TWI338044B - Saccharomyces cerevisiae yeast strain with functional expression of a glut transporter - Google Patents

Description

IX. Description of the invention: [Technical field to which the invention pertains] The present invention relates to a strain of Saccharomyces cerevisiae (8) (IV) 'The result is that the hexasaccharide transporter is no longer synthesized due to deletion of the gene sequence' and cannot be used as a carbon source Growth on the substrate, only after the expression of the GLUT4 gene, its ability to grow on a substrate with six carbon sugar as the only carbon source. [Prior Art] Most of the singular secrets are transported through the special transport egg, and (4) the sugar is transported into the cells. In various organisms, different mechanisms have been developed to mediate glucose transport, namely, proton-to-co-transportation of pure, Na+ (tetra) sugar co-transporters, dependent binding protein systems, Wei metastasis, and diffusion-promoting systems. In the case of eukaryotic cells, the mammals are referred to by the (four) Γ gene and the sputum yeast by the / ΑΤ 基 基 ( 四 四 四 四 四 四 四 四 四 ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ ’ 葡萄糖 葡萄糖 葡萄糖. These transporters belong to the larger sugar transport superfamily', which is characterized by the presence of 12 trans-spin and Xuxiang (iv) amino acid residues and major structures. Since the understanding of the transport process is important for diseases associated with the lack of glucose homeostasis, such as diabetes or Fanconi-Bickel syndrome, the transport of glucose in the feeding is the focus of many studies. To date, eight glucose transporters [GLUT1 to GLUT5 'GLUT8, GLUT9/SLC2A9, GLUT9 (GenBank Accession No. Y17803)] have been identified which contribute to the promotion of glucose exposure. Important tasks of these transporters include glucose uptake into various tissues, storage in the liver, insulin-dependent uptake into muscle cells and adipocytes, and glucose measurement using pancreatic 0 cells. GLUT1 mediates the importation of glucose into the red blood cells and across the brain barrier, but also in many other tissues; whereas GLUT4 is restricted to insulin-dependent tissues, mainly muscle and adipose tissue. In these insulin-dependent tissues, control of the GLUT4 transporter to the intracellular or protoplasmic membrane segment constitutes an important mechanism for regulating glucose uptake. In the presence of insulin, intracellular GLUT4 is redistributed to the plasma membrane to promote glucose uptake. GLUT1 is also expressed in these insulin-dependent tissues, and its distribution in cells is also affected by the effect of temsin. In addition, the relative efficiency of GLUT1 or GLUT4 catalyzed sugar transport is determined not only by the extent to which each transporter directs to the cell surface, but also by its kinetic properties. Since the different glucose transporters of the isoforms will behave together, and the glucose will be rapidly metabolized, the study of the role and detailed properties of each glucose transporter isoform in these insulin-dependent tissues becomes a complicated task. Heterologous expression systems, such as Xenopus egg cells, tissue culture cells, insect cells, and yeast cells, have been used to solve these problems. However, these systems have some difficulty. The heterologously expressed transporter activity is too weak, the glucose transporters inherent in these systems, the intracellular retention of many transporters, or the production of physically inactive transporters. In addition to the heterologous and functional Glut4 glucose transporters, it is not known that organisms can express additional hexose transporters, particularly the intrinsic hexose transporters. This has led to a series of disadvantages in the search for compounds that modify the transport properties of the Giut4 protein. Since GIut4 is known to play an important role in reducing blood glucose levels in combination with islet 8 * 6 - and other factors, it is of considerable interest to use these compounds as ingredients in pharmaceutical preparations. An organism exhibiting a functional Glut4 transporter protein can be used to find compounds that directly affect the Glut4 transporter. Since the side effects of signal factoring do not occur, the side effects of these compounds are less pronounced. In addition, if a yeast strain can be used, it will be more advantageous in the handling and supply of materials. SUMMARY OF THE INVENTION The present invention relates to a Saccharomyces cerevisiae strain which is not capable of growing on a substrate having six carbon sugar as the sole carbon source, but after it exhibits the GLUT4 gene, it is restored to a substrate having six carbon sugar as the sole carbon source. The ability to grow on. Such strains can be produced, for example, by mutation or deletion of the relevant gene body sequence. The hexose sugar system means an aldose having 6 carbon atoms, such as glucose, galactose or mannose, and a ketose having 6 carbon atoms, such as sugar or sorbose. The invention further relates to a Saccharomyces cerevisiae strain comprising the GLUT4 gene as described above. In S. cerevisiae, 17 hexasaccharide transporters and three other maltose transporters are known, and if they exhibit sufficient money, the hexoses are transported into the yeast. It is known that all the transporters of the -strain which can take up the six-carbon sugar are removed by the deletion method, so this ride contains only two genes, Μ(4) and ΜΡΗ3, which are homologous to the maltose transporter. ΜρΗ2 and delete ^2 base = (4) sugar (4) present in the medium towel. The production and identification of the mother g strain Wieczorke et al., FEBS Lett. 464, 123-128 (1999) Γ This strain cannot grow in the base f where glucose is the only carbon source. According to function 1338044 ^GhUKhxtfgyW _) of the appropriate #龙 selection mutant strain. Money, ribbons have (four) dumping (four) ^ this ^ in the gene plastid vector transformed into the yeast strain hxtfgyM ^ L coffee = glucose transport. - Functional performance requires this leave d -: Shishi: to use Glut4 for a significant amount of glucose transport. This n uses the early-glucose transporter Glut4 to ingest glucose into the cell tenth, which can be used to separate glucose from the substrate that is the only source of carbon. For this, the GLW4-based yeast strain hxtfgyl-Ι with functional control under the yeast promoter was transformed. The bifed cells have been plated in this way and cultured on a medium containing glucose as the sole carbon source. For example, after 3 〇t: after several days of culture, it can be observed that individual: 15 20 long's turned off. If the yeast (4) was removed, no growth occurred on the medium with glucose as the sole carbon source. If the yeast plastid with the GLUT4 gene under the functional control of the yeast promoter is reshaped into a strain that no longer contains the vector plastid, the strain will be re-extracted on the medium with glucose as the sole carbon source. The ability to grow. The production of Saccharomyces cerevisiae strains which may take up glucose using (7) hoppers will be described in detail in the examples. This strain does not exhibit a yeast hexasaccharide transporter, but can utilize a Glut4 transporter gene, such as a gene that has been transformed into this strain, to take hexoses, particularly glucose, into the cells. For the purposes of the patent procedure, the yeast strains of this nature have been deposited under the Budapest Treaty for the international recognition of microbiological deposits in the German Physician Conservation Center (Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH, DSMZ) in Braunschweig, Germany. DSM 14035, DSM 14036 or DSM 14037 (Table 1). 8 25 P38044 The heterozygous control τ of the promoter of the Saccharomyces cerevisiae in the present invention, the table material 'sound yeast artist must be familiar with the appropriate yeast table ^ this technology _ _ disproducibility enzymes), fine start = ^ go It is a face-to-face start ΗΥΤ〇ό ^ 勒 ( ( 知 虱 ) )), acid phosphatase sugar transporter 7) = child (four) sugar transporter 2), HXT7 promoter (grape = child (galactose transporter), etc. In order to achieve the expression of the mover and the composition of the GLUT4 gene, the yeast vector can be self-replicating age independent of the yeast genome, or stable and continuous mother silk. In principle, the appropriate material is poured into all the nucleotide sequences that can be propagated in the yeast towel. Particularly useful yeast yeast (4) squama human chromosomes. In general, the yeast vector contains a replication source W for starting replication. (d), and usually selected from the auxotrophic face or the antibiotic resistant group red group. The yeast vectors known to those skilled in the art are, for example, BM272, pcsl9, pEMBCYe23, pFL26, pG6, pNN414, pTV3, etc. In principle, GLUT4 gene of any species Preferably, the GLUT4 gene is obtained from human, mouse or rat. The polynucleotide and amino acid sequence of Glut4 can be obtained from the following gene bank registration data: M2〇747 (cDNA; human), EMBL: D28561 (cDNA; rat), EMBL: M23382 (cDNA; mouse), Swissprot: P14672 (protein; human), swiSSpr〇t: pi9357 (protein; rat) and Swissprot: P14142 (protein; mouse). GLUT4 The gene is particularly preferably expressed by the vector YEp4H7-HsGlut4 (SEQ ID No. 9.) The GLUT4 gene of this vector is derived from humans. Those skilled in the art will be familiar with the production of a yeast vector containing the GLUT4 gene expressed in cells. The production of the vector is described in the examples. The yeast containing the gene of 8 25 1338044, which is intended to express the gene, is transformed into a fermented material. The method for this purpose is, for example, electroporation using vector DNA or Competent cell culture method. 'Transformation is a technique known to those skilled in the art, which is used to introduce foreign DNA, especially f or (4), into microorganisms such as yeast or fine. Yeast is transferred to 5%, yeast carrier, Yeast mutant Screening or protein expression in yeast and other detailed experimental procedures were seen _f this skill who kept informed of the "melting heart in Yeast Genetics, 1997: A Cold Spring Harbor Laboratory Course

Manud; Ad painting Alis〇n (rat); c〇id Spring _〇γ ub〇 ry; ISBN: 0-87969-508-0" in the manual. Evidence that the GLUT4 gene has been expressed in yeast according to the present invention can be specifically provided by Northern blotting, Western blotting, glucose uptake, glucose conversion studies, or other greens. The northern ink point method involves applying RNA from the biomass to the collateral body (such as nitrocellulose), and then setting the plexus body with the biological RNA to the GLUT4 Place the (four) marker or fluorescently labeled 15 DNA together. The expression of GLUT4-mRNA according to the present invention in yeast is evidenced by the appearance of a black band. In contrast, in the same, but not containing the GUJT4 • expression vector (four) _ mother RNA, the financial system from the belt. The Western blot method involves the application of a protein extract of a living organism to a membrane support (e.g., nitrocellulose) to demonstrate the expressed protein via an antibody. 20 Glut4 Protein Antibodies are available from, for example, Alpha Diagnostic

International, Inc" 5415 Lost Lane, San Antonio, TX 78238 USA. The analysis of the binding antibody was also obtained from this supplier. The Glut4 egg is expressed in the same strain but the yeast containing the Glut4 egg. The strains were compared and tested. In the case of the study, the test was performed on the purely radioactively labeled glucose as the only source of 4. In contrast to the control strains of the same but without the 10-G]ut4 transporter, The yeast, the sole glucose transporter, carries the radiolabeled glucose into the interior of the cell. The conversion of glucose can be tested on a nutrient medium containing glucose as the sole carbon source. In contrast to the control group of the same but not Glut4 transporter, Ghjt4 The yeast strain of the transporter as the sole glucose transporter can be grown on a nutrient medium with glucose as the sole carbon source. Those skilled in the art will be familiar with the method just described. For details, see, for example, "Current Pr〇t〇 C〇ls in Molecular Biology; Edited by : FM Ausubel, R. Brent, RE Kingston, DM Moore, JG Seidman, JA Smith, K. Struhl; published by John Wiley & Sons in 2000 (currently in the update). The present invention is preferably a Saccharomyces cerevisiae strain of the Glm4 gene obtained from human, mouse or rat in relation to Table j. More particularly, the present invention relates to a Saccharomyces cerevisiae strain which exhibits a polynucleotide sequence comprising a coding region of the human Glut4 gene. In a further preferred embodiment, the invention relates to one or more strains of yeast yeast deposited in, for example, the German Species Preservation Center of Braunschweig, Germany, with accession number DSM 14038, DSM 14039 or DSM 14040. These strains are listed in Table 1. This table provides information on the yeast strains, plastids, and growth conditions of these yeasts used for plastid transformation. The invention also relates to the production of a strain of Saccharomyces cerevisiae according to the invention, which is obtained by the following procedure: a) providing a yeast which is no longer grown on a substrate having hexose carbon as the sole carbon source; b) containing soluble yeast The plastids of the GLUT4 gene under the functional control of the promoter are transformed into the yeast; c) will be obtained according to b), and the plate is cultured on a medium containing hexose carbon as the sole carbon source; d) selecting a strain which is cultured on the medium according to c) and is supported by the Glut4 gene to support the uptake of the six carbon sugars. The present invention also relates to the growth and growth of such strains. Providing the yeast according to the invention, the first step will no longer be grown on a substrate which is a six-only carbon source, but in which the wine of the expression gene is reconstituted on a substrate which is a carbon source of six carbon sugars. The growth ability of the growth can be separated. This can be carried out by mutating or deleting the relevant gene sequence of the cyclamate hexose transporter. After providing the yeast, it is further necessary to make the yeast grow. Method, in appropriate cultivation The medium to be used for the growth of the yeast is, for example, a complete medium, a YPD medium (fermented material (4), and a selective medium, etc.). The yeast cells are grown in these mediums, and after growth, centrifuged. Separating it from the medium 'and then' it is suspended in an aqueous medium containing, for example, a buffer, salt or other additive, for the purpose of forming an aqueous suspension. Those skilled in the art can "Methods in Yeast Genetics, 1997: A Cold Spring Harbor"

Information on the growth of yeast is found in the Laboratory Course Manual; Adams Alison (Edt.); Cold Spring Harbor Laboratory; ISBN: 0-87969-508-0. In a preferred embodiment of the above-described yeast strain, the GLUT4 gene which can be expressed in the yeast under the functional control of the promoter is used for transformation 8 , 12 · 1338044 . For the transformation, please refer to the “Methods in Yeast Genetics j 前” mentioned above.

10 In particular, the GLUT4 gene for transformation to produce such yeast strains is derived from the GLUT4 gene of human, mouse or rat. Further, the GLUT4 gene line which is particularly useful for transformation is present in the polynucleotide sequence as shown in SEQ ID No. 9, or SEQ ID No. 10. SEQ ID No. 9 discloses the polynucleotide sequence of the yeast vector Yep4H7-HsGLUT4. This vector contains a polynucleotide sequence functionally controlled by the HXT7 promoter, which encodes the amino acid sequence of the human GLUT4 gene. SEQ ID No. 1 多 contains the polynucleotide sequence of the vector H2rg4g2. The yeast vector H2rg4g2 carries the rat GLUT4 gene under the control of the HXT2 promoter function. Functional control of the GLUT4 gene by the promoter means that the promoter is transcribed to mRNA which can be translated into the Ghlt4 protein. For the disclosure of the GLUT4 sequence and the method used, reference is made to the literature already mentioned above. The present invention further relates to a method for identifying a compound for increasing or decreasing the amount of hexose carbon saccharide transported by Glut4 protein using the following procedure: a) providing a Saccharomyces cerevisiae strain according to the present invention which exhibits the GLUT4 gene; b) determining its six carbon Sugar intake; 'c) providing a compound; 20 w π low-capture yeast strain and compound provided according to 〇 e) measuring the amount of hexose ingested in the yeast strain after contact according to d) f) The compound which increases or decreases the amount of hexose carbon occluded by _ protein transport is determined by the peak according to the amount measured by d)_managen _ hexose. 8 -13- 25 In order to provide the yeast according to (4), the first step will no longer grow on the substrate with the hexoseose as the carbon source, but after the Giut4 gene is expressed, it will be restored to the six carbon The Saccharomyces cerevisiae strains that are capable of growing on a carbon-like substrate are isolated. The mother strain was transformed into a yeast vector containing the GUJT4 gene functionally controlled by the yeast promoter. The production of this transgenic strain is described in the implementation of the financial. After the yeast is provided, it is necessary to further grow the yeast. The growth line is carried out in a suitable medium using microbiological standard methods. A suitable medium for growing the yeast is, for example, a complete medium, particularly 疋YPD medium (yeast extract/gland/dextrose medium) or a selective medium. The yeast cells are grown in these media, grown, centrifuged to separate them from the culture medium, and then suspended in an aqueous medium containing, for example, a buffer substance, a salt or other additives, for the purpose of the method used to form Aqueous suspension. Those skilled in the art can refer to "Meth〇ds in Yeast Genetics, 1997: A Cold Spring Harbor Laboratory Course Manual; Adams Alison (Edt.); Cold Spring Harbor Laboratory; ISBN·0-87969-508. Find information on yeast growth in -0". The amount of hexose carbon uptake by the yeast strain supplied by k as described above can be measured by a radiolabeled glucose uptake study. To achieve this, a specific amount of yeast cells, for example, 60 mg wet weight per ml, is suspended in, for example, 100 μl of buffer and quantified as 14 c- or 3H-labeled glucose as the sole source of destruction. deal with. Cell culture is carried out to sample a specific amount of cells at a specific time. LSC (liquid scintillation counting) is used to help determine glucose uptake. However, the hexose uptake of the yeast strain as provided above can also be determined by a growth test on a medium having glucose as the sole carbon source. To achieve this, after the addition of the compound, the growth rate of the strain of the strain - 14 - 丄 JJ0U44 compound is determined, or the separation from the organism is completed. Chemical synthesis can also be automated. The compound obtained by synthesis or separation can be dissolved in the solvent. Properly dissolved (4) is an aqueous solution containing a specific amount of an organic solvent 'e.g., 'DMS 〇 (dimethyl (tetra)). The contact of the yeast strain with a compound for increasing or decreasing the identification of a compound utilizing the (3) (10) protein transport of the six carbon stalk is accomplished, in particular, in an automated laboratory system for this purpose. This material can be composed of specially prepared rooms, microplates, microcentrifuge tubes or laboratory glassware. The automated laboratory system - the general system is set to achieve high productivity. Therefore, the procedure described by means of automated laboratory secrets is also referred to as HTS (High Productivity Screening). To make the yeast and the compound smooth, under these conditions, by the yeast fine 15

The amount of hexose (especially glucose) that is transported into the cells by the cells is measured. For this purpose, the same procedure as for the glucose uptake of the compound has been used. The identification of the compound 20 which increases or decreases the amount of the six-carbon sugar transported by the Glut4 protein is carried out in comparison with the amount of hexose carbon in the compound copper. Further, the present invention relates to a medicament for the treatment of diabetes and obesity which comprises a compound which has been identified and, if appropriate, developed using the above-mentioned method using the Ghit4 gene, and an adjuvant comprising the preparation of the drug . The further development of the identified compound means that the target protein (in this case, the specificity of G_ is improved; secondly, the availability in the animal or human organism 25 is increased; and Third, any side effects that are already present and not desired are reduced. To this end, those skilled in the art can use a range of methods including, for example, but not limited to, the use of pharmacological animal models such as diabetic rats or obese mice. The use of in vitro biochemical assays, the use of compounds and the actual structural mode of the G丨ut4 protein. The adjuvants for the formulation of the active substances can be used for the application, the distribution of the active ingredients to the intended application, and the development of the active substances. The examples of such adjuvants are fillers, binders, disintegrating agents or glidants such as lactose, sucrose, mannitol, sorbitol, cellulose, temple powder, dicalcium phosphate, polyglycols. , alginate, polyvinylpyrrolidine, j, methyl cellulose, talc or cerium oxide. Diabetes is caused by a lack of insulin or a decrease in the metabolic state of insulin There is a sign that glucose is secreted together with urine and at the same time has an abnormal increase in blood glucose (hyperglycemia). Insufficient or reduced insulin action will cause somatic cells to be completely absorbed and transformed into glucose and absorbed into the blood. In the tissue, the hormone that antagonizes insulin has the effect of increasing the decomposition of fat, so that the content of free fatty acids in the blood is inevitably increased. ..., e Obesity is caused by excessive caloric intake, resulting in an imbalance of energy, resulting in an abnormal increase in body weight. In addition, the present invention relates to the use of a compound which has been identified and, where appropriate, further developed by the method of using Glut4 protein for the preparation of a medicament for the treatment of diabetes and obesity. The medicaments are pharmacologically active. A dosage form of a substance for treating diseases or bodily functions of humans and animals. Examples of dosage forms known for oral treatment are powders, granules, lozenges, pills, elixirs, dragees, capsules, and extracts. , tincture and syrup. For external (S), spray, gel, soft Powder or parenteral administration, the dosage form of these and other dosage forms of the present invention is further related to the use of those skilled in

20 Examples of dosage forms for administration are aerosols and agents. Injectable or infused solutions are for syringes that are not via sputum, bottled or prefilled. This is known to pharmaceutical professionals. a) Saccharomyces cerevisiae strain which is capable of growing on a substrate which is not a carbon source, but only on its LT1 base, and which is capable of growing on a substrate having a hexose carbon-only source. Containing the &gluti gene under the functional control of the promoter in yeast; b) determining the hexose intake of the strain according to a); d) making the yeast strain according to a) according to c) Contacting the provided compound; e) measuring the amount of hexoses ingested in the yeast strain after contact according to d); f) by comparing the amount of hexoses in the human strain before and after exposure to d), ie Comparing the amounts determined according to b) and e), identify compounds that increase or decrease the amount of hexose carbon _g(d) protein transport. In order to provide the yeast according to the present invention, the first step will be due to the deletion of the genome sequence; f will form a hexacarbon transporter, and as a result, the hexacarbose will no longer grow on the substrate of the only carbon source, but in its expression Glutl After the gene, it is then isolated from the S. cerevisiae strain which is capable of growing on a substrate having six carbon sugar as the sole carbon source. These strains are deposited in the German Culture Collection Center under the registration number DSM 14031, DSM 14032 or DSM 14034. Among S. cerevisiae, 17 hexasaccharide transporters and three other: maltose transporters are known to transport hexoses into yeast. Two =_1.28 If: 9) There are ^ Transformation of the second-class 3=, :^ The plastid of the G_ gene of the sugar-making Korean H: The car series does not make the sugar transport still does not occur. Glutl gamma H b is further adapted to the glucose/sugar=yellow=strain to make use of the glucose-based::di-(tetra) sugar transporter-the scorpion (10), which is used as the sole carbon source for glucose 15 For this purpose, a yeast strain carrying the functional sugar gene of the yeast promoter is transformed into a yeast strain which no longer exhibits the intact six carbon protein. The yeast cells which have been transformed according to this method are plated in a medium 2 containing (tetra) glucose as a carbon source and then cultured. For example, after 3 days of rc culture, the growth of individual mycelium can be observed, and the -g can be isolated. If the yeast is removed, then the medium with glucose as the sole carbon source is not See growth. Now if the yeast plastid with the GLUT1 gene under the functional control of the yeast promoter is transformed into a g strain that no longer contains the lysine, the strain will be re-expressed with glucose. The ability to grow on a medium of carbon source. 20 To transform a yeast strain, the GLUT1 gene from human, mouse or rat can be used in particular. The polynucleotide and amino acid sequence of Glutl are disclosed in the indicated library. The following numbers are: EMBL: M20653 (cDNA; human), EMBL.M13979 (cDNA; rat), EMBL: M23384 (cDNA; mouse), 8\¥185 mouth 1'〇1:.?11166 (protein; Human), 8 188 卩 1 > 〇 [: 1) 11167 (protein; rat) and Swissprot: P17809 (protein; mouse). -18- 25 1338044 Liquid order, and quantified by 14. Or 3h • labeled glucose as a “dislike” to handle. Cell culture is performed to sample a specific amount of cells at a specific time. LSC (liquid scintillation counting) is used to help determine glucose uptake. The supply of the compound is accomplished by chemical synthesis or separation from the organism. Chemical synthesis can also be self-healing. The compound synthesized or isolated can be dissolved in a suitable solvent.适#Solution, is an aqueous solution containing a specific organic solvent, for example, DMS hydrazine (dimethyl sulfoxide). in

The contact of the 10 曰 yeast strain with the compound used to identify or increase the hexose of the Glutl protein transport is specifically accomplished in an automated laboratory system provided for this purpose. These systems can be composed of specially prepared concealed parts (4), micro-cultures, micro-dissociation (five) or laboratory glassware. Automated laboratory systems are typically designed to achieve high productivity. Therefore, the procedure as described immediately by means of an automated laboratory system is also referred to as HTS (High Productivity Screening). 15

After the yeast was brought into contact with the compound, the amount of hexose (especially glucose) which was carried into the cells by the yeast cells under these conditions was measured. For this purpose, the same procedures as described for determining the glucose uptake of yeast strains that are not in contact with the compound can be used. The identification of a compound which increases or decreases the amount of hexose carbon transported by the Glutl protein is carried out by using the amount of the hexasaccharide in the strain before and after the contact of the comparative strain with the compound. Further, the present invention relates to an agent for the treatment of diabetes and obesity comprising an agent which has been identified and, if appropriate, further developed using the above-described procedure using the Gkltl gene' and an adjuvant for formulating the agent. This further development of the identified compound means that the first 'target protein (in this case * 20 - the specificity is improved; the second, the small in the riding or human object = increase, and the third 'Anything that is already present and not intended to be reduced'. For this purpose, a person skilled in the art can use the -series method, including, if not limited to, 'pharmacological animal models such as diabetic atmosphere or obese mice. , the use of in vitro biochemical assays, compounds and G (four) protein = the use of the structure of the structure. For the general use of _ can be used for adaptation, active ingredients (four) distribution distribution, such as the active substance: tune: Examples of such adjuvants are fillers, binders, disintegrators or glidants such as lactose, mannitol, sorbitol, cellulose, temple powder, machinery 2, polyglycols, sea salt Polyethylene sulphate, slow methylcellulose, talc or cerium oxide. Diabetes is caused by the lack of a chronic metabolic state caused by the reduction of the effect of temsin or temsine. Abnormal increase in glucose in the blood ( Signs of hyperglycemia) Insufficient or reduced insulin action will cause somatic cells to be ingested and converted into glucose in the absence of complete absorption and conversion of glucose. In adipose tissue, hormones that antagonize the oxytocin increase fat decomposition. The effect is that the content of free fatty acids in the blood is inevitably increased. 曰 Obesity is caused by excessive calorie intake, resulting in an imbalance of energy, resulting in an abnormal increase in body weight and damage to health. Further, the use of the present invention has been identified and, where appropriate, The compound developed by the method of Glutl protein is used to prepare the treatment of urinary diseases and obesity symptoms. The _ is in the form of a pharmacologically active substance to treat diseases and physical disorders of humans and animals. Examples of dosage forms known for oral treatment are powders, granules, lozenges, pills, dragees, capsules, liquid extracts, elixirs and syrups. Examples of dosage forms for external administration are aerosols. , spray, gel, ointment or powder. Injectable or infused solutions are allowed to be administered parenterally, the dose used The invention is in the form of a vial, a bottle or a prefilled syringe. These and other pharmaceutical forms are known to those skilled in the pharmaceutical arts. Further, the present invention relates to a polynucleotide sequence of SEQ ID No. 13 and a polynucleotide of SEQ ID No. 14. The acid sequence. SEqID No. 13 and the SEQ ID No. 14 polynucleotide sequence encode a mutant of the rat Giuti gene which results in the substitution of the individual amino acid of the protein in question. SEq ID Νο· 13 polynucleotide sequence encodes its amino acid a Glutl protein in which the proline is replaced by guanidine thiocyanate at position 69; the SEQ ID NO: 14 polynucleotide sequence encodes a Glutl protein in which the alanine of the amino acid chain position 70 is substituted with methionine. Even in the case where the hexose-carbon transporter has been shut down due to deletion, glucose uptake is maintained in the strain in which glucose uptake is not maintained by the wild-type Glutl protein. Such mutants can be obtained, for example, by selective mutation or inhibitor mutations' or by in vitro mutation formation. The invention further relates to a Glut 1 protein encoded by the SEQ ID No. 13 or SEQ ID No. 14 polynucleic acid sequence.

The invention also relates to a polynucleotide sequence comprising SEQ ID No. 13 and a SEQ

Yeast strain of ID No. 14 polynucleotide sequence. These yeast strains were deposited at the German Culture Collection Center under the registration numbers of DSM 14026 and DSM 14027. In order to produce these strains, the yeast vector corresponding to SEqIDn〇13 or seqidn〇14 can be transformed into a single growth without hexose, but only when the GLUT1 gene is expressed in this strain; The only strain of yeast that grows on the substrate of the carbon source. Next, after -22-8, the cells were plated in a medium containing glucose as the sole carbon source. The colonies grown in this medium were isolated. The yeast strain transformed in this way is suitable as an example of a method for identifying a compound which increases or decreases the amount of carbon sugar above the transport of Glutl protein. Abbreviation HXT hexasaccharide transporter ORF open translation architecture PCR polymerase chain reaction example yeast strain of the county The yeast strain described herein was obtained from strain CEN.PK2-lC〇^47^ leu2-3, 112um3-52trpl- 289his3-Al MAL2-8C SUC2). The production of a yeast strain with a deletion of the hexose glucose transporter gene (// especially 7) is described in Wieczorke et al., FEBS Lett. 464, 123-128 (1999): EBY. 18ga (MATa Ahxtl-17 Δ gal2 AagtlAstll Leu2-3, 112 ura3-52 trpl-289 his3-Al MAL2-8C SUC2), EBY.VW4000 {ΜΑΤαΔ hxtl-17 Agal2 Δ agtl Δ mph2 Amph3 Astll leu2-3, 112 um3-52 irpJ-289 his3-d! MAL2-8C SUC2). The medium is based on Wo yeast extract and 2% gland (YP), while the minimal medium consists of 0.67% Difco yeast nitrogen-containing base (ΥΝΒ) without amino acid, and contains additives for nutrient requirements and different carbons. source. The yeast cells were aerobically grown on an orbital shaker or a canola dish at 30 °C. The optical density (OD_) of 6 nanometers was measured to monitor the growth of cells. -23- 1338044 Glucose uptake tears The glucose kinetics of the D-[U-C]-glucose (Amersham) uptake was measured by the Eadie_H〇fstee plot. The cells were centrifuged, washed with sulphate buffer, and resuspended at a concentration of 60 mg (fresh weight) per ml. 5 floated in discate buffer. Glucose uptake was determined at a glucose concentration between 0.2 and 100 mM. The specific activity of the matrix was between 0 1 and 55 5 kBq//m 〇 rl. The cells and glucose solution were preincubated at 30 ° C for 5 minutes. Glucose uptake begins with treatment of cells with radioactive glucose. After 5 seconds of incubation, 10 ml of ice-cold stop buffer (1) 丨M KiP04, pH 6.5, 500 10 · mM glucose) was added to rapidly filter the cells into a glass fiber filter (0=24 mm,

Whatman). The filter was immediately washed three times with ice-cold buffer and the incorporated radioactivity was measured with a liquid scintillation counter. The inhibition of cytochalasin B (final concentration 2 〇Μ, dissolved in ethanol) was determined by incubating the cells for 15 minutes in the presence of an inhibitor or in the presence of a solvent, followed by 15 mM of 50 mM or 100 mM radioactive grape 15 saccharide. Clock uptake test.

20 H2rg4g2(SE〇ID No. 10)舆H2m1g2fSEO ID No. 12) The mH2rg4g2 and H2rglg2 lines comprise the GLUT4 gene (in SEQ ID No. 10) or the GLUT1 gene derived from rat (in SEQ ID No. 12) DNA construct of the functionally linked HXT2 promoter (promoter of yeast glucose transporter 2). The plastids GLUT1-pTV3 and GLUT4-pTV3 (Kasahara and Kasahara, Biochem J. 315, 177-182 (1996); Kasahara and Kasahara, Biochim. Biophys. Acta 857, 146-154 (1997)) 0.5 kb &/ The I/five promoter fragment was replaced by a 0.5 kb DNA fragment containing the yeast ΤΓ2 promoter from -452 bp to +9 bp, 24-25 (Genbank: P23585). YEMH7-HsGLUT 1 rSEO ID No. ΊΠ^ YF, p4H7-HsGLUT4 (gEQ ID No. 9) is constructed by YEp4H7-HsGLUT1 and YEp4H7-HsGLUT4 as plastids, wherein the HXT7 promoter (the promoter of the HXT7 gene) is located at -392 to The promoter fragment of -1 is functionally linked to the human GLUT1 gene (in SEQ ID No. 11) or the GLUT4 gene (in SEQ ID No. 9). Since the intact HXT7 promoter is inhibited by glucose, a promoter fragment is used. The 0.4 kb promoter fragment from p426MET25 (Mumberg et al., Nucleic Acids Res. 22, 5767-5768 (1994)) was replaced by a 7/AT7 promoter fragment containing position _392 to -1, which was lined with the HXT7 gene. (Genbank: P39004) As a template, primers P426H7-1 (SEQ ID No. 1) and P426H7-2 (SEQ ID No. 2) were used to amplify by PCR to obtain plastid YEp4H7 (SEQ ID No. 15). HSG1-F7/T2-HSG1 (SEQ ID No, 3'4) and HSG4-F7/T2-HSG4 (SEQ ID No. 5.6) were used as primers for Glutl and Glut4, respectively, to human GLUT1 (EMBL: M20653) and humans. GLUT4 c DNA (Genbank: M20747) was used as a template to amplify human GLUT1 and GLUT4 ORFs (open translation architecture) by PCR. This PCR product was re-amplified by primers T71-ORF (SEQ ID No. 7) and T2-HSG1 (SEQ ID No. 4) or T2-HSG4 (SEQ ID No. 6) for 1 cycle. The PCR end product, upstream or downstream of the GLUT ORF sequence, contains a sequence homologous to the promoter region of the YEp4H7//_ΛΤ7 promoter region or the crc/terminator region (iso-cytochrome cl). It was transformed into the yeast strain EBY.F4-1 with the oRI linearized (Hnearlized) YEp4H7, and then, in the yeast, -25-'8 homologous recombination, in 2% strength maltose medium Uracil prototrophism was performed to select for the transformed product. Excellent J_ rat. GLUT1 and GLUT4 in the absence of hexa-glucose transport yeast strains _ expression of yeast multicopy expression plastids GLUT1-pTV3e and GLUT4-pTV3e with yeast promoter controlled by galactose-induced and glucose inhibition The next is derived from the rat glucose transporter gene GLUT and GLUT4. In this construct, the promoter is replaced by a glucose-induced yeast promoter. These vectors were transformed into the yeast strain ΕΒγ18, which was unable to ingest any hexoses and therefore could not grow on medium with glucose or other hexoses as the sole carbon source. Using maltose as a carbon source and no tryptophan-containing synthetic medium. Transfected plates were cultured in a mold-free culture method without maltose, but with different glucose concentrations (5 mM, 10 mM, 50 mM, l mM On the same basic medium, even if cultured at 30 ° C for more than - weeks, the transgenic plants could not grow on the medium with different glucose concentrations. This proves that Glutl and Giut4 glucose transporters are not supported in normal S. cerevisiae strains. Glucose uptake. Glucose uptake strain of EBY, a Glutl transgenic strain of 18ga, grows on glucose medium for a long period of time, and the growth of these colonies is apparently ingested by g colony (hereinafter referred to as saddle mutant or suppressor colony). And transforming glucose. Therefore, the plated and GLUT4 transgenic plants were plated on a dish of 1 () mM glucose as the only carbon source and s YNB medium. The dose was carried out - -26- 3 with i-狡 cultured cells for 7·14 days. Although the number of transgenic plants in GLUT4 was detected by the number of mutants, the GLUT1 transgenic strain had 1 #糖上The growth inhibitor (4) was grown in the aquaculture dish. Several GLirn inhibitor mutants were cultured in non-selective γρ maltose: more than 5 generations. All cells that lost their plastids could no longer be (4) sugar. On the medium of carbon source. It can be proved that the growth of glucose as the only carbon source is GLUT1 dependent. After the original wild type Η2 ring 2 plastid is transformed into these cells, several yeast strains are obtained again. The ability to grow on the sugar = sugar. This confirms that the gene body towel contains a suppressor (tetra) mutation that eliminates functional GLUT1 New Zealand. The mutation read the gene name plus 7·/ (represents the functional performance of GLUT1 in yeast towel "), the strain was named EBY.S7.

The H2rglg2 plastid was isolated from other suppressor mutants and amplified in E. coli and transformed into the yeast strain EBY.18ga (zlta) which originally lacked glucose transport. Several of these plastids are allowed to grow on synthetic media with 1 mM glucose as the sole carbon source. Thus, their GLUT(R) sequences contain mutations that convert the corresponding GLUTl protein in yeast to a functional glucose transporter. For example, the mutants comprise a proline acid substituted with hydrazine thione at position 69 of the amino acid chain (SEQ ID No. 13), or alanine at position 70 of the amino acid chain substituted with methionine. (SEQ ID No. 14). As described above, the mutant of SEQ ID No. 13 was found during screening of the mutant strain. The mutant of SEq ID No. 14 was obtained as described above using in vitro mutation formation. The principle of the in vitro mutation method used is described by B〇les and Miosga (1995) (Boles and Miosga, Curr Genet. 28, 197-198 (1995)). In the first PCR reaction, the plastid YEpH2-rGLUTl (20 Ng) was used as -27-1338044

For the DNA template, and the primers seqhxt2 (SEQ ID No. 16) and glutmet2 (SEQ ID No. 17) (each 100 picomoles (pmol)) (pCR conditions: 95 ° C 45 seconds ' 5 (TC 30 seconds, 72 ° C for 2 minutes, 25 cycles, taq polymerase.) The primer glutmet2 contains a base sequence in which the normal GLUT1 gene is modified and the alanine of the GLUT1 amino acid position 70 in the rat is replaced by guanidine thiocyanate. The PCR fragment was purified by agarose gel electrophoresis followed by gel extraction. In the second PCR reaction, the purified pCR was used.

10 tablets · ^ (20 Nike) and plastid GLUT1-pTV3 (Kasahara and Kasahara, Biochem J. 315, 177-182 (1996)) as DNA template (50 Ng), and primers seqhxt2 and seq2gal2 (SEQ ID No) 18) (100 picomoles each) (PCR conditions · 95 ° C 45 seconds, 54 ° C 30 seconds, 72 ° C 2 minutes, 20 weeks 15

Period 'taq polymerase). Since the primer seqhxt2 binds only to the fragment of the first pcr reaction, thus only the DNA sequence which causes the amino acid position of the amino acid to be substituted with methionine is amplified in this second PCR reaction. The resulting PCR fragment having the mutant GLUT1 gene was purified by cyclolipogel electrophoresis followed by gel extraction and exchanged with the wild-type GLUT1 gene in the plastid YEpH2-rGLUT1. This plastid (SEQ is still N〇 14) was transformed into yeast strain EBY.18ga (Zl to) lacking glucose transport, and grown on synthetic medium with glucose as the sole carbon source. 20 total Glock4 transporter uptake of glucose _|_ 梓 strain £8丫.87 (/11^/ illusion; plant /) apparently contains a genetic mutation, 1 ^ 2 / take the plant / 'it makes GluU in yeast It becomes functional to support the uptake of glucose into the cell through the plasma membrane. After transforming the strain EBY.S7 ("to plant/) with H2rg4g2, it will be able to grow for 15 generations on -28-' 25 ^ GLUT4 胄 胄 胄 胄 胄 胄 γ γ 麦 麦 = = sugar medium All of the cells that lost their bodies were no longer grown on the 1 〇 glucose medium, thus confirming that the growth was first GLUT4-dependent. From these nine inhibitory strains, _fH2rg4g2 shellfish was further subdivided into E. coli and transformed into yeast strain EBYS7 which was originally deficient in glucose transport. None of these plastids were allowed to grow on synthetic media with 10 mM glucose as the sole carbon source. This proves that it does not contain the form of the "activated" GLUT4 mutant. The original wild-type H2rg4g2 plastid was re-transformed into the nine inhibitory strains without plastids, and all of these strains were further grown on glucose relative to the control vector containing the Glut4-free transporter gene. ability. The corresponding mutant of this strain was named /illusion^-Χ(Χ=1-9). The mutated dual gene fgy4-x induces a functional representation of the GLUT4 gene in these strains. Thus the object of the invention is achieved. A review of the yeast strains used in accordance with the present invention is provided in the attached table. Listed in the table is a review of yeast strains used in accordance with the present invention, including genotypes, desired growth conditions, and their storage numbers at a German strain preservation center. 5 -29- 1338044

&#中κ^»βο.67%«*呤iL»Dif8>:PHS.20«*#s#.^i«·Wear:0·»ίίρ44 ms.£.mat(0.19 ms)-e- »*M0.25 3M), privately by AP443M). 3⁄4$荈珅^冷访1 »285Λ 3 ;»尥薄冷萃P2 靼 2% Λ3 (汸* 痒»}»阡珈**). Ro Ο to 00 -vj σι οι CO ro S ΕΒΥ·Μ·7 s -< m CD < S < s < S ks < S *< I δ δ s ξ δ § S -< s To m 09 < s ύ» SS 0» m Bamboo shoots|| MATa ΔΗχΐ1·17 0gai2 0agt1 AsU1 fgy4~ 7 fgy4-1 leu2-3,112 ura3-52 ίφ 1-289 his3-^1 MAL2-SF SUC2 =>* δ Hi _ δί| MATa Zihxtf-17^gai2/isgt1 AstH fgy4~ 1 fgy4-UBu2,3,112 ura3,52trp1289 hls3-A1 MAL2-iF SUC2 S' NJ > δ^Ι A & _ 〇-* 1 III Ill <3a ^ N o-^ fck 111 ^5>& 1! win 3* -* > Its irl O-* fck 11 •B fell _ S|l lls Isi _ 1 1^1 〇> S CK3 ^ β|Ν Pi fi» ί?»5 召泛11客S Find ^ r iyl °^| 〇>r S % It 3* ct li —r °^| c ^ S* δΙ! 迠上Η Sr MATa Ahxt1-170ga!2 Mgt1 0st!1 bu2-3,112 ura3-52 ίφ 1 -289 his3-^ 1 MAL2^ 8^ SUC2 δ | ^ fer 11 11 <1> ^ §1 乂·Γ m η ϊ I i choose P brother St ti Φ· and dissolve 莩琛Ρ ίί «Ρ 渚Φ咐 and » 'JK pregnancy (B>^ sound·, h 珩? ▲ a taste® At private At stain; thin (3> 傅弟Bud r- b 漭厗A cold at珈. 农淳*啪(»·亵Age 筅μ “V Age A St St. Acoustic Eh Order (»为锫抹* ύ A 埤忤琯琴•择 s 漭 疼 ύ If If If If If If If If If If If If If If If If If If If If If If If If If If If If If If If If * * * * * S sleeve skirt 翱fi Ρ 瑢 猱 猱 猱 啭 啭 啭 啭 嵙 P P P P P P 择 择 ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( ( 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 1 Window | I? fi X cg>>E II Toshi ip Η i ! 1 1 * « 1 « *5 FS ir〇-ms〇R c Pen: ί Ξ > κ < Gossip 1 A! 3) Η 1< •-· 〇> C0 | ! Ϊ imhf :3⁄4 DSM 14040 S δ Min δ •mJi δ CO SS δ 2 δ 2 is •U § 2: 1 SSXS Ό C0 S δ g 03 % S II·p -30- 1338044 Sequence Listing <110, Avantis Pharmaceuticals Deutsche GmbH <12〇> Saccharomyces cerevisiae strain containing functional expression of GLUT transporter <13〇> AVE D-2001/ A002 <140><141><150> 10106718.6 <151> 2001-02-14

<160> 18

<170> Patentln Ver. 2.1 <210> 1 <211> 25 <212> DNA wise man <400>

Ctagagctcg taggaacaat ttcgg <210> 2 <211> 60 <212> DNA <213> wise man. <400> 2 cgactagtgt gatggtgatg gtgatgcatg ttaacttttt gattaaaatt aaaaaaactt 60 <210> 3 <211> 3S <212&gt DNA <213>Wisdom Man<400? 3 ttaattttaa tcaaaaaatg gagcccagca gcaag 35 <210> 4 <211> 52 1338044 <212> DMA <213> Wisdom Man <400> 4 acatgactcg aggtcgacgg tatcgataag cttatcacac ttgsrgaatca Gc

<210> 5 <211? 35 <212> DMA Wisdom

<400> S ttaattctaa tcaaaaaatg ccgtcgggct tccaa

<210> 6 <211> 52 <212> DNA c 213 > wise man

<400> C 52 acatgactcg aggtcgacgg tatcgataag cttatcagtc gttctcatcc gg

<210> 7 <211> 73 <212> DNA <400> 7 caaagaataa acacsaaaac aaaaagtttt tttaatttta atcaaaaaat gtctgaattc 60 agcagcaaga agg 73

<210> 8 <211> 71 <212> DNA brown rat <4〇o> e aagtttcttt gtctccgtcc cactcaactt tetgagaaca aatgatcgac aaataatagg 60 tctaggtaag a 71

<210^ 9 <211> 782Θ <212> DNA

(D -2 - 1338044 < 213 > wise < 400 > 9 atgccgtcgg gcttccaaca gataggctcc gaagatgggg aaccccctca gcagcgagtg 60 actgggaccc tggtccttgc tgtgccctct gcggtgcttg gctccctgca gtttgggtac 120 aacattgggg tcatcaatgc cccccagaag gtgattgaac agagctacaa tgagacgtgg 1Θ0 ctgg9ga99c aggggcctga gggacccagc tccatccctc caggcaccct caccaccctc 240 tgggccctct ccgtggccat cttttccgtg ggcggcatga tttcctcctt cctcattggt 300 atcatctctc agtggcttgg aaggaaaagg gccatgctgg tcaacaatgt cctggcggtg 360 ctggggggca gcctcatggg cccggccaac gctgctgcct cctatgaaat gctcaccctt 42o ggacgattcc tcatcggcgc ctactcaggg ctgacatcag ggctggtgcc catgtacgtg 4Θ0 ggggagattg ctcccactca cctgcggggc gccctgggga cgctcaacca actggccatc S40 gttatcggca ttctgatcgc ccaggtgctg ggcttggagt ccctcctggg cactgccagc 600 ctgtggccac tgctcctggg cctcacagtg ctacctgccc tcctgcagct ggtcctgctg 660 cccttctgtc ccgagagccc ccgctacctc tacatcatcc agaatctcga ggggcctgcc 720

^gaaagagtc tgaagcgcct gacaggctgg gccgatgttt ctggagtgct ggctgagctg 780 aaggatgaga agcggaagct ggagcgtgag cggccactgt ccctgcccca gctcctgggc 840 agccgtaccc accggcagcc cctgatcatt gcggtcgtgc tgcagctgag ccagcagctc 900

tctggcatca atgctgtttt ctattattcg accagcacct tcgagacagc aggggtaggc SSO cagcctgcct atgccaccat aggagctggt gtggtcaaca cagtcttcac cttggtctcg 1020 gtgttgttgg tggagcgggc ggggcgccgg acgctccatc tcctgggcct ggcgggcatg 1060 tgtggctgtg ccatcctgat gactgtggct ccgctcctgc tggagcgagt tccagccatg 1140 agctacgtct ccattgtggc catctttggc ttcgtggcat tttttgagat tggccctggc 1200 cccattcctt ggttcatcgt ggccgagctc ttcagccagg gaccccgccc ggcagccatg 1260 gctgtggctg gtttctccaa ctggacgagc aacttcatca ttggcatggg tttccagtat 1320 gttgcggagg ctatggggcc ctacgtcttc ctcctatttg cggtcctcct gctgggcttc 12Q0 ttcatcttca cctccttaag aotacctgaa actcgaggcc ggacgtttga ccagatctca 1440 gctgccttcc accggacacc ctctctttta gagcaggagg tgaaacccag cacagaactt 1500 gagtacctag ggccagatga gaacgactga taagcttatc gataccgtcg acctcgagtc 1560 atgtaattag ttatgtcacg cttacattca cgccctcccc ccacatccgc tctaaccgaa 1620 aaggaaggag ttagacaacc tgaagtctag gtccctattt atttttttat agttatgtta 1680 gtattaagaa cgttatttat acttcaaatt tttccttttt ttctgtacag acgcgtgcac 1740 gcatgta aca ctatactgaa aaccttgctt gagaaggttt gaaggcttta 1800 atttgcggcc ggtacccaat tcgccctata gtgagtcgta ttacgcgcgc tcactggccg I860 tcgttttaca acgtcgtgac tgggaaaacc ctggcgttac ccaacttaat cgccttgcag 1920 cacatccccc tttcgccagc tggcgtaata gcgaagaggc ccgcaccgat cgcccttccc 1980 aacagrtgcg cagcctgaat ggcgaatggc gcgacgcgcc ctgtagcggc gcattaagcg 2040 cggcgggtgt ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ctagcgcccg 2100 ctcctttcgc tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagccc 2160 taaatcgggg tgggacgctc gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa 2220 aacttgatta gggtgatggt tcacgtagtg ggccatcgcc ctgatagacg gtttttcgcc 2280 ctccgacgtt ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac 2340 tcaaccctat ctcggcctat tcttttgatt tataagggat tctgccgatt tcggcctatt 2400 ggttaaaaaa tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt 24 ^ 0 ttacaatttc ctgatgcggt attttctcct tacgcatctg tgcggtattt cacaccgcat 2520 agggtaataa ctgatataat taaattgaag ctctaatttg tgagtttagt atacatgcat 2580 Ttacttataa ta Cagttttt tagttttgct ggccgcatct tctcaaatat gcttcccagc 2640 ctgcttttct gtaacgttca ccctctacct tagcacccct tcccttcgca aatagtcctc 2700 1338044

ttccaacaat aataatgtca gatcctgtag agaccacatc atccacggtt ctatactgtt 2760 gacccaacgc gtctccctcg tcatctaaac ccacaccggg tgtcataatc aaccaatcgt 2B20 aaccttcatc tcttccaccc atgtctctrc gagcaataaa gc ^ gataaca aaatctcrgt 2880 cgctctccgc aatgtcaaca gtacccccag tatattcccc agtagatagg gagcccttgc 2940 atgacaattc tgctaacatc aaaaggcctc taggttcctt tgttactcct tctgccgcct 3000 gcttcaaacc gctaacaata cctgggccca ccacaccgtg tgcattcgca atgtctgccc 3060 attctgctat tctgtataca cccgcagagr accgcaatcc gactgtatta ccaatgccag 3120 caaattttct gtcttcgaag agtaaaaaat tgtacttggc ggataatgcc tttagcggct 3180 taactgtgcc ctccatggaa aaatcagtca agatatccac atgtgttttir agtaaacaaa 3.240 ttttgggacc taatgcttca actaactcca gtaattcctt ggtggtacga acatccaatg 3300 aagcacacaa gtttgtttgc tcttcgtgca cgatattaaa tagcttggca gcaacaggac 3360 taggatgagt agcagcacgt tccttatatg tagctttcga catgatttac cttcgttccc 3420 tgcaggcttt tgttctgtgc agttgggtta agaatactgg gcaattccat gtttccccaa 34B0 cactacatat gcgtatatat accaatctaa gtctgtgctc cttccttcgt tcttccttct 3540 Gttc ggagat taccgaatca aaaaaatttc aaagaaaccg aaatcaaaaa aaagaataaa 3600 aaaaaaatga tgaattgaat tgaaaagctg tggtatggtg cactctcagt acaatctgct 3660 ctgatgccgc atagttaagc cagccccgac acccgccaac acccgctgac gcgccctgac 3720 gggcttgtct gctcccggca tccgctcaca gacaagctgt gaccgtctcc gggagctgca 3780 tgtgtcagag gttttceccg tcatcaccga aacgcgcgag acgaaagggc ctcgtgatac 3840 gcctattttt ataggtcaat gtcatgataa taatggtttc ttagtatgat ccaatatcaa 3900 aggaaatgat agcattgaag gatgagacta atccaattga ggagtggcag cat & tagaac 3960 agctaaaggg tagtgctgaa ggaagcacac gataccccgc atggaatggg ataatatcac 4020 aggaggtact agactacctt tcatcctaca taaatagacg catataagta cgcatttaag 4080 cataaacacg cactatgccg ttcttctcat gtatatatac atacaggcaa cacgcagata 4140 taggtgcgac gtgaacagtg agctgtatgt gcgcagctcg cgttgcattt tcggaagcgc 4200 tcgttttcgg aaacgctttg aagttcctat cccgaagttc ctattctcta gaaagtatag 4260 gaacttcaga gcgcttttge aaaccaaaag cgctctgaag acgcactttc aaaaaaccaa 4320 aaacgcaccg gaccgtaacg agctactaaa atattgcgaa taccgcttcc acaaacattg 43 €0 ctca aaagta tctctttgct anatatctct gtgctatatc cctatataac ctacccatcc 4440 acctttcgct ccttgaactt gcatccaaac tcgacctcta caccttttat gtttatctct 4500 agtattaccc tttagacaaa aaaattgtag taagaactat tcatagagtg aatcgaaaac 45 $ 〇aatacgaaaa tgtaaacatt tcctat & cgc agtatataga gacaaaatag aagaaaccgt 4620 tcataatttt ctgaccaatg aagaatcatc aacgctatca ctttctgttc acaaagtatg 4660 cgcaatccac atcggtatag aatataatcg gggatgcctt taccttgaaa aaatgcaccc 4740 gcagcttcgc tagtaatcag caaacgcggg aagtggagtc aggctttttt tatggaagag 4Θ00 aaaatagaca ccaaagtagc cctcctctaa ccttaacgga cc ^ acagtgc aaaaagctat 4 $ 6〇caagagactg cattatagag cgcacaaagg agaaaaaaag taatctaaga tgctttgtta 4920 gaaaaatagc gctctcggga tgcacttttg tagaacaaaa aagaagcata g & ttctttgt 4360 tggtaaaata gcgctctcgc gccgcatttc tgttctgtaa aaatgcagct cagattcttt 5040 gtttgaaaaa ctagcgctct cgcgttgcat ttttgtttta caaaaacgaa gcacagatcc 51〇〇tccgttggta aaatagcgct ctcgcgttgc atttctgttc Tgtaaaaatg cagctcagat 5160 tccttgtttg aaaaattagc gctctcgcgt tgcatttttg ttccacaaaa tgaagcaca g 5220 acgcttcgtt caggtggcac ttttcgggga aatgtgcgcg gaacccctat ctgtttattt 52Θ0 ttctaaatac attcaaatat gtatccgctc atgagacaat aaccctgata aatgcttcaa S340 taatattgaa aaaggaagag tatgagtatt caacatttcc gtgtcgccct tatccccttt 5400 ttcgcggcat tttgccttcc tgtttttgcc cacccagaaa cgctggtgaa agtaaaagat 5460 gctgaagatc agttgggtgc acgagtgggt tacatcgaac tggatctcaa cagcggtaag 5520 atccttgaga gttrtcgccc cgaagaacgt tttccaatga tgagcacttt taaagttctg 55B0 -4- 1338044 ctatgtggcg cggtattatc ccgtattgac gccgggcaag agcaactcgg tcgccgcata 5640 cactattctc agaatgactt ggttgagtac tcaccagtca cagaaaagca ccntacggat S700 ggcatgacag taagagaatt atgcagtgct gccataacca tgagtgataa cactgcggcc 5760 aacttactcc tgacaacgat cggaggaccg aaggagctaa ccgctttttt gcacaacatg 5Θ20 ggggatcatg taactcgcct tgatcgttgg gaaccggagc tgaatgaagc cataccaaac 58ΘΟ gacgagcgtg acaccacgat gcctgtagca atggcaacaa cgttgcgcaa actattaact 5940 ggcgaactac tcaccccagc tccccggcaa caattaatag actggatgga ggcggataaa 6〇〇〇 Gccgcaggac cacttccgcg ctcggccctt ccggctggct ggtt tattgc tgataaatct 6060 ggagccggtg agcgtgggtc tcgcggtatc attgcagcac tgsggccaga tggtaagccc 6120 tcccgtatcg tagttatcta cacgacgggg agtcaggcaa ctatggatga acgaaataga 61Θ0 cagatcgctg agataggtgc ctcactgatt aagcattggt aactgtcaga ccadgtttac tcatatatac cttagattoa tttaaaactt catttttaat ttaaaaggat ctaggt ^ aag 6300 atcctttttg ataatctcat gaccaaaatc cctcaacgtg agttttcgtt ccactgagcg 6360 tcagaccccg tagaaaagat caaaggatct tcttgagatc ctccttctct gcgcgtaatc £ 420 tgctgcttgc aaacaaaaaa accaccgcta Ccaggggtgg tttgcttgcc ggatcaagag £480

ctaccaactc tttttccgaa ggtaactggc ttcagcagag cgcagatacc aaatactgtc 6540 cttctagtgt agccgtagtt aggccaccac ttcaagaacc ctgtagcacc gcctacatac 6600 ctcgctctgc taatcctgtt 6660 accagtggcc gctgccagtg gcgataagtc gtgtcttacc gagcttccag ggggaaacgc ctggtacctt gggtcggact caagacgata gttaccggat aaggcgcagc ggtrcgggctg aacggggggt 6720 tcgtgcacac agcccagctt ggagcgaacg acctacaccg aactgagata cctacagcgt 67Θ0 gagctatgag aaagcgccac gcttcccgaa gggagaaagg cggacaggta tccggtaagc 6840 ggcagggtcg gaacaggaga gcgcacgagg 6900 tatagtcctg tcgggtttca ccacctctga cttgagcgtc gaccctcgtg atgctcgtca ¢ 960 ggggggcgga gcctatggaa aaacgccagc aacgcggcct ttttacggtt cctggccttt 7020 tgctggcctt ttgctcacat gttctttcct gcgttatccc ctgattctgt ggataaccgt 7030 attaccgcct ttgagtgagc tgataccgct cgccgcagcc gaacgaccga gcgcagcgag 7140 tcagtgagcg aggaagcgga taggcacccc aggctttaca ctttatgctt agagcgccca atacgcaaac cgcctctccc cgcgcgttgg 7200 ccgattcatt aatgcagctg gcacgacagg tttcccgact ggaaagcggg cagtgagcgc 7260 aacgcaatta atgtgagtta cctcactcat 7320 ccg gctccta tgttgtgtgg aattgtgagc ggataacaak ttcacacagg aaacagctat 7380 gaccatgatt acgccaagcg cgcaattaac cctcactaaa gggaacaaaa gctggagctc 7440 gtaggaacaa tttcgggccc ctgcgtgttc ttctgaggtt catctttcac atctgcttct 7500 gctggataat tttcagaggc aacaaggaaa aattagatgg caaaaagtcg tctttcaagg 7560 aaaaatcccc accatctttc gagatcccct gtaacttatt ggcaactgaa agaatgaaaa 7620 ggaggaaaat acaaaatata ctagaactga aaaaaaaaaa gbataaatag agacgatata 7ββ0 tgccaatact tcacaatgtt cgaatctatt cttcatttgc agctattgta aaataataaa 7740 acatcaagaa caaacaagct caacttgcct ttcctaagaa caaagaacaa acacaaaaac 7800 aaaaagttct tttaatttca atcaaaaa 7826 < 210 > 10 < 211 > 2366 < 212 > DNA < 213 > Rattus norvegicus < 400 > 10 tcgactctag aggatcccct taagctaatc cttatgaatc cggagaaaag cggggtcttt 60 taactcaata aaattttccg aaatcctttt tcctacgcgt tttcttcggg aactagatag 120 5 -5 - 1338044 gtggctcttc cacccgtttt tecatcattt cagtttttcg caagccatgc gtgccttttc 180 gttttngcga tggcgaacga gggctggaaa aatfcaacggt acgccgccta acgatagtaa 240 taggccacgc aactggcg tggctataac attggagtca € 00 tcaacgcccc acagaaagtg attgaacaga gctacaatgc aacttggctg tcccggattc acaaatgata taaaaagcga 360 ttacaattct acattctaac cagatttgag atttcctctt tctcaattcc tcttatatta 420 gattataaga acaacaaact aaattacaaa aagacttata aagcaacata atgtctgaat 460 tccagcagat cggctctgaa gatggggaac ccccccagca gcgagtgact gggacactgg 540 cccttgctgt attctcagcc gtgcttggct cccttcagtt: cc cccaccaaac ttgtcatcgt; tg gacgacaaca ataagtcgcc cattttttat gtttccaaaa 300 cctagca & amp ggtaggcagg S € 0 gtcctggggg accggactcc accccacaag gcaccctcac taccctttgg gctctctccg 720 tggccatctt ctccgtgggt ggcatgattt cctcctttct cattggcatc atttctcaat 7fi〇ggttgggaag gaaaagggct atgctggcca acaatgtctt ggctgtgctg gggggcgccc 840 tcatgggcct agccaatgcc gcggcctcct atgagacact cattctcgga cggtccctca 900 ttggcgccta ctcagggcta acatcagggt tggtgcctat gtatgtggga gaaatcgccc 960 ccactcatct tcggggtgcc ttgggaacac tcaaccaatt ggccatcgtc atcggcattc 1020

tggttgccca ggtgttgggt ttggagtcta tgctgggcac agctaccctg tggccattgc 1060 ttctggccat cacagtactc cctgctctcc tgcagctgct tctgttgccc ctctgtcctg 1140 agagcccccg acacctctac atcatccgga acctggaggg gcctgcccga aagagtctaa 1200 agcgcctgac aggctgggct gatgtgtctg atgcactggc tgagctgaag gatgagaaac 1260 ggaagttgga aagagagcgt ccactgtcct tgctgcagct cctgggcagc cgcacccacc 1320 ggcagcctct gatcattgca gtggrgctgc agccgagcca gcagctctca ggcatcaatg 13Θ0 ctgtcttcta ctattcaacc agcatctttg agttagctgg ggtggaacag ccagcctacg 1440 ccaccatagg agccggtgtg gtcaataccg tcttcacgtt ggtctcggtg ctcttagtag 1500 agcgagctgg gcgacggaca ctccatctcc tgggcctggc aggcatgtgt ggctgtgcca 1560 tcttgatgac ggtggctctg ctgctgctgg agcgggttcc atccatgagt tatgtgtcca 1620 tcgtggccat atttggctct gtggccttct ttgagattgg tcctggcccc atcccctggt 1680 ccattgtggc cgagctcttc agccagggcc cccgcccagc agccatggct gtagctggtt 1740 tctccaactg gacctgtaac ttcatcgttg gcatgggttt ccagtatgtt gcggatgcta 1800 tgggtcccta cgtcttcctt ctatttgccg tcctcctgct tggcctcctc atcttcacct 1Θ60 tcct aagagt gcctgaaacc agaggccgga catttgacca gatctcggcc acctcccgac 1920 ggacaccccc tctctcagag caggaggtga aacccagtac agaacttgaa tacccagggc 1980 cagacgagaa tgaccaatcg attcgaagtg agacgctcca tcatctctct taatttttca 2040 tgactgacgt tttttcttca ttttaattat catagtattt gtttgaaaaa aaaaaaaaaa 2100 aatttccctt atcaatgata tccttacgat catataaatt ccttacctaa acctattatr 2160 tgtgtacata tatcagagta ttattacata tataaccttt ttctctaa a caggaaaaaa 2220 aaaagaaadc gataacatgc tctgccatcc tttgttcacc gagcaaaatt aaaaacgcaa in 2280 aatgaattgt ccctatgaaa tcattaaagg accacatcac cagacttatc cctggggggt 2340 cctctagaaa ataagtcagg tacttgcctg gactttcttc cagtng 2366 < 210 > 11 < 211 > 7777 < 212 > DNA < 213 > wise < 400 > 11 acggagccca gcagcaagaa gctgacgggt cgcctcatgc tggctgtggg aggagcagtg 60 cttggctccc tgcagcttgg ctacaacact Ggagtcatca atgcccccca gaaggtgatc 120 -6 - 1338044 gaggagttct acaaccagac atgggtccac cgctatgggg agagcatcct gcccaccacg 180 ctcaccacgc cctggtccct ctcagtggcc atcttttctg ttgggggcat gattggctcc 240 tt ctctgtgg gccttttcgt taaccgcttt ggccggcgga attcaatgct gatgatgaac 300 ctgctggcct tcgtgtccgc cgtgctcatg ggcttctcga aactgggcaa gtcctttgag 3 € 〇atgctgatcc tgggccgctt catcatcggt gtgtactgcg gcctgaccac aggcttcgtg 420 cccatgtatg tgggtgaagt gtcacccaca gcctttcgtg gggccctggg caccctgcac 4Θ0 cagctgggca tcgtcgtcgg catcctcatc gcccaggtgc tcggcctgga ctccatcatg 540 ggcaacaagg acctgtggcc cctgctgctg agcatcatct tcatcccggc cctgctgcag 600 tgcatcgtgc cgcccttctg ccccgagagt ccccgcttcc tgctcatcaa ccgcaacgag 660 gagaaccggg ccaagagtgt gctaaagaag ctgcgcggga cagctgacgt gacccacgac 720 ctgcaggaga tgaaggaaga gagtcggcag atgatgcggg agaagaaggt caccatcctg 780 gagctgttcc gctcccccgc ctaccgccag cccatcctca tcgctgtggt gctgcagctg 640 tcccagcagc tgtctggcat caacgctgtc ttctattact ccacgagcat cttcgagaag 900 gcgggggtgc agcagcctgt gtatgccacc attggctccg gtatcgtcaa cacggccttc 960 actgtcgtgt cgctgtttgt ggtggagcga gcaggccggc ggaccctgca cctcataggc 1020

cccgctggca cggcgggttg cgccatactc atgaccatcg cgctagcact gctggagcag 1060 ctaccctgga tgtcctatct gagcatcgtg gccatctttg gctctgtggc cttctttgaa 1140 gtgggtcctg gccccatccc atggttcatc gtggctgaac tcttcagcca gggtccacgt 1200 ccagctgcca ttgccgttgc aggcttctcc aactggaccc caaatttcat tgtgggcatg 1260 tgcttccagt atgtggagca actgtgtggt ccctacgtct tcatcatcct cactgtgctc 1320 ctggttctgt tcttcacctt cacctacntc aaagttcccg agactaaagg ccggaccttc 1360 gatgagaccg cttccggctc ccggcagggg ggagccagcc aaagtgacaa gacacccgag 1440 gagctgttcc atcccctggg ggctgattcc caagtgtgat aagcttatcg ataccgtcga 1500 cctcgagtca tgtaattagt tatgtcacgc ttacattcac gccctccccc cacatccgct 1560 ctaaccgaaa aggaaggagt tiagacaacct gaagtctagg tccctattta ttcttttata 1620 gttatgttag tattaagaac gttatttata tttcaaattt ttcttttttt tctgtacaga 1600 cgcgtgtacg catgtaacat tacactgaaa accttgcttg agaaggtttt gggacgctcg 1740 aaggctttaa tctgcagccg gcacccaatt cgccctatag tgagtcgtat tacgcgcgct ΙβΟΟ cactggccgt cgttttacaa cgtcgtgact gggaaaaccc cggcgttacc caacttaatc I860 g ccttgcagc acatccccct ttcgccagct ggcgtaatag cgaagaggcc cgcaccgatc 1920 gcccttccca acagttgcgc agcctgaatg gcgaatggcg cgacgcgccc ngtagcggcg 1980 cattaagcgc ggcgggtgtg gtggttacgc gcagcgtgac cgctacactt gccagcgccc 2040 tagcgcccgc ccctttcgct tccrtccctt cccttctcgc cacgttcgcc ggctctcccc 2100 gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg 2160 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc tgatagacgg 2220 tttttcgccc tttgacgttg gagtccacgt tctttaatag tggactcttg ttccaaactg 22Θ0 gaacaacacc caaccctatc tcggtctatt cttttgattt ataagggatt ttgccgattt 2340 cggcctattg gttaaaaaat gagctgattt aacaaaaatt taacgcgaat ttcaacaaaa 2400 tactaacgtt tacaatttcc tgatgcggta ttttctcctt acgcatctgt: gcggtatttc 2460 acaccgcata gggtaataac tgatataatt aaattgaagc tctaatttgn gagtttagta 2520 tacatgcatt tacttataat acagtttctt agttttgctg gccgcatctt ctcaaatatg 25Θ0 cttcccagcc tgcttttctg taacgttcac cctctacctt agcatccctt ccctttgcaa 2640 atagtcctct tccaacaaca ataatgtcag atcctgtaga gaccacatca tccacggttc 2700 tata ctgttg acccaatgcg tctcccttgt catctaaacc cacaccgggt gtcataatca 27 ^ 0 accaatcgta accttcatct cttccaccca tgtctctttg agcaataaag ccgataacaa 2Θ20 aatccttgtc gctcttcgca atgtcaacag tacccttagt atattctcca gtagataggg 2Θ60 agcccttgca tgacaattct gctaacatca aaaggcctct aggttcctct gttacttctt 2940 ctgccgcctg cttcaaaccg ctaacaatac ctgggcccac cacaccgtgt gcattcgtaa 3000 ⑤ -7 - 1338044

tgtctgccca ttctgctatt ctgtatacac ccgcagagta ctgcaatttg actgtattac 3060 caatgtcagc aaattttctg tcttcgaaga gtaaaaaatt gtacttggcg gataatgcct 3120 ttagcggctt aactgcgccc tccatggaaa aatcagtcaa gatatccaca tgtgtttcta gtaaacaa & amp 31Θ0; gcaggttttt gttctgtgca gttgggttaa t tttgggacct aatgcttcaa ctaactccag taattccttg gtggtacgaa 3240 catccaatga agcacacaag ttcgtttgct tttcgtgcat gacatcaaat agcttggcag 3300 caacaggact aggatgagta gcagcacgtt ccttatatgt agctttcgac atgatttarc 3360 tccgcttcct gaatactggg caacttcacg 3420 tttcttcaac actacatatg cgcatatata ccaatctaag tctgtgctcc ttccttcgtt 3480 cttccttctg ttcggagatt accgaatcaa aaaaatttca aagaaaccga aaccaaaaaa 3540 dagaataaaa aaaaaatcfat gaattgaatt gaaaagctgt ggtatggtgc actctcagta 3600 caatctgccc tgatgccgca tagttaagcc agccccgaca cccgccaaca cccgctgacg 3660 cgccctgacg ggcttgtctg ctcccggcat ccgcttacag acaagccgtg accgtctccg 372〇ggagctgcat gtgtcagagg ttttcaccgt catcaccgaa acgcgcgaga cgaaagggcc 3 * 780 tcgtgatacg cctaccctta taggttaatg tcatgataat Aacggctcct tagtatgatc 38 40 caatatcaaa ggaaatgata gcattgaagg atgagactaa tccaatcgag gagtggcagc 3900 atatagaaca gctaaagggt agtgccgaag gaagcatacg at ^ ccccgca tggaatggga 39β〇taatatcaca ggaggtacta gactaccttt catcctacat aaatagacgc atatdagtac 4020 gcatttaagc ataaacacgc actatgccgt tcttctcatg taratatata tacaggcaac 4060 acgcagatat aggtgcgacg tgaacagtga gctgtatgtg cgcagctcgc gttgcatttt 4140 cggaagcgct cgttctcgga aacgctttga agttcctatt ccgaagttcc tattctctag 4200 aaagtatagg aacttcagag cgcttttgaa aaccaaaagc gctctgaaga cgcactttca 4260 aaaaacca ^ a ^ acgcaccgg actgtaacga gctactaaaa tattgcgaat accgctccca 4320 caaacattgc tcaaaagtat ctctttgcta tatatctctg tgctatatcc ctatataacc 4380 tacccatcca cctttcgctc cttgaacttg catctaaact cgacctctac attttttatg 4440 tttatctcta gtattacccc ttagacaaaa aaattgtagt aagaactatt catagagtga 4500 atcgaaaaca atacgaaaat gcaaacattt cctatacgta gtatatagag acaaaataga 4560 agaaaccgtt cataattttc tgaccaatga agaatcatca acgctatcac tttctgttca 4620 caaagtatgc gcaatccaca Tcggtataga atacaatcgg ggatgccttt atcttgaaaa 4680 aatgcacccg cagcttcgct agtaatcagt aaacgcggga agtggagtca ggcttttttt 4740 atggaagaga aaatagacac caaagtagcc ttcttctaac cttaacggac ctacagtgca 4800 aaaagctatc aagagactgc attatagagc gcacaaagga gaaaaaaagt aacctaagac 4860 gctttgttag aaaaatagcg ctctcgggat gcatttttgc. agaacaaaaa agaagtatag 4920 attctttgtt ggtaaaatag cgctctcgcg ttgcatttct gtcctgtaaa aatgcagctc 4980 agattctttg tttgaaaaat tagcgctctc gcgttgcatt tctgttttac aaaaatgaag 5040 cacagactct tcgttggtaa aatagcgctt tcgcgttgca tttctgttct gtaaaaatgc 5100 agctcagatt ctttgtttga aaaatcagcg ctctcgcgtt gcatttttgc tctacaaaat 5160 gaagcacaga tgcttcgttc aggtggcact tctcggggaa atgtgcgcgg aacccctatt 5220 tgtttatttt tctaaataca ttcaaatatg tatccgctca tgagacaata accctgataa 5280 atgcttcaat aatatrgaaa aaggaagagt atgagtattc aacatttccg tgtcgccctt 5340 attccctttt ctgcggcatt ttgccttcct gttcttgctc acccagaaac gctggtgaaa 5400 gtaaaagatg ctgaagatca gttgggtgca cgagtgggtt acatcgaact ggatctcaac 5460 agcggtaaga tccttgagag ttttcgcccc gaagaacgtt ttccaatgat gagcactttt 5520 aaagc tctgc tatgtggcgc ggtactatcc cgtattgacg ccgggcaaga gcaactcggt 5580 cgccgcatac actatcctca gaatgacttg gccgagcact caccagtcac agaaaagcac 5640 cttacggatg gcatgacagt aagagaatta tgcagtgctg ccataaccat gagtgataac S700 actgcggcca acttacttct gacaacgatc ggaggaccga aggagetaac cgcttttttg 5760 cacaacatgg gggatcatgt aacccgcctc gatcgttggg aaccggagct gaatgaagcc 5820 ataccaaacg acgagcgtga caccacgatg cctgtagcaa tggca & caac gttgcgcaaa 5880 1338044

ctattaactg gcggataaag gataaatctg ggtaagccct cgaaatagac caagtttact taggtgaaga cactgagcgt cgcgtaatct gaccaagagc aatactgtcc cctacatacc tgtcttaccg acggggggtt ctacagcgtg ccggtaagcg tggtatcttt tgctcgtcag ctgocctttt gataaccgta cgcagcgagt gcgcgttggc agtgagcgca tttatgcttc aacagctatg ctggagctcg tttgcttctg ctttcaagga gaatgaaaag gacgatatac aataataaaa cacaaaaaca gcgaactact ttgcaggacc gagccggtga cccgtatcgt agatcgctga catatatact tcctttttga cagaccccgt gctgcttgca taccaactct ttctagtgta tcgctctgct ggttggaccc cgtgcacaca agctatgaga gcagggtcgg atagtcctgt gggggcggag gctggccttt ttaccgcctt. cagcgagcga cgatccatta acgcaattaa cggctcctac accacgatts taggaacaat ctggataatfc aaaatcccca gaggaaaata gccaacactt catcaagaac aaaagttttt cactctagct acttctgcgc gcgtgggtct agttatctac gataggtgcc ttagattgat taatctcatg agaaaagatc aacaaaaaaa ttttccgaag gccgtagtta aatcctgtta aagacgatag gcccagcttg aagcgccacg aacaggagag cgggtttcgc cctatggaaa cgctcacatg cgagtgagct ggaagcggaa atgcagctgg tgtgagttac gttgcgtgga cgccaagcgc ttcgggcccc ttcagagg ca ccatctctcg caaaatatac cacaatgttc aaacaagctc ttaattctaa tcccggcaac tcggcccttc cgcggtatca acgacgggga tcactgatta ttaaaacttc acca & aaccc aaaggatctt ccaccgctac gtaactggct ggccaccact ccagtggctg ttaccggata gagcgaacga cctcccgaag cgcacgaggg cacctctgac aacgccagca ttctttcctg gataccgctc gagcgcccaa cacgacaggt ctcactcatt attgtgagcg gcaattaacc tgcgtgttct acaaggaaaa agatcccctg tagaactgaa gaatccactc aactcgtctt tcaaaaa aattaataga cggctggctg ttgcagcact gtcaggcaac agcattggta atttttaatc cttaacgtga cttgagatcc cagcggcggt tcagcagagc tcaagaactc ctgccagtgg aggcgcagcg cctacaccga ggagaaaggc agcrcccagg ttgagcgtcg acgcggcctt cgttaccccc gccgcagccg tacgcaaacc ttcccgactg aggcacccca gataacaatt ctcactaaag cctgaggttc attagatggc taacttattg aaaaaaaaag ttcatttgca ttct & agaac ctggatggag gtttatbgct ggggccagat tatggatgaa actigtcagac taaaagg & tc gttttcgttc cttttttctg ttgtttgccg gcagatacca tgtagcaccg cgataagtcg gtcgggctga actgagatac ggacaggtat gggaaacgcc atttttgtga tccacggttc tgattctgtg aacgaccgag gcctctcccc gaaagcgg Gc ggctttacac tcacacagga ggaacaeaag atcctttaca aaaaagtcgt gcaactigaaa tataaataga gctatcgtaa aaagaataaa 5940 ^000 6060 6120 6160 6240 ^300 63€0 6420 64B0 6540 6600 6660 ¢720 67Θ0 6840 6900 6960 7020 70β0 7140 7200 7260 7320 7380 7440 7S00 7560 7620 7S80 7740 7777

≪ 210 > 12 < 211i > 2338 < 212 > DNA C13 > Rattus norvegicus < 40〇 > 12 tcgactctag aggatcccct taagctaatc ctcatgaatc cggagaaaag cggggtcttc 60 taactcaata aaattttccg aaatcctttt ccctacgcgt tttcttcggg aactagatag 120 gtggctcttc cacctgtttt tccatcattt tagttttccg caagccatgc gtgccttttc 18〇gtttttgcga tggcgaacga gggctggaaa aattaacggt acgccgccta acgatagtaa 240 taggccacgc aactggcgtg gacgacaaca ataagtcgcc cattttttat gttttcaaaa 300 cctagcaacc cccaccaaac ttgccatcgt tcccggattc acaaaCgata taaaaagcga 360 ttacaatcct acattctaac cagatttgag atttcctctt tctcaattcc tcttatatta 420 gattataaga acaacaaatt aaattacaaa aagacttata aagcaacata atgtctgaat 460 -9 - 1338044 tcagcaagaa ggtgacgggc cgccttatgt tggccgtggg aggggcagtg ctcggatccc 540 tgcagttcgg ctataacacc ggtgtcatca acgcccccca gaaggtaatc gaggagttct 6〇〇acaatcaaac atggaaccac cgctatggag agcccatccc acccaccaca ctcaccacac 660 tctggtctct ctccgtggcc atcttctctg tcgggggcat gattggttcc ttctctgtgg 720 gcctctttgt taatcgcttt ggcaggcgga accccatgct gatgatgaac ctgttgg cct 780 ttgtgtctgc cgtgcttatg g9tt: tctcca aactgggcaa gtcctttgag atgctgatcc S40 tgggccgctt caecattgga gtgtactgtg gcctgaccac cggctttgcg cccetgtatg 9〇o tgggggaggt: gtcacccaca gctcttcgtg gagccctggg caccccgcac cagctgggca 960 tcgtcgttgg gatccttatt gcccaggtgt tcggcttaga ctccatcatg ggcaatgcag 1020 acttgtggcc tctactgctc agtgtcatct tcatcccagc cctgctacag tgtatcctgt 1060 tgcccttctg ccctgagagc ccccgcttcc tgctcatcaa ccgtaacgag gagaaccggg 1140 ccaagagtgt Gctgaaaaag ctccgaggga cagccgatgt gacccgagac ctgcaggaga 1200 tgaaagaaga gggccggcag atgacgcggg agaagaaggt caccatcttg gagctgttcc 1260 gctcacccgc ctaccgccag cccatcctca tcgccgtggt gctgcagctg ccccagcagc 1320 tgtcgggcat caatgccgtg ttctactact caacgagcat cttcgagaag gcaggtgtgc 1380

agcagcctgt gtatgccacc atcggctcgg gcatcgtcaa cacggccttc actgtggtgt 1440 cgctgttcgt cgtggagcga gctggccgtc ggaccctgca tctcattggt ctggctggca 1500 tggcgggctg tgctgtgctc atgaccatcg ccctggccct gctggagcag ctgccccgga 1560 tgtcctatct gagtatcgtg gccatctctg gctttgtggc cttcttcgaa gtaggccctg 1620 gtcctattcc acggttcatt gtggccgagc tgttcagcca ggggccccga cctgctgctg 1 € 80 ttgctgtggc cggcttctct aactggacct caaacttcat cgcgggcatg tgcttccaat 1740 atgtggagca accgtgtggc ccctacgtct tcatcatctt cacggtgctg ccggtactct 1800 tcttcatctt cacctacttc aaagttcctg agaccaaagg ccggaccttc gatgagatcg Ιβ € 0 cttccggctt ccggcagggg ggcgccagcc agagcgacaa gacacctgag gagctcttcc 1920 accccctggg ggccgactcc caagtgtaat cgatttgaag tgagacgctc catcatctct 1980 cttaattttt catgactgac gttttttctt cattttaatt atcatagtat ttgtttgaaa 2040 aaaaaaaaaa aaaatttccc ttatcaacga tatccttacg attatataaa ttccttacct 2100 aaacctatta tttgtgtaca tatatcagag tattattaca tatataacct ttttctctaa 2160 aacaggaaaa aaaaaagaaa acgataacat gctctgccat cctttgttca Ccgagcaaaa 2220 Ttaaaaacgc aaaatgaatt gtccctatga aattattaaa ggaccacatc accagactta 2280 tctccggggg gtcctctaga aaataagtca ggtacttgcc tggactttct tccagttg 2336

≪ 210 > 13 < 211 > 2336 < 212 > DNA < 213 > Rattus norvegicus < 400 > 13 ccgactctag aggatcccct taagctaatc cttatgaatc cggagaaaag cggggtcttt 60 taactcaata aaactttccg aaaccccttt tcctacgcgt tttcttcggg aactagatag 120 gtggctcttc cacctgtttt tccatcattt tagtttttcg caagccatgc gtgccttttc 1Θ0 gtttttgcga tggcgaacga gggctggaaa aattaacggt acgccgccta acgatagtaa 240 taggccacgc aactggcgtg gaegacaaca ataagtcgcc cattttttat gttttcaaaa 300 cccagcaacc cccaccaaac ttgtcatcgt tcccggattc acaaatgata taaaaagcga 360 ttacaattct acattctaac cagatttgag atctcctctt tctcaattcc tetcatacta 420 gattataaga acaacaaatt aaattacaaa aagacttata aagcaacata atgtctgaat 480 tcagcaagaa ggtgacgggc cgccttatgt tggccgtggg aggggcagtg ctcggatccc 540 ⑤ -10- 1338044 tgcagttcgg acaarcaaac tctggtctct gcctctttgt ttgtgtctgc tgggccgctt Tgggggaggt ccgtcgttgg acttgtggcc tgcccttctg ccaagagtgt tgaaagaaga gctcacccgc tgtcgggcat agcagcctgt cgctgttcgt: tggcgggctg cgtcctatct gtcccattcc ttgctgtggc atgtggagca tcctcatctt cttccggctt accctctggg cttaatt ttt aaaaaaaaaa aaacctatta aacaggaaaa ttaaaaacgc tctccggggg ctat & acacc atggaaccac ctccatggcc taatcgcttt cgtgcttatg caccattgga gtcacccaca gatccttatt tctactgctc ccctgagagc gctgaaaaag gggtcggcag ctaccgccag caatgctgtg gtatgccacc cgtggagcga tgctgcgctc gagtatcgtg atggttcatt tggcttctct actgtgtggc cacctactcc ccggcagggg ggctgacccc catgactgac aaaatttccc tttgtgtaca aaaaaagaa ^ aaaatgaatt gtcctctaga ggtgtcatca cgctatggag atcttctctg ggcaggcgga ggtttctcca gtgtactgtg gctcttcgtg gcccaggtgt; agtgtcatct ccccgcttcc cttcgaggga atgatgcggg cccatcctca ttctactact atcggctcgg gctggccgtc atgaccatcg gccatctttg gtggccgagc aactggacct ccctacgtct aaagttcctg ggtgccagcc caagtgtaat gttttttctc ttatcaatga tatatcagag acgataacat gtccctatga aaataagtca acgcccccca agtccatccc ccgggggcat actccatgct aactgggcaa gcctgaccac gagccctggg tcggcttaga tcatcccagc tgcccatcaa cagccgatgt agaagaaggt tcgccgtggt caacgagcat gtatcgtcaa ggaccctgca ccctggccct gctttgtggc tgttcagcca caaacctcat tcatcatctt agaccaaagg agagcgacaa cgatttgaag cattttaatt t atccttacg tattattaca gctctgccat aattattaaa ggtacttgcc gaaggtaatt atccaccaca gatcggttcc gatgatgaac gtcctttgag cggctttgtg caccctgcac ctccaccatg cctgctacag tcgtaacgag gacccgagac caccatcttg gctgcagctg cttcgagaag cacggccttc tctcattggt gctggagcag cttctttgaa ggggccccga cgtgggcatg cacggtoctg ccggacctcc gacacccgag tgagacgccc atcacagrat attatataaa tatataacct cctttgttca ggaccacatc tggaccttct gaggagttct ctcaccacac ttctctgtgg ctgttggcct atgctgatcc cccatgtatg cagctgggca ggcaatgcag tgtatcccgt gagaaccggg ctgcaggaga gagctgttcc tcccagcagc gcaggcgtgc actgtggtgt ctggctggca ctgccctgga gtaggccctg cctgctgctg tgcttccaat ctggtactct gatgagatcg gagctcttcc catcatctct ttgtttgaaa ttccttacct ttttctctaa ccgagcaaaa accagactta tccagttg 600 660 720 780 840 900 3 € 0 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 I860 1920 I960 2040 2100 2160 2220 2280 2338 < 210 & gt 14 <211> 2338 <212> DNA <213>Brown Rat <400> 14 tcgactctag aggatccccc taagctaatc cttatgaatc cggagaaaag cggggtc ttt 60 taactcaata aaaccctccg aaatcctttt tcctacgcgt tttcttcggg aactagatag 120 gtggctcttc cacctgtttt tccatcattt tagtttttcg caagccatgc gtgccttttc 180 gttttcgcga tggcgaacga gggctggaaa aattaacggt acgccgccta acgatagtaa 240 taggccacgc aactggcgtg gacgacaaca ataagtcgcc cattttttat gttttcaaaa 300 cctagcaacc cccaccaaac ttgtcatcgt tcccggattc acaaatgata taaaaagcga 360 ttacaattct acattctaac cagatttgag atttcctctt tctcaattcc tcttatatta 420 gattataaga acaacaaatt daattacaaa aagacttata aagcaacata atgtctgaat 460 tcagcaagaa ggtgacgggc cgccttatgt tggccgtggg aggggcagtg ctcggatccc 540 tgcagttcgg ctataacacc ggtgtcatca acgcccccca gaaggtaatt gaggagttct € 00 -11 - 1338044 acaatcaaac atggaaccac cgccatggag agtccatccc atccaccaca ctcaccacac 66〇tctggtctct ctccgtgatg arcttctctg tcgggggcat gattggttcc tcctctgtgg 720 gcctctttgt taatcgcttt ggcaggcgga actccatgct gatgatgaac ccgttggcct 7e〇ttgtgtctgc cgtgcccatg ggtttctcca aactgggcaa gtcctttgag acgctgatcc Θ40 Tgggccgctt catcattgga gtgtactgtg gcctgaccac cggctttgcg cccatgt atg 900 tgggggaggt gccacccaca gctcttcgcg gagccctggg caccctgcac cagctgggca 960 tcgtcgttgg gatccttatt gcccaggtgt tcggcttaga ctccatcacg ggcaacgcag 1020 acttgtggcc tctactgctc agtgtcacct tcatcccagc cctgctacag tgtatcctgt 1ΟΘ0 tgcccttctg ccctgagagc ccccgcctcc tgctcatcaa tcgtaacgag gagaaccggg 1140 ccaagagtgr gctgaaaaag cttcgaggga cagccgatgc gacccgagac ctgcaggaga 1200 tgaaagaAga gggtcggcag atgatgcggg agaagaaggt caccatcttg gagctgttcc 1260 gctcacccgc ctaccgccag cccatcctca tcgccgtggc gctgcagctg tcccagcagc 1320 tgtcgggcat caatgctgtg ttctactact caacgagcar cttcgagaag gcaggtgtgc 1380 agcagcctgt gtatgccacc atcggctcgg gtatcgtcaa cacggccttc actgtggtgt 1440 cgctgttcgt cgcggagcga gctggccgtc ggaccctgca tctcattggt ctggctggca 1500

tggcgggctg cgctgtgctc atgaccatcg ccctggccct gctggagcag ctgccctgga 1560 tgtcctatct gagtatcgtg gccatctctg gctttgtggc cttctttgaa gtaggccctg 1620 gtcctattcc atggttcatc gtggccgagc tgttcagcca ggggccccga cctgctgctg 1680 ctgctgtggc tggcttctct aaccggacct caaacttcat cgtgggcatg tgcttccaat 1740 atgtggagca actgtgtggc ccctacgtct tcatcatctt cacggtgctg ctggtactct 1800 tcttcatctt cacctacttc aaagttcctg agaccaaagg ccggaccttc gatgagatcg I860 cttccggctt ccggcagggg ggtgccagcc agagcgacaa gacacccgaa gagctcttcc 1920 accctctggg ggctgacccc caagtgtaat tgagacgctc catcatctct I960 cttaattttt catgactgac gttttttctt catttcaatt atcatagtat ctgtttgaaa 2040 aaaaaaaaaa aaaatttccc ttatcaatga tatccttacg attacataaa tcccctacct 2100 aaacctatta tctgtgtaca tatdtcagag tattattaca tatataacct ttttctctaa 2160 aacaggaaaa aaaaaagaaa acgataacat gctctgccat cccrtgttca ccgagcaaaa 2220 ttaaaaacgc aaaatgaatt gtccctatga aattactaaa ggaccacatc accagactta 2260 tctctggggg gtcctctaga aaataagtca ggtacttgcc tggactttct tccagttg 2338 & lt cgatttgaag; 210> 15 < 211 > 6360 < 212 > DNA < 213 > carrier < 400 > 15 cgtaggaaca atttcgggcc cctgcgtgrt tgctggacaa ttttcagagg caacaaggaa gaaaaatccc caccatcttt cgagatcccc aggaggaaaa tacaaaatat actagaactg atgccaatac ttcacaatgt tcgaatctat aacatcaaga acaaacaagc tcaacttgtc ceaaaagttt ttttaatttt aatcaaaaag crtggatcccc cgggctgcag gaattcgata catgtaatta gttatgtcac gcttacattc aaaggaagga gctagacaac ctgaagtcta agtattaaga acgttattta tatttcaaat cttctgaggt tcatctttta catttgcttc € 0 aaatcagatg gcaaaaagtc gtctttcaag 120 cgtaacttat cggcaactga aagaatgaaa 180 aaaaaaaaaa agtataaaca gagacgatat 240 tcttcatttg cagctattgt aaaatadcaa 300 tttcctaaga acaaagaata aacacaaaaa 360 ttadcatgca tcaccatcac catcacacca 420 tcaagcttat cgataccgtc gacctcgagt 480 acgccctccc cccacatccg ctctaaccga 540 ggtccctact tattttttta tagttatgtt 600 ttttcttttt tttctgtaca gacgcgtgta 660 -12 - 1338044 cgcatgtaac attacactga aaaccccgct tgagaaggtt ttgggacgct cgaaggcttt 720 aatttgcggc cggtacccaa ttcgccctat agtgagtcgt attacgcgcg ctcactggcc 760 g tcgtttcac aacgtcgtga ctgggaaaac cctggcgcta cccaacttaa ccgccttgca Θ40 gcacaccccc ctttcgccag ctggcgtaat agcgaagagg cccgcaccga tcgcccttcc 900 caacagttgc gcagcctgaa tggcgaatgg cgcgacgcgc cctgtagcgg cgcattaagc 960 gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc cctagcgccc 1020 gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctctcc ccgtcaagcc 10Θ0 ctaaatcggg ggctcccttt agggttccga tttagcgctt tacggcacct cgaccccaaa 1140 aaacttgatt agggtgatgg tccacgtagt gggccatcgc cctgatagac ggtttttcgc 1200 ccttcgacgt tggagtccac gctctttaat agtggacccc tgttccaaac tggaacaaca 1260 ctcaacccta tctcggtcta ttcctttgat ttataaggga tttcgccgat ttcggcctat 1320 tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attttaacaa aarattaacg 1380 tttacaattt cctgatgcgg tactttctcc ttacgcatct gtgcggtatt tcacaccgca 1440 tagggtaata actgatataa ttaaattgaa gctctaattt gtgagtttag tatacatgca 1500 tttacttata atacagtttt ttagttttgc tggccgcatc ttctcaaaca tgcttcccag 1560

cctgcttttc tgtaacgttc accctctacc ttagcatccc ttccctttgc aaatagtcct 1620 cttccaacaa taataatgtc agatcctgta gagaccacat catccacggt tctatactgt 1600 tgacccaatg cgtctccctt gtcatctaaa cccacaccgg gtgtcataat caaccaatcg 1740 taaccttcac ctcttccacc cacgtctctt tgagcaataa agccgataac aaaatctttg 1600 tcgctcttcg caatgccaac agtaccctta gtatattctc cagtagatag ggagcccttg I860 catgacaatc ctgctaacat caaaaggcct ctaggttcct ttgttacttc ttctgccgcc 1920 tgcttcaaac cgctaacaat acccgggccc accacaccgt gtgcattcgt aatgtctgcc 1980 cattccgcta ttctgtatac acccgcagag tactgcaact tgactgtatt accaatgtca 2040 gcaaattttc tgtcttcgaa gagtaaaaaa ttgtacttgg cggataatgc ccttagcggc 2100 ttaactgtgc cctccatgga aaaatcagtc aagatatcda catgtgtttt cagtaaacaa 2160 attttgggac ctaatgcttc aactaactcc agcaattcct tggtggtacg aacatccaat 2220 gaagcacaca agtttgtttg cttttcgtgc atgatattaa atagcttggc agcaacagga 2280 ctaggatgag tagcagcacg ccccttatat gtagctttcg acatgattta tcttcgtttc 2340 ctgcaggctt ttgttctgtg cagttgggtt aagaatactg ggcaatttca tgtttcctca 2400 acacta cata tgcgtatata taccaatcta agtctgtgct ccttccttcg ttcttccttc 2460 tgttcggaga ttaccgaatc aaaaaaattt caaagaaacc gaaatcaaaa aaaagaataa 2520 aaaaaaaacg atgaattgaa ttgaaaagct gtggtatggt gcactctcag tacaacctgc 2580 tctgatgccg catagttaag ccagccccga cacccgccaa cacccgctga cgcgccctga 2640 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc 2700 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga gacgaaaggg cctcgtgata 2760 cgcctatttt tataggttaa tgccatgata ataatggttt cttagtatga tccaacatca 2620 aaggaaatga tagcattgaa ggatgagact aatccaattg aggagtggca gcatatagaa 2ΘΘ0 cagctaaagg gtagtgctga aggaagcata cgataccccg catggaatgg gataatatca 2940 caggaggtac tagactacct ttcatcctac ataaatagac gcatacaagt acgcatttaa 3000 gcataaacac gcactatgcc gttcttctca tgtatatata tatacaggca acacgcagat 3060 ataggtgcga cgtgaacagt gagctgtatg tgcgcagctc gcgttgcatt ttcggaagcg 3120 ctcgttttcg gaaacgcttt gaagttccta ttccgaagtt cctattctct agaaagtata 3180 ggaacttcag agcgcttttg aaaaccaaaa gcgctctgaa gacgcacttt caaaaaacca 3240 aaaacgcacc ggactgtaac gagctactaa aatattgcga ataccgcttc cacaaacatt 3300 gctcaaaagt atctctttgc tatatatctc tgtgctatat cccxratataa cctacccatc 33 ^ 0 caccttncgc tccttgaact tgcatctaaa ctcgacctct acarctttta tgtttatctc 3420 tagtattact ctttagacaa aaaaattgta gtaagaacta ttcacagagt gaatcgaaaa 34Θ0 caatacgaaa atgtaaacat ttcctatacg tagtatatag agacaaaata gaagaaaccg 3540 -13 - 1338044

ttcataattt tctgaccaat gaagaaticat caacgctatc actttctgtt cacaaagtat 3600 gcgcaatcca catcggtata gaatataatc ggggatgcct ttatcttgaa daaatgcacc 3660 cgcagcttcg ccagtaatca gcaaecgcgg gaagtggagt caggcttttt ttatggaaga 3720 gaaaatagac accaaagtag ccttcttcta accttaacgg acctacagtg caaaaagcta 37Θ0 tcaaigagact gcattatag in gcgcacaaag gagaaaaaaa gtaatctaag atgctttgct 3640 agaaaaatag cgctctcggg atgcattttt gtagaacaaa aaagaagtat agattctttg 3900 ttggtaaaac agcgctctcg cgttgcattt ctgttctgta aaaatgcagc tcagattctt 3960 tgtttgaaaa attagcgccc tcgcgttgca tttttgtttt scaaaaatga agcacagatt 4020 cttcgttggc aaaatagcgc tttcgcgttg catttctgtt ctgtaaaaat gcagctcaga 4080 ttctttgttt gaaaaattag cgctctcgcg ttacactttt gttctacaaa atgaagcaca 4140 gatgcttcgt tcaggtggca cttttcgggg aaatgtgcgc ggaaccccta tttgtttatc 4200 tttctaaata cattcaaata tgtatccgct catgagacaa taaccctgat aaatgcttca 4260 ataatattga aaaaggaaga gtatgagtat tcaacatttc cgtgtcgccc ttattccctt 4320 tttcgcggca ttttgccttc ctgttttitgc tcacccagaa acgctggtga aagtaaaaga 4380 tgctgaagat cagttgggtg cacgagtggg ttacatcgaa ctggatctca acagcggtaa 4440 gatccttgag agttttcgcc ccgaagaacg ttttccaatg atgagcactt ttaaagttct 4500 gccatgtggc gcggtattat cccgtattga cgccgggcaa gagcaactcg gtcgccgcat 4560 acactattct cagaatgact tggttgagta ctcaccagtc acagaaaagc atcctacgga 4 € 20 tggcatgaca gtaagagaat tatgcagtgc tgccataacc atgagtgata acactgcggc 4680 eaacttactt ctgacaacga tcggaggacc gaaggagcta accgcttctt tgcacaacat 4740 gggggatcac gtaactcgcc ttgatcgttg ggaaccggag ctgaatgaag ccataccaaa 4800 cgacgagcgt gacaccacga tgcctgtagc aatggcaaca acgttgcgca aaccattaac 4860 tggcgaacta cttactctag cttcccggca acaattaatsi gactggaitgg aggcggataa 4920 agttgcagga ccacttctgc gctcggccct tccggctggc tggtttatcg ctgataaatc 49Θ0 tggagccggt gagcgtgggt ctcgcggtat cattgcagca ctggggccag atggtaagcc 5040 ctcccgtatc gtagttatct acacgacggg gagtcaggca actatggacg aacgaaatag 5100 acagatcgct gagataggtg ccccactgat taagcattgg taactgtcag accaagttta 5160 ctcatatata ctttagattg atttaaaact tcatttttaa tttaaaagga tctaggtgaa 5220 g acccttttt gataatctca cgaccaaaat cccttaacgt gagttttcgt tccaccgagc 5280 gtcagacccc gtagaaaaga ccaaaggatc ttcttgagat cctttttttc tgcgcgtaat 5340 ctgctgcttg caaacaaaaa aaccaccgct accagcggtg gtttgtttgc cggatcaaga 5400 gctaccaact ctttttccga aggtaactgg cttcagcaga gcgcagatac caaatactgt 5460 ccttctagtg tagccgtagt taggccacca cctcaagaac tctgtagcac cgcctacata 5520 cctcgctctg ctaancctgt taccagtggc tgctgccagt ggcgataagc cgtgtcttac 558〇cgggttggac tcaagacgat agttaccgga taaggcgcag cggtcgggct gaacgggggg 5640 ttcgtgcaca cagcccagct tggagcgaac gacctacacc gaactgagac acctacagcg 5700 tgagctatga gaaagcgcca cgcttcccga agggagaaag gcggacaggt atccggcaag 5760 cggcagggtc ggaacaggag agcgcacgag ggagcttcca gggggaaacg cctggtatct 5820 ttatagtcct gtcgggtttc gccacccctg acttgagcgt cgatccttgt gatgctcgtc 5860 aggggggcgg agcctatgga aaaacgccag caacgcggcc tttttacggt tcctggcctt 5940 ttgctggcct tttgctcaca tgttctttcc tgcgttatcc cctgatcctg tggataaccg 6000 tattaccgcc tttgagtgag ctgataccgc tcgccgcagc cgaacgaccg agcgcagcga 6060 gtcag tgagc gaggaagcgg aagagcgccc aatacgcaaa ccgcctctcc ccgcgcgttg 6120 gccgattcat taatgcagct ggcacgacag gtttcccgac tggaaagcgg gcagcgagcg 61Θ0 caacgcaatt aatgtgagtt acctcactca ttaggcaccc caggctttac actttatgct 6240 tccggctcct atgttgtgtg gaattgtgag cggataacaa tttcacacag gaaacagcta 6300 tgaccatgat tacgccaagc gcgcaattaa ccctcactaa agggaacaaa agctggagct 6360 ⑤ Shu -14 - 1338044 < 210 > 16 < 211 > 24 <212> DNA <213>Brown Rat <400> 16 cctcctcaat tcctcttata ttag 24 <210> 17 <211> 36 <212> DNA <213>

<400> 17 cccgacagag aagatcatca cggagagaga ccagag 36 <210> 18 <211> 24 <212> DNA <213>Brown Rat <400> 18 aacgtcagtc atgaaaaatt aaga 24

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Claims (1)

----------------- Li application No. 竽 ⁄ ⁄ ⁄ ⁄ 928 知 知 ( ( tent tent tent tent tent tent tent tent tent tent tent tent tent tent tent tent tent tent tent tent tent tent tent tent tent tent tent tent tent tent tent tent tent tent Anicuduil^laims in Chinese - Enel. II (Submitted on Julyv^, 2010) (Submitted on Julyv^, 2010) X. Patent application scope: 1. The use of a polynucleotide sequence encoding the GLUT1 protein, which makes Restoring the Saccharomyces cerevisiae strain (Sflcc/zarowyca cerevw/ae) grown on a substrate with six carbon sugar as the sole carbon source by deletion or mutation of the genome sequence of all the six carbon sugar transporters of the yeast The growth ability on a substrate having hexose carbon as the sole carbon source, wherein the polynucleotide sequence comprises SEQ ID No. 13. 2. The use of a GLUT1 protein which enables the growth of Saccharomyces cerevisiae on a substrate having six carbon sugars as the sole carbon source by deletion or mutation of the genome sequence of all six carbon sugar transporters of the yeast The strain is restored to its ability to grow on a substrate having hexose monosaccharide as the sole carbon source, wherein the protein is encoded by the polynucleotide sequence of claim 1 of the patent application. 3. Use of a polynucleotide sequence encoding a GLUT1 protein, which is such that the deletion or mutation of the genome sequence of all six carbon sugar transporters of the yeast does not allow the substrate to have a six carbon sugar as the sole carbon source. The growing Saccharomyces cerevisiae strain restores its ability to grow on a substrate having hexose carbon as the sole carbon source, wherein the polynucleotide sequence comprises SEQ ID No. 14. 4. Use of a GLUT1 protein for the deletion or mutation of the genome sequence of all six carbon sugar transporters of the yeast, and I338Q44 is incapable of growing on a substrate having six carbon sugar as the sole carbon source The strain is restored to its ability to grow on a substrate having hexose monosaccharide as the sole carbon source, wherein the protein is encoded by the polynucleotide sequence as in claim 3 of the patent application.