTGTTGTAAAAAG(SEQ?IDNO：13)

Domesticated dog TRPML3 (XM_547306) (SEQ ID NO:14)

MTPFGSLASAKASNSRAAWKIVVDSFSSQHTPQVGTGGPQESDKALTFGELTFSHSSPFFICASPSLPPLHSSGLDDEPYCWTGFHCIKYLAGPASVPNSLERGSKILVSQASFPIRTSPYLTLLSRGEKKPLCSSVEKRPLGVLEMGSLTLLSEELKRQLPGTLLLGQPPLLRERGGSSAEIDKTPLPIGRLLSGRLFKAALVIGCCRADVTQLEPPRDPGFSPARQEVCGLGSRVEGAAGSNVNNLFFFLPLEMANPTVVISSCSSHEEENRCTFSQHTSPSEELLLEDQIRRKLKFFFMNPCEKFWARGRKPWKLAIQILKIAMVTIQLVFFGLSNQMVVAFKEENTIAFKHLFLKGYMDRMDDTYAVYTQRDVYDQIIFAVNQYLLLRNTSVGNHAYENKGTEQSAMAICQHFYKQGNICPGNDTFDIDPEIETECFSVEPAEPFHVGTLEENKLNLTLDFHRLLTVDLQFKLKAINLQTIRHHELPDCYDFTLTITFDNKAHSGRIKISLDNDISIRECKDWHVSGSIQKNTHYMMIFDAFVILTCLASLTLCLRSVIRGLQLQQEFVNFFLLHYKKEVSVSDRMEFVNGWYIMIIISDMLTIIGSILKMEIQAKSLTSYDVCSILLGTSTMLVWLGVIRYLGFFQKYNLLILTLQAALPSVIRFCCCAAMIYLGYCFCGWIVLGPYHDKFRSLNMVSECLFSLINGDDMFATFAKMQQKSYLVWLFSRIYLYSFISLFIYMILSLFIALITDTYETIKHYQQDGFPETELRTFISECKDLPNSGKYRLEDDTPISIFCCCKK(SEQ?ID?NO：14)

Zebra fish (danio rerio) TRPML3 (XM_688418) (SEQ ID NO:15)

ATGTCTGACCGAGCGTCACACACTCATGAAAGCGCAACACTTCTGGACCCGGAGTGTGTGGAAAGCTTAAGGAGAAAACTCAAGTATTTCTTCATGAGTCCGTGTCAGAAATACAGCACTAGAGGACGGATACCATGGAAGATGATGCTTCAGATACTCAAGATTTGTTTAGTATTCATCTACCTGGTCTCTTTTGGATTGAGCAACGAGATGATGGTGACGTTCAAAGAGGAAAATCTCATCGCCTTCAAGCACTTCTTTCTGAAAAACTACAAGGACAGCAATAAACATTACGCCTTGTACACAAAACATGAAGTTCACGACCACATCCTCTACACCATCAGACGGTATCTACAGCTACAAAACCTGACGATTGGCAATCAAGCGCTGGAGATGATCGATGGTCTGGCGACTCCTCTGTCTCTCTGTCAGCAGTTGTATCGACATGCGCGCGTCGTGCCGTCTAATGAGACGTTTGAAATCGATCCACATGTAGAGACAGAGTGTGTTTCTGTGTATCCCCTTTCTCCCATCACGACTGACAGTCTGGAAAACTCCCTGAACTTGACTTTAGATTTTCAAAGGTTGTTAGCGGTAAACATTTATCTGAAGATCAAGGCTATCAACATTCAGACGGTTCGCCATCAAGAGTTACCAGACTGCTACGACTTCAGCATTAATATCATGTTTGACAATCGTGCACACAGCGGACAGATCAAGATCTCTCTCAGCAGCGGCGTGCAGATAAACGTCTGTAAGGACTGGAACATTTCTGGCTCAAGTAAGTTGAACAGCCACTTTGCGCTGATTGTGGTGTTTGACTGTTTGATCATCTGCTTCTGTCTGCTGTCACTCATCCTCTGCACGCGCTCAGTCCACACAGGATTTCTCCTACAGACTGAATACAGAAGATTCATGTCCAGTCAGCACAGTAAAAGCGTCTCATGGTCTGAGAGGCTGGAGTTCATCAACGGCTGGTACATCCTCATCATCATCAGCGATGCGCTGACTATTGCAGGCTCAATCCTCAAAATCTGCATACAGAGCAAAGAACTGACGAGCTATGACGTGTGCAGTATTCTGCTGGGCACTGCAACAATGCTGGTGTGGATTGGAGTAATGCGCTACCTCAGTTTCTTCCAGAAATATTATATCCTCATCCTCACCCTGAAGGCTGCACTTCCCAATGTGATTCGATTCTCCATCTGCGCTGTTATGATCTACCTGAGTTACTGCTTCTGCGGATGGATCGTTTTGGGGCCACACCATGAAAATTTTCGCACATTCAGTAGGGTTGCTGGCTGTCTTTTCTCCATGATTAATGGGGATGAAATCTACTCCACGTTCACCAAGCTCCGGGAATACAGCACTCTGGTGTGGCTGTTCAGCAGACTCTACGTCTACAGCTTCATCCCGGTCTTCACATACATGGTTCTGAGTGTCTTCATCGCCCTCATCACAGACACGTATGAAACCATCAGGGTGAGTTATTTCAGCTTCAGTGAGAGTAGCTGCAAA(SEQ?ID?NO：15)

Zebra fish TRPML3 (XM_688418) (SEQ ID NO:16)

MSDRASHTHESATLLDPECVESLRRKLKYFFMSPCQKYSTRGRIPWKMMLQILKICLVFIYLVSFGLSNEMMVTFKEENLIAFKHFFLKNYKDSNKHYALYTKHEVHDHILYTIRRYLQLQNLTIGNQALEMIDGLATPLSLCQQLYRHARVVPSNETFEIDPHVETECVSVYPLSPITTDSLENSLNLTLDFQRLLAVNIYLKIKAINIQTVRHQELPDCYDFSINIMFDNRAHSGQIKISLSSGVQINVCKDWNISGSSKLNSHFALIVVFDCLIICFCLLSLILCTRSVHTGFLLQTEYRRFMSSQHSKSVSWSERLEFINGWYILIIISDALTIAGSILKICIQSKELTSYDVCSILLGTATMLVWIGVMRYLSFFQKYYILILTLKAALPNVIRFSICAVMIYLSYCFCGWIVLGPHHENFRTFSRVAGCLFSMINGDEIYSTFTKLREYSTLVWLFSRLYVYSFIPVFTYMVLSVFIALITDTYETIRVSYFSFSESSCK(SEQ?ID?NO：16)

Homo sapiens TRPML3 codon optimized (SEQ ID NO:17)

ATGGCCGACCCTGAGGTGGTGGTGTCCTCCTGCTCTAGCCACGAGGAAGAGAACCGGTGCAACTTCAACCAGCAGACCAGCCCCAGCGAGGAACTGCTGCTGGAAGATCAGATGCGGCGGAAGCTGAAGTTCTTCTTCATGAACCCCTGCGAGAAGTTCTGGGCCAGAGGCCGGAAGCCTTGGAAGCTGGCCATCCAGATCCTGAAGATCGCCATGGTGACCATCCAGCTGGTGCTGTTCGGCCTGAGCAACCAGATGGTGGTGGCCTTCAAAGAGGAAAACACAATCGCCTTCAAGCACCTGTTTCTGAAGGGCTACATGGACCGGATGGACGACACCTACGCCGTGTACACCCAGAGCGACGTGTACGACCAGCTGATCTTCGCCGTGAACCAGTACCTGCAGCTGTACAACGTGAGCGTGGGCAACCACGCCTACGAGAACAAGGGCACCAAGCAGAGCGCCATGGCCATCTGCCAGCACTTCTACAAGCGGGGCAACATCTACCCCGGCAACGACACCTTCGACATCGACCCCGAGATCGAGACAGAGTGCTTCTTCGTGGAGCCCGACGAGCCTTTCCACATCGGCACCCCTGCCGAGAACAAGCTGAACCTGACCCTGGACTTCCACCGGCTGCTGACCGTGGAGCTGCAGTTCAAGCTGAAGGCCATCAACCTGCAGACCGTGCGGCACCAGGAACTGCCCGACTGCTACGACTTCACCCTGACCATCACCTTCGATAACAAGGCCCACAGCGGCCGGATCAAGATCAGCCTGGACAACGACATCAGCATCCGGGAGTGCAAGGACTGGCACGTGAGCGGCAGCATCCAGAAAAACACCCACTACATGATGATCTTCGACGCCTTCGTGATCCTGACCTGCCTGGTGTCCCTGATCCTGTGCATCAGAAGCGTCATCAGGGGCCTGCAGCTCCAGCAGGAATTCGTCAACTTCTTCCTGCTGCACTACAAGAAAGAAGTGTCCGTCAGCGACCAGATGGAATTTGTGAACGGCTGGTACATCATGATCATCATCAGCGACATCCTGACAATCATCGGCAGCATTCTGAAGATGGAAATCCAGGCCAAGAGCCTGACCAGCTACGACGTGTGCAGCATTCTGCTGGGAACCTCCACCATGCTGGTCTGGCTCGGCGTGATCAGATACCTGGGCTTCTTCGCCAAGTACAACCTGCTGATTCTGACACTGCAGGCCGCCCTGCCCAACGTGATCCGGTTCTGCTGCTGCGCCGCCATGATCTACCTGGGCTACTGCTTCTGCGGCTGGATCGTGCTGGGCCCCTACCACGACAAGTTCCGGTCCCTGAACATGGTGTCCGAGTGCCTGTTCAGCCTGATCAACGGCGACGACATGTTCGCCACCTTCGCCAAGATGCAGCAGAAAAGCTACCTGGTCTGGCTGTTCAGCCGGATCTACCTGTACAGCTTCATCAGCCTGTTCATCTACATGATCCTGAGCCTGTTTATCGCCCTGATCACCGATACCTACGAGACAATCAAGCAGTACCAGCAGGACGGCTTCCCCGAGACAGAGCTGCGGACCTTCATCAGCGAGTGTAAGGACCTGCCCAACAGCGGCAAGTACCGGCTGGAAGATGACCCCCCCGTGTCCCTGTTCTGCTGTTGCAAGAAG(SEQ?ID?NO：17)

Homo sapiens TRPML3 CDS variant C1149T silent mutation (SEQ ID NO:18)

ATGGCAGATCCTGAGGTAGTTGTGAGTAGCTGCAGCTCTCATGAAGAGGAAAATCGCTGCAATTTTAACCAGCAAACATCTCCATCTGAGGAGCTTCTATTAGAAGACCAGATGAGGCGAAAACTCAAATTTTTTTTCATGAATCCCTGTGAGAAGTTCTGGGCTCGAGGTAGAAAACCATGGAAACTTGCCATACAAATTCTAAAAATTGCAATGGTGACTATCCAGCTGGTCTTATTTGGGCTAAGTAACCAGATGGTGGTAGCTTTCAAGGAAGAGAATACTATAGCATTCAAACACCTTTTCCTAAAAGGATATATGGACCGAATGGATGACACATATGCAGTGTACACACAAAGTGACGTGTATGATCAGTTAATCTTCGCAGTAAACCAGTACTTGCAGCTATACAATGTCTCCGTTGGGAATCATGCTTATGAGAACAAAGGTACCAAGCAATCTGCTATGGCAATCTGTCAGCACTTCTACAAGCGAGGAAACATCTACCCTGGAAATGATACCTTTGACATCGATCCAGAAATTGAAACTGAGTGTTTCTTTGTGGAGCCAGATGAACCTTTTCACATTGGGACACCAGCAGAAAATAAACTGAACTTAACACTGGACTTCCACAGACTCCTAACAGTGGAGCTTCAGTTTAAACTGAAAGCCATTAATCTGCAGACAGTTCGTCATCAAGAACTCCCTGACTGTTATGACTTTACTCTGACTATAACATTTGACAACAAGGCCCATAGTGGAAGAATTAAAATAAGTTTAGATAATGACATTTCCATCAGAGAATGTAAAGACTGGCATGTATCTGGATCAATTCAGAAGAACACTCATTACATGATGATCTTTGATGCCTTTGTCATTCTGACTTGCTTGGTTTCATTAATCCTCTGCATTAGATCTGTGATTAGAGGACTTCAGCTTCAGCAGGAGTTTGTCAATTTTTTCCTCCTCCATTATAAGAAGGAAGTTTCTGTTTCTGATCAAATGGAATTTGTCAATGGATGGTACATTATGATTATTATTAGTGACATATTGACAATCATTGGATCAATTCTAAAAATGGAAATCCAAGCTAAGAGTCTAACTAGTTATGATGTCTGTAGCATACTTCTTGGGACTTCTACCATGCTTGTGTGGCTTGGAGTCATCCGATACCTCGGTTTCTTTGCAAAGTACAACCTCCTCATTTTGACCCTTCAGGCAGCGCTGCCCAATGTCATCAGGTTCTGCTGCTGTGCAGCTATGATTTACTTAGGTTACTGCTTCTGTGGATGGATCGTGCTGGGGCCTTACCATGACAAGTTTCGTTCTCTGAACATGGTCTCTGAGTGCCTTTTCTCTCTGATAAATGGAGATGATATGTTTGCCACGTTTGCAAAAATGCAGCAAAAAAGTTACTTAGTCTGGCTGTTTAGTAGAATTTACCTCTACTCATTCATCAGCCTCTTTATATATATGATTTTAAGTCTTTTCATTGCACTGATCACTGATACATACGAAACAATTAAGCAATACCAACAAGATGGCTTCCCAGAGACTGAACTTCGTACATTTATATCAGAATGCAAAGATCTACCCAACTCTGGAAAATACAGATTAGAAGATGACCCTCCAGTATCTTTATTCTGCTGTTGTAAAAAG(SEQ?ID?NO：18)

Brown rat (rattus norevigus) is (GI:58865683) (SEQ ID NO:19) (NM_00102059.1)

gttcgacaga?agcttgtgat?ttatggtcca?aggaatccag?tgtcagatca?atagacaaaa

61?tgccccaggg?aagttgtgtg?tgcattctac?tggacagatc?agagactggt?cagaacaggt

121?gcttggctgg?cggtgcgtcc?aaacctcaga?gatggcaaat?cctgaggtgg?tggtaagcag

181?ctgcagttct?caccaggatg?aaagtccctg?cactttctac?ccgagctcat?cccagtccga

241?gcagcttctc?ttagaagatc?agatgaggcg?gaaactcaaa?ttctttttta?tgaatccttg

301?cgagaagttc?tgggctcggg?gtaggaagcc?atggaaactt?gccatacaga?ttctgaaaat

361?cgctatggtg?actatccagc?tggttctgtt?tggactaagt?aaccagatgg?tagtagcttt

421?caaggaagag?aacacgatag?ccttcaaaca?cctcttcctg?aaaggctaca?tggaccgaat

481?ggacgacacc?tacgcggtgt?acactcagaa?tgatgtgtac?gaccagatca?tctttgcagt

541?gacccggtac?ttgcaacttc?gaaacatctc?cgtcggcaac?catgcttatg?agaacaaggg

601?gactaagcag?tcagcaatgg?cagtctgtca?gcacttctac?aggcaaggca?ccatctgccc

661?cgggaacgat?accttcgaca?tcgatccaga?agtcgaaaca?gactgtttcc?ttatagagcc

721?agaggaagct?ttccacatgg?gaacacctgg?agaaaacaaa?ctcaacctga?ccctggactt

781?ccacagactt?ctgacagtgg?agctccaatt?taagctcaaa?gccatcaacc?tgcagacagt

841?tcgccaccag?gagcttcctg?actgttacga?ctttaccctg?actataacat?tcgacaacaa

901?ggcacacagt?ggaagaatca?aaataagttt?agacaacgac?atttctatca?gagaatgcaa

961?agattggcac?gtgtctggat?caattcagaa?gaacacccac?tacatgatga?tcttcgatgc

1021?ctttgttatc?ctgacctgct?tgtcctcgct?ggtgctctgc?gccaggtctg?tgattcgggg

1081?tcttcagctt?cagcaggagt?ttgtcaactt?tttccttctt?cactacaaga?aggaagtttc

1141?ggcctctgat?cagatggagt?tcatcaacgg?gtggtacatt?atgatcatcg?ttagtgacat

1201?actgacgatc?gttggatcga?ttctgaaaat?ggaaatccaa?gccaagagtc?ttacaagcta

1261?cgatgtctgt?agcatacttc?ttgggacttc?caccatgctc?gtgtggcttg?gcgttatccg

1321?atacctgggt?ttctttgcga?agtacaatct?ccttatcctg?accctccagg?cagcgctgcc

1381?caatgtcatc?aggttctgtt?gctgtgcggc?tatgatctat?cttgggtatt?gcttttgcgg

1441?atggattgtg?ctgggccctt?accatgagaa?gttccgctct?ctgaacaagg?tctctgagtg

1501?cctattctca?ctgataaatg?gagacgacat?gttttccacg?ttcgcgaaaa?tgcagcagaa

1561?aagttacctg?gtgtggctgt?tcagccgcgt?ctacctgtac?tcgttcatca?gcctcttcat

1621?ctacatgatc?ttgagccttt?tcatcgcgct?catcacagac?acgtacgaaa?ccattaagca

1681?ctaccagcaa?gatggcttcc?cagagacgga?acttcgaaag?tttatcgctg?aatgcaaaga

1741?cctccccaac?tctggaaaat?acagattgga?agatgaccct?ccaggttctt?tattctgctg

1801?ctgcaaaaag?taactgtcgg?gtttctctgt?gcctgggagg?aaaatacagt?gtgtggatga

1861?gtcagagaca?atatggatta?ttggtaatca?cgcaacagtg?tgttcagata?ctagtgttct

1921?gagttaactc?acagctatga?ctttgcgggg?cctgttaaat?atatttttaa?atattaaaaa

1981?aaaaaaaaaa?aaaaaaaaaa

Brown rat (NP_00102059.1) is (SEQ ID NO:20) (GI:58865684)

MANPEVVVSSCSSHQDESPCTFYPSSSQSEQLLLEDQMRRKLKFFFMNPCEKFWARGRKPWKLAIQILKIAMVTIQLVLFGLSNQMVVAFKEENTIAFKHLFLKGYMDRMDDTYAVYTQNDVYDQIIFAVTRYLQLRNISVGNHAYENKGTKQSAMAVCQHFYRQGTICPGNDTFDIDPEVETDCFLIEPEEAFHMGTPGENKLNLTLDFHRLLTVELQFKLKAINLQTVRHQELPDCYDFTLTITFDNKAHSGRIKISLDNDISIRECKDWHVSGSIQKNTHYMMIFDAFVILTCLSSLVLCARSVIRGLQLQQEFVNFFLLHYKKEVSASDQMEFINGWYIMIIVSDILTIVGSILKMEIQAKSLTSYDVCSILLGTSTMLVWLGVIRYLGFFAKYNLLILTLQAALPNVIRFCCCAAMIYLGYCFCGWIVLGPYHEKFRSLNKVSECLFSLINGDDMFSTFAKMQQKSYLVWLFSRVYLYSFISLFIYMILSLFIALITDTYETIKHYQQDGFPETELRKFIAECKDLPNSGKYRLEDDPPGSLFCCCKK

Wild boar (sus scrofa) is (GI:146741299) (SEQ ID NO:21) (part) (AB271930)

1?gtgaaatggc?agatcctgag?cctgtcataa?gtagctgcag?ctctcgtgaa?gaggaaaatc

61?gctgcacttt?taaccagcac?acatgtccct?ctgaggagcg?tctattagaa?gaccagatga

121?ggcgaaaact?caaatttttt?ttcatgactc?cttgtgagaa?gttctggact?cgaggtcgaa

181?aaccatggaa?acttgccatg?caagttctaa?aaattgcgat?ggtgactatc?cagctgatct

241?ttttcgggct?aagtaaccag?atggtggtag?ctttcaagga?agagaacacg?atagcattta

301?aacacctctt?tctaaagggc?tatgtggacc?agatggatga?cacatatgcc?gtgtacaccc

361?aaagcgacgt?atacgatcgg?atcgtcttcg?cagtgaacca?gtacttgcag?ctacgcagca

421?tctcggttgg?gaaccacgct?tacgagaaca?agggcgcgga?gcagtcggcc?atggcgatct

481?gttggcactt?ctacaagcaa?ggaaacatct?gtcctggaaa?tgacaccttt?gacgttgatc

541?cagaagtaaa?aactgaatgt?ttctttgttg?agccggatga?agctgttgac?actggaacac

601?tggaggagaa?taagctcaac?ttaacccttg?actttcacag?actcctaacg?gtggagctgc

661?agtttaaact?caaggccatt?aatctgcaga?cgattcgcca?tcacgaactc?cctgactgtt

721?atgacttcac?cctgaccata?acatttgaca?a

Wild boar (BAF62305.1) is (SEQ ID NO:22) (part) (GI:14671300)

MADPEPVISSCSSREEENRCTFNQHTCPSEERLLEDQMRRKLKFFFMTPCEKFWTRGRKPWKLAMQVLKIAMVTIQLIFFGLSNQMVVAFKEENTIAFKHLFLKGYVDQMDDTYAVYTQSDVYDRIVFAVNQYLQLRSISVGNHAYENKGAEQSAMAICWHFYKQGNICPGNDTFDVDPEVKTECFFVEPDEAVDTGTLEENKLNLTLDFHRLLTVELQFKLKAINLQTIRHHELPDCYDFTLTITFD

Chimpanzee (pan troglytes) TRPML3 (XM_001140555.1) is (SEQ ID NO:23) (GI:114557485)

1?cttactccaa?tcaagcctct?gcccgccagg?aataggtaac?ctgtgtgtgt?ccgtttgctc

61?cttctaagag?catgcctgat?agatacttcg?gtagcctctc?cggatggccc?cttcgtcggg

121?tagcctctcc?tgatggggtc?cttcgcccac?cctgcctccc?gcgccggcgc?tccgggtgaa

181?tgtcaagggt?ggctggctgc?gaatacctcc?ttcagctgct?ggggttcccg?acagtttgca

241?gtttttaaaa?gtgcaccctc?ggaagggctt?ttcagactgg?gtaaagctga?cttttccaag

301?agatggcaga?tcctgaggta?gttgtgagta?gctgcagctc?tcatgaagag?gaaaatcgct

361?gcaattttaa?ccagcaaaca?tctccatctg?aggagcttct?attagaagac?cagatgaggc

421?gaaaactcaa?attttttttc?atgaatccct?gtgagaagtt?ctgggctcga?ggtagaaaac

481?catggaaact?tgccatacaa?attctaaaaa?ttgcaatggt?gactatccag?ctggtcttat

541?ttgggctaag?taaccagatg?gtggtagctt?tcaaggaaga?gaatactata?gcattcaaac

601?accttttcct?aaaaggatat?atggaccgaa?tggatgacac?atatgcagtg?tacacacaaa

661?gtgacgtgta?tgatcagtta?atcttcgcag?taaaccagta?cttgcagcta?tacaatgtct

721?ccgttgggaa?tcatgcttat?gagaacaaag?gtaccaagca?atctgctatg?gcaatctgtc

781?agcacttcta?caagcgagga?aacatctacc?ctggaaatga?tacctttgac?atcgatccag

841?aaattgaaac?tgagtgtttc?tttgtggagc?cagatgaacc?ttttcacatt?gggacaccag

901?cagaaaataa?actgaactta?acactggact?tccacagact?cctaacagtg?gagcttcagt

961?ttaaactgaa?agccattaat?ctgcagacag?ttcgtcatca?agaactccct?gactgttatg

1021?actttactct?gactataaca?tttgacaaca?aggcccatag?tggaagaatt?aaaataagtt

1081?tagataatga?catttccatc?agagaatgta?aagactggca?tgtatctgga?tcaattcaga

1141?agaacactca?ttacatgatg?atctttgatg?cctttgtcat?tctgacttgc?ttggtttcat

1201?taatcctctg?cattagatct?gtgattagag?gacttcagct?tcagcaggta?gggaacgttg

1261?ctttctagga?atgctactga?cattttgatt?gacagagaca?ttcactgtgc?ctcccctctt

1321?ttccctaaag?gagtttgtca?attttttcct?cctccattat?aagaagggag?tttctgtttc

1381?tgatcaaatg?gaatttgtca?atggatggta?cattatgatt?attattagtg?acatattgac

1441?aatcattgga?tcaattctaa?aaatggaaat?ccaagctaag?gtaatttttt?tcctaatcat

1501?gctattgtta?gtgtcagatt?tgcactaatg?gtaatgtatt?tttccagaat?gtaagaattt

1561?tcagaatgaa?ttgtttcttc?caaactgtat?atcaagtaga?cttgaaattg?gtaatggtaa

1621?ttttcttaaa?tctagtcagg?aggtctctta?ggcagagttt?ttcaaagtgt?gatccacaaa

1681?ccattgcatc?agaatcattg?ggtgcctggt?aaagtgtacc?atgttagacc?tactgaattc

1741?agactcttcg?gcggggcctg?tgaattctta?cacacaccaa?aattcataca?caaccaaggt

1801?aactaaggta?agagtttttt?ttttttttaa?atcttacaag?aaatgctcaa?atctttaaca

1861?aaaatgagtg?ggtctatagg?ggaaagtgag?gtcaaggcac?tatggtgtgc?atgcttgcat

1921?ttgtttcctc?cgtccattca?aagtgagaat?gctcccattt?tcttacttta?ccattgatgt

1981?gctacaagct?tatttatttt?aagactaacc?tagcctaaaa?atcaactgtc?cccacaaaat

2041?aaaaatcaca?ttaaaaaaac?taatagtgtt?cagactaatc?ttgctcaaac?ttatgtttcc

2101?ctagtcttga?tgcgactgat?tgagtcacct?ggggagttgg?ttataaacct?gggcagagac

2161?cccaaatgca?atggctcaga?gaagatagga?gcttatttct?gtcttatgca?atagtcagaa

2221?tgggttttac?agactggtga?gtagctcaac?atctcacagt?cattcaggca?cccatgttcc

2281?tcccattttg?tttctctgcc?atcccttaag?gacttgccct?gactgcatga?ttattgctgt

2341?gttgcctcaa?acaggttgca?gcttatggga?agcaaaaaca?cggtatggtg?gaagctctcc

2401?catagactga?tggcttggct?caagagtggc?cgactttatt?tctgtacata?tcccactgga

2461?tagaatttag?tcaatcctaa?ctgcagaggg?agccagggaa?cacagcccag?gcatgtgcct

2521?aggaagggga?gaatgggttt?aggttgacac?ttagcagctg?ccactatatg?tggctatagt

2581?atgtatcatt?ggaatagatg?tttaacttta?gggacaaata?aacaaaaaac?caaaacaaaa

2641?aaaggagtaa?ggggagagat?ttgcagcaaa?tctttatttt?taccaacctc?aactatcatt

2701?aatttcagtg?aaccctaaat?ggtgtccaac?aaaatatctt?tctagaccat?tcaccgtctc

2761?tgcctcatag?atgatcatat?catgttttct?tctcttctga?aacctctaat?acccttgtcc

2821?tatcctcatt?ctaagctgat?gaccttactt?cctatttcac?aaaaataata?gaaaaaaaaa

Chimpanzee TRPML3 (XP_001140555.1) is (SEQ ID NO:24) (GI:469371)

MADPEVVVSSCSSHEEENRCNFNQQTSPSEELLLEDQMRRKLKFFFMNPCEKFWARGRKPWKLAIQILKIAMVTIQLVLFGLSNQMVVAFKEENTIAFKHLFLKGYMDRMDDTYAVYTQSDVYDQLIFAVNQYLQLYNVSVGNHAYENKGTKQSAMAICQHFYKRGNIYPGNDTFDIDPEIETECFFVEPDEPFHIGTPAENKLNLTLDFHRLLTVELQFKLKAINLQTVRHQELPDCYDFTLTITFDNKAHSGRIKISLDNDISIRECKDWHVSGSIQKNTHYMMIFDAFVILTCLVSLILCIRSVIRGLQLQQVGNVAF

Chimpanzee TRPML3 (XM_001140401.1) is (SEQ ID NO:25) (GI:114557483)

1?cctctagaga?tggcagatcc?tgaggtagtt?gtgagtagct?gcagctctca?tgaagaggaa

61?aatcgctgca?attttaacca?gcaaacatct?ccatctgagg?agcttctatt?agaagaccag

121?atgaggcgaa?aactcaaatt?ttttttcatg?aatccctgtg?agaagttctg?ggctcgaggt

181?agaaaaccat?ggaaacttgc?catacaaatt?ctaaaaattg?caatggtgac?tatccagctg

241?gtcttatttg?ggctaagtaa?ccagatggtg?gtagctttca?aggaagagaa?tactatagca

301?ttcaaacacc?ttttcctaaa?aggatatatg?gaccgaatgg?atgacacata?tgcagtgtac

361?acacaaagtg?acgtgtatga?tcagttaatc?ttcgcagtaa?accagtactt?gcagctatac

421?aatgtctccg?ttgggaatca?tgcttatgag?aacaaaggta?ccaagcaatc?tgctatggca

481?atctgtcagc?acttctacaa?gcgaggaaac?atctaccctg?gaaatgatac?ctttgacatc

541?gatccagaaa?ttgaaactga?gtgtttcttt?gtggagccag?atgaaccttt?tcacattggg

601?acaccagcag?aaaataaact?gaacttaaca?ctggacttcc?acagactcct?aacagtggag

661?cttcagttta?aactgaaagc?cattaatctg?cagacagttc?gtcatcaaga?actccctgac

721?tgttatgact?ttactctgac?tataacattt?gacaacaagg?cccatagtgg?aagaattaaa

781?ataagtttag?ataatgacat?ttccatcaga?gaatgtaaag?actggcattc?tccctccgtc

841?gcccagcctg?gaaacactca?ttacatgatg?atctttgatg?cctttgtcat?tctgacttgc

901?ttggtttcat?taatcctctg?cattagatct?gtgattagag?gacttcagct?tcagcaggag

961?tttgtcaatt?ttttcctcct?ccattataag?aagggagttt?ctgtttctga?tcaaatggaa

1021?tttgtcaatg?gatggtacat?tatgattatt?attagtgaca?tattgacaat?cattggatca

1081?attctaaaaa?tggaaatcca?agctaagagt?ctaactagtt?atgatgtctg?tagcatactt

1141?cttgggactt?ctaccatgct?tgtgtggctt?ggagtcatcc?gatacctcgg?tttctttgca

1201?aagtacaatc?tcctcatttt?gacccttcag?gcagcactgc?ccaatgtcat?caggttctgc

1261?tgctgtgcag?ctatgattta?cttaggttac?tgcttctgtg?gatggatcgt?gctggggcct

1321?taccatgaca?agtttcgttc?tctgaacatg?gtctctgagt?gccttttctc?tctgataaat

1381?ggagatgata?tgtttgccac?gtttgcaaaa?atgcagcaaa?aaagttactt?agtctggctg

1441?tttagtagaa?tttacctcta?ctcattcatc?agcctcttta?tatatatgat?tttaagtctt

1501?ttcattgcac?tgatcactga?tacatacgaa?acaattaagc?aataccaaca?agatggcttc

1561?ccagagactg?aacttcgtac?atttatatca?gaatgcaaag?atctacccaa?ctctggaaaa

1621?tacagattag?aagatgaccc?tccagtatct?ttattctgct?gttgtaaaaa?gtagctatca

1681?ggtttatctg?tactttagag?gaaaatataa?tgtgtagctg?agttagaaca?ctgtggatat

1741?tctgagatca?gatgtagtat?gtttgaagac?tgttattttg?agctaattga?gacctataat

1801?tcaccaataa?ctgtttatat?ttttaaaagc?aatatttaat?gtctttgcag?ctttatgctg

1861?ggcttgtt

Chimpanzee TRPML3 XP_001140401.1 (GI:114557484) (SEQ ID NO:26)

MADPEVVVSSCSSHEEENRCNFNQQTSPSEELLLEDQMRRKLKFFFMNPCEKFWARGRKPWKLAIQILKIAMVTIQLVLFGLSNQMVVAFKEENTIAFKHLFLKGYMDRMDDTYAVYTQSDVYDQLIFAVNQYLQLYNVSVGNHAYENKGTKQSAMAICQHFYKRGNIYPGNDTFDIDPEIETECFFVEPDEPFHIGTPAENKLNLTLDFHRLLTVELQFKLKAINLQTVRHQELPDCYDFTLTITFDNKAHSGRIKISLDNDISIRECKDWHSPSVAQPGNTHYMMIFDAFVILTCLVSLILCIRSVIRGLQLQQEFVNFFLLHYKKGVSVSDQMEFVNGWYIMIIISDILTIIGSILKMEIQAKSLTSYDVCSILLGTSTMLVWLGVIRYLGFFAKYNLLILTLQAALPNVIRFCCCAAMIYLGYCFCGWIVLGPYHDKFRSLNMVSECLFSLINGDDMFATFAKMQQKSYLVWLFSRIYLYSFISLFIYMILSLFIALITDTYETIKQYQQDGFPETELRTFISECKDLPNSGKYRLEDDPPVSLFCCCKK

Chimpanzee TRPML3 (XM_524756.2) is (SEQ ID NO:27) (GI:114557481)

1?tcctctagag?atggcagatc?ctgaggtagt?tgtgagtagc?tgcagctctc?atgaagagga

61?aaatcgctgc?aattttaacc?agcaaacatc?tccatctgag?gagcttctat?tagaagacca

121?gatgaggcga?aaactcaaat?tttttttcat?gaatccctgt?gagaagttct?gggctcgagg

181?tagaaaacca?tggaaacttg?ccatacaaat?tctaaaaatt?gcaatggtga?ctatccagta

241?cttgcagcta?tacaatgtct?ccgttgggaa?tcatgcttat?gagaacaaag?gtaccaagca

301?atctgctatg?gcaatctgtc?agcacttcta?caagcgagga?aacatctacc?ctggaaatga

361?tacctttgac?atcgatccag?aaattgaaac?tgagtgtttc?tttgtggagc?cagatgaacc

421?ttttcacatt?gggacaccag?cagaaaataa?actgaactta?acactggact?tccacagact

481?cctaacagtg?gagcttcagt?ttaaactgaa?agccattaat?ctgcagacag?ttcgtcatca

541?agaactccct?gactgttatg?actttactct?gactataaca?tttgacaaca?aggcccatag

601?tggaagaatt?aaaataagtt?tagataatga?catttccatc?agagaatgta?aagactggca

661?tgtatctgga?tcaattcaga?agaacactca?ttacatgatg?atctttgatg?cctttgtcat

721?tctgacttgc?ttggtttcat?taatcctctg?cattagatct?gtgattagag?gacttcagct

781?tcagcaggag?tttgtcaatt?ttttcctcct?ccattataag?aagggagttt?ctgtttctga

841?tcaaatggaa?tttgtcaatg?gatggtacat?tatgattatt?attagtgaca?tattgacaat

901?cattggatca?attctaaaaa?tggaaatcca?agctaagagt?ctaactagtt?atgatgtctg

961?tagcatactt?cttgggactt?ctaccatgct?tgtgtggctt?ggagtcatcc?gatacctcgg

1021?tttctttgca?aagtacaatc?tcctcatttt?gacccttcag?gcagcactgc?ccaatgtcat

1081?caggttctgc?tgctgtgcag?ctatgattta?cttaggttac?tgcttctgtg?gatggatcgt

1141?gctggggcct?taccatgaca?agtttcgttc?tctgaacatg?gtctctgagt?gccttttctc

1201?tctgataaat?ggagatgata?tgtttgccac?gtttgcaaaa?atgcagcaaa?aaagttactt

1261?agtctggctg?tttagtagaa?tttacctcta?ctcattcatc?agcctcttta?tatatatgat

1321?tttaagtctt?ttcattgcac?tgatcactga?tacatacgaa?acaattaagc?aataccaaca

1381?agatggcttc?ccagagactg?aacttcgtac?atttatatca?gaatgcaaag?atctacccaa

1441?ctctggaaaa?tacagattag?aagatgaccc?tccagtatct?ttattctgct?gttgtaaaaa

1501?gtagctatca?ggtttatctg?tactttagag?gaaaatataa?tgtgtagctg?agttagaaca

1561?ctgtggatat?tctgagatca?gatgtagtat?gtttgaagac?tgttattttg?agctaattga

1621?gacctataat?tcaccaataa?ctgtttatat?ttttaaaagc?aatatttaat?gtctttgcag

1681?ctttatgctg?ggcttgtttt?taaaacaact?ttaatgagga?aagctattgg?attattatta

1741?tttcttgttt?attttgccat?ggctttagaa?tgtattctgt?atgcctctct?tttgctctga

1801?tactcttgct?tcctgctatt?ctgattgtgc?agactgtgta?attagtggaa?aacaatcctt

1861?ggtctgactg?tgactttgga?caactcagta?accctggctt?ggaccactct?caggagtcca

1921?tccttgagag?agtgggtgta?gttaccattt?atacagtaat?cattgcattt?taaaatctgc

1981?tcttgaaagg?aagaataaga?gtgcaccaga?ataagagcgc?accagaataa?gagcgcacca

2041?gctaacaatg?tgatacggcc?atatgtcact?taaggataga?gatatgttct?gagaaatgtg

2101?tcattaggcg?attttgtcat?taaacatcat?agcatgtact?tccacaaacc?tagatggtat

2161?agcctactac?acacctaggc?tatttggtat?agcctgttga?tcctggggta?caaatctgta

2221?caacatgtta?ctgtattgaa?tacagtaggc?aattgtaaca?caatggtaag?tatctaaaca

2281?tagaaaaggg?acagtaaaaa?tatggtttta?taatcttctg?ggaccaccat?tgtatatgcg

2341?gtacatcgtt?gaccaaaaca?tcgttatcca?gcatatgact?gtatttggtt?atgaaagcca

2401?actgttactt?gattctgctt?ttagttctta?agaggatcag?gtttttaaat?actcatttac

2461?aagttttcta?tcctccttca?gtgttaaagt?agaaagtaaa?aagagtatct?tatacatgca

2521?tgaaattaaa?gcatatacca?aatgca

Chimpanzee TRPML3 (XP_524756.2) is (SEQ ID NO:28) (GI:114557482)

MADPEVVVSSCSSHEEENRCNFNQQTSPSEELLLEDQMRRKLKFFFMNPCEKFWARGRKPWKLAIQILKIAMVTIQYLQLYNVSVGNHAYENKGTKQSAMAICQHFYKRGNIYPGNDTFDIDPEIETECFFVEPDEPFHIGTPAENKLNLTLDFHRLLTVELQFKLKAINLQTVRHQELPDCYDFTLTITFDNKAHSGRIKISLDNDISIRECKDWHVSGSIQKNTHYMMIFDAFVILTCLVSLILCIRSVIRGLQLQQEFVNFFLLHYKKGVSVSDQMEFVNGWYIMIIISDILTIIGSILKMEIQAKSLTSYDVCSILLGTSTMLVWLGVIRYLGFFAKYNLLILTLQAALPNVIRFCCCAAMIYLGYCFCGWIVLGPYHDKFRSLNMVSECLFSLINGDDMFATFAKMQQKSYLVWLFSRIYLYSFISLFIYMILSLFIALITDTYETIKQYQQDGFPETELRTFISECKDLPNSGKYRLEDDPPVSLFCCCKK

Chimpanzee TRPML3 (XM_00114082.1) is (SEQ ID NO:29) (GI:114557479)

1?tcctctagag?atggcagatc?ctgaggtagt?tgtgagtagc?tgcagctctc?atgaagagga

61?aaatcgctgc?aattttaacc?agcaaacatc?tccatctgag?gagcttctat?tagaagacca

121?gatgaggcga?aaactcaaat?tttttttcat?gaatccctgt?gagaagttct?gggctcgagg

181?tagaaaacca?tggaaacttg?ccatacaaat?tctaaaaatt?gcaatggtga?ctatccagct

241?ggtcttattt?gggctaagta?accagatggt?ggtagctttc?aaggaagaga?atactatagc

301?attcaaacac?cttttcctaa?aaggatatat?ggaccgaatg?gatgacacat?atgcagtgta

361?cacacaaagt?gacgtgtatg?atcagttaat?cttcgcagta?aaccagtact?tgcagctata

421?caatgtctcc?gttgggaatc?atgcttatga?gaacaaaggt?accaagcaat?ctgctatggc

481?aatctgtcag?cacttctaca?agcgaggaaa?catctaccct?ggaaatgata?cctttgacat

541?cgatccagaa?attgaaactg?agtgtttctt?tgtggagcca?gatgaacctt?ttcacattgg

601?gacaccagca?gaaaataaac?tgaacttaac?actggacttc?cacagactcc?taacagtgga

661?gcttcagttt?aaactgaaag?ccattaatct?gcagacagtt?cgtcatcaag?aactccctga

721?ctgttatgac?tttactctga?ctataacatt?tgacaacaag?gcccatagtg?gaagaattaa

781?aataagttta?gataatgaca?tttccatcag?agaatgtaaa?gactggcatg?tatctggatc

841?aattcagaag?aacactcatt?acatgatgat?ctttgatgcc?tttgtcattc?tgacttgctt

901?ggtttcatta?atcctctgca?ttagatctgt?gattagagga?cttcagcttc?agcaggagtt

961?tgtcaatttt?ttcctcctcc?attataagaa?gggagtttct?gtttctgatc?aaatggaatt

1021?tgtcaatgga?tggtacatta?tgattattat?tagtgacata?ttgacaatca?ttggatcaat

1081?tctaaaaatg?gaaatccaag?ctaagagtct?aactagttat?gatgtctgta?gcatacttct

1141?tgggacttct?accatgcttg?tgtggcttgg?agtcatccga?tacctcggtt?tctttgcaaa

1201?gtacaatctc?ctcattttga?cccttcaggc?agcactgccc?aatgtcatca?ggttctgctg

1261?ctgtgcagct?atgatttact?taggttactg?cttctgtgga?tggatcgtgc?tggggcctta

1321?ccatgacaag?tttcgttctc?tgaacatggt?ctctgagtgc?cttttctctc?tgataaatgg

1381?agatgatatg?tttgccacgt?ttgcaaaaat?gcagcaaaaa?agttacttag?tctggctgtt

1441?tagtagaatt?tacctctact?cattcatcag?cctctttata?tatatgattt?taagtctttt

1501?cattgcactg?atcactgata?catacgaaac?aattaagcaa?taccaacaag?atggcttccc

1561?agagactgaa?cttcgtacat?ttatatcaga?atgcaaagat?ctacccaact?ctggaaaata

1621?cagattagaa?gatgaccctc?cagtatcttt?attctgctgt?tgtaaaaagt?agctatcagg

1681?tttatctgta?ctttagagga?aaatataatg?tgtagctgag?ttagaacact?gtggatattc

1741?tgagatcaga?tgtagtatgt?ttgaagactg?ttattttgag?ctaattgaga?cctataattc

1801?accaataact?gtttatattt?ttaaaagcaa?tatttaatgt?ctttgcagct?ttatgctggg

1861?cttgttttta?aaacaacttt?aatgaggaaa?gctattggat?tattattatt?tcttgtttat

1921?tttgccatgg?ctttagaatg?tattctgtat?gcctctcttt?tgctctgata?ctcttgcttc

1981?ctgctattct?gattgtgcag?actgtgtaat?tagtggaaaa?caatccttgg?tctgactgtg

2041?actttggaca?actcagtaac?cctggcttgg?accactctca?ggagtccatc?cttgagagag

2101?tgggtgtagt?taccatttat?acagtaatca?ttgcatttta?aaatctgctc?ttgaaaggaa

2161?gaataagagt?gcaccagaat?aagagcgcac?cagaataaga?gcgcaccagc?taacaatgtg

2221?atacggccat?atgtcactta?aggatagaga?tatgttctga?gaaatgtgtc?attaggcgat

2281?tttgtcatta?aacatcatag?catgtacttc?cacaaaccta?gatggtatag?cctactacac

2341?acctaggcta?tttggtatag?cctgttgatc?ctggggtaca?aatctgtaca?acatgttact

2401?gtattgaata?cagtaggcaa?ttgtaacaca?atggtaagta?tctaaacata?gaaaagggac

2461?agtaaaaata?tggttttata?atcttctggg?accaccattg?tatatgcggt?acatcgttga

2521?ccaaaacatc?gttatccagc?atatgactgt?atttggttat?gaaagccaac?tgttacttga

2581?ttctgctttt?agttcttaag?aggatcaggt?ttttaaatac?tcatttacaa?gttttctatc

2641?ctccttcagt?gttaaagtag?aaagtaaaaa?gagtatctta?tacatgcatg?aaattaaagc

2701?atataccaaa?tgca

Chimpanzee TRPML3 (XP_00114082.1) is (SEQ ID NO:30) (GI:114557480)

MADPEVVVSSCSSHEEENRCNFNQQTSPSEELLLEDQMRRKLKFFFMNPCEKFWARGRKPWKLAIQILKIAMVTIQLVLFGLSNQMVVAFKEENTIAFKHLFLKGYMDRMDDTYAVYTQSDVYDQLIFAVNQYLQLYNVSVGNHAYENKGTKQSAMAICQHFYKRGNIYPGNDTFDIDPEIETECFFVEPDEPFHIGTPAENKLNLTLDFHRLLTVELQFKLKAINLQTVRHQELPDCYDFTLTITFDNKAHSGRIKISLDNDISIRECKDWHVSGSIQKNTHYMMIFDAFVILTCLVSLILCIRSVIRGLQLQQEFVNFFLLHYKKGVSVSDQMEFVNGWYIMIIISDILTIIGSILKMEIQAKSLTSYDVCSILLGTSTMLVWLGVIRYLGFFAKYNLLILTLQAALPNVIRFCCCAAMIYLGYCFCGWIVLGPYHDKFRSLNMVSECLFSLINGDDMFATFAKMQQKSYLVWLFSRIYLYSFISLFIYMILSLFIALITDTYETIKQYQQDGFPETELRTFISECKDLPNSGKYRLEDDPPVSLFCCCKK

Torrid zone Xenopus laevis (Xenopus tropicalis) (tropical Xenopus laevis Silurana tropicalis) (NM_001016804.2) (GI:77682897) (SEQ ID NO:31)

1?ttgcaactag?gtctgacagt?aggacaatgt?ggcaggtcac?gtgacagcag?tgctgatggt

61?agagatgcgc?cagcattcag?gtctgagagc?agaaagaaaa?gctggccaaa?acaaaggaca

121?ttctctttgc?tgcttcgcta?gctgagacgc?tgctatagta?tagcagacat?ggaaaaccca

181?gagctaataa?agacatgcaa?ctctttggat?gaacatgatg?gtccctactg?ctgcaagcag

241?tgccctatga?ctgatgagct?acttatggaa?gaccagctac?gaaggaaact?taaattcttt

301?ttcatgaacc?catgtgagaa?gttccgtgcc?cgtggacgaa?agccttggaa?gctttgtatt

361?caaattttaa?aaattgcaat?ggtgacaatc?caattagttt?tatttggact?cagtaatgaa

421?atggtagtca?cctttaaaga?ggagaacact?gtagctttta?agcatctgtt?tttgaaagga

481?tataaggatg?gacatgatga?cacatatgct?atctacagtc?aagaagatgt?tcatgctcat

541?ataaacttta?caattaaaag?gtacctagag?ctacaaaaca?tatctgttgg?aaatcatgca

601?tatgaaagta?atggtaaagg?tcaaactgga?atgtcattat?gtcaacatta?ctataaacaa

661?gggagtatct?ttcctggaaa?tgaaacattt?gaaattgacc?cacaaataga?tactgaatgt

721?ttccatattg?atccatcaac?tctgtgttct?aatgacacac?ctgcagaata?ctactggtct

781?aatatgacac?tagacttcta?tagacttgtt?tcagttgaaa?ttatgtttaa?gcttaaagca

841?attaatcttc?aaaccattcg?tcatcatgaa?cttccagact?gctatgactt?catggttata

901?ataacatttg?ataataaggc?acacagtgga?aggataaaaa?tcagcttaga?taatgatgtt

961?ggaatccagg?aatgcaaaga?ctggcatgtg?tctggatcta?ttcaaaaaaa?tactcattac

1021?atgatgattt?ttgatgctgc?tgttattttg?gtctgcttat?cttccataac?actctgcatt

1081?cgctccgtgg?ttaaaggaat?tcacctacaa?aaagaatatg?taaacttttt?ccagcatcgt

1141?tttgcaagga?ctgtgtcctc?agctgatcgc?atggaatttg?tcaatggctg?gtacattatg

1201?ataatcatca?gtgatgtttt?gtcaattatt?ggctcaatct?tgaagatgga?gatccaagca

1261?aagagcctca?ccagttatga?tgtctgcagt?attctcttgg?gaacatccac?cttattagtg

1321?tggcttggag?ttattcgcta?cttgggattt?tttaagaaat?acaatcttct?gatcctgaca

1381?cttagggcag?ccttacctaa?tgtgatacga?ttctgctgtt?gtgctgctat?gatctacctg

1441?ggctactgct?tctgtggctg?gattgttctg?gggccttacc?atgtaaagtt?caggtccctg

1501?aacatggttt?cagagtgcct?gttctccctt?attaatggag?acgatatgtt?tacaacgttt

1561?tcaatcatgc?aggagaagag?ctacttggtt?tggctgttta?gtcgcattta?tttgtattcc

1621?tttataagtc?tcttcatata?catggttctg?agtctcttca?ttgcacttat?tactgacaca

1681?tacgatacaa?tcaagaatta?ccagatcgat?ggctttccag?aatcagaact?tcacacattt

1741?gtatccgagt?gcaaagattt?gccaacctct?ggtcgatata?gggaacaaga?cgagacctcc

1801?tgtttgtcta?tgctgtgttg?taatcggtaa?aaaagaatcc?cagaagaagc?actttatcca

1861?tggcctttaa?aaatctgcaa?aaaaaaaaaa?aaaaaa

Torrid zone Xenopus laevis (Xenopus tropicalis) (tropical Xenopus laevis Silurana tropicalis) (NP_001016804.2) (GI:62857333) (SEQ ID NO:32)

MENPELIKTCNSLDEHDGPYCCKQCPMTDELLMEDQLRRKLKFFFMNPCEKFRARGRKPWKLCIQILKIAMVTIQLVLFGLSNEMVVTFKEENTVAFKHLFLKGYKDGHDDTYAIYSQEDVHAHINFTIKRYLELQNISVGNHAYESNGKGQTGMSLCQHYYKQGSIFPGNETFEIDPQIDTECFHIDPSTLCSNDTPAEYYWSNMTLDFYRLVSVEIMFKLKAINLQTIRHHELPDCYDFMVIITFDNKAHSGRIKISLDNDVGIQECKDWHVSGSIQKNTHYMMIFDAAVILVCLSSITLCIRSVVKGIHLQKEYVNFFQHRFARTVSSADRMEFVNGWYIMIIISDVLSIIGSILKMEIQAKSLTSYDVCSILLGTSTLLVWLGVIRYLGFFKKYNLLILTLRAALPNVIRFCCCAAMIYLGYCFCGWIVLGPYHVKFRSLNMVSECLFSLINGDDMFTTFSIMQEKSYLVWLFSRIYLYSFISLFIYMVLSLFIALITDTYDTIKNYQIDGFPESELHTFVSECKDLPTSGRYREQDETSCLSMLCCNR

Torrid zone Xenopus laevis (Xenopus tropicalis) (tropical Xenopus laevis Silurana tropicalis) (CR855599.2) (GI:77623768) (SEQ ID NO:33)

1?ttgcaactag?gtctgacagt?aggacaatgt?ggcaggtcac?gtgacagcag?tgctgatggt

61?agagatgcgc?cagcattcag?gtctgagagc?agaaagaaaa?gctggccaaa?acaaaggaca

121?ttctctttgc?tgcttcgcta?gctgagacgc?tgctatagta?tagcagacat?ggaaaaccca

181?gagctaataa?agacatgcaa?ctctttggat?gaacatgatg?gtccctactg?ctgcaagcag

241?tgccctatga?ctgatgagct?acttatggaa?gaccagctac?gaaggaaact?taaattcttt

301?ttcatgaacc?catgtgagaa?gttccgtgcc?cgtggacgaa?agccttggaa?gctttgtatt

361?caaattttaa?aaattgcaat?ggtgacaatc?caattagttt?tatttggact?cagtaatgaa

421?atggtagtca?cctttaaaga?ggagaacact?gtagctttta?agcatctgtt?tttgaaagga

481?tataaggatg?gacatgatga?cacatatgct?atctacagtc?aagaagatgt?tcatgctcat

541?ataaacttta?caattaaaag?gtacctagag?ctacaaaaca?tatctgttgg?aaatcatgca

601?tatgaaagta?atggtaaagg?tcaaactgga?atgtcattat?gtcaacatta?ctataaacaa

661?gggagtatct?ttcctggaaa?tgaaacattt?gaaattgacc?cacaaataga?tactgaatgt

721?ttccatattg?atccatcaac?tctgtgttct?aatgacacac?ctgcagaata?ctactggtct

781?aatatgacac?tagacttcta?tagacttgtt?tcagttgaaa?ttatgtttaa?gcttaaagca

841?attaatcttc?aaaccattcg?tcatcatgaa?cttccagact?gctatgactt?catggttata

901?ataacatttg?ataataaggc?acacagtgga?aggataaaaa?tcagcttaga?taatgatgtt

961?ggaatccagg?aatgcaaaga?ctggcatgtg?tctggatcta?ttcaaaaaaa?tactcattac

1021?atgatgattt?ttgatgctgc?tgttattttg?gtctgcttat?cttccataac?actctgcatt

1081?cgctccgtgg?ttaaaggaat?tcacctacaa?aaagaatatg?taaacttttt?ccagcatcgt

1141?tttgcaagga?ctgtgtcctc?agctgatcgc?atggaatttg?tcaatggctg?gtacattatg

1201?ataatcatca?gtgatgtttt?gtcaattatt?ggctcaatct?tgaagatgga?gatccaagca

1261?aagagcctca?ccagttatga?tgtctgcagt?attctcttgg?gaacatccac?cttattagtg

1321?tggcttggag?ttattcgcta?cttgggattt?tttaagaaat?acaatcttct?gatcctgaca

1381?cttagggcag?ccttacctaa?tgtgatacga?ttctgctgtt?gtgctgctat?gatctacctg

1441?ggctactgct?tctgtggctg?gattgttctg?gggccttacc?atgtaaagtt?caggtccctg

1501?aacatggttt?cagagtgcct?gttctccctt?attaatggag?acgatatgtt?tacaacgttt

1561?tcaatcatgc?aggagaagag?ctacttggtt?tggctgttta?gtcgcattta?tttgtattcc

1621?tttataagtc?tcttcatata?catggttctg?agtctcttca?ttgcacttat?tactgacaca

1681?tacgatacaa?tcaagaatta?ccagatcgat?ggctttccag?aatcagaact?tcacacattt

1741?gtatccgagt?gcaaagattt?gccaacctct?ggtcgatata?gggaacaaga?cgagacctcc

1801?tgtttgtcta?tgctgtgttg?taatcggtaa?aaaagaatcc?cagaagaagc?actttatcca

1861?tggcctttaa?aaatctgcaa?aaaaaaaaaa?aaaaaa

//

Torrid zone Xenopus laevis (Xenopus tropicalis) (tropical Xenopus laevis Silurana tropicalis) (CAJ83717.1) (GI:89273945) (SEQ ID NO:34)

MENPELIKTCNSLDEHDGPYCCKQCPMTDELLMEDQLRRKLKFFFMNPCEKFRARGRKPWKLCIQILKIAMVTIQLVLFGLSNEMVVTFKEENTVAFKHLFLKGYKDGHDDTYAIYSQEDVHAHINFTIKRYLELQNISVGNHAYESNGKGQTGMSLCQHYYKQGSIFPGNETFEIDPQIDTECFHIDPSTLCSNDTPAEYYWSNMTLDFYRLVSVEIMFKLKAINLQTIRHHELPDCYDFMVIITFDNKAHSGRIKISLDNDVGIQECKDWHVSGSIQKNTHYMMIFDAAVILVCLSSITLCIRSVVKGIHLQKEYVNFFQHRFARTVSSADRMEFVNGWYIMIIISDVLSIIGSILKMEIQAKSLTSYDVCSILLGTSTLLVWLGVIRYLGFFKKYNLLILTLRAALPNVIRFCCCAAMIYLGYCFCGWIVLGPYHVKFRSLNMVSECLFSLINGDDMFTTFSIMQEKSYLVWLFSRIYLYSFISLFIYMVLSLFIALITDTYDTIKNYQIDGFPESELHTFVSECKDLPTSGRYREQDETSCLSMLCCNR

Homo sapiens's sodium leakage passage, non-selective (NALCN), mRNa (NM_052867.2) GI:93277089 (SEQ ID NO:35)

Gi|93277089|ref|NM_052867.2| homo sapiens's sodium leakage passage, non-selective (NALCN), mRNA

CGCGCTGCCTGAGCTGAGCCGCCGTAGGTGAGGGGCCCGCGTCCCCGCCCGCCCTGGGCGCCGCGCCTGGCACTGATCCTGCCGGTCGCCCACTGTCGCCGCCGCCGCCGCCCGCGGGCACCATGACAGCTCTGAGCGCTGGGGTTACAGACTGTGGTTTTGTGCTTGCTCACCAAAGCTAACCTCAGCATGCTCAAAAGGAAGCAGAGTTCCAGGGTGGAAGCCCAGCCAGTCACTGACTTTGGTCCTGATGAGTCTCTGTCGGATAATGCTGACATCCTCTGGATTAACAAACCATGGGTTCACTCTTTGCTGCGCATCTGTGCCATCATCAGCGTCATTTCTGTTTGTATGAATACGCCAATGACCTTCGAGCACTATCCTCCACTTCAGTATGTGACCTTCACTTTGGATACATTATTGATGTTTCTCTACACGGCAGAGATGATAGCAAAAATGCACATCCGGGGCATTGTCAAGGGGGATAGTTCCTATGTGAAAGATCGCTGGTGTGTTTTTGATGGATTTATGGTCTTTTGCCTTTGGGTTTCTTTGGTGCTACAGGTGTTTGAAATTGCTGATATAGTTGATCAGATGTCACCTTGGGGCATGTTGCGGATTCCACGGCCACTGATTATGATCCGAGCATTCCGGATTTATTTCCGATTTGAACTGCCAAGGACCAGAATTACAAATATTTTAAAGCGATCGGGAGAACAAATATGGAGTGTTTCCATTTTTCTACTTTTCTTTCTACTTCTTTATGGAATTTTAGGAGTTCAGATGTTTGGAACATTTACTTATCACTGTGTTGTAAATGACACAAAGCCAGGGAATGTAACCTGGAATAGTTTAGCTATTCCAGACACACACTGCTCACCAGAGCTAGAAGAAGGCTACCAGTGCCCACCTGGATTTAAATGCATGGACCTTGAAGATCTGGGACTTAGCAGGCAAGAGCTGGGCTACAGTGGCTTTAATGAGATAGGAACTAGTATATTCACCGTCTATGAGGCCGCCTCACAGGAAGGCTGGGTGTTCCTCATGTACAGAGCAATTGACAGCTTTCCCCGTTGGCGTTCCTACTTCTATTTCATCACTCTCATTTTCTTCCTCGCCTGGCTTGTGAAGAACGTGTTTATTGCTGTTATCATTGAAACATTTGCAGAAATCAGAGTACAGTTTCAACAAATGTGGGGATCGAGAAGCAGCACTACCTCAACAGCCACCACCCAGATGTTTCATGAAGATGCTGCTGGAGGTTGGCAGCTGGTAGCTGTGGATGTCAACAAGCCCCAGGGACGCGCCCCAGCCTGCCTCCAGAAAATGATGCGGTCATCCGTTTTCCACATGTTCATCCTGAGCATGGTGACCGTGGACGTGATCGTGGCGGCTAGCAACTACTACAAAGGAGAAAACTTCAGGAGGCAGTACGACGAGTTCTACCTGGCGGAGGTGGCTTTTACAGTACTTTTTGATTTGGAAGCACTTCTGAAGATATGGTGTTTGGGATTTACTGGATATATTAGCTCATCTCTCCACAAATTCGAACTACTACTCGTAATTGGAACTACTCTTCATGTATACCCAGATCTTTATCATTCACAATTCACGTACTTTCAGGTTCTCCGAGTAGTTCGGCTGATTAAGATTTCACCTGCATTAGAAGACTTTGTGTACAAGATATTTGGTCCTGGAAAAAAGCTTGGGAGTTTGGTTGTATTTACTGCCAGCCTCTTGATTGTTATGTCAGCAATTAGTTTGCAGATGTTCTGCTTTGTCGAAGAACTGGACAGATTTACTACGTTTCCGAGGGCATTTATGTCCATGTTCCAGATCCTCACCCAGGAAGGATGGGTGGACGTAATGGACCAAACTCTAAATGCTGTGGGACATATGTGGGCACCCGTGGTTGCCATCTATTTCATTCTCTATCATCTTTTTGCCACTCTGATCCTCCTGAGTTTGTTTGTTGCTGTTATTTTGGACAACTTAGAACTTGATGAAGACCTAAAGAAGCTTAAACAATTAAAGCAAAGTGAAGCAAATGCGGACACCAAAGAAAAGCTCCCTTTACGCCTGCGAATCTTTGAAAAATTTCCAAACAGACCTCAAATGGTGAAAATCTCAAAGCTTCCTTCAGATTTTACAGTTCCTAAAATCAGGGAGAGTTTTATGAAGCAGTTTATTGACCGCCAGCAACAGGACACATGTTGCCTCCTGAGAAGCCTCCCGACCACCTCTTCCTCCTCCTGCGACCACTCCAAACGCTCAGCAATTGAGGACAACAAATACATCGACCAAAAACTTCGCAAGTCTGTTTTCAGCATCAGGGCAAGGAACCTTCTGGAAAAGGAGACCGCAGTCACTAAAATCTTAAGAGCTTGCACCCGACAGCGCATGCTGAGCGGATCATTTGAGGGGCAGCCCGCAAAGGAGAGGTCAATCCTCAGCGTGCAGCATCATATCCGCCAAGAGCGCAGGTCACTAAGACATGGATCAAACAGCCAGAGGATCAGCAGGGGAAAATCTCTTGAAACTTTGACTCAAGATCATTCCAATACAGTGAGATATAGAAATGCACAAAGAGAAGACAGTGAAATAAAGATGATTCAGGAAAAAAAGGAGCAAGCAGAGATGAAAAGGAAAGTGCAAGAAGAGGAACTCAGAGAGAACCACCCATACTTCGATAAGCCACTGTTCATTGTCGGGCGAGAACACAGGTTCAGAAACTTTTGCCGGGTGGTGGTCCGAGCACGCTTCAACGCATCTAAAACAGACCCTGTCACAGGAGCTGTGAAAAATACAAAGTACCATCAACTTTATGATTTGCTGGGATTGGTCACTTACCTGGACTGGGTCATGATCATCGTAACCATCTGCTCTTGCATTTCCATGATGTTTGAGTCCCCGTTTCGAAGAGTCATGCATGCACCTACTTTGCAGATTGCTGAGTATGTGTTTGTGATATTCATGAGCATTGAGCTTAATCTGAAGATTATGGCAGATGGCTTATTTTTCACTCCAACTGCTGTCATCAGGGACTTCGGTGGAGTAATGGACATATTTATATATCTTGTGAGCTTGATATTTCTTTGTTGGATGCCTCAAAATGTACCTGCTGAATCGGGAGCTCAGCTTCTAATGGTCCTTCGGTGCCTGAGACCTCTGCGCATATTCAAACTGGTGCCCCAGATGAGGAAAGTTGTTCGAGAACTTTTCAGCGGCTTCAAGGAAATTTTTTTGGTCTCCATTCTTTTGCTGACATTAATGCTCGTTTTTGCAAGCTTTGGAGTTCAGCTTTTTGCTGGAAAACTGGCCAAGTGCAATGATCCCAACATTATTAGAAGGGAAGATTGCAATGGCATATTCAGAATTAATGTCAGTGTGTCAAAGAACTTAAATTTAAAATTGAGGCCTGGAGAGAAAAAACCTGGATTTTGGGTGCCCCGTGTTTGGGCGAATCCTCGGAACTTTAATTTCGACAATGTGGGAAACGCTATGCTGGCGTTGTTTGAAGTTCTCTCCTTGAAAGGCTGGGTGGAAGTGAGAGATGTTATTATTCATCGTGTGGGGCCGATCCATGGAATCTATATTCATGTTTTTGTATTCCTGGGTTGCATGATTGGACTGACCCTTTTTGTTGGAGTAGTTATTGCTAATTTCAATGAAAACAAGGGGACGGCTTTGCTGACCGTCGATCAGAGAAGATGGGAAGACCTGAAGAGCCGACTGAAGATCGCACAGCCTCTTCATCTTCCGCCTCGCCCGGATAATGATGGTTTTAGAGCTAAAATGTATGACATAACCCAGCATCCATTTTTTAAGAGGACAATCGCATTACTCGTCCTGGCCCAGTCGGTGTTGCTCTCTGTCAAGTGGGACGTCGAGGACCCGGTGACCGTACCTTTGGCAACAATGTCAGTTGTTTTCACCTTCATCTTTGTTCTGGAGGTTACCATGAAGATCATAGCAATGTCGCCTGCTGGCTTCTGGCAAAGCAGAAGAAACCGATACGATCTCCTGGTGACGTCGCTTGGCGTTGTATGGGTGGTGCTTCACTTTGCCCTCCTGAATGCATATACTTACATGATGGGCGCTTGTGTGATTGTATTTAGGTTTTTCTCCATCTGTGGAAAACATGTAACGCTAAAGATGCTCCTCTTGACAGTGGTCGTCAGCATGTACAAGAGCTTCTTTATCATAGTAGGCATGTTTCTCTTGCTGCTGTGTTACGCTTTTGCTGGAGTTGTTTTATTTGGTACTGTGAAATATGGGGAGAATATTAACAGGCATGCAAATTTTTCTTCGGCTGGAAAAGCTATTACCGTACTGTTCCGAATTGTCACAGGTGAAGACTGGAACAAGATTATGCATGACTGTATGGTTCAGCCTCCGTTTTGTACTCCAGATGAATTTACATACTGGGCAACAGACTGTGGAAATTATGCTGGGGCACTTATGTATTTCTGTTCATTTTATGTCATCATTGCCTACATCATGCTAAATCTGCTTGTAGCCATAATTGTGGAGAATTTCTCCTTGTTTTATTCCACTGAGGAGGACCAGCTTTTAAGTTACAATGATCTTCGCCACTTTCAAATAATATGGAACATGGTGGATGATAAAAGAGAGGGGGTGATCCCCACGTTCCGCGTCAAGTTCCTGCTGCGGCTACTGCGTGGGAGGCTGGAGGTGGACCTGGACAAGGACAAGCTCCTGTTTAAGCACATGTGCTACGAAATGGAGAGGCTCCACAATGGCGGCGACGTCACCTTCCATGATGTCCTGAGCATGCTTTCATACCGGTCCGTGGACATCCGGAAGAGCTTGCAGCTGGAGGAACTCCTGGCGAGGGAGCAGCTGGAGTACACCATAGAGGAGGAGGTGGCCAAGCAGACCATCCGCATGTGGCTCAAGAAGTGCCTGAAGCGCATCAGAGCTAAACAGCAGCAGTCGTGCAGTATCATCCACAGCCTGAGAGAGAGTCAGCAGCAAGAGCTGAGCCGGTTTCTGAACCCGCCCAGCATCGAGACCACCCAGCCCAGTGAGGACACGAATGCCAACAGTCAGGACAACAGCATGCAACCTGAGACAAGCAGCCAGCAGCAGCTCCTGAGCCCCACGCTGTCGGATCGAGGAGGAAGTCGGCAAGATGCAGCCGACGCAGGGAAACCCCAGAGGAAATTTGGGCAGTGGCGTCTGCCCTCAGCCCCAAAACCAATAAGCCATTCAGTGTCCTCAGTCAACTTACGGTTTGGAGGAAGGACAACCATGAAATCTGTCGTGTGCAAAATGAACCCCATGACTGACGCGGCTTCCTGCGGTTCTGAAGTTAAGAAGTGGTGGACCCGGCAGCTGACTGTGGAGAGCGACGAAAGTGGGGATGACCTTCTGGATATTTAGGTGGATGTCAATGTAGATGAATTTCTAGTGGTGGAAACCGTTTTCTAATAATGTCCTTGATTGTCCAGTGAGCAATCTGTAATTGATCTATAACTGAATTCCAGCTTGTCACAAGATGTTTATAAATTGATTTTCATCCTGCCACAGAAAGGCATAAGCTGCATGTATGATGGGTTACTATCAATCATTGCTCAAAAAAATTTTTGTATAATGACAGTACTGATAATATTAGAAATGATACCGCAAGCAAATGTATATCACTTAAAAATGTCATATATTCTGTCTGCGTAAACTAAGGTATATATTCATATGTGCTCTAATGCAGTATTATCACCGCCCCGCAAAAGAGTGCTAAGCCCAAAGTGGCTGATATTTAGGGTACAGGGGTTATAGCTTTAGTTCACATCTTTCCCATTTCCACTAGAAATATTTCTCTTGAGAGAATTTATTATTTATGATTGATCTGAAAAGGTCAGCACTGAACTTATGCTAAAATGATAGTAGTTTTACAAACTACAGATTCTGAATTTTAAAAAGTATCTTCTTTTTCTCGTGTTATATTTTTAAATATACACAAGACATTTGGTGACCAGAACAAGTTGATTTCTGTCCTCAGTTATGTTAATGAAACTGTTGCCTCCTTCTAAGAAAATTGTGTGTGCAAGCACCAGGCAAAGAAATGGACTCAGGATGCTTAGCGGTTTAAAACAAACCTGTAGATAAATCACTTGAGTGACATAGTTGCGCAAAGATGTTAAGTTTCTTAAGAAACCTTTTAATAACTGAGTTTAGCAAAAAGAATAAAACTATATAGCTCAATTTATTTAAAAAAATCTTTGCATGTGTGATGTTATCATTGGCTTCATTTCTTACCCAAGGTATGTCTGTTTTGCCATAAATCAGCAGAGTCATTTCATTCTGGGTGATCCTAACACACCATTGCTACGTTAGATTTGAAATGACATCTCTGTTAAAAGAATCTTCTATGGAAATAATGGTGCCCTGCAAAATCTTCCTTTGAACTCACAGGTTAGGGATCACACAACTTACTTAATCGTTTTTTGTTTTTGTTTTTTTTCCTTATATGTCAATGGCCCATGTCCTCCGGGAAAATTAGAAAAGCAAAATGATTACAAAGTGCTGTTAGATTTCTTGTGCTGGGCCAGCCAAGTAGAAGTGGACTTGACTTGGACCTTTAACTATTTTATTACAGATTGGACATTTGCTGTTCAGATGTTTTTTAACAGAGGGATTATCTCAGAATCCTGTGACCTCCAGGTTGTTTTATAATCTATTTTTCTCTATTTAACATTCCTCAGATAGATAGGCAAATAGGACATTCCTTCTGTGTCACAGAAGTATCGTGGTAGTGGCAGTCTACAGTTTATATGATTCATTGTAACTATGAGATAAAGAACAACCAGTCATGTGGCCAAAAGGATTAGATTTGATTTGATGTTCACTTGGAGTTTACTTTTTGTACATACAAGATAAAATAAATATTGGATTTGTAAAAT

Homo sapiens's sodium leakage passage, non-selective (NALCN), polypeptide

(NP_443099.1)GI：24119274(SEQ?ID?NO：36)

MLKRKQSSRVEAQPVTDFGPDESLSDNADILWINKPWVHSLLRICAIISVISVCMNTPMTFEHYPPLQYVTFTLDTLLMFLYTAEMIAKMHIRGIVKGDSSYVKDRWCVFDGFMVFCLWVSLVLQVFEIADIVDQMSPWGMLRIPRPLIMIRAFRIYFRFELPRTRITNILKRSGEQIWSVSIFLLFFLLLYGILGVQMFGTFTYHCVVNDTKPGNVTWNSLAIPDTHCSPELEEGYQCPPGFKCMDLEDLGLSRQELGYSGFNEIGTSIFTVYEAASQEGWVFLMYRAIDSFPRWRSYFYFITLIFFLAWLVKNVFIAVIIETFAEIRVQFQQMWGSRSSTTSTATTQMFHEDAAGGWQLVAVDVNKPQGRAPACLQKMMRSSVFHMFILSMVTVDVIVAASNYYKGENFRRQYDEFYLAEVAFTVLFDLEALLKIWCLGFTGYISSSLHKFELLLVIGTTLHVYPDLYHSQFTYFQVLRVVRLIKISPALEDFVYKIFGPGKKLGSLVVFTASLLIVMSAISLQMFCFVEELDRFTTFPRAFMSMFQILTQEGWVDVMDQTLNAVGHMWAPVVAIYFILYHLFATLILLSLFVAVILDNLELDEDLKKLKQLKQSEANADTKEKLPLRLRIFEKFPNRPQMVKISKLPSDFTVPKIRESFMKQFIDRQQQDTCCLLRSLPTTSSSSCDHSKRSAIEDNKYIDQKLRKSVFSIRARNLLEKETAVTKILRACTRQRMLSGSFEGQPAKERSILSVQHHIRQERRSLRHGSNSQRISRGKSLETLTQDHSNTVRYRNAQREDSEIKMIQEKKEQAEMKRKVQEEELRENHPYFDKPLFIVGREHRFRNFCRVVVRARFNASKTDPVTGAVKNTKYHQLYDLLGLVTYLDWVMIIVTICSCISMMFESPFRRVMHAPTLQIAEYVFVIFMSIELNLKIMADGLFFTPTAVIRDFGGVMDIFIYLVSLIFLCWMPQNVPAESGAQLLMVLRCLRPLRIFKLVPQMRKVVRELFSGFKEIFLVSILLLTLMLVFASFGVQLFAGKLAKCNDPNIIRREDCNGIFRINVSVSKNLNLKLRPGEKKPGFWVPRVWANPRNFNFDNVGNAMLALFEVLSLKGWVEVRDVIIHRVGPIHGIYIHVFVFLGCMIGLTLFVGVVIANFNENKGTALLTVDQRRWEDLKSRLKIAQPLHLPPRPDNDGFRAKMYDITQHPFFKRTIALLVLAQSVLLSVKWDVEDPVTVPLATMSVVFTFIFVLEVTMKIIAMSPAGFWQSRRNRYDLLVTSLGVVWVVLHFALLNAYTYMMGACVIVFRFFSICGKHVTLKMLLLTVVVSMYKSFFIIVGMFLLLLCYAFAGVVLFGTVKYGENINRHANFSSAGKAITVLFRIVTGEDWNKIMHDCMVQPPFCTPDEFTYWATDCGNYAGALMYFCSFYVIIAYIMLNLLVAIIVENFSLFYSTEEDQLLSYNDLRHFQIIWNMVDDKREGVIPTFRVKFLLRLLRGRLEVDLDKDKLLFKHMCYEMERLHNGGDVTFHDVLSMLSYRSVDIRKSLQLEELLAREQLEYTIEEEVAKQTIRMWLKKCLKRIRAKQQQSCSIIHSLRESQQQELSRFLNPPSIETTQPSEDTNANSQDNSMQPETSSQQQLLSPTLSDRGGSRQDAADAGKPQRKFGQWRLPSAPKPISHSVSSVNLRFGGRTTMKSVVCKMNPMTDAASCGSEVKKWWTRQLTVESDESGDDLLDI

Homo sapiens's sodium/potassium transhipment ATP enzyme interacting 3 (NKAIN3)

(NP_173688.2)GI：14538007(SEQ?ID?NO：3)

1?agccgcgagc?ggcggccgcg?gggccgagga?gcctgggccg?ggccgggcgg?ggactactcc

61?ggagtcagga?ggcagcagcg?gcggaggacg?aggatctctg?gcagtcagcg?ccgctcggac

121?gccgccggca?ccatgggctg?ctgcaccgga?cgctgctcgc?tcatctgcct?ctgcgcgctg

181?cagttggtct?cagcattaga?gaggcagatc?tttgacttcc?ttggtttcca?gtgggcgcct

241?attcttggaa?attttctaca?cataatagtt?gtcatattgg?gtttgtttgg?gaccattcag

301?tacagacctc?gatacataat?ggtgtataca?gtgtggactg?ccctctgggt?cacctggaat

361?gtgttcatta?tctgctttta?tttggaagta?ggtggactct?caaaggacac?cgatctaatg

421?acattcaata?tctctgtaca?tcggtcatgg?tggagagaac?atgggcctgg?ttgtgtcaga

481?agagtgctgc?ctccctcagc?ccatggcatg?atggacgatt?acacgtacgt?ctctgtcaca

541?ggctgcatcg?ttgacttcca?gtacctggag?gtcatccaca?gtgctgtcca?aatactactc

601?tctttggtgg?gttttgtgta?tgcctgttat?gtgatcagta?tttccatgga?agaagaagac

661?acatattcat?gtgatctgca?agtatgcaaa?catcttttta?tccagatgct?gcaaattatt

721?gaataagcaa?gaattagtaa?gatattatca?ccaaattgtc?acatcagtca?agcctcatgt

781?gcttcctaag?aactgaggtg?atgcattatt?ttagagtgtc?attctaaacc?ccagattcaa

841?catcttccta?atctttctag?tgcagtctaa?tatataaatt?ttatgaaaag?cataggtttt

901?tttttaacca?gcagtgctct?ttgagaattt?acattgattc?ctaaagattg?ccattgcttt

961?gtataaaatg?ttataaatta?tcttagcatc?ttacctggaa?tttccactaa?attcaccaat

1021?ttatgatttg?tgaaatctga?ttttactttt?tgaaaatttt?catgtgaatt?tcccattttc

1081?agtgttgtag?cacctctctc?ttcctctaag?atcctccaag?ctcatcaaaa?gccatgatct

1141?tattatacca?gcagttttat?ttattcaatc?tttcaacaag?tagttattga?acttctataa

1201?tgtgccaggc?tctggagctc?gccttacacc?aaacagacac?aatcgatcca?ttcgaagtgt

1261?cgtaattaca?cattgaggga?ccaactagac?cttttctcat?tgtaaacttg?gagcaaaagt

1321?aaattcatta?aaataaattt?acattatagt?gccacaaaaa?aatgaacaga?accagaaagc

1381?attttttaca?aaaattaaca?gaacagtgtg?atagagggga?aaaggatgtg?agatcatggt

1441?gccctacctt?caatagggtg?gccagaaaac?acctctctga?agaagcagca?tttgagctga

1501?gacctgaaga?acgaggagtc?agtgatgcag?agaacctcag?gagatgcctt?ccaatctgag

1561?aaaaga

Homo sapiens's sodium/potassium transhipment ATP enzyme interacting 3 (NKAIN3) polypeptide (NP_775959.1) GI:27735000 (SEQ IDNO:38)

MGCCTGRCSLICLCALQLVSALERQIFDFLGFQWAPILGNFLHIIVVILGLFGTIQYRPRYIMVYTVWTALWVTWNVFIICFYLEVGGLSKDTDLMTFNISVHRSWWREHGPGCVRRVLPPSAHGMMDDYTYVSVTGCIVDFQYLEVIHSAVQILLSLVGFVYACYVISISMEEEDTYSCDLQVCKHLFIQMLQIIE

〉3,mRNA gi|38174237|gb|BC060765.1| ( SEQ ID NO：39 ) TGGAGTCGCTCGCTGACTCGCCCTGCGCCCTCGCCGCGGACACCGGAGCTGCGGCCGCTCCCCGCTGTCCCCCAGCTTACTCCAATCAAGCCTCTGCCCGCCAGGAACAGGTAACCTGTGTGTGTCCGTTTGCTCCTTCTAAGAGCATGCCTGATAGATACTTCGGTAGCCTCTCCGGATGGCCCCTTCGTCGGGTAGCCTCTCCTGATGGGGTCCTTCGCCCACCCTGCCTCCCGCGCCGGCGCTCCGGGTGAATGTCAAGGGTGGCTGGCTGCGAATACCTCCTTCAGCTGCTGGGGTTCCCGACAGTTTGCAGTTTTTAAAAGTGCACCCTCGGAAGGGCTTTTCAGACTGGGTAAACCTGACTTTTCCAAGAGATGGCAGATCCTGAGGTAGTTGTGAGTAGCTGCAGCTCTCATGAAGAGGAAAATCGCTGCAATTTTAACCAGCAAACATCTCCATCTGAGGAGCTTCTATTAGAAGACCAGATGAGGCGAAAACTCAAATTTTTTTTCATGAATCCCTGTGAGAAGTTCTGGGCTCGAGGTAGAAAACCATGGAAACTTGCCATACAAATTCTAAAAATTGCAATGGTGACTATCCAGCTGGTCTTATTTGGGCTAAGTAACCAGATGGTGGTAGCTTTCAAGGAAGAGAATACTATAGCATTCAAACACCTTTTCCTAAAAGGATATATGGACCGAATGGATGACACATATGCAGTGTACACACAAAGTGACGTGTATGATCAGTTAATCTTCGCAGTAAACCAGTACTTGCAGCTATACAATGTCTCCGTTGGGAATCATGCTTATGAGAACAAAGGTACCAAGCAATCTGCTATGGCAATCTGTCAGCACTTATACAAGCGAGGAAACATCTACCCTGGAAATGATACCTTTGACATCGATCCAGAAATTGAAACTGAGTGTTTCTTTGTGGAGCCAGATGAACCTTTTCACATTGGGACACCAGCAGAAAATAAACTGAACTTAACACTGGACTTCCACAGACTCCTAACAGTGGAGCTTCAGTTTAAACTGAAAGCCATTAATCTGCAGACAGTTCGTCATCAAGAACTCCCTGACTGTTATGACTTTACTCTGACTATAACATTTGACAACAAGGCCCATAGTGGAAGAATTAAAATAAGTTTAGATAATGACATTTCCATCAGAGAATGTAAAGACTGGCATGTATCTGGATCAATTCAGAAGAACACTCATTACATGATGATCTTTGATGCCTTTGTCATTCTGACTTGCTTGGTTTCATTAATCCTCTGCATTAGATCTGTGATTAGAGGACTTCAGCTTCAGCAGGTAGGGAACGTTGCTTTCTAGGAATGCTACTGACATTTTGATTGACAGAGACATTCACTGTGCCTCCCCTCTTTTCCCTAAAGGAGTTTGTCAATTTTTTCCTCCTCCATTATAAGAAGGAAGTTTCTGTTTCTGATCAAATGGAATTTGTCAATGGATGGTACATTATGATTATTATTAGTGACATATTGACAATCATTGGATCAATTCTAAAAATGGAAATCCAAGCTAAGGTAATTTTTTTCCTAATCATGCTATTGTTAGTGTCAGATTTGCACTAATGGTAATGTATTTTTCCAGAATGTAAGAATTTTCAGAATGAATTGTTTCTTCCAAACTGTATATCAAGTAGACTTGAAATTGGTAATGGTAATTTTCTTAAATCTAGTCAGGAGGTCTCTTAGGCAGAGTTTTTCAAAGTGTGATCCACAAACCATTGCATCAGAATCATTGGGTGCCTGGTAAAGTGTACCATGTTAGACCTACTGAATTCAGACTCTTCGGCGGGGCCTGTGAATTCTTACACACACCAAAATTCATACACAACCAAGGTAACTAAGGTAAGAGTTTTTTTTTTTTTTTAATCTTACAAGAAATGCTCGAATCTTTAACAAAAATGAGTGGGGCTATAGGGGAAAGTGAGGTCAAGGCACTATGGTGTGCATGCTTGCATTTGTTTCCTCCGTCCATTCAAAGTGAGAATGCTCCCATTTTCTTACTTTACCATTGATGTGCTACAAGCTTATTTATTTTAAGACTAACCTAGCCTAAAAATCAACTGTCCCCACAAAATAAAAATCACATTAAAAAAACTAATAGTGTTCAGACTAATCTTGCTCAAACTTATGTTTCCCTAGTCTTGATGCAACTGATTGAGTCACCTGGGGAGTTGGTTATAAACCTGGGCAGAGACCCCAAATGCAATGGCTCAGAGAAGATAGGAGCTTATTTCTGTCTTATGCAATAGTCAGAATGGGTTTTACAGACTGGTGAGTAGCTCAACATCTCACAGTCATTCAGGCACCCATGTTCCTCCCATTTTGTTTCTCTGCCACCCCTTAAGGACTTGCCCTGACTGCATGATTATTGCCGTGTTGCCTCAAACAGGTTGCAGCTTATGGGAAGCAAAAACACGGTATGGTAGAAGCTCTCCCATAGACTGATGGCTTGGCTCAAGAGTGGCCGACTTTATTTCTGTACATATCCCACTGGATAGAATTTAGTCAATCCTAACTGCAGAGGGAGCCAGGGAACACAGCCCAGGCATGTGCCTAGGAAGGGGAGAATGGGTTTAGGTTGACACTTAGCAGCTGCCACTATATGTGGCTATAGTATGTATCATTGGAATAGATGTTTAACTTTAGGGACAAATAAAAAACCAAAACAAAAAAAGGAGTAAGGGGAGAGATTTGCAGCAAATCTTTATTTTTACCAACCTCAACTATCATTAATTTCAGTGAACCCTAAATGGTATCCAACAAAATATCTTTCTAGACCATTCACCGTCTCTGCCTCATAGATGATCATATCATGTTTTCTTCTCTTCTGAAACCTCTAATACCCTTGTCCTATCCTCATTCTAAGCTGATGACCTTACTTCCTATTTCACAAAAATAATAGAAAAAAAAAAAAAAAA

The glutinous fat 3 of homo sapiens, cDNA gi|21752911|dbj|AK093948.1| homo sapiens cDNA FLJ36629 (SEQ ID N0:40)

ATAGCCTTTCAAATTTCGGTTAATGGTAACTCTCATCAGTTACTCAAGCCAAAAATCTTGGATTCATCACAGACTCCTCTCTTTCACTAATCTCTTCTTCCCCACATCTAATCCAAGAGGAAATCCTATTGTTCTACCTTCATTGGCCAGGCACTGTGACTCATATAATCCCAGCACATTGGGAGGCTGAGGCGGGAGGATGGCTTGAGCCCAGGAGTTCCAGACCAGCCTGGGAAACATAGTGAGGCCCCATCTCTACAAAAAATTAAAAAAATTAGCAGTCAAGGTGGCATGTGTCTGTAATCCAAGCTACTTGGGGGGCTGAGGTGGGAGGATTTCTTGAGCCCGGGAAGTCGAGGCTGCATTGAGCCACGGTTGTGCCACGGCACTCCAGCCTGGGTGACAGAGTGAGACCCCTGTTTCCAAAAAAAAAAAAAAAAAAAAAGAATAAAAATCACCTGGTCAACTCCACTATTACCAGCCTGGTCCAAGCTACTATCTCTCATTTATATTATTGCAATAGCCTCCTCACTCCTCCAACAACCTGCTGTCAACCACAGCAGCCAACATCTGATCATATCACTTCTGTTTGTGGTTCTCAAATCTCCCCAATTGAGTTACAGTAAAAGACAAACTTGGTGAGTGCCACCTTATCTCTATAACTGTATACCCTTTCTATTGCTCACTCCAGCCAGATGCAATATCCTTGCCAAGCACCCTCCTGTCTCAGGGCCTTTGCACTTGCCAGTCCCTGTGCCTGGAAGGCTTCTCCCCTAGATTTTTGCATGACTTCTCCCTCCCTCCCTTCAGATCTTTGCTCAAATGCCTTCTTTTTAGTGTATGTAAAATGACAAACCCATACCCATTCCTTATCCCCTCCTCTGAATTTTCTCTTCAGCAATTATCAGCAGCAAGTGTCCCAAAGTTTCTATTAACTTATTTCTGTTGTCTCTTTCTTCCCTCCACTAGAATGTAAGCTTTATGAGAGCAGAGACTTTTGTTTGTTCACTGCTTTATCCTTAGCACCTAAAACAGTGCCTTACTCATAGTTACCTCAATATTTATTGCCAAATGAATTTCTGCTTTATAATCTGATTATATTTTTCCACTCTCTCTTAGAGTCTAACTAGTTATGATGTCTGTAGCATACTTCTTGGGACTTCTACCATGCTTGTGTGGCTTGGAGTCATCCGATACCTCGGTTTCTTTGCAAAGTACAACCTCCTCATTTTGACCCTTCAGGCAGCGCTGCCCAATGTCATCAGGTTCTGCTGCTGTGCAGCTATGATTTACTTAGGTTACTGCTTCTGTGGATGGATCGTGCTGGGGCCTTACCATGACAAGTTTCGTTCTCTGAACATGGTCTCTGAGTGCCTTTTCTCTCTGATAAATGGAGATGATATGTTTGCCACGTTTGCAAAAATGCAGCAAAAAAGTTACTTAGTCTGGCTGTTTAGTAGAATTTACCTCTACTCATTCATCAGCCTCTTTATATATATGATTTTAAGTCTTTTCATTGCACTGATCACTGATACATACGAAACAATTAAGCAATACCAACAAGATGGCTTCCCAGAGACTGAACTTCGTACATTTATATCAGAATGCAAAGATCTACCCAACTCTGGAAAATACAGATTAGAAGATGACCCTCCAGTAACTTTATTCTGCTGTTGTAAAAAGTAGCTATCAGGTTTATCTGTACTTTAGAGGAAAATATAATGTGTAGCTGAGTTGGAACACTGTGGATATTCTGAGATCAGATGTAGTATGTTTGAAGACTGTTATTTTGAGCTAATTGAGACCTATAATTCACCAATAACTGTTTATATTTTTAAAAGCAATATTTAATGTCTTTGCAACTTTATGCTGGGATTGTTTTTAAAAAAAACTTTAATGAGGAAAGCTATTGGATTATTATTATTTCTTGTTTATTTTGCCATGGCTTTAGAATGTATTCTGTATGCCTCTCTTTTGCTCTGATACTGTTGCTCCTGCTATTCTGATTGTGCAGACTGTATAATTAGTGGAAAACAATCCTTGGTCTGACTGTGACTTTGGACAACTCAGTAACCCTGGCTTGGACCACTCTCAGGAGTCCATCCTTGAGAGAGTGGGTGTAGTTACCATTTATACAGTAATCATTGCATTTTAAAATCTTCTCTTGAAAGGAAGAATAAGAGTGCACCAGAATAAGAGCGCACCAGAATAAGAGCACACCAGCTAACAATGTGATACGGCCATATGTCACTTAAGGATGGAGATATGTTCTGAGAAATGTGTCATTAGGCGATTTTGTCATTAAACATCATAGCATGTACTTCCACAAACCTAGATGGTATAGCCTACTACACACCTAGGCTATTTGGTATAGCCTGTTGGTCCTGGGGTACAAATCTGTACAACATGTTACTGTATTGAATACAGTAGGCAATTGTAACTCAATGGTAAGTATCTAAACATAGAAAAGGGACAGTAAAAATATGGTTTTATAATCTTCTGGGACCACCATTGTATATGCGGTACATCATTGACCAAAACATCGTTATCCAGCATATGACTGTATTTGGTTATGAAAGCCAACTGTTACTTGATTCTGCTTTTAGTTCTTAAGAGGATCAGGCTTTTAAATACTCATTTACAAGCTTTCTATCCTCCTTCAGTGTTAAAGTAGAAAGTAAAAAGAGTATCTTATACATGCATGAAATTAAAGCATATACCAAATGC

Sequence table

<110>Moyer，Bryan

Zlotnik，Albert

Hevezi，Peter

Soto，Hortensia

Kalabat，Dalia

Lu，Min

Gao，Na

White，Evan?Carl

Servant，Guy

Brust，Paul

Williams，Mark

 

＜120〉TRPML3 (MC0LN3) is as the evaluation of saline taste acceptor and in the purposes of the mensuration of the sense of taste (saline taste) conditioning agent of generation that is used for identifying regulation and control sodium transport, absorption or drainage and/or aldosterone and/or vasopressing or release and/or therapeutical agent

 

<130>67824.703302

 

<140>PCT/US08/07101

<141>2008-06-06

 

<150>11/808,356

<151>2007-06-08

 

<150>12/134,302

<151>2008-06-06

 

<150>60/929,007

<151>2007-06-08

 

<150>60/929,017

<151>2007-06-08

 

<150>60/935,297

<151>2007-08-03

 

<150>60/947,052

<151>2007-06-29

 

<150>60/987,611

<151>2007-11-13

 

<150>60/988,938

<151>2007-11-19

 

<150>60/991,274

<151>2007-11-30

 

<150>60/991,289

<151>2007-11-30

 

<150>60/992,502

<151>2007-12-05

 

<150>60/992,517

<151>2007-12-05

 

<150>61/017,244

<151>2007-12-28

 

<150>61/021,437

<151>2008-01-16

 

<150>61/043,257

<151>2008-04-08

 

<150>61/053,310

<151>2008-05-15

 

<160>40

 

<170>PatentIn?version?3.5

 

<210>1

<211>1659

<212>DNA

＜213〉homo sapiens (Homo sapiens)

 

<400>1

atggcagatc?ctgaggtagt?tgtgagtagc?tgcagctctc?atgaagagga?aaatcgctgc 60

aattttaacc?agcaaacatc?tccatctgag?gagcttctat?tagaagacca?gatgaggcga 120

aaactcaaat?tttttttcat?gaatccctgt?gagaagttct?gggctcgagg?tagaaaacca 180

tggaaacttg?ccatacaaat?tctaaaaatt?gcaatggtga?ctatccagct?ggtcttattt 240

gggctaagta?accagatggt?ggtagctttc?aaggaagaga?atactatagc?attcaaacac 300

cttttcctaa?aaggatatat?ggaccgaatg?gatgacacat?atgcagtgta?cacacaaagt 360

gacgtgtatg?atcagttaat?cttcgcagta?aaccagtact?tgcagctata?caatgtctcc 420

gttgggaatc?atgcttatga?gaacaaaggt?accaagcaat?ctgctatggc?aatctgtcag 480

cacttctaca?agcgaggaaa?catctaccct?ggaaatgata?cctttgacat?cgatccagaa 540

attgaaactg?agtgtttctt?tgtggagcca?gatgaacctt?ttcacattgg?gacaccagca 600

gaaaataaac?tgaacttaac?actggacttc?cacagactcc?taacagtgga?gcttcagttt 660

aaactgaaag?ccattaatct?gcagacagtt?cgtcatcaag?aactccctga?ctgttatgac 720

tttactctga?ctataacatt?tgacaacaag?gcccatagtg?gaagaattaa?aataagttta 780

gataatgaca?tttccatcag?agaatgtaaa?gactggcatg?tatctggatc?aattcagaag 840

aacactcatt?acatgatgat?ctttgatgcc?tttgtcattc?tgacttgctt?ggtttcatta 900

atcctctgca?ttagatctgt?gattagagga?cttcagcttc?agcaggagtt?tgtcaatttt 960

ttcctcctcc?attataagaa?ggaagtttct?gtttctgatc?aaatggaatt?tgtcaatgga 1020

tggtacatta?tgattattat?tagtgacata?ttgacaatca?ttggatcaat?tctaaaaatg 1080

gaaatccaag?ctaagagtct?aactagttat?gatgtctgta?gcatacttct?tgggacttct 1140

accatgctcg?tgtggcttgg?agtcatccga?tacctcggtt?tctttgcaaa?gtacaacctc 1200

ctcattttga?cccttcaggc?agcgctgccc?aatgtcatca?ggttctgctg?ctgtgcagct 1260

atgatttact?taggttactg?cttctgtgga?tggatcgtgc?tggggcctta?ccatgacaag 1320

tttcgttctc?tgaacatggt?ctctgagtgc?cttttctctc?tgataaatgg?agatgatatg 1380

tttgccacgt?ttgcaaaaat?gcagcaaaaa?agttacttag?tctggctgtt?tagtagaatt 1440

tacctctact?cattcatcag?cctctttata?tatatgattt?taagtctttt?cattgcactg 1500

atcactgata?catacgaaac?aattaagcaa?taccaacaag?atggcttccc?agagactgaa 1560

cttcgtacat?ttatatcaga?atgcaaagat?ctacccaact?ctggaaaata?cagattagaa 1620

gatgaccctc?cagtatcttt?attctgctgt?tgtaaaaag 1659

 

<210>2

<211>553

<212>PRT

＜213〉homo sapiens

 

<400>2

 

Met?Ala?Asp?Pro?Glu?Val?Val?Val?Ser?Ser?Cys?Ser?Ser?His?Glu?Glu

1 5 10 15

 

Glu?Asn?Arg?Cys?Asn?Phe?Asn?Gln?Gln?Thr?Ser?Pro?Ser?Glu?Glu?Leu

20 25 30

Leu?Leu?Glu?Asp?Gln?Met?Arg?Arg?Lys?Leu?Lys?Phe?Phe?Phe?Met?Asn

35 40 45

 

Pro?Cys?Glu?Lys?Phe?Trp?Ala?Arg?Gly?Arg?Lys?Pro?Trp?Lys?Leu?Ala

50 55 60

 

Ile?Gln?Ile?Leu?Lys?Ile?Ala?Met?Val?Thr?Ile?Gln?Leu?Val?Leu?Phe

65 70 75 80

 

Gly?Leu?Ser?Asn?Gln?Met?Val?Val?Ala?Phe?Lys?Glu?Glu?Asn?Thr?Ile

85 90 95

 

Ala?Phe?Lys?His?Leu?Phe?Leu?Lys?Gly?Tyr?Met?Asp?Arg?Met?Asp?Asp

100 105 110

 

Thr?Tyr?Ala?Val?Tyr?Thr?Gln?Ser?Asp?Val?Tyr?Asp?Gln?Leu?Ile?Phe

115 120 125

 

Ala?Val?Asn?Gln?Tyr?Leu?Gln?Leu?Tyr?Asn?Val?Ser?Val?Gly?Asn?His

130 135 140

 

Ala?Tyr?Glu?Asn?Lys?Gly?Thr?Lys?Gln?Ser?Ala?Met?Ala?Ile?Cys?Gln

145 150 155 160

 

His?Phe?Tyr?Lys?Arg?Gly?Asn?Ile?Tyr?Pro?Gly?Asn?Asp?Thr?Phe?Asp

165 170 175

 

Ile?Asp?Pro?Glu?Ile?Glu?Thr?Glu?Cys?Phe?Phe?Val?Glu?Pro?Asp?Glu

180 185 190

 

Pro?Phe?His?Ile?Gly?Thr?Pro?Ala?Glu?Asn?Lys?Leu?Asn?Leu?Thr?Leu

195 200 205

 

Asp?Phe?His?Arg?Leu?Leu?Thr?Val?Glu?Leu?Gln?Phe?Lys?Leu?Lys?Ala

210 215 220

 

Ile?Asn?Leu?Gln?Thr?Val?Arg?His?Gln?Glu?Leu?Pro?Asp?Cys?Tyr?Asp

225 230 235 240

 

Phe?Thr?Leu?Thr?Ile?Thr?Phe?Asp?Asn?Lys?Ala?His?Ser?Gly?Arg?Ile

245 250 255

 

Lys?Ile?Ser?Leu?Asp?Asn?Asp?Ile?Ser?Ile?Arg?Glu?Cys?Lys?Asp?Trp

260 265 270

 

His?Val?Ser?Gly?Ser?Ile?Gln?Lys?Asn?Thr?His?Tyr?Met?Met?Ile?Phe

275 280 285

 

Asp?Ala?Phe?Val?Ile?Leu?Thr?Cys?Leu?Val?Ser?Leu?Ile?Leu?Cys?Ile

290 295 300

 

Arg?Ser?Val?Ile?Arg?Gly?Leu?Gln?Leu?Gln?Gln?Glu?Phe?Val?Asn?Phe

305 310 315 320

 

Phe?Leu?Leu?His?Tyr?Lys?Lys?Glu?Val?Ser?Val?Ser?Asp?Gln?Met?Glu

325 330 335

Phe?Val?Asn?Gly?Trp?Tyr?Ile?Met?Ile?Ile?Ile?Ser?Asp?Ile?Leu?Thr

340 345 350

 

Ile?Ile?Gly?Ser?Ile?Leu?Lys?Met?Glu?Ile?Gln?Ala?Lys?Ser?Leu?Thr

355 360 365

 

Ser?Tyr?Asp?Val?Cys?Ser?Ile?Leu?Leu?Gly?Thr?Ser?Thr?Met?Leu?Val

370 375 380

 

Trp?Leu?Gly?Val?Ile?Arg?Tyr?Leu?Gly?Phe?Phe?Ala?Lys?Tyr?Asn?Leu

385 390 395 400

 

Leu?Ile?Leu?Thr?Leu?Gln?Ala?Ala?Leu?Pro?Asn?Val?Ile?Arg?Phe?Cys

405 410 415

 

Cys?Cys?Ala?Ala?Met?Ile?Tyr?Leu?Gly?Tyr?Cys?Phe?Cys?Gly?Trp?Ile

420 425 430

 

Val?Leu?Gly?Pro?Tyr?His?Asp?Lys?Phe?Arg?Ser?Leu?Asn?Met?Val?Ser

435 440 445

 

Glu?Cys?Leu?Phe?Ser?Leu?Ile?Asn?Gly?Asp?Asp?Met?Phe?Ala?Thr?Phe

450 455 460

 

Ala?Lys?Met?Gln?Gln?Lys?Ser?Tyr?Leu?Val?Trp?Leu?Phe?Ser?Arg?Ile

465 470 475 480

 

Tyr?Leu?Tyr?Ser?Phe?Ile?Ser?Leu?Phe?Ile?Tyr?Met?Ile?Leu?Ser?Leu

485 490 495

 

Phe?Ile?Ala?Leu?Ile?Thr?Asp?Thr?Tyr?Glu?Thr?Ile?Lys?Gln?Tyr?Gln

500 505 510

 

Gln?Asp?Gly?Phe?Pro?Glu?Thr?Glu?Leu?Arg?Thr?Phe?Ile?Ser?Glu?Cys

515 520 525

 

Lys?Asp?Leu?Pro?Asn?Ser?Gly?Lys?Tyr?Arg?Leu?Glu?Asp?Asp?Pro?Pro

530 535 540

 

Val?Ser?Leu?Phe?Cys?Cys?Cys?Lys?Lys

545 550

 

<210>3

<211>1659

<212>DNA

＜213〉homo sapiens

 

<400>3

atggcagatc?ctgaggtagt?tgtgagtagc?tgcagctctc?atgaagagga?aaatcgctgc 60

aattttaacc?agcaaacatc?tccatctgag?gagcttctat?tagaagacca?gatgaggcga 120

aaactcaaat?tttttttcat?gaatccctgt?gagaagttct?gggctcgagg?tagaaaacca 180

tggaaacttg?ccatacaaat?tctaaaaatt?gcaatggtga?ctatccagct?ggtcttattt 240

gggctaagta?accagatggt?ggtagctttc?aaggaagaga?atactatagc?attcaaacac 300

cttttcctaa?aaggatatat?ggaccgaatg?gatgacacat?atgcagtgta?cacacaaagt 360

gacgtgtatg?atcagttaat?cttcgcagta?aaccagtact?tgcagctata?caatgtctcc 420

gttgggaatc?atgcttatga?gaacaaaggt?accaagcaat?ctgctatggc?aatctgtcag 480

cacttctaca?agcgaggaaa?catctaccct?ggaaatgata?cctttgacat?cgatccagaa 540

attgaaactg?agtgtttctt?tgtggagcca?gatgaacctt?ttcacattgg?gacaccagca 600

gaaaataaac?tgaacttaac?actggacttc?cacagactcc?taacagtgga?gcttcagttt 660

aaactgaaag?ccattaatct?gcagacagtt?cgtcatcaag?aactccctga?ctgttatgac 720

tttactctga?ctataacatt?tgacaacaag?gcccatagtg?gaagaattaa?aataagttta 780

gataatgaca?tttccatcag?agaatgtaaa?gactggcatg?tatctggatc?aattcagaag 840

aacactcatt?acatgatgat?ctttgatgcc?tttgtcattc?tgacttgctt?ggtttcatta 900

atcctctgca?ttagatctgt?gattagagga?cttcagcttc?agcaggagtt?tgtcaatttt 960

ttcctcctcc?attataagaa?ggaagtttct?gtttctgatc?aaatggaatt?tgtcaatgga 1020

tggtacatta?tgattattat?tagtgacata?ttgacaatca?ttggatcaat?tctaaaaatg 1080

gaaatccaag?ctaagagtct?aactagttat?gatgtctgta?gcatacttct?tgggacttct 1140

accatgctcg?tgtggcttgg?agtcatccga?tacctcggtt?tctttgcaaa?gtacaacctc 1200

ctcattttga?cccttcaggc?agcgctgccc?aatgtcatca?ggttctgctg?ctgtccagct 1260

atgatttact?taggttactg?cttctgtgga?tggatcgtgc?tggggcctta?ccatgacaag 1320

tttcgttctc?tgaacatggt?ctctgagtgc?cttttctctc?tgataaatgg?agatgatatg 1380

tttgccacgt?ttgcaaaaat?gcagcaaaaa?agttacttag?tctggctgtt?tagtagaatt 1440

tacctctact?cattcatcag?cctctttata?tatatgattt?taagtctttt?cattgcactg 1500

atcactgata?catacgaaac?aattaagcaa?taccaacaag?atggcttccc?agagactgaa 1560

cttcgtacat?ttatatcaga?atgcaaagat?ctacccaact?ctggaaaata?cagattagaa 1620

gatgaccctc?cagtatcttt?attctgctgt?tgtaaaaag 1659

<210>4

<211>553

<212>PRT

＜213〉homo sapiens

 

<400>4

Met?Ala?Asp?Pro?Glu?Val?Val?Val?Ser?Ser?Cys?Ser?Ser?His?Glu?Glu

1 5 10 15

 

Glu?Asn?Arg?Cys?Asn?Phe?Asn?Gln?Gln?Thr?Ser?Pro?Ser?Glu?Glu?Leu

20 25 30

 

Leu?Leu?Glu?Asp?Gln?Met?Arg?Arg?Lys?Leu?Lys?Phe?Phe?Phe?Met?Asn

35 40 45

 

Pro?Cys?Glu?Lys?Phe?Trp?Ala?Arg?Gly?Arg?Lys?Pro?Trp?Lys?Leu?Ala

50 55 60

 

Ile?Gln?Ile?Leu?Lys?Ile?Ala?Met?Val?Thr?Ile?Gln?Leu?Val?Leu?Phe

65 70 75 80

 

Gly?Leu?Ser?Asn?Gln?Met?Val?Val?Ala?Phe?Lys?Glu?Glu?Asn?Thr?Ile

85 90 95

 

Ala?Phe?Lys?His?Leu?Phe?Leu?Lys?Gly?Tyr?Met?Asp?Arg?Met?Asp?Asp

100 105 110

 

Thr?Tyr?Ala?Val?Tyr?Thr?Gln?Ser?Asp?Val?Tyr?Asp?Gln?Leu?Ile?Phe

115 120 125

 

Ala?Val?Asn?Gln?Tyr?Leu?Gln?Leu?Tyr?Asn?Val?Ser?Val?Gly?Asn?His

130 135 140

 

Ala?Tyr?Glu?Asn?Lys?Gly?Thr?Lys?Gln?Ser?Ala?Met?Ala?Ile?Cys?Gln

145 150 155 160

 

His?Phe?Tyr?Lys?Arg?Gly?Asn?Ile?Tyr?Pro?Gly?Asn?Asp?Thr?Phe?Asp

165 170 175

 

Ile?Asp?Pro?Glu?Ile?Glu?Thr?Glu?Cys?Phe?Phe?Val?Glu?Pro?Asp?Glu

180 185 190

 

Pro?Phe?His?Ile?Gly?Thr?Pro?Ala?Glu?Asn?Lys?Leu?Asn?Leu?Thr?Leu

195 200 205

 

Asp?Phe?His?Arg?Leu?Leu?Thr?Val?Glu?Leu?Gln?Phe?Lys?Leu?Lys?Ala

210 215 220

 

Ile?Asn?Leu?Gln?Thr?Val?Arg?His?Gln?Glu?Leu?Pro?Asp?Cys?Tyr?Asp

225 230 235 240

 

Phe?Thr?Leu?Thr?Ile?Thr?Phe?Asp?Asn?Lys?Ala?His?Ser?Gly?Arg?Ile

245 250 255

 

Lys?Ile?Ser?Leu?Asp?Asn?Asp?Ile?Ser?Ile?Arg?Glu?Cys?Lys?Asp?Trp

260 265 270

 

His?Val?Ser?Gly?Ser?Ile?Gln?Lys?Asn?Thr?His?Tyr?Met?Met?Ile?Phe

275 280 285

 

Asp?Ala?Phe?Val?Ile?Leu?Thr?Cys?Leu?Val?Ser?Leu?Ile?Leu?Cys?Ile

290 295 300

 

Arg?Ser?Val?Ile?Arg?Gly?Leu?Gln?Leu?Gln?Gln?Glu?Phe?Val?Asn?Phe

305 310 315 320

 

Phe?Leu?Leu?His?Tyr?Lys?Lys?Glu?Val?Ser?Val?Ser?Asp?Gln?Met?Glu

325 330 335

 

Phe?Val?Asn?Gly?Trp?Tyr?Ile?Met?Ile?Ile?Ile?Ser?Asp?Ile?Leu?Thr

340 345 350

 

Ile?Ile?Gly?Ser?Ile?Leu?Lys?Met?Glu?Ile?Gln?Ala?Lys?Ser?Leu?Thr

355 360 365

 

Ser?Tyr?Asp?Val?Cys?Ser?Ile?Leu?Leu?Gly?Thr?Ser?Thr?Met?Leu?Val

370 375 380

 

Trp?Leu?Gly?Val?Ile?Arg?Tyr?Leu?Gly?Phe?Phe?Ala?Lys?Tyr?Asn?Leu

385 390 395 400

 

Leu?Ile?Leu?Thr?Leu?Gln?Ala?Ala?Leu?Pro?Asn?Val?Ile?Arg?Phe?Cys

405 410 415

 

Cys?Cys?Pro?Ala?Met?Ile?Tyr?Leu?Gly?Tyr?Cys?Phe?Cys?Gly?Trp?Ile

420 425 430

 

Val?Leu?Gly?Pro?Tyr?His?Asp?Lys?Phe?Arg?Ser?Leu?Asn?Met?Val?Ser

435 440 445

 

Glu?Cys?Leu?Phe?Ser?Leu?Ile?Asn?Gly?Asp?Asp?Met?Phe?Ala?Thr?Phe

450 455 460

 

Ala?Lys?Met?Gln?Gln?Lys?Ser?Tyr?Leu?Val?Trp?Leu?Phe?Ser?Arg?Ile

465 470 475 480

 

Tyr?Leu?Tyr?Ser?Phe?Ile?Ser?Leu?Phe?Ile?Tyr?Met?Ile?Leu?Ser?Leu

485 490 495

 

Phe?Ile?Ala?Leu?Ile?Thr?Asp?Thr?Tyr?Glu?Thr?Ile?Lys?Gln?Tyr?Gln

500 505 510

 

Gln?Asp?Gly?Phe?Pro?Glu?Thr?Glu?Leu?Arg?Thr?Phe?Ile?Ser?Glu?Cys

515 520 525

 

Lys?Asp?Leu?Pro?Asn?Ser?Gly?Lys?Tyr?Arg?Leu?Glu?Asp?Asp?Pro?Pro

530 535 540

 

Val?Ser?Leu?Phe?Cys?Cys?Cys?Lys?Lys

545 550

 

<210>5

<211>1740

<212>DNA

＜213〉homo sapiens

 

<400>5

atgacagccc?cggcgggtcc?gcgcggctca?gagaccgagc?ggcttctgac?ccccaacccc 60

gggtatggga?cccaggcggg?gccttcaccg?gcccctccga?cacccccaga?agaggaagac 120

cttcgccgtc?gtctcaaata?ctttttcatg?agtccctgcg?acaagtttcg?agccaagggc 180

cgcaagccct?gcaagctgat?gctgcaagtg?gtcaagatcc?tggtggtcac?ggtgcagctc 240

atcctgtttg?ggctcagtaa?tcagctggct?gtgacattcc?gggaagagaa?caccatcgcc 300

ttccgacacc?tcttcctgct?gggctactcg?gacggagcgg?atgacacctt?cgcagcctac 360

acgcgggagc?agctgtacca?ggccatcttc?catgctgtgg?accagtacct?ggcgttgcct 420

gacgtgtcac?tgggccggta?tgcgtatgtc?cgtggtgggg?gtgacccttg?gaccaatggc 480

tcagggcttg?ctctctgcca?gcggtactac?caccgaggcc?acgtggaccc?ggccaacgac 540

acatttgaca?ttgatccgat?ggtggttact?gactgcatcc?aggtggatcc?ccccgagcgg 600

ccccctccgc?cccccagcga?cgatctcacc?ctcttggaaa?gcagctccag?ttacaagaac 660

ctcacgctca?aattccacaa?gctggtcaat?gtcaccatcc?acttccggct?gaagaccatt 720

aacctccaga?gcctcatcaa?taatgagatc?ccggactgct?ataccttcag?cgtcctgatc 780

acgtttgaca?acaaagcaca?cagtgggcgg?atccccatca?gcctggagac?ccaggcccac 840

atccaggagt?gtaagcaccc?cagtgtcttc?cagcacggag?acaacagctt?ccggctcctg 900

tttgacgtgg?tggtcatcct?cacctgctcc?ctgtccttcc?tcctctgcgc?ccgctcactc 960

cttcgaggct?tcctgctgca?gaacgagttt?gtggggttca?tgtggcggca?gcggggacgg 1020

gtcatcagcc?tgtgggagcg?gctggaattt?gtcaatggct?ggtacatcct?gctcgtcacc 1080

agcgatgtgc?tcaccatctc?gggcaccatc?atgaagatcg?gcatcgaggc?caagaacttg 1140

gcgagctacg?acgtctgcag?catcctcctg?ggcacctcga?cgctgctggt?gtgggtgggc 1200

gtgatccgct?acctgacctt?cttccacaac?tacaatatcc?tcatcgccac?actgcgggtg 1260

gccctgccca?gcgtcatgcg?cttctgctgc?tgcgtggctg?tcatctacct?gggctactgc 1320

ttctgtggct?ggatcgtgct?ggggccctat?catgtgaagt?tccgctcact?ctccatggtg 1380

tctgagtgcc?tgttctcgct?catcaatggg?gacgacatgt?ttgtgacgtt?cgccgccatg 1440

caggcgcagc?agggccgcag?cagcctggtg?tggctcttct?cccagctcta?cctttactcc 1500

ttcatcagcc?tcttcatcta?catggtgctc?agcctcttca?tcgcgctcat?caccggcgcc 1560

tacgacacca?tcaagcatcc?cggcggcgca?ggcgcagagg?agagcgagct?gcaggcctac 1620

atcgcacagt?gccaggacag?ccccacctcc?ggcaagttcc?gccgcgggag?cggctcggcc 1680

tgcagccttc?tctgctgctg?cggaagggac?ccctcggagg?agcattcgct?gctggtgaat 1740

 

<210>6

<211>580

<212>PRT

＜213〉homo sapiens

 

<400>6

 

Met?Thr?Ala?Pro?Ala?Gly?Pro?Arg?Gly?Ser?Glu?Thr?Glu?Arg?Leu?Leu

1 5 10 15

 

Thr?Pro?Asn?Pro?Gly?Tyr?Gly?Thr?Gln?Ala?Gly?Pro?Ser?Pro?Ala?Pro

20 25 30

 

Pro?Thr?Pro?Pro?Glu?Glu?Glu?Asp?Leu?Arg?Arg?Arg?Leu?Lys?Tyr?Phe

35 40 45

 

Phe?Met?Ser?Pro?Cys?Asp?Lys?Phe?Arg?Ala?Lys?Gly?Arg?Lys?Pro?Cys

50 55 60

 

Lys?Leu?Met?Leu?Gln?Val?Val?Lys?Ile?Leu?Val?Val?Thr?Val?Gln?Leu

65 70 75 80

 

Ile?Leu?Phe?Gly?Leu?Ser?Asn?Gln?Leu?Ala?Val?Thr?Phe?Arg?Glu?Glu

85 90 95

 

Asn?Thr?Ile?Ala?Phe?Arg?His?Leu?Phe?Leu?Leu?Gly?Tyr?Ser?Asp?Gly

100 105 110

 

Ala?Asp?Asp?Thr?Phe?Ala?Ala?Tyr?Thr?Arg?Glu?Gln?Leu?Tyr?Gln?Ala

115 120 125

 

Ile?Phe?His?Ala?Val?Asp?Gln?Tyr?Leu?Ala?Leu?Pro?Asp?Val?Ser?Leu

130 135 140

 

Gly?Arg?Tyr?Ala?Tyr?Val?Arg?Gly?Gly?Gly?Asp?Pro?Trp?Thr?Asn?Gly

145 150 155 160

 

Ser?Gly?Leu?Ala?Leu?Cys?Gln?Arg?Tyr?Tyr?His?Arg?Gly?His?Val?Asp

165 170 175

 

Pro?Ala?Asn?Asp?Thr?Phe?Asp?Ile?Asp?Pro?Met?Val?Val?Thr?Asp?Cys

180 185 190

 

Ile?Gln?Val?Asp?Pro?Pro?Glu?Arg?Pro?Pro?Pro?Pro?Pro?Ser?Asp?Asp

195 200 205

 

Leu?Thr?Leu?Leu?Glu?Ser?Ser?Ser?Ser?Tyr?Lys?Asn?Leu?Thr?Leu?Lys

210 215 220

 

Phe?His?Lys?Leu?Val?Asn?Val?Thr?Ile?His?Phe?Arg?Leu?Lys?Thr?Ile

225 230 235 240

 

Asn?Leu?Gln?Ser?Leu?Ile?Asn?Asn?Glu?Ile?Pro?Asp?Cys?Tyr?Thr?Phe

245 250 255

 

Ser?Val?Leu?Ile?Thr?Phe?Asp?Asn?Lys?Ala?His?Ser?Gly?Arg?Ile?Pro

260 265 270

 

Ile?Ser?Leu?Glu?Thr?Gln?Ala?His?Ile?Gln?Glu?Cys?Lys?His?Pro?Ser

275 280 285

 

Val?Phe?Gln?His?Gly?Asp?Asn?Ser?Phe?Arg?Leu?Leu?Phe?Asp?Val?Val

290 295 300

 

Val?Ile?Leu?Thr?Cys?Ser?Leu?Ser?Phe?Leu?Leu?Cys?Ala?Arg?Ser?Leu

305 310 315 320

 

Leu?Arg?Gly?Phe?Leu?Leu?Gln?Asn?Glu?Phe?Val?Gly?Phe?Met?Trp?Arg

325 330 335

 

Gln?Arg?Gly?Arg?Val?Ile?Ser?Leu?Trp?Glu?Arg?Leu?Glu?Phe?Val?Asn

340 345 350

 

Gly?Trp?Tyr?Ile?Leu?Leu?Val?Thr?Ser?Asp?Val?Leu?Thr?Ile?Ser?Gly

355 360 365

 

Thr?Ile?Met?Lys?Ile?Gly?Ile?Glu?Ala?Lys?Asn?Leu?Ala?Ser?Tyr?Asp

370 375 380

 

Val?Cys?Ser?Ile?Leu?Leu?Gly?Thr?Ser?Thr?Leu?Leu?Val?Trp?Val?Gly

385 390 395 400

 

Val?Ile?Arg?Tyr?Leu?Thr?Phe?Phe?His?Asn?Tyr?Asn?Ile?Leu?Ile?Ala

405 410 415

 

Thr?Leu?Arg?Val?Ala?Leu?Pro?Ser?Val?Met?Arg?Phe?Cys?Cys?Cys?Val

420 425 430

 

Ala?Val?Ile?Tyr?Leu?Gly?Tyr?Cys?Phe?Cys?Gly?Trp?Ile?Val?Leu?Gly

435 440 445

 

Pro?Tyr?His?Val?Lys?Phe?Arg?Ser?Leu?Ser?Met?Val?Ser?Glu?Cys?Leu

450 455 460

 

Phe?Ser?Leu?Ile?Asn?Gly?Asp?Asp?Met?Phe?Val?Thr?Phe?Ala?Ala?Met

465 470 475 480

 

Gln?Ala?Gln?Gln?Gly?Arg?Ser?Ser?Leu?Val?Trp?Leu?Phe?Ser?Gln?Leu

485 490 495

 

Tyr?Leu?Tyr?Ser?Phe?Ile?Ser?Leu?Phe?Ile?Tyr?Met?Val?Leu?Ser?Leu

500 505 510

 

Phe?Ile?Ala?Leu?Ile?Thr?Gly?Ala?Tyr?Asp?Thr?Ile?Lys?His?Pro?Gly

515 520 525

 

Gly?Ala?Gly?Ala?Glu?Glu?Ser?Glu?Leu?Gln?Ala?Tyr?Ile?Ala?Gln?Cys

530 535 540

 

Gln?Asp?Ser?Pro?Thr?Ser?Gly?Lys?Phe?Arg?Arg?Gly?Ser?Gly?Ser?Ala

545 550 555 560

 

Cys?Ser?Leu?Leu?Cys?Cys?Cys?Gly?Arg?Asp?Pro?Ser?Glu?Glu?His?Ser

565 570 575

 

Leu?Leu?Val?Asn

580

 

<210>7

<211>1698

<212>DNA

＜213〉homo sapiens

 

<400>7

atggcccggc?agccttatcg?ttttccccag?gcaaggattc?cggagagagg?atcaggtgtt 60

ttcaggttaa?ccgtcagaaa?cgcaatggca?catcgtgatt?ctgagatgaa?agaagaatgt 120

ctaagggaag?acctgaagtt?ttacttcatg?agcccttgtg?aaaaataccg?agccagacgc 180

cagattccgt?ggaaactggg?tttgcagatt?ttgaagatag?tcatggtcac?cacacagctt 240

gttcgttttg?gtttaagtaa?ccagctggtg?gttgctttca?aagaagataa?cactgttgct 300

tttaagcact?tgtttttgaa?aggatattct?ggtacagatg?aagatgacta?cagctgcagt 360

gtatatactc?aagaggatgc?ctatgagagc?atcttttttg?ctattaatca?gtatcatcag 420

ctaaaggaca?ttaccctggg?gacccttggt?tatggagaaa?atgaagacaa?tagaattggc 480

ttaaaagtct?gtaagcagca?ttacaagaaa?gggaccatgt?ttccttctaa?tgagacactg 540

aatattgaca?acgacgttga?gctcgattgt?gttcaattag?accttcagga?cctctccaag 600

aagcctccgg?actggaagaa?ctcatcattc?ttcagactgg?aattttatcg?gctcttacag 660

gttgaaatct?cctttcatct?taaaggcatt?gacctacaga?caattcattc?ccgtgagtta 720

ccagactgtt?atgtctttca?gaatacgatt?atctttgaca?ataaagctca?cagtggcaaa 780

atcaaaatct?attttgacag?tgatgccaaa?attgaagaat?gtaaagactt?gaacatattt 840

ggatctactc?agaaaaatgc?tcagtatgtc?ctggtgtttg?atgcatttgt?cattgtgatt 900

tgcttggcat?ctcttattct?gtgtacaaga?tccattgttc?ttgctctaag?gttacggaag 960

agatttctaa?atttcttcct?ggagaagtac?aagcggcctg?tgtgtgacac?cgaccagtgg 1020

gagttcatca?acggctggta?tgtcctggtg?attatcagcg?acctaatgac?aatcattggc 1080

tccatattaa?aaatggaaat?caaagcaaag?aatctcacaa?actatgatct?ctgcagcatt 1140

tttcttggaa?cctctacgct?cttggtttgg?gttggagtca?tcagatacct?gggttatttc 1200

caggcatata?atgtgctgat?tttaacaatg?caggcctcac?tgccaaaagt?tcttcggttt 1260

tgtgcttgtg?ctggtatgat?ttatctgggt?tacacattct?gtggctggat?tgtcttagga 1320

ccataccatg?acaagtttga?aaatctgaac?acagttgctg?agtgtctgtt?ttctctggtc 1380

aacggtgatg?acatgtttgc?aacctttgcc?caaatccagc?agaagagcat?cttggtgtgg 1440

ctgttcagtc?gtctgtattt?atattccttc?atcagccttt?ttatatatat?gattctcagt 1500

ctttttattg?cacttattac?agattcttat?gacaccatta?agaaattcca?acagaatggg 1560

tttcctgaaa?cggatttgca?ggaattcctg?aaggaatgca?gtagcaaaga?agagtatcag 1620

aaagagtcct?cagccttcct?gtcctgcatc?tgctgtcgga?ggaggaaaag?aagtgatgat 1680

cacttgatac?ctattagc 1698

 

<210>8

<211>566

<212>PRT

＜213〉homo sapiens

 

<400>8

Met?Ala?Arg?Gln?Pro?Tyr?Arg?Phe?Pro?Gln?Ala?Arg?Ile?Pro?Glu?Arg

1 5 10 15

 

Gly?Ser?Gly?Val?Phe?Arg?Leu?Thr?Val?Arg?Asn?Ala?Met?Ala?His?Arg

20 25 30

 

Asp?Ser?Glu?Met?Lys?Glu?Glu?Cys?Leu?Arg?Glu?Asp?Leu?Lys?Phe?Tyr

35 40 45

 

Phe?Met?Ser?Pro?Cys?Glu?Lys?Tyr?Arg?Ala?Arg?Arg?Gln?Ile?Pro?Trp

50 55 60

 

Lys?Leu?Gly?Leu?Gln?Ile?Leu?Lys?Ile?Val?Met?Val?Thr?Thr?Gln?Leu

65 70 75 80

 

Val?Arg?Phe?Gly?Leu?Ser?Asn?Gln?Leu?Val?Val?Ala?Phe?Lys?Glu?Asp

85 90 95

 

Asn?Thr?Val?Ala?Phe?Lys?His?Leu?Phe?Leu?Lys?Gly?Tyr?Ser?Gly?Thr

100 105 110

 

Asp?Glu?Asp?Asp?Tyr?Ser?Cys?Ser?Val?Tyr?Thr?Gln?Glu?Asp?Ala?Tyr

115 120 125

 

Glu?Ser?Ile?Phe?Phe?Ala?Ile?Asn?Gln?Tyr?His?Gln?Leu?Lys?Asp?Ile

130 135 140

 

Thr?Leu?Gly?Thr?Leu?Gly?Tyr?Gly?Glu?Asn?Glu?Asp?Asn?Arg?Ile?Gly

145 150 155 160

 

Leu?Lys?Val?Cys?Lys?Gln?His?Tyr?Lys?Lys?Gly?Thr?Met?Phe?Pro?Ser

165 170 175

 

Asn?Glu?Thr?Leu?Asn?Ile?Asp?Asn?Asp?Val?Glu?Leu?Asp?Cys?Val?Gln

180 185 190

 

Leu?Asp?Leu?Gln?Asp?Leu?Ser?Lys?Lys?Pro?Pro?Asp?Trp?Lys?Asn?Ser

195 200 205

 

Ser?Phe?Phe?Arg?Leu?Glu?Phe?Tyr?Arg?Leu?Leu?Gln?Val?Glu?Ile?Ser

210 215 220

 

Phe?His?Leu?Lys?Gly?Ile?Asp?Leu?Gln?Thr?Ile?His?Ser?Arg?Glu?Leu

225 230 235 240

 

Pro?Asp?Cys?Tyr?Val?Phe?Gln?Asn?Thr?Ile?Ile?Phe?Asp?Asn?Lys?Ala

245 250 255

 

His?Ser?Gly?Lys?Ile?Lys?Ile?Tyr?Phe?Asp?Ser?Asp?Ala?Lys?Ile?Glu

260 265 270

 

Glu?Cys?Lys?Asp?Leu?Asn?Ile?Phe?Gly?Ser?Thr?Gln?Lys?Asn?Ala?Gln

275 280 285

 

Tyr?Val?Leu?Val?Phe?Asp?Ala?Phe?Val?Ile?Val?Ile?Cys?Leu?Ala?Ser

290 295 300

 

Leu?Ile?Leu?Cys?Thr?Arg?Ser?Ile?Val?Leu?Ala?Leu?Arg?Leu?Arg?Lys

 

305 310 315 320

 

Arg?Phe?Leu?Asn?Phe?Phe?Leu?Glu?Lys?Tyr?Lys?Arg?Pro?Val?Cys?Asp

325 330 335

 

Thr?Asp?Gln?Trp?Glu?Phe?Ile?Asn?Gly?Trp?Tyr?Val?Leu?Val?Ile?Ile

340 345 350

 

Ser?Asp?Leu?Met?Thr?Ile?Ile?Gly?Ser?Ile?Leu?Lys?Met?Glu?Ile?Lys

355 360 365

 

Ala?Lys?Asn?Leu?Thr?Asn?Tyr?Asp?Leu?Cys?Ser?Ile?Phe?Leu?Gly?Thr

370 375 380

 

Ser?Thr?Leu?Leu?Val?Trp?Val?Gly?Val?Ile?Arg?Tyr?Leu?Gly?Tyr?Phe

385 390 395 400

 

Gln?Ala?Tyr?Asn?Val?Leu?Ile?Leu?Thr?Met?Gln?Ala?Ser?Leu?Pro?Lys

405 410 415

 

Val?Leu?Arg?Phe?Cys?Ala?Cys?Ala?Gly?Met?Ile?Tyr?Leu?Gly?Tyr?Thr

420 425 430

 

Phe?Cys?Gly?Trp?Ile?Val?Leu?Gly?Pro?Tyr?His?Asp?Lys?Phe?Glu?Asn

435 440 445

 

Leu?Asn?Thr?Val?Ala?Glu?Cys?Leu?Phe?Ser?Leu?Val?Asn?Gly?Asp?Asp

450 455 460

 

Met?Phe?Ala?Thr?Phe?Ala?Gln?Ile?Gln?Gln?Lys?Ser?Ile?Leu?Val?Trp

465 470 475 480

 

Leu?Phe?Ser?Arg?Leu?Tyr?Leu?Tyr?Ser?Phe?Ile?Ser?Leu?Phe?Ile?Tyr

485 490 495

 

Met?Ile?Leu?Ser?Leu?Phe?Ile?Ala?Leu?Ile?Thr?Asp?Ser?Tyr?Asp?Thr

500 505 510

 

Ile?Lys?Lys?Phe?Gln?Gln?Asn?Gly?Phe?Pro?Glu?Thr?Asp?Leu?Gln?Glu

515 520 525

 

Phe?Leu?Lys?Glu?Cys?Ser?Ser?Lys?Glu?Glu?Tyr?Gln?Lys?Glu?Ser?Ser

530 535 540

 

Ala?Phe?Leu?Ser?Cys?Ile?Cys?Cys?Arg?Arg?Arg?Lys?Arg?Ser?Asp?Asp

545 550 555 560

 

His?Leu?Ile?Pro?Ile?Ser

565

 

<210>9

<211>1659

<212>DNA

＜213〉mouse (Mus musculus)

 

<400>9

atggcaaatc?ccgaggtgct?ggttagcagc?tgcagagctc?gccaagatga?aagcccctgc 60

actttccacc?cgagctcgtc?cccgtcagag?cagcttctct?tagaagacca?gatgaggcgg 120

aaactcaagt?tcttttttat?gaatccttgt?gagaagttct?gggctcgggg?taggaagcca 180

tggaaacttg?ccatacagat?tctgaaaatc?gcgatggtga?ctatccagct?ggttctgttt 240

ggactaagta?accagatggt?agtagctttc?aaagaggaga?acactatagc?cttcaaacac 300

ctcttcctaa?agggctacat?ggatcgaatg?gacgacacct?atgcagtgta?cactcagagt 360

gaagtgtatg?accagatcat?ctttgcagtg?acccagtact?tgcagcttca?gaacatctcc 420

gtgggcaatc?acgcttatga?gaacaagggg?actaagcagt?cggcgatggc?aatctgtcag 480

cacttctaca?ggcaaggaac?catctgcccc?gggaacgaca?cctttgacat?cgatccagaa 540

gttgaaacag?aatgtttcct?tgtagagcca?gatgaagctt?cccaccttgg?aacgcctgga 600

gaaaataaac?tcaacctgag?cctggacttc?cacagacttc?tgacggtgga?gctccagttt 660

aagctcaaag?ccatcaatct?gcagacagtt?cgacaccagg?agcttcctga?ctgttacgac 720

tttacgctga?ctataacatt?cgacaacaag?gctcacagtg?gaagaatcaa?aataagctta 780

gacaacgaca?tttctatcaa?agaatgcaaa?gactggcatg?tgtctggatc?aattcagaag 840

aacacacact?acatgatgat?ctttgatgcc?tttgtcattc?tgacctgctt?ggcctcactg 900

gtgctgtgtg?ccaggtctgt?gattaggggt?cttcagcttc?agcaggagtt?tgtcaacttc 960

ttccttcttc?actacaagaa?ggaagtttcg?gcctctgatc?agatggagtt?catcaacggg 1020

tggtacatta?tgatcatcat?tagtgacata?ttgacaatcg?ttggatcagt?tctgaaaatg 1080

gaaatccaag?ccaagagtct?cacaagctat?gatgtctgca?gcatacttct?cgggacgtca 1140

actatgctcg?tgtggcttgg?agttatccga?tacctgggtt?tctttgcgaa?gtacgatctc 1200

cttattctga?ccctccaggc?agcgctgccc?aacgtcatga?ggttctgttg?ctgcgctgct 1260

atgatctatc?taggctattg?cttttgcgga?tggattgtgc?tgggccctta?ccatgagaag 1320

ttccgttccc?tgaacagggt?ctccgagtgc?ctgttctcgc?tgataaacgg?agacgatatg 1380

ttttccacat?ttgcgaaaat?gcagcagaag?agttacctgg?tgtggctgtt?cagccgagtc 1440

tacctgtact?cgttcatcag?cctcttcatt?tacatgattc?tgagcctttt?catcgcgctc 1500

atcacagaca?catacgaaac?aattaagcac?taccagcaag?atggcttccc?agagacggga 1560

cttcgaaagt?ttatagcgga?atgcaaagac?ctccccaact?ccggaaaata?cagattagaa 1620

gatgaccctc?cgggttcttt?actctgctgc?tgcaaaaag 1659

 

<210>10

<211>553

<212>PRT

＜213〉mouse

 

<400>10

 

Met?Ala?Asn?Pro?Glu?Val?Leu?Val?Ser?Ser?Cys?Arg?Ala?Arg?Gln?Asp

1 5 10 15

 

Glu?Ser?Pro?Cys?Thr?Phe?His?Pro?Ser?Ser?Ser?Pro?Ser?Glu?Gln?Leu

20 25 30

 

Leu?Leu?Glu?Asp?Gln?Met?Arg?Arg?Lys?Leu?Lys?Phe?Phe?Phe?Met?Asn

35 40 45

 

Pro?Cys?Glu?Lys?Phe?Trp?Ala?Arg?Gly?Arg?Lys?Pro?Trp?Lys?Leu?Ala

50 55 60

 

Ile?Gln?Ile?Leu?Lys?Ile?Ala?Met?Val?Thr?Ile?Gln?Leu?Val?Leu?Phe

65 70 75 80

 

Gly?Leu?Ser?Asn?Gln?Met?Val?Val?Ala?Phe?Lys?Glu?Glu?Asn?Thr?Ile

85 90 95

 

Ala?Phe?Lys?His?Leu?Phe?Leu?Lys?Gly?Tyr?Met?Asp?Arg?Met?Asp?Asp

100 105 110

 

Thr?Tyr?Ala?Val?Tyr?Thr?Gln?Ser?Glu?Val?Tyr?Asp?Gln?Ile?Ile?Phe

115 120 125

 

Ala?Val?Thr?Gln?Tyr?Leu?Gln?Leu?Gln?Asn?Ile?Ser?Val?Gly?Asn?His

130 135 140

 

Ala?Tyr?Glu?Asn?Lys?Gly?Thr?Lys?Gln?Ser?Ala?Met?Ala?Ile?Cys?Gln

145 150 155 160

 

His?Phe?Tyr?Arg?Gln?Gly?Thr?Ile?Cys?Pro?Gly?Asn?Asp?Thr?Phe?Asp

165 170 175

 

Ile?Asp?Pro?Glu?Val?Glu?Thr?Glu?Cys?Phe?Leu?Val?Glu?Pro?Asp?Glu

180 185 190

 

Ala?Ser?His?Leu?Gly?Thr?Pro?Gly?Glu?Asn?Lys?Leu?Asn?Leu?Ser?Leu

195 200 205

 

Asp?Phe?His?Arg?Leu?Leu?Thr?Val?G1u?Leu?Gln?Phe?Lys?Leu?Lys?Ala

210 215 220

 

Ile?Asn?Leu?Gln?Thr?Val?Arg?His?Gln?Glu?Leu?Pro?Asp?Cys?Tyr?Asp

225 230 235 240

 

Phe?Thr?Leu?Thr?Ile?Thr?Phe?Asp?Asn?Lys?Ala?His?Ser?Gly?Arg?Ile

245 250 255

Lys?Ile?Ser?Leu?Asp?Asn?Asp?Ile?Ser?Ile?Lys?Glu?Cys?Lys?Asp?Trp

260 265 270

 

His?Val?Ser?Gly?Ser?Ile?Gln?Lys?Asn?Thr?His?Tyr?Met?Met?Ile?Phe

275 280 285

 

Asp?Ala?Phe?Val?Ile?Leu?Thr?Cys?Leu?Ala?Ser?Leu?Val?Leu?Cys?Ala

290 295 300

 

Arg?Ser?Val?Ile?Arg?Gly?Leu?Gln?Leu?Gln?Gln?Glu?Phe?Val?Asn?Phe

305 310 315 320

 

Phe?Leu?Leu?His?Tyr?Lys?Lys?Glu?Val?Ser?Ala?Ser?Asp?Gln?Met?Glu

325 330 335

 

Phe?Ile?Asn?Gly?Trp?Tyr?Ile?Met?Ile?Ile?Ile?Ser?Asp?Ile?Leu?Thr

340 345 350

 

Ile?Val?Gly?Ser?Val?Leu?Lys?Met?Glu?Ile?Gln?Ala?Lys?Ser?Leu?Thr

355 360 365

 

Ser?Tyr?Asp?Val?Cys?Ser?Ile?Leu?Leu?Gly?Thr?Ser?Thr?Met?Leu?Val

370 375 380

 

Trp?Leu?Gly?Val?Ile?Arg?Tyr?Leu?Gly?Phe?Phe?Ala?Lys?Tyr?Asn?Leu

385 390 395 400

 

Leu?Ile?Leu?Thr?Leu?Gln?Ala?Ala?Leu?Pro?Asn?Val?Met?Arg?Phe?Cys

405 410 415

 

Cys?Cys?Ala?Ala?Met?Ile?Tyr?Leu?Gly?Tyr?Cys?Phe?Cys?Gly?Trp?Ile

420 425 430

 

Val?Leu?Gly?Pro?Tyr?His?Glu?Lys?Phe?Arg?Ser?Leu?Asn?Arg?Val?Ser

435 440 445

 

Glu?Cys?Leu?Phe?Ser?Leu?Ile?Asn?Gly?Asp?Asp?Met?Phe?Ser?Thr?Phe

450 455 460

 

Ala?Lys?Met?Gln?Gln?Lys?Ser?Tyr?Leu?Val?Trp?Leu?Phe?Ser?Arg?Val

465 470 475 480

 

Tyr?Leu?Tyr?Ser?Phe?Ile?Ser?Leu?Phe?Ile?Tyr?Met?Ile?Leu?Ser?Leu

485 490 495

 

Phe?Ile?Ala?Leu?Ile?Thr?Asp?Thr?Tyr?Glu?Thr?Ile?Lys?His?Tyr?Gln

500 505 510

 

Gln?Asp?Gly?Phe?Pro?Glu?Thr?Glu?Leu?Arg?Lys?Phe?Ile?Ala?Glu?Cys

515 520 525

 

Lys?Asp?Leu?Pro?Asn?Ser?Gly?Lys?Tyr?Arg?Leu?Glu?Asp?Asp?Pro?Pro

530 535 540

 

Gly?Ser?Leu?Leu?Cys?Cys?Cys?Lys?Lys

545 550

<210>11

<211>1773

<212>DNA

＜213〉Hongyuan chicken (Gallus gallus)

 

<400>11

atgatcaccc?gtggtttccg?ttcaggcttt?tacggctgta?gcaatcgata?tagtggtgct 60

aagtgccagt?tagttgctca?gacgtttgtc?atttcagcaa?tggagactcc?tgaagtggct 120

gtaagcagct?gcagtgctcg?ggatgacgaa?gggctctgca?gctacggaca?acacctattg 180

ctgccacaag?agctggtggc?ggaagaccag?ctgaggagga?agctgaagtt?cttcttcatg 240

aacccatgtg?aaaaattctg?ggctcggggc?agaaaacctt?ggaaacttgg?gattcagctg 300

ctcaaaatag?caatggttac?cattcagctg?gtgctttttg?gattgagcaa?tcaaatggtg 360

gttgctttca?aagaagagaa?cactattgca?ttcaaacatc?tcttcttgaa?agggtacatg 420

gacagaatgg?atgataccta?tgcggtatac?acacagacag?atgtctatga?ccaaatattc 480

tttgccatca?atcagtactt?acagttgccc?aacatttctg?ttggaaacca?tgcttatgag 540

aagaaaggag?cagaagagac?agctctggct?gtatgtcaac?agttctacaa?gcaaggaacc 600

atctgtcctg?gaaatgacac?ctttgatata?gacccagaga?ttgtgactga?ctgcttgtac 660

attgagccga?tgatgtcttt?agacaacaga?acagtgggaa?agcacaattt?gaatttcact 720

ctggatttcc?acaggctcgt?ggcagtgcaa?ctcatgttca?atctgaaggc?aatcaacctc 780

cagaccgtcc?gtcaccacga?gctccctgac?tgttacgatt?tcaccctgac?gatagtgttt 840

gataataaag?cccacagtgg?aagaatcaaa?atcagtctag?acaacgacat?agagatcagg 900

gaatgtaaag?actggcacgt?ttctggatca?atacagaaga?atacgcatta?catgatgatc 960

ttcgatgctt?ttgtcatact?gatctgtctg?agctcattga?tcctttgcac?tcgatcagta 1020

gtcaaaggaa?ttcggctcca?aagagaattt?gtaagttttt?tcctatatta?ttacaagaaa 1080

gaggtatctt?acaatgatca?gatggaattt?gtcaatggct?ggtatatcct?cattatggtt 1140

agtgatgtcc?tcactatcgt?tggatcaact?ctcaaaatgg?agatacaggc?caagagtctg 1200

acaagttacg?acgtctgtag?catactctta?ggaacatcca?ctatgctggt?gtggcttgga 1260

gtcattcgct?acctcggttt?ctttcagaag?tataatcttc?tcattctaac?gctgcgagca 1320

gcactaccca?acgtcatgag?gttctgctgt?tgtgctgcta?tgatctatct?aggttattgt 1380

ttctgcggat?ggattgtact?ggggccatac?cacgtgaagt?tccgttctct?gaatgtggtt 1440

tctgaatgcc?tcttttcatt?gataaatgga?gatgacatgt?ttgccacttt?tgcaaaaatg 1500

cagcagaaaa?gttacttggt?ttggttattc?agtagaatct?acctctactc?cttcatcagc 1560

ctgttcatct?acatggtgct?aagtctcttc?attgcactca?ttacagatac?atatgaaact 1620

atcaagcact?accaacaaga?tggctttcca?gagacagaac?ttcagagatt?tatatcacag 1680

tgcaaagact?taccaaactc?tggaaggtac?agattagaag?aggaaggttc?tgtatctctc 1740

ttctgttgtt?gcagtggtcc?tagtgaacat?atc 1773

 

<210>12

<211>591

<212>PRT

＜213〉Hongyuan chicken

 

<400>12

 

Met?Ile?Thr?Arg?Gly?Phe?Arg?Ser?Gly?Phe?Tyr?Gly?Cys?Ser?Asn?Arg

1 5 10 15

 

Tyr?Ser?Gly?Ala?Lys?Cys?Gln?Leu?Val?Ala?Gln?Thr?Phe?Val?Ile?Ser

20 25 30

 

Ala?Met?Glu?Thr?Pro?Glu?Val?Ala?Val?Ser?Ser?Cys?Ser?Ala?Arg?Asp

35 40 45

 

Asp?Glu?Gly?Leu?Cys?Ser?Tyr?Gly?Gln?His?Leu?Leu?Leu?Pro?Gln?Glu

50 55 60

 

Leu?Val?Ala?Glu?Asp?Gln?Leu?Arg?Arg?Lys?Leu?Lys?Phe?Phe?Phe?Met

65 70 75 80

 

Asn?Pro?Cys?Glu?Lys?Phe?Trp?Ala?Arg?Gly?Arg?Lys?Pro?Trp?Lys?Leu

85 90 95

 

Gly?Ile?Gln?Leu?Leu?Lys?Ile?Ala?Met?Val?Thr?Ile?Gln?Leu?Val?Leu

100 105 110

 

Phe?Gly?Leu?Ser?Asn?Gln?Met?Val?Val?Ala?Phe?Lys?Glu?Glu?Asn?Thr

115 120 125

 

Ile?Ala?Phe?Lys?His?Leu?Phe?Leu?Lys?Gly?Tyr?Met?Asp?Arg?Met?Asp

130 135 140

 

Asp?Thr?Tyr?Ala?Val?Tyr?Thr?Gln?Thr?Asp?Val?Tyr?Asp?Gln?Ile?Phe

145 150 155 160

 

Phe?Ala?Ile?Asn?Gln?Tyr?Leu?Gln?Leu?Pro?Asn?Ile?Ser?Val?Gly?Asn

165 170 175

 

His?Ala?Tyr?Glu?Lys?Lys?Gly?Ala?Glu?Glu?Thr?Ala?Leu?Ala?Val?Cys

180 185 190

 

Gln?Gln?Phe?Tyr?Lys?Gln?Gly?Thr?Ile?Cys?Pro?Gly?Asn?Asp?Thr?Phe

195 200 205

 

Asp?Ile?Asp?Pro?Glu?Ile?Val?Thr?Asp?Cys?Leu?Tyr?Ile?Glu?Pro?Met

210 215 220

 

Met?Ser?Leu?Asp?Asn?Arg?Thr?Val?Gly?Lys?His?Asn?Leu?Asn?Phe?Thr

225 230 235 240

 

Leu?Asp?Phe?His?Arg?Leu?Val?Ala?Val?Gln?Leu?Met?Phe?Asn?Leu?Lys

245 250 255

 

Ala?Ile?Asn?Leu?Gln?Thr?Val?Arg?His?His?Glu?Leu?Pro?Asp?Cys?Tyr

260 265 270

 

Asp?Phe?Thr?Leu?Thr?Ile?Val?Phe?Asp?Asn?Lys?Ala?His?Ser?Gly?Arg

275 280 285

 

Ile?Lys?Ile?Ser?Leu?Asp?Asn?Asp?Ile?Glu?Ile?Arg?Glu?Cys?Lys?Asp

290 295 300

 

Trp?His?Val?Ser?Gly?Ser?Ile?Gln?Lys?Asn?Thr?His?Tyr?Met?Met?Ile

305 310 315 320

 

Phe?Asp?Ala?Phe?Val?Ile?Leu?Ile?Cys?Leu?Ser?Ser?Leu?Ile?Leu?Cys

325 330 335

 

Thr?Arg?Ser?Val?Val?Lys?Gly?Ile?Arg?Leu?Gln?Arg?Glu?Phe?Val?Ser

340 345 350

 

Phe?Phe?Leu?Tyr?Tyr?Tyr?Lys?Lys?Glu?Val?Ser?Tyr?Asn?Asp?Gln?Met

355 360 365

 

Glu?Phe?Val?Asn?Gly?Trp?Tyr?Ile?Leu?Ile?Met?Val?Ser?Asp?Val?Leu

370 375 380

 

Thr?Ile?Val?Gly?Ser?Thr?Leu?Lys?Met?Glu?Ile?Gln?Ala?Lys?Ser?Leu

385 390 395 400

 

Thr?Ser?Tyr?Asp?Val?Cys?Ser?Ile?Leu?Leu?Gly?Thr?Ser?Thr?Met?Leu

405 410 415

 

Val?Trp?Leu?Gly?Val?Ile?Arg?Tyr?Leu?Gly?Phe?Phe?Gln?Lys?Tyr?Asn

420 425 430

 

Leu?Leu?Ile?Leu?Thr?Leu?Arg?Ala?Ala?Leu?Pro?Asn?Val?Met?Arg?Phe

435 440 445

 

Cys?Cys?Cys?Ala?Ala?Met?Ile?Tyr?Leu?Gly?Tyr?Cys?Phe?Cys?Gly?Trp

450 455 460

 

Ile?Val?Leu?Gly?Pro?Tyr?His?Val?Lys?Phe?Arg?Ser?Leu?Asn?Val?Val

465 470 475 480

 

Ser?Glu?Cys?Leu?Phe?Ser?Leu?Ile?Asn?Gly?Asp?Asp?Met?Phe?Ala?Thr

485 490 495

 

Phe?Ala?Lys?Met?Gln?Gln?Lys?Ser?Tyr?Leu?Val?Trp?Leu?Phe?Ser?Arg

500 505 510

 

Ile?Tyr?Leu?Tyr?Ser?Phe?Ile?Ser?Leu?Phe?Ile?Tyr?Met?Val?Leu?Ser

515 520 525

 

Leu?Phe?Ile?Ala?Leu?Ile?Thr?Asp?Thr?Tyr?Glu?Thr?Ile?Lys?His?Tyr

530 535 540

 

Gln?Gln?Asp?Gly?Phe?Pro?Glu?Thr?Glu?Leu?Gln?Arg?Phe?Ile?Ser?Gln

545 550 555 560

 

Cys?Lys?Asp?Leu?Pro?Asn?Ser?Gly?Arg?Tyr?Arg?Leu?Glu?Glu?Glu?Gly

565 570 575

 

Ser?Val?Ser?Leu?Phe?Cys?Cys?Cys?Ser?Gly?Pro?Ser?Glu?His?Ile

580 585 590

 

<210>13

<211>2424

<212>DNA

＜213〉dog (Canis familiaris)

<400>13

atgacccctt?ttggcagctt?ggcttctgca?aaggcttcaa?actcaagagc?tgcctggaag 60

attgtggtgg?attcattcag?ctcgcagcac?acgccccagg?tgggcactgg?tggtccccag 120

gaatcagaca?aggcccttac?cttcggagag?ttaacgttct?cccactcatc?tccattcttc 180

atctgcgcca?gcccttctct?cccaccgttg?cacagcagtg?ggctagacga?tgagccatac 240

tgctggacag?gttttcactg?catcaagtac?ctcgcgggcc?cagcgagtgt?cccaaactcc 300

cttgaaagag?ggagtaagat?attggtttcc?caagcctcct?ttcccatccg?gacctcccct 360

tacctgacac?tgctaagccg?aggggaaaag?aagcctctct?gcagttccgt?ggagaaaagg 420

cctttggggg?ttttggagat?gggaagtctg?actctcctct?cggaagagct?caaacggcag 480

ctccccggca?cgctgttgct?aggacaacct?ccgttgctaa?gggaaagagg?cggctcctct 540

gctgagattg?acaagacgcc?gctaccaata?gggcgtctac?tctccggccg?cctcttcaag 600

gccgcacttg?tgattggctg?ctgtcgtgct?gacgtcacgc?aactcgaacc?gccgagagat 660

cctgggtttt?cgcccgctcg?gcaggaggtg?tgcggtttgg?gttcccgcgt?ggagggtgct 720

gctggctcga?atgtaaacaa?tctttttttt?tttctccccc?tagagatggc?aaatcctacg 780

gttgttataa?gtagctgcag?ctctcatgaa?gaggaaaatc?gttgcacttt?tagccagcac 840

acatcgccct?ctgaggagct?tctgttagaa?gaccagataa?ggcgaaaact?caaatttttt 900

ttcatgaatc?cttgtgaaaa?gttctgggct?cgaggtagaa?aaccatggaa?gcttgccata 960

caaattctaa?aaattgcaat?ggtgactatc?cagctggtct?tttttgggct?aagtaaccag 1020

atggttgtag?ctttcaagga?agaaaacact?atagcattca?aacacctctt?cttaaaagga 1080

tatatggacc?gaatggatga?cacatatgca?gtgtacacac?aacgtgatgt?atatgatcag 1140

atcatctttg?cagtgaacca?gtacttgctt?ctacgcaata?cctcggttgg?gaatcatgct 1200

tatgagaaca?aggggacgga?acagtctgct?atggcaatct?gtcagcactt?ctacaagcag 1260

ggaaacatct?gtcctggaaa?tgataccttc?gacattgatc?cagagattga?aactgagtgt 1320

ttctctgtag?agccagctga?gcctttccac?gtcggaacac?tggaagaaaa?taaactcaac 1380

ttaacgctgg?actttcacag?actcctcacg?gtggacctgc?agtttaagct?gaaggccatt 1440

aatctgcaga?ccattcggca?tcacgagctc?cctgactgtt?atgactttac?tctcactata 1500

acatttgaca?ataaggccca?tagtggaaga?attaagataa?gtttagataa?tgatatttcc 1560

atcagagaat?gtaaagactg?gcatgtatcg?ggatcaattc?agaagaacac?tcactacatg 1620

atgatctttg?atgcctttgt?tattctgaca?tgcttggctt?cactaaccct?gtgccttcga 1680

tctgtaatta?gaggacttca?gcttcaacag?gaatttgtca?attttttcct?cctccattat 1740

aagaaggaag?tttctgtttc?tgatcgaatg?gaatttgtca?atggatggta?cattatgatt 1800

attattagtg?acatgttgac?aattattgga?tcaattctga?aaatggaaat?tcaagctaag 1860

agtctaacaa?gttatgatgt?ttgtagcata?cttcttggga?cttccaccat?gcttgtgtgg 1920

cttggagtta?ttcgatacct?cggcttcttt?cagaagtaca?atctccttat?tctgaccctg 1980

caggcagcac?tgcccagtgt?catcaggttc?tgttgctgtg?ccgctatgat?ttatttaggc 2040

tattgcttct?gtggatggat?tgtgctgggg?ccgtaccatg?ataagttccg?ttctctgaac 2100

atggtctctg?agtgcctttt?ctctctgata?aatggagatg?atatgtttgc?cacatttgca 2160

aaaatgcaac?aaaaaagtta?cttggtetgg?ctgtttagca?gaatttatct?ctactcattc 2220

atcagcctct?ttatatatat?gattttaagt?cttttcatcg?cactgatcac?tgatacgtat 2280

gaaacaatta?agcattacca?acaagatggc?tttccagaga?ctgaacttcg?tacatttata 2340

tcagagtgca?aagatctacc?caattctgga?aaatacagat?tagaagatga?cactccaata 2400

tctatattct?gctgttgtaa?aaag 2424

 

<210>14

<211>808

<212>PRT

＜213〉dog

 

<400>14

Met?Thr?Pro?Phe?Gly?Ser?Leu?Ala?Ser?Ala?Lys?Ala?Ser?Asn?Ser?Arg

1 5 10 15

 

Ala?Ala?Trp?Lys?Ile?Val?Val?Asp?Ser?Phe?Ser?Ser?Gln?His?Thr?Pro

20 25 30

 

Gln?Val?Gly?Thr?Gly?Gly?Pro?Gln?Glu?Ser?Asp?Lys?Ala?Leu?Thr?Phe

35 40 45

 

Gly?Glu?Leu?Thr?Phe?Ser?His?Ser?Ser?Pro?Phe?Phe?Ile?Cys?Ala?Ser

50 55 60

 

Pro?Ser?Leu?Pro?Pro?Leu?His?Ser?Ser?Gly?Leu?Asp?Asp?Glu?Pro?Tyr

65 70 75 80

 

Cys?Trp?Thr?Gly?Phe?His?Cys?Ile?Lys?Tyr?Leu?Ala?Gly?Pro?Ala?Ser

85 90 95

 

Val?Pro?Asn?Ser?Leu?Glu?Arg?Gly?Ser?Lys?Ile?Leu?Val?Ser?Gln?Ala

100 105 110

 

Ser?Phe?Pro?Ile?Arg?Thr?Ser?Pro?Tyr?Leu?Thr?Leu?Leu?Ser?Arg?Gly

115 120 125

 

Glu?Lys?Lys?Pro?Leu?Cys?Ser?Ser?Val?Glu?Lys?Arg?Pro?Leu?Gly?Val

130 135 140

 

Leu?Glu?Met?Gly?Ser?Leu?Thr?Leu?Leu?Ser?Glu?Glu?Leu?Lys?Arg?Gln

145 150 155 160

 

Leu?Pro?Gly?Thr?Leu?Leu?Leu?Gly?Gln?Pro?Pro?Leu?Leu?Arg?Glu?Arg

165 170 175

 

Gly?Gly?Ser?Ser?Ala?Glu?Ile?Asp?Lys?Thr?Pro?Leu?Pro?Ile?Gly?Arg

180 185 190

 

Leu?Leu?Ser?Gly?Arg?Leu?Phe?Lys?Ala?Ala?Leu?Val?Ile?Gly?Cys?Cys

195 200 205

 

Arg?Ala?Asp?Val?Thr?Gln?Leu?Glu?Pro?Pro?Arg?Asp?Pro?Gly?Phe?Ser

210 215 220

 

Pro?Ala?Arg?Gln?Glu?Val?Cys?Gly?Leu?Gly?Ser?Arg?Val?Glu?Gly?Ala

225 230 235 240

 

Ala?Gly?Ser?Asn?Val?Asn?Asn?Leu?Phe?Phe?Phe?Leu?Pro?Leu?Glu?Met

245 250 255

 

Ala?Asn?Pro?Thr?Val?Val?Ile?Ser?Ser?Cys?Ser?Ser?His?Glu?Glu?Glu

260 265 270

 

Asn?Arg?Cys?Thr?Phe?Ser?Gln?His?Thr?Ser?Pro?Ser?Glu?Glu?Leu?Leu

275 280 285

 

Leu?Glu?Asp?Gln?Ile?Arg?Arg?Lys?Leu?Lys?Phe?Phe?Phe?Met?Asn?Pro

290 295 300

 

Cys?Glu?Lys?Phe?Trp?Ala?Arg?Gly?Arg?Lys?Pro?Trp?Lys?Leu?Ala?Ile

305 310 315 320

 

Gln?Ile?Leu?Lys?Ile?Ala?Met?Val?Thr?Ile?Gln?Leu?Val?Phe?Phe?Gly

325 330 335

 

Leu?Ser?Asn?Gln?Met?Val?Val?Ala?Phe?Lys?Glu?Glu?Asn?Thr?Ile?Ala

340 345 350

 

Phe?Lys?His?Leu?Phe?Leu?Lys?Gly?Tyr?Met?Asp?Arg?Met?Asp?Asp?Thr

355 360 365

 

Tyr?Ala?Val?Tyr?Thr?Gln?Arg?Asp?Val?Tyr?Asp?Gln?Ile?Ile?Phe?Ala

370 375 380

 

Val?Asn?Gln?Tyr?Leu?Leu?Leu?Arg?Asn?Thr?Ser?Val?Gly?Asn?His?Ala

385 390 395 400

 

Tyr?Glu?Asn?Lys?Gly?Thr?Glu?Gln?Ser?Ala?Met?Ala?Ile?Cys?Gln?His

405 410 415

 

Phe?Tyr?Lys?Gln?Gly?Asn?Ile?Cys?Pro?Gly?Asn?Asp?Thr?Phe?Asp?Ile

420 425 430

 

Asp?Pro?Glu?Ile?Glu?Thr?Glu?Cys?Phe?Ser?Val?Glu?Pro?Ala?Glu?Pro

435 440 445

 

Phe?His?Val?Gly?Thr?Leu?Glu?Glu?Asn?Lys?Leu?Asn?Leu?Thr?Leu?Asp

450 455 460

 

Phe?His?Arg?Leu?Leu?Thr?Val?Asp?Leu?Gln?Phe?Lys?Leu?Lys?Ala?Ile

465 470 475 480

 

Asn?Leu?Gln?Thr?Ile?Arg?His?His?Glu?Leu?Pro?Asp?Cys?Tyr?Asp?Phe

485 490 495

 

Thr?Leu?Thr?Ile?Thr?Phe?Asp?Asn?Lys?Ala?His?Ser?Gly?Arg?Ile?Lys

500 505 510

 

Ile?Ser?Leu?Asp?Asn?Asp?Ile?Ser?Ile?Arg?Glu?Cys?Lys?Asp?Trp?His

515 520 525

 

Val?Ser?Gly?Ser?Ile?Gln?Lys?Asn?Thr?His?Tyr?Met?Met?Ile?Phe?Asp

530 535 540

 

Ala?Phe?Val?Ile?Leu?Thr?Cys?Leu?Ala?Ser?Leu?Thr?Leu?Cys?Leu?Arg

545 550 555 560

 

Ser?Val?Ile?Arg?Gly?Leu?Gln?Leu?Gln?Gln?Glu?Phe?Val?Asn?Phe?Phe

565 570 575

 

Leu?Leu?His?Tyr?Lys?Lys?Glu?Val?Ser?Val?Ser?Asp?Arg?Met?Glu?Phe

580 585 590

 

Val?Asn?Gly?Trp?Tyr?Ile?Met?Ile?Ile?Ile?Ser?Asp?Met?Leu?Thr?Ile

595 600 605

 

Ile?Gly?Ser?Ile?Leu?Lys?Met?Glu?Ile?Gln?Ala?Lys?Ser?Leu?Thr?Ser

610 615 620

 

Tyr?Asp?Val?Cys?Ser?Ile?Leu?Leu?Gly?Thr?Ser?Thr?Met?Leu?Val?Trp

625 630 635 640

 

Leu?Gly?Val?Ile?Arg?Tyr?Leu?Gly?Phe?Phe?Gln?Lys?Tyr?Asn?Leu?Leu

645 650 655

 

Ile?Leu?Thr?Leu?Gln?Ala?Ala?Leu?Pro?Ser?Val?Ile?Arg?Phe?Cys?Cys

660 665 670

 

Cys?Ala?Ala?Met?Ile?Tyr?Leu?Gly?Tyr?Cys?Phe?Cys?Gly?Trp?Ile?Val

675 680 685

 

Leu?Gly?Pro?Tyr?His?Asp?Lys?Phe?Arg?Ser?Leu?Asn?Met?Val?Ser?Glu

690 695 700

 

Cys?Leu?Phe?Ser?Leu?Ile?Asn?Gly?Asp?Asp?Met?Phe?Ala?Thr?Phe?Ala

705 710 715 720

 

Lys?Met?Gln?Gln?Lys?Ser?Tyr?Leu?Val?Trp?Leu?Phe?Ser?Arg?Ile?Tyr

725 730 735

 

Leu?Tyr?Ser?Phe?Ile?Ser?Leu?Phe?Ile?Tyr?Met?Ile?Leu?Ser?Leu?Phe

740 745 750

 

Ile?Ala?Leu?Ile?Thr?Asp?Thr?Tyr?Glu?Thr?Ile?Lys?His?Tyr?Gln?Gln

755 760 765

 

Asp?Gly?Phe?Pro?Glu?Thr?Glu?Leu?Arg?Thr?Phe?Ile?Ser?Glu?Cys?Lys

770 775 780

 

Asp?Leu?Pro?Asn?Ser?Gly?Lys?Tyr?Arg?Leu?Glu?Asp?Asp?Thr?Pro?Ile

785 790 795 800

 

Ser?Ile?Phe?Cys?Cys?Cys?Lys?Lys

805

 

<210>15

<211>1518

<212>DNA

＜213〉zebra fish (Danio rerio)

 

<400>15

atgtctgacc?gagcgtcaca?cactcatgaa?agcgcaacac?ttctggaccc?ggagtgtgtg 60

gaaagcttaa?ggagaaaact?caagtatttc?ttcatgagtc?cgtgtcagaa?atacagcact 120

agaggacgga?taccatggaa?gatgatgctt?cagatactca?agatttgttt?agtattcatc 180

tacctggtct?cttttggatt?gagcaacgag?atgatggtga?cgttcaaaga?ggaaaatctc 240

atcgccttca?agcacttctt?tctgaaaaac?tacaaggaca?gcaataaaca?ttacgccttg 300

tacacaaaac?atgaagttca?cgaccacatc?ctctacacca?tcagacggta?tctacagcta 360

caaaacctga?cgattggcaa?tcaagcgctg?gagatgatcg?atggtctggc?gactcctctg 420

tctctctgtc?agcagttgta?tcgacatgcg?cgcgtcgtgc?cgtctaatga?gacgtttgaa 480

atcgatccac?atgtagagac?agagtgtgtt?tctgtgtatc?ccctttctcc?catcacgact 540

gacagtctgg?aaaactccct?gaacttgact?ttagattttc?aaaggttgtt?agcggtaaac 600

atttatctga?agatcaaggc?tatcaacatt?cagacggttc?gccatcaaga?gttaccagac 660

tgctacgact?tcagcattaa?tatcatgttt?gacaatcgtg?cacacagcgg?acagatcaag 720

atctctctca?gcagcggcgt?gcagataaac?gtctgtaagg?actggaacat?ttctggctca 780

agtaagttga?acagccactt?tgcgctgatt?gtggtgtttg?actgtttgat?catctgcttc 840

tgtctgctgt?cactcatcct?ctgcacgcgc?tcagtccaca?caggatttct?cctacagact 900

gaatacagaa?gattcatgtc?cagtcagcac?agtaaaagcg?tctcatggtc?tgagaggctg 960

gagttcatca?acggctggta?catcctcatc?atcatcagcg?atgcgctgac?tattgcaggc 1020

tcaatcctca?aaatctgcat?acagagcaaa?gaactgacga?gctatgacgt?gtgcagtatt 1080

ctgctgggca?ctgcaacaat?gctggtgtgg?attggagtaa?tgcgctacct?cagtttcttc 1140

cagaaatatt?atatcctcat?cctcaccctg?aaggctgcac?ttcccaatgt?gattcgattc 1200

tccatctgcg?ctgttatgat?ctacctgagt?tactgcttct?gcggatggat?cgttttgggg 1260

ccacaccatg?aaaattttcg?cacattcagt?agggttgctg?gctgtctttt?ctccatgatt 1320

aatggggatg?aaatctactc?cacgttcacc?aagctccggg?aatacagcac?tctggtgtgg 1380

ctgttcagca?gactctacgt?ctacagcttc?atcccggtct?tcacatacat?ggttctgagt 1440

gtcttcatcg?ccctcatcac?agacacgtat?gaaaccatca?gggtgagtta?tttcagcttc 1500

agtgagagta?gctgcaaa 1518

 

<210>16

<211>506

<212>PRT

＜213〉zebra fish

 

<400>16

Met?Ser?Asp?Arg?Ala?Ser?His?Thr?His?Glu?Ser?Ala?Thr?Leu?Leu?Asp

1 5 10 15

 

Pro?Glu?Cys?Val?Glu?Ser?Leu?Arg?Arg?Lys?Leu?Lys?Tyr?Phe?Phe?Met

20 25 30

 

Ser?Pro?Cys?Gln?Lys?Tyr?Ser?Thr?Arg?Gly?Arg?Ile?Pro?Trp?Lys?Met

35 40 45

 

Met?Leu?Gln?Ile?Leu?Lys?Ile?Cys?Leu?Val?Phe?Ile?Tyr?Leu?Val?Ser

50 55 60

 

Phe?Gly?Leu?Ser?Asn?Glu?Met?Met?Val?Thr?Phe?Lys?Glu?Glu?Asn?Leu

65 70 75 80

Ile?Ala?Phe?Lys?His?Phe?Phe?Leu?Lys?Asn?Tyr?Lys?Asp?Ser?Asn?Lys

85 90 95

 

His?Tyr?Ala?Leu?Tyr?Thr?Lys?His?Glu?Val?His?Asp?His?Ile?Leu?Tyr

100 105 110

 

Thr?Ile?Arg?Arg?Tyr?Leu?Gln?Leu?Gln?Asn?Leu?Thr?Ile?Gly?Asn?Gln

115 120 125

 

Ala?Leu?Glu?Met?Ile?Asp?Gly?Leu?Ala?Thr?Pro?Leu?Ser?Leu?Cys?Gln

130 135 140

 

Gln?Leu?Tyr?Arg?His?Ala?Arg?Val?Val?Pro?Ser?Asn?Glu?Thr?Phe?Glu

145 150 155 160

 

Ile?Asp?Pro?His?Val?Glu?Thr?Glu?Cys?Val?Ser?Val?Tyr?Pro?Leu?Ser

165 170 175

 

Pro?Ile?Thr?Thr?Asp?Ser?Leu?Glu?Asn?Ser?Leu?Asn?Leu?Thr?Leu?Asp

180 185 190

 

Phe?Gln?Arg?Leu?Leu?Ala?Val?Asn?Ile?Tyr?Leu?Lys?Ile?Lys?Ala?Ile

195 200 205

 

Asn?Ile?Gln?Thr?Val?Arg?His?Gln?Glu?Leu?Pro?Asp?Cys?Tyr?Asp?Phe

210 215 220

 

Ser?Ile?Asn?Ile?Met?Phe?Asp?Asn?Arg?Ala?His?Ser?Gly?Gln?Ile?Lys

225 230 235 240

 

Ile?Ser?Leu?Ser?Ser?Gly?Val?Gln?Ile?Asn?Val?Cys?Lys?Asp?Trp?Asn

245 250 255

 

Ile?Ser?Gly?Ser?Ser?Lys?Leu?Asn?Ser?His?Phe?Ala?Leu?Ile?Val?Val

260 265 270

 

Phe?Asp?Cys?Leu?Ile?Ile?Cys?Phe?Cys?Leu?Leu?Ser?Leu?Ile?Leu?Cys

275 280 285

 

Thr?Arg?Ser?Val?His?Thr?Gly?Phe?Leu?Leu?Gln?Thr?Glu?Tyr?Arg?Arg

290 295 300

 

Phe?Met?Ser?Ser?Gln?His?Ser?Lys?Ser?Val?Ser?Trp?Ser?Glu?Arg?Leu

305 310 315 320

 

Glu?Phe?Ile?Asn?Gly?Trp?Tyr?Ile?Leu?Ile?Ile?Ile?Ser?Asp?Ala?Leu

325 330 335

 

Thr?Ile?Ala?Gly?Ser?Ile?Leu?Lys?Ile?Cys?Ile?Gln?Ser?Lys?Glu?Leu

340 345 350

 

Thr?Ser?Tyr?Asp?Val?Cys?Ser?Ile?Leu?Leu?Gly?Thr?Ala?Thr?Met?Leu

355 360 365

 

Val?Trp?Ile?Gly?Val?Met?Arg?Tyr?Leu?Ser?Phe?Phe?Gln?Lys?Tyr?Tyr

370 375 380

Ile?Leu?Ile?Leu?Thr?Leu?Lys?Ala?Ala?Leu?Pro?Asn?Val?Ile?Arg?Phe

385 390 395 400

 

Ser?Ile?Cys?Ala?Val?Met?Ile?Tyr?Leu?Ser?Tyr?Cys?Phe?Cys?Gly?Trp

405 410 415

 

Ile?Val?Leu?Gly?Pro?His?His?Glu?Asn?Phe?Arg?Thr?Phe?Ser?Arg?Val

420 425 430

 

Ala?Gly?Cys?Leu?Phe?Ser?Met?Ile?Asn?Gly?Asp?Glu?Ile?Tyr?Ser?Thr

435 440 445

 

Phe?Thr?Lys?Leu?Arg?Glu?Tyr?Ser?Thr?Leu?Val?Trp?Leu?Phe?Ser?Arg

450 455 460

 

Leu?Tyr?Val?Tyr?Ser?Phe?Ile?Pro?Val?Phe?Thr?Tyr?Met?Val?Leu?Ser

465 470 475 480

 

Val?Phe?Ile?Ala?Leu?Ile?Thr?Asp?Thr?Tyr?Glu?Thr?Ile?Arg?Val?Ser

485 490 495

 

Tyr?Phe?Ser?Phe?Ser?Glu?Ser?Ser?Cys?Lys

500 505

 

<210>17

<211>1659

<212>DNA

＜213〉homo sapiens

 

<400>17

atggccgacc?ctgaggtggt?ggtgtcctcc?tgctctagcc?acgaggaaga?gaaccggtgc 60

aacttcaacc?agcagaccag?ccccagcgag?gaactgctgc?tggaagatca?gatgcggcgg 120

aagctgaagt?tcttcttcat?gaacccctgc?gagaagttct?gggccagagg?ccggaagcct 180

tggaagctgg?ccatccagat?cctgaagatc?gccatggtga?ccatccagct?ggtgctgttc 240

ggcctgagca?accagatggt?ggtggccttc?aaagaggaaa?acacaatcgc?cttcaagcac 300

ctgtttctga?agggctacat?ggaccggatg?gacgacacct?acgccgtgta?cacccagagc 360

gacgtgtacg?accagctgat?cttcgccgtg?aaccagtacc?tgcagctgta?caacgtgagc 420

gtgggcaacc?acgcctacga?gaacaagggc?accaagcaga?gcgccatggc?catctgccag 480

cacttctaca?agcggggcaa?catctacccc?ggcaacgaca?ccttcgacat?cgaccccgag 540

atcgagacag?agtgcttctt?cgtggagccc?gacgagcctt?tccacatcgg?cacccctgcc 600

gagaacaagc?tgaacctgac?cctggacttc?caccggctgc?tgaccgtgga?gctgcagttc 660

aagctgaagg?ccatcaacct?gcagaccgtg?cggcaccagg?aactgcccga?ctgctacgac 720

ttcaccctga?ccatcacctt?cgataacaag?gcccacagcg?gccggatcaa?gatcagcctg 780

gacaacgaca?tcagcatccg?ggagtgcaag?gactggcacg?tgagcggcag?catccagaaa 840

aacacccact?acatgatgat?cttcgacgcc?ttcgtgatcc?tgacctgcct?ggtgtccctg 900

atcctgtgca?tcagaagcgt?catcaggggc?ctgcagctcc?agcaggaatt?cgtcaacttc 960

ttcctgctgc?actacaagaa?agaagtgtcc?gtcagcgacc?agatggaatt?tgtgaacggc 1020

tggtacatca?tgatcatcat?cagcgacatc?ctgacaatca?tcggcagcat?tctgaagatg 1080

gaaatccagg?ccaagagcct?gaccagctac?gacgtgtgca?gcattctgct?gggaacctcc 1140

accatgctgg?tctggctcgg?cgtgatcaga?tacctgggct?tcttcgccaa?gtacaacctg 1200

ctgattctga?cactgcaggc?cgccctgccc?aacgtgatcc?ggttctgctg?ctgcgccgcc 1260

atgatctacc?tgggctactg?cttctgcggc?tggatcgtgc?tgggccccta?ccacgacaag 1320

ttccggtccc?tgaacatggt?gtccgagtgc?ctgttcagcc?tgatcaacgg?cgacgacatg 1380

ttcgccacct?tcgccaagat?gcagcagaaa?agctacctgg?tctggctgtt?cagccggatc 1440

tacctgtaca?gcttcatcag?cctgttcatc?tacatgatcc?tgagcctgtt?tatcgccctg 1500

atcaccgata?cctacgagac?aatcaagcag?taccagcagg?acggcttccc?cgagacagag 1560

ctgcggacct?tcatcagcga?gtgtaaggac?ctgcccaaca?gcggcaagta?ccggctggaa 1620

gatgaccccc?ccgtgtccct?gttctgctgt?tgcaagaag 1659

 

<210>18

<211>1659

<212>DNA

＜213〉homo sapiens

 

<400>18

atggcagatc?ctgaggtagt?tgtgagtagc?tgcagctctc?atgaagagga?aaatcgctgc 60

aattttaacc?agcaaacatc?tccatctgag?gagcttctat?tagaagacca?gatgaggcga 120

aaactcaaat?tttttttcat?gaatccctgt?gagaagttct?gggctcgagg?tagaaaacca 180

tggaaacttg?ccatacaaat?tctaaaaatt?gcaatggtga?ctatccagct?ggtcttattt 240

gggctaagta?accagatggt?ggtagctttc?aaggaagaga?atactatagc?attcaaacac 300

cttttcctaa?aaggatatat?ggaccgaatg?gatgacacat?atgcagtgta?cacacaaagt 360

gacgtgtatg?atcagttaat?cttcgcagta?aaccagtact?tgcagctata?caatgtctcc 420

gttgggaatc?atgcttatga?gaacaaaggt?accaagcaat?ctgctatggc?aatctgtcag 480

cacttctaca?agcgaggaaa?catctaccct?ggaaatgata?cctttgacat?cgatccagaa 540

attgaaactg?agtgtttctt?tgtggagcca?gatgaacctt?ttcacattgg?gacaccagca 600

gaaaataaac?tgaacttaac?actggacttc?cacagactcc?taacagtgga?gcttcagttt 660

aaactgaaag?ccattaatct?gcagacagtt?cgtcatcaag?aactccctga?ctgttatgac 720

tttactctga?ctataacatt?tgacaacaag?gcccatagtg?gaagaattaa?aataagttta 780

gataatgaca?tttccatcag?agaatgtaaa?gactggcatg?tatctggatc?aattcagaag 840

aacactcatt?acatgatgat?ctttgatgcc?tttgtcattc?tgacttgctt?ggtttcatta 900

atcctctgca?ttagatctgt?gattagagga?cttcagcttc?agcaggagtt?tgtcaatttt 960

ttcctcctcc?attataagaa?ggaagtttct?gtttctgatc?aaatggaatt?tgtcaatgga 1020

tggtacatta?tgattattat?tagtgacata?ttgacaatca?ttggatcaat?tctaaaaatg 1080

gaaatccaag?ctaagagtct?aactagttat?gatgtctgta?gcatacttct?tgggacttct 1140

accatgcttg?tgtggcttgg?agtcatccga?tacctcggtt?tctttgcaaa?gtacaacctc 1200

ctcattttga?cccttcaggc?agcgctgccc?aatgtcatca?ggttctgctg?ctgtgcagct 1260

atgatttact?taggttactg?cttctgtgga?tggatcgtgc?tggggcctta?ccatgacaag 1320

tttcgttctc?tgaacatggt?ctctgagtgc?cttttctctc?tgataaatgg?agatgatatg 1380

tttgccacgt?ttgcaaaaat?gcagcaaaaa?agttacttag?tctggctgtt?tagtagaatt 1440

tacctctact?cattcatcag?cctctttata?tatatgattt?taagtctttt?cattgcactg 1500

atcactgata?catacgaaac?aattaagcaa?taccaacaag?atggcttccc?agagactgaa 1560

cttcgtacat?ttatatcaga?atgcaaagat?ctacccaact?ctggaaaata?cagattagaa 1620

gatgaccctc?cagtatcttt?attctgctgt?tgtaaaaag 1659

 

<210>19

<211>2000

<212>DNA

＜213〉rat (Rattus norvegicus)

 

<400>19

gttcgacaga?agcttgtgat?ttatggtcca?aggaatccag?tgtcagatca?atagacaaaa 60

tgccccaggg?aagttgtgtg?tgcattctac?tggacagatc?agagactggt?cagaacaggt 120

gcttggctgg?cggtgcgtcc?aaacctcaga?gatggcaaat?cctgaggtgg?tggtaagcag 180

ctgcagttct?caccaggatg?aaagtccctg?cactttctac?ccgagctcat?cccagtccga 240

gcagcttctc?ttagaagatc?agatgaggcg?gaaactcaaa?ttctttttta?tgaatccttg 300

cgagaagttc?tgggctcggg?gtaggaagcc?atggaaactt?gccatacaga?ttctgaaaat 360

cgctatggtg?actatccagc?tggttctgtt?tggactaagt?aaccagatgg?tagtagcttt 420

caaggaagag?aacacgatag?ccttcaaaca?cctcttcctg?aaaggctaca?tggaccgaat 480

ggacgacacc?tacgcggtgt?acactcagaa?tgatgtgtac?gaccagatca?tctttgcagt 540

gacccggtac?ttgcaacttc?gaaacatctc?cgtcggcaac?catgcttatg?agaacaaggg 600

gactaagcag?tcagcaatgg?cagtctgtca?gcacttctac?aggcaaggca?ccatctgccc 660

cgggaacgat?accttcgaca?tcgatccaga?agtcgaaaca?gactgtttcc?ttatagagcc 720

agaggaagct?ttccacatgg?gaacacctgg?agaaaacaaa?ctcaacctga?ccctggactt 780

ccacagactt?ctgacagtgg?agctccaatt?taagctcaaa?gccatcaacc?tgcagacagt 840

tcgccaccag?gagcttcctg?actgttacga?ctttaccctg?actataacat?tcgacaacaa 900

ggcacacagt?ggaagaatca?aaataagttt?agacaacgac?atttctatca?gagaatgcaa 960

agattggcac?gtgtctggat?caattcagaa?gaacacccac?tacatgatga?tcttcgatgc 1020

ctttgttatc?ctgacctgct?tgtcctcgct?ggtgctctgc?gccaggtctg?tgattcgggg 1080

tcttcagctt?cagcaggagt?ttgtcaactt?tttccttctt?cactacaaga?aggaagtttc 1140

ggcctctgat?cagatggagt?tcatcaacgg?gtggtacatt?atgatcatcg?ttagtgacat 1200

actgacgatc?gttggatcga?ttctgaaaat?ggaaatccaa?gccaagagtc?ttacaagcta 1260

cgatgtctgt?agcatacttc?ttgggacttc?caccatgctc?gtgtggcttg?gcgttatccg 1320

atacctgggt?ttctttgcga?agtacaatct?ccttatcctg?accctccagg?cagcgctgcc 1380

caatgtcatc?aggttctgtt?gctgtgcggc?tatgatctat?cttgggtatt?gcttttgcgg 1440

atggattgtg?ctgggccctt?accatgagaa?gttccgctct?ctgaacaagg?tctctgagtg 1500

cctattctca?ctgataaatg?gagacgacat?gttttccacg?ttcgcgaaaa?tgcagcagaa 1560

aagttacctg?gtgtggctgt?tcagccgcgt?ctacctgtac?tcgttcatca?gcctcttcat 1620

ctacatgatc?ttgagccttt?tcatcgcgct?catcacagac?acgtacgaaa?ccattaagca 1680

ctaccagcaa?gatggcttcc?cagagacgga?acttcgaaag?tttatcgctg?aatgcaaaga 1740

cctccccaac?tctggaaaat?acagattgga?agatgaccct?ccaggttctt?tattctgctg 1800

ctgcaaaaag?taactgtcgg?gtttctctgt?gcctgggagg?aaaatacagt?gtgtggatga 1860

gtcagagaca?atatggatta?ttggtaatca?cgcaacagtg?tgttcagata?ctagtgttct 1920

gagttaactc?acagctatga?ctttgcgggg?cctgttaaat?atatttttaa?atattaaaaa 1980

aaaaaaaaaa?aaaaaaaaaa 2000

 

<210>20

<211>553

<212>PRT

＜213〉rat

 

<400>20

Met?Ala?Asn?Pro?Glu?Val?Val?Val?Ser?Ser?Cys?Ser?Ser?His?Gln?Asp

1 5 10 15

 

Glu?Ser?Pro?Cys?Thr?Phe?Tyr?Pro?Ser?Ser?Ser?Gln?Ser?Glu?Gln?Leu

20 25 30

 

Leu?Leu?Glu?Asp?Gln?Met?Arg?Arg?Lys?Leu?Lys?Phe?Phe?Phe?Met?Asn

35 40 45

 

Pro?Cys?Glu?Lys?Phe?Trp?Ala?Arg?Gly?Arg?Lys?Pro?Trp?Lys?Leu?Ala

50 55 60

 

Ile?Gln?Ile?Leu?Lys?Ile?Ala?Met?Val?Thr?Ile?Gln?Leu?Val?Leu?Phe

65 70 75 80

 

Gly?Leu?Ser?Asn?Gln?Met?Val?Val?Ala?Phe?Lys?Glu?Glu?Asn?Thr?Ile

85 90 95

 

Ala?Phe?Lys?His?Leu?Phe?Leu?Lys?Gly?Tyr?Met?Asp?Arg?Met?Asp?Asp

100 105 110

 

Thr?Tyr?Ala?Val?Tyr?Thr?Gln?Asn?Asp?Val?Tyr?Asp?Gln?Ile?Ile?Phe

115 120 125

 

Ala?Val?Thr?Arg?Tyr?Leu?Gln?Leu?Arg?Asn?Ile?Ser?Val?Gly?Asn?His

130 135 140

 

Ala?Tyr?Glu?Asn?Lys?Gly?Thr?Lys?Gln?Ser?Ala?Met?Ala?Val?Cys?Gln

145 150 155 160

 

His?Phe?Tyr?Arg?Gln?Gly?Thr?Ile?Cys?Pro?Gly?Asn?Asp?Thr?Phe?Asp

165 170 175

 

Ile?Asp?Pro?Glu?Val?Glu?Thr?Asp?Cys?Phe?Leu?Ile?Glu?Pro?Glu?Glu

180 185 190

 

Ala?Phe?His?Met?Gly?Thr?Pro?Gly?Glu?Asn?Lys?Leu?Asn?Leu?Thr?Leu

195 200 205

 

Asp?Phe?His?Arg?Leu?Leu?Thr?Val?Glu?Leu?Gln?Phe?Lys?Leu?Lys?Ala

210 215 220

 

Ile?Asn?Leu?Gln?Thr?Val?Arg?His?Gln?Glu?Leu?Pro?Asp?Cys?Tyr?Asp

225 230 235 240

 

Phe?Thr?Leu?Thr?Ile?Thr?Phe?Asp?Asn?Lys?Ala?His?Ser?Gly?Arg?Ile

245 250 255

Lys?Ile?Ser?Leu?Asp?Asn?Asp?Ile?Ser?Ile?Arg?Glu?Cys?Lys?Asp?Trp

260 265 270

 

His?Val?Ser?Gly?Ser?Ile?Gln?Lys?Asn?Thr?His?Tyr?Met?Met?Ile?Phe

275 280 285

 

Asp?Ala?Phe?Val?Ile?Leu?Thr?Cys?Leu?Ser?Ser?Leu?Val?Leu?Cys?Ala

290 295 300

 

Arg?Ser?Val?Ile?Arg?Gly?Leu?Gln?Leu?Gln?Gln?Glu?Phe?Val?Asn?Phe

305 310 315 320

 

Phe?Leu?Leu?His?Tyr?Lys?Lys?Glu?Val?Ser?Ala?Ser?Asp?Gln?Met?Glu

325 330 335

 

Phe?Ile?Asn?Gly?Trp?Tyr?Ile?Met?Ile?Ile?Val?Ser?Asp?Ile?Leu?Thr

340 345 350

 

Ile?Val?Gly?Ser?Ile?Leu?Lys?Met?Glu?Ile?Gln?Ala?Lys?Ser?Leu?Thr

355 360 365

 

Ser?Tyr?Asp?Val?Cys?Ser?Ile?Leu?Leu?Gly?Thr?Ser?Thr?Met?Leu?Val

370 375 380

 

Trp?Leu?Gly?Val?Ile?Arg?Tyr?Leu?Gly?Phe?Phe?Ala?Lys?Tyr?Asn?Leu

385 390 395 400

 

Leu?Ile?Leu?Thr?Leu?Gln?Ala?Ala?Leu?Pro?Asn?Val?Ile?Arg?Phe?Cys

405 410 415

 

Cys?Cys?Ala?Ala?Met?Ile?Tyr?Leu?Gly?Tyr?Cys?Phe?Cys?Gly?Trp?Ile

420 425 430

 

Val?Leu?Gly?Pro?Tyr?His?Glu?Lys?Phe?Arg?Ser?Leu?Asn?Lys?Val?Ser

435 440 445

 

Glu?Cys?Leu?Phe?Ser?Leu?Ile?Asn?Gly?Asp?Asp?Met?Phe?Ser?Thr?Phe

450 455 460

 

Ala?Lys?Met?Gln?Gln?Lys?Ser?Tyr?Leu?Val?Trp?Leu?Phe?Ser?Arg?Val

465 470 475 480

 

Tyr?Leu?Tyr?Ser?Phe?Ile?Ser?Leu?Phe?Ile?Tyr?Met?Ile?Leu?Ser?Leu

485 490 495

 

Phe?Ile?Ala?Leu?Ile?Thr?Asp?Thr?Tyr?Glu?Thr?Ile?Lys?His?Tyr?Gln

500 505 510

 

Gln?Asp?Gly?Phe?Pro?Glu?Thr?Glu?Leu?Arg?Lys?Phe?Ile?Ala?Glu?Cys

515 520 525

 

Lys?Asp?Leu?Pro?Asn?Ser?Gly?Lys?Tyr?Arg?Leu?Glu?Asp?Asp?Pro?Pro

530 535 540

 

Gly?Ser?Leu?Phe?Cys?Cys?Cys?Lys?Lys

545 550

<210>21

<211>751

<212>DNA

＜213〉pig (Sus scrofa)

 

<400>21

gtgaaatggc?agatcctgag?cctgtcataa?gtagctgcag?ctctcgtgaa?gaggaaaatc 60

gctgcacttt?taaccagcac?acatgtccct?ctgaggagcg?tctattagaa?gaccagatga 120

ggcgaaaact?caaatttttt?ttcatgactc?cttgtgagaa?gttctggact?cgaggtcgaa 180

aaccatggaa?acttgccatg?caagttctaa?aaattgcgat?ggtgactatc?cagctgatct 240

ttttcgggct?aagtaaccag?atggtggtag?ctttcaagga?agagaacacg?atagcattta 300

aacacctctt?tctaaagggc?tatgtggacc?agatggatga?cacatatgcc?gtgtacaccc 360

aaagcgacgt?atacgatcgg?atcgtcttcg?cagtgaacca?gtacttgcag?ctacgcagca 420

tctcggttgg?gaaccacgct?tacgagaaca?agggcgcgga?gcagtcggcc?atggcgatct 480

gttggcactt?ctacaagcaa?ggaaacatct?gtcctggaaa?tgacaccttt?gacgttgatc 540

cagaagtaaa?aactgaatgt?ttctttgttg?agccggatga?agctgttgac?actggaacac 600

tggaggagaa?taagctcaac?ttaacccttg?actttcacag?actcctaacg?gtggagctgc 660

agtttaaact?caaggccatt?aatctgcaga?cgattcgcca?tcacgaactc?cctgactgtt 720

atgacttcac?cctgaccata?acatttgaca?a 751

 

<210>22

<211>248

<212>PRT

＜213〉pig

 

<400>22

Met?Ala?Asp?Pro?Glu?Pro?Val?Ile?Ser?Ser?Cys?Ser?Ser?Arg?Glu?Glu

1 5 10 15

 

Glu?Asn?Arg?Cys?Thr?Phe?Asn?Gln?His?Thr?Cys?Pro?Ser?Glu?Glu?Arg

20 25 30

 

Leu?Leu?Glu?Asp?Gln?Met?Arg?Arg?Lys?Leu?Lys?Phe?Phe?Phe?Met?Thr

35 40 45

 

Pro?Cys?Glu?Lys?Phe?Trp?Thr?Arg?Gly?Arg?Lys?Pro?Trp?Lys?Leu?Ala

50 55 60

 

Met?Gln?Val?Leu?Lys?Ile?Ala?Met?Val?Thr?Ile?Gln?Leu?Ile?Phe?Phe

65 70 75 80

 

Gly?Leu?Ser?Asn?Gln?Met?Val?Val?Ala?Phe?Lys?Glu?Glu?Asn?Thr?Ile

85 90 95

 

Ala?Phe?Lys?His?Leu?Phe?Leu?Lys?Gly?Tyr?Val?Asp?Gln?Met?Asp?Asp

100 105 110

 

Thr?Tyr?Ala?Val?Tyr?Thr?Gln?Ser?Asp?Val?Tyr?Asp?Arg?Ile?Val?Phe

115 120 125

 

Ala?Val?Asn?Gln?Tyr?Leu?Gln?Leu?Arg?Ser?Ile?Ser?Val?Gly?Asn?His

130 135 140

Ala?Tyr?Glu?Asn?Lys?Gly?Ala?Glu?Gln?Ser?Ala?Met?Ala?Ile?Cys?Trp

145 150 155 160

 

His?Phe?Tyr?Lys?Gln?Gly?Asn?Ile?Cys?Pro?Gly?Asn?Asp?Thr?Phe?Asp

165 170 175

 

Val?Asp?Pro?Glu?Val?Lys?Thr?Glu?Cys?Phe?Phe?Val?Glu?Pro?Asp?Glu

180 185 190

 

Ala?Val?Asp?Thr?Gly?Thr?Leu?Glu?Glu?Asn?Lys?Leu?Asn?Leu?Thr?Leu

195 200 205

 

Asp?Phe?His?Arg?Leu?Leu?Thr?Val?Glu?Leu?Gln?Phe?Lys?Leu?Lys?Ala

210 215 220

 

Ile?Asn?Leu?Gln?Thr?Ile?Arg?His?His?Glu?Leu?Pro?Asp?Cys?Tyr?Asp

225 230 235 240

 

Phe?Thr?Leu?Thr?Ile?Thr?Phe?Asp

245

 

<210>23

<211>2880

<212>DNA

＜213〉orangutan (Pan troglodytes)

 

<400>23

cttactccaa?tcaagcctct?gcccgccagg?aataggtaac?ctgtgtgtgt?ccgtttgctc 60

cttctaagag?catgcctgat?agatacttcg?gtagcctctc?cggatggccc?cttcgtcggg 120

tagcctctcc?tgatggggtc?cttcgcccac?cctgcctccc?gcgccggcgc?tccgggtgaa 180

tgtcaagggt?ggctggctgc?gaatacctcc?ttcagctgct?ggggttcccg?acagtttgca 240

gtttttaaaa?gtgcaccctc?ggaagggctt?ttcagactgg?gtaaagctga?cttttccaag 300

agatggcaga?tcctgaggta?gttgtgagta?gctgcagctc?tcatgaagag?gaaaatcgct 360

gcaattttaa?ccagcaaaca?tctccatctg?aggagcttct?attagaagac?cagatgaggc 420

gaaaactcaa?attttttttc?atgaatccct?gtgagaagtt?ctgggctcga?ggtagaaaac 480

catggaaact?tgccatacaa?attctaaaaa?ttgcaatggt?gactatccag?ctggtcttat 540

ttgggctaag?taaccagatg?gtggtagctt?tcaaggaaga?gaatactata?gcattcaaac 600

accttttcct?aaaaggatat?atggaccgaa?tggatgacac?atatgcagtg?tacacacaaa 660

gtgacgtgta?tgatcagtta?atcttcgcag?taaaccagta?cttgcagcta?tacaatgtct 720

ccgttgggaa?tcatgcttat?gagaacaaag?gtaccaagca?atctgctatg?gcaatctgtc 780

agcacttcta?caagcgagga?aacatctacc?ctggaaatga?tacctttgac?atcgatccag 840

aaattgaaac?tgagtgtttc?tttgtggagc?cagatgaacc?ttttcacatt?gggacaccag 900

cagaaaataa?actgaactta?acactggact?tccacagact?cctaacagtg?gagcttcagt 960

ttaaactgaa?agccattaat?ctgcagacag?ttcgtcatca?agaactccct?gactgttatg 1020

actttactct?gactataaca?tttgacaaca?aggcccatag?tggaagaatt?aaaataagtt 1080

tagataatga?catttccatc?agagaatgta?aagactggca?tgtatctgga?tcaattcaga 1140

agaacactca?ttacatgatg?atctttgatg?cctttgtcat?tctgacttgc?ttggtttcat 1200

taatcctctg?cattagatct?gtgattagag?gacttcagct?tcagcaggta?gggaacgttg 1260

ctttctagga?atgctactga?cattttgatt?gacagagaca?ttcactgtgc?ctcccctctt 1320

ttccctaaag?gagtttgtca?attttttcct?cctccattat?aagaagggag?tttctgtttc 1380

tgatcaaatg?gaatttgtca?atggatggta?cattatgatt?attattagtg?acatattgac 1440

aatcattgga?tcaattctaa?aaatggaaat?ccaagctaag?gtaatttttt?tcctaatcat 1500

gctattgtta?gtgtcagatt?tgcactaatg?gtaatgtatt?tttccagaat?gtaagaattt 1560

tcagaatgaa?ttgtttcttc?caaactgtat?atcaagtaga?cttgaaattg?gtaatggtaa 1620

ttttcttaaa?tctagtcagg?aggtctctta?ggcagagttt?ttcaaagtgt?gatccacaaa 1680

ccattgcatc?agaatcattg?ggtgcctggt?aaagtgtacc?atgttagacc?tactgaattc 1740

agactcttcg?gcggggcctg?tgaattctta?cacacaccaa?aattcataca?caaccaaggt 1800

aactaaggta?agagtttttt?ttttttttaa?atcttacaag?aaatgctcaa?atctttaaca 1860

aaaatgagtg?ggtctatagg?ggaaagtgag?gtcaaggcac?tatggtgtgc?atgcttgcat 1920

ttgtttcctc?cgtccattca?aagtgagaat?gctcccattt?tcttacttta?ccattgatgt 1980

gctacaagct?tatttatttt?aagactaacc?tagcctaaaa?atcaactgtc?cccacaaaat 2040

aaaaatcaca?ttaaaaaaac?taatagtgtt?cagactaatc?ttgctcaaac?ttatgtttcc 2100

ctagtcttga?tgcgactgat?tgagtcacct?ggggagttgg?ttataaacct?gggcagagac 2160

cccaaatgca?atggctcaga?gaagatagga?gcttatttct?gtcttatgca?atagtcagaa 2220

tgggttttac?agactggtga?gtagctcaac?atctcacagt?cattcaggca?cccatgttcc 2280

tcccattttg?tttctctgcc?atcccttaag?gacttgccct?gactgcatga?ttattgctgt 2340

gttgcctcaa?acaggttgca?gcttatggga?agcaaaaaca?cggtatggtg?gaagctctcc 2400

catagactga?tggcttggct?caagagtggc?cgactttatt?tctgtacata?tcccactgga 2460

tagaatttag?tcaatcctaa?ctgcagaggg?agccagggaa?cacagcccag?gcatgtgcct 2520

aggaagggga?gaatgggttt?aggttgacac?ttagcagctg?ccactatatg?tggctatagt 2580

atgtatcatt?ggaatagatg?tttaacttta?gggacaaata?aacaaaaaac?caaaacaaaa 2640

aaaggagtaa?ggggagagat?ttgcagcaaa?tctttatttt?taccaacctc?aactatcatt 2700

aatttcagtg?aaccctaaat?ggtgtccaac?aaaatatctt?tctagaccat?tcaccgtctc 2760

tgcctcatag?atgatcatat?catgttttct?tctcttctga?aacctctaat?acccttgtcc 2820

tatcctcatt?ctaagctgat?gaccttactt?cctatttcac?aaaaataata?gaaaaaaaaa 2880

 

<210>24

<211>321

<212>PRT

＜213〉orangutan

 

<400>24

Met?Ala?Asp?Pro?Glu?Val?Val?Val?Ser?Ser?Cys?Ser?Ser?His?Glu?Glu

1 5 10 15

 

Glu?Asn?Arg?Cys?Asn?Phe?Asn?Gln?Gln?Thr?Ser?Pro?Ser?Glu?Glu?Leu

20 25 30

 

Leu?Leu?Glu?Asp?Gln?Met?Arg?Arg?Lys?Leu?Lys?Phe?Phe?Phe?Met?Asn

35 40 45

 

Pro?Cys?Glu?Lys?Phe?Trp?Ala?Arg?Gly?Arg?Lys?Pro?Trp?Lys?Leu?Ala

50 55 60

 

Ile?Gln?Ile?Leu?Lys?Ile?Ala?Met?Val?Thr?Ile?Gln?Leu?Val?Leu?Phe

65 70 75 80

 

Gly?Leu?Ser?Asn?Gln?Met?Val?Val?Ala?Phe?Lys?Glu?Glu?Asn?Thr?Ile

85 90 95

 

Ala?Phe?Lys?His?Leu?Phe?Leu?Lys?Gly?Tyr?Met?Asp?Arg?Met?Asp?Asp

100 105 110

 

Thr?Tyr?Ala?Val?Tyr?Thr?Gln?Ser?Asp?Val?Tyr?Asp?Gln?Leu?Ile?Phe

115 120 125

 

Ala?Val?Asn?Gln?Tyr?Leu?Gln?Leu?Tyr?Asn?Val?Ser?Val?Gly?Asn?His

130 135 140

 

Ala?Tyr?Glu?Asn?Lys?Gly?Thr?Lys?Gln?Ser?Ala?Met?Ala?Ile?Cys?Gln

145 150 155 160

 

His?Phe?Tyr?Lys?Arg?Gly?Asn?Ile?Tyr?Pro?Gly?Asn?Asp?Thr?Phe?Asp

165 170 175

 

Ile?Asp?Pro?Glu?Ile?Glu?Thr?Glu?Cys?Phe?Phe?Val?Glu?Pro?Asp?Glu

180 185 190

 

Pro?Phe?His?Ile?Gly?Thr?Pro?Ala?Glu?Asn?Lys?Leu?Asn?Leu?Thr?Leu

195 200 205

 

Asp?Phe?His?Arg?Leu?Leu?Thr?Val?Glu?Leu?Gln?Phe?Lys?Leu?Lys?Ala

210 215 220

 

Ile?Asn?Leu?Gln?Thr?Val?Arg?His?Gln?Glu?Leu?Pro?Asp?Cys?Tyr?Asp

225 230 235 240

 

Phe?Thr?Leu?Thr?Ile?Thr?Phe?Asp?Asn?Lys?Ala?His?Ser?Gly?Arg?Ile

245 250 255

 

Lys?Ile?Ser?Leu?Asp?Asn?Asp?Ile?Ser?Ile?Arg?Glu?Cys?Lys?Asp?Trp

260 265 270

 

His?Val?Ser?Gly?Ser?Ile?Gln?Lys?Asn?Thr?His?Tyr?Met?Met?Ile?Phe

275 280 285

 

Asp?Ala?Phe?Val?Ile?Leu?Thr?Cys?Leu?Val?Ser?Leu?Ile?Leu?Cys?Ile

290 295 300

 

Arg?Ser?Val?Ile?Arg?Gly?Leu?Gln?Leu?Gln?Gln?Val?Gly?Asn?Val?Ala

305 310 315 320

 

Phe

 

<210>25

<211>1868

<212>DNA

＜213〉orangutan

<400>25

cctctagaga?tggcagatcc?tgaggtagtt?gtgagtagct?gcagctctca?tgaagaggaa 60

aatcgctgca?attttaacca?gcaaacatct?ccatctgagg?agcttctatt?agaagaccag 120

atgaggcgaa?aactcaaatt?ttttttcatg?aatccctgtg?agaagttctg?ggctcgaggt 180

agaaaaccat?ggaaacttgc?catacaaatt?ctaaaaattg?caatggtgac?tatccagctg 240

gtcttatttg?ggctaagtaa?ccagatggtg?gtagctttca?aggaagagaa?tactatagca 300

ttcaaacacc?ttttcctaaa?aggatatatg?gaccgaatgg?atgacacata?tgcagtgtac 360

acacaaagtg?acgtgtatga?tcagttaatc?ttcgcagtaa?accagtactt?gcagctatac 420

aatgtctccg?ttgggaatca?tgcttatgag?aacaaaggta?ccaagcaatc?tgctatggca 480

atctgtcagc?acttctacaa?gcgaggaaac?atctaccctg?gaaatgatac?ctttgacatc 540

gatccagaaa?ttgaaactga?gtgtttcttt?gtggagccag?atgaaccttt?tcacattggg 600

acaccagcag?aaaataaact?gaacttaaca?ctggacttcc?acagactcct?aacagtggag 660

cttcagttta?aactgaaagc?cattaatctg?cagacagttc?gtcatcaaga?actccctgac 720

tgttatgact?ttactctgac?tataacattt?gacaacaagg?cccatagtgg?aagaattaaa 780

ataagtttag?ataatgacat?ttccatcaga?gaatgtaaag?actggcattc?tccctccgtc 840

gcccagcctg?gaaacactca?ttacatgatg?atctttgatg?cctttgtcat?tctgacttgc 900

ttggtttcat?taatcctctg?cattagatct?gtgattagag?gacttcagct?tcagcaggag 960

tttgtcaatt?ttttcctcct?ccattataag?aagggagttt?ctgtttctga?tcaaatggaa 1020

tttgtcaatg?gatggtacat?tatgattatt?attagtgaca?tattgacaat?cattggatca 1080

attctaaaaa?tggaaatcca?agctaagagt?ctaactagtt?atgatgtctg?tagcatactt 1140

cttgggactt?ctaccatgct?tgtgtggctt?ggagtcatcc?gatacctcgg?tttctttgca 1200

aagtacaatc?tcctcatttt?gacccttcag?gcagcactgc?ccaatgtcat?caggttctgc 1260

tgctgtgcag?ctatgattta?cttaggttac?tgcttctgtg?gatggatcgt?gctggggcct 1320

taccatgaca?agtttcgttc?tctgaacatg?gtctctgagt?gccttttctc?tctgataaat 1380

ggagatgata?tgtttgccac?gtttgcaaaa?atgcagcaaa?aaagttactt?agtctggctg 1440

tttagtagaa?tttacctcta?ctcattcatc?agcctcttta?tatatatgat?tttaagtctt 1500

ttcattgcac?tgatcactga?tacatacgaa?acaattaagc?aataccaaca?agatggcttc 1560

ccagagactg?aacttcgtac?atttatatca?gaatgcaaag?atctacccaa?ctctggaaaa 1620

tacagattag?aagatgaccc?tccagtatct?ttattctgct?gttgtaaaaa?gtagctatca 1680

ggtttatctg?tactttagag?gaaaatataa?tgtgtagctg?agttagaaca?ctgtggatat 1740

tctgagatca?gatgtagtat?gtttgaagac?tgttattttg?agctaattga?gacctataat 1800

tcaccaataa?ctgtttatat?ttttaaaagc?aatatttaat?gtctttgcag?ctttatgctg 1860

ggcttgtt 1868

 

<210>26

<211>554

<212>PRT

＜213〉orangutan

 

<400>26

Met?Ala?Asp?Pro?Glu?Val?Val?Val?Ser?Ser?Cys?Ser?Ser?His?Glu?Glu

1 5 10 15

Glu?Asn?Arg?Cys?Asn?Phe?Asn?Gln?Gln?Thr?Ser?Pro?Ser?Glu?Glu?Leu

20 25 30

 

Leu?Leu?Glu?Asp?Gln?Met?Arg?Arg?Lys?Leu?Lys?Phe?Phe?Phe?Met?Asn

35 40 45

 

Pro?Cys?Glu?Lys?Phe?Trp?Ala?Arg?Gly?Arg?Lys?Pro?Trp?Lys?Leu?Ala

50 55 60

 

Ile?Gln?Ile?Leu?Lys?Ile?Ala?Met?Val?Thr?Ile?Gln?Leu?Val?Leu?Phe

65 70 75 80

 

Gly?Leu?Ser?Asn?Gln?Met?Val?Val?Ala?Phe?Lys?Glu?Glu?Asn?Thr?Ile

85 90 95

 

Ala?Phe?Lys?His?Leu?Phe?Leu?Lys?Gly?Tyr?Met?Asp?Arg?Met?Asp?Asp

100 105 110

 

Thr?Tyr?Ala?Val?Tyr?Thr?Gln?Ser?Asp?Val?Tyr?Asp?Gln?Leu?Ile?Phe

115 120 125

 

Ala?Val?Asn?Gln?Tyr?Leu?Gln?Leu?Tyr?Asn?Val?Ser?Val?Gly?Asn?His

130 135 140

 

Ala?Tyr?Glu?Asn?Lys?Gly?Thr?Lys?Gln?Ser?Ala?Met?Ala?Ile?Cys?Gln

145 150 155 160

 

His?Phe?Tyr?Lys?Arg?Gly?Asn?Ile?Tyr?Pro?Gly?Asn?Asp?Thr?Phe?Asp

165 170 175

 

Ile?Asp?Pro?Glu?Ile?Glu?Thr?Glu?Cys?Phe?Phe?Val?Glu?Pro?Asp?Glu

180 185 190

 

Pro?Phe?His?Ile?Gly?Thr?Pro?Ala?Glu?Asn?Lys?Leu?Asn?Leu?Thr?Leu

195 200 205

 

Asp?Phe?His?Arg?Leu?Leu?Thr?Val?Glu?Leu?Gln?Phe?Lys?Leu?Lys?Ala

210 215 220

 

Ile?Asn?Leu?Gln?Thr?Val?Arg?His?Gln?Glu?Leu?Pro?Asp?Cys?Tyr?Asp

225 230 235 240

 

Phe?Thr?Leu?Thr?Ile?Thr?Phe?Asp?Asn?Lys?Ala?His?Ser?Gly?Arg?Ile

245 250 255

 

Lys?Ile?Ser?Leu?Asp?Asn?Asp?Ile?Ser?Ile?Arg?Glu?Cys?Lys?Asp?Trp

260 265 270

 

His?Ser?Pro?Ser?Val?Ala?Gln?Pro?Gly?Asn?Thr?His?Tyr?Met?Met?Ile

275 280 285

 

Phe?Asp?Ala?Phe?Val?Ile?Leu?Thr?Cys?Leu?Val?Ser?Leu?Ile?Leu?Cys

290 295 300

 

Ile?Arg?Ser?Val?Ile?Arg?Gly?Leu?Gln?Leu?Gln?Gln?Glu?Phe?Val?Asn

305 310 315 320

Phe?Phe?Leu?Leu?His?Tyr?Lys?Lys?Gly?Val?Ser?Val?Ser?Asp?Gln?Met

325 330 335

 

Glu?Phe?Val?Asn?Gly?Trp?Tyr?Ile?Met?Ile?Ile?Ile?Ser?Asp?Ile?Leu

340 345 350

 

Thr?Ile?Ile?Gly?Ser?Ile?Leu?Lys?Met?Glu?Ile?Gln?Ala?Lys?Ser?Leu

355 360 365

 

Thr?Ser?Tyr?Asp?Val?Cys?Ser?Ile?Leu?Leu?Gly?Thr?Ser?Thr?Met?Leu

370 375 380

 

Val?Trp?Leu?Gly?Val?Ile?Arg?Tyr?Leu?Gly?Phe?Phe?Ala?Lys?Tyr?Asn

385 390 395 400

 

Leu?Leu?Ile?Leu?Thr?Leu?Gln?Ala?Ala?Leu?Pro?Asn?Val?Ile?Arg?Phe

405 410 415

 

Cys?Cys?Cys?Ala?Ala?Met?Ile?Tyr?Leu?Gly?Tyr?Cys?Phe?Cys?Gly?Trp

420 425 430

 

Ile?Val?Leu?Gly?Pro?Tyr?His?Asp?Lys?Phe?Arg?Ser?Leu?Asn?Met?Val

435 440 445

 

Ser?Glu?Cys?Leu?Phe?Ser?Leu?Ile?Asn?Gly?Asp?Asp?Met?Phe?Ala?Thr

450 455 460

 

Phe?Ala?Lys?Met?Gln?Gln?Lys?Ser?Tyr?Leu?Val?Trp?Leu?Phe?Ser?Arg

465 470 475 480

 

Ile?Tyr?Leu?Tyr?Ser?Phe?Ile?Ser?Leu?Phe?Ile?Tyr?Met?Ile?Leu?Ser

485 490 495

 

Leu?Phe?Ile?Ala?Leu?Ile?Thr?Asp?Thr?Tyr?Glu?Thr?Ile?Lys?Gln?Tyr

500 505 510

 

Gln?Gln?Asp?Gly?Phe?Pro?Glu?Thr?Glu?Leu?Arg?Thr?Phe?Ile?Ser?Glu

515 520 525

 

Cys?Lys?Asp?Leu?Pro?Asn?Ser?Gly?Lys?Tyr?Arg?Leu?Glu?Asp?Asp?Pro

530 535 540

 

Pro?Val?Ser?Leu?Phe?Cys?Cys?Cys?Lys?Lys

545 550

 

<210>27

<211>2546

<212>DNA

＜213〉orangutan

 

<400>27

tcctctagag?atggcagatc?ctgaggtagt?tgtgagtagc?tgcagctctc?atgaagagga 60

aaatcgctgc?aattttaacc?agcaaacatc?tccatctgag?gagcttctat?tagaagacca 120

gatgaggcga?aaactcaaat?tttttttcat?gaatccctgt?gagaagttct?gggctcgagg 180

tagaaaacca?tggaaacttg?ccatacaaat?tctaaaaatt?gcaatggtga?ctatccagta 240

cttgcagcta?tacaatgtct?ccgttgggaa?tcatgcttat?gagaacaaag?gtaccaagca 300

atctgctatg?gcaatctgtc?agcacttcta?caagcgagga?aacatctacc?ctggaaatga 360

tacctttgac?atcgatccag?aaattgaaac?tgagtgtttc?tttgtggagc?cagatgaacc 420

ttttcacatt?gggacaccag?cagaaaataa?actgaactta?acactggact?tccacagact 480

cctaacagtg?gagcttcagt?ttaaactgaa?agccattaat?ctgcagacag?ttcgtcatca 540

agaactccct?gactgttatg?actttactct?gactataaca?tttgacaaca?aggcccatag 600

tggaagaatt?aaaataagtt?tagataatga?catttccatc?agagaatgta?aagactggca 660

tgtatctgga?tcaattcaga?agaacactca?ttacatgatg?atctttgatg?cctttgtcat 720

tctgacttgc?ttggtttcat?taatcctctg?cattagatct?gtgattagag?gacttcagct 780

tcagcaggag?tttgtcaatt?ttttcctcct?ccattataag?aagggagttt?ctgtttctga 840

tcaaatggaa?tttgtcaatg?gatggtacat?tatgattatt?attagtgaca?tattgacaat 900

cattggatca?attctaaaaa?tggaaatcca?agctaagagt?ctaactagtt?atgatgtctg 960

tagcatactt?cttgggactt?ctaccatgct?tgtgtggctt?ggagtcatcc?gatacctcgg 1020

tttctttgca?aagtacaatc?tcctcatttt?gacccttcag?gcagcactgc?ccaatgtcat 1080

caggttctgc?tgctgtgcag?ctatgattta?cttaggttac?tgcttctgtg?gatggatcgt 1140

gctggggcct?taccatgaca?agtttcgttc?tctgaacatg?gtctctgagt?gccttttctc 1200

tctgataaat?ggagatgata?tgtttgccac?gtttgcaaaa?atgcagcaaa?aaagttactt 1260

agtctggctg?tttagtagaa?tttacctcta?ctcattcatc?agcctcttta?tatatatgat 1320

tttaagtctt?ttcattgcac?tgatcactga?tacatacgaa?acaattaagc?aataccaaca 1380

agatggcttc?ccagagactg?aacttcgtac?atttatatca?gaatgcaaag?atctacccaa 1440

ctctggaaaa?tacagattag?aagatgaccc?tccagtatct?ttattctgct?gttgtaaaaa 1500

gtagctatca?ggtttatctg?tactttagag?gaaaatataa?tgtgtagctg?agttagaaca 1560

ctgtggatat?tctgagatca?gatgtagtat?gtttgaagac?tgttattttg?agctaattga 1620

gacctataat?tcaccaataa?ctgtttatat?ttttaaaagc?aatatttaat?gtctttgcag 1680

ctttatgctg?ggcttgtttt?taaaacaact?ttaatgagga?aagctattgg?attattatta 1740

tttcttgttt?attttgccat?ggctttagaa?tgtattctgt?atgcctctct?tttgctctga 1800

tactcttgct?tcctgctatt?ctgattgtgc?agactgtgta?attagtggaa?aacaatcctt 1860

ggtctgactg?tgactttgga?caactcagta?accctggctt?ggaccactct?caggagtcca 1920

tccttgagag?agtgggtgta?gttaccattt?atacagtaat?cattgcattt?taaaatctgc 1980

tcttgaaagg?aagaataaga?gtgcaccaga?ataagagcgc?accagaataa?gagcgcacca 2040

gctaacaatg?tgatacggcc?atatgtcact?taaggataga?gatatgttct?gagaaatgtg 2100

tcattaggcg?attttgtcat?taaacatcat?agcatgtact?tccacaaacc?tagatggtat 2160

agcctactac?acacctaggc?tatttggtat?agcctgttga?tcctggggta?caaatctgta 2220

caacatgtta?ctgtattgaa?tacagtaggc?aattgtaaca?caatggtaag?tatctaaaca 2280

tagaaaaggg?acagtaaaaa?tatggtttta?taatcttctg?ggaccaccat?tgtatatgcg 2340

gtacatcgtt?gaccaaaaca?tcgttatcca?gcatatgact?gtatttggtt?atgaaagcca 2400

actgttactt?gattctgctt?ttagttctta?agaggatcag?gtttttaaat?actcatttac 2460

aagttttcta?tcctccttca?gtgttaaagt?agaaagtaaa?aagagtatct?tatacatgca 2520

tgaaattaaa?gcatatacca?aatgca 2546

<210>28

<211>497

<212>PRT

＜213〉orangutan

 

<400>28

Met?Ala?Asp?Pro?Glu?Val?Val?Val?Ser?Ser?Cys?Ser?Ser?His?Glu?Glu

1 5 10 15

 

Glu?Asn?Arg?Cys?Asn?Phe?Asn?Gln?Gln?Thr?Ser?Pro?Ser?Glu?Glu?Leu

20 25 30

 

Leu?Leu?Glu?Asp?Gln?Met?Arg?Arg?Lys?Leu?Lys?Phe?Phe?Phe?Met?Asn

35 40 45

 

Pro?Cys?Glu?Lys?Phe?Trp?Ala?Arg?Gly?Arg?Lys?Pro?Trp?Lys?Leu?Ala

50 55 60

 

Ile?Gln?Ile?Leu?Lys?Ile?Ala?Met?Val?Thr?Ile?Gln?Tyr?Leu?Gln?Leu

65 70 75 80

 

Tyr?Asn?Val?Ser?Val?Gly?Asn?His?Ala?Tyr?Glu?Asn?Lys?Gly?Thr?Lys

85 90 95

 

Gln?Ser?Ala?Met?Ala?Ile?Cys?Gln?His?Phe?Tyr?Lys?Arg?Gly?Asn?Ile

100 105 110

 

Tyr?Pro?Gly?Asn?Asp?Thr?Phe?Asp?Ile?Asp?Pro?Glu?Ile?Glu?Thr?Glu

115 120 125

 

Cys?Phe?Phe?Val?Glu?Pro?Asp?Glu?Pro?Phe?His?Ile?Gly?Thr?Pro?Ala

130 135 140

 

Glu?Asn?Lys?Leu?Asn?Leu?Thr?Leu?Asp?Phe?His?Arg?Leu?Leu?Thr?Val

145 150 155 160

 

Glu?Leu?Gln?Phe?Lys?Leu?Lys?Ala?Ile?Asn?Leu?Gln?Thr?Val?Arg?His

165 170 175

 

Gln?Glu?Leu?Pro?Asp?Cys?Tyr?Asp?Phe?Thr?Leu?Thr?Ile?Thr?Phe?Asp

180 185 190

 

Asn?Lys?Ala?His?Ser?Gly?Arg?Ile?Lys?Ile?Ser?Leu?Asp?Asn?Asp?Ile

195 200 205

 

Ser?Ile?Arg?Glu?Cys?Lys?Asp?Trp?His?Val?Ser?Gly?Ser?Ile?Gln?Lys

210 215 220

 

Asn?Thr?His?Tyr?Met?Met?Ile?Phe?Asp?Ala?Phe?Val?Ile?Leu?Thr?Cys

225 230 235 240

 

Leu?Val?Ser?Leu?Ile?Leu?Cys?Ile?Arg?Ser?Val?Ile?Arg?Gly?Leu?Gln

245 250 255

 

Leu?Gln?Gln?Glu?Phe?Val?Asn?Phe?Phe?Leu?Leu?His?Tyr?Lys?Lys?Gly

260 265 270

Val?Ser?Val?Ser?Asp?Gln?Met?Glu?Phe?Val?Asn?Gly?Trp?Tyr?Ile?Met

275 280 285

 

Ile?Ile?Ile?Ser?Asp?Ile?Leu?Thr?Ile?Ile?Gly?Ser?Ile?Leu?Lys?Met

290 295 300

 

Glu?Ile?Gln?Ala?Lys?Ser?Leu?Thr?Ser?Tyr?Asp?Val?Cys?Ser?Ile?Leu

305 310 315 320

 

Leu?Gly?Thr?Ser?Thr?Met?Leu?Val?Trp?Leu?Gly?Val?Ile?Arg?Tyr?Leu

325 330 335

 

Gly?Phe?Phe?Ala?Lys?Tyr?Asn?Leu?Leu?Ile?Leu?Thr?Leu?Gln?Ala?Ala

340 345 350

 

Leu?Pro?Asn?Val?Ile?Arg?Phe?Cys?Cys?Cys?Ala?Ala?Met?Ile?Tyr?Leu

355 360 365

 

Gly?Tyr?Cys?Phe?Cys?Gly?Trp?Ile?Val?Leu?Gly?Pro?Tyr?His?Asp?Lys

370 375 380

 

Phe?Arg?Ser?Leu?Asn?Met?Val?Ser?Glu?Cys?Leu?Phe?Ser?Leu?Ile?Asn

385 390 395 400

 

Gly?Asp?Asp?Met?Phe?Ala?Thr?Phe?Ala?Lys?Met?Gln?Gln?Lys?Ser?Tyr

405 410 415

 

Leu?Val?Trp?Leu?Phe?Ser?Arg?Ile?Tyr?Leu?Tyr?Ser?Phe?Ile?Ser?Leu

420 425 430

 

Phe?Ile?Tyr?Met?Ile?Leu?Ser?Leu?Phe?Ile?Ala?Leu?Ile?Thr?Asp?Thr

435 440 445

 

Tyr?Glu?Thr?Ile?Lys?Gln?Tyr?Gln?Gln?Asp?Gly?Phe?Pro?Glu?Thr?Glu

450 455 460

 

Leu?Arg?Thr?Phe?Ile?Ser?Glu?Cys?Lys?Asp?Leu?Pro?Asn?Ser?Gly?Lys

465 470 475 480

 

Tyr?Arg?Leu?Glu?Asp?Asp?Pro?Pro?Val?Ser?Leu?Phe?Cys?Cys?Cys?Lys

485 490 495

 

Lys

 

<210>29

<211>2714

<212>DNA

＜213〉orangutan

 

<400>29

tcctctagag?atggcagatc?ctgaggtagt?tgtgagtagc?tgcagctctc?atgaagagga 60

aaatcgctgc?aattttaacc?agcaaacatc?tccatctgag?gagcttctat?tagaagacca 120

gatgaggcga?aaactcaaat?tttttttcat?gaatccctgt?gagaagttct?gggctcgagg 180

tagaaaacca?tggaaacttg?ccatacaaat?tctaaaaatt?gcaatggtga?ctatccagct 240

ggtcttattt?gggctaagta?accagatggt?ggtagctttc?aaggaagaga?atactatagc 300

attcaaacac?cttttcctaa?aaggatatat?ggaccgaatg?gatgacacat?atgcagtgta 360

cacacaaagt?gacgtgtatg?atcagttaat?cttcgcagta?aaccagtact?tgcagctata 420

caatgtctcc?gttgggaatc?atgcttatga?gaacaaaggt?accaagcaat?ctgctatggc 480

aatctgtcag?cacttctaca?agcgaggaaa?catctaccct?ggaaatgata?cctttgacat 540

cgatccagaa?attgaaactg?agtgtttctt?tgtggagcca?gatgaacctt?ttcacattgg 600

gacaccagca?gaaaataaac?tgaacttaac?actggacttc?cacagactcc?taacagtgga 660

gcttcagttt?aaactgaaag?ccattaatct?gcagacagtt?cgtcatcaag?aactccctga 720

ctgttatgac?tttactctga?ctataacatt?tgacaacaag?gcccatagtg?gaagaattaa 780

aataagttta?gataatgaca?tttccatcag?agaatgtaaa?gactggcatg?tatctggatc 840

aattcagaag?aacactcatt?acatgatgat?ctttgatgcc?tttgtcattc?tgacttgctt 900

ggtttcatta?atcctctgca?ttagatctgt?gattagagga?cttcagcttc?agcaggagtt 960

tgtcaatttt?ttcctcctcc?attataagaa?gggagtttct?gtttctgatc?aaatggaatt 1020

tgtcaatgga?tggtacatta?tgattattat?tagtgacata?ttgacaatca?ttggatcaat 1080

tctaaaaatg?gaaatccaag?ctaagagtct?aactagttat?gatgtctgta?gcatacttct 1140

tgggacttct?accatgcttg?tgtggcttgg?agtcatccga?tacctcggtt?tctttgcaaa 1200

gtacaatctc?ctcattttga?cccttcaggc?agcactgccc?aatgtcatca?ggttctgctg 1260

ctgtgcagct?atgatttact?taggttactg?cttctgtgga?tggatcgtgc?tggggcctta 1320

ccatgacaag?tttcgttctc?tgaacatggt?ctctgagtgc?cttttctctc?tgataaatgg 1380

agatgatatg?tttgccacgt?ttgcaaaaat?gcagcaaaaa?agttacttag?tctggctgtt 1440

tagtagaatt?tacctctact?cattcatcag?cctctttata?tatatgattt?taagtctttt 1500

cattgcactg?atcactgata?catacgaaac?aattaagcaa?taccaacaag?atggcttccc 1560

agagactgaa?cttcgtacat?ttatatcaga?atgcaaagat?ctacccaact?ctggaaaata 1620

cagattagaa?gatgaccctc?cagtatcttt?attctgctgt?tgtaaaaagt?agctatcagg 1680

tttatctgta?ctttagagga?aaatataatg?tgtagctgag?ttagaacact?gtggatattc 1740

tgagatcaga?tgtagtatgt?ttgaagactg?ttattttgag?ctaattgaga?cctataattc 1800

accaataact?gtttatattt?ttaaaagcaa?tatttaatgt?ctttgcagct?ttatgctggg 1860

cttgttttta?aaacaacttt?aatgaggaaa?gctattggat?tattattatt?tcttgtttat 1920

tttgccatgg?ctttagaatg?tattctgtat?gcctctcttt?tgctctgata?ctcttgcttc 1980

ctgctattct?gattgtgcag?actgtgtaat?tagtggaaaa?caatccttgg?tctgactgtg 2040

actttggaca?actcagtaac?cctggcttgg?accactctca?ggagtccatc?cttgagagag 2100

tgggtgtagt?taccatttat?acagtaatca?ttgcatttta?aaatctgctc?ttgaaaggaa 2160

gaataagagt?gcaccagaat?aagagcgcac?cagaataaga?gcgcaccagc?taacaatgtg 2220

atacggccat?atgtcactta?aggatagaga?tatgttctga?gaaatgtgtc?attaggcgat 2280

tttgtcatta?aacatcatag?catgtacttc?cacaaaccta?gatggtatag?cctactacac 2340

acctaggcta?tttggtatag?cctgttgatc?ctggggtaca?aatctgtaca?acatgttact 2400

gtattgaata?cagtaggcaa?ttgtaacaca?atggtaagta?tctaaacata?gaaaagggac 2460

agtaaaaata?tggttttata?atcttctggg?accaccattg?tatatgcggt?acatcgttga 2520

ccaaaacatc?gttatccagc?atatgactgt?atttggttat?gaaagccaac?tgttacttga 2580

ttctgctttt?agttcttaag?aggatcaggt?ttttaaatac?tcatttacaa?gttttctatc 2640

ctccttcagt?gttaaagtag?aaagtaaaaa?gagtatctta?tacatgcatg?aaattaaagc 2700

atataccaaa?tgca 2714

 

<210>30

<211>553

<212>PRT

＜213〉orangutan

 

<400>30

Met?Ala?Asp?Pro?Glu?Val?Val?Val?Ser?Ser?Cys?Ser?Ser?His?Glu?Glu

1 5 10 15

 

Glu?Asn?Arg?Cys?Asn?Phe?Asn?Gln?Gln?Thr?Ser?Pro?Ser?Glu?Glu?Leu

20 25 30

 

Leu?Leu?Glu?Asp?Gln?Met?Arg?Arg?Lys?Leu?Lys?Phe?Phe?Phe?Met?Asn

35 40 45

 

Pro?Cys?Glu?Lys?Phe?Trp?Ala?Arg?Gly?Arg?Lys?Pro?Trp?Lys?Leu?Ala

50 55 60

 

Ile?Gln?Ile?Leu?Lys?Ile?Ala?Met?Val?Thr?Ile?Gln?Leu?Val?Leu?Phe

65 70 75 80

 

Gly?Leu?Ser?Asn?Gln?Met?Val?Val?Ala?Phe?Lys?Glu?Glu?Asn?Thr?Ile

85 90 95

 

Ala?Phe?Lys?His?Leu?Phe?Leu?Lys?Gly?Tyr?Met?Asp?Arg?Met?Asp?Asp

100 105 110

 

Thr?Tyr?Ala?Val?Tyr?Thr?Gln?Ser?Asp?Val?Tyr?Asp?Gln?Leu?Ile?Phe

115 120 125

 

Ala?Val?Asn?Gln?Tyr?Leu?Gln?Leu?Tyr?Asn?Val?Ser?Val?Gly?Asn?His

130 135 140

 

Ala?Tyr?Glu?Asn?Lys?Gly?Thr?Lys?Gln?Ser?Ala?Met?Ala?Ile?Cys?Gln

145 150 155 160

 

His?Phe?Tyr?Lys?Arg?Gly?Asn?Ile?Tyr?Pro?Gly?Asn?Asp?Thr?Phe?Asp

165 170 175

 

Ile?Asp?Pro?Glu?Ile?Glu?Thr?Glu?Cys?Phe?Phe?Val?Glu?Pro?Asp?Glu

180 185 190

 

Pro?Phe?His?Ile?Gly?Thr?Pro?Ala?Glu?Asn?Lys?Leu?Asn?Leu?Thr?Leu

195 200 205

 

Asp?Phe?His?Arg?Leu?Leu?Thr?Val?Glu?Leu?Gln?Phe?Lys?Leu?Lys?Ala

210 215 220

 

Ile?Asn?Leu?Gln?Thr?Val?Arg?His?Gln?Glu?Leu?Pro?Asp?Cys?Tyr?Asp

225 230 235 240

 

Phe?Thr?Leu?Thr?Ile?Thr?Phe?Asp?Asn?Lys?Ala?His?Ser?Gly?Arg?Ile

245 250 255

 

Lys?Ile?Ser?Leu?Asp?Asn?Asp?Ile?Ser?Ile?Arg?Glu?Cys?Lys?Asp?Trp

260 265 270

 

His?Val?Ser?Gly?Ser?Ile?Gln?Lys?Asn?Thr?His?Tyr?Met?Met?Ile?Phe

275 280 285

 

Asp?Ala?Phe?Val?Ile?Leu?Thr?Cys?Leu?Val?Ser?Leu?Ile?Leu?Cys?Ile

290 295 300

 

Arg?Ser?Val?Ile?Arg?Gly?Leu?Gln?Leu?Gln?Gln?Glu?Phe?Val?Asn?Phe

305 310 315 320

 

Phe?Leu?Leu?His?Tyr?Lys?Lys?Gly?Val?Ser?Val?Ser?Asp?Gln?Met?Glu

325 330 335

 

Phe?Val?Asn?Gly?Trp?Tyr?Ile?Met?Ile?Ile?Ile?Ser?Asp?Ile?Leu?Thr

340 345 350

 

Ile?Ile?Gly?Ser?Ile?Leu?Lys?Met?Glu?Ile?Gln?Ala?Lys?Ser?Leu?Thr

355 360 365

 

Ser?Tyr?Asp?Val?Cys?Ser?Ile?Leu?Leu?Gly?Thr?Ser?Thr?Met?Leu?Val

370 375 380

 

Trp?Leu?Gly?Val?Ile?Arg?Tyr?Leu?Gly?Phe?Phe?Ala?Lys?Tyr?Asn?Leu

385 390 395 400

 

Leu?Ile?Leu?Thr?Leu?Gln?Ala?Ala?Leu?Pro?Asn?Val?Ile?Arg?Phe?Cys

405 410 415

 

Cys?Cys?Ala?Ala?Met?Ile?Tyr?Leu?Gly?Tyr?Cys?Phe?Cys?Gly?Trp?Ile

420 425 430

 

Val?Leu?Gly?Pro?Tyr?His?Asp?Lys?Phe?Arg?Ser?Leu?Asn?Met?Val?Ser

435 440 445

 

Glu?Cys?Leu?Phe?Ser?Leu?Ile?Asn?Gly?Asp?Asp?Met?Phe?Ala?Thr?Phe

450 455 460

 

Ala?Lys?Met?Gln?Gln?Lys?Ser?Tyr?Leu?Val?Trp?Leu?Phe?Ser?Arg?Ile

465 470 475 480

 

Tyr?Leu?Tyr?Ser?Phe?Ile?Ser?Leu?Phe?Ile?Tyr?Met?Ile?Leu?Ser?Leu

485 490 495

 

Phe?Ile?Ala?Leu?Ile?Thr?Asp?Thr?Tyr?Glu?Thr?Ile?Lys?Gln?Tyr?Gln

500 505 510

 

Gln?Asp?Gly?Phe?Pro?Glu?Thr?Glu?Leu?Arg?Thr?Phe?Ile?Ser?Glu?Cys

515 520 525

 

Lys?Asp?Leu?Pro?Asn?Ser?Gly?Lys?Tyr?Arg?Leu?Glu?Asp?Asp?Pro?Pro

530 535 540

 

Val?Ser?Leu?Phe?Cys?Cys?Cys?Lys?Lys

545 550

 

<210>31

<211>1896

<212>DNA

＜213〉Xenopus laevis (Silurana tropicalis)

 

<400>31

ttgcaactag?gtctgacagt?aggacaatgt?ggcaggtcac?gtgacagcag?tgctgatggt 60

agagatgcgc?cagcattcag?gtctgagagc?agaaagaaaa?gctggccaaa?acaaaggaca 120

ttctctttgc?tgcttcgcta?gctgagacgc?tgctatagta?tagcagacat?ggaaaaccca 180

gagctaataa?agacatgcaa?ctctttggat?gaacatgatg?gtccctactg?ctgcaagcag 240

tgccctatga?ctgatgagct?acttatggaa?gaccagctac?gaaggaaact?taaattcttt 300

ttcatgaacc?catgtgagaa?gttccgtgcc?cgtggacgaa?agccttggaa?gctttgtatt 360

caaattttaa?aaattgcaat?ggtgacaatc?caattagttt?tatttggact?cagtaatgaa 420

atggtagtca?cctttaaaga?ggagaacact?gtagctttta?agcatctgtt?tttgaaagga 480

tataaggatg?gacatgatga?cacatatgct?atctacagtc?aagaagatgt?tcatgctcat 540

ataaacttta?caattaaaag?gtacctagag?ctacaaaaca?tatctgttgg?aaatcatgca 600

tatgaaagta?atggtaaagg?tcaaactgga?atgtcattat?gtcaacatta?ctataaacaa 660

gggagtatct?ttcctggaaa?tgaaacattt?gaaattgacc?cacaaataga?tactgaatgt 720

ttccatattg?atccatcaac?tctgtgttct?aatgacacac?ctgcagaata?ctactggtct 780

aatatgacac?tagacttcta?tagacttgtt?tcagttgaaa?ttatgtttaa?gcttaaagca 840

attaatcttc?aaaccattcg?tcatcatgaa?cttccagact?gctatgactt?catggttata 900

ataacatttg?ataataaggc?acacagtgga?aggataaaaa?tcagcttaga?taatgatgtt 960

ggaatccagg?aatgcaaaga?ctggcatgtg?tctggatcta?ttcaaaaaaa?tactcattac 1020

atgatgattt?ttgatgctgc?tgttattttg?gtctgcttat?cttccataac?actctgcatt 1080

cgctccgtgg?ttaaaggaat?tcacctacaa?aaagaatatg?taaacttttt?ccagcatcgt 1140

tttgcaagga?ctgtgtcctc?agctgatcgc?atggaatttg?tcaatggctg?gtacattatg 1200

ataatcatca?gtgatgtttt?gtcaattatt?ggctcaatct?tgaagatgga?gatccaagca 1260

aagagcctca?ccagttatga?tgtctgcagt?attctcttgg?gaacatccac?cttattagtg 1320

tggcttggag?ttattcgcta?cttgggattt?tttaagaaat?acaatcttct?gatcctgaca 1380

cttagggcag?ccttacctaa?tgtgatacga?ttctgctgtt?gtgctgctat?gatctacctg 1440

ggctactgct?tctgtggctg?gattgttctg?gggccttacc?atgtaaagtt?caggtccctg 1500

aacatggttt?cagagtgcct?gttctccctt?attaatggag?acgatatgtt?tacaacgttt 1560

tcaatcatgc?aggagaagag?ctacttggtt?tggctgttta?gtcgcattta?tttgtattcc 1620

tttataagtc?tcttcatata?catggttctg?agtctcttca?ttgcacttat?tactgacaca 1680

tacgatacaa?tcaagaatta?ccagatcgat?ggctttccag?aatcagaact?tcacacattt 1740

gtatccgagt?gcaaagattt?gccaacctct?ggtcgatata?gggaacaaga?cgagacctcc 1800

tgtttgtcta?tgctgtgttg?taatcggtaa?aaaagaatcc?cagaagaagc?actttatcca 1860

tggcctttaa?aaatctgcaa?aaaaaaaaaa?aaaaaa 1896

 

<210>32

<211>553

<212>PRT

＜213〉Xenopus laevis

 

<400>32

Met?Glu?Asn?Pro?Glu?Leu?Ile?Lys?Thr?Cys?Asn?Ser?Leu?Asp?Glu?His

1 5 10 15

 

Asp?Gly?Pro?Tyr?Cys?Cys?Lys?Gln?Cys?Pro?Met?Thr?Asp?Glu?Leu?Leu

20 25 30

 

Met?Glu?Asp?Gln?Leu?Arg?Arg?Lys?Leu?Lys?Phe?Phe?Phe?Met?Asn?Pro

35 40 45

 

Cys?Glu?Lys?Phe?Arg?Ala?Arg?Gly?Arg?Lys?Pro?Trp?Lys?Leu?Cys?Ile

50 55 60

 

Gln?Ile?Leu?Lys?Ile?Ala?Met?Val?Thr?Ile?Gln?Leu?Val?Leu?Phe?Gly

65 70 75 80

 

Leu?Ser?Asn?Glu?Met?Val?Val?Thr?Phe?Lys?Glu?Glu?Asn?Thr?Val?Ala

85 90 95

 

Phe?Lys?His?Leu?Phe?Leu?Lys?Gly?Tyr?Lys?Asp?Gly?His?Asp?Asp?Thr

100 105 110

 

Tyr?Ala?Ile?Tyr?Ser?Gln?Glu?Asp?Val?His?Ala?His?Ile?Asn?Phe?Thr

115 120 125

 

Ile?Lys?Arg?Tyr?Leu?Glu?Leu?Gln?Asn?Ile?Ser?Val?Gly?Asn?His?Ala

130 135 140

 

Tyr?Glu?Ser?Asn?Gly?Lys?Gly?Gln?Thr?Gly?Met?Ser?Leu?Cys?Gln?His

145 150 155 160

 

Tyr?Tyr?Lys?Gln?Gly?Ser?Ile?Phe?Pro?Gly?Asn?Glu?Thr?Phe?Glu?Ile

165 170 175

 

Asp?Pro?Gln?Ile?Asp?Thr?Glu?Cys?Phe?His?Ile?Asp?Pro?Ser?Thr?Leu

180 185 190

 

Cys?Ser?Asn?Asp?Thr?Pro?Ala?Glu?Tyr?Tyr?Trp?Ser?Asn?Met?Thr?Leu

195 200 205

 

Asp?Phe?Tyr?Arg?Leu?Val?Ser?Val?Glu?Ile?Met?Phe?Lys?Leu?Lys?Ala

210 215 220

 

Ile?Asn?Leu?Gln?Thr?Ile?Arg?His?His?Glu?Leu?Pro?Asp?Cys?Tyr?Asp

225 230 235 240

 

Phe?Met?Val?Ile?Ile?Thr?Phe?Asp?Asn?Lys?Ala?His?Ser?Gly?Arg?Ile

245 250 255

 

Lys?Ile?Ser?Leu?Asp?Asn?Asp?Val?Gly?Ile?Gln?Glu?Cys?Lys?Asp?Trp

260 265 270

 

His?Val?Ser?Gly?Ser?Ile?Gln?Lys?Asn?Thr?His?Tyr?Met?Met?Ile?Phe

275 280 285

Asp?Ala?Ala?Val?Ile?Leu?Val?Cys?Leu?Ser?Ser?Ile?Thr?Leu?Cys?Ile

290 295 300

 

Arg?Ser?Val?Val?Lys?Gly?Ile?His?Leu?Gln?Lys?Glu?Tyr?Val?Asn?Phe

305 310 315 320

 

Phe?Gln?His?Arg?Phe?Ala?Arg?Thr?Val?Ser?Ser?Ala?Asp?Arg?Met?Glu

325 330 335

 

Phe?Val?Asn?Gly?Trp?Tyr?Ile?Met?Ile?Ile?Ile?Ser?Asp?Val?Leu?Ser

340 345 350

 

Ile?Ile?Gly?Ser?Ile?Leu?Lys?Met?Glu?Ile?Gln?Ala?Lys?Ser?Leu?Thr

355 360 365

 

Ser?Tyr?Asp?Val?Cys?Ser?Ile?Leu?Leu?Gly?Thr?Ser?Thr?Leu?Leu?Val

370 375 380

 

Trp?Leu?Gly?Val?Ile?Arg?Tyr?Leu?Gly?Phe?Phe?Lys?Lys?Tyr?Asn?Leu

385 390 395 400

 

Leu?Ile?Leu?Thr?Leu?Arg?Ala?Ala?Leu?Pro?Asn?Val?Ile?Arg?Phe?Cys

405 410 415

 

Cys?Cys?Ala?Ala?Met?Ile?Tyr?Leu?Gly?Tyr?Cys?Phe?Cys?Gly?Trp?Ile

420 425 430

 

Val?Leu?Gly?Pro?Tyr?His?Val?Lys?Phe?Arg?Ser?Leu?Asn?Met?Val?Ser

435 440 445

 

Glu?Cys?Leu?Phe?Ser?Leu?Ile?Asn?Gly?Asp?Asp?Met?Phe?Thr?Thr?Phe

450 455 460

 

Ser?Ile?Met?Gln?Glu?Lys?Ser?Tyr?Leu?Val?Trp?Leu?Phe?Ser?Arg?Ile

465 470 475 480

 

Tyr?Leu?Tyr?Ser?Phe?Ile?Ser?Leu?Phe?Ile?Tyr?Met?Val?Leu?Ser?Leu

485 490 495

 

Phe?Ile?Ala?Leu?Ile?Thr?Asp?Thr?Tyr?Asp?Thr?Ile?Lys?Asn?Tyr?Gln

500 505 510

 

Ile?Asp?Gly?Phe?Pro?Glu?Ser?Glu?Leu?His?Thr?Phe?Val?Ser?Glu?Cys

515 520 525

 

Lys?Asp?Leu?Pro?Thr?Ser?Gly?Arg?Tyr?Arg?Glu?Gln?Asp?Glu?Thr?Ser

530 535 540

 

Cys?Leu?Ser?Met?Leu?Cys?Cys?Asn?Arg

545 550

 

<210>33

<211>1896

<212>DNA

＜213〉Xenopus laevis

 

<400>33

ttgcaactag?gtctgacagt?aggacaatgt?ggcaggtcac?gtgacagcag?tgctgatggt 60

agagatgcgc?cagcattcag?gtctgagagc?agaaagaaaa?gctggccaaa?acaaaggaca 120

ttctctttgc?tgcttcgcta?gctgagacgc?tgctatagta?tagcagacat?ggaaaaccca 180

gagctaataa?agacatgcaa?ctctttggat?gaacatgatg?gtccctactg?ctgcaagcag 240

tgccctatga?ctgatgagct?acttatggaa?gaccagctac?gaaggaaact?taaattcttt 300

ttcatgaacc?catgtgagaa?gttccgtgcc?cgtggacgaa?agccttggaa?gctttgtatt 360

caaattttaa?aaattgcaat?ggtgacaatc?caattagttt?tatttggact?cagtaatgaa 420

atggtagtca?cctttaaaga?ggagaacact?gtagctttta?agcatctgtt?tttgaaagga 480

tataaggatg?gacatgatga?cacatatgct?atctacagtc?aagaagatgt?tcatgctcat 540

ataaacttta?caattaaaag?gtacctagag?ctacaaaaca?tatctgttgg?aaatcatgca 600

tatgaaagta?atggtaaagg?tcaaactgga?atgtcattat?gtcaacatta?ctataaacaa 660

gggagtatct?ttcctggaaa?tgaaacattt?gaaattgacc?cacaaataga?tactgaatgt 720

ttccatattg?atccatcaac?tctgtgttct?aatgacacac?ctgcagaata?ctactggtct 780

aatatgacac?tagacttcta?tagacttgtt?tcagttgaaa?ttatgtttaa?gcttaaagca 840

attaatcttc?aaaccattcg?tcatcatgaa?cttccagact?gctatgactt?catggttata 900

ataacatttg?ataataaggc?acacagtgga?aggataaaaa?tcagcttaga?taatgatgtt 960

ggaatccagg?aatgcaaaga?ctggcatgtg?tctggatcta?ttcaaaaaaa?tactcattac 1020

atgatgattt?ttgatgctgc?tgttattttg?gtctgcttat?cttccataac?actctgcatt 1080

cgctccgtgg?ttaaaggaat?tcacctacaa?aaagaatatg?taaacttttt?ccagcatcgt 1140

tttgcaagga?ctgtgtcctc?agctgatcgc?atggaatttg?tcaatggctg?gtacattatg 1200

ataatcatca?gtgatgtttt?gtcaattatt?ggctcaatct?tgaagatgga?gatccaagca 1260

aagagcctca?ccagttatga?tgtctgcagt?attctcttgg?gaacatccac?cttattagtg 1320

tggcttggag?ttattcgcta?cttgggattt?tttaagaaat?acaatcttct?gatcctgaca 1380

cttagggcag?ccttacctaa?tgtgatacga?ttctgctgtt?gtgctgctat?gatctacctg 1440

ggctactgct?tctgtggctg?gattgttctg?gggccttacc?atgtaaagtt?caggtccctg 1500

aacatggttt?cagagtgcct?gttctccctt?attaatggag?acgatatgtt?tacaacgttt 1560

tcaatcatgc?aggagaagag?ctacttggtt?tggctgttta?gtcgcattta?tttgtattcc 1620

tttataagtc?tcttcatata?catggttctg?agtctcttca?ttgcacttat?tactgacaca 1680

tacgatacaa?tcaagaatta?ccagatcgat?ggctttccag?aatcagaact?tcacacattt 1740

gtatccgagt?gcaaagattt?gccaacctct?ggtcgatata?gggaacaaga?cgagacctcc 1800

tgtttgtcta?tgctgtgttg?taatcggtaa?aaaagaatcc?cagaagaagc?actttatcca 1860

tggcctttaa?aaatctgcaa?aaaaaaaaaa?aaaaaa 1896

 

<210>34

<211>553

<212>PRT

＜213〉Xenopus laevis

 

<400>34

Met?Glu?Asn?Pro?Glu?Leu?Ile?Lys?Thr?Cys?Asn?Ser?Leu?Asp?Glu?His

1 5 10 15

 

Asp?Gly?Pro?Tyr?Cys?Cys?Lys?Gln?Cys?Pro?Met?Thr?Asp?Glu?Leu?Leu

20 25 30

 

Met?Glu?Asp?Gln?Leu?Arg?Arg?Lys?Leu?Lys?Phe?Phe?Phe?Met?Asn?Pro

35 40 45

 

Cys?Glu?Lys?Phe?Arg?Ala?Arg?Gly?Arg?Lys?Pro?Trp?Lys?Leu?Cys?Ile

50 55 60

 

Gln?Ile?Leu?Lys?Ile?Ala?Met?Val?Thr?Ile?Gln?Leu?Val?Leu?Phe?Gly

65 70 75 80

 

Leu?Ser?Asn?Glu?Met?Val?Val?Thr?Phe?Lys?Glu?Glu?Asn?Thr?Val?Ala

85 90 95

 

Phe?Lys?His?Leu?Phe?Leu?Lys?Gly?Tyr?Lys?Asp?Gly?His?Asp?Asp?Thr

100 105 110

 

Tyr?Ala?Ile?Tyr?Ser?Gln?Glu?Asp?Val?His?Ala?His?Ile?Asn?Phe?Thr

115 120 125

 

Ile?Lys?Arg?Tyr?Leu?Glu?Leu?Gln?Asn?Ile?Ser?Val?Gly?Asn?His?Ala

130 135 140

 

Tyr?Glu?Ser?Asn?Gly?Lys?Gly?Gln?Thr?Gly?Met?Ser?Leu?Cys?Gln?His

145 150 155 160

 

Tyr?Tyr?Lys?Gln?Gly?Ser?Ile?Phe?Pro?Gly?Asn?Glu?Thr?Phe?Glu?Ile

165 170 175

 

Asp?Pro?Gln?Ile?Asp?Thr?Glu?Cys?Phe?His?Ile?Asp?Pro?Ser?Thr?Leu

180 185 190

 

Cys?Ser?Asn?Asp?Thr?Pro?Ala?Glu?Tyr?Tyr?Trp?Ser?Asn?Met?Thr?Leu

195 200 205

 

Asp?Phe?Tyr?Arg?Leu?Val?Ser?Val?Glu?Ile?Met?Phe?Lys?Leu?Lys?Ala

210 215 220

 

Ile?Asn?Leu?Gln?Thr?Ile?Arg?His?His?Glu?Leu?Pro?Asp?Cys?Tyr?Asp

225 230 235 240

 

Phe?Met?Val?Ile?Ile?Thr?Phe?Asp?Asn?Lys?Ala?His?Ser?Gly?Arg?Ile

245 250 255

 

Lys?Ile?Ser?Leu?Asp?Asn?Asp?Val?Gly?Ile?Gln?Glu?Cys?Lys?Asp?Trp

260 265 270

 

His?Val?Ser?Gly?Ser?Ile?Gln?Lys?Asn?Thr?His?Tyr?Met?Met?Ile?Phe

275 280 285

 

Asp?Ala?Ala?Val?Ile?Leu?Val?Cys?Leu?Ser?Ser?Ile?Thr?Leu?Cys?Ile

290 295 300

 

Arg?Ser?Val?Val?Lys?Gly?Ile?His?Leu?Gln?Lys?Glu?Tyr?Val?Asn?Phe

305 310 315 320

 

Phe?Gln?His?Arg?Phe?Ala?Arg?Thr?Val?Ser?Ser?Ala?Asp?Arg?Met?Glu

325 330 335

 

Phe?Val?Asn?Gly?Trp?Tyr?Ile?Met?Ile?Ile?Ile?Ser?Asp?Val?Leu?Ser

340 345 350

 

Ile?Ile?Gly?Ser?Ile?Leu?Lys?Met?Glu?Ile?Gln?Ala?Lys?Ser?Leu?Thr

355 360 365

 

Ser?Tyr?Asp?Val?Cys?Ser?Ile?Leu?Leu?Gly?Thr?Ser?Thr?Leu?Leu?Val

370 375 380

 

Trp?Leu?Gly?Val?Ile?Arg?Tyr?Leu?Gly?Phe?Phe?Lys?Lys?Tyr?Asn?Leu

385 390 395 400

 

Leu?Ile?Leu?Thr?Leu?Arg?Ala?Ala?Leu?Pro?Asn?Val?Ile?Arg?Phe?Cys

405 410 415

 

Cys?Cys?Ala?Ala?Met?Ile?Tyr?Leu?Gly?Tyr?Cys?Phe?Cys?Gly?Trp?Ile

420 425 430

 

Val?Leu?Gly?Pro?Tyr?His?Val?Lys?Phe?Arg?Ser?Leu?Asn?Met?Val?Ser

435 440 445

 

Glu?Cys?Leu?Phe?Ser?Leu?Ile?Asn?Gly?Asp?Asp?Met?Phe?Thr?Thr?Phe

450 455 460

 

Ser?Ile?Met?Gln?Glu?Lys?Ser?Tyr?Leu?Val?Trp?Leu?Phe?Ser?Arg?Ile

465 470 475 480

 

Tyr?Leu?Tyr?Ser?Phe?Ile?Ser?Leu?Phe?Ile?Tyr?Met?Val?Leu?Ser?Leu

485 490 495

 

Phe?Ile?Ala?Leu?Ile?Thr?Asp?Thr?Tyr?Asp?Thr?Ile?Lys?Asn?Tyr?Gln

500 505 510

 

Ile?Asp?Gly?Phe?Pro?Glu?Ser?Glu?Leu?His?Thr?Phe?Val?Ser?Glu?Cys

515 520 525

 

Lys?Asp?Leu?Pro?Thr?Ser?Gly?Arg?Tyr?Arg?Glu?Gln?Asp?Glu?Thr?Ser

530 535 540

 

Cys?Leu?Ser?Met?Leu?Cys?Cys?Asn?Arg

545 550

 

<210>35

<211>6923

<212>DNA

＜213〉homo sapiens

 

<400>35

cgcgctgcct?gagctgagcc?gccgtaggtg?aggggcccgc?gtccccgccc?gccctgggcg 60

ccgcgcctgg?cactgatcct?gccggtcgcc?cactgtcgcc?gccgccgccg?cccgcgggca 120

ccatgacagc?tctgagcgct?ggggttacag?actgtggttt?tgtgcttgct?caccaaagct 180

aacctcagca?tgctcaaaag?gaagcagagt?tccagggtgg?aagcccagcc?agtcactgac 240

tttggtcctg?atgagtctct?gtcggataat?gctgacatcc?tctggattaa?caaaccatgg 300

gttcactctt?tgctgcgcat?ctgtgccatc?atcagcgtca?tttctgtttg?tatgaatacg 360

ccaatgacct?tcgagcacta?tcctccactt?cagtatgtga?ccttcacttt?ggatacatta 420

ttgatgtttc?tctacacggc?agagatgata?gcaaaaatgc?acatccgggg?cattgtcaag 480

ggggatagtt?cctatgtgaa?agatcgctgg?tgtgtttttg?atggatttat?ggtcttttgc 540

ctttgggttt?ctttggtgct?acaggtgttt?gaaattgctg?atatagttga?tcagatgtca 600

ccttggggca?tgttgcggat?tccacggcca?ctgattatga?tccgagcatt?ccggatttat 660

ttccgatttg?aactgccaag?gaccagaatt?acaaatattt?taaagcgatc?gggagaacaa 720

atatggagtg?tttccatttt?tctacttttc?tttctacttc?tttatggaat?tttaggagtt 780

cagatgtttg?gaacatttac?ttatcactgt?gttgtaaatg?acacaaagcc?agggaatgta 840

acctggaata?gtttagctat?tccagacaca?cactgctcac?cagagctaga?agaaggctac 900

cagtgcccac?ctggatttaa?atgcatggac?cttgaagatc?tgggacttag?caggcaagag 960

ctgggctaca?gtggctttaa?tgagatagga?actagtatat?tcaccgtcta?tgaggccgcc 1020

tcacaggaag?gctgggtgtt?cctcatgtac?agagcaattg?acagctttcc?ccgttggcgt 1080

tcctacttct?atttcatcac?tctcattttc?ttcctcgcct?ggcttgtgaa?gaacgtgttt 1140

attgctgtta?tcattgaaac?atttgcagaa?atcagagtac?agtttcaaca?aatgtgggga 1200

tcgagaagca?gcactacctc?aacagccacc?acccagatgt?ttcatgaaga?tgctgctgga 1260

ggttggcagc?tggtagctgt?ggatgtcaac?aagccccagg?gacgcgcccc?agcctgcctc 1320

cagaaaatga?tgcggtcatc?cgttttccac?atgttcatcc?tgagcatggt?gaccgtggac 1380

gtgatcgtgg?cggctagcaa?ctactacaaa?ggagaaaact?tcaggaggca?gtacgacgag 1440

ttctacctgg?cggaggtggc?ttttacagta?ctttttgatt?tggaagcact?tctgaagata 1500

tggtgtttgg?gatttactgg?atatattagc?tcatctctcc?acaaattcga?actactactc 1560

gtaattggaa?ctactcttca?tgtataccca?gatctttatc?attcacaatt?cacgtacttt 1620

caggttctcc?gagtagttcg?gctgattaag?atttcacctg?cattagaaga?ctttgtgtac 1680

aagatatttg?gtcctggaaa?aaagcttggg?agtttggttg?tatttactgc?cagcctcttg 1740

attgttatgt?cagcaattag?tttgcagatg?ttctgctttg?tcgaagaact?ggacagattt 1800

actacgtttc?cgagggcatt?tatgtccatg?ttccagatcc?tcacccagga?aggatgggtg 1860

gacgtaatgg?accaaactct?aaatgctgtg?ggacatatgt?gggcacccgt?ggttgccatc 1920

tatttcattc?tctatcatct?ttttgccact?ctgatcctcc?tgagtttgtt?tgttgctgtt 1980

attttggaca?acttagaact?tgatgaagac?ctaaagaagc?ttaaacaatt?aaagcaaagt 2040

gaagcaaatg?cggacaccaa?agaaaagctc?cctttacgcc?tgcgaatctt?tgaaaaattt 2100

ccaaacagac?ctcaaatggt?gaaaatctca?aagcttcctt?cagattttac?agttcctaaa 2160

atcagggaga?gttttatgaa?gcagtttatt?gaccgccagc?aacaggacac?atgttgcctc 2220

ctgagaagcc?tcccgaccac?ctcttcctcc?tcctgcgacc?actccaaacg?ctcagcaatt 2280

gaggacaaca?aatacatcga?ccaaaaactt?cgcaagtctg?ttttcagcat?cagggcaagg 2340

aaccttctgg?aaaaggagac?cgcagtcact?aaaatcttaa?gagcttgcac?ccgacagcgc 2400

atgctgagcg?gatcatttga?ggggcagccc?gcaaaggaga?ggtcaatcct?cagcgtgcag 2460

catcatatcc?gccaagagcg?caggtcacta?agacatggat?caaacagcca?gaggatcagc 2520

aggggaaaat?ctcttgaaac?tttgactcaa?gatcattcca?atacagtgag?atatagaaat 2580

gcacaaagag?aagacagtga?aataaagatg?attcaggaaa?aaaaggagca?agcagagatg 2640

aaaaggaaag?tgcaagaaga?ggaactcaga?gagaaccacc?catacttcga?taagccactg 2700

ttcattgtcg?ggcgagaaca?caggttcaga?aacttttgcc?gggtggtggt?ccgagcacgc 2760

ttcaacgcat?ctaaaacaga?ccctgtcaca?ggagctgtga?aaaatacaaa?gtaccatcaa 2820

ctttatgatt?tgctgggatt?ggtcacttac?ctggactggg?tcatgatcat?cgtaaccatc 2880

tgctcttgca?tttccatgat?gtttgagtcc?ccgtttcgaa?gagtcatgca?tgcacctact 2940

ttgcagattg?ctgagtatgt?gtttgtgata?ttcatgagca?ttgagcttaa?tctgaagatt 3000

atggcagatg?gcttattttt?cactccaact?gctgtcatca?gggacttcgg?tggagtaatg 3060

gacatattta?tatatcttgt?gagcttgata?tttctttgtt?ggatgcctca?aaatgtacct 3120

gctgaatcgg?gagctcagct?tctaatggtc?cttcggtgcc?tgagacctct?gcgcatattc 3180

aaactggtgc?cccagatgag?gaaagttgtt?cgagaacttt?tcagcggctt?caaggaaatt 3240

tttttggtct?ccattctttt?gctgacatta?atgctcgttt?ttgcaagctt?tggagttcag 3300

ctttttgctg?gaaaactggc?caagtgcaat?gatcccaaca?ttattagaag?ggaagattgc 3360

aatggcatat?tcagaattaa?tgtcagtgtg?tcaaagaact?taaatttaaa?attgaggcct 3420

ggagagaaaa?aacctggatt?ttgggtgccc?cgtgtttggg?cgaatcctcg?gaactttaat 3480

ttcgacaatg?tgggaaacgc?tatgctggcg?ttgtttgaag?ttctctcctt?gaaaggctgg 3540

gtggaagtga?gagatgttat?tattcatcgt?gtggggccga?tccatggaat?ctatattcat 3600

gtttttgtat?tcctgggttg?catgattgga?ctgacccttt?ttgttggagt?agttattgct 3660

aatttcaatg?aaaacaaggg?gacggctttg?ctgaccgtcg?atcagagaag?atgggaagac 3720

ctgaagagcc?gactgaagat?cgcacagcct?cttcatcttc?cgcctcgccc?ggataatgat 3780

ggttttagag?ctaaaatgta?tgacataacc?cagcatccat?tttttaagag?gacaatcgca 3840

ttactcgtcc?tggcccagtc?ggtgttgctc?tctgtcaagt?gggacgtcga?ggacccggtg 3900

accgtacctt?tggcaacaat?gtcagttgtt?ttcaccttca?tctttgttct?ggaggttacc 3960

atgaagatca?tagcaatgtc?gcctgctggc?ttctggcaaa?gcagaagaaa?ccgatacgat 4020

ctcctggtga?cgtcgcttgg?cgttgtatgg?gtggtgcttc?actttgccct?cctgaatgca 4080

tatacttaca?tgatgggcgc?ttgtgtgatt?gtatttaggt?ttttctccat?ctgtggaaaa 4140

catgtaacgc?taaagatgct?cctcttgaca?gtggtcgtca?gcatgtacaa?gagcttcttt 4200

atcatagtag?gcatgtttct?cttgctgctg?tgttacgctt?ttgctggagt?tgttttattt 4260

ggtactgtga?aatatgggga?gaatattaac?aggcatgcaa?atttttcttc?ggctggaaaa 4320

gctattaccg?tactgttccg?aattgtcaca?ggtgaagact?ggaacaagat?tatgcatgac 4380

tgtatggttc?agcctccgtt?ttgtactcca?gatgaattta?catactgggc?aacagactgt 4440

ggaaattatg?ctggggcact?tatgtatttc?tgttcatttt?atgtcatcat?tgcctacatc 4500

atgctaaatc?tgcttgtagc?cataattgtg?gagaatttct?ccttgtttta?ttccactgag 4560

gaggaccagc?ttttaagtta?caatgatctt?cgccactttc?aaataatatg?gaacatggtg 4620

gatgataaaa?gagagggggt?gatccccacg?ttccgcgtca?agttcctgct?gcggctactg 4680

cgtgggaggc?tggaggtgga?cctggacaag?gacaagctcc?tgtttaagca?catgtgctac 4740

gaaatggaga?ggctccacaa?tggcggcgac?gtcaccttcc?atgatgtcct?gagcatgctt 4800

tcataccggt?ccgtggacat?ccggaagagc?ttgcagctgg?aggaactcct?ggcgagggag 4860

cagctggagt?acaccataga?ggaggaggtg?gccaagcaga?ccatccgcat?gtggctcaag 4920

aagtgcctga?agcgcatcag?agctaaacag?cagcagtcgt?gcagtatcat?ccacagcctg 4980

agagagagtc?agcagcaaga?gctgagccgg?tttctgaacc?cgcccagcat?cgagaccacc 5040

cagcccagtg?aggacacgaa?tgccaacagt?caggacaaca?gcatgcaacc?tgagacaagc 5100

agccagcagc?agctcctgag?ccccacgctg?tcggatcgag?gaggaagtcg?gcaagatgca 5160

gccgacgcag?ggaaacccca?gaggaaattt?gggcagtggc?gtctgccctc?agccccaaaa 5220

ccaataagcc?attcagtgtc?ctcagtcaac?ttacggtttg?gaggaaggac?aaccatgaaa 5280

tctgtcgtgt?gcaaaatgaa?ccccatgact?gacgcggctt?cctgcggttc?tgaagttaag 5340

aagtggtgga?cccggcagct?gactgtggag?agcgacgaaa?gtggggatga?ccttctggat 5400

atttaggtgg?atgtcaatgt?agatgaattt?ctagtggtgg?aaaccgtttt?ctaataatgt 5460

ccttgattgt?ccagtgagca?atctgtaatt?gatctataac?tgaattccag?cttgtcacaa 5520

gatgtttata?aattgatttt?catcctgcca?cagaaaggca?taagctgcat?gtatgatggg 5580

ttactatcaa?tcattgctca?aaaaaatttt?tgtataatga?cagtactgat?aatattagaa 5640

atgataccgc?aagcaaatgt?atatcactta?aaaatgtcat?atattctgtc?tgcgtaaact 5700

aaggtatata?ttcatatgtg?ctctaatgca?gtattatcac?cgccccgcaa?aagagtgcta 5760

agcccaaagt?ggctgatatt?tagggtacag?gggttatagc?tttagttcac?atctttccca 5820

tttccactag?aaatatttct?cttgagagaa?tttattattt?atgattgatc?tgaaaaggtc 5880

agcactgaac?ttatgctaaa?atgatagtag?ttttacaaac?tacagattct?gaattttaaa 5940

aagtatcttc?tttttctcgt?gttatatttt?taaatataca?caagacattt?ggtgaccaga 6000

acaagttgat?ttctgtcctc?agttatgtta?atgaaactgt?tgcctccttc?taagaaaatt 6060

gtgtgtgcaa?gcaccaggca?aagaaatgga?ctcaggatgc?ttagcggttt?aaaacaaacc 6120

tgtagataaa?tcacttgagt?gacatagttg?cgcaaagatg?ttaagtttct?taagaaacct 6180

tttaataact?gagtttagca?aaaagaataa?aactatatag?ctcaatttat?ttaaaaaaat 6240

ctttgcatgt?gtgatgttat?cattggcttc?atttcttacc?caaggtatgt?ctgttttgcc 6300

ataaatcagc?agagtcattt?cattctgggt?gatcctaaca?caccattgct?acgttagatt 6360

tgaaatgaca?tctctgttaa?aagaatcttc?tatggaaata?atggtgccct?gcaaaatctt 6420

cctttgaact?cacaggttag?ggatcacaca?acttacttaa?tcgttttttg?tttttgtttt 6480

ttttccttat?atgtcaatgg?cccatgtcct?ccgggaaaat?tagaaaagca?aaatgattac 6540

aaagtgctgt?tagatttctt?gtgctgggcc?agccaagtag?aagtggactt?gacttggacc 6600

tttaactatt?ttattacaga?ttggacattt?gctgttcaga?tgttttttaa?cagagggatt 6660

atctcagaat?cctgtgacct?ccaggttgtt?ttataatcta?tttttctcta?tttaacattc 6720

ctcagataga?taggcaaata?ggacattcct?tctgtgtcac?agaagtatcg?tggtagtggc 6780

agtctacagt?ttatatgatt?cattgtaact?atgagataaa?gaacaaccag?tcatgtggcc 6840

aaaaggatta?gatttgattt?gatgttcact?tggagtttac?tttttgtaca?tacaagataa 6900

aataaatatt?ggatttgtaa?aat 6923

 

<210>36

<211>1738

<212>PRT

＜213〉homo sapiens

 

<400>36

Met?Leu?Lys?Arg?Lys?Gln?Ser?Ser?Arg?Val?Glu?Ala?Gln?Pro?Val?Thr

1 5 10 15

 

Asp?Phe?Gly?Pro?Asp?Glu?Ser?Leu?Ser?Asp?Asn?Ala?Asp?Ile?Leu?Trp

20 25 30

 

Ile?Asn?Lys?Pro?Trp?Val?His?Ser?Leu?Leu?Arg?Ile?Cys?Ala?Ile?Ile

35 40 45

 

Ser?Val?Ile?Ser?Val?Cys?Met?Asn?Thr?Pro?Met?Thr?Phe?Glu?His?Tyr

50 55 60

 

Pro?Pro?Leu?Gln?Tyr?Val?Thr?Phe?Thr?Leu?Asp?Thr?Leu?Leu?Met?Phe

65 70 75 80

 

Leu?Tyr?Thr?Ala?Glu?Met?Ile?Ala?Lys?Met?His?Ile?Arg?Gly?Ile?Val

85 90 95

 

Lys?Gly?Asp?Ser?Ser?Tyr?Val?Lys?Asp?Arg?Trp?Cys?Val?Phe?Asp?Gly

100 105 110

 

Phe?Met?Val?Phe?Cys?Leu?Trp?Val?Ser?Leu?Val?Leu?Gln?Val?Phe?Glu

115 120 125

 

Ile?Ala?Asp?Ile?Val?Asp?Gln?Met?Ser?Pro?Trp?Gly?Met?Leu?Arg?Ile

130 135 140

 

Pro?Arg?Pro?Leu?Ile?Met?Ile?Arg?Ala?Phe?Arg?Ile?Tyr?Phe?Arg?Phe

145 150 155 160

 

Glu?Leu?Pro?Arg?Thr?Arg?Ile?Thr?Asn?Ile?Leu?Lys?Arg?Ser?Gly?Glu

165 170 175

 

Gln?Ile?Trp?Ser?Val?Ser?Ile?Phe?Leu?Leu?Phe?Phe?Leu?Leu?Leu?Tyr

180 185 190

 

Gly?Ile?Leu?Gly?Val?G1n?Met?Phe?Gly?Thr?Phe?Thr?Tyr?His?Cys?Val

195 200 205

 

Val?Asn?Asp?Thr?Lys?Pro?Gly?Asn?Val?Thr?Trp?Asn?Ser?Leu?Ala?Ile

210 215 220

 

Pro?Asp?Thr?His?Cys?Ser?Pro?Glu?Leu?Glu?Glu?Gly?Tyr?Gln?Cys?Pro

225 230 235 240

 

Pro?Gly?Phe?Lys?Cys?Met?Asp?Leu?Glu?Asp?Leu?Gly?Leu?Ser?Arg?Gln

245 250 255

 

Glu?Leu?Gly?Tyr?Ser?Gly?Phe?Asn?Glu?Ile?Gly?Thr?Ser?Ile?Phe?Thr

260 265 270

 

Val?Tyr?Glu?Ala?Ala?Ser?Gln?Glu?Gly?Trp?Val?Phe?Leu?Met?Tyr?Arg

275 280 285

 

Ala?Ile?Asp?Ser?Phe?Pro?Arg?Trp?Arg?Ser?Tyr?Phe?Tyr?Phe?Ile?Thr

290 295 300

Leu?Ile?Phe?Phe?Leu?Ala?Trp?Leu?Val?Lys?Asn?Val?Phe?Ile?Ala?Val

305 310 315 320

 

Ile?Ile?Glu?Thr?Phe?Ala?Glu?Ile?Arg?Val?Gln?Phe?Gln?Gln?Met?Trp

325 330 335

 

Gly?Ser?Arg?Ser?Ser?Thr?Thr?Ser?Thr?Ala?Thr?Thr?Gln?Met?Phe?His

340 345 350

 

Glu?Asp?Ala?Ala?Gly?Gly?Trp?Gln?Leu?Val?Ala?Val?Asp?Val?Asn?Lys

355 360 365

 

Pro?Gln?Gly?Arg?Ala?Pro?Ala?Cys?Leu?Gln?Lys?Met?Met?Arg?Ser?Ser

370 375 380

 

Val?Phe?His?Met?Phe?Ile?Leu?Ser?Met?Val?Thr?Val?Asp?Val?Ile?Val

385 390 395 400

 

Ala?Ala?Ser?Asn?Tyr?Tyr?Lys?Gly?Glu?Asn?Phe?Arg?Arg?Gln?Tyr?Asp

405 410 415

 

Glu?Phe?Tyr?Leu?Ala?Glu?Val?Ala?Phe?Thr?Val?Leu?Phe?Asp?Leu?Glu

420 425 430

 

Ala?Leu?Leu?Lys?Ile?Trp?Cys?Leu?Gly?Phe?Thr?Gly?Tyr?Ile?Ser?Ser

435 440 445

 

Ser?Leu?His?Lys?Phe?Glu?Leu?Leu?Leu?Val?Ile?Gly?Thr?Thr?Leu?His

450 455 460

 

Val?Tyr?Pro?Asp?Leu?Tyr?His?Ser?Gln?Phe?Thr?Tyr?Phe?Gln?Val?Leu

465 470 475 480

 

Arg?Val?Val?Arg?Leu?Ile?Lys?Ile?Ser?Pro?Ala?Leu?Glu?Asp?Phe?Val

485 490 495

 

Tyr?Lys?Ile?Phe?Gly?Pro?Gly?Lys?Lys?Leu?Gly?Ser?Leu?Val?Val?Phe

500 505 510

 

Thr?Ala?Ser?Leu?Leu?Ile?Val?Met?Ser?Ala?Ile?Ser?Leu?Gln?Met?Phe

515 520 525

 

Cys?Phe?Val?Glu?Glu?Leu?Asp?Arg?Phe?Thr?Thr?Phe?Pro?Arg?Ala?Phe

530 535 540

 

Met?Ser?Met?Phe?Gln?Ile?Leu?Thr?Gln?Glu?Gly?Trp?Val?Asp?Val?Met

545 550 555 560

 

Asp?Gln?Thr?Leu?Asn?Ala?Val?Gly?His?Met?Trp?Ala?Pro?Val?Val?Ala

565 570 575

 

Ile?Tyr?Phe?Ile?Leu?Tyr?His?Leu?Phe?Ala?Thr?Leu?Ile?Leu?Leu?Ser

580 585 590

 

Leu?Phe?Val?Ala?Val?Ile?Leu?Asp?Asn?Leu?Glu?Leu?Asp?Glu?Asp?Leu

595 600 605

Lys?Lys?Leu?Lys?Gln?Leu?Lys?Gln?Ser?Glu?Ala?Asn?Ala?Asp?Thr?Lys

610 615 620

 

Glu?Lys?Leu?Pro?Leu?Arg?Leu?Arg?Ile?Phe?Glu?Lys?Phe?Pro?Asn?Arg

625 630 635 640

 

Pro?Gln?Met?Val?Lys?Ile?Ser?Lys?Leu?Pro?Ser?Asp?Phe?Thr?Val?Pro

645 650 655

 

Lys?Ile?Arg?Glu?Ser?Phe?Met?Lys?Gln?Phe?Ile?Asp?Arg?Gln?Gln?Gln

660 665 670

 

Asp?Thr?Cys?Cys?Leu?Leu?Arg?Ser?Leu?Pro?Thr?Thr?Ser?Ser?Ser?Ser

675 680 685

 

Cys?Asp?His?Ser?Lys?Arg?Ser?Ala?Ile?Glu?Asp?Asn?Lys?Tyr?Ile?Asp

690 695 700

 

Gln?Lys?Leu?Arg?Lys?Ser?Val?Phe?Ser?Ile?Arg?Ala?Arg?Asn?Leu?Leu

705 710 715 720

 

Glu?Lys?Glu?Thr?Ala?Val?Thr?Lys?Ile?Leu?Arg?Ala?Cys?Thr?Arg?Gln

725 730 735

 

Arg?Met?Leu?Ser?Gly?Ser?Phe?Glu?Gly?Gln?Pro?Ala?Lys?Glu?Arg?Ser

740 745 750

 

Ile?Leu?Ser?Val?G1n?His?His?Ile?Arg?Gln?Glu?Arg?Arg?Ser?Leu?Arg

755 760 765

 

His?Gly?Ser?Asn?Ser?Gln?Arg?Ile?Ser?Arg?Gly?Lys?Ser?Leu?Glu?Thr

770 775 780

 

Leu?Thr?Gln?Asp?His?Ser?Asn?Thr?Val?Arg?Tyr?Arg?Asn?Ala?Gln?Arg

785 790 795 800

 

Glu?Asp?Ser?Glu?Ile?Lys?Met?Ile?Gln?Glu?Lys?Lys?Glu?Gln?Ala?Glu

805 810 815

 

Met?Lys?Arg?Lys?Val?Gln?Glu?Glu?Glu?Leu?Arg?Glu?Asn?His?Pro?Tyr

820 825 830

 

Phe?Asp?Lys?Pro?Leu?Phe?Ile?Val?Gly?Arg?Glu?His?Arg?Phe?Arg?Asn

835 840 845

 

Phe?Cys?Arg?Val?Val?Val?Arg?Ala?Arg?Phe?Asn?Ala?Ser?Lys?Thr?Asp

850 855 860

 

Pro?Val?Thr?Gly?Ala?Val?Lys?Asn?Thr?Lys?Tyr?His?Gln?Leu?Tyr?Asp

865 870 875 880

 

Leu?Leu?Gly?Leu?Val?Thr?Tyr?Leu?Asp?Trp?Val?Met?Ile?Ile?Val?Thr

885 890 895

 

Ile?Cys?Ser?Cys?Ile?Ser?Met?Met?Phe?Glu?Ser?Pro?Phe?Arg?Arg?Val

900 905 910

Met?His?Ala?Pro?Thr?Leu?Gln?Ile?Ala?Glu?Tyr?Val?Phe?Val?Ile?Phe

915 920 925

 

Met?Ser?Ile?Glu?Leu?Asn?Leu?Lys?Ile?Met?Ala?Asp?Gly?Leu?Phe?Phe

930 935 940

 

Thr?Pro?Thr?Ala?Val?Ile?Arg?Asp?Phe?Gly?Gly?Val?Met?Asp?Ile?Phe

945 950 955 960

 

Ile?Tyr?Leu?Val?Ser?Leu?Ile?Phe?Leu?Cys?Trp?Met?Pro?Gln?Asn?Val

965 970 975

 

Pro?Ala?Glu?Ser?Gly?Ala?Gln?Leu?Leu?Met?Val?Leu?Arg?Cys?Leu?Arg

980 985 990

 

Pro?Leu?Arg?Ile?Phe?Lys?Leu?Val?Pro?Gln?Met?Arg?Lys?Val?Val?Arg

995 1000 1005

 

Glu?Leu?Phe?Ser?Gly?Phe?Lys?Glu?Ile?Phe?Leu?Val?Ser?Ile?Leu

1010 1015 1020

 

Leu?Leu?Thr?Leu?Met?Leu?Val?Phe?Ala?Ser?Phe?Gly?Val?Gln?Leu

1025 1030 1035

 

Phe?Ala?Gly?Lys?Leu?Ala?Lys?Cys?Asn?Asp?Pro?Asn?Ile?Ile?Arg

1040 1045 1050

 

Arg?Glu?Asp?Cys?Asn?Gly?Ile?Phe?Arg?Ile?Asn?Val?Ser?Val?Ser

1055 1060 1065

 

Lys?Asn?Leu?Asn?Leu?Lys?Leu?Arg?Pro?Gly?Glu?Lys?Lys?Pro?Gly

1070 1075 1080

 

Phe?Trp?Val?Pro?Arg?Val?Trp?Ala?Asn?Pro?Arg?Asn?Phe?Asn?Phe

1085 1090 1095

 

Asp?Asn?Val?Gly?Asa?Ala?Met?Leu?Ala?Leu?Phe?Glu?Val?Leu?Ser

1100 1105 1110

 

Leu?Lys?Gly?Trp?Val?Glu?Val?Arg?Asp?Val?Ile?Ile?His?Arg?Val

1115 1120 1125

 

Gly?Pro?Ile?His?Gly?Ile?Tyr?Ile?His?Val?Phe?Val?Phe?Leu?Gly

1130 1135 1140

 

Cys?Met?Ile?Gly?Leu?Thr?Leu?Phe?Val?Gly?Val?Val?Ile?Ala?Asn

1145 1150 1155

 

Phe?Asn?Glu?Asn?Lys?Gly?Thr?Ala?Leu?Leu?Thr?Val?Asp?Gln?Arg

1160 1165 1170

 

Arg?Trp?Glu?Asp?Leu?Lys?Ser?Arg?Leu?Lys?Ile?Ala?Gln?Pro?Leu

1175 1180 1185

 

His?Leu?Pro?Pro?Arg?Pro?Asp?Asn?Asp?Gly?Phe?Arg?Ala?Lys?Met

1190 1195 1200

Tyr?Asp?Ile?Thr?Gln?His?Pro?Phe?Phe?Lys?Arg?Thr?Ile?Ala?Leu

1205 1210 1215

 

Leu?Val?Leu?Ala?Gln?Ser?Val?Leu?Leu?Ser?Val?Lys?Trp?Asp?Val

1220 1225 1230

 

Glu?Asp?Pro?Val?Thr?Val?Pro?Leu?Ala?Thr?Met?Ser?Val?Val?Phe

1235 1240 1245

 

Thr?Phe?Ile?Phe?Val?Leu?Glu?Val?Thr?Met?Lys?Ile?Ile?Ala?Met

1250 1255 1260

 

Ser?Pro?Ala?Gly?Phe?Trp?Gln?Ser?Arg?Arg?Asn?Arg?Tyr?Asp?Leu

1265 1270 1275

 

Leu?Val?Thr?Ser?Leu?Gly?Val?Val?Trp?Val?Val?Leu?His?Phe?Ala

1280 1285 1290

 

Leu?Leu?Asn?Ala?Tyr?Thr?Tyr?Met?Met?Gly?Ala?Cys?Val?Ile?Val

1295 1300 1305

 

Phe?Arg?Phe?Phe?Ser?Ile?Cys?Gly?Lys?His?Val?Thr?Leu?Lys?Met

1310 1315 1320

 

Leu?Leu?Leu?Thr?Val?Val?Val?Ser?Met?Tyr?Lys?Ser?Phe?Phe?Ile

1325 1330 1335

 

Ile?Val?Gly?Met?Phe?Leu?Leu?Leu?Leu?Cys?Tyr?Ala?Phe?Ala?Gly

1340 1345 1350

 

Val?Val?Leu?Phe?Gly?Thr?Val?Lys?Tyr?Gly?Glu?Asn?Ile?Asn?Arg

1355 1360 1365

 

His?Ala?Asn?Phe?Ser?Ser?Ala?Gly?Lys?Ala?Ile?Thr?Val?Leu?Phe

1370 1375 1380

 

Arg?Ile?Val?Thr?Gly?Glu?Asp?Trp?Asn?Lys?Ile?Met?His?Asp?Cys

1385 1390 1395

 

Met?Val?Gln?Pro?Pro?Phe?Cys?Thr?Pro?Asp?Glu?Phe?Thr?Tyr?Trp

1400 1405 1410

 

Ala?Thr?Asp?Cys?Gly?Asn?Tyr?Ala?Gly?Ala?Leu?Met?Tyr?Phe?Cys

1415 1420 1425

 

Ser?Phe?Tyr?Val?Ile?Ile?Ala?Tyr?Ile?Met?Leu?Asn?Leu?Leu?Val

1430 1435 1440

 

Ala?Ile?Ile?Val?Glu?Asn?Phe?Ser?Leu?Phe?Tyr?Ser?Thr?Glu?Glu

1445 1450 1455

 

Asp?Gln?Leu?Leu?Ser?Tyr?Asn?Asp?Leu?Arg?His?Phe?Gln?Ile?Ile

1460 1465 1470

 

Trp?Asn?Met?Val?Asp?Asp?Lys?Arg?Glu?Gly?Val?Ile?Pro?Thr?Phe

1475 1480 1485

Arg?Val?Lys?Phe?Leu?Leu?Arg?Leu?Leu?Arg?Gly?Arg?Leu?Glu?Val

1490 1495 1500

 

Asp?Leu?Asp?Lys?Asp?Lys?Leu?Leu?Phe?Lys?His?Met?Cys?Tyr?Glu

1505 1510 1515

 

Met?Glu?Arg?Leu?His?Asn?Gly?Gly?Asp?Val?Thr?Phe?His?Asp?Val

1520 1525 1530

 

Leu?Ser?Met?Leu?Ser?Tyr?Arg?Ser?Val?Asp?Ile?Arg?Lys?Ser?Leu

1535 1540 1545

 

Gln?Leu?Glu?Glu?Leu?Leu?Ala?Arg?Glu?Gln?Leu?Glu?Tyr?Thr?Ile

1550 1555 1560

 

Glu?Glu?Glu?Val?Ala?Lys?Gln?Thr?Ile?Arg?Met?Trp?Leu?Lys?Lys

1565 1570 1575

 

Cys?Leu?Lys?Arg?Ile?Arg?Ala?Lys?Gln?Gln?Gln?Ser?Cys?Ser?Ile

1580 1585 1590

 

Ile?His?Ser?Leu?Arg?Glu?Ser?Gln?Gln?Gln?Glu?Leu?Ser?Arg?Phe

1595 16001 605

 

Leu?Asn?Pro?Pro?Ser?Ile?Glu?Thr?Thr?Gln?Pro?Ser?Glu?Asp?Thr

1610 1615 1620

 

Asn?Ala?Asn?Ser?Gln?Asp?Asn?Ser?Met?Gln?Pro?Glu?Thr?Ser?Ser

1625 1630 1635

 

Gln?Gln?Gln?Leu?Leu?Ser?Pro?Thr?Leu?Ser?Asp?Arg?Gly?Gly?Ser

1640 1645 1650

 

Arg?Gln?Asp?Ala?Ala?Asp?Ala?Gly?Lys?Pro?Gln?Arg?Lys?Phe?Gly

1655 1660 1665

 

Gln?Trp?Arg?Leu?Pro?Ser?Ala?Pro?Lys?Pro?Ile?Ser?His?Ser?Val

1670 1675 1680

 

Ser?Ser?Val?Asn?Leu?Arg?Phe?Gly?Gly?Arg?Thr?Thr?Met?Lys?Ser

1685 1690 1695

 

Val?Val?Cys?Lys?Met?Asn?Pro?Met?Thr?Asp?Ala?Ala?Ser?Cys?Gly

1700 1705 1710

 

Ser?Glu?Val?Lys?Lys?Trp?Trp?Thr?Arg?Gln?Leu?Thr?Val?Glu?Ser

1715 1720 1725

 

Asp?Glu?Ser?Gly?Asp?Asp?Leu?Leu?Asp?Ile

1730 1735

 

<210>37

<211>1566

<212>DNA

＜213〉homo sapiens

 

<400>37

agccgcgagc?ggcggccgcg?gggccgagga?gcctgggccg?ggccgggcgg?ggactactcc 60

ggagtcagga?ggcagcagcg?gcggaggacg?aggatctctg?gcagtcagcg?ccgctcggac 120

gccgccggca?ccatgggctg?ctgcaccgga?cgctgctcgc?tcatctgcct?ctgcgcgctg 180

cagttggtct?cagcattaga?gaggcagatc?tttgacttcc?ttggtttcca?gtgggcgcct 240

attcttggaa?attttctaca?cataatagtt?gtcatattgg?gtttgtttgg?gaccattcag 300

tacagacctc?gatacataat?ggtgtataca?gtgtggactg?ccctctgggt?cacctggaat 360

gtgttcatta?tctgctttta?tttggaagta?ggtggactct?caaaggacac?cgatctaatg 420

acattcaata?tctctgtaca?tcggtcatgg?tggagagaac?atgggcctgg?ttgtgtcaga 480

agagtgctgc?ctccctcagc?ccatggcatg?atggacgatt?acacgtacgt?ctctgtcaca 540

ggctgcatcg?ttgacttcca?gtacctggag?gtcatccaca?gtgctgtcca?aatactactc 600

tctttggtgg?gttttgtgta?tgcctgttat?gtgatcagta?tttccatgga?agaagaagac 660

acatattcat?gtgatctgca?agtatgcaaa?catcttttta?tccagatgct?gcaaattatt 720

gaataagcaa?gaattagtaa?gatattatca?ccaaattgtc?acatcagtca?agcctcatgt 780

gcttcctaag?aactgaggtg?atgcattatt?ttagagtgtc?attctaaacc?ccagattcaa 840

catcttccta?atctttctag?tgcagtctaa?tatataaatt?ttatgaaaag?cataggtttt 900

tttttaacca?gcagtgctct?ttgagaattt?acattgattc?ctaaagattg?ccattgcttt 960

gtataaaatg?ttataaatta?tcttagcatc?ttacctggaa?tttccactaa?attcaccaat 1020

ttatgatttg?tgaaatctga?ttttactttt?tgaaaatttt?catgtgaatt?tcccattttc 1080

agtgttgtag?cacctctctc?ttcctctaag?atcctccaag?ctcatcaaaa?gccatgatct 1140

tattatacca?gcagttttat?ttattcaatc?tttcaacaag?tagttattga?acttctataa 1200

tgtgccaggc?tctggagctc?gccttacacc?aaacagacac?aatcgatcca?ttcgaagtgt 1260

cgtaattaca?cattgaggga?ccaactagac?cttttctcat?tgtaaacttg?gagcaaaagt 1320

aaattcatta?aaataaattt?acattatagt?gccacaaaaa?aatgaacaga?accagaaagc 1380

attttttaca?aaaattaaca?gaacagtgtg?atagagggga?aaaggatgtg?agatcatggt 1440

gccctacctt?caatagggtg?gccagaaaac?acctctctga?agaagcagca?tttgagctga 1500

gacctgaaga?acgaggagtc?agtgatgcag?agaacctcag?gagatgcctt?ccaatctgag 1560

aaaaga 1566

 

<210>38

<211>197

<212>PRT

＜213〉homo sapiens

 

<400>38

Met?Gly?Cys?Cys?Thr?Gly?Arg?CysSer?Leu?Ile?Cys?Leu?Cys?Ala?Leu

1 5 10 15

 

Gln?Leu?Val?Ser?Ala?Leu?Glu?Arg?Gln?Ile?Phe?Asp?Phe?Leu?Gly?Phe

20 25 30

 

Gln?Trp?Ala?Pro?Ile?Leu?Gly?Asn?Phe?Leu?His?Ile?Ile?Val?Val?Ile

35 40 45

 

Leu?Gly?Leu?Phe?Gly?Thr?Ile?Gln?Tyr?Arg?Pro?Arg?Tyr?Ile?Met?Val

50 55 60

Tyr?Thr?Val?Trp?Thr?Ala?Leu?Trp?Val?Thr?Trp?Asn?Val?Phe?Ile?Ile

65 70 75 80

 

Cys?Phe?Tyr?Leu?Glu?Val?Gly?Gly?Leu?Ser?Lys?Asp?Thr?Asp?Leu?Met

85 90 95

 

Thr?Phe?Asn?Ile?Ser?Val?His?Arg?Ser?Trp?Trp?Arg?Glu?His?Gly?Pro

100 105 110

 

Gly?Cys?Val?Arg?Arg?Val?Leu?Pro?Pro?Ser?Ala?His?Gly?Met?Met?Asp

115 120 125

 

Asp?Tyr?Thr?Tyr?Val?Ser?Val?Thr?Gly?Cys?Ile?Val?Asp?Phe?Gln?Tyr

130 135 140

 

Leu?Glu?Val?Ile?His?Ser?Ala?Val?Gln?Ile?Leu?Leu?Ser?Leu?Val?Gly

145 150 155 160

 

Phe?Val?Tyr?Ala?Cys?Tyr?Val?Ile?Ser?Ile?Ser?Met?Glu?Glu?Glu?Asp

165 170 175

 

Thr?Tyr?Ser?Cys?Asp?Leu?Gln?Val?Cys?Lys?His?Leu?Phe?Ile?Gln?Met

180 185 190

 

Leu?Gln?Ile?Ile?Glu

195

 

<210>39

<211>2958

<212>DNA

＜213〉homo sapiens

 

<400>39

tggagtcgct?cgctgactcg?ccctgcgccc?tcgccgcgga?caccggagct?gcggccgctc 60

cccgctgtcc?cccagcttac?tccaatcaag?cctctgcccg?ccaggaacag?gtaacctgtg 120

tgtgtccgtt?tgctccttct?aagagcatgc?ctgatagata?cttcggtagc?ctctccggat 180

ggccccttcg?tcgggtagcc?tctcctgatg?gggtccttcg?cccaccctgc?ctcccgcgcc 240

ggcgctccgg?gtgaatgtca?agggtggctg?gctgcgaata?cctccttcag?ctgctggggt 300

tcccgacagt?ttgcagtttt?taaaagtgca?ccctcggaag?ggcttttcag?actgggtaaa 360

cctgactttt?ccaagagatg?gcagatcctg?aggtagttgt?gagtagctgc?agctctcatg 420

aagaggaaaa?tcgctgcaat?tttaaccagc?aaacatctcc?atctgaggag?cttctattag 480

aagaccagat?gaggcgaaaa?ctcaaatttt?ttttcatgaa?tccctgtgag?aagttctggg 540

ctcgaggtag?aaaaccatgg?aaacttgcca?tacaaattct?aaaaattgca?atggtgacta 600

tccagctggt?cttatttggg?ctaagtaacc?agatggtggt?agctttcaag?gaagagaata 660

ctatagcatt?caaacacctt?ttcctaaaag?gatatatgga?ccgaatggat?gacacatatg 720

cagtgtacac?acaaagtgac?gtgtatgatc?agttaatctt?cgcagtaaac?cagtacttgc 780

agctatacaa?tgtctccgtt?gggaatcatg?cttatgagaa?caaaggtacc?aagcaatctg 840

ctatggcaat?ctgtcagcac?ttatacaagc?gaggaaacat?ctaccctgga?aatgatacct 900

ttgacatcga?tccagaaatt?gaaactgagt?gtttctttgt?ggagccagat?gaaccttttc 960

acattgggac?accagcagaa?aataaactga?acttaacact?ggacttccac?agactcctaa 1020

cagtggagct?tcagtttaaa?ctgaaagcca?ttaatctgca?gacagttcgt?catcaagaac 1080

tccctgactg?ttatgacttt?actctgacta?taacatttga?caacaaggcc?catagtggaa 1140

gaattaaaat?aagtttagat?aatgacattt?ccatcagaga?atgtaaagac?tggcatgtat 1200

ctggatcaat?tcagaagaac?actcattaca?tgatgatctt?tgatgccttt?gtcattctga 1260

cttgcttggt?ttcattaatc?ctctgcatta?gatctgtgat?tagaggactt?cagcttcagc 1320

aggtagggaa?cgttgctttc?taggaatgct?actgacattt?tgattgacag?agacattcac 1380

tgtgcctccc?ctcttttccc?taaaggagtt?tgtcaatttt?ttcctcctcc?attataagaa 1440

ggaagtttct?gtttctgatc?aaatggaatt?tgtcaatgga?tggtacatta?tgattattat 1500

tagtgacata?ttgacaatca?ttggatcaat?tctaaaaatg?gaaatccaag?ctaaggtaat 1560

ttttttccta?atcatgctat?tgttagtgtc?agatttgcac?taatggtaat?gtatttttcc 1620

agaatgtaag?aattttcaga?atgaattgtt?tcttccaaac?tgtatatcaa?gtagacttga 1680

aattggtaat?ggtaattttc?ttaaatctag?tcaggaggtc?tcttaggcag?agtttttcaa 1740

agtgtgatcc?acaaaccatt?gcatcagaat?cattgggtgc?ctggtaaagt?gtaccatgtt 1800

agacctactg?aattcagact?cttcggcggg?gcctgtgaat?tcttacacac?accaaaattc 1860

atacacaacc?aaggtaacta?aggtaagagt?tttttttttt?ttttaatctt?acaagaaatg 1920

ctcgaatctt?taacaaaaat?gagtggggct?ataggggaaa?gtgaggtcaa?ggcactatgg 1980

tgtgcatgct?tgcatttgtt?tcctccgtcc?attcaaagtg?agaatgctcc?cattttctta 2040

ctttaccatt?gatgtgctac?aagcttattt?attttaagac?taacctagcc?taaaaatcaa 2100

ctgtccccac?aaaataaaaa?tcacattaaa?aaaactaata?gtgttcagac?taatcttgct 2160

caaacttatg?tttccctagt?cttgatgcaa?ctgattgagt?cacctgggga?gttggttata 2220

aacctgggca?gagaccccaa?atgcaatggc?tcagagaaga?taggagctta?tttctgtctt 2280

atgcaatagt?cagaatgggt?tttacagact?ggtgagtagc?tcaacatctc?acagtcattc 2340

aggcacccat?gttcctccca?ttttgtttct?ctgccacccc?ttaaggactt?gccctgactg 2400

catgattatt?gccgtgttgc?ctcaaacagg?ttgcagctta?tgggaagcaa?aaacacggta 2460

tggtagaagc?tctcccatag?actgatggct?tggctcaaga?gtggccgact?ttatttctgt 2520

acatatccca?ctggatagaa?tttagtcaat?cctaactgca?gagggagcca?gggaacacag 2580

cccaggcatg?tgcctaggaa?ggggagaatg?ggtttaggtt?gacacttagc?agctgccact 2640

atatgtggct?atagtatgta?tcattggaat?agatgtttaa?ctttagggac?aaataaaaaa 2700

ccaaaacaaa?aaaaggagta?aggggagaga?tttgcagcaa?atctttattt?ttaccaacct 2760

caactatcat?taatttcagt?gaaccctaaa?tggtatccaa?caaaatatct?ttctagacca 2820

ttcaccgtct?ctgcctcata?gatgatcata?tcatgttttc?ttctcttctg?aaacctctaa 2880

tacccttgtc?ctatcctcat?tctaagctga?tgaccttact?tcctatttca?caaaaataat 2940

agaaaaaaaa?aaaaaaaa 2958

 

<210>40

<211>2727

<212>DNA

＜213〉homo sapiens

 

<400>40

atagcctttc?aaatttcggt?taatggtaac?tctcatcagt?tactcaagcc?aaaaatcttg 60

gattcatcac?agactcctct?ctttcactaa?tctcttcttc?cccacatcta?atccaagagg 120

aaatcctatt?gttctacctt?cattggccag?gcactgtgac?tcatataatc?ccagcacatt 180

gggaggctga?ggcgggagga?tggcttgagc?ccaggagttc?cagaccagcc?tgggaaacat 240

agtgaggccc?catctctaca?aaaaattaaa?aaaattagca?gtcaaggtgg?catgtgtctg 300

taatccaagc?tacttggggg?gctgaggtgg?gaggatttct?tgagcccggg?aagtcgaggc 360

tgcattgagc?cacggttgtg?ccacggcact?ccagcctggg?tgacagagtg?agacccctgt 420

ttccaaaaaa?aaaaaaaaaa?aaaaagaata?aaaatcacct?ggtcaactcc?actattacca 480

gcctggtcca?agctactatc?tctcatttat?attattgcaa?tagcctcctc?actcctccaa 540

caacctgctg?tcaaccacag?cagccaacat?ctgatcatat?cacttctgtt?tgtggttctc 600

aaatctcccc?aattgagtta?cagtaaaaga?caaacttggt?gagtgccacc?ttatctctat 660

aactgtatac?cctttctatt?gctcactcca?gccagatgca?atatccttgc?caagcaccct 720

cctgtctcag?ggcctttgca?cttgccagtc?cctgtgcctg?gaaggcttct?cccctagatt 780

tttgcatgac?ttctccctcc?ctcccttcag?atctttgctc?aaatgccttc?tttttagtgt 840

atgtaaaatg?acaaacccat?acccattcct?tatcccctcc?tctgaatttt?ctcttcagca 900

attatcagca?gcaagtgtcc?caaagtttct?attaacttat?ttctgttgtc?tctttcttcc 960

ctccactaga?atgtaagctt?tatgagagca?gagacttttg?tttgttcact?gctttatcct 1020

tagcacctaa?aacagtgcct?tactcatagt?tacctcaata?tttattgcca?aatgaatttc 1080

tgctttataa?tctgattata?tttttccact?ctctcttaga?gtctaactag?ttatgatgtc 1140

tgtagcatac?ttcttgggac?ttctaccatg?cttgtgtggc?ttggagtcat?ccgatacctc 1200

ggtttctttg?caaagtacaa?cctcctcatt?ttgacccttc?aggcagcgct?gcccaatgtc 1260

atcaggttct?gctgctgtgc?agctatgatt?tacttaggtt?actgcttctg?tggatggatc 1320

gtgctggggc?cttaccatga?caagtttcgt?tctctgaaca?tggtctctga?gtgccttttc 1380

tctctgataa?atggagatga?tatgtttgcc?acgtttgcaa?aaatgcagca?aaaaagttac 1440

ttagtctggc?tgtttagtag?aatttacctc?tactcattca?tcagcctctt?tatatatatg 1500

attttaagtc?ttttcattgc?actgatcact?gatacatacg?aaacaattaa?gcaataccaa 1560

caagatggct?tcccagagac?tgaacttcgt?acatttatat?cagaatgcaa?agatctaccc 1620

aactctggaa?aatacagatt?agaagatgac?cctccagtaa?ctttattctg?ctgttgtaaa 1680

aagtagctat?caggtttatc?tgtactttag?aggaaaatat?aatgtgtagc?tgagttggaa 1740

cactgtggat?attctgagat?cagatgtagt?atgtttgaag?actgttattt?tgagctaatt 1800

gagacctata?attcaccaat?aactgtttat?atttttaaaa?gcaatattta?atgtctttgc 1860

aactttatgc?tgggattgtt?tttaaaaaaa?actttaatga?ggaaagctat?tggattatta 1920

ttatttcttg?tttattttgc?catggcttta?gaatgtattc?tgtatgcctc?tcttttgctc 1980

tgatactgtt?gctcctgcta?ttctgattgt?gcagactgta?taattagtgg?aaaacaatcc 2040

ttggtctgac?tgtgactttg?gacaactcag?taaccctggc?ttggaccact?ctcaggagtc 2100

catccttgag?agagtgggtg?tagttaccat?ttatacagta?atcattgcat?tttaaaatct 2160

tctcttgaaa?ggaagaataa?gagtgcacca?gaataagagc?gcaccagaat?aagagcacac 2220

cagctaacaa?tgtgatacgg?ccatatgtca?cttaaggatg?gagatatgtt?ctgagaaatg 2280

tgtcattagg?cgattttgtc?attaaacatc?atagcatgta?cttccacaaa?cctagatggt 2340

atagcctact?acacacctag?gctatttggt?atagcctgtt?ggtcctgggg?tacaaatctg 2400

tacaacatgt?tactgtattg?aatacagtag?gcaattgtaa?ctcaatggta?agtatctaaa 2460

catagaaaag?ggacagtaaa?aatatggttt?tataatcttc?tgggaccacc?attgtatatg 2520

cggtacatca?ttgaccaaaa?catcgttatc?cagcatatga?ctgtatttgg?ttatgaaagc 2580

caactgttac?ttgattctgc?ttttagttct?taagaggatc?aggcttttaa?atactcattt 2640

acaagctttc?tatcctcctt?cagtgttaaa?gtagaaagta?aaaagagtat?cttatacatg 2700

catgaaatta?aagcatatac?caaatgc 2727

Claims (259)

1. isolating gustatory cell, adrenal cells, pituicyte, parathyroid gland cell, melanocyte or uropoiesis device cell, or the gustatory cell sample of enrichment, the cell sample of wherein said separation or enrichment comprises the cell of expressing TRPML3 ionic channel polypeptide.

2. the gustatory cell sample of isolated cell as claimed in claim 1 or enrichment, wherein said TRPML3 are selected from Mammals, fowl, Amphibians, fish and Reptilia TRPML3.

3. the gustatory cell sample of isolated cell as claimed in claim 1 or enrichment, it expresses Mammals TRPML3 or chicken, zebra fish or Xenopus laevis TRPML3.

4. the group that the cell sample of isolated cell as claimed in claim 3 or enrichment, wherein said Mammals TRPML 3 select freeman, mouse, rat, dog, cat, cavy, pig, horse, ox, goat, sheep, bear, monkey, gorilla, chimpanzee, orangutan, macaque, cynomolgus monkey, gibbon, gazelle and zebra TRPML3 to form.

5. the cell sample of isolated cell as claimed in claim 1 or enrichment, wherein said TRPML3 ionic channel polypeptide be selected from have SEQ ID NO:2, the TRPML3 polypeptide of 4,10,12,14,16,22,24,26,28,30,32 and 34 polypeptide or have at least 90% sequence identity by the TRPML3 polypeptide of SEQ ID NO:39 or 40 codings.

6. the cell sample of isolated cell as claimed in claim 5 or enrichment, wherein said TRPML3 ionic channel polypeptide be selected from have SEQ ID NO:2, TRPML 3 polypeptide of 4,10,12,14,16,22,24,26,28,30,32 and 34 polypeptide or have at least 95% sequence identity by the TRPML3 polypeptide of SEQ ID NO:39 or 40 codings.

7. the cell sample of isolated cell as claimed in claim 5 or enrichment, wherein said TRPML3 ionic channel polypeptide have the sequence that is contained among the SEQ ID NO:2,4 or 10 or by TRPML 3 polypeptide of SEQ ID NO:39 or 40 codings.

8. isolated cell as claimed in claim 1 or cell sample, but the TRPML3 polypeptide that the cell sample of wherein said cell or enrichment is expressed has and makes susceptibility or the perviousness lower sudden change of this ionic channel to sodium.

9. isolated cell as claimed in claim 1 or cell sample, but the TRPML3 polypeptide that the cell sample of wherein said cell or enrichment is expressed has and makes susceptibility or the perviousness higher sudden change of this ionic channel to sodium.

10. isolated cell as claimed in claim 1 or cell sample, the TRPML3 polypeptide that the cell sample of wherein said cell or enrichment is expressed have the sudden change that this ionic channel of maintenance is in open to the outside world or " closing " orientation.

11. isolated cell as claimed in claim 1 or cell sample, the TRPML3 polypeptide that the cell sample of wherein said cell or enrichment is expressed has Cytotoxic sudden change.

12. isolated cell as claimed in claim 1 or cell sample, TRPML 3 polypeptide that the cell sample of wherein said cell or enrichment is expressed have the sudden change that causes wherein sodium inflow and flow out uncontrolled cell.

13. isolated cell as claimed in claim 1 or cell sample, it comprises the gustatory cell in response to the saline taste sense of taste.

14. isolated cell as claimed in claim 13 or cell sample, wherein said gustatory cell is selected from the gustatory cell of people, non-human primate, rodent, dog or cat.

15. isolated cell as claimed in claim 14 or cell sample, it is the gustatory cell of people or non-human primate.

16. isolated cell as claimed in claim 1 or cell sample, it comprises uropoiesis device cell.

17. isolated cell as claimed in claim 1 or cell sample, it comprises pituicyte.

18. isolated cell as claimed in claim 1 or cell sample, it comprises adrenal cells.

19. isolated cell as claimed in claim 1 or cell sample, it comprises the parathyroid gland cell.

20. isolated cell as claimed in claim 1 or cell sample, it comprises gustatory cell, adrenal cells, pituicyte, parathyroid gland cell, melanocyte or the uropoiesis device cell that participates in saline taste impression, sodium metabolism, aldosterone generation and/or vasopressing release.

21. isolated cell as claimed in claim 1 or cell sample, it is in solution.

22. isolated cell as claimed in claim 1 or cell sample, it is on solid support.

23. isolated cell as claimed in claim 22 or cell sample, wherein said upholder is on pillar.

24. the cell sample of isolating gustatory cell as claimed in claim 1 or enrichment, wherein said gustatory cell are people's saline taste gustatory celies.

25. the gustatory cell sample of isolating gustatory cell as claimed in claim 1 or enrichment, it is contained in upper part of people or non-human primate taste bud.

26. the gustatory cell sample of isolating gustatory cell as claimed in claim 25 or enrichment, people that wherein said TRPML3 gene is a wild-type or macaque TRPML3.

27. the gustatory cell sample of isolating gustatory cell as claimed in claim 26 or enrichment, wherein said TRPML3 gene is a people's gene, and has the sequence among the SEQ ID NO:2.

28. the cell sample of isolated cell as claimed in claim 1 or enrichment, it further expresses NALCN, NKAIN3, TRPML1 and/or TRPML2.

29. an isolating Taste Receptors of regulating and control the saline taste impression, it comprises TRPML3 polypeptide or its variant of the regulation and control Mammals saline taste sense of taste.

30. isolating Taste Receptors as claimed in claim 29, it is same aggressiveness.

31. isolating Taste Receptors as claimed in claim 29, it is a monomer.

32. isolating Taste Receptors as claimed in claim 29, it is different aggressiveness.

33. isolating Taste Receptors as claimed in claim 29, it comprises Mammals, fowl, Amphibians, fish or Reptilia TRPML3 ionic channel.

34. isolating Taste Receptors as claimed in claim 29, it is the Taste Receptors of Mammals or chicken, zebra fish or Xenopus laevis.

35. isolating Taste Receptors as claimed in claim 29, it comprises the TRPML3 that is selected from people, mouse, rat, dog, cat, cavy, pig, horse, ox, goat, sheep, bear, monkey, gorilla, chimpanzee, orangutan, cynomolgus monkey, gibbon, gazelle, macaque and zebra TRPML3.

36. isolating Taste Receptors as claimed in claim 29, wherein said TRPML3 ionic channel polypeptide be selected from have SEQ ID NO:2, the TRPML3 polypeptide of 4,10,12,14,16,22,24,26,28,30,32 and 34 polypeptide or have at least 90% sequence identity by the TRPML3 polypeptide of SEQ ID NO:39 or 40 codings.

37. isolating Taste Receptors as claimed in claim 29, wherein said TRPML3 ionic channel polypeptide be selected from have SEQ ID NO:2, the TRPML3 polypeptide of 4,10,12,14,16,22,24,26,28,30,32 and 34 polypeptide or have at least 95% sequence identity by the TRPML3 polypeptide of SEQ ID NO:39 or 40 codings.

38. having, isolating Taste Receptors as claimed in claim 29, the described TRPML3 polypeptide of wherein being expressed make the more responsive or more insensitive sudden change of this ionic channel to sodium.

39. isolating Taste Receptors as claimed in claim 29, the described TRPML3 polypeptide of wherein being expressed have this ionic channel more can be seen through or the more sudden change of impermeable sodium.

40. having, isolating Taste Receptors as claimed in claim 29, described TRPML 3 polypeptide of wherein being expressed keep this ionic channel to be in the sudden change of open to the outside world or " closing " orientation.

41. isolating Taste Receptors as claimed in claim 29, the described TRPML3 polypeptide of wherein being expressed has the virose sudden change of the cell that contains this ionic channel.

42. isolating Taste Receptors as claimed in claim 29, the described TRPML3 polypeptide of wherein being expressed have the inflow of sodium when this ionic channel is expressed or calcium and flow out uncontrolled sudden change in cell.

43. isolating Taste Receptors as claimed in claim 29, it comprises another ionic channel polypeptide.

44. isolating Taste Receptors as claimed in claim 43, wherein said other ionic channels are NKAIN3, NALCN, TRPML1 or TRPML2.

45. isolating Taste Receptors as claimed in claim 29, the saline taste acceptor of its behaviour or non-human primate.

46. isolating Taste Receptors as claimed in claim 45, it comprises people's saline taste acceptor.

47. a transgenic nonhuman animal, it is the expression that knocks out or damage endogenous TRPML3 by genetic modification, and condition is that described transgenic animal are not the Varitint mouse.

48. a transgenic nonhuman animal, it is an expressing heterologous TRPML3 polypeptide by genetic modification, and condition is that described transgenic animal are not the Varitint mouse.

49. transgenic animal as claimed in claim 48, wherein said TRPML3 polypeptide are Mammals, fowl, fish, Amphibians or Reptilia TRPML3.

50. transgenic animal as claimed in claim 48, wherein said TRPML3 is Mammals TRPML3.

51. transgenic animal as claimed in claim 50, wherein said TRPML3 is selected from people, mouse, rat, dog, cat, cavy, pig, horse, ox, goat, sheep, bear, monkey, gorilla, chimpanzee, orangutan, cynomolgus monkey, gibbon, gazelle, macaque and zebra TRPML3.

52. transgenic animal as claimed in claim 48, wherein said allos TRPML3 ionic channel polypeptide be selected from have SEQ ID NO:2, the TRPML3 polypeptide of 4,10,12,14,16,22,24,26,28,30,32 and 34 polypeptide or have at least 90% sequence identity by the TRPML3 polypeptide of SEQ ID NO:39 or 40 codings.

53. transgenic animal as claimed in claim 48, wherein said allos TRPML3 ionic channel polypeptide be selected from have SEQ ID NO:2, the TRPML3 polypeptide of 4,10,12,14,16,22,24,26,28,30,32 and 34 polypeptide or have at least 95% sequence identity by the TRPML3 polypeptide of SEQ ID NO:39 or 40 codings.

54. having, transgenic animal as claimed in claim 48, wherein said allos TRPML 3 ionic channel polypeptide, the described allos TRPML3 polypeptide of wherein being expressed make the more responsive or more insensitive sudden change of this ionic channel to sodium.

55. transgenic animal as claimed in claim 48, the described allos TRPML3 polypeptide of wherein being expressed have this ionic channel more can be seen through or the more sudden change of impermeable sodium.

56. having, transgenic animal as claimed in claim 48, the described allos TRPML3 polypeptide of wherein being expressed keep this ionic channel to be in the sudden change of open to the outside world or " closing " orientation.

57. transgenic animal as claimed in claim 48, the described TRPML3 polypeptide of wherein being expressed has the virose sudden change of the cell of expressing this ionic channel.

Go into and flow out uncontrolled sudden change 58. transgenic animal as claimed in claim 48, the described allos TRPML3 polypeptide of wherein being expressed have when this ionic channel is expressed sodium or calcium current in cell.

59. transgenic animal as claimed in claim 48, the described allos TRPML3 polypeptide of wherein being expressed causes the excision of particular cell types.

60. transgenic animal as claimed in claim 59, the described allos TRPML3 polypeptide of wherein being expressed cause being selected from the excision of one or more cell types of gustatory cell, pituicyte, adrenal cells, melanocyte, parathyroid gland cell and uropoiesis device cell.

61. transgenic animal as claimed in claim 60, described allos TRPML 3 polypeptide of wherein being expressed are the people TRPML3 polypeptide of sudden change, it has when this ionic channel is expressed sodium or calcium current and goes into and flow out uncontrolled sudden change in cell.

62. method of in the screening of the compound of identifying the regulation and control saline taste sense of taste, using each described transgenic animal of claim 47-61.

63. method of using the varitint-waddler mouse to measure TRPM13 function in gustatory cell, pituicyte, adrenal cells, uropoiesis device cell or melanocyte.

64. as the described method of claim 63, it comprises the gene that uses gene chip to come comparison specifically expressing in the saline taste gustatory cell of normal mouse and varitint waddler mouse, to identify the gene of specifically expressing in the gustatory cell that detects the saline taste sense of taste.

65. detected gene or the detected corresponding polypeptide application in the mensuration that detects saline taste sense of taste conditioning agent in the claim 64.

66. as the described mensuration of claim 64, it is used for detecting regulation and control TRPML 3 functions or works or regulate and control saline taste sense of taste signal and grow from the transmission of TRPML3 neuralward fiber and/or control saline taste gustatory cell and break up or the gene of apoptosis as the saline taste acceptor.

67. as the described method of claim 65, it is used for identifying antagonist, agonist or the toughener of TRPML3.

68. as the described method of claim 65, wherein certified compound is further assessed in the test of people's sense of taste.

69. the method for the influence that 3 pairs of aldosterone generations of TRPML, sodium metabolism, saline taste impression or vasopressing discharge is illustrated in a use according to each transgenic animal of claim 47-61.

70. transgenic animal (inhuman) of using the TRPML3 gene of expressing the coding ionic channel assess relate to that unusual aldosterone produces, vasopressing discharges, the method for the potential treatment plan of the disease of sodium metabolism and/or melanocyte loss or the patient's condition, described ionic channel is toxic to the cell of expressing this ionic channel.

71. as the described method of claim 70, the wherein said disease or the patient's condition are selected from oedema, blood pressure (high or low blood pressure), liver cirrhosis, the primary hyperaldosteronemia, renal tubal dysfunction, diabetes (I or II type) and relative pathological symptom, comprise circulatory problems, oedema, the illness in eye relevant with Cycle Difference, high cortisone mass formed by blood stasis, the atherosclerosis or the obesity of abdominal obesity for example, and hepatopathy, sexual dysfunction (sex), cerebrovascular disease, vascular disease, retinopathy, neuropathy, Insulinopathy, endothelial function disturbance, pressure function of receptors obstacle, migraine, hectic fever and premenstrual tension and other cardiovascular patient's condition are such as atherosclerosis, in heart failure, congestive heart failure, vascular disease, apoplexy, myocardial infarction, endothelial function disturbance, ventricular hypertrophy, renal tubal dysfunction, target organ damage, thrombosis, arrhythmia, plaque rupture and aneurysma or can be by the another kind of patient's condition of aldosterone agonist or antagonist for treating.

72. as the described method of claim 70, the wherein said disease or the patient's condition be selected from diabetes, obesity, such as the ephrosis of cystic kidney disease, acquired cystic kidney disease, such as the eye circular correlation sufferer of myopia; Feel sick, vomiting, sexual dysfunction (sex), oedema, hypertension, congestive heart failure (scope is from the II class of New York heart association to ruddy pulmonary edema), the spontaneous oedema of periodicity, nephrotic syndrome, the ascites that causes by liver cirrhosis or other reasons, the cerebral edema of a variety of causes, dilutional hyponatremia is with the metabolism change that is referred to as inappropriate ADH secretion syndrome and wherein treat the other diseases or the patient's condition that needs vasorelaxation and/or anti-oxytocic activity or use vasopressing agonist or antagonist.

73. a reconstitution cell of expressing the saline taste acceptor, described saline taste acceptor comprises TRPML3 or its variant.

74. as the described reconstitution cell of claim 73, it is yeast, Amphibians, insect, bacterium, Reptilia, fowl or mammalian cell.

75. as the described reconstitution cell of claim 74, it is Amphibians ovocyte or mammalian cell.

76. as the described reconstitution cell of claim 75, it is selected from HEK-293, COS, CHO and bhk cell.

77. as the described reconstitution cell of claim 73, the described TRPML3 polypeptide of its transient expression.

78. as the described reconstitution cell of claim 73, the described TRPML3 polypeptide of its stably express.

79. as the described reconstitution cell of claim 73, it comprises rhabdovirus expression vector.

80. as the described reconstitution cell of claim 79, wherein said carrier is the BacMam carrier.

81. as the described reconstitution cell sample of claim 73, wherein said TRPML3 is selected from Mammals, fowl, Amphibians, fish and Reptilia TRPML3.

82. as the described reconstitution cell of claim 73, it expresses Mammals TRPML3 or chicken, zebra fish or Xenopus laevis TRPML3.

83. as the described reconstitution cell of claim 73, it expresses Mammals TRPML3, the group of selecting freeman, mouse, rat, dog, cat, cavy, pig, horse, ox, goat, sheep, bear, monkey, gorilla, chimpanzee, orangutan, macaque, cynomolgus monkey, gibbon, gazelle and zebra TRPML3 to form.

84. as the described reconstitution cell of claim 73, wherein said TRPML3 ionic channel polypeptide be selected from have SEQ ID NO:2, the TRPML3 polypeptide of 4,10,12,14,16,22,24,26,28,30,32 and 34 polypeptide or have at least 90% sequence identity by the TRPML3 polypeptide of SEQ ID NO:39 or 40 codings.

85. as the described reconstitution cell of claim 73, wherein said TRPML3 ionic channel polypeptide be selected from have SEQ ID NO:2, the TRPML3 polypeptide of 4,10,12,14,16,22,24,26,28,30,32 and 34 polypeptide or have at least 95% sequence identity by the TRPML3 polypeptide of SEQ ID NO:39 or 40 codings.

86. as the described reconstitution cell of claim 73, wherein said TRPML3 ionic channel polypeptide has the sequence that is contained in SEQ ID NO:2 or 4 or comprises by SEQ ID NO:39 or the 40 TRPML3 polypeptide of encoding.

87. as the described reconstitution cell of claim 73, the TRPML3 polypeptide of wherein said cell expressing has makes the more responsive or more insensitive sudden change to sodium of this ionic channel.

88. as the described reconstitution cell of claim 73, the TRPML3 polypeptide of wherein said cell expressing has more can see through or the more sudden change of impermeable sodium this ionic channel.

89. as the described reconstitution cell of claim 73, the TRPML3 polypeptide of wherein said cell expressing has the sudden change that this ionic channel of maintenance is in the open to the outside world orientation.

90. as the described reconstitution cell of claim 73, the TRPML3 polypeptide that wherein said cell sample is expressed has the virose sudden change of some cells.

91. as the described reconstitution cell of claim 73, the TRPML3 polypeptide that the cell sample of wherein said cell or enrichment is expressed has the sudden change that causes wherein sodium inflow and flow out uncontrolled cell.

92. as the described reconstitution cell of claim 73, wherein said TRPML 3 ion channel response are in saline taste sense of taste conditioning agent.

93. as the described reconstitution cell of claim 73, it expresses another sodium-ion channel polypeptide.

94. as the described reconstitution cell of claim 89, wherein said other sodium channels are NALCN, NKAIN3, TRPML1 or TRPML2.

95. a mensuration that is used to identify excitement, antagonism or strengthens the active compound of TRPML3, it comprise make according to the cell of the reconstitution cell of one of claim 73-94 or endogenous blastema of expressing TRPML3 ionic channel polypeptide, acute isolation or other cells contact with TRPML3 toughener, agonist or the antagonist of supposition and definite its to TRPML 3 active influences.

96. as the described mensuration of claim 95, wherein endogenous cell is selected from adrenal cortical cell, parathyroid gland cell, taste bud cell, uropoiesis device cell, melanocyte, suprarenal gland or parathyroid gland cell.

97. as the described mensuration of claim 95, wherein said cell is nascent, acute isolation or other endogenous cells of expressing TRPML3.

98. as the described mensuration of claim 97, wherein said cell is adrenal cortical cell, pituicyte, parathyroid gland cell, gustatory cell or melanocyte.

99. as the described mensuration of claim 98, the TRPML3 sequence of wherein said cell expressing rodent or people or non-human primate.

100. as the described mensuration of claim 98, wherein said TRPML3 is supported in the sequence encoding that strengthens the optimizing codon of expressing in the recombinant host cell by having.

101. as the described mensuration of claim 98, the sequence of wherein said TRPML3 be selected from the peptide sequence that is contained in SEQ ID NO:2,4,10,12,14,16,22,24,26,28,30,32 and 34 polypeptide or identical by the peptide sequence at least 90% of SEQ ID NO:39 or 40 peptide codings.

102. as the described mensuration of claim 98, the sequence of wherein said TRPML3 be selected from the peptide sequence that is contained in SEQ ID NO:2,4,10,12,14,16,22,24,26,28,30,32 and 34 polypeptide or identical by the peptide sequence at least 95% of SEQ ID NO:39 or 40 peptide codings.

103. as the described mensuration of claim 98, wherein said TRPML3 is wild-type human sequence or rodent or the human sequence who comprises Varitint-waddler sudden change (A419P).

104. as the described mensuration of claim 98, wherein said TRPML3 is the human sequence by SEQ IDNO:1,3,17 or 18 codings.

105. as each described mensuration of claim 98-103, it is an electrophysiological detection.

106. as the described mensuration of claim 105, wherein said reconstitution cell is the Amphibians ovocyte.

107. as the described mensuration of claim 105, wherein said reconstitution cell is a mammalian cell.

108. as the described mensuration of claim 105, wherein said mensuration is to use the electrophysiological detection of ion-sensitive dyestuff or fluorophore.

109. as the described mensuration of claim 105, wherein said mensuration is that two electrode voltages pincers are measured.

110. as the described mensuration of claim 109, wherein said test cell is an xenopus leavis oocytes.

111. as the described mensuration of claim 107, wherein said mammalian cell is selected from the group of being made up of HEK293, HEK293T, Swiss3T3, CHO, BHK, NIH3T3 and COS cell.

112. as the described mensuration of claim 105, wherein said cell is an xenopus leavis oocytes.

113. as the described mensuration of claim 105, wherein said mensuration is that patch clamp is measured.

114. as the described mensuration of claim 105, it uses the membrane potential dyestuff to be selected from the group of being made up of following: Molecular Devices membrane potential test kit (catalog number (Cat.No.) R8034), Di-4-ANEPPS (4-(2-(6-(dibutylamino)-2-naphthalene-yl) vinyl)-1-(3-sulfopropyl) hydroxide pyridine, inner salt, DiSBACC4 (2) (two-(1,2-two barbituric acids)-three acetylene oxprenolols), Cc-2-DMPE (Pacific Ocean indigo plant 1,2-two mnyristoyls-sn-glycerol-3 phosphorylethanolamine, triethylammonium salts) and SBFI-AM (1, the 3-benzene dicarboxylic acid, 4,4-[1,4,10-trioxa-7,13-diazacyclo pentadecane-7, two (the 5-methoxyl groups-6 of 13-two bases, 1,2-cumarone two bases) two-four { (acetyl oxygen) methyl } ester (Molecular Probes).

115. as the described method of claim 105, it uses the sodium sensitive dye.

116. as the described method of claim 115, wherein said sodium sensitive dye is tetraacethyl sodium green (Molecular Probes) or Na-sensitive dye test kit (MolecularDevices).

117. as the described mensuration of claim 97, wherein said mensuration is measured by ionic current and is measured activity.

118. as the described mensuration of claim 117, it uses atomic absorption spectrum to detect ionic current.

119. as the described mensuration of claim 105, it uses fluorescent plate reader (FLIPR).

120. as the described mensuration of claim 105, its working voltage imaging plate reader (VIPR).

121. as the described mensuration of claim 105, it uses automatic electrophysiology instrument device.

122. as the described mensuration of claim 121, it uses IonWorks to measure system.

123. as the described mensuration of claim 105, its use is selected from the membrane potential dyestuff by the following group of forming: Molecular Devices membrane potential test kit (catalog number (Cat.No.) 8034), Di-4-ANEPPS (4-(2-(6-(dibutylamino)-2-naphthalene-yl) vinyl)-1-(3-sulfopropyl)-hydroxide pyridine, inner salt); DiSBACC4 (2) (two-(1.2-two barbituric acids)-cyclonite oxprenolol); DiSBAC4 (3) (two-(1,3-two barbituric acids)-cyclonite oxprenolol); CC-2-DPME (Pacific Ocean indigo plant 1,2-two mnyristoyls-sn-glycerol-3-phosphorylethanolamine, triethylammonium salts) and SBFI-AM (1, the 3-benzene dicarboxylic acid, 4,4 '-[1,4,10-trioxa-7,13-diazacyclo pentadecane-7,13-two bases two (5-methoxyl group-6,1,2-cumarone two bases)] two-four [(acetyl oxygen) methyl] ester (Molecular Probes).

124. as each described mensuration of claim 99-123, wherein certified agonist, antagonist or toughener compound are assessed in taste test.

125. as each described mensuration of claim 99-123, wherein certified agonist, antagonist or toughener are tested in animal the influence that aldosterone produces.

126. as each described mensuration of claim 99-123, wherein certified agonist, antagonist or toughener are tested in animal the influence that vasopressing discharges.

127. as each described mensuration of claim 99-123, wherein said certified antagonist, agonist or toughener compound are tested in animal at least a influence that Addison's disease, trichomadesis, hair or fur fade, gustatory cell regeneration, pituicyte regeneration, adrenal cells regeneration, melanocyte cell regeneration, blood pressure, fluid retention, sodium metabolism and urine produce.

128. as each described mensuration of claim 99-123, wherein certified compound is assessed in the animal model in suitable external or body the influence of the treatment disease or the patient's condition, the wherein said disease or the patient's condition are selected from oedema, blood pressure (high or low blood pressure), liver cirrhosis, the primary hyperaldosteronemia, renal tubal dysfunction, diabetes (I or II type) and relative pathological symptom, comprise circulatory problems, oedema, the illness in eye relevant with Cycle Difference, high cortisone mass formed by blood stasis, the atherosclerosis or the obesity of abdominal obesity for example, and hepatopathy, sexual dysfunction (sex), cerebrovascular disease, vascular disease, retinopathy, neuropathy, Insulinopathy, endothelial function disturbance, pressure function of receptors obstacle, migraine, hectic fever and premenstrual tension and other cardiovascular patient's condition are such as atherosclerosis, in heart failure, congestive heart failure, vascular disease, apoplexy, myocardial infarction, endothelial function disturbance, ventricular hypertrophy, renal tubal dysfunction, target organ damage, thrombosis, arrhythmia, plaque rupture and aneurysma or can be by the another kind of patient's condition of aldosterone agonist or antagonist for treating.

129. as each described mensuration of claim 99-123, wherein certified compound is tested in suitable external or body inner model the influence of treatment disease or the patient's condition, and the wherein said disease or the patient's condition be selected from cystic kidney disease, acquired cystic kidney disease, such as the eye circular correlation sufferer of myopia; Feel sick, vomiting, sexual dysfunction (sex), oedema, hypertension, congestive heart failure (scope is from the II class of New York heart association to ruddy pulmonary edema), the spontaneous oedema of periodicity, nephrotic syndrome, the ascites that causes by liver cirrhosis or other reasons, the cerebral edema of a variety of causes, dilutional hyponatremia changes with the metabolism that is referred to as inappropriate ADH secretion syndrome and wherein treats the other diseases or the patient's condition that needs vasorelaxation and/or anti-oxytocic activity or use vasopressing agonist or antagonist.

Produce maybe and can comprise the compound of the regulation and control TRPML3 of administering therapeutic significant quantity 130. a treatment relates to aldosterone by using the disease that aldosterone agonist or antagonist treat or the method for the patient's condition.

131. as the described method of claim 130, the wherein said disease or the patient's condition are selected from oedema, blood pressure (high or low blood pressure), liver cirrhosis, the primary hyperaldosteronemia, renal tubal dysfunction, diabetes (I or II type) and relative pathological symptom, comprise circulatory problems, oedema, the illness in eye relevant with Cycle Difference, high cortisone mass formed by blood stasis, the atherosclerosis or the obesity of abdominal obesity for example, and hepatopathy, sexual dysfunction (sex), cerebrovascular disease, vascular disease, retinopathy, neuropathy, Insulinopathy, endothelial function disturbance, pressure function of receptors obstacle, migraine, hectic fever and premenstrual tension and other cardiovascular patient's condition are such as atherosclerosis, in heart failure, congestive heart failure, vascular disease, apoplexy, myocardial infarction, endothelial function disturbance, ventricular hypertrophy, renal tubal dysfunction, target organ damage, thrombosis, arrhythmia, plaque rupture and aneurysma.

132. a treatment relates to the disease of vasopressing release or the method for the patient's condition, it comprises the compound of the regulation and control TRPML3 that uses significant quantity.

133. as the described method of claim 132, the wherein said disease or the patient's condition be selected from cystic kidney disease, acquired cystic kidney disease, such as the eye circular correlation sufferer of myopia; Feel sick, vomiting, sexual dysfunction (sex), oedema, hypertension, congestive heart failure (scope is from the II class of New York heart association to ruddy pulmonary edema), the spontaneous oedema of periodicity, nephrotic syndrome, the ascites that causes by liver cirrhosis or other reasons, the cerebral edema of a variety of causes, dilutional hyponatremia changes with the metabolism that is referred to as inappropriate ADH secretion syndrome and wherein needs vasorelaxation and/or the anti-oxytocic other diseases or the patient's condition or wherein treat the another kind of patient's condition that needs to use vasopressing agonist or antagonist in the treatment.

134. regulation and control have in requisition for curee's blood pressure or the method for fluid retention, it comprises the compound of the regulation and control TRPML3 that uses significant quantity.

135. regulation and control have in requisition for curee's urine produce and/or the excretory method, it comprises the compound of the regulation and control TRPML3 that uses significant quantity.

136. a treatment have in requisition for curee's Addison's disease or the method for the glutinous lipidosis of IV type, it comprises the compound of the regulation and control TRPML3 that uses significant quantity.

137. as each described method of claim 120-126, wherein said compound is polypeptide, antibody, small molecules, siRNA, sense-rna or ribozyme.

138. a method for the treatment of the disease of parathyroid glands or the patient's condition, it comprises uses TRPML 3 conditioning agents.

139. as the described method of claim 138, wherein said disease is selected from that calcium homeostasis, hyperthyroidism, hypothyroidism, hypercalcemia, cryptomere osteitis, false hypothyroidism, Jansen metaphysis cartilage generate, the Blomstrand cartilage generates and the osteoporosis of different reasons, and described reason comprises that for example the age is relevant, menopause or medicine, chemotherapy or radiotherapy-induced.

140. isolating saline taste gustatory cell as claimed in claim 1, it does not express T1R.

141. isolating saline taste gustatory cell as claimed in claim 1, it does not express T2R.

142. isolating saline taste gustatory cell as claimed in claim 1, it does not express PKD2L1/PKD1L3 and/or TRPM5.

143. the probe that a use is specific to TRPML3 gene or gene product is identified and/or separation and/or enriched sample in the method for saline taste sense of taste specific cell.

144. as the described method of claim 143, wherein said gustatory cell is people or macaque gustatory cell.

145. as the described method of claim 142, wherein said isolating gustatory cell is not expressed PKD2L1, PKD1L3 or TRPM5.

146. as the described method of claim 142, wherein said cell is not expressed T1R or T2R.

147. as the described method of claim 142, wherein said gustatory cell is not expressed transducer or gusducin.

148. as the described method of claim 142, wherein said saline taste gustatory cell hypotype or gustatory cell pedigree by comprise that the method for using fluorescence-activated cell sorter (FACS) is separated, purifying, enrichment or mark.

149. as the described method of claim 142, the method for wherein said gustatory cell hypotype or the gustatory cell pedigree magnetic bead by comprising applying marking is separated, purifying, enrichment or mark.

150. as the described method of claim 142, the wherein enzymic digestion of blended cell colony or the cell suspension tissue by containing gustatory cell and/or organize depolymerization to produce.

151. as the described method of claim 150, wherein said cell-derived at least one from tongue, oral cavity, gi tract or relevant organ, suprarenal gland, hypophysis, melanocyte and urethra.

152. as the described method of claim 142, wherein said saline taste gustatory cell hypotype or gustatory cell pedigree by the method that comprises negative cells selection technology separate, purifying, enrichment or mark, described negative cells selects technology based on the expression of at least one other gustatory cell specific gene or do not express and eliminate at least one non-target gustatory cell hypotype or pedigree.

153. as the described method of claim 152, wherein said method uses cytotoxic antibody to kill at least one non-target cell type or pedigree specifically.

154. one kind is used, and TRPML3 and/or TRPML1 or TRPML2 as a token of identify, the method for separation or enrichment saline taste recipient cell.

155. one kind in conjunction with the method for identifying the saline taste sense of taste conditioning agent of inferring in measuring, it comprises provides the TRPML3 polypeptide or expresses the cell of TRPML3 and make described polypeptide or cell contacts with the TRPML3 adjusting compound of supposition and identify potential TRPML3 conditioning agent based on the specific combination of itself and TRPML3 polypeptide.

156. described in conjunction with measuring as claim 155, it is directly in conjunction with measuring.

157. described in conjunction with measuring as claim 155, it is measured for the competition combination.

158. described in conjunction with measuring as claim 155, it uses Mammals TRPML3 polypeptide.

159. described in conjunction with measuring as claim 158, its use be selected from be contained in SEQID NO:2,4,10,12,14,16,22,24,26,28,30,32 and 34 those the TRPML3 polypeptide or the TRPML3 polypeptide that has at least 90% sequence identity by SEQ ID NO:39 or 40 encoded polypeptides.

160. described in conjunction with measuring as claim 158, its use be selected from be contained in SEQID NO:2,4,10,12,14,16,22,24,26,28,30,32 and 34 those the TRPML3 polypeptide or the TRPML3 polypeptide that has at least 95% sequence identity by SEQ ID NO:39 or 40 encoded polypeptides.

161. it is described in conjunction with measuring the TRPML3 polypeptide that it uses wild-type people, non-human primate or rodent TRPML3 polypeptide or has the A419P sudden change as claim 160.

162. described in conjunction with measuring as claim 158, it uses radionuclide, fluorophore or enzyme to help bonded and detects.

163. described in conjunction with measuring as claim 158, wherein said ionic channel is by mammalian cell expression.

164. described in conjunction with measuring as claim 158, wherein said TRPML3 polypeptide is monomer or polymer.

165. described in conjunction with measuring as claim 158, wherein said TRPML3 is different aggressiveness and comprises TRPML1 or TRPML2 that perhaps described TRPML3 expresses with TRPML1 or TRPML2.

166. a method for the treatment of the melanocyte related conditions, it TRPML3 that comprises administering therapeutic or beauty treatment significant quantity regulates compound.

167. as the described method of claim 166, the wherein said patient's condition is melanoma or pigmentation or cell generation disorders.

168. as the described method of claim 166, the wherein said patient's condition is alopecia, bald or hair graying or skin color (" gloss ") forfeiture.

169. as the described method of claim 168, it is the result of aging, hormone imbalances, radiation or chemotherapy.

170. as the described method of claim 166, wherein said conditioning agent is an agonist.

171. as the described method of claim 166, wherein said conditioning agent is an antagonist.

172. as the described method of claim 166, wherein said conditioning agent is selected from small molecules, carbohydrate, sense-rna, dsRNA, ribozyme, antibody, polypeptide and glycopeptide.

173. a method of utilizing mammalian cell or the ovocyte and the supposition TRPML3 adjusting compound identification TRPML3 conditioning agent of expressive function TRPML3 sodium channel, it comprises:

(i) measure of the influence of described compound to the sodium transport by the TRPML3 passage; And

(ii) identify based on its enhancing or restraining effect whether described compound is the TRPML3 conditioning agent to sodium transport.

174. as the described method of claim 173, it also comprises (iii) confirms the certified compound regulation and control saline taste sense of taste in people or Mammals taste test.

175. as the described method of claim 173, wherein certified compound is tested in people or Mammals influence in the body of sodium excretion or urinary function.

176. as the described method of claim 173, wherein said TRMPL3 is Mammals TRMPL3.

177. as the described method of claim 164, wherein said TRMPL3 is people, non-human primate, rodent, ox, pig, horse or sheep TRMPL3.

178. the method for claim 1, wherein said mammalian cell are selected from the group of being made up of HEK293, HEK293T, Swiss3T3, CHO, BHK, NIH3T3 and COS cell.

179. as the described method of claim 173, wherein said ovocyte is Mammals, Amphibians, fowl or Reptilia ovocyte.

180. as the described method of claim 179, wherein said Amphibians ovocyte is an xenopus leavis oocytes.

181. as the described method of claim 173, other genes or ionic channel that wherein said cell expressing is expressed in gustatory cell.

182. method of using compounds identified in the claim 173 to regulate and control or strengthen the saline taste impression.

183. method of using compounds identified in the claim 173 to regulate and control people or mammiferous sodium excretion or urinary function.

184. as the described method of claim 183, wherein said mensuration is electrophysiological detection.

185. as the described method of claim 184, wherein said mensuration is two electrode voltage tongs technologies.

186. as the described method of claim 185, wherein said ovocyte is the Amphibians ovocyte.

187. as the described method of claim 186, wherein said ovocyte is the frog ovocyte.

188. as the described method of claim 187, wherein said mensuration is electrophysiological detection.

189. as the described method of claim 188, wherein said mensuration is two electrode voltage tongs technologies.

190. as the described method of claim 189, it is used for identifier TRPML3 toughener.

191. as the described method of claim 189, it is used for identifier TRPML3 inhibitor.

192. as the described method of claim 188, wherein said ovocyte contacted with people TRPML3 inhibitor before the people TRPML3 toughener with supposition contacts.

193. as the described method of claim 190, it comprises that also use is not by the negative control of the ovocyte of microinjection people TRPML3 cRNA.

194. as the described method of claim 190, the conditioning agent of wherein said supposition is used with the concentration of scope from about 1nM to about 1mM.

195. as the described method of claim 190, the reinforcing coefficient of wherein said people TRPML3 toughener performance at least 20%.

196. as the described method of claim 190, the reinforcing coefficient of wherein said people TRPML3 toughener performance at least 50%.

197. as the described method of claim 190, the reinforcing coefficient of wherein said people TRPML3 toughener performance at least 100%.

198. as the described method of claim 191, the rejection coefficient of wherein said people TRPML3 co-inhibitor performance at least 20%.

199. as the described method of claim 191, the rejection coefficient of wherein said people TRPML3 co-inhibitor performance at least 50%.

200. as the described method of claim 191, the rejection coefficient of wherein said people TRPML3 co-inhibitor performance at least 100%.

201. the high throughput assay of TRPML3 conditioning agent that is used to analyze and screen supposition based on Mammals or frog ovocyte, it comprises: make the expression TRPML3 of preload membrane potential fluorescence dye or sodium fluorescence dye or the test cell of variant, fragment or functional equivalent infer that with at least one TRPML3 conditioning agent compound contacts in the presence of sodium or lithium; And in the interact change of the cation mediated test cell fluorescence of monitoring when existing of the conditioning agent of inferring/TRPML3, and the regulation and control degree of definite TRPML3 recently mutually of the change when lacking described conditioning agent.

202. as the described measuring method of claim 201, wherein said positively charged ion is a sodium.

203. as the described measuring method of claim 201, wherein said positively charged ion is a lithium.

204. as the described mensuration of claim 201, wherein said positively charged ion is a potassium.

205. as the described measuring method of claim 201, wherein said test cell is selected from the group of being made up of MDCK, HEK293, HEK293 T, BHK, COS, NIH3T3, U2OS, Swiss3T3 and CHO.

206. as the described measuring method of claim 205, wherein said test cell is the HEK293 cell.

207. as the described measuring method of claim 201, wherein said method is used for changing the compound of compound identification for the special regulation and control sense of taste based on detectable fluorescence.

208. as the described measuring method of claim 207, the wherein said sense of taste is the saline taste sense of taste.

209. as the described measuring method of claim 201, wherein said test cell is seeded on the hole of porous test panel.

210., wherein be added in the hole of described porous test panel described test cell contacted with the conditioning agent of described supposition by the conditioning agent that will infer as the described measuring method of claim 209.

211. as the described measuring method of claim 210, wherein said test cell loads and allows fluorescence to change detected membrane potential dyestuff.

212. as the described measuring method of claim 201, wherein TRPML3 is the people TRPML3 by the people TRPML3DNA sequence encoding of cloning from people's sense of taste cell cDNA.

213. as the described measuring method of claim 201, wherein said TRPML3 DNA is TRPML3 DNA codon optimized or wild-type or sudden change.

214. as the described measuring method of claim 213, wherein said TRPML3 DNA is selected from and is contained among the SEQ ID NO:1,3,17,18,39 and 40 those.

215. as the described mensuration of claim 201, wherein the fluorescent plate reader is used for monitoring the change of fluorescence.

216. as the described mensuration of claim 201, wherein the voltage imaging plate reader is used for monitoring the change of fluorescence.

217. as the described mensuration of claim 201, wherein said membrane potential dyestuff is selected from the group of being made up of following: Di-4-ANEPPS (4-(2-(6-(dibutylamino)-2-naphthalene-yl) vinyl)-1-(3-sulfopropyl))-hydroxide pyridine, inner salt), DiSBACC4 (2) (two-(1,2-two barbituric acids)-the cyclonite oxprenolol), DiSBAC4 (3) (two-(1,3-two barbituric acids)-the cyclonite oxprenolol), CC-2-DMPE (1,2-two mnyristoyls-sn-glycerol-3-phosphorylethanolamine, triethylammonium salts) and SBFI-AM (1, the 3-benzene dicarboxylic acid, 4,4 '-[1,4,10-trioxa-7,13-diazacyclo pentadecane-7,13-two bases two (5-methoxyl group-6,1-2-cumarone two bases)] two-, four [(acetyl oxygen) methyl] ester.

218. one kind is used to monitor TRPML 3 active methods, it comprises: the test cell with functional TRPML3 splice variant and fragment transfection thereof is provided; Described test cell is inoculated in the hole of porous plate; In the hole of porous plate the test cell of inoculation being carried out dyestuff with the membrane potential dyestuff loads; The test cell that dyestuff is loaded contacts in the hole of described porous plate with sodium with the regulating compound of at least one supposition; And any fluorescence that uses fluorescent plate reader or voltage strength plate reader to monitor by conditioning agent/membrane potential dyestuff that TRPML3 interaction causes changes.

219. as the described method of claim 218, wherein said test cell is the HEK293 cell.

220. as the described method of claim 219, wherein said test cell is the HEK293T cell.

221. as the described method of claim 218, wherein said membrane potential dyestuff is selected from the group of being made up of following: Di-4-ANEPPS (4-(2-(6-(dibutylamino)-2-naphthalene-yl) vinyl)-1-(3-sulfopropyl)-hydroxide pyridine, inner salt), DiSBAC4 (2) (two-(1,2-two barbituric acids)-the cyclonite oxprenolol), DiSBAC4 (3) (two-(1,3-two barbituric acids)-the cyclonite oxprenolol), CC-2-DMPE (1,2-two mnyristoyls-sn-glycerol-3-phosphorylethanolamine, triethylammonium salts) and SBFI-AM (1, the 3-benzene dicarboxylic acid, 4,4 '-[1,4,10-trioxa-7,13-diazacyclo pentadecane-7,13-two bases two (5-methoxyl group-6,1-2-cumarone two bases)] two-, four [(acetyl oxygen) methyl] ester.

222. as the described method of claim 218, wherein said TRPML3 is the people TRPML3 from people's sense of taste cell cDNA clone.

223. as the described method of claim 218, wherein said TRPML3 is selected from by the people TRPML3 of naturally occurring people TRPML3, alternative splicing and the group that functional variant is formed thereof.

224. as the described method of claim 218, wherein said test cell is selected from the group of being made up of MDCK, HEK293, HEK293T, COS, BHK, NIH3T3, Swiss3T3, U2OS and Chinese hamster ovary celI.

225. a method that is used to identify the compound of the regulation and control saline taste sense of taste, it comprises: the test cell with functional human TRPML3, its splice variant, mosaic or fragment transfection or conversion is provided; Described test cell is inoculated in the hole of porous plate; In the hole of described porous plate the test cell of inoculation being carried out dyestuff with the membrane potential dyestuff loads; The test cell that dyestuff is loaded contacts in the hole of described porous plate with sodium with the adjusting compound of at least one supposition; Any fluorescence that uses fluorescent plate reader or voltage strength plate reader to monitor by conditioning agent/membrane potential dyestuff that TRPML3 interaction causes changes; And change the compound that the conditioning agent of at least one supposition is accredited as the regulation and control saline taste sense of taste based on the fluorescence of monitoring.

226. as the described method of claim 225, its TRPML3 that comprises that also assessment is identified regulates the influence of compound to the saline taste impression.

227. as the described method of claim 212, wherein said test cell is selected from the group of being made up of MDCK, HEK293, HEK293T, COS, BHK, NIH3T3, Swiss3T3, U2OS and CHO.

228. as the described method of claim 227, wherein said test cell is the HEK293 cell.

229. as the described method of claim 228, wherein said test cell is the HEK293T cell.

230. as the described method of claim 225, wherein said method is used for changing the compound of compound identification for the special regulation and control sense of taste based on detectable fluorescence.

231. as the described method of claim 225, the wherein said sense of taste is the saline taste sense of taste.

232., wherein be added in the hole of porous test panel described test cell contacted with the conditioning agent of described supposition by the conditioning agent that will infer as the described method of claim 225.

233. as the described method of claim 225, wherein said test cell is loaded and allows fluorescence to change detected membrane potential dyestuff.

234. as the described method of claim 225, wherein said test cell is expressed TRPML3 or its fragment or variant.

235. as the described method of claim 225, wherein said TRPML3 is selected from the group of being made up of the people TRPML3 of naturally occurring people TRPML3, alternative splicing or its functional variant.

236. as the described method of claim 225, wherein the fluorescent plate reader is used for monitoring the change of fluorescence.

237. as the described method of claim 225, wherein the voltage imaging plate reader is used for monitoring the change of fluorescence.

238. as the described method of claim 233, wherein said membrane potential dyestuff is selected from the group of being made up of following: Di-4-ANEPPS (4-(2-(6-(dibutylamino)-2-naphthalene-yl) vinyl)-1-(3-sulfopropyl))-hydroxide pyridine, inner salt), DiSBACC4 (2) (two-(1,2-two barbituric acids)-the cyclonite oxprenolol), DiSBAC4 (3) (two-(1,3-two barbituric acids)-the cyclonite oxprenolol), CC-2-DMPE (1,2-two mnyristoyls-sn-glycerol-3-phosphorylethanolamine, triethylammonium salts) and SBFI-AM (1,3-benzene-dicarboxylic acid, 4,4 '-[1,4,10-trioxa-7,13-diazacyclo pentadecane-7,13-two bases two (5-methoxyl group-6,1-2-cumarone two bases)] two-, four [(acetyl oxygen) methyl] ester.

239. one kind by using high-throughput diaphragm pincers electrophysiological detection system to identify the method for TRPML3 conditioning agent.

240. as the described method of claim 239, it comprises the automatic patch clamp of IonWorks system.

241. as the described method of claim 239, it uses the mammalian cell of expressing TRPML3 DNA.

242. as the described method of claim 241, wherein said mammalian cell is the CHO-K1 cell.

243. as the described method of claim 239, it is used for identifying the compound of open (activation) TRPML3 ionic channel.

244. as the described method of claim 226, it is used for identifying the compound of closing (preventing) TRPML3 ionic channel.

245. as the described method of claim 243, wherein said compound is tested as saline taste sense of taste toughener.

246. as the described method of claim 243, wherein said compound is tested as saline taste sense of taste co-inhibitor.

247. as the described method of claim 239, wherein said being determined under the mode standard moved, wherein each hole is corresponding to individual cells.

248. as the described method of claim 239, wherein said being determined under colony's patch clamp (PPC) pattern moved, wherein each hole provides the mean current of 64 cells, the otherness that this has strengthened overall success ratio (" positive is hit ") potentially and has reduced Kong Yukong.

249. as the described method of claim 239, its use comprises the 384-hole form of the Mammals test cell of expressing TRPML 3.

250. as the described method of claim 239, it uses the CHO-K1 cell of transient transfection.

251. as the described method of claim 239, it uses the CHO-K1 cell of stable transfection.

252. as the described method of claim 239, it introduces the CHO-K1 cell by using Ba cMam expression system with the TRPML3 gene.

253. as the described method of claim 239, the TRPML3DNA sequence that wherein said mammalian cell expression wild-type people TRPML3 DNA, codon (people) optimize or comprise the TRPML3 mutant of the modification that causes the A419P sudden change.

254. as the described method of claim 226, wherein the TRPML3 function is to go up the determined TRPML3 function of using the perforation patch clamp technique to measure at IonWorksQuattro instrument (MDS Analytical Technologies).

255. a use is accredited as the method for the compound of TRPML3 conditioning agent as the flavor additives useful of the composition that consumes for the human or animal.

256. as the described method of claim 255, wherein said composition is food, beverage, makeup, nutritious prod, the medicine that consumes for the human or animal.

257. as the described method of claim 255, wherein said composition is selected from potato flakes, corn flakes, crispbread, soup, the meat product that dips in material, soft drink, packing and pretzel.

258. as the described method of claim 242, wherein said composition is selected from: beverage, such as nectar, sports beverages, vegetables juice, lactic acid-fermenting beverage, soda pop, coffee, cocoa drink, black tea, oolong tea, green tea, pure mellow wine, alcohol and powder-form drink; Confectionery comprise candy, chewing gum, piece candy, gelatin-gum, soda pop sugar and chocolate; Bake comprises cookies, biscuit and bread; Dessert is such as sour milk and ice-creams; Snack comprises potato flakes and crispbread; Stew, curry, soup, condiment, dip in material, noodle soup, soup-stock, miso, fast food meat soup, dip, meat soup, jam, sprinkling water material, Japanese flapjack, miso soup, pickles, rice dumpling water material, ordinary food and drink water material, such as half-mature food or prepared food or its refrigeration and the frozen food of wheat, buckwheat and Chinese style noodles; Fast food food comprises the instinct noodles; And condiments, comprise blended powdery condiments and mayonnaise.

259. as the described method of claim 255, wherein said composition is selected from nutritional supplement or sports beverages and snack products.

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