CA2341148A1 - Human membrane channel proteins - Google Patents

Abstract

The invention provides new human membrane channel proteins (MECHP) and polynucleotides which identify and encode MECHP. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating, or preventing disorders associated with expression of MECHP.

Description

HUMAN MEMBRANE CHANNEL PROTEINS
TECHNICAL FIELD
This invention relates to nucleic acid and amino acid sequences of human membrane S channel proteins and to the use of these sequences in the diagnosis, treatment, and prevention of cell proliferative, immune/inflammatory, transport/secretory, osmoregulatory, muscular, cardiovascular, and neurological disorders.
BACKGROUND OF THE INVENTION
Channel proteins facilitate the transport of hydrophilic molecules across membranes by forming aqueous pores that can perforate a lipid bilayer. Many channels consist of protein complexes formed by the assembly of multiple subunits, at least one of which is an integral membrane protein that contributes to formation of the pore. In some cases, the pore is constructed to selectively allow passage of only one or a few molecular species. Distinct types of membrane t S channels that differ greatly in their distribution and selectiivity include: ( 1 ) aquaporins, which transport water; (2) protein-conducting channels, which transport proteins across the endoplasmic reticulum membrane; (3) gap junctions, which facilitate diiffusion of ions and small organic molecules between neighboring cells; and {4) ion channels, which regulate ion flux through various membranes.
Aquaporins Aquaporins (AQP) are channels that transport water and, in some cases, nonionic small solutes such as urea and glycerol. Water movement is important for a number of physiological processes including renal fluid filtration, aqueous humor generation in the eye, cerebrospinal fluid production in the brain, and appropriate hydration of the lung. A variety of aquaporins have been found in higher animals, plants and microorganisms. The .mammalian aquaporins appear to have selective expression in particular tissues, with AQPO localized to lens epithelium; AQPl localized to many tissues including red blood cells, kidney, eye, lunl;, choroid plexus, bile duct, and vascular epithelium; AQP2 localized to the apical membrane of kidney collecting duct cells; AQP3 localized to kidney, colon, trachea, urinary bladder, skin, and sclera of eye;
AQP4 localized to kidney, colon, trachea, stomach, skeletal muscle, spinal cord, brain, and retina; AQPS localized to the apical membranes of exocrine tissues; AQP6 localized to kidney; and AQP7 localized to testis (King, L.S. and P. Agre (1996) Annu. Rev. Physiol. 58:619-648; Ishibashi, K.
et al. (1997) J. Biol.
Chem. 272:20782-20786). AQP9 is expressed in peripheral leukocytes, less abundantly in liver, even less in lung and spleen, and not at al) in thymus tissue (Ishibashi, K.
et al. ( 1998) Biochem.

Biophys. Res. Commun. 244:268-274).
Aquaporins are members of the major intrinsic protein (MIP) family of membrane transporters. MIP family proteins are composed of four s~ubunits, each of which may span the membrane six times, and have their N-and C-termini facing the cell cytoplasm.
Proteins from bacteria, yeast, plants, and animals have been shown to bt: members of the MIP
family {Reizer, J.
et al. {I993) Crit. Rev. Biochem. 28:235-257). Aquaporin subunits are integral membrane proteins with six transmembrane regions and two conserved Asn-Pro-Ala (NPA) boxes (which are sometimes found as Asn-Pro-Ser) found in loop regions between the transmembrane regions {King, supra; Ishibashi, ( 1997) supra). The, study of aquaporins may have relevance to understanding edema formation and fluid balance in both normal physiological and disease states (King, supra). Mutations in AQP2 cause autosomal recessive nephrogenic diabetes insipidus (Online Mendelian Inheritance in Man (OM1M) *107777 Aquaporin 2; AQP2).
Reduced AQP4 expression in skeletal muscle may be associated with Duchenne muscular dystrophy (Frigeri, A. et al. (1998) J. Clin. Invest. I02:695-703). Mutations in AQPO cause autosomal dominant cataracts in mice (OMIM * 154050 Major Intrinsic Protein of Lens :Fiber; MIP}.
Protein-Conducting Channels Secreted and integral membrane proteins are transported from the cytoplasm to the endoplasmic reticuium (ER) through protein-conducting channels in the ER
membrane. The channel is used for both co- and post-translational translocation. In the co-translational process, transport is initiated by the action of a cytopiasmic signal .recognition particle (SRP) which recognizes a signal sequence on a growing, nascent polype;ptide and binds the polypeptide and its ribosome complex to.the ER membrane through an SRP re;ceptor located on the membrane. The ribosome complex, together with the attached polypeptide" becomes membrane bound. As the nascent chain emerges from the ribosome, it is fed into the channel and across the ER membrane.
The post-translational process also requires a signal sequence on the protein to be translocated, but does not require an SRP. The protein enters the channel and is driven across the ER membrane by the hydrolysis of adenosine triphosphate (ATP) by BiP, an ATPase and molecular chaperone in the ER lumen.
The protein-conducting channel, termed the Secd l p complex, is composed of multiple, probably two, heterotrimers of three membrane proteins, the alpha, beta, and gamma subunits of Sec6lp. The Sec6lp complex forms a ring structure visible by electron microscopy (EM). EM
and quenching experiments indicate a channel diameter of 20 to 60 A.
Association of the Sec61 p complex with the ribosome and with the proteins Secb2p, Sec63p, Sec71 p, Sec72p, BiP, and TRAM (translocating chain-associating membrane protein;l is required for some of the channel's functions. The Sect 1 p alpha subunit contains ten membrane-spanning segments and has been found to line the path of the translocating polypeptide chain from one side of the membrane to the other. The sequences of dog and rat Sec61 p alpha genes have been determined.
Homologs of the mammalian Sec6lp alpha are found in the yeast Saccharomvces cerevisiae (Sec6lp) and in bacteria (SecYp). (See Giirlich, D. et al. ( 1992) Cell 71:489-503; Matlack, K.E.S. et al. ( 1998) CeII 92:381-390.) Defects in protein trafficking to organelles or to 'the cell surface are involved in numerous human diseases and disorders including cystic fibrosis, glucose-galactose malabsorption syndrome, hypercholesterolemia, diabetes mellitus, diabc;tes insipidus, hyper-and hypoglycemia, Grave's disease, goiter, Cushing's disease, and Addison';s disease. Cancer cells secrete excessive amounts of hormones or other biologically active peptides.
Gap Junctions Gap junctions (also called eonnexons} are channels that function chemically and electrically to couple the cytoplasms of neighboring cells in many tissues.
Gap junctions function as electrical synapses for intercellular propagation of actuon potentials in excitable tissues. In nonexcitable tissues, gap junctions have roles in tissue homeostasis, coordinated physiological response, metabolic cooperation, growth control, and the regulation of development and differentiation. Gap junctions help to synchronize heart and smooth muscle contraction, speed neural transmission, and propagate extracellular signals. Gap junctions can open and close in response to particular stimuli (e.g., pH, Ca+Z, and cAMP). The effective pore size of a gap junction is approximately 1.5 nm, which enables small molecules (e.g., those under 1000 daltons) to diffuse freely through the pore. Transported molecules include ions, small metabolites, and second messengers (e.g., Ca+2 and cAMP).
Each connexon is composed of six identical subunits called connexins. At least thirteen distinct connexin proteins exist, with each having similar structures but differing tissue distributions. Structurally, the connexins are integral membrane proteins with four putative membrane spanning regions and N- and C-termini oriented towards the cell cytoplasm. Conserved regions include the membrane spanning regions and two e:xtracellular loops.
The variable regions, which are two cytoplasmic loops and the C-terminal region, may be responsible for the regulation of different connexins. (See Hennemann, H. et ai. ( 1992) .1. Biol. Chem.
267:17225-17233;
PRINTS PR00206 connexin signature.) Connexins have many disease associations. Female mice lacking connexin 37 (Cx37) are infertile due to the absence of the oocyte-granuIosa cell signaling pathway.
Mice lacking Cx43 die shortfy after birth and show cardiac defects reminiscent of some forms of stenosis of the _3_ pulmonary artery in humans. Mutations in Cx32 are associated with the X-linked form of Charcot-Marie-Tooth disease, a motor and sensory neuropathy of the peripheral nervous system.
Cx26 is expressed in the placenta, and Cx26-deficient mi<;e show decreased transplacental transport of a glucose analog from the maternal to the fetal circulation. In humans, Cx26 has been identified as the first susceptibility gene for non-syndromic sensorineurai autosomal deafness.
Cx46 is expressed in lens fiber cells, and Cx46-deficient mice develop early-onset cataracts that resemble human nuclear cataracts. (See Nicholson, S.M. and R. Bruzzone (1997) Curr. Biol.
7:8340-8344.) Ion Channels The electrical potential of a cell is generated and maintained by controlling the movement of ions across the plasma membrane. The movement of ions requires ion channels, which form an ion-selective pore within the membrane. There are two basic types of ion channel: ion transporters and gated ion channels. Ian transporters utili:ae the energy obtained from ATP
hydrolysis to actively transport an ion against the ion's concentration gradient. Gated ion channels allow passive flow of an ion down the ion's electrochemical gradient under restricted conditions.
Together, these types of ion channels generate, maintain, and utilize an electrochemical gradient that is used in 1) electrical impulse conduction down the axon of a nerve cell, 2} transport of molecules into cells against concentration gradients, 3) initiation of muscle contraction, and 4) endocrine cell secretion.
Ion channels share common structural and mechanistic themes. The channel consists of four or five subunits or protein monomers that are arranged like a barrel in the plasma membrane.
Each subunit typically constists of six potential transmemlbrane segments (Sl, S2, S3, S4, S5, and S6). The center ofthe barrel forms a pore lined by a-helices or (3-strands.
The side chains ofthe amino acid residues comprising the a-helices or p-strands establish the charge (cation or anion) selectivity of the channel. The degree of selectivity, or what specific ions are allowed to pass through the channel, depends on the diameter of the narrowest part of the pore.
Ion Transporters Ion transporters generate and maintain the resting electrical potential of a cell. Utilizing the energy derived from ATP hydrolysis, they transport ions against the ion's concentration gradient. These transmembrane ATPases are divided into three families. The vacuolar (V) class of ion transporters includes H+ pumps on intracellular organelles, such as lysosomes and Golgi.
V-class ion transporters are responsible for generating the low pH within the lumen of these organelles that is required for function. The coupling factor (F) class consists of Hr pumps in the mitochondria. F-class ion transporters utilize a proton gradient to generate ATP from ADP and inorganic phosphate (P;). The phosphorylated (P) class ion transporters, including Na+-K+
ATPase, Ca+2-ATPase, and H+-ATPase, are activated by a phosphorylation event.
P-class ion transporters are responsible for maintaining resting potential distributions such that cytosolic concentrations ofNa~ and Ca+2 are low and cytosolic concentration of K+ is high. The resting potential of the cell is utilized in many processes involving carrier proteins and gated ion channels.
Carrier proteins utilize the resting potential to transport molecules into and out of the cell. Amino acid and glucose transport into many cells is linked to sodium ion co-transport (symport) so that the movement of Na+ down an electrochemical gradient drives transport of the other molecule up a concentration gradient. Similarly, cardiac muscle links transfer of Ca+= out of the cell with transport of Na+ into the cell (antiport).
Gated Ion Channels Gated ion channels control ion flow by regulating the opening and closing of pores. The ability to control ion flux through various gating mechanisms allows ion channels to mediate such diverse signaling and homeostatic functions as neuronal and endocrine signaling, muscle contraction, fertilization; and regulation of ion and pH balance. Gated ion channels are categorized according to the manner of regulating the gating function.
Mechanically-gated channels open their pores in response to mechanical stress.; voltage-gated channels (e.g., Na+, K+, Ca+2, and Cf channels) open their pores in response to changes in membrane potential; and ligand-gated channels (e.g., acetyicholine-, serotonin-, and glutamate- gated cation channels, and GABA-and glycine- gated chloride channels) open their pores in tl'ne presence of a specific ion, nucleotide, or neurotransmitter. The gating properties of a particular ion channel ( i.e., its threshold forand duration of opening and closing) are sometimes modulated! by association with auxiliary channel proteins and/or post translational modifications, such as phospharylation. The pore forming subunits of voltage-gated and transmitter-gated cation channels form two distinct superfamilies of conserved multipass membrane proteins.
Voltage-gated Na+ and K+ channels are necessary for the function of electrically excitable cells such as nerve, endocrine, and muscle cells. Action potentials, which lead to neurotransmitter release and muscle contraction, arise from large, transient changes in the permeability of the membrane to Na+ and K+ ions. Depolarization of the membrane beyond the threshold level opens voltage-gated Na+ channels. Sodium ions flow into the cell, further depolarizing the membrane and opening more voltage-gated Na+ channels, thus propagating the depolarization down the length of the cell. Depolarization also opens voltage-gated K+ channels.
Consequently, potassium ions flow outward, leading to repolarization of the membrane. Voltage-gated channels utilize charged residues in the fourth transmembrane segment (S4) to sense voltage change. The open state lasts only about 1 millisecond, at which time the channel spontaneously converts into an inactive state that cannot be opened irrespective of the membrane potential.
Inactivation is mediated by the channel's N-terminus, which acts as a plug that closes the pore. The transition from an inactive to a closed state requires a return to restiing potential.
Na+ channels isolated from rat brain tissue are heterotrimeric complexes composed of a 260 kDa pore-forming a subunit that associates with two smaller auxiliary subunits, pl and ~i2.
The [32 subunit is an integral membrane glycoprotein that: contains an extracellular Ig domain, and its association with a and X31 subunits correlates with increased function of the channel, a change in the channel's gating properties, as well as an increase iin whole cell capacitance (Isom, L.L. et al. (1995) Cell 83:433-442).
K+ channels are located in a!l cell types, and may be regulated by voltage, ATP
concentration, or second messengers such as Ca'~'' and cA.MP. In non-excitable tissue, K' channels are involved in protein synthesis, control of endocrine secretions, and the maintenance of osmotic equilibrium across membranes. In neurons and other excitable cells, in addition to regulating IS action potentials and repolarizing membranes, K+ channels are responsible for setting resting membrane potential. The cytosol contains non-diffusible; anions and, to balance this net negative charge, the cell contains a Na+-K+ pump and ion channel; that provide the redistribution ofNa+, K+, and Cf. The pump actively transports Na+ out of the cell and K+ into the cell in a 3:2 ratio.
Ion channels in the plasma membrane allow K+ and Cl' to flow by passive diffusion. Because of the high negative charge within the cytosol, C1' flows out of the cell. The flow of K+ is balanced by an electromotive force pulling K+ into the cell, and a 1C+ concentration gradient pushing K+ out of the cell. Thus, the resting membrane potential is primarily regulated by K+flow (Salkoff, L.
and T. Jegla ( 1995) Neuron 15:489-492).
K+ pore-forming subunits generally have six transrnembrane-spanning domains with a short region between the ffth and sixth transmembrane regions that senses membrane potential;
and the amino and carboxy termini are located intracellularly. In mammalian heart, the duration of ventricular action potential is controlled by a K+ current. Thus, the K+
channel is central to the control of heart rate and rhythm. K+ channel dysfunctions are associated with a number of renal diseases including hypertension, hypokalemia, and the a ssociated Banter's syndrome and Getelman's syndrome, as well as neurological disorders including epilepsy. K+
channels have been implicated in Alzheimer's disease by observations that a significant component of senile plaques, beta amyioid or A beta, also blocks voltage-gated potassium channels in hippocampal neurons. {See Antes, L.M. et al. (1998) Seminar Nephrol. 18:31-45; Stoffel, M.
and L.Y. Jan {1998) Nat. Genet. 18:6-8; Madeja, M. et al. (1997) Eur. J. Neurosci: 9:390-395; Good, T.A. et al.

( 1996) Biophys. J. 70:296-304.) Voltage-gated Ca~' channels are involved in presynaptic neurotransmitter release, and heart and skeletal muscle contraction. The voltage-gated Ca+'- channels from skeletal muscle (L-type) and brain (N-type) have been purified and, though l:heir functions differ dramatically, they have similar subunit compositions. The channels are composed of three subunits. The a, subunit forms the membrane pore and voltage sensor, while the oN& and ~i subunits modulate the voltage-dependence, gating properties, and the current amplitude of the channel. These subunits are encoded by at least six a,, one a,8, and four ~ genes. A fourth subunit, y, has been identified in skeletal muscle. (See Walker, D. et al. ( 1998) J. Biol. Chem. 273:2361-2367;
Say, S.D. et al.
( 1990) Science 248:490-492.) Chloride channels are necessary in endocrine secretion and in regulation of cytosolic and organelle pH. In secretory epithelial cells, Cl' enters the. cell across a basolateral membrane through an Na+, K+/Cl' cotransporter, accumulating in the cell above its electrochemical equilibrium concentration. Secretion ofCl' from the apical surface, in response to hormonal stimulation; leads to flow of Na'~ and water into the secretory lumen. The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloriide channel.encoded by the gene for cystic fibrosis, a common fatal genetic disorder in humans. Loss of CFTR
function decreases transepithelial water secretion and, as a result, the layers of mucus that coat the respiratory tree, pancreatic ducts, and intestine are dehydrated and difficult to clear. The resulting blockage of ~ these sites leads to pancreatic insufficiency, "meconium ileus," and devastating "chronic obstructive pulmonary disease" (AI-Awqatii, Q. et al. ( 1992) J. Exp. Biol.
172:245-266).
Many intracellular organelles contain H+-ATPase pumps that generate transmembrane pH
and electrochemical differences by moving protons from the cytosol to the organelle lumen. If the membrane of the organelle is permeable to other ions, then the electrochemical gradient can be abrogated without affecting the pH differential. In fact, removal of the electrochemical barrier allows more H+ to be pumped across the membrane, increasing the pH
differential. Cl' is the sole counterion of H+ translocation in a number of organelles,, including chromaffin granules, Golgi vesicles, lysosomes, and endosomes. Functions that require a low vacuolar pH
include uptake of small molecules such as biogenic amines in chromaffin I;ranules, processing of vacuolar constituents such as pro-hormones by proteolytic enrym~es, and protein degradation in lysosomes (Al-Awqati, supra).
Liigand-gated channels open their pores when an extracellular or intracellular mediator binds to the channel. Neurotransmitter-gated channels are channels that open when a neurotransmitter binds to their extraceilular domain. These channels exist in the postsynaptiic membrane of nerve or muscle cells. There are two types of neurotransmitter-gated channels.
Sodium channels open in response to excitatory neurotransmitters, such as acetylchoiine, glutamate, and serotonin. This opening causes an influx of Na+ and produces the initial localized depolarization that activates the voltage-gated channels and starts the action potential. Chloride channels open in response to inhibitory neurotransmitters, such as y-aminobutyric acid (GABA) and glycine, leading to hyperpofarization of the membrane and the subsequent generation of an action potential.
Ligand-gated channels can be regulated by intracellular second messengers.
Calcium-activated K+ channels are gated by internat calcium ions.. In nerve cells, an influx of calcium during depolarization opens K+ channels to modulate the. magnitude of the action potential (Ishi, T.M. et al. (1997) Proc. Natl. Acad. Sci. USA 94:11651-11656). Cyclic nucleotide-gated (CNG) channels are gated by cytosoiic cyclic nucleotides. The best examples of these are the cAMP-gated Nat channels involved in olfaction and the cGMP-gated cation channels involved in vision.
Both systems involve iigand-activation of a G-protein coupled receptor which then alters the level of cyclic nucleotide within the cell. In olfaction, binding; of an odorant to the receptor activates adenylate cyclase, leading to a rise in cytosolic cAMP. 'The cAMP binds to the cAMP-gated Na+
channel causing an influx of Na+, depolarization of the nnembrane, and initiation of a nerve impulse that travels along the axon to the brain. In vision, light activation of rhodopsin leads to activation of cGMP phosphodiesterase, which hydrolyzes cGMP. As a result, cytosolic cGMP
levels drop, cGMP dissociates from cGMP-gated cation channels, and the channels close, resulting in hyperpolarization of the membrane. (See Zagotta, W.M. and S.A. Siegelbaum ( 1996) Annu.
Rev. Neurosci. 19:235-263; Molday, R.S. and L.L. Molday (1998) Vision Res.
38:1315-1323.) The subunits or monomers of an ion channel may be identical or different. CNG
channels, for example, consist of a and p subunits that differ from each other at the N-terminal cytoplasmic tail. The central pore formed by the barrel arrangement is lined by an antiparallel (3-sheet, the pore (P) region, contained within each subunit. This region also contains information specifying the ion selectivity for the channel. In the case of K~ channels, a GYG tripeptide is involved in this selectivity (Ishi et al., supra). In voltage-gated channels;
one of the transmembrane domains contains regularly spaced, positively charged amino acids that act as a voltage-sensor. In CNG channels, a region in the C-terminal cytoplasmic domain acts as a cyclic nucleotide binding site (Zagotta and Siegelbaum, supra). Ion channels also have a domain that functions in inactivation of the channel. In CNG K+ channels, the inactivation domain is on the N-terminal cytoplasmic tail of the (i-subunit. This domain acts as a tethered ball to block ion flow through the pore. This domain is also expressed as a separate protein, a glutamic acid-rich protein _g_ (GARP), by alternative splicing and may act as an independent regulator of pore activity (Sautter, A. et ai. ( 1997) Molec. Brain Res. 48:171-175).
Ion channels are essential to a wide range of physiological functions including neuronal signaling, muscle contraction, cardiac pacemaking, hormone secretion, and cell proliferation. Ion channels are expressed in a number of tissues where they are implicated in a variety of processes.
CNG channels, while abundantly expressed in photoreceptor and olfactory sensory cells, are also found in kidney, lung, pineal, retinal ganglion cells, testis, aorta, and brain. Calcium-activated K' channels may be responsible for the vasodilatory effects of bradykinin in the kidney and for shunting excess K+ from brain capillary endothelial cells into the blood. They are also implicated in repolarizing granulocytes after agonist-stimulated depolarization (Ishi et al., supra). Ion channels have been the target for many drug therapies. Neurotransmitter-gated channels have been targeted in therapies for treatment of insomnia, anxiety, depression, and schizophrenia.
Voltage-gated channels have been targeted in therapies for arrhythmia, ischemic stroke, head trauma, and neurodegenerative disease (Taylor, C.P. and L.S. Narasimhan (1997) Adv.
Pharmacol.39:47-98).
The discovery of new human membrane channell proteins and the polynucleotides encoding them satisfies a need in the art by providing new compositions which are useful in the diagnosis, prevention, and treatment of cell proliferative, immune/inflammatory, transpartJsecretory, osmoregulatory, muscular, cardiovascular, and neurological disorders.
SUMMARY OF THE II~fVENTION
The invention features substantially purified polypeptides, human membrane channel proteins, referred to collectively as "MECHP" and individually as "MECHP-1,"
"MECHP-2,"
"MECHP-3," "MECHP-4," "MECHP-5," "MECHP-6," "MECHP-7," "MECHP-8," "MECHP-9,"
"MECHP-10," "MECHP-11," "MECHP-12," "MECHP-~13," "MECHP-14," "MECHP-15,"
"MECHP-16", "MECHP-17", and "MECHP-18." In one aspect, the invention provides a substantially purified polypeptide comprising an amino .acid sequence selected from the group consisting of SEQ ID NO:1-18, and fragments thereof.
The invention further provides a substantially purifed variant having at least 95% amino acid sequence identity to at least one of the amino acid sequences selected from the group consisting of SEQ ID NO:I-I 8 and fragments thereof. 'lflhe invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-18 and fragments thereof. The invention also includes an isolated and purified polynucleotide variant having at least 95%
polynucleotide sequence -g-identity to the polynucieotide encoding the poiypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO:1-18 and fragments thereof.
Additionally, the invention provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-18 and fragments thereof. The invention also provides an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide encoding the poiypeptiide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: I-18 and fragments thereof.
The invention also provides a method for detectiing a polynucleotide in a sample containing nucleic acids, the method comprising the steps of (a) hybridizing the complement of the polynucleotide sequence to at least one of the polynucleotides of the sample, thereby forming a hybridization complex; and {b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of a poiynucleotide in the sample. In one aspect, the method further comprises amplifying the poiynucleotide prior to hybridization.
The invention also provides an isolated and puriified polynucieotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID N0:19-36, and fragments thereof. The invention further provides an isolated and purified polynucleotide variant having at least 95% polynucleotide sequence identity to the polyn~ucieotide sequence selected from the group consisting of SEQ ID N0:19-36 and fragments thereof. The invention also provides an isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ
ID N0:19-36 and fragments thereof.
The invention further provides an expression vector containing at least a fragment of the polynucieotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:1-18 and fragments thereof. In another aspect, the expression vector is contained within a host cell.
The invention also provides a method for prodn~cing a polypeptide, the method comprising the steps of-. (a} culturing the host cell containing an expression vector containing at least a fragment of a polynucleotide under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.
The invention also provides a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ
ID NO:I-18 and fragments thereof, in conjunction widh a suitable pharmaceutical carrier.
The invention further includes a purified antibody which binds to a polypeptide selected from the group consisting of SEQ iD NO:1-18 and fragments thereof. The invention also provides a purified agonist and a purified antagonist to the polypeptide.
'The invention also provides a method for treating or preventing a disorder associated with decreased expression or activity of MECHP, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO:1-18 and fragments thereof, in conjunction with a suitable pharmaceutical carrier.
The invention also provides a method for treating or preventing a disorder associated with increased expression or activity of MECHP, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:1-I8 and fragments thereof.
BRIEF DESCRIPTION OF THE FIGURES AND TABLES
IS Figure 1 shows the amino acid sequence alignment between MECHP-1 (1568324;
SEQ ID
NO:1 ) and rat glutamic acid-rich protein (GI 2924369; S~EQ ID N0:37), produced using the BLAST search tool.
Figure 2 shows the amino acid sequence alignment among MECHP-2 {4094907; SEQ
ID
N0:2), Drosophila voltage-gated potassium channel (GI 116443; SEQ ID N0:38), and P.
penicillatus potassium channel a-subunit (GI 1763619; SEQ ID N0:39), produced using the multisequence alignment program of LASERGENE software (DNASTAR, Madison WI).
Figures 3A and 3B show the amino acid sequence alignment between MECHP-3 (518158;
SEQ ID N0:3) and rat calcium-activated potassium channel rSK3 (GI 2564072; SEQ
ID N0:40), produced using the multisequence alignment program o1F LASERGENE software.
Figures 4A, 4B, and 4C show the amino acid sequence alignment among MECHP-4 (602926; SEQ ID N0:4), Droso~hila voltage-gated potassium channel (GI 116443;
SEQ ID
N0:38) and P. penicillatus potassium channel a-subunit (Gi 1763619; SEQ ID
N0:39), produced using the multisequence alignment program of LASERCiENE software.
Figures SA and SB show the amino acid sequence alignment between MECHP-5 {922119;
SEQ ID NO:S) and rat aquaporin 7 (GI 2350843; SEQ ID N0:41 ), produced using the multisequence alignment program of LASERGENE software.
Figures 6A and 6B show the amino acid sequence alignment between MECHP-7 (2731369; SEQ ID N0:7) and mouse connexin 30.3 (GI 192647; SEQ ID N0:42), produced using the muitisequence alignment program of LASERGENE software.

WO 00/12711 PCT/US99/204ti8 Figure 7 shows the amino acid sequence alignment between MECHP-16 (2069907;
SEQ
ID N0:16) and human beta subunit of Ca' activated K+ channel (GI 1055345; SEQ
ID N0:43);
produced using the multisequence alignment program of LASERGENE software.
Figures 8A and 8B show the amino acid sequence alignment between MECHP-17 (2243917; SEQ iD N0:17) and a homolog of Caenorhabditis elesans K+ channel protein (GI
3292929; SEQ ID N0:44), produced using the multiseque~nce alignment program of LASERGENE software.
Figures 9A and 9B show the amino acid sequence alignment between MECHP-18 (2597476; SEQ ID N0:18) and human aquaporin 9 {GI 2887407; SEQ ID N0:45), produced using the multisequence alignment program of LASERGENE software.
Table 1 shows polypeptide and nucleotide sequence identification numbers (SEQ
ID
NOs), clone identification numbers (clone IDs); cDNA libraries, and cDNA
fragments used to assemble full-length sequences encoding MECHP.
Table 2 shows features of each polypeptide sequence, including potential motifs, homologous sequences, and methods and algorithms used for identification of MECHP.
Table 3 shows the tissue-specific expression patterns of each nucleic acid sequence as determined by northern analysis, diseases, disorders, or conditions associated with these tissues, and the vector into which each cDNA was cloned.
Table 4 describes the tissues used to construct the cDNA libraries from which cDNA
clones encoding MECHP were isolated.
Table 5 shows the tools, programs, and algorithms used to analyze MECHP, along with applicable descriptions, references, and threshold parameters.
DESCRIPTION OF THE II'JVENTION
Before the present proteins, nucleotide sequences" and methods are described, it is understood that this invention is not limited to the particullar machines, materials and methods described, as these may vary. It is also to be understood tlhat the terminology used herein is for the purpose of describing particular embodiments only, and i<.; not intended to limit the scope of the present invention which will be limited only by the appended claims.
It must be noted that as used herein and in the appended claims, the singular forms "a,"
"an," and "the" include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "a host cell" includes a plurality of such host cells, and a reference to "an antibody" is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.

WO 00/12'I11 PCT/US99/20468 Unless deftned otherwise, all technical and scientifc terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any machines, materials, and method:. similar or equivalent to those described herein can be used to practice or test the present invention, the preferred machines, materials and methods are now described. All publications mentioned herein are cited for the purpose of describing and disclosing the cell lines, protocols, reagemts and vectors which are reported in the publications and which might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.
DEFINITIONS
"MECHP" refers to the amino acid sequences of substantially purified MECHP
obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine, equine, and preferably the human species, from any source, whether natural, synthetic, semi-synthetic, or recombinant.
The term "agonist" refers to a molecule which, when bound to MECHP, increases or prolongs the duration of the effect of MECHP. Agonists may include proteins, nucleic acids, carbohydrates, or any other molecules which bind to and modulate the effect of MECHP.
An "allelic variant" is an alternative fonm of the gene encoding MECHP.
Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. Any given natural or recombinant gene may have none, one, or many allelic forms. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
"Altered" nucleic acid sequences encoding MECI';IP include those sequences with deletions, insertions, or substitutions of different nucleotidles, resulting in a polypeptide the same as MECHP or a polypeptide with at least one functional characteristic of MECHP. Tncluded within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding MECHP, and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding MECHP. The encoded protein may also be "altered,"
and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent MECHP. Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues. as long as the biological or immunological activity of MECHP is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid, positively charged amino acids may include lysine and arginine, and amino acids with uncharged polar head groups having similar hydrophilicity values may include leucine, isoleucine, and valine; glycine and allanine; asparagine and glutamine; seriile and threonine; and phenylalanine and tyrosine.
The terms "amino acid" and "amino acid sequence:" refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. In this context, "fragments," "immun~ogenic fragments,"
or "antigenic fragments" refer to fragments of MECHP which are preferably at least 5 to about I S amino acids in length, most preferably at least 14 amino acids, and which retain some biological activity or immunological activity of MECHP. Where "amino acid sequence" is recited to refer to an amino acid sequence of a naturally occurring protein molecule, "aunino acid sequence" and like terms are not meant to limit the amino acid sequence to the complete; native amino acid sequence associated with the recited protein molecule.
"Amplification" relates to the production of additional copies of a nucleic acid sequence.
Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known in the art.
The term "antagonist" refers to a molecule which, when bound to MECHP, decreases the amount or the duration of the effect of the biological or immunological activity of MECHP.
Antagonists may include proteins, nucleic acids, carbohydrates, antibodies, or any other molecules which decrease the effect of MECHP.
The term "antibody" refers to intact molecules as well as' to fragments thereof, such as Fab, F(ab')2, and Fv fragments, which are capable of binding the epitopic determinant. Antibodies that bind MECHP polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen. The polypeptide or oiigopeptide used to immunize an animal (e.g., a mouse, a rat, or a rabbit) can be derived from the translation of RNA, or synthesized chemically, and can be conjugated to a carrier protein if desired. Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (ICLHI). The coupled peptide is then used to immunize the animal.
The term "antigenic determinant" refers to that fragment of a molecule (i.e., an epitope) that makes contact with a particular antibody. When a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies which bind specifically to antigenic determinants {given regions or three-dimensional structures on _14_ WO 00/12711 PCT/US99/2046$
the protein). An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
The term "antisense" refers to any composition containing a nucleic acid sequence which is complementary to the "sense" strand of a specific nucleic acid sequence.
Antisense molecules may be produced by any method including synthesis or transcription. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form duplexes and to block either transcription or translation. The designation "negative" can refer to the antisense strand, and the designation "positive" can refer to the sense strand.
The term "biologically active" refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule. Likewise, "immunologically active"
refers to the capability of the natural, recombinant, or synthetic MECHP, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
The terms "complementary" and "complementarily" refer to the natural binding of polynucleotides by base pairing. For example, the sequence "5' A-G-T 3"' bonds to the complementary sequence "3' T-C-A 5'." Complementarit~,~ between two single-stranded molecules may be "partial," such that only some of the nucleic acids bind, or it may be "complete," such that total complementarity exists between the single stranded molecules. The degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acids strands, and in the design and use of peptide nucleic acid {PNA) molecules.
A "composition comprising a given polynucleotide sequence" and a "composition comprising a given amino acid sequence" refer broadly to any composition containing the given polynucleotide or amino acid sequence. The composition may comprise a dry formulation or an aqueous solution. Compositions comprising polynucleotide sequences encoding MECHP or fragments of MECHP may be employed as hybridization probes. The probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate. In hybridizations, the probe may be deployed in an aqueous solution containing salts (e.g., NaCI), detergents {e.g., sodium dodecyi sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
"Consensus sequence" refers to a nucleic acid sequence which has been resequenced to resolve uncalled bases, extended using the XL-PCR kit (Pe:rkin-Elmer, Norwalk CT) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from the overlapping sequences of more than one Incyte Clone using a computer program for fragment assembly, such as the GELVIEW fragment assembly system (GCG, Madison WI). Some sequences have been both extended and assembled to produce the consensus sequence.
The term "correlates with expression of a polynuc:leotide" indicates that the detection of the presence of nucleic acids, the same or related to a nucleic acid sequence encoding MECHP, by northern analysis is indicative of the presence of nucleic acids encoding MECHP in a sample, and thereby correlates with expression of the transcript from the polynucteotide encoding MECHP.
A "deietion" refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues. or nucleotides.
The term "derivative" refers to the chemical modification of a polypeptide sequence, yr a polynucleotide sequence. Chemical modifications of a polynucleotide sequence can include, for example, replacement of hydrogen by an alkyl, acyl, or amino group. A
derivative pvlynucleotide encodes a polypeptide which retains at least one biological or immunvlogical function of the natural molecule. A derivative polypeptide is one modified by glycosylation, pegylation, or any similar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
The term "similarity" refers to a degree of complementarity. There may be partial similarity or complete similarity. The word "identity" maw substitute for the word "similarity." A
partially complementary sequence that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid is referred to as "substantially similar." The inhibition of hybridization ofthe completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or northern blot, solution hybridization, and the like) under conditions of reduced stringency. A substantially similar sequence or hybridization probe will compete for and inhibit the binding of a completely similar (identical) sequence to the target sequence under conditions of reduced stringency. This is mot to say that conditions of reduced stringency are such that non-specific binding is permitted, as reduced stringency conditions require that the binding of two sequences to one another be a specific (i.e., a selective) interaction.
The absence of non-specific binding may be tested by the u.se of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30%
similarity or identity).
In the absence of non-specific binding, the substantially similar sequence or probe will not hybridize to the second non-complementary target sequence.
The phrases "percent identity" and "% identity" refi~r to the percentage of sequence similarity found in a comparison of two or more amino acidl or nucleic acid sequences. Percent identity can be determined electronically, e.g., by using the MEGALIGN program (DNASTAR) WO OO/i2711 PCT/US99/20468 which creates alignments between two or more sequences according to methods selected by the user, e.g., the clustal method. (See, e.g., Higgins, D.G. arrd P.M. Sharp ( 1988) Gene 73:237-244.) Parameters for each method may be the default parameters provided by MEGALIGN
or may be specified by the user. The clustal algorithm groups sequences into clusters by examining the distances between all pairs. The clusters are aligned painwise and then in groups. The percentage similarity between two amino acid sequences, e.g., sequence A and sequence B, is calculated by dividing the length of sequence A, minus the number of g,ap residues in sequence A, minus the number of gap residues in sequence B, into the sum of the residue matches between sequence A
and sequence B, times one hundred. Gaps of low or of no similarity between the two amino acid sequences are not included in determining percentage similarity. Percent identity between nucleic acid sequences can also be counted or calculated by other methods known in the art, e.g., the Jotun Hein method. (See, e.g., Hein, J. ( 1990) Methods Enzymol. 183:626-645.) Identity between sequences can also be determined by other methods know~a in the art, e.g., by varying hybridization conditions.
"Human artificial chromosomes" (HACs) are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size, and which contain all of the elements required for stable mitotic chromosome segregation and maintenance.
The term "humanized antibody" refers to antibody molecules in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original blinding ability.
"Hybridization" refers to any process by which a strand of nucleic acid binds with a complementary strand through base pairing.
The term "hybridization complex" refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases. A
hybridization complex may be formed in solution (e.g., Cot or Rot analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, flters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
The words "insertion" and "addition" refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively, to the sequence found in the naturally occurring molecule.
"Immune response" can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, wind other signaling molecules, which WO 00/12711 PCT/US99i20468 may affect cellular and systemic defense systems.
The term "microarray" refers to an arrangement of distinct polynucleotides on a substrate.
The terms "element" and "array element" in a microarray context, refer to hybridizable polynucleotides arranged on the surface of a substrate.
The term "modulate" refers to a change in the activity of MECHP. For example, modulation may cause an increase or a decrease in proteiin activity, binding characteristics, or any other biological, functional, or irnrnunological properties of MECHP.
The phrases "nucleic acid" or "nucleic acid seque;nce," as used herein. refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may Ibe single-stranded or double-stranded and may represent the sense or the antisense strand, to pelptide nucleic acid (PNA), or to any DNA-like or RNA-like material. In this context; "fragments" refers to those nucleic acid sequences which comprise a region of unique polynucleotide sequence that specifically identifies SEQ ID N0:19-36, for example, as distinct from any other sequence in the same genome. For IS example, a fragment of SEQ ID N0:19-36 is useful in hybridization and amplification technologies and in analogous methods that distinguish S1:Q ID N0:19-36 from related polynucleotide sequences. A fragment of SEQ ID N0:19-36 is at least about 15-20 nucleotides in length. The precise length of the fragment of SEQ ID NO~:19-36 and the region of SEQ ID
N0:19-36 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment. In some cases, a fragment, when translated, would produce polypeptides retaining some functional characteristic, e.g., antigenicity, or structural domain characteristic, e.g., ATP-binding site, of the full-length polypeptide.
The terms "operably associated" and "operably linked" refer to functionally related nucleic acid sequences. A promoter is operably associated or operably linked with a coding sequence if the promoter controls the translation of the encoded polyp~eptide. While operably associated or operably linked nucleic acid sequences can be contiguous and in the same reading frame, certain genetic elements, e.g., repressor genes, are not contiguousily linked to the sequence encoding the polypeptide but still bind to operator sequences that contrcd expression of the polypeptide.
The term "oligonucleotide" refers to a nucleic acidl sequence of at least about 6 nucleotides to 60 nucleotides, preferably about 15 to 30 nucleotides, and most preferably about 20 to 25 nucleotides, which can be used in PCR amplification or in a hybridization assay or microarray.
"Oligonucleotide" is substantially equivalent to the terms "'amplimer,"
"primer," "oligomer," and "probe," as these terms are commonly defined in the art.
"Peptide nucleic acid" (PNA) refers to an antisense; molecule or anti-gene agent which comprises an oligonucleotide of at least about S nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition.
PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
The term "sample" is used in its broadest sense. ,A sample suspected of containing nucleic acids encoding MECHP, or fragments thereof, or MECHI' itself, may comprise a bodily fluid; an extract from a cell, chromosome, organelle, or membrane isolated from a cell;
a cell; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
The terms "specific binding" and "specifically binding" refer to that interaction between a protein or peptide and an agonist, an antibody, or an antagonist. The interaction is dependent upon the presence of a particular structure of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A," the presence of a polypeptide containing the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds 1S to the antibody.
The term "stringent conditions" refers to conditions which permit hybridization between polynucleotides and the claimed polynucleotides. Stringent conditions can be defined by salt concentration, the concentration of organic solvent, e.g., forrnamide, temperature, and other conditions well known in the art. In particular, stringency can be increased by reducing the concentration of salt, increasing the concentration of form~amide, or raising the hybridization temperature.
The term "substantially purified" refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least about 60%
free, preferably about 7S% free, and most preferably aboul: 90% free from other components with which they are naturally associated.
A "substitution" refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.
"Substrate" refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beadis, gels, tubing, plates, polymers, microparticles and capillaries. The substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotide;s or polypeptides are bound.
"Transformation" describes a process by which exogenous DNA enters and changes a recipient cell. Transformation may occur under natural or ;artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell. The method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to, viral infection, electroporation, heat shock, lipofection, and particle bombardment.
The term "transformed" cells includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods oftime.
A "variant" of MECHP polypeptides refers to an. amino acid sequence that is altered by one or more amino acid residues. The variant may have "conservative" changes, wherein a I0 substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have "nonconservative" changes (e.g., replacement of glycine with tryptophan). Analogous minor variations m,ay also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological or immunological activity may be found using 15 computer programs well known in the art, for example, L,ASERGENE software (DNASTAR).
The term "variant," when used in the context of a~ po(ynucleotide sequence, may encompass a polynucleotide sequence related to MECHP. This defnition may also include, for example, "allelic" (as defined above}, "splice," "species," or "polymorphic"
variants. A splice variant may have significant identity to a reference molecule, but will generally have a greater or 20 lesser number of polynucleotides due to alternate splicing; of exons during mRNA processing. The corresponding polypeptide may possess additional functional domains or an absence of domains.
Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides generally will have significant amino acid identity relative to each other. A
polymorphic variant is a variation in the polynucleotide sequence of a particular gene between 25 individuals of a given species. Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the poiynucleotide sequence varies by one base.
The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
THE INVENTION
30 The invention is based on the discovery of new human membrane channel proteins (MECHP), the polynucleotides encoding MECHP, and the. use of these compositions for the diagnosis, treatment, or prevention of cell proliferative, im~munelinflammatory, transport/secretory, osmoregulatory, muscular, cardiovascular, and neurological disorders.
Table 1 lists the Incyte clones used to assemble full length nucleotide sequences encoding WO 00/i2711 PCTIUS99/20468 MECHP. Columns 1 and 2 show the sequence identification numbers (SEQ ID NOs}
of the polypeptide and nucleotide sequences, respectively. Column 3 shows the clone IDs of the Incyte clones in which nucleic acids encoding each MECHP were identified, and column 4 shows the cDNA libraries from which these clones were isolated. Column 5 shows Incyte clones and their corresponding cDNA libraries. Clones for which cDNA libraries are not indicated were derived from pooled cDNA libraries. The clones in column S were used to assemble the consensus nucleotide sequence of each MECHP and are useful as fragments in hybridization technologies.
The columns of Table 2 show various properties of each of the polypeptides of the invention: column 1 references the SEQ ID.NO; column 2 shows the number of amino acid residues in each polypeptide; column 3 shows potential phosphorylation sites;
column 4 shows potential glycosylation sites; column 5 shows the amino ;acid residues comprising signature sequences and motifs; column 6 shows the identity of each polypeptide; and column 7 shows analytical methods used to identify each polypeptide through sequence homology and protein motifs.
IS MECHP-1 has chemical and structural similarity with rat glutamic acid-rich protein (GI
2924369; SEQ ID N0:37). In particular, MECHP-1 and rat glutamic acid-rich protein share 15%
overall identity. As shown in Figure 1, BLAST analysis identifies regions of MECHP-1 and rat glutamic acid-rich protein which share 27-30% identity. 'These regions extend from residue V12 through T163, P266 through 6344, P461 through E548, and E653 through 6709 in MECHP-i.
As shown in Figure 2, MECHP-2 has chemical and structural similarity with Drosonhila voltage-gated potassium channel (GI 116443; SEQ ID NO:38) and P, penicillatus potassium channel a-subunit (GI 1763619; SEQ ID N0:39). In particular, MECHP-2 shares 18% identity with Drosophila voltage-gated K+ channel, and 17% identity with P.
penicillatus K+ channel a-subunit. In particular, MECHP-2 shares 27% identity with Drosophila voltage-gated potassium channel and P. penicillatus potassium channel a-subunit over the first 133 residues, from M1 through T 133 in MECHP-2.
As shown in Figures 3A and 3B, MECHP-3 has chemical and structural similarity with rat calcium-activated potassium channel rSK3 (GI 2564072; SEQ ID N0:40). In particular, MECHP-3 and rat rSk;3 share 40% identity. MECHP-3 and rat rSk;3 also share a canonical ion pore (P) region, including a GYG potassium ion selectivity sequence, from residue W192 through 6213 in MECHP-3.
As shown in Figures 4A, 4B, and 4C, MECHP-4 h;as chemical and structural similarity with Drosophila voltage-gated potassium channel (GI 116f.43; SEQ ID N0:38) and P. penicillatus potassium channel a-subunit (GI 1763619; SEQ ID N0:39). In particular, MECHP-4 shares 28%

identity with Drosophila voltage-gated K* channel, and 2Ei% identity with P.,penicillatus K*
channel a-subunit, respectively. MECHP-4, Droso~hila voltage-gated K* channel, and_P.
penicillatus K+ channel a-subunit also share a GYG potassium ion selectivity sequence from residue 6372 through 6374 in MECHP-4.
As shown in Figures SA and SB, MECHP-5 has chemical and structural similarity with rat aquaporin 7 (Gi 2350843; SEQ ID N0:41). In particular, MECHP-5 and rat aquaporin 7 share 74% identity.
As shown in Figures 6A arid 6B, MECHP-7 has chemical and structural similarity with mouse connexin 30.3 (GI 192647; SEQ ID N0:42). In particular, MECHP-7 and mouse connexin 30.3 (GI 192647) share 84% identity.
As shown in Figure 7, MECHP-16 has chemical and structural similarity with human beta subunit of Ca* activated K* channel (GI 1055345; SEQ ID N0:43). In particular, MECHP-16 and human beta subunit of Ca* activated K+ channel share 40%. identity.
As shown in Figures 8A and 8B, MECHP-17 has chemical and structural similarity with a homolog of C. elegans K* channel protein (GI 3292929; SEQ ID N0:44). In particular, MECHP-17 and the specified homolog of C. eleQans K* channel protein share 47%
identity.
As shown in Figures 9A and 9B, MECHP-I8 has chemical and structural similarity with human aquaporin 9 (GI 2887407; SEQ ID N0:45). In particular, MECHP-18 and human aquaporin 9 share 46% identity.
The columns of Table 3 show the tissue-specificity and diseases, disorders, or conditions associated with nucleotide sequences encoding MECHP. The first column of Table 3 lists the nucleotide SEQ ID NOs. Column 2 lists tissue categories which express MECHP as a fraction of total tissue categories expressing MECHP. Column 3 lists diseases, disorders, or conditions associated with those tissues expressing MECHP. Column 4 lists the vectors used to subclone the cDNA library. Northern analysis shows the expression of SEQ ID N0:34 in only 7 libraries, of which 6 (86%} are associated with cell proliferation. Two of these libraries are associated with brain tissue, one with pancreatic islet cells, one with kidney tissue, one with fetal lung tissue, one with ovarian tissue, and one with adrenal tissue. Northern analysis shows the expression of SEQ
ID N0:36 in only 3 libraries, one of which is associated with ovarian tumor tissue, one with developing lung tissue, and one with gastrointestinal tissue associated with inflammation. Of particular note is the enriched expression of MECHP in neural and neuroendocrine tissue, most prominently the neural tissue-specific expression of SEQ IL) N0:30.
The columns of Table 4 show descriptions of the tissues used to construct the cDNA
libraries from which cDNA clones encoding MECHP were ;isolated. Column 1 references the nucleotide SEQ ID NOs, column 2 shows the cDNA libraries from which these clones were isolated, and column 3 shows the tissue origins and other descriptive information relevant to the cDNA libraries in column 2.
The following fragments of the nucleotide sequences encoding MECHP are useful, for example, in hybridization or amplification technologies to identify SEQ ID
N0:19-36, and to distinguish between SEQ ID NO: f 9-36 and related polynucleotide sequences.
The useful fragments include the fragment of SEQ ID N0:19 from about nucleotide 764 to about nucleotide 808; the fragment of SEQ ID N0:20 from about nucleotide 523 to about nucleotide 582; the fragment of SEQ ID N0:21 from about nucleotide 628 to .about nucleotide 669;
the fragment of SEQ ID N0:22 from about nucleotide 779 to about nucleotide 826; the fragment of SEQ ID
N0:23 from about nucleotide 64 to about nucleotide 108; 'the fragment of SEQ
ID N0:24 from about nucleotide 1133 to about nucleotide 1180; the fragment of SEQ ID N0:25 from about nucleotide 656 to about nucleotide 700; the fragment of SEQ ID N0:26 from about nucleotide 153 to about nucleotide 197; the fragment of SEQ ID N0:27 from about nucleotide 2160 to about nucleotide 22 i 9; the fragment of SEQ ID N0:28 from about nucleotide 1275 to about nucleotide 1322; the fragment of SEQ ID N0:29 from about nucleotide 313 to about nucleotide 348; the fragment of SEQ ID N0:30 from about nucleotide 994 to about nucleotide 1041;
the fragment of SEQ ID N0:31 from about nucleotide 443 to about nucleotide 478; the fragment of SEQ ID
N0:32 from about nucleotide l I75 to about nucleotide 12Ct7; the fragment of SEQ ID N0:34 from about nucleotide 381 to about nucleotide 425; the fragment: of SEQ ID N0:35 from about nucleotide 17 to about nucleotide 61; and the fragment of SEQ ID N0:36 from about nucleotide 54 to about nucleotide 98. The polypeptides encoded by th.e fragments of SEQ
ID N0:19, SEQ ID
N0:20, SEQ ID N0:21, SEQ ID N0:22, SEQ ID N0:23, SEQ ID N0:24, SEQ ID N0:25, SEQ
ID N0:26, SEQ ID N0:27, SEQ ID N0:28, SEQ ID N0:2~>, SEQ ID N0:30, SEQ ID
N0:34, SEQ ID N0:35, AND SEQ ID N0:36 are useful, for example, as immunogenic peptides.
The invention also encompasses MECHP variants. A preferred MECHP variant is one which has at least about 80%, more preferably at least abou~,t 90%, and most preferably at least about 95% amino acid sequence identity to the MECHP amino acid sequence, and which contains at least one functional or structural characteristic of MECHP.
The invention also encompasses polynucleotides which encode MECHP. In a particular embodiment, the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID N0:19-36, which encodes MECHP.
The invention also encompasses a variant,of a polynucleotide sequence encoding MECHP. In particular, such a variant polynucleotide sequence will have at least about 70%, more preferably at least about 85%. and most preferably at iea;st about 95%
polynucleotide sequence identity to the polynucleotide sequence encoding MECHf. A particular aspect of the invention encompasses a variant of a sequence selected from the group consisting of SEQ
ID N0:19-36 which has at least about 70%, more preferably at least about 85%, and most preferably at least about 95% poiynucleotide sequence identity to a sequence selected from the group consisting of SEQ ID N0:19-36. Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of MECHP.
It will be appreciated by those skilled in the art that as a result of the degeneracy of the genetic code, a multitude of polynucleotide, sequences encoding~MECHP, some bearing minimal similarity to the polynucleotide sequences of any known ;and naturally occurring gene, may be produced. Thus, the invention contemplates each and every possible variation of polynucieotide sequence that could be made by selecting combinations biased on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide sequence of naturally occurring MECHP, ;and ail such variations are to be considered as being specifically disclosed.
Although nucleotide sequences which encode ME',CHP and its variants are preferably capable of hybridizing to the nucleotide sequence of the naturally occurring MECHP under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding MECHP or its derivatives possessing a substantially different codon usage, e.g., inclusion of non-naturally occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prol<:aryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host.
Other reasons for substantially altering the nucleotide sequence encoding M:ECHP and its derivatives without altering the encoded amino acid sequences include the production of RNA
transcripts having more desirable properties, such as a greater half life, than transcripts produced from the naturally occurring sequence.
The invention also encompasses production of DNA sequences which encode MECHP
and MECHP derivatives, or fragments thereof, entirely by synthetic chemistry.
After production, the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art. Moreover, synthetic chemistry may be used to introduce mutations into a sequence encoding MECHP or any fragment thereof.
Also encompassed by the invention are polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, iin particular, to those shown in SEQ ID
NO: I9-36, or to a fragment of SEQ ID N0:19-36, under various conditions of stringency. (See, e.g., Wahi, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods Enzymol. 152:507-51 I .) For example, stringent: salt concentration will ordinarily be less than about 750 mM NaCI and 75 mM trisodium citrate, preferably less than about 500 mM NaCI
and 50 mM trisodium citrate, and most preferably less than about 250 mM NaCI
and 25 mM
trisodium citrate. Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, while high stringency hybridization can Ibe obtained in the presence of at least about 35% formamide, and most preferably at least about SO% formamide.
Stringent temperature conditions will ordinarily include temperatures of at least about 30°C, more preferably of at least about 37°C, and most preferably of at least about 42°C. Varying additional parameters, such as i0 hybridization time, the concentration of detergent, e.g:, sodium dodecyl sulfate {SDS), and the inclusion or exclusion of carrier DNA, are well known to chose skilled in the art. Various levels of stringency are accomplished by combining these various conditions as needed.
In a preferred embodiment, hybridization will occur at 30°C in 750 mM NaCI, 75 mM
trisodium citrate, and I%
SDS. In a more preferred embodiment, hybridization will occur at 37°C
in 500 mM NaCI, S0 mM
IS trisodium citrate, l% SDS, 35% formamide, and 100 p.g/rn~l denatured salmon sperm DNA
(ssDNA). In a most preferred embodiment, hybridization will occur at 42°C in 250 mM NaCI, 25 mM trisodium citrate, 1% SDS, 50 % formamide, and 200 pg/ml ssDNA. Useful variations on these conditions will be readily apparent to those skilled in the art.
The washing steps which follow hybridization can also vary in stringency. Wash 20 stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature. For example, stringent salt concentration for the wash steps willl preferably be less than about 30 mM
NaCI and 3 mM trisodium citrate, and most preferably less than about 15 mM
NaCI and 1.5 mM
trisodium citrate. Stringent temperature conditions for the wash steps will ordinarily include 25 temperature of at least about 25°C, more preferably of at least about 42°C, and most preferably of at least about 68°C. In a preferred embodiment, wash steps. will occur at 25°C in 30 mM NaCI, 3 mM trisodium citrate, and 0. I % SDS. In a more preferred embodiment, wash steps will occur at 42°C in 1 S mM NaCI, 1.5 mM trisodium citrate, and 0.1 % SDS. In a most preferred embodiment, wash steps will occur at 68°C in 15 mM NaCI, 1.5 mM trisodium citrate, and 0.1 % SDS.
30 Additional variations on these conditions will be readily apparent to those skilled in the art.
Methods for DNA sequencing are well known in the art arid may be used to practice any of the embodiments of the invention. The methods may employ such enzymes as the Klenow fragment of DNA polymerise I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerise (Perkin-Elmer), thetrnostable T7 polymerise (Amersham Pharmacia Biotech, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Life Technologies, Gaithersburg MD).
Preferably, sequence preparation is automated with machines such as the Robbins Hydra microdispenser (Robbins Scientific, Sunnyvale CA), Hamilton MICROLAB 2200 (Hamilton, Reno NV), Pettier Thermal S Cycler 200 (PTC200; MJ Research, Watertown MA) and the ABI CATALYST 800 (Perkin-Elmer). Sequencing is then carried out using either ABI a73 or 377 DNA
sequencing systems (Perkin-Eimer), the MEGABACE 1000 DNA sequencing system (Molecular Dynamics, Sunnyvale CA}, or other systems known in the art. The resulting sequences are analyzed using a variety of algorithms which are well known, in the art. (See, e.g., Ausubel, F.M. ( I 997) Short Protocols in Molecular Biolo v, John Wiley & Sons, New York NY, unit 7.7;
Meyers, R.A.
( 1995) Molecular Biology and Biotechnolo~v, Wiley VCH, New York NY, pp. 856-853.) The nucleic acid sequences encoding MECHP may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements. For example, one method which IS may be employed, restriction-site PCR, uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector. (Se:e, e.g., Sarkar, G.
(1993} PCR Methods Applic. 2:318-322.) Another method, inverse PCR, uses primers that extend in divergent directions to amplify unknown sequence from a circularized template. The template is derived from restriction fragments comprising a known genomic locus and surrounding sequences. (See, e.g., Triglia, T. et al. ( 1988) Nucleic Acids Res. 16:8186.) A third method, capture PCR, involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al. (1991 ) PCR Methods Applic.
1: i 11-119.) In this method, multiple restriction enzyme digestions and ligations may be used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR.
2S Other methods which may be used to retrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (I991) Nucleic Acids Res. 19:30SS-3060). Additionally, one may use PCR, nested primers, and PROMOTERFINDER libraries (Clonte,ch, Palo Alto CA) to walk genomic DNA. This procedure avoids the need to screen libraries arid is useful in finding intron/exon junctions. For ail PCR-based methods, primers may be designed using commercially available software, such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about SO% or more, and to anneal to the template at temperatures of about 68°C to 72°C.
When screening for full-length cDNAs, it is preferable to use libraries that have been size-selected to include larger cDNAs. In addition, random-primed libraries, which often include sequences containing the 5' regions of genes, are preferable for situations in which an oiigo d(T) library does not yield a full-length cDNA. Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
Capillary electrophoresis systems which are commercially available may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR
products. In particular, capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide-specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths. Output/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQIJENCE NAVIGATOR, Perkin-Elmer), IO and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled. Capillary electrophoresis is especially preferable for sequencing small DNA fragments which may be present in limited amounts in a particular sample.
In another embodiment of the invention, polynucleotide sequences or fragments thereof which encode MECHP may be cloned in recombinant DNA molecules that direct expression of MECHP, or fragments or functional equivalents thereof, in appropriate host cells. Due to the inherent degeneracy of the genetic code, other DNA sequences which encode substantially the same or a functionally equivalent amino acid sequence may be produced and used to express MECHP.
The nucleotide sequences of the present invention can be engineered using methods generally known in the art in order to alter MECHP-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product. DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences. For example, oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce splice variants, and so forth.
In another embodiment, sequences encoding MECFtP may be synthesized, in whole or in part, using chemical methods well known in the art. (See, e.g., Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223, and Horn, T. et al. (1f80) Nucleic Acids Symp. Ser.
7:225-232.) Alternatively, MECHP itself or a fragment thereof may be synthesized using chemical methods. For example, peptide synthesis can be performed using various solid-phase techniques. (See, e.g., Roberge, J.Y. et al. (1995) Science 26.9:202-204.) Automated synthesis may be achieved using the ABI 431A peptide synthesizer (Perkin-Elmer).
Additionally, the amino acid sequence of MECHP, or any part thereof, may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant poiypeptide.
The peptide may be substantially purified by preparative high performance liquid chromatography. (See, e.g, Chiez, R.M. and F.Z. Regnier ( 1990) Methods Enzymol. 182:392-421.) The composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton, T. ( 1984) Proteins. Structures and Molecular Properties, WH
Freeman, New York NY.) In order to express a biologically active MECHP, the nucleotide sequences encoding MECHP or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translationai control of the inserted coding sequence in a suitable host. These elements inclucle regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions in the vector and in pofynucleotide sequences encoding MECHP. Such elements may vary in their strength and specificity. Specific initiation signals may also be used to achieve more efficient translation of sequences encoding MECHP. Such signals include the A'CG initiation codon and adjacent sequences, e.g. the Kozak sequence. In cases where sequences encoding MECHP
and its initiation codon and upstream regulatory sequences are inserted into. the appropriate expression vector, no additional transcriptional or translational control signals m;ay be needed.
However, in cases where only coding sequence, or a fragment thereof, is inserted, exogenous translational control signals including an in-frame ATG initiation codon should be provided by the vector.
Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic.
The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host cell system used. (See, e.g., Scharf, D. et al. ( 1994) Results Probl. Cell Differ.
20:125-162.) Methods which are well known to those skilled in Nhe art may be used to construct expression vectors containing sequences encoding MECHF' and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA
techniques, synthetic techniques, and in vivo genetic recombination. (See, e.g., Sambrook, J. et al. (1989) Molecular Cloning A Laboratory Manual, Cold Spring Harbor Press, Flainview NY, ch. 4, 8, and 16-17; Ausubel, F.M. et al. (1995) Current Protocols in Molecular Biolosv, John Wiley & Sons, New York NY, ch. 9, 13, and 16.) A variety of expression vector/host systems may be. utilized to contain and express sequences encoding MECHP. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasrnid, or cosmid DNA
expression vectors; yeast transformed with yeast expression vectors.; insect cell systems infected with viral expression vectors (e.g., bacuiovirus}; plantceli systems. transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g:, Ti or pBR322'piasmids); or animal cell systems. The invention is not limited by the host cell employed.
In bacterial systems, a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding MECHP.
For example, routine cloning, subcloning, and propagation of polynucleotide sequences encoding MECHP can be achieved using a multifunctional E. coli.vector such a;s PBLUESCRIPT
(Stratagene, La Jolla CA) or pSPORTI plasmid (Life Technologies). Ligation of sequences encoding MECHP into the vector's multiple cloning site disrupts the IacZ gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules.
In addition, these vectors may be useful for in vitro transcription. dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence. (See, e.g., Van Heeke, G.
and S.M. Schuster.(1989) J. Biol. Chem. 264:5503-5509.;1 When large quantities of MECHP are needed, e:g. for the production of antibodies, vectors which direct high level expression of MECHP may be used. For example, vectors containing the strong, inducible TS or bacteriophage promoter may be used.
Yeast expression systems may be used for production of MECHP. A number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH
promoters, may be used in the yeast Saccharomvces cerevisiae or Pichia pastoris. In addition, such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for ;stable propagation.
(See, e.g., Ausubel, 1995, supra; Bitter, G.A. et al. ( 1987) Methods Enzymol. i 53:516-544; and Scorer, C.A. et al.
(1994) Bio/Technology 12:181-184.}
Plant systems may also be used for expression of 1VIECHP. Transcription of sequences encoding MECHP may be driven by viral promoters, e.g., the 35S and 19S
promoters of CaMV
used alone or in combination with the omega leader sequence from TMV
(Takamatsu, N. (1987) EMBO J. 6:307-311 ). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used. (See, e:g., Coruzzi, G. et aL (1984) EMBO J.
3:1671-1680;
Broglie, R. et al. (1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105.) These constructs can be introduced into plant cells by direct DNA
transformation or pathogen-mediated transfection. (See, e.g., The McGraw Hill Yearbook of Science and Tech~ nolo~y (1992) McGraw Hill, New York rdY, pp. 191-196.) WO 00/12711 PCTIUS99/204b$
In mammalian cells, a number of viral-based expression systems may be utilized. In cases where an adenovirus is used as an expression vector, sequences encoding MECHP
may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential E1 or E3 region of the viral genome may be used to obtain infective virus which expresses MECHP in host cells. (See, e.g., Logan, J. and T. Shenk ( 1984) Proc. Natl. Acad. Sci. USA 81:3655-3659.) In addition, transcription enhancers, such as the Rous sarcoma virus {RSV) enhancer, may be used to increase expression in mammalian host cells. SV40 or EBV-based vectors may also be used for hiigh-level protein expression.
Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of DNA than can be contained in and expressed from a plasmid. HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes. (See, e.g., Harrington, J.3.
et al. ( 1997) Nat.
Genet. 15:345-355.) For long term production of recombinant proteins iin mammalian systems, stable expression of MECHP in cell lines is preferred. For example, sequences encoding MECHP can be transformed into cell lines using expression vectors which :may contain viral origins of replication and/or endogenous expression element's and a selectable marker gene on the same or on a separate vector. Following the introduction of the vector, cells may be al lowed to grow for about 1 to 2 days in enriched media before being switched to selective media. The purpose of the selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences. Resistant clones of stably transformed cells may be propagated using tissue culture techniques apI>ropriate to the cell type.
Any number of selection systems may be used to recover transformed cell lines.
These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk or apP cells, respectively.
(See, e.g., Wigler, M. et al. ( 1977) Cell 1 I :223-232; Lowy, I. et al. ( I980) Cell 22:817-823.) Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection.
For example, dhfr confers resistance to methotrexate; neo confers resistance to the aminoglycosides neomycin and G-418;
and als or pat confer resistance to chlorsulfuron and phasphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M. et al. ( 1980) Proc. Natl. Acad. Sci. USA
77:3567-3570;
Colbere-Garapin, F. et al. (1981) J. Mol. Biol. 150:1-14.) Additional selectable genes have been described, e.g., trpB and hisD, which alter cellular requirements for metabolites. (See, e.g., Hartman, S.C. and R.C. Mulligan ( 1988) Proc. Natl. Acad. Sci. USA 85:8047-8051.) Visible markers, e.g., anthocyanins, green fluorescent proteins (GFP; Clontech), (3 glucuronidase and its substrate f3-glucuronide, or luciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system. (See, e.g., Rhodes, C.A. (1995) Methods Mol. Biol. 55:121-131.) Although the presencelabsence of marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed. For example, if the sequence encoding MECHP is inserted within a marker gene sequence, transformed cells containing sequences encoding MECHP' can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding MECHP under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
In general, host cells that contain the nucleic acid aequence encoding MECHP
and that express MECHP may be identifed by a variety of procedures known to those of skill in the art.
These procedures include, but are not limited to, DNA-DN'A or DNA-RNA
hybridizations, PCR
amplification, and protein bioassay or immunoassay techniiques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences.
immunological methods for detecting and measuring the expression of MECHP
using either specific polyclonal or monoclonal antibodies are known in the art.
Examples of such techniques include enzyme-linked immunosorbent assays (.ELISAs);
radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on MECHP is preferred, but a competitive binding assay may be employed. These a.nd other assays are well known in the art. (See, e.g., Hampton, R. et al. ( 1990) Serological Methods. a Laboratory Manual, APS Press, St. Paul MN, Sect. IV; Coligan, J.E. et ai. (1997) Current Protocols in Immunolo y, Greene Pub.
Associates and Wiley-Interscience, New York NY; and Pound, J.D. ( 1998) Immunochemical Protocols, Humana Press, Totowa NJ.) A wide variety of labels and conjugation techniques. are known by those skilled in the art and may be used in various nucleic acid and amino acid assays. Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding MECHP
include oligolabeling, nick translation, end-labeling, or PCR. amplification using a labeled nucleotide. Alternatively, the sequences encoding MECHP, or any fragments thereof, may be cloned into a vector for the production of an mRNA probe. .Such vectors are known in the art, are commercially available, and may be used to synthesize RNA, probes in vitro by addition of an appropriate RNA potymerase such as T7, T3, or SP6 and labeled nucleotides.
These procedures may be conducted using a variety of commercially availalble kits, such as those provided by Amersham Pharmacia Biotech, Promega (Madison WI), and US Biochemical. Suitable reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the tike.
Host cells transformed with nucleotide sequences encoding MECHP maybe cultured under conditions suitable for the expression and recovery .of the protein from cell culture. The protein produced by a transformed cell may be secreted or' retained intracel lularly depending on the sequence and/or the vector used. As will be understood by those of skill in the art, expression vectors containing polynucleotides which encode MECHP' may be designed to contain signal sequences which direct secretion of MECHP through a prokaryotic or eukaryotic cell membrane.
In addition, a host cell strain may be chosen for its. ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion.
Such modifications IS ofthe polypeptide include, but are not limited to, acetylation, carboxylation, glycosyiation, phosphorylation, lipidation, and acylation. Post-transtational processing which cleaves a "prepro"
form of the protein may also be used to specify protein tarl;eting, folding, and/or activity.
Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HE:K293, and WI38), are available from the American Type Culture Collection (ATCC, Manassas 'JA) and may be chosen to ensure the correct modification and processing of the foreign protein.
In another embodiment ofthe invention, natural, modified, or recombinant nucleic acid sequences encoding MECHP may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems. For example, a chimeric MECHP
protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of MECHP activity.
Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available amity matrices. Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodufin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (H,A). GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione, maltose, phenylarsine oxide, calmodulin, and metal-chelate resins, respectively. FLAG, c-myc; and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commercially available monoclonal and polyclonai antibodies that specifically recognize these epitope tags. A

fusion protein may also be engineered to contain a proteolytic cleavage site located between the MECHP encoding sequence and the heterologous protein sequence, so that MECHP
may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel ( 1955, supra, ch 10). A
variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
In a further embodiment of the invention, synthesis of radiolabeled MECHP may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract systems (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, preferably 'SS-methionine.
Fragments of MECHP may be produced not only by recombinant production, but also by direct peptide synthesis using solid-phase techniques. (See, e.g., Creighton, supra pp. 55-60.) Protein synthesis may be performed by manual techniques. or by automation.
Automated synthesis 1 S may be achieved, for example, using the ABI 43 I A Peptife Synthesizer (Perkin-Elmer). Various fragments of MECHP may be synthesized separately and then combined to produce the full length molecule.
THERAPEUTICS
Chemical and structural similarity, e.g., in the context of sequences and motifs, exists between regions of MECHP and human membrane channel proteins. In addition, the expression of MECHP is closely associated with nervous, reproductive, and gastrointestinal tissues; fetal development; and neurological, immune/inflammatory, and cell proliferative disorders, including cancer. Therefore, MECHP appears to play a role in cell proliferative, immune/inflammatory, transportlsecretory, osmoregulatory, muscular, cardiovascular, and neurological disorders. In the treatment of disorders associated with increased MECHP expression or activity, it is desirable to decrease the expression or activity of MECHP. In the treatment of disorders associated with decreased MECHP expression or activity, it is desirable to increase the expression or activity of MECHP.
Therefore, in one embodiment, MECHP or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of MECHP. Examples of such disorders include, but are not limited to, a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, WO 00112711 PCT/US99/2046$
leukemia, lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma, and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix; gall bladder, ganglia, gastrointestinal tract, heart, kidney, Liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus;
an immune/inflammatory disorder such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmmne thyroiditis, autoimmune polyenodocrinopathy-candidiasis-ectodermal dystrophy (:APECED), bronchitis, choiecystitis, contact dermatitis, Crohn's disease, atopie dermatitis, derrnatomyositis, diabetes mellitus, emphysema, episodic lymphopenia with lymphocytotoxins, erythroblastosis fetaiis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpasture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophiIia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma; a transport/secretory diisorder such as akinesia, amyotrophic lateral sclerosis, ataxia telangiectasia, cystic fibrosis, Becker's muscular dystrophy, Bell's palsy, Charcot-Marie Tooth disease, Chediak-Higashi syndrome., diabetes mellitus, diabetes insipidus, diabetic neuropathy, hyperkalemic periodic paralysis, nonnokalemic periodic paralysis, malignant hyperthermia, multidrug resistance, myotonic dystrophy, <;atatonia, dystonias, peripheral neuropathy, neuroftbromatosis, postherpetic neuralgia, tril;eminal neuropathy, sarcoidosis, sickle cell anemia, toxic shock syndrome, Wilson's disease, cataracts, infertility, pulmonary artery stenosis, sensorineural autosomal deafness, hyperglycemia, hypoglycemia, goiter, Cushing's disease, glucose-galactose malabsorption syndrome, hypercholesterolemia, and allergies, including hay fever, asthma, and urticaria (hives); an osmoregulatory disorder such as diabetes insipidus, diarrhea, peritonitis, chronic renal failure, Addison's disease, SIADH, hypoaldosteronism, hyponatremia, adrenal insufficiency, hypothyroidism, hypernatremia, hypokalemia, Barter's syndrome, metabolic acidosis, metabolic alkalosis, encephalopathy, edema, hypotension, and hypertension; a muscular disorder such ass cardiomyopathy, myocarditis, Duchenne's muscular dystrophy, Becker's muscular dystrophy, myotonic dystrophy, central core disease, nemaline myopathy, centronuclear myopathy, lipid myopathy, mitochondrial myopathy, infectious myositis, polymyositis, dermatornyositis, inclusion body myositis, thyrotoxic myopathy, and ethanol myopathy; a cardiovascular disorder such as~ arteriovenous fistula, atherosclerosis, hypertension, vasculitis, Raynaud's disease, aneurysms, ;arterial dissections, varicose veins, thrombophlebitis and phlebothrombosis, vascular tumors, and complications of thrombolysis, balloon angioplasty, vascular replacement, and coronary artery bypass graft surgery; congestive S heart failure, ischemic heart disease, angina pectoris, myocardial infaretian, hypertensive heart disease, degenerative valvular heart disease, calcific aortic valve stenosis, congenitally bicuspid aortic valve, mitral annular calcification, rnitral valve prolapse, rheumatic fever and rheumatic heart disease, infective endocarditis, nonbacterial thromb~otic endocarditis, endocarditis of systemic lupus erythematosus, carcinoid heart disease, ca~rdiomyopathy, myocarditis, pericarditis, neoplastic heart disease, congenital heart disease, cornpiications of cardiac transplantation;
congenital lung anomalies, atelectasis, pulmonary congestion and edema, pulmonary embolism, pulmonary hemorrhage, pulmonary infarction, pulmonan~ hypertension, vascular sclerosis, obstructive pulmonary disease, restrictive pulmonary disease, chronic obstructive pulmonary disease, emphysema, chronic bronchitis, bronchial asthma, bronchiectasis, bacterial pneumonia, IS viral and mycoplasmal pneumonia, lung abscess, pulmonary tuberculosis, diffuse interstitial diseases, pneumoconioses, sarcoidosis, idiopathic pulmonary fibrosis, desquarnative interstitial pneumonitis, hypersensitivity pneumonitis, pulmonary eosinophilia bronchiolitis obliterans-organizing pneumonia, diffuse pulmonary hemorrhage syndromes, Goodpasture's syndromes, idiopathic pulmonary hemosiderosis, pulmonary involvement in collagen-vascular disorders, pulmonary alveolar proteinosis,:lung tumors, inflammatory and noninflammatory pleural effusions, pneuntothorax, pleural tumors, drug-induced lung disease, radiation-induced lung disease, and complications of lung transplantation; and a neurological disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms, Alzheimer's disease, Pick's disease, Down syndrome, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis arnd other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidura) abscess, suppurative intracraniaI thrombophlebitis, myelitis and radiculitis, viral central nervous system disease; prion diseases including kuru, Cre;utzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome; fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neuroflbromatosis, tuberous sclerosis, cer~ebelloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system, cerebral palsy; neuroskeletal disorders, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases; neuromuscular disorders including spinal muscular atrophy, carpal tunnel syndrome, monomeuriti:~ multiplex; muscular dystrophies such as Duchenne's, myotonic facioscapulohumeral, oculopharyngeal, scapuloperoneal, congenital. distal, and ocular; congenital and metabolic myopathies, myotonia, peripheral nervous system disorders, dermatomyositis and polymyositis; inherited, metabolic, endocrine, and toxic myopathies;
myasthenia gravis, periodic paralysis; mental disorders including depression and bipolar disorder, and mood, anxiety, and schizophrenic disorders; seasonal affective disorder (SAD); akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesi.a, dystonias, paranoid psychoses, postherpetic neuralgia, and Tourette's disorder; abnormalities in electrolytes such as calcium, phosphate, magnesium, and potasium; hypo- and hyperfunction of the thyroid, adrenal, parathyroid, and pituitary; and primary and metastatic neaplasms.
In another embodiment, a vector capable of expressing MECHP or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of MECHP including, but: not limited to, those described above.
In a further embodiment, a pharmaceutical composition comprising a substantially purified MECHP in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of MECHP
including, but not limited to, those provided above.
In still another embodiment, an agonist which modulates the activity of MECHP
may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of MECHP including, but not limited to, those Iiste~d above.
In a further embodiment, an antagonist of MECHP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of MECHP: Such disorders may include, but are not limited to, those discussed above. In one aspect, an antibody which specifically binds MECHP may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to cells or tissue which express MECHP.
In an additional embodiment, a vector expressing the complement of the polynucleotide encoding MECHP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of MECHP including, but not limited to, those described above.
In other embodiments, any of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention may Ibe administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art, according to conventional pharmaceutical principles. The combination of therapeutic agents may act synergistically to effect the treatment WO 00/12711 I'CT/US99120468 or prevention of the various disorders described above. Using this approach.
one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
An antagonist of MECHP may be produced using methods which are generally known in the art. In particular, purified MECHP may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind MECHP.
Antibodies to MECHP
may also be generated using methods that are well known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, a:nd single chain antibodies, Fab fragments, and fragments produced by a Fab, expression library. Neutralizing antibodies (i.e., those which inhibit dimer formation) are especially preferred for therapeutic use.
For the production of antibodies, various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with MECHP or with any fragment or oligopeptide thereof which has immunogenic properties. Depending on the host species, various adjuvants may be used to increase immunological response;. Such adjuvants include, but are not IS limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.
Among adjuvants used in humans, BCG (bacilli CaImette-Guerin) and Corvnebacterium parvum are especially preferable.
It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to MECHP have an amino acid sequence consisting of at least: about 5 amino acids, and, more preferably, of at least about 10 amino acids. It is also preferable that these oligopeptides, peptides, or fragments are identical to a portion of the amino acid sequence of the natural protein and contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of MECHP amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
Monoclonal antibodies to MECHP may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture.
These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique. (See, e.g., Kohler, G. et al. ( 1975) Nature 256:495-497;
Kozbor, D. et al.
(1985) J. Immunol. Methods 81:31-42; Cote, R.J. et al. (1983) Proc. Natl.
Acad. Sci. USA
80:2026-2030; and Cole, S.P, et al. (1984) Moi. Cell Biol. 6:2:109-120.) In addition, techniques developed for the production of "chimeric antibodies,"
such as the splicing of mouse antibody genes to human antibody genes 1:0 obtain a molecule with appropriate antigen specificity and biological activity, can be used. (See;, e.g., Morrison, S.L. et al. (1984) WO 00/12'111 PCT/US99/20468 Proc. Natl. Acad. Sci. USA 81:6851-6855; Neuberger, M.S. et al. (1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature 3 I4:452-454.) Alternativeoiy, techniques described for the production.of single chain antibodies may be adapted, using methods known in the art, to produce MECHP-specific single chain antibodies. Antibodies with related specificity, but of distinct idiotypic composition, may be generated by chain shuffling from random combinatorial immunoglobulin libraries. (See, e.g., Burton D.R. (1991) Proc. Natl. Acad.
Sci. USA 88:10134-10137.) Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin.libraries or panels of highly specific binding reagents t0 as disclosed in the literature. (See, e.g., (?rlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:
3833-3837; Winter, G. et al. (1991) Nature 349:293-299.) Antibody fragments which contain specific binding sites for MECHP may also be generated. For example, such fragments include, but are not limited to, F(ab')2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (See, e.g., Huse, W.D. et al. (1989) Science 246:1275-1281.) Various immunoassays may be used for screening to identify antibodies having the desired speeifcity. Numerous protocols for competitive blinding or immunoradiometric assays using either polyclonal or monoclonal antibodies with estalblished specificities are well known in the art. Such immunoassays typically involve the measurement of complex formation between MECHP and its specific antibody. A two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering MEt:HP epitopes is preferred, but a competitive binding assay may also be employed (Pound,_sa~).
Various methods such as Scatchard analysis in conjunction with radioimmunoassay techniques may be used to assess the affinity of antibodies for MECHP.
Affinity is expressed as an association constant, Ke, which is defined as the molar concentration of MECHP-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions. The Ka determined for a preparation of polyclonal antibodies, which are heterogeneous in their affinities for multiple MECHP epito~pes, represents the average affnity, or avidity, of the antibodies for MECHP. The Ka determined i°or a preparation of monoclonal antibodies, which are monospeci$c for a particular MECHf epitope, represents a true measure of affinity. High-affinity antibody preparations with Ka ranging from about 109 to 10'2 I/mole are preferred for use in immunoassays in which the MECHP-antibody complex must withstand rigorous manipulations. Low-affinity antibody preparations with Ka ranging from about 106 to 10' I/mole are preferred for use in immunopurification and similar procedures which ultimately require dissociation of MECHP, preferably in active forrri, from the antibody (Catty, D. (1988) Antibodies. Volume I: A Practical Approach, IRL Press, Washington DC; Liddell, J.E. and Cryer, A. ( 1991 ) A Practical Guide to Monoclonal Antibodies, John Wiley & Sons, New York NY).
The titer and avidity of polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications. For example; a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml;
preferably 5-10 mg specific antibody/ml, is.preferred for use in procedures requiring precipitation of MECHP-antibody complexes. Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various applications, are generally available.
(See, e.g.,.Catty, supra, and Coligan et al. supra.) In another embodiment of the invention; the polynucleotides encoding MECHP, or any fragment or complement thereof, may be used for therapeutic purposes. In one aspect, the complement of the polynucleotide encoding MECHP may be used in situations in which it would be desirable to block the transcription of the mRNA. In particular, cells may be transformed with sequences complementary to polynucleotides encoding MIECHP. Thus, complementary molecules or fragments may be used to modulate MECHP activity, or to achieve regulation of gene function.
Such technology is now well known in the art, and sense or antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding MECHP.
Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids, may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. Methods which are well known to those skilled in the art can be used to construct vectors to express nucleic acid seqiuences complementary to the polynucieotides encoding MECHP. (See, e.g., Sambrook, ;~unra; Ausubel, 1995, supra.) Genes encoding MECHP can be turned off by transforming a cell or tissue with expression vectors which express high levels of a polynucleotide, or fragment thereof, encoding MECHP. Such constructs may be used to introduce untranslatable sense or antisense sequences into a cell. Even in the absence of integration into the DNA, such vectors may continue to transcribe RNA molecules until they are disabled by endogenous nucleases.
Transient expression may last for a month or more with a non-replicating vector, and may last even longer if appropriate replication elements are part of the vector system.
As mentioned above, modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the control, 5', or regulatory regions ofthe gene encoding MECHP. Oligonucleotides derived from the transcription initiation site, e.g., between about positions -10 and +10 :from the start site, are preferred.
Similarly, inhibition can be achieved using triple helix base-pairing methodology. Triple helix pairing is useful because it causes inhibition of the ability of the double helix to open sufficiently for the binding of polymerises, transcription factors, or rE:gulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature. (See, e.g., Gee, J.E. et al.
(1994) in Huber, B.E. and B.I. Carr, Molecular and Immuinologic Approaches, Futura Publishing, Mt. Kisco NY, pp. 163-177.) A complementary sequence; or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
Ribozymes, enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA. The mechanism of ribozyme action involves seduence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. For example, engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleoiytic cleavage of sequences encoding MECHP.
Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, :including the following sequences:
GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides, corresponding to the region of the tauge;t gene containing the cleavage site, may be evaluated for secondary structural features which may render the oligonucleotide inoperable.
The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucieotides using ribonuclease protection assays.
Complementary ribonucleic acid molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of nucleic acid molecules. These include techniques for chemically synthesizing oligonucIeotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding MEC;HP. Such DNA sequences may be incorporated into a wide variety of vectors with suitable Rr~fA polymerise promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be introduced into cell lines, cells, or tissues., RNA molecules may be modified to increase intracellular stability and half life. Possible modifications include, but are not limited to; the addition of"flanking sequences at the 5' andlor 3' ends of the molecule, or the use of phosphorothioate or 2' 0~-methyl rather than phosphodiesterase linkages within the backbone of the molecule. This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-, and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by .
endogenous endonucIeases.
Many methods for introducing vectors into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo. For ex vivp therapy, vectors may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection, by liposome injections, or by polycationic amino polymers may be.achieved using methods which are well known in the art. (See, e.g., Goldman, C.K. et aI.
( 1997) Nat. Biotech. 15:462-466.) Any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
An additional embodiment of the invention relates to the administration of a pharmaceutical or sterile composition, in conjunction with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above. Such pharmaceutical compositions may consist of MECHP, antibodies to MECHP, and mimetics, agonist:>, antagonists, or inhibitors of MECHP.
The compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to, saline, buffered saline, dextrose, and water.
The compositions may be administered to a patient alone, or in combination with other agents, drugs, or hormones.
The pharmaceutical compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial;
intramedullary, intrathecal, intraventricular; transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising e:~ccipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remin~ton's Pharmaceutical Sciences (Maack Publishing, Easton PA).
Pharmaceutical compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, -4 l-pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, far ingestion by the patient:
Pharmaceutical preparations for oral use can be obtained through combining active compounds with solid excipient and processing the resultant mixture of granules (optionally, after grinding) to obtain tablets or dragee cores. Suitable auxiliaries can be added, if desired. Suitable excipients include carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, and sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropylmethyf-cellulose, or sodium carboxymethylcellulose; gums, including arabic and tragacanth; and proteins, such as gelatin and collagen.
If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, and alginic acid or a salt thereof, such as sodium alginate.
Dragee cores may be used in conjunction with suiitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
Push-fit capsules can contain active ingredients mixed with fillers or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
Pharmaceutical formulations suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline. Aqu<;ous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally, suspensions o~f the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate, triglycerides, or liposomes. Non-lipid polycationic amino polymers may also be used for delivery. Optionally, the suspension may also contain suitable stabilizers or agents to increase the solubility of the compounds and allow for the preparation of highly concentrated solutions.
For topical or nasal administration, penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are; generally known in the art.

The pharmaceutical compositions of the present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
The pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric, acetic, lactic, tartaric, malic, and succinic acids. Salts tend to be more soluble in aqueous or other pratonic solvents than are the corresponding free base forms. In other cases, the prefewed preparation may be a lyophilized powder which may contain any or all of the following: I mM to 50 mM histidine, O.I% to 2%
sucrose, and 2% to 7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. For administration of MECHP, such labeling would include amount, frequency, and method of administration.
Pharmaceutical compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose. The determination of an effective dose is well within the capability of those skilled in the art.
For any compound, the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells or in animal models such as mice, rats, rabbits, dogs, or pigs. An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
A therapeutically effective dose refers to that amount of active ingredient, for example MECHP or fragments thereof, antibodies of MECHP, and ;~gonists, antagonists or inhibitors of MECHP, which ameliorates the symptoms or condition. Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the EDS° (the dose therapeutically effective in 50% of the population) or LDs° (the dose lethal to 50% of the population) statistics. 7.'he dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LDS°/EDS° ratio. Pharmaceutical compositions which exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies are used to formulate a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that includes the EDS° with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.

The exact dosage will be determined by the practitioner, in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general healtlh of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Long-acting phar~r~aceutical compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half life and clearance rate of the particular formulation.
Normal dosage amounts may vary from about 0.1 pg to 100,000 ug, up to a total dose of about I gram, depending upon the route of administration. Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art:
Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polyheptides will be specific to particular cells, conditions, locations, etc.
DIAGNOSTICS
In another embodiment, antibodies which specifically bind MECHP may be used for the diagnosis of disorders characterized by expression of MEC~HP, or in assays to monitor patients being treated with MECHP or agonists, antagonists, or inhibitors of MECHP.
Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics.
Diagnostic assays for MECHP include methods which utilize the antibody and a label to detect MECHP in human body fluids or in extracts of cells or tissues. The antibodies may be used with or without modification, and may be labeled by covalent o:r non-covalent attachment of a reporter molecule. A wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
A variety of protocols for measuring MECHP, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of MECHP
expression. Normal or standard values for MECHP expression are established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to MECHP under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, preferably by photometric means. Quantities of MECHP expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.
In another embodiment of the invention, the polynu.cieotides encoding MECHP
may be WO OO/i2711 PCT/US99/20468 used for diagnostic purposes. The polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, anal PNAs. The polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which expression of MECHP
may be correlated with disease. The diagnostic assay ma;y be used to determine absence, presence, and excess expression of MECHP, and to monitor regulation of MECHP
levels during therapeutic intervention.
In one aspect, hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding MECHP or closely related molecules may be used to identify nucleic acid sequences which encode MECHP.
The specificity 1 a of the probe, whether it is made from a highly specific re8;ion, e.g., the 5' regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or axnpiification (maximal, high, intermediate, or low), will determine whether the probe identifies only naturally occurring sequences encoding MECHP, allelic variants, or related sequences.
Probes may also be used for the detection of related sequences, and should preferably have at least 50% sequence identity to any of the MECHP encoding sequences.
The hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID N0:19-36 or from genomic sequences including promoters, enhancers, and introns of the gene encoding MECHP.
Means for producing specific hybridization probes for DNAs encoding MECHP
include the cloning of polynueleotide sequences encoding MECHP' or MECHP derivatives into vectors for the production of mRNA probes. Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides. Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclidles such as'ZP or 355, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
Polynucleotide sequences encoding MECHP may be used for the diagnosis of disorders associated with expression of MECHP. Examples of such disorders include, but are not limited to, a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD~), myelofibrosis, paroxysmal nocturnal hemoglobinuria, polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma, melanoma, myelom,a, sarcoma, teratocarcinoma., and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, hung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus; an immune/inflammatory disorder such as acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, ankylosing spondyiitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyenodocrinopathy-candidiasis-ectodermal dystrophy (APECED), bronchitis, cholecystitis, contact dermatitis, Crohn's disease, atopic dermatitis, de~matomyositis, diabetes mellitus, emphysema, episodic lymphopenia with tymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophic gastritis, glomerulonephritis, Goodpa,sture's syndrome, gout, Graves' disease, Hashimoto's thyroiditis, hypereosinophilia, irritable bowel syndrome, multiple sclerosis, myasthenia gravis, myocardial or pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis, polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis, scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupus erythematosus, systemic sclerosis;
thrombocytopenic purpura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extracorporeal circulation, viral, bacterial, fungal, parasitic, protozoal, and helminthic infections, and trauma; a transportlsecretory disorder such as akinesia, amyotrophic lateral sclerosis, ataxia telangiectasia, cystic fibrosis, Becker's muscular dystrophy, Bell's palsy, Charcot-Marie Tooth disease, Chediak-Higashi syndrome, diabetes mellitus, diabetes insipidus, diabetic neuropathy, hyperkalemic periodic paralysis, normokalemic periodic paralysis, malignant hyperthermia, multidrug resistance, myotonic dystrophy, catatonia, dystonias, peripheral neuropathy, neurofibromatosis, postherpetic neuralgia, tri,geminal neuropathy, sarcoidosis, sickle cell anemia, toxic shock syndrome, Wilson's disease, cataracts, infertility, pulmonary artery stenosis, sensorineural autosomal deafness, hyperglycemia, hypoglycemia, goiter, Cushing's disease, glucose-gaIactose malabsorption syndrome, hype:rcholesterolemia, and allergies, including hay fever, asthma, and urticaria {hives); an osmoregulatory disorder such as diabetes insipidus, diarrhea, peritonitis, chronic renal failure, Addison's disease, SIADH, hypoaldosteronism, hyponatremia, adrenal insufficiency, hypothyroidism, hypernatremia, hypokalemia, Barter's syndrome, metabolic acidosis, metabolic alkalosis, encephalopathy, edema, hypotension, and hypertension; a muscular disorder such ass cardiomyopathy, myocarditis, Duchenne's muscular dystrophy, Becker's muscular dystrophy, myotonic dystrophy, central core disease, nemaline myopathy, centronuclear myopathy, lipid myopathy, mitochondrial myopathy, infectious myositis, polymyositis, dermatomyositis, inclusion body myositis, thyrotoxic myopathy, and ethanol myopathy; a cardiovascular disorder such as alrteriovenous fistula, atherosclerosis, hypertension, vasculitis, Raynaud's disease, aneurysms, arterial dissections, varicose veins, thrombophlebitis and phlebothrombosis, vascular tumors, and complications of thrombolysis, WO 00112711 PCT/US99/204b8 balloon angioplasty, vascular replacement, and coronary artery bypass graft surgery; congestive heart failure, ischemic heart disease, angina pectoris, myocardial infarction, hypertensive heart disease, degenerative valvular heart disease, calcific aortic valve stenosis, congenitally bicuspid aortic valve, mitral annular calcification, mitral valve prolapse, rheumatic fever and rheumatic heart disease, infective endocarditis, nonbacterial thrombotic endocarditis, endocarditis of systemic lupus erythematosus, carcinoid heart disease, cardiomyopathy, myocarditis, pericarditis, neopiastic heart disease, congenital heart disease, complications of cardiac transplantation;
congenital lung anomalies, atelectasis, pulmonary congestion and edema, pulmonary embolism, pulmonary hemorrhage, pulmonary infarction, pulmonary hypertension, vascular sclerosis, obstructive pulmonary disease, restrictive pulmonary disease, chronic obstructive pulmonary disease, emphysema, chronic bronchitis, bronchial asthma, bronchiectasis, bacterial pneumonia, viral and mycoplasmal pneumonia, lung abscess, pulmonary tuberculosis, diffuse interstitial diseases, pneumoconioses, sarcoidosis, idiopathic pulmonary frbrosis, desquamative interstitial pneumonitis, hypersensitivity pneumonitis, pulmonary eo;>inophilia bronchiolitis obliterans-organizing pneumonia, diffuse pulmonary hemorrhage syndromes, Goodpasture's syndromes, idiopathic pulmonary hemosiderosis, pulmonary involvement in collagen-vascular disorders, pulmonary alveolar proteinosis, lung tumors, inflammatory and noninflammatory pleural effusions, pneumothorax, pleural tumors, drug-induced lung disease, radiation-induced lung disease, and complications of lung transplantation; and a neurological disorder such as epilepsy, ischemic cerebrovascular disease, stroke, cerebra.i neopiasms, Alzheimer's disease, Pick's disease, Down syndrome, Huntington's disease, dementia, Parkinson's disease and other extrapyramidal disorders, amyotrophic lateral sclerosis and other motor neuron disorders, progressive neural muscular atrophy, retinitis pigmentosa, hereditary ataxias, multiple sclerosis and other demyelinating diseases, bacterial and viral meningitis, brain abscess, subdural empyema, epidural abscess, suppurative intracranial thrombophlebitis, myelitis and radiculitis, viral central nervous system disease; prion diseases including kunz, Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome; fatal familial insomnia, nutritional and metabolic diseases of the nervous system, neurofibromatosis, tuberous sclerosis, cere;belloretinal hemangioblastomatosis, encephalotrigeminal syndrome, mental retardation and other developmental disorders of the central nervous system, cerebral palsy, neuroskeletal disordlers, autonomic nervous system disorders, cranial nerve disorders, spinal cord diseases; neuromuscular disorders including spinal muscular atrophy, carpal tunnel syndrome, monomeuritis multiplex; muscular dystrophies such as Duchenne's, myotonic facioscapulohumeral, oculopharyngc;al, scapuloperoneal, congenital, distal, and ocular; congenital and metabolic myopathies, myotonia., peripheral nervous system disorders, dermatomyositis and polymyositis; inherited, metabolic, endocrine, and toxic myopathies;
myasthenia gravis, periodic paralysis; mental disorders including depression arid bipolar disorder, arid mood,. anxiety, and schizophrenic disorders; seasonal affective disorder (SAD); akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia, dystonias, paranoid psychoses, postherpetic neuralgia, and Tourette's disorder; abnormalities in electrolytes such as calcium, phosphate, magnesium, and potasium; hypo- and hyperfu.nction of the thyroid, adrenal, parathyroid, and pituitary; and primary and metastatic ne~oplasms. The polynucleotide sequences encoding MECHP may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-like i0 assays; and in microarrays utilizing fluids or tissues from patients to detect altered MECHP
expression. Such qualitative or quantitative methods are well known in the art.
In a particular aspect, the nucleotide sequences encoding MECHP may be useful in assays that detect the presence of associated disorders, particularly those mentioned above. The nucleotide sequences encoding MECHP may be labeled by standard methods and added to a fluid t 5 or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample; is washed and the signal is quantitated and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding MECHP in the sample indicates the presence of the associated disorder. Such 20 assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.
In order to provide a basis for the diagnosis of a diisorder associated with expression of MECHP, a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a 25 sequence, or a fragment thereof, encoding MECHP, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are sympl:omatic for a disorder. Deviation from 30 standard values is used to establish the presence of a disorder.
Once the presence of a disorder is established and a treatment protocol is initiated, hybridization assays may be repeated on a regular basis to .determine if the level of expression in the patient begins to approximate that which is observed in the normal subject. The results obtained from successive assays may be used to show the efficacy of treatment over a period WO 00/12711 PCT/US99/2046$
ranging fram several days to months.
With respect to cancer, the presence of an abnormal amount of transcript (either under- or over-expressed) in biopsied tissue from an individual mar indicate a predisposition for the development of the disease, or may provide a means for dietecting the disease prior to the appearance of actual clinical symptoms. A more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development ar further progression of the cancer.
Additional diagnostic uses for oligonucleotides designed from the sequences encoding MECHP may involve the use of PCR. These oligomers may be chemically synthesized, generated enzymatically, or produced in vitro. Oligomers will preferably contain a fragment of a polynucleotide encoding MECHP, or a fragment of a polynucieotide complementary to the polynucleotide encoding MECHP, and will be employed under optimized conditions for identi$cation of a specific gene or condition. Oligomers rnay also be employed under less stringent conditions for detection or quantitation of closely related DNA or RNA sequences.
Methods which may also be used to quantitate the expression of MECHP include radiolabeIing or biotinylating nucleotides, coampIification of a control nucleic acid, and interpolating results from standard curves. (See, e.g., Melby, P.C. et al.
{1993) J. Immunol.
Methods 159:235-244; Duplaa, C. et al. (1993) Anal. Bioclhem. 212:229-236.) The speed of quantitation of multiple samples may be accelerated by running the assay in an ELISA format where the oligomer of interest is presented in various dilutions and a spectraphotometric or colorimetric response gives rapid quantitation.
In further embodiments, oligonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as targets in a microarray. The mieroarray can be used to monitor the expression level of large numbers of genes simultaneously and to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, and to develop and monitor the activities of therapeutic agents.
Microarrays may be prepared, used, and analyzed wising methods known in the art. (See, e.g., Brennan, T.M. et al. ( 1995) U.S. Patent No. 5,474,796;; Schena, M. et al. ( 1996) Proc. Natl.
Acad. Sci. 93:10614-10619; Baldeschweiler et al. ( 1995) PCT application W0951251116; Shalon, D. et al. (1995) PCT application W095135505; Heller, R.A. et al. (1997) Proc.
Natl. Acad. Sci.
USA 94:2150-2155; and Heller, M.J. et al. (1997) U.S. Pate:nt No. 5,605,662.) In another embodiment of the invention; nucleic aciid sequences encoding MECHP
may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence. The sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs}, yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial P 1 constructions, or single chromosome cDNA libraries,. (See, e.g., Harrington, J.J. et al. ( 1997) Nat. Genet. i 5:345-355; Price, C.M. ( 1993) Blood Rev. 7:127-134; and Trask, B.J. ( 1991 ) Trends Genet. 7: I 49-154.) Fluorescent in situ hybridization (FISH) may be correlated with other physical chromosome mapping techniques and genetic map data. (See, e.g., Heinz-Ulrich, et al. (1995) in Meyers, supra, pp. 965-968.) Examples of genetic map data can be found .in various scientific journals or at the Online Mendelian Inheritance in Man {OMIM) site.
Correlation between the location of the gene encoding MECHP on a physical chromosomal map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA
associated with that disorder. The nucleotide sequences of the invention may be used to detect differences in gene sequences among normal, carrier, and affected individuals.
IS In situ hybridization of chromosomal preparations and physical mapping techniques, such as linkage analysis using established chromosomal markers, may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the number or arm of a particular human chromosome is not known. New sequences can be assigned to chromosomal arms by physical 24 mapping. This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques. Once the disease or syndrome has been crudely localized by genetic linkage to a particular genomie region, e.g., ataxia-teiangiectasia to I 1q22-23, any sequences mapping to that area may represent associated or regulatory genes for further investigation. (See, e.g., Gatti, R.A. et al. (1988) rJature 336:577-580.) The nucleotide 2S sequence ofthe subject invention may also be used to detect differences in the chromosomal location due to translocation, inversion, ete., among normal, carrier, or affected individuals.
In another embodiment of the invention, MECHP, its cataiyt'tc or immunagenic fragments, or oligopeptides thereof can be used for screening libraries of compounds in any of a variety of drug screening techniques. The fragment employed in such screening may be free in solution, 30 affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes between MECHP and the agent being tested may be measured.
Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest. (See, e.g., Geysen, et al.
(1984) PCT application W084/03564.) In this method, large numbers of different small test -SO-WO 00!12711 PCT/US99120468 compounds are synthesized on a solid substrate. The test compounds are reacted with MECHP, or fragments thereof, and washed. Bound MECHP is then detected by methods well known in the art. Purified MECHP can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
In another embodiment, one may use competitive; drug screening assays in which neutralizing antibodies capable of binding MECHP specifically compete with a test compound for binding MECHP. In this manner, antibodies can be used to detect the presence of any peptide which shares one or mare antigenic determinants with MECHP.
In additional embodiments, the nucleotide sequences which encode MECHP may be used in any molecular biology techniques that have yet to be dE;veloped, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
Without further elaboration, it is believed that one; skilled in the art can, using the i5 preceding description, utilize the present invention to its fiullest extent. The following preferred specific embodiments are, therefore, to be construed as m<~rely illustrative, and not iimitative of the remainder of the disclosure in any way whatsoever.
The disclosures of ail patents, applications, and publications mentioned above and below, in particular U.S. Ser. No. [Attorney Docket No. PF-0589 P, filed September 2, 1998], U.S. Ser.
ZO No. [Attorney Docket No. PF-0632 P, filed November 12, 1998], U.S. Ser. No.
[Attorney Docket No. PF-0648 P, filed December 9, 199$), U.S. Ser. No. [Attorney Docket No. PF-0664 P, filed January 26, 1999], and U.S. Ser. No. [Attorney Docket No. PF-0671 P, filed February 10, 1999], are hereby expressly incorporated by reference.

I. Construction of cDNA Libraries RNA was purchased from Clontech or isolated from tissues described in Table 4.
Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Life 30 Technologies), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCI cushions or extracted with chloroform. RNA
was precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.
Phenol extraction and precipitation of RNA were repeated as necessary to increase RNA

purity. In some cases, RNA was treated with DNase. For most libraries, poly(A+) RNA was isolated using oligo d(Trcoupled paramagnetic particles (Promega), OLIGOTEX
latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA ;purification kit (QIAGEN).
Alternatively, RNA was isolated directly from tissue lysates using other RNA isolation kits, e.g., the POLY(A)PURE mRNA purification kit (Ambion, Austin TX).
In some cases, Stratagene was provided with RNA and constructed the corresponding cDNA libraries. Otherwise, cDNA was synthesized and cDNA libraries were constructed with the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system {Life Technologies), using the recommended procedures or similar methods known in the art. (See, e.g., Ausubel, 1997, supra, units 5.1-6.6.) Reverse transcription was initiated using oligo d(T) or random primers. Synthetic o(igonucieotide adapters were iigatedl to double stranded cDNA, and the cDNA
was digested with the appropriate restriction enzyme or enzymes. For most libraries, the cDNA
was size-selected (300-1000 bp) using SEPHACRYL S 1 iD00, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis. cDNAs were iigated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBL;UESCR1PT plasmid (Stratagene), pSPORTI piasmid (Life Technologies), or pINCY (Incyte Pharmaceuticals, Palo Alto CA).
Recombinant plasmids were transformed into competent E. coli cells including ~:L1-Blue, XLl-BIueMRF, or SOLR from Stratagene or DHSa, DH10B, or ElectroMAX DHI OB from Life Technologies.
II. Isola#ion of cDNA Clones Plasmids were recovered from host cells by in vivo excision, using the UNIZAP
vector system (Stratagene) or cell lysis. Plasmids were purified using at feast one of the following: a Magic or WIZARD Minipreps DNA purification system ('Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L.
PREP 96 plasmid purifcation kit from QIAGEN. Following precipitation, plasmids were resuspended in 0.1 ml of distilled water and stored, with or without lyophilization, at 4 °C.
Alternatively, plasmid DNA was amplified from host cell lysates using direct link PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host cell lysis and thermal cycling steps were carried out in a single reaction mixture. Samples were processed and stored in 384-well plates, and the concentration of amplified plasmid DNA was quantified fluorometrically using PICOGREEN dye (Molecular Probes, Eugene OR) and a Fluoroskan II
fluorescence scanner (Labsystems Oy, Helsinki, Finland).
III. Sequencing and Analysis cDNA sequencing reactions were processed using; standard methods or high-throughput instrumentation such as the AB1 CATALYST 800 (Perkin-Elmer) thermal cycler or the PTC-200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton) liquid transfer system. cDNA
sequencing S reactions were prepared using reagents provided by Amersham Pharmacia Biotech or supplied in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-Elmer). Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA
sequencing system (Molecular Dynamics); the ABI PRISPrI 373 or 377 sequencing systems (Perkin-Elmer) in conjunction With standard ABI protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA
sequences were identified using standard methods (reviewed in Ausubel, 1997, supra, unit 7.7}. Some of the cDNA sequences were selected for extension using the techniques disclosed in Example V.
The polynucleotide sequences derived from cDNA, sequencing were assembled and analyzed using a combination of software programs which utilize algorithms well known to those skilled in the art. Table 5 summarizes the tools, programs, and algorithms used and provides applicable descriptions, references, and threshold parameters. The first column of Table 5 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, all of which are incorporated by reference herein in their entirety, and the fourth column presents, where app~iicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score, the greater the homology between two sequences). Sequences were analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR). Polypeptide sequence alignments were generated using the default parameters specified by the clustal algorithm as incorporated into the MEGALIGN
multisequence alignment program (DNASTAR), which also calculates the percent identity between aligned sequences.
The polynucleotide sequences were validated by removing vector, linker, and polyA
sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programing, and dinucleotide nearest neighbor analysis. The sequences were then queried against a selection of public databases such as the GienBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS to acquire annotation using programs based on BLAST, FASTA, and BLIMPS. The sequences were assembled into full length polynucleotide sequences using programs based on Phred, Phrap, and Consed, and were screened for open reading frames using programs based on GeneMark, BLAST, and FASTA. The full length polynucleotide sequences were translated to derive the corresponding full length amino acid sequences,. and these full length sequences were subsequE;ntly analyzed by querying against databases such as the GenBank databases (described above), SwissProt, BLOCKS, PRINTS, S Prosite, and Hidden Markov Model (HMM)-based proteim family databases such as PFAM.
HMM is a probabilistic approach which analyzes consensus primary structures of gene families.
(See, e.g., Eddy, S.R. (1996) Curr. Opin. Struct. Biol. 6:361-365.) The programs described above for the assembly a.nd analysis of full length polynucleotide and amino acid sequences were also used to. identify polynucieotide sequence fragments from SEQ ID N0:19-36. Fragments from about 20 to about 4040 nucleotides which are useful in hybridization and amplification technologies were described in The Invention section above.
IV. Northern Analysis Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which 1S RNAs from a particular cell type or tissue have been bound. (See, e.g., Sambrook, suura, ch. 7;
Ausubel, 1995, supra, ch. 4 and 16.) Analogous computer techniques applying BLAST were used to search for identical or related molecules in nucleotide databases such as GenBanlk or LIFESEQ (Incyte Pharmaceuticals, Palo Alto CA). This analysis is much faster than multiple membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or similar. The basis of the search is the product score, which is defined as:
se4uence identity x % maXimurn BLAST score 2S The product score takes into account both the degree of similarity between two sequences and the length of the sequence match. For example, with a produce: score of 40, the match will be exact within a 1 % to 2% error, and, with a product score of 70, the match will be exact. Similar molecules are usually identified by selecting those which show product scores between 1S and 40, although lower scores may identify related molecules.
The results of northern analyses are reported as a percentage distribution of libraries in which the transcript encoding MECHP occurred. Analysis involved the categorization of cDNA
libraries by.organ/tissue and disease. The organ/tissue categories included cardiovascular, dermatologic, developmental, endocrine, gastrointestinal, h~ematopoietic/immune, musculoskeletal, nervous, reproductive, and urologic. The disease/condition categories included cancer, inflammation/trauma, cell proliferation, neurological, and pooled. For each category, the number of libraries expressing the sequence of interest was counted and divided by the total number of libraries across all categories. Percentage values of tissue-specific and disease- or condition-specific expression are reported in the description of the invention.
V. Extension of MECHP Encoding Polynucleotidles The full length nucleic acid sequences of SEQ II) N0:19-36 was produced by extension of an appropriate fragment of the full length molecule using oligonucleotide primers designed from this fragment. One primer was synthesized to initiate 5' extension of the known fragment, and the other primer, to initiate 3' extension of the known fragment. The initial primers were designed using OLIGO 4.06 software (National Biasciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of abut 50% or more, and to anneal to the target sequence at temperatures of about 68 °C to about 72°C.
Any stretch of nucleotides which would result in hairpin structures and primer-primer dimf:rizations was avoided.
Selected human eDNA libraries were used to extend the sequence. If more than one extension was necessary or desired, additional or nested sets of primers were designed.
High fidelity amplification was obtained by PCR using methods well known in the art.
PCR was performed in 96-well plates using the PTC-200 thermal cycler (MJ
Research, Inc.). The reaction mix contained DNA template, 200 nmol of each primer, reaction buffer containing Mgz+, (NH4)zSO4, and ~i-mercaptoethanol, Taq DNA polymerise; (Amersham Pharmacia Biotech), ELONGASE enzyme (Life Technologies), and Pfu DNA polymerise (Stratagene), with the following parameters for primer pair PCI A and PCI B: Step 1: 94 °C, 3 min; Step 2: 94°C, 1 S sec;
Step 3: 60°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2., 3, and 4 repeated 20 times; Step 6:
68°C, 5 min; Step 7: storage at 4°C. In the alternative, the parameters for primer pair T7 and SK+
were as follows: Step I: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 57°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Steg 6: 6.8°C, S min;
Step 7: storage at 4°C.
The concentration of DNA in each well was determined by dispensing 100 p.l PICO
GREEN quantitation reagent (0.25% (v/v) PICO GREEN; Molecular Probes, Eugene OR) dissolved in IX TE and 0.5 pl of undiluted PCR product into each well of an opaque fluorimeter plate (Corning Costar, Acton MA), allowing the DNA to bind to the reagent. The plate was scanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA. A S ul to 10,u1 aliquot of the reaction mixture was analyzed by electrophoresis on a 1 % agarose mini-gel to determine which reactions were successful in extending the sequence.
The extended nucleotides were desalted and concentrated, transferred to 384-well plates, digested with CviJI cholera virus endonucfease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to relegation into pUC 18 vector (Amersham Pharmacia Biotech). For shotgun sequencing, the digested nucleotides were separated on low concentration (0.6 to 0.8%) agarose gels, fragments were excised, and agar digested with Agar ACE
(Promega). Extended clones were relegated using T4 ligase {New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Pharmacia Biotech), treated with Pfu DNA polymerase {Stratagene) to fill-in restriction site overhangs, and transfected into competent E. coli cells.
Transformed cells were selected on antibiotic-containing media, individual colonies were picked and cultured overnight at 37°C in 384-well plates in LB/2x carb liquid media.
I0 The cells were lysed, and DNA was amplified by PCR using Taq DNA polymerase (Amersham Pharmacia Biotech) and Pfu DNA polymerase (Stratagene) with the following parameters: Step I: 94°C, 3 min; Step 2: 94°C, i5 sec; Step 3:
60°C, I min; Step 4: 72°C, 2 min;
Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, S min; Step 7: storage at 4°C. DNA was quantified by PICOGREEN reagent (Molecular Probes) a.s described above.
Samples with low IS DNA recoveries were reamplified using the same conditions as described above. Samples were diluted with 20% dimethysulphoxide (1:2, v/v), and sequenced using DYENAMIC
energy transfer sequencing primers and the DYENAMIC DIRECT kit (Amersham Pharmacia Biotech) or the ABI
PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-Elmer).
In like manner, the nucleotide sequence of SEQ 1fD N0:19-36 is used to obtain 5' 20 regulatory sequences using the procedure above, oligonueleotides designed for such extension, and an appropriate genomic library.
VI. Labeling and Use of Individual Hybridization Probes Hybridization probes derived from SEQ ID N0:19-36 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of olig;onucieotides, consisting of about 20 25 base pairs, is specifically described,,essentially the same procedure is used with larger nucleotide fragments. Oligonucleotides are designed using state-of the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 prnol of each oligomer, 250 p.Ci of tszp]-adenosine triphosphate (Amersham Pharmacia Biotec;h), and T4 polynucleotide kinase (DuPont NEN, Boston MA). The labeled oligonucleotides are substantially purified using a 30 SEPI-IADEX G-25 supe~ne size exclusion dextran bead column (Amersham Pharmacia Biotech).
An aliquot containing 10'counts per minute ofthe labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the following endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xba 1, or Pvu iI (DuPont NEN).
The DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon WO 00/12711 . PCT/US99120468 membranes (Nytran Plus, Schleicher & Schueli, Durham NH). Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots are ;sequentially washed at room temperature under increasingly stringent conditions up to 0.1 x saline sodium citrate and 0.5% sodium dodecyl sulfate. Hybridization patterns are visualized using autoradiography and compared.
VII. Microarrays A chemical coupling procedure and an ink jet device can be used to synthesize array elements on the surface of a substrate. (See, e.g., Baldesc:hweiler, supra.) An array analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, L1V, chemical, or mechanical bonding procedures. A typical array may be produced by l0 hand or using available methods and machines and contain any appropriate number of elements.
After hybridization, nonhybridized probes are removed and a scanner used to determine the levels and patterns of fluorescence. The degree of complementarity and the relative abundance of each probe which hybridizes to an element on the microarray may be assessed through analysis of the scanned images.
Full-length cDNAs, Expressed Sequence Tags (E;STs), or fragments thereof may comprise the elements of the micraarray. Fragments suitable for hybridization can be selected using software well known in the art such as LASERGEN1E software (DNASTAR).
Full-length cDNAs, ESTs, or fragments thereof corresponding to one of the nucleotide sequences of the present invention, or selected at random from a cDNA library relevant to the present invention, are arranged on an appropriate substrate, e.g., a glass slide. The cDNA is fixed to the slide using, e.g., W cross-linking followed by thermal and chemical treatments and subsequent drying. (See, e.g., Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645.) Fluorescent probes are prepared and used for hybridization. to the elements on the substrate. The substrate is analyzed by procedures described above.
VIII. Complementary Polynucleotides Sequences complementary to the MECHP-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring MECHP.
Although use of oligonucleotides comprising from about 15 to 30 base pairs. is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of MECHP. To inhibit transcription, a complementary oligonucieotide is designed from the most unique 5' sequence and used to prevent promoter binding toy the coding sequence. To inhibit translation, a complementary oligonucieotide is designed to~ prevent ribosomal binding to the MECHP-encoding transcript.

WO 00!12711 PCT/US99120468 IX. Expression of MECHP
Expression and purification of MECHP are achieved using bacterial or virus-based expression systems. Far expression of MECHP in bacteria, cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription. Examples of such promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacterioplhage promoter in conjunction with the lac operator regulatory element. Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3). Antibiotic resistant bacteria express MECHP upon induction with isopropyl beta-D-thiogalactopyranoside {1PTG). Expression of MECHP in eukaryotic cells is achieved by infecting insect or mammalian cell lines with recombinant Auto~raphica catifornica nuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. The nonessential polyhedrin gene of bacutovirus is replaced with cDNA encoding MECHP by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA
transcription. Recombinant bacuiovirus is used to infect,Spodoptera fruaiperda (Sf~) insect cells in most cases, or human hepatocytes, in some cases. Infection ofthe latter requires additional genetic modifications to baculovirus. (See Engelhard, E. K. et al. (1994) Proc. Natl. Acad. Sci.
USA 91:3224-3227; Sandig, V. et al. (1996) Hum. Gene'Ther. 7:1937-1945.) In most expression systems, MECHP is synthesiized as a fusion protein with, e.g., giutathione S-transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell lysates.
GST, a 26-kilodalton enzyme from Schistosoma iaponicu:m, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Pharmacia Biotech). Following purification, the GST
moiety can be proteolytically cleaved from MECHP at specifically engineered sites. FLAG, an 8-amino acid peptide, enables immunoaffinity purification using commercially available monoclonal and polyclonai anti-FLAG antibodies (Eastman Kodak). 6-Hiis, a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QLAGEN). Methods for protein expression and purification are discussed in Ausubel (1995, supra, ch 10 and 16).
Purified MECHP obtained by these methods can be used directly in the following activity assay.
X. Demonstration of MECHP Activity Aquaporin Activit~of MECHP
Aquaporin activity of MECHP is demonstrated .as the ability to induce osmotic water permeability in Xenopus laevis oocytes injected with MECHP cRNA (Ishibashi, K.
et al. (1994) Proc. Natl. Acad. Sci. USA 91:6269-6273). Oocytes injiected with water are used as the control.
Injected oocytes are given a hypotonic shock by being transferred from 200 mosM to 70 mosM
modified Barth's buffer. The increase in osmotic volume of the oocytes, observed at 24 °C by videomicroscopy, is proportional to the MECHP aquaporin activity in the injected oocytes.
Protein Transport Activity of MECHP
Protein transport activity of MECHP is demonstrated by its ability to catalyze the translocation of newly synthesized preprolactin into proteoliposomes in an in vitro system (Gorlich, D. and T.A. Rapoport (1993) Cell 75:615-630;1. Proteoliposomes are prepared containing purified MECHP, purified dog Sec61 p beta and gamma, purified dog SRP receptor, and a mixture of phospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and phosphatidylinositol) corresponding approximately to those found in native microsomes. The proteoliposomes are incubated in a wheat germ in vitro translation system in which a secretory protein (preprolactin) is synthesized in the presence of SRP and radioactive amino acids. After translation and synthesis of preprolactin, half of the sample is treated with S00 IS llg/ml proteinase K while the other half remains untreated. Any translocated preprolactin will be inaccessible to proteinase K while any untranslocated preprolactin will be degraded. The amount of preprolactin in the samples with and without proteinase K treatment is determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by phosphor image analysis. The amount of preprolactin protected from proteinase K digestion in the proteinase K-treated sample is proportional to the protein transport activity of MECHP.
Gap Junction Activity of MECHP
Gap junction activity of MECHP is demonstrated as the ability to induce the formation of intercellular channels between paired Xenopus laevis oocytes injected with MECHP cRNA
(Hennemann, supra). One week prior to the experimental injection with MECHP
cRNA, oocytes are injected with antisense oligonucleotide to MECHP to reduce background.
MECHP cRNA-injected oocytes are incubated overnight, stripped of viteelline membranes, and paired for recording of functional currents by dual cell voltage clamp. The measured conductances are proportional to gap junction activity of MECHP.
ton Channel Activity of MECHP
Ion channel activity of MECHP is demonstrated using an electrophysiological assay for ion conductance. MECHP can be expressed by transfornning a mammalian cell line such as COS7, HeLa or CHO with a eukaryotic expression vector encoding MECHP.
Eukaryotic expression vectors are commercially available, and the techniques to introduce them into cells are well known to those skilled in the art. A second plasmid which expresses any one of a number of marker genes, such as I3-galactosidase, is co-transformec! into the cells to allow rapid identification of those cells which have taken up and expressed the foreign DNA. The cells are incubated for 48-72 hours after transformation under conditions appropriate for the cell line to allow expression and accumulation of MECHP and Q-galactosidase.
Transformed cells expressing I3-galactosidase are stained blue when a suitable colorimetric substrate is added to the culture media under conditions that are well known in the art.
Stained cells are tested for differences in membrane conductance due to potassium ions by electrophysiological techniques that are well known in the art. Untransformed cells, andlor cells transformed with either vector sequences alone or 13-gala~ctosidase sequences alone, are used as controls and tested in parallel. Cells expressing MECHP will have higher cation conductance relative to control cells. The contribution of MECHP to conductance can be confirmed by incubating the cells using antibodies specific for MECH.'P. The antibodies will bind to the extracellular side of MECHP, thereby blocking the pore in the ion channel, and the associated conductance.
IS Ion channel activity of MECHP is also measured as current flow across a MECHP-containing Xenopus oocyte membrane using the two-electrode voltage-clamp technique (Ishi et al., supra; Jegla, T. and L. Salkoff ( 1997) J. Neurosci. 1 T:32-44}. MECHP is subcloned into an appropriate Xenopus oocyte expression vector, such as p~BF, and 0.5-5 ng of mRNA is injected into mature stage IV oocytes. Injected oocytes are incubated at 18°C
for 1-5 days. Inside-out macropatches are excised into an intracellular solution containing 116 mM K-gluconate, 4 mM
KCI, and 10 mM Hepes (pH 7.2). The intracellular solution is supplemented with varying concentrations of the MECHP mediator, such as cAMP, cGMP, or Ca+Z (in the form of CaCh}, where appropriate. Electrode resistance is set at 2-5 MSS! and electrodes are filled with the intracellular solution lacking mediator. Experiments are performed at room temperature from a holding potential of 0 mV. Voltage ramps (2.5 s) from -100 to 100 mV are acquired at a sampling frequency of 500 Hz. Current measured is proportional fro the activity of MECHP in the assay.
XI. Functional Assays MECHP function is assessed by expressing the sequences encoding MECHP at physiologically elevated levels in mammalian cell culture systems. cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA
expression. Vectors of choice include pCMV SPORT (L,ife Technologies) and pCR3.1 (Invitrogen, Carlsbad CA), both of which contain the cyt:omegalovirus promoter. 5-10 pg of recombinant vector are transiently transfected into a hurr~an cell line, preferably of endothelial or hematopoietic origin, using either liposome formulations or electroporation. 1-2 pg of an additional plasmid containing sequences encoding a marker protein are co-transfected. Expression of a marker protein provides a means to distinguish transfected cells from nontransfected cells and is a reliable predictor of cDNA expression from the recombinant vector. Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; Clontech), CD64, or a CD64-GFP fusion protein. Flow cytometry (FCM), an automated, laser optics-based technique, is used to identify trans~ected cells expressing GFP or CD64-GFP, and to evaluate the apoptotic state of the cells and other cellular properties. FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with cell death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in cell size and granularity as measured by forward light scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeo:Kyuridine uptake;
alterations in expression of cell surface and intracellular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the cell surface. Methods in flow cytometry are discussed in Ormerod, M.G. ( 1994) Flow Cytometry, Oxford, New York NY.
The influence of MECHP on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding; MECHP and either CD64 or CD64-GFP.
CD64 and CD64-GFP are expressed on the surface of tra~nsfected cells and bind to conserved regions of human immunoglobulin G (IgG). Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with eiither human IgG or antibody against CD64 (DYNAL, Lake Success NY). mRNA can be purified from the cells using methods well known by those of skill in the art. Expression of mRNA encoding MECHP and other genes of interest can be analyzed by northern analysis or rnicroarray techniques.
XII. Production of MECHP Specific Antibodies MECHP substantially purified using polyacrylamide gel electrophoresis (PAGE;
see, e.g., Harrington, M.G. (1990) Methods Enzymol. 182:488-495), or other purification techniques, is used to immunize rabbits and to produce antibodies u;>ing standard protocols.
Alternatively, the MECHP amino acid sequence is analyzed using LASERGENE
software (DNASTAR) to determine regions of high imrrmnogenicity, and a corresponding oligopeptide is synthesized and used to raise antibodies 'by means known to those of skill in the art. Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions are well described in the art. (See, e.g., Ausubel, 1995, su ra, ch. i l.) Typically, oligopeptides 15 residues in length are synthesized using an ABI
431A peptide synthesizer (Perkin-Elmer) using fmoc-chemistry and coupled to ICLH (Sigma-Aldrich, St. Louis MO} by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity. {See, e.g., Ausubel, 1995, su ra.) Rabbits are immunized with the oligopeptide-ICLH complex in complete Freund's adjuvant. Resulting autisera are tested for antipeptide activity by, for example, binding the peptide to plastic, blocking with 1% BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit igG.
XIII. Purification of Naturally Occurring MECHP Using Specific Antibodies Naturally occurring or recombinant MECHP is substantially purified by immunoaffinity chromatography using antibodies specific for MECHP. A.n immunoa~nity column is constructed by covalently coupling anti-MECHP antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech). After the coupling, the resin is blocked and washed according to the manufacturer's instructions:
Media containing MECHP are passed over the irnmunoaffinity column, and the column is washed under conditions that allow the preferential abs~orbance of MECHP
(e.g., high ionic IS strength buffers in the presence of detergent). The column is eluted under conditions that disrupt antibodyIMECHP binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and MECHP is collected.
XIV. Identification of Molecules Which Interact with MECHP
MECHP, or biologically active fragments thereof, are labeled with''-SI Bolton-Hunter reagent. {See, e.g., Bolton, A.E. and W.M. Hunter (1973;1 Biochem. J. 133:529-539.) Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled MECHP, washed, and any wells with labeled MECHP complex are assayed. Data obtained using different concentrations of MECHP are used to calculate values for the number, affinity, and association of MECHP with the candidate molecules.
Various modifications and variations of the described methods and systems of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific preferred embodiments, it should be understood that the invention .as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention which are obvious to those skilled in molecular biology or related fields are intended to be within the scope of the following claims.

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_77_ SEQUENCE LISTING
<110> INCYTE PHARMACEUTICALS, TNC.
AU-YOUNG, Janice SANDMAN, Olga TANG, Y. Tam REDDY, Roopa HILLMAN, Jennifer L.
YUE, Henry T~AT,, Preeti CORLEY, Neil C.
GUEGLER, Kari J.
GORGONE, Gina BAUGHN, Mariah R.
AZIMZAI, Yalda <120> HUMAN MEMBRANE CHANNEL PROTEINS
<130> PF-OS89 PCT
<140> To Be Assigned <141> Herewith <150> 09/145,815; unassigned; 09/191,283; unas:aigned; 09/208,821; unassigned 09/237,506; unassigned; 09/247;891; unas:aigned <151> 1998-09-02; 1998-09-02; 1998-hl-12; 199 8-11-12; 1998-12-09; 1998-12-09 1999-01-26; 1999-O1-26; 1999-02-10; 199 9-02-10 <160> 45 <170> PERL Program <210> 1 <211> 724 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1568324CD1 <400> 1 Met Ser Phe Glu Ser Ile Ser Ser Leu Pro Glu Val Glu Pro Asp Pro Glu Ala Gly Ser Glu Gln Glu Val Phe Ser Ala Val Glu Giy Pro Ser Ala Glu Glu Thr Pro Ser Asp Thr Glu Ser Pro GIu Val Leu Glu Thr Gln Leu Asp Ala His Gln Gly Leu Leu Gly Met Asp Pro Pro G1y Asp Met Va1 Asp Phe Val Ala Ala Glu Ser Thr Glu Asp Leu Lys Ala Leu Ser Ser Glu Glu Glu Glu Glu Met Gly Gly Ala Ala Gln Glu Pro Glu Ser Leu Leu Pra Pro Ser Val Leu Asp Gln Ala Ser Val Ile Ala Glu Arg Phe Val Ser S'~er Phe Ser Arg Arg Ser Ser Val AIa Gln Glu Asp Ser Lys Ser Ser Gly Fhe Gly Ser Pro Arg Leu Val Ser Arg Ser Ser Ser Val Leu Ser Leu Glu Gly Ser GIu Lys Gly Leu Ala Arg His Gly Ser Ala Thr Asp Ser Leu Ser Cys Gln Leu Ser Pro Glu Val Asp Ile Ser Val Gly VaI

Ala Thr Glu Asp Ser Pro Ser Val Asn Gly Met G:lu Pro Pro Ser Pro Gly Cys Pro Val Glu Pro Asp Arg Ser Ser Cys Lys Lys Lys Glu Ser Ala Leu Sex Thr Arg Asp Arg Leu Leu L<au Asp Lys IIe Lys Ser Tyr Tyr Glu Asn AIa GIu His His Asp AIa Gly Phe Sex Val Arg Arg Arg Glu Ser Leu Ser Tyr IIe Pro Lys Gly Leu Val Arg Asn Ser Ile Ser Arg Phe Asn Ser Leu Pro Arg Pro Asp Pro Glu Pro Val Pro Pro Val Gly Ser Lys Arg Gln Va:l Gly Ser Arg Pro Thr Ser Trp Ala Leu Phe Glu Leu Pro Gly Pro Ser Gln Ala Val Lys Gly Asp Pro Pro Pro Ile Ser Asp AIa Glu Phe Arg Pro Ser Ser Glu Ile Val Lys Ile Trp Glu GIy Met GIu Ser Ser Gly Gly Ser Pro G1y Lys GIy Pro Gly Gln GIy Gln Ala Asn Gly Phe Asp Leu His Glu Pro Leu Phe Ile Leu Glu GIu Hi,s Glu Leu Gly Ala Ile Thr Glu Glu Ser Ala Thr Ala Ser Pro Glu Ser Ser Ser Pro Thr Glu Gly Arg Ser Pro Ala His Leu Ala Ar<fi Glu Leu Lys Glu Leu VaI Lys Glu Leu Ser Ser Ser Thr Gln Gly Glu Leu Val Ala Pro Leu His Pro Arg Ile Val Gln Leu Ser His Val Met Asp Ser His Val Ser Glu Arg Val Lys Asn Lys Val Tyx- Gln Leu AIa Arg Gln Tyr Ser Leu Arg Ile Lys Ser Asn Lys Pro Val Met Ala Arg Pro Pro Leu Gln Trp Glu Lys Val Ala Pro Glu; Arg Asp Gly Lys Ser Pro Thr Val Pro Cys Leu G1n Glu Glu Ala Gly Glu Pra Leu Gly Gly Lys Gly Lys Arg Lys Pro Val Leu Ser Leu Phe Asp Tyr Glu Gln Leu Met Ala Gln Glu His Ser Pro Pro Lys Pro Ser Ser AIa Gly Glu Met Ser Pro Gln Arg Phe Phe Phe Asn Pro Pro 2/~3 Ala Val Ser Gln Arg Thr Thr Ser Pro Gly Gly Arg Pro Ser Ala Arg Ser Pro Leu Ser Pro Thr Glu Thr Phe Sex Trp Pro Asp Val Arg Glu Leu Cys Ser Lys Tyr Ala Ser Arg Asp G:Lu Ala Arg Arg Ala Gly Gly Gly Arg Pro Arg Gly Pro Pro Val A:an Arg Ser His Ser Val Pro Glu Asn Met Val Glu Pro Pro Leu Se:r Gly Arg Val Gly Arg Cys Arg Ser Leu Ser Thr Lys Arg Gly Ai:g Gly Gly Gly Glu Ala Ala Gln Ser Pro Gly Pro Leu Pro Gln Se:r Lys Pro Asp Gly Gly Glu Thr Leu Tyr Val Thr Ala Asp Leu Thr Leu Glu Asp Asn Arg Arg Val Ile Val Met Glu Lys Gly Pro Le:u Pro Ser Pro Thr Ala Gly Leu Glu Glu Ser Ser Gly Gln Gly Pro Ser Ser Pro Vai Ala Leu Leu Gly Gln Val Gln Asp Phe Gln G1n Ser Ala Glu Cys Gln Pro Lys Glu Glu Gly Ser Arg Asp Pro Ala Asp Pro Ser Gln Gln Gly Arg Val Arg Asn Leu Arg Glu Lys Phe Gln Ala Leu Asn Ser Val Gly <210> 2 <211> 257 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 4094907CD1 <400> 2 Met Ser Arg Pro Leu Ile Thr Arg Ser Pro Ala Ser Pro Leu Asn Asn Gln Gly Ile Pro Thr Pro Ala Gln Leu Thr Lys Ser Asn Ala Pro Val His Ile Asp Val Gly Gly His Met Tyr Thr Ser Ser Leu Ala Thr Leu Thr Lys Tyr Pro Glu Ser Arg Ile Gly Arg Leu Phe Asp Gly Thr Glu Pro Ile Val Leu Asp Ser Leu Lys~ Gln His Tyr Phe Ile Asp Arg Asp Gly Gln Met Phe Arg Tyr Ile Leu Asn Phe Leu Arg Thr Ser Lys Leu Leu Ile Pro Asp Asp Phe Lys Asp Tyr Thr Leu Leu Tyr Glu Glu Ala Lys Tyr Phe Gln Leu Gln Pro Met Leu Leu Glu Met Glu Arg Trp Lys Gln Asp Arg C~lu Thr Gly Arg Phe Ser Arg Pro Cys Glu Cys Leu Val Val Arg Val AIa Pro Asp Leu Gly Glu Arg Ile Thr Leu Ser Gly Asp Lys Ser Leu Ile Glu Glu Val Phe Pro GIu Ile GIy Asp Val Met Cys A.sn Ser Val Asn Ala Gly Trp Asn His Asp Ser Thr His Val Ile Arg Phe Pro Leu Asn Gly Tyr Cys His Leu Asn Ser Val Gln Val Leu Glu Arg Leu Gln Gln Arg Gly Phe Glu Ile Val Gly Ser Cys G.ly Gly Gly Val Asp Ser Ser Gln Phe Ser Glu Tyr Vah Leu Arg A:rg Glu Leu Arg Arg Thr Pro Arg Val Pro Ser Val Ile Arg Ile Lys Gln Glu Pro 245 250 ° 255 Leu Asp <210> 3 <211> 377 <212 > PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 518158CD1 <400> 3 Met Gly Gly Asp Leu Gly Leu Gly Leu Arg Arg Val Leu Ala Arg Lys Arg Leu Leu Glu Lys Ser Leu Gly Trp Ala Gln Glu Ala Leu Val Leu Ala Gly Thr Gly Leu Met Leu His Ala Gly Ile Val Glu Met Leu Trp Phe Gly Ser Trp Ala Ty:r Leu Phe Gly Cys Leu Leu Val Lys Cys Thr Ile Ser Thr Phe Leu Leu Cys Ser Ile Leu Leu Ile Val Ala Phe His Glu Val Gln Phe= Met Thr Ala Lys Leu Asp Asn Gly Leu Arg Asp Val Ala Leu Gly Arg GIn Trp Arg Thr Ala Ala Gln Ile Val Leu Val Val Cys Leu Has Pro Glu Leu Gly Ala Pro Val Arg GIy Pro Val Gln Asp Gly Ala Pro Pro Cys Leu Leu Thr Ser Pro GIn Pre GIy Phe Leu Gln Gly Glu Trp Pro Gly AIa Leu Leu Ser Leu AIa Leu Leu Gly Thr Leu Gly Met Leu Leu Leu Txp Leu Thr Thr AIa Leu Ser Val Glu. Arg Gln Trp Val, Ala AIa Val Asn AIa Thr Gly Ser Asp Thr Txp Leu Ile His Leu Leu Pro Ile Thr Phe Leu Thr Ile Gly Tyr Gly Asp Val Val Pro Gly Thr Met Trp Gly Lys Ile Val Cys Leu Cys Thr Gly teal Met Gly Val Cys Cys Thr Ala Leu Leu.Val Ala Val Val Ala 1?xg Lys Leu Glu Phe Asn Lys Ala GIu Lys His Val His Asn Phe Nfet Met Asp Ile Gln Tyr Thr Lys Glu Met Lys Glu Ser Ala AIa A.rg Vai Leu Gln Glu Ala Trp Met Phe Tyr Lys His Thr Arg Arg Lys Glu Sex His Ala Ala Arg Arg His Gln Arg Lys Leu Leu Ala Ala I1e Asn Ala Phe Arg Gln Val Arg Leu Lys His Arg Lys Leu Arg Glu Gln Val Asn Ser Met Val Asp Ile Ser Lys Met His Met Lle Leu Tyr Asp Leu Gln Gln Asn Leu Ser Ser Ser His Arg Ala Leu Glu Lys Gln Ile Asp Thr Leu Ala Gly Lys Leu Asp Ala Leu Tlar Glu Leu Leu Ser Thr Ala Leu GIy Pro Arg Gln Leu Pro Glu Pro Ser Gln Gln Ser Lys <220> 4 <211> 49I
<212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 602926CD1 <400> 4 Met Val Phe Gly Glu Phe Phe His Arg Pro Gly Gln Asp Glu Glu Leu Val Asn Leu Asn Val Gly Gly Phe Lys Gln Ser Val Asp Gln Ser Thr Leu Leu Arg Phe Pro His Thr Arg Leu Gly Lys Leu Leu Thr Cys His Ser GIu Glu Ala Ile Leu Glu Leu Cys Asp Asp Tyr Ser Val Ala Asp Lys Glu Tyr Tyr Phe Asp Arg Assn Pro Ser Ser Phe Arg Tyr Val Leu Asn Phe Tyr Tyr Thr G1y Ly;s Leu His VaI

Met Glu Glu Leu Cys Val Phe Ser Phe Cys Gln Glu Tle Glu Tyr Trp Gly Ile Asn Glu Leu Phe Ile Asp Ser Cys Cys Ser Asn Arg Tyr Gln Glu Arg Lys Glu Glu Asn His Glu Lys Asp Trp Asp Gln Lys Ser His Asp Val Ser Thr Asp Ser Ser Phe Glu Glu Ser Ser Leu Phe Glu Lys Glu Leu Glu Lys Phe Asp Thr Leu Arg Phe Gly Gln Leu Arg Lys Lys Ile Trp Ile Arg Met GIu A,sn Pro Ala Tyr Cys Leu Ser Ala Lys Leu Ile Ala Ile Ser Ser Leu Ser Val Val Leu Ala Ser Ile Val Ala Met Cys Val His Ser Met Ser Glu Phe 200 205 2Ip Gln Asn Glu Asp Gly Glu Val Asp Asp Pro Val Lesu Glu Gly Val Glu Ile Ala Cys Ile Ala Trp Phe Thr Gly Glu Le:u Ala Val Arg Leu Ala Ala Ala Pro Cys Gln Lys Lys Phe Trp Lys Asn Pro Leu Asn Ile Ile Asp Phe Val Ser Ile Ile Pro Phe Tyr Ala Thr Leu Ala Val Asp Thr Lys Glu Glu Glu Ser Glu Asp Il.e Glu Asn Met Gly Lys Val Val Gln Ile Leu Arg Leu Met Arg Il.e Phe Arg Ile Leu Lys Leu Ala Arg His Sex Val Gly Leu Arg Se.r Leu Gly Ala Thr Leu Arg His Ser Tyr His G1u Va1 Gly Leu Leu Leu Leu Phe Leu Ser Val GIy Ile Ser Ile Phe Ser Val Leu Ile Tyr Ser Val Glu Lys Asp Asp His Thr Ser Ser Leu Thr Ser Ile Pro Ile Cys Trp Trp Trp Ala Thr Tle Ser Met Thr Thr Val Gly Tyr Gly Asp 3.65 370 375 Thr His Pro Val Thr Leu Ala Gly Lys Leu Ile Al;a Ser Thr Cys Ile Ile Cys Gly Ile Leu Val Val AIa Leu Pro Ile Thr Ile Ile Phe Asn Lys Phe Ser Lys Tyr Tyr Gln Lys Gln Lya Asp Ile Asp Val Asp Gln Cys Ser Glu Asp Ala Pro Glu Lys Cy:a His Glu Leu Pro Tyr Phe Asn IIe Arg Asp IIe Tyr Ala Gln Arch Met His Ala Phe Ile Thr Ser Leu Ser Ser Val Gly Ile Val Va7L Ser Asp Pro Asp Ser Thr Asp Ala Ser Ser Ile Glu Asp Asn Glu Asp Ile Cys Asn Thr Thr Ser Leu Glu Asn Cys Thr Ala Lys <210> 5 <211> 341 <212> PRT
<213> Homo sapiens <220>

<221> misc_feature <223> Incyte ID No: 922119CD1 <400> 5 Met Gly Ser Gly His Cys Leu Arg Ser Thr Arg G:Ly Ser Lys Met Val Ser Trp Ser Val Ile Ala Lys Ile Gln Glu I7Le Leu Gln Arg Lys Met Val Arg Glu Phe Leu Ala Glu Phe Met Se:r Thr Tyr Val Met Met Val Phe Gly Leu Gly Ser Val Ala His Me;t Val Leu Asn Lys Lys Tyr Gly Ser Tyr Leu Gly VaI Asn Leu Gl.y Phe Gly Phe Gly Vai Thr Met Gly Val His Val Ala~Gly Arg Ile Ser Gly Ala His Met Asn Ala Ala Val Thr Phe Ala Asn Cys Ala Leu Gly Arg g5 100 105 Val Pro Trp Arg Lys Phe Pro Val Tyr Val Leu Gly Gln Phe Leu Gly Ser Phe Leu Ala Ala Ala Thr Ile Tyr Ser Leu Phe Tyr Thr Ala Ile Leu His Phe Ser Gly Gly Gln Leu Met Val Thr Gly Pro Val Ala Thr Ala Gly Ile Phe Ala Thr Tyr Leu Pro Asp His Met Thr Leu Trp Arg Gly Phe Leu Asn Glu Ala Trp Leu Thr Gly Met Leu G1n Leu Cys Leu Phe Ala Ile Thr Asp Gln Glu Asn Asn Pro Ala Leu Pro Gly Thr Glu Ala Leu Val Ile Gly Ilea Leu Val Val 2.00 205 Ile Ile Gly Val Ser Leu Gly Met Asn Thr Gly Tyr Ala Ile Asn Pro Ser Arg Asp Leu Pro Pro Arg Tle Phe Thr Phe: Ile Ala Gly Trp GIy Lys Gln Val Phe Ser Asn Gly Glu Asn Trp Trp Trp Val Pro Val Val Ala Pro Leu Leu Gly Ala Tyr Leu Gly Gly Ile Ile 270 .
Tyr Leu Val Phe Ile Gly Ser Thr Ile Pro Arg Glue Pro Leu Lys Leu Glu Asp Ser Val Ala Tyr Glu Asp His Gly Ile Thr Va1 Leu Pro Lys Met Gly Ser His Giu Pro Thr Ile Ser Pra Leu Thr Pro Val Ser Val Ser Pro Ala Asn Arg Ser Ser Val His Pro Ala Pro Pro Leu His G1u Ser Met Ala Leu Glu His Phe <210> 6 <211> 476 <212> PRT
<213> Homo sapiens <220>
<22I> misc_feature <223> Incyte ID No: 2666782CD1 <400> 6 Met Gly Ile Lys Phe Leu Glu Val Ile Lys Pro Phe Cys Ala Val Leu Pro Glu Ile Gln Lys pro GIu Arg Lys Ile Gln Phe Arg Glu Lys Val Leu Trp Thr Ala Ile Thr Leu Phe Ile Phe Leu Val Cys Cys Gln Ile Pro Leu Phe Gly Ile Met Ser Ser Asp Ser AIa Asp Pro Phe Tyr Trp Met Arg Val IIe Leu Ala Ser Aan Arg Gly Thr Leu Met Glu Leu Gly Ile Ser Pro Ile Val Thr Se=r Gly Leu IIe Met GIn Leu Leu Ala Gly Ala Lys Ile Ile Glu Val Gly Asp Thr Pro Lys Asp Arg Ala Leu Phe Asn Gly Ala Gln Lys Leu Phe Gly Met Ile Ile Thr Ile Gly Gln Ala Ile Val Tyr Val Met Thr Gly Met Tyr Gly Asp Pro Ala Glu Met Gly Ala Gly Il.e Cys Leu Leu Ile Ile Ile Gln Leu phe Val Thr Ser Leu Ile Va.l Leu Leu Leu Asp Glu Leu Leu Gln Thr Gly Tyr Ser Leu Gly Ser Gly Ile Ser Leu Val Ile Ala Thr Asn Ile Cys Glu Thr Ile Val Trp Lys Ala Phe Ser Pro Thr Thr Ile Asn Thr Gly Arg Gly Thr Glu Phe Glu Gly Ala Val Ile AIa Leu Phe His Leu Leu AIa Th:r Arg Thr Asp Lys Val Arg Ala Leu Arg Glu Ala Phe Tyr Arg Gln Asn Leu Pro Asn Leu Met Asrl Leu Ile Ala Thr Val Phe Val Phe A1a Val Val Ile Tyr Phe Gln Gly Phe Arg Val Asp Leu Pro Ilea Lys Ser Ala Arg Tyr Arg Gly Gln Tyr Ser Ser Tyr Pro Ile Ly.; Leu Phe Tyr Thr Ser Asn Ile Pro Ile Ile Leu Gln Ser Ala Leu Val Ser Asn Leu Tyr Val Ile Ser G1n Met Leu Ser Val Arg PhE: Ser Gly Asn Phe Leu Val Asn Leu Leu Gly Gln Trp Ala Asp Val. Ser Gly Gly Gly pro Ala Arg Ser Tyr Pro Va1 Gly Gly Leu Cys Tyr Tyr Leu Ser Pro pro Glu Ser Met Gly Ala Ile Phe Glu Asp Pro Val His VaI Val Val Tyr Ile Ile Phe Met Leu Gly Ser Cys Ala Phe Phe Ser Lys Thr Trp Ile Glu Val Sex Gly Ser Ser Ala Lys Asp Val Ala Lys Gln Leu Lys Glu Gln Gln Met Val Met Ax~g Gly His Arg Asp Thr Ser Met Val His Glu Leu Asn Arg Tyr Ile Pro Thr Ala Aia Ala Phe Gly Gly Leu Cys Ile Gly Ala Leu Ser Val Leu Ala Asp Phe Leu Gly Ala Ile Gly Ser Gly Thr Gly Ile Leu Leu Ala Val Thr Ile Ile Tyr Gln Tyr Phe Glu Ile Phe Val Lys Glu Gln Ala Glu Val Gly Gly Met Gly Ala Leu Phe Phe <210> 7 <211> 266 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2731369CD1 <400> 7 Met Asn Ala Phe Leu Gln Leu Gly Asn Trp Gly Leu Ser Val Lys Tyr Ser Val Leu Ser Arg Leu Va7. Phe Thr Ile Trp Ser Val Ile Phe Arg Leu Val Tyr Val Ala Glu Trp Val Val Ala Glu Val Asp Asp Glu Lys Asp Phe Asp Thr Gln Gly Gln Cys Asn Lys Pro Cys Thr Asn Cys Tyr Asp Asn Pro Sex' Ile Val Tyr Phe Ile Asn Arg Leu Trp Leu Gln Leu Ile Thr Pro Leu Ala Leu Val Cys Ser Leu Val Val His Val Ala Tyr Glu Glu. Lys Met Arg Glu Arg Arg His His Leu His Gly Pro Asn Ser Tyr Asn Lys Ala Pro Leu Asp Lew Ser Lys Arg Gly Gly Leu Thr Leu Ser Lys Trp Trp Tyr Leu Leu 125 I3 0 3.3 Ile Phe Ala Ala Val Asp Phe Tyr Phe Lys Ala Gly Leu Ile His Arg Leu Lys Asp Tyr Asp Arg Val Cys Tyr Met Pro Val Ala Ser Val Glu Cys Pro His Thr Cys Ile Arg Pro Val Asp Tyr Ser Pro Thr Glu Lys Val Phe Thr Met Thr.ThrAla Lys Tyr Phe Val Ala Ile Cys Leu Leu Asn Leu Val Tyr Val Ile Ser Glu Phe Leu Gly Lys Arg Met G1u Ile Phe Arg Arg Pro Cys Gly Pro His Arg Arg Cys Arg Cys Leu Pro Asp Pro Tyr Leu Glu Thr Cys Pro Val Ser WO 00//2711 PCT/US99/204b8 Gln Gly Gly His Pro Glu Asp Gly Asn Ser Val L~~u Met Lys Ala Gly Ber Ala Pro Val Asp Ala Gly Gly Tyr Pro <210> 8 <211> 182 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1375415CD1 <400> 8 Met Ala Glu Phe Pro Ser Lys Val Ser Thr Arg Thr Ser Ser Pro Ala Gln Gly Ala Glu Ala Ser Val Ser Ala Leu Arg Pro Asp Leu Gly Phe Val Arg Ser Arg Leu Gly Ala Leu Met Leu Leu Gln Leu Val Leu Gly Leu Leu Val Trp Ala Leu Ile Ala Asp Thr Pro Tyr His Leu Tyr Pro Ala Tyr Gly Trp Val Met Phe Val Ala Val Phe Leu Trp Leu Val Thr Ile Val Leu Phe Asn Leu Tyr Leu Phe Gln Leu His Met Lys Leu Tyr Met Val Pro Trp Pro Leu Val Leu Met Ile Phe Asn Ile Ser Ala Thr Val Leu Tyr Ile Th:r Ala Phe Ile Ala Cys Ser Ala Ala Val Asp Leu Thr Ser Leu Ar<1 Gly Thr Arg 125 130 , 135 Pro Tyr Asn Gln Arg Ala Ala Ala Ser Phe Phe Al:~ Cys Leu Val Met Ile Ala Tyr Gly Va3 Ser Ala Phe Phe Ser Tyr Gln Ala Trp Arg Gly Val Gly Ser Asn Ala Ala Thr Ser Gln Met: Ala Gly Giy Tyr Ala <210> 9 <211> 942 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2733282CD1 <400> 9 Met Thr Gln Arg Ser Ile Ala Gly Pro Ile Cys Asn Leu Lys Phe Val Thr Leu Leu Val Ala Leu Ser Ser GIu Leu P:ro Phe Leu Gly AIa Gly Val Gln Leu GIn Asp Asn Gly Tyr Asn G:Ly Leu Leu Ile Ala Ile Asn Pro Gln Val Pro GIu Asn Gln Asn Leu IIe Ser Asn Ile Lys Glu Met IIe Thr Glu Ala Ser Phe Tyr Le:u Phe Asn Ala Thr Lys Arg Arg Val Phe Phe Arg Asn IIe Lys I:Le Leu Ile Pro Ala Thr Trp Lys AIa Asn Asn Asn Ser Lys Ile Lys Gln Glu Ser Tyr GIu Lys Ala Asn Val Ile Val Thr Asp Trp Tyr Gly Ala His Gly Asp Asp Pro Tyr Thr Leu Gln Tyr~Arg Gly C~~s Gly Lys Glu Gly Lys Tyr Ile His Phe Thr Pro Asn Phe Leu Le:u Asn Asp Asn 3.40 145 150 Leu Thr Ala Gly Tyr Gly Ser Arg Gly Arg Val Phe Val His Glu Trg Ala His Leu Arg Trp Gly Val Phe Asp Glu Tyr Asn Asn Asp Lys Pro Phe Tyr Ile Asn Gly Gln Asn Gln Ile Lys Val Thr Arg Cys Ser Ser Asp Ile Thr Gly Ile Phe VaI Cys Glu Lys Gly Pro Cys Pro Gln Glu Asn Cys IIe Iie Ser Lys Leu Phe Lys Glu Gly Cys Thr Phe Ile Tyr Asn Ser Thr Gln Asn Ala Thr Ala Ser Ile Met Phe Met Gln Ser Tyr Leu Cys Gly Glu IIe Cy,s Asn Ala Ser Thr His Asn Gln Glu Ala Pro Asn Leu Gln Asn GIn Met Cys Ser Leu Arg Ser Ala Trp Asp Val Ile Thr Asp Ser Ala Asp Phe His His Ser Phe Pro Met Asn Gly Thr Glu Leu Pro Pro Pro Pro Thr Phe Ser Leu VaI Glu Ala Gly Asp Lys Val Val Cy:a Leu Val Leu Asp Val Ser Ser Lys Met Ala Glu Ala Asp Arg Leu Leu Gln Leu Gln Gln AIa Ala Glu Phe Tyr Leu Met Gln Ile Va7L Glu Ile His Thr Phe Val Gly Ile Ala Ser Phe Asp Ser Lys Gly Glu Ile Arg 350 . 355 360 Ala Gln Leu His GIn Ile Asn Ser Asn Asp Asp Arch Lys Leu Leu Val Ser Tyr Leu Pro Thr Thr Val Ser Ala Lys Thr Asp Ile Ser Ile Cys Ser Gly Leu Lys Lys Gly Phe GIu Val Val. Glu Lys Leu Asn Gly Lys Ala Tyr GIy Ser VaI Met Ile Leu Val Thr Ser Gly Asp Asp Lys Leu Leu Gly Asn Cys Leu Pro Thr Val Leu Ser Ser Gly Ser Thr Ile His Ser Ile Ala Leu Gly Ser S'~er Ala Ala Pro Asn Leu Glu Glu Leu Ser Arg Leu Thr Gly Gly Leu Lys Phe Phe VaI Pro Asp Ile Ser Asn Ser Asn Ser Met Ile Asp Ala Phe Ser Arg Ile Ser Ser Gly Thr Gly Asp IIe Phe Gln Gln His Ile Glri Leu Glu Ser Thr Gly Glu Asn Val Lys Pro His His Gln Leu Lys Asn Thr Val Thr Val Asp Asn Thr Val Gly Asn A;sp Thr Met Phe Leu Val Thr Trp Gln Ala Ser Gly Fro Pro Glu I:Le Ile Leu Phe Asp Pro Asp Gly Arg Lys Tyr Tyr Thr Asn Asn Plze Ile Thr Asn Leu Thr Phe Arg Thr Ala Ser Leu Trp Ile Pro G:Ly Thr Ala Lys Pro Gly His Trp Thr Tyr Thr Leu Asn Asn Thr His His Ser Leu Gln Ala Leu Lys Val Thr Val Thr Ser Arg Ala Se:r Asn Ser Ala Val Pro Pro Ala Thr Val Glu Ala Phe Val Glu Az~g Asp Ser Leu His Phe Pro His Pro Val Met Ile Tyr Ala Asn Va~.l Lys Gln Gly Phe Tyr Pro Ile Leu Asn Ala Thr Val Thr Ala Th.r Val Glu Pro Glu Thr Gly Asp Pro Val Thr Leu Arg Leu Leu Asp Asp Gly AIa Gly AIa Asp Val Ile Lys Asn Asp Gly Ile Tyr Ser Arg Tyr Phe Phe Ser Phe Ala Ala Asn Gly Arg Tyr Ser Leu Lys Val His Val Asn His Ser Pro Ser Ile Ser Thr Pro Ala His Ser Ile Pro Gly Ser His Ala Met Tyr Val Pro Gly Tyr Thr Ala Assn Gly Asn Ile Gln Met Asn Ala Pro Arg Lys Ser Val Gly Arg Asn Glu Glu Glu Arg Lys Tzp Gly Phe Ser Arg Val Ser Ser Gly Gly Ser Phe Ser Val Leu Gly Val Pro Ala Gly Pro His Fro Asp Va:l Phe Pro Pro Cys Lys Ile IIe Asp Leu Glu Ala Val Lys VaI Glu Glu GIu Leu 770 ?75 780 Thr Leu Ser Trp Thr Ala Pro Gly Glu Asp Phe Asp Gln Gly Gln Ala Thr Ser Tyr Glu Ile Arg Met Ser Lys Ser Leu Gln Asn Ile Gln Asp Asp Phe Asn Asn Ala I1e Leu VaI Asn Thzv Ser Lys Arg Asn Pro Gln Gln Ala Gly Ile Arg Glu Ile Phe Thr Phe Ser Fro 830 $3S 840 G1n Ile Ser Thr Asn Gly Pro Glu His Gln Pro Asn, Gly Glu Thr His Glu Ser His Arg Ile Tyr Val Ala Ile Arg Ala Met Asp Arg Asn Ser Leu Gln Ser Ala Val Ser Asn Ile Ala Gln Ala Pro Leu.

Phe Ile Pro Pro Asn Ser Asp Pro Val Pro Ala A.rg Asp Tyr Leu Ile Leu Lys Gly Val Leu Thr Ala Met Gly Leu Ile Gly Ile Ile Cys Leu Ile Ile Val Val Thr His His Thr Leu Ser Arg Lys Lys Arg Ala Asp Lys Lys Glu Asn Gly Thr Lys Leu L~eu <210> 10 <211> 519 <212> PI2T
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 3148427CDI
<400> 10 Met Glu Glu Met Phe His Lys Lys Ser Glu Ala Va,l Arg Arg Leu VaI Glu Ala AIa Glu Glu Ala His Leu Lys His Glu Phe Asp Ala Asp Leu Gln Tyr Glu Tyr Phe Asn Ala Val Leu Ile Asn Glu Arg Asp Lys Asp Gly Asn Phe Leu Glu Leu Gly Lys Glu Phe IIe Leu AIa Pro Asn Asp His Phe Asn Asn Leu Pro Val Assn Ile Ser Leu Ser Asp Val Gln Val Pro Thr Asn Met Tyr Asn Ly,s Asp Pro Ala Ile Val Asn Gly Val Tyr Trp Ser Glu Sex Leu Asn Lys Val Phe Val Asp Asn Phe Asp Arg Asp Pro Ser Leu Ile Trp Gln Tyr Phe Gly Ser Ala Lys Gly Phe Phe Arg Gln Tyr Pro Gly Ile Lys Trp Glu Pro Asp Glu Asn Gly Val Ile Ala Phe Asp Cys Arg Asn Arg Lys Trp Tyr Ile Gln Ala Ala Thr Ser Pro Lys Asp Val Val Ile Leu Val Asp Val Ser Gly Ser Met Lys Gly Leu Arg~ Leu Thr Ile Ala Lys Gln Thr Val Ser Ser Ile Leu Asp Thr Leu~. Gly Asp Asp Asp Phe Phe Asn Ile Ile Ala Tyr Asn Glu Glu Leu. His Tyr Val Glu Pro Cys Leu Asn Gly Thr Leu Val G1n Ala Asp Arg Thr Asn Lys Glu His Phe Arg GIu His Leu Asp Lys Leu Phe Ala Lys Gly Ile Gly Met Leu Asp Ile Ala Leu Asn Glu Ala F?he Asn Tle Leu Ser Asp Phe Asn His Thr Gly Gln Gly Ser Ile C'ys Ser Gln Ala Ile Met Leu Ile Thr Asp Gly Ala Val Asp Thr Tyr Asp Thr Ile Phe Ala Lys Tyr Asn Trp Pro Asp Arg Lys Val A.rg Ile Phe Thr Tyr Leu Ile Gly Arg Glu Ala Ala Phe Ala Asp A,sn Leu Lys Trp Met Ala Cys Ala Asn Lys Gly Phe Phe Thr Gln Ile Ser Thr Leu Ala Asp Val Gln Glu Asn Val Met Glu Tyr Leu His Val Leu Ser Arg Pro Lys Val Ile Asp Gln Glu His~Asp Val Vai Trp Thr Glu Ala Tyr Ile Asp Ser Thr Leu Pro Gln Ala Gln Lvys Leu Thr Asp Asp Gln Gly Pro Val Leu Met Thr Thr Val Ala Met Pro Val Phe Ser Lys Gln Asn Glu Thr Arg Ser Lys Gly Tle Le~u Leu Gly Val Val Gly Thr Asp Val Pro Val Lys Glu Leu Leu L;rs Thr Tle Pro Lys Tyr Lys Leu Gly Ile His Gly Tyr Ala Phe Ala Ile Thr Asn Asn Gly Tyr Ile Leu Thr His Pro Glu Leu Arg Le:u Leu Tyr Glu Glu Gly Lys Lys Arg Arg Lys Pro Asn Tyr Ser Se:r Val Asp Leu Ser Glu Val Glu Trp Glu Asp Arg Asp Asp Val Le:u Arg Asn Ala Met Val Asn Arg Lys Thr Gly Lys Phe Ser Met Glu Val Lys Lys Thr Val Asp Lys Gly Val His Phe Ser Gln Thr Ph.e Leu Leu Leu Asn Leu Lys Gln Thr Thr Val Lys Asn <210> 11 <211> 251 <212> PRT
<213> Homo sapiens <220>
<221> miso_feature <223> Incyte ID p'o: 3342358CD1 <400> 11 Met Thr Asp Ser Ala Thr Ala Asn Gly Asp Asp Arch Asp pra Glu 1 5 10 ~ 15 Ile Glu Leu Phe VaI Lys Ala Gly Ile Asp Gly Glu Ser Ile Gly Asn Cys Pro Phe Ser Gln Arg Leu Phe Met Ile Leu Trp Leu Lys WO 00/1271 i PCT/US99I20468 Gly Val Val Phe Asn Val Thr Thr Val Asp Leu L~rs Arg Lys Pro Ala Asp Leu His Asn Leu Ala Pro Gly Thr His Px-o Pro Phe Leu Thr Phe Asn Gly Asp Val Lys Thr Asp Val Asn Lys Ile Glu Glu Phe Leu Glu Glu Thr Leu Thr Pro Glu Lys Tyr Pro Lys Leu Ala Ala Lys His Arg Glu Ser Asn Thr Ala Gly Ile Asp Ile Phe Ser Lys Phe Ser Ala Tyr Ile Lys Asn Thr Lys Gln Gln Asn Asn A1a Ala Leu Glu Arg Gly Leu Thr Lys AIa Leu Lys Lys Leu Asp Asp Tyr Leu Asn Thr Pro Leu Pro Glu Glu~Ile Asp Ala Asn Thr Cys Gly Glu Asp Lys Gly Ser Arg Arg Lys Phe Leu As;p Gly Asp Glu Leu Thr Leu Ala Asp Cys Asn Leu Leu Pro Lys Leu His Val Val Lys Ile Val Ala Lys Lys Tyr Arg Asn Tyr Asp Ile Pro Ala Glu Met Thr Gly Leu Trp Arg Tyr Leu Lys Asn Ala Tyr Ala Arg Asp Glu Phe Thr Asn Thr Cys Ala Ala Asp Ser Glu Ile: Glu Leu Ala Tyr Ala Asp Val Ala Lys Arg Leu Ser Arg Ser <210> 12 <211> 323 <212> PRT
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1267774CD1 <400> 12 Met Gly Leu Phe Asp Arg Gly Val Gln Met Leu Leu Thr Thr Val Gly Ala Phe Ala Ala Phe Ser Leu Met Thr Ile Ala Val Gly Thr Asp Tyr Trp Leu Tyr Ser Arg Gly Val Cys Lys Thr Lys 5er Val Ser Glu Asn Glu Thr Ser Lys Lys Asn Glu Glu Val Met Thr His Ser Gly Leu Trp Arg Thr Cys Cys Leu Glu Gly Asn Ser Lys Gly Leu Cys Lys Gln Ile Asp His Phe Pro Glu Asp Ala Asp Tyr GIu Ala Asp Thr Ala Glu Tyr Phe Leu Arg Ala Va1 Arg Ala Ser Ser Ile Phe Pro Ile Leu Ser Val Ile Leu Leu Phe Met Gly Gly Leu WO 00/12711 PCT/US99/2046$
Cys Ile Ala Ala Ser Phe Tyr Lys Thr Arg Ile Glu Hi.s Asn Ile Leu Ser Ala Gly Ile Phe Val Ser Ala Gly Asn Phe Le:u Ser Ile Ile Gly Ile Ile Val Ile Ser Ala Asn Ala Pro Tyr Gly Asp Ser Lys Ser Asp Ser Lys Asn Ser Tyr Ser Tyr Ser Lys Gly Trp Phe Tyr Phe Gly Ala Leu Phe Ile Ile Ala Glu Gly Ser Met Val Val Leu Ala Val His Met IIe Asp Arg His Lys Arg Phe Gl:n Leu Ala Thr Ala Arg Ala Thr Tyr Leu Gln Ala Ser Thr Asp Al,a Ile Arg IIe Pro Ser Tyr Arg Arg Tyr Gln Arg Arg Ser Tyr Se:r Arg Ser Ser Arg Ser Thr Glu Ser His Ser Arg Asp Pro Pro A1<~ Ser Val Gly Ile Lys Gly Phe Thr Leu Pro Ser Thr Ser Asn Glu Ile Met Tyr Thr Leu Ser Arg Pro Leu Lys Ala Ala Pro Asp Thr Thr Thr Ala Thr Tyr Asn Ser Arg Asp Asn Ser Phe Val Asp Leu Gln His Asn Cys Ile Gln Lys Glu Asn Lys Asp Ser Leu His. Ser Asn Thr Ala Asn Arg Arg Thr Pro Val Thr <210> 13 <211> 51 <212> PRT
<213> Homa Sapiens <220>
<221> misc_feature <223> Incyte ID No: I8I7329CD1 <400> 13 Met Asn Gln Gly Ser Gly Leu Asp Leu Leu Lys Ile Ser Tyr Gly Lys Gly Ala Arg Arg Lys Asn Arg Phe Lys Gly Ser Asp Gly Ser Thr Ser Ser Asp Thr Thr Ser Asn Ser Phe Val Arg Gln Val Arg Val Leu Ser Ser Trp Phe <210> 14 <211> 113 <212> PRT
<213> Homo Sapiens <220>

<221> misc_feature <223> Incyte ID No: 3273307CD1 <400> 14 Met Glu Gln Arg Lys Leu Asn Asp Gln Ala Asn Thr Leu Val Asp Leu Ala Lys Thr Gln Asn Ile Met Tyr Asp Met Il~e Ser Asp Leu Asn Glu Arg Ser Glu Asp Phe Glu Lys Arg Ile Va.l Thr Leu Glu Thr Lys Leu Glu Thr Leu Ile Gly Ser Ile His Ala Leu Pro Gly Leu Ile Ser Gln Thr Ile Arg Gin Gln Gln Arg Asp Phe Ile Glu Ala Gln Met Glu Ser Tyr Asp Lys His Val Thr Tyr Asn Ala Glu Arg Sex Arg Ser Ser Ser Arg Arg Arg Arg Ser Ser Ser Thr Ala Pro Pro Thr Ser Ser Glu Ser Ser <210> 15 c211> 215 < 212 > PTtT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 3824833CD1 <400> 15 Met His Arg Asp Ala Trp Leu Pro Arg Pro Ala Phe Ser Leu Thr Gly Leu Ser Leu Phe Phe Ser Leu Val Pro Pro Gly Arg Ser Met Glu Val Thr Val Pro Ala Thr Leu Asn Val Leu Asn Gly Ser Asp Ala Arg Leu Pro Cys Thr Phe Asn Ser Cys Tyr Thr Val Asn His Lys Gln Phe Ser Leu Asn Trp Thr Tyr Gln Glu Cys Asn Asn Cys Ser Glu GIu Met Phe Leu Gln Phe Arg Met Lys Ile Ile Asn Leu Lys Leu Glu Arg Phe Gln Asp Arg Val Glu Phe Ser Gly Asn Pro Ser Lys Tyr Asp Val Ser Val Met Leu Arg Asn Val Gln Pro Glu Asp Glu Gly Ile Tyr Asn Cys Tyr Ile Met Asn Pro Pro Asp Arg His Arg Gly His Gly Lys Ile His Leu Gln Val Leu Met Glu Glu Pro Pro Glu Arg Asp Ser Thr Val Ala Val Ile Val Gly Ala Ser Val Gly Gly Phe Leu Ala Val Val Ile Leu Val Leu Met Val Val i ~r4~

Lys Cys Val Arg Arg Lys Lys GIu Gln Lys Leu Se:r Thr Asp Asp Leu Lys Thr Glu Glu GIu Gly Lys Thr Asp GIy Glu Gly Asn Pro Asp Asp Gly Ala Lys 2 3.5 <210> 16 <211> 235 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2069907CD1 <400> 16 Met Phe Ile Trp Thr Ser Gly Arg Thr Ser Ser Sexy Tyr Arg His Asp Glu Lys Arg Asn Ile Tyr Gln Lys Ile Arg Asp His Asp Leu Leu Asp Lys Arg Lys Thr Val Thr AIa Leu Lys Ala. Gly Glu Asp Arg Ala Ile Leu Leu Gly Leu Ala Met Met Val Cys Ser IIe Met Met Tyr Phe Leu Leu Gly Ile Thr Leu Leu Arg Ser Tyr Met Gln Ser Val Trp Thr Glu Glu Ser Gln Cys Thr Leu Leu Asn Ala Ser Ile Thr Glu Thr Phe Asn Cys Ser Phe Ser Cys Gly Pro Asp Cys Trp Lys Leu Ser Gln Tyr pro Cys Pro Gln Val Tyr Va1 Asn Leu Thr Ser Ser Gly Glu Lys Leu Leu Leu Tyr His Thr Glu Glu Thr Ile Lys Ile Asn Gln Lys Cys Ser Tyr Ile Pro Lys Cys Gly Lys Asn Phe Glu Glu Ser Met Ser Leu Val Asn Val Val Met Glu Asn Phe Arg Lys Tyr Gln His Phe Ser Cys Tyr Ser Asp Pro Glu Gly 170 175 1$0 Asn Gln Lys Ser Val Ile Leu Thr Lys Leu Tyr Ser Ser Asn Val Leu Phe His Ser Leu Phe Trp Pro Thr Cys Met Met Ala Gly Gly Val Ala Ile Val Ala Met Val Lys Leu Thr Gln Tyr Leu Ser Leu Leu Cys Glu Arg Ile Gln Arg Ile Asn Arg <210> 17 <211> 234 <212> PRT
<213> Homo Sapiens <220>
<22i> misc_feature <223> Incyte ID No: 2243917CDI
<400> 17 Met Ala Glu Asn His Cys Glu Leu Leu Ser Pro A.la Arg Gly Gly Ile Gly Ala Gly Leu Gly Gly Gly Leu Cys Arg A.rg Cys Ser Ala Gly Leu Gly Ala Leu Ala Gln Arg Pro Gly Ser Val Ser Lys Trp VaI Arg Leu Asn Val Gly Gly Thr Tyr Phe Leu T.hr Thr Arg Gln Thr Leu Cys Arg Asp Pro Lys Ser Phe.Leu Tyr A:rg Leu Cys Gln Ala Asp Pro Asp Leu Asp Ser Asp Lys Asp Glu Thr Gly Ala Tyr Leu IIe Asp Arg Asp Pro Thr Tyr Phe Gly Pro V<~1 Leu Asn Tyr Leu Arg His Gly Lys Leu Vai Ile Asn Lys Asp Le~u Ala Glu G1u Gly Val Leu Glu Glu Ala Glu Phe Tyr Asn Ile Thr Ser Leu Ile Lys Leu Val Lys Asp Lys Ile Arg Glu Arg Asp Se:r Lys Thr Ser Gln Val Pro Val Lys His Val Tyr Arg Val Leu Gl.n Cys Gln Glu Glu Glu Leu Thr Gln Met Val Ser Thr Met Ser As;p Gly Trp Lys Phe Glu Gln Leu Val Ser Ile Gly Ser Ser Tyr Asn Tyr Gly Asn Glu Asp G1n Ala Glu Phe Leu Cys VaI Val Ser Lys Glu Leu His Asn Thr Pro Tyr Gly Thr Ala Ser Glu Pro Ser Glu Lys Ala Lys Ile Leu Gln Glu Arg Gly Ser Arg Met <210> 18 <211> 301 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2597476CD1 <400> 18 Met Val Phe Thr Gln Ala Pro Ala Glu Ile Met Gly His Leu Arg 1 5 i0 15 Ile Arg Sex Leu Leu Ala Arg Gln Cys Leu Ala Glu Phe Leu Gly Val Phe Val Leu Met Leu Leu Thr Gln Gly Ala Val Ala Gln Ala Va1 Thr Ser Gly Glu Thr Lys Gly Asn Phe Phe Thr Met Phe Leu Ala Gly Ser Leu Ala Val Thr.Ile Ala Ile Tyr Val Gly Gly Asn Val Ser G1y Ala His Leu Asn Pro Ala Phe Ser Leu Ala Met Cys Ile Val Gly Arg Leu Pro Trp Val Lys Leu Pro Il,e Tyr Ile Leu Val Gln Leu Leu Ser Ala Phe Cys Ala Ser Gly Ala Thr Tyr Val Leu Tyr His Asp Ala Leu Gln Asn Tyr Thr Gly Gly Asn Leu Thr Val Thr Gly Pro Lys Glu Thr Ala Ser Ile Phe Ala Thr Tyr Pro Ala Pro Tyr Leu Ser Leu Asn Asn Gly.Phe Leu Asp Gln Val Leu GIy Thr Gly Met Leu Ile Val Gly Leu Leu Ala Il~e Leu Asp Arg Arg Asn Lys Gly Val Pro Ala Gly Leu Glu Pro Va:l Val Val Gly Met Leu Ile Leu Ala Leu Gly Leu Ser Met Gly A1;~ Asn Cys Gly Ile Pro Leu Asn Pro Ala Arg Asp Leu Gly Pro Arg Leu Phe Thr Tyr Val Ala Gly Trp Gly Pro Glu Val Phe Ser Ala Gly Asn Gly Trp Trp Trp Val Pro Val Val Ala Pro Leu Val Gly Ala Thr Val Gly Thr Ala Thr Tyr Gln Leu Leu Val Ala Leu Hi.o His Pro Glu Gly Pro Glu Pro Ala Gln Asp Leu Val Ser Ala Glr.~ His Lys A1a Sex Glu Leu Glu Thr Pro Ala Ser Ala Gln Met Leu Glu Cys Lys Leu <210> 19 <211> 2994 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1568324CB1 <400> 19 gggaggcact gcccgtgttg ggatgcagaa gggatcagct tcc<~gttgtc ttggagttga 60 tgactgacct ctacctctgc agggaaaggg gcgcagggag tctc;aaagct ccaggagcag 120 cagaaggccc 'agtggccggt ctccaaccag tactgagaag cgca~tgagct tcgagtccat 180 ttcttccctg ccagaggttg agccggaccc tgaggctggg agtc~agcaag aggtattttc 240 tgctgtggaa gggcccagtg ccgaggagac gccttcagac acac~aatctc cagaagtcct 300 ggagacacag 'cttgatgccc accagggcct tctggggatg gaccccccag gtgacatggt 360 ggacttcgtg gcagctgaga gcactgagga ccttaaggcc ctgagcagcg aggaggaaga 420 agaaatggga ggtgccgccc aggagcctga gagccttctg ccac;cctctg tgctggacca 480 ggccagcgtc attgcggagc gatttgtcag cagcttctct cggc:ggagca gcgtggcaca 540 ggaggacagc aagtccagtg gctttgggag cccgcggctg gtcaegecgga gcagcagcgt 600 gctcagcctg gagggcagcg agaagggcct ggcccggcat gc~cagtgcca cagactccct 660 cagctgtcag ctctccccag aagtggacat cagtgtgggg gt~ggccacag aggacagccc 720 ttctgtcaat gggatggagc ccccaagccc aggctgccca gt~ggagcctg accggtcttc 780 ctgcaagaag aaggaatcag cactctccac ccgagaccgg ct:gttgctag acaagattaa 840 gagctattat gaaaatgcag aacaccatga tgcaggcttc ac~cgtccgtc gccgggagag 900 cctctcctac atccccaaag gactggtaag aaactccatc tc:caggttca acagccttcc 960 ccggccagac ccagagccag tacctccagt ggggagcaag ac~acaggtgg gctcccggcc 1020 gacttcgtgg gccctgtttg agctcccagg accaagccag gc:agtcaaag gggacccacc 1080 tcccatctca gatgctgagt tccgcccatc ttcagaaatt gtgaagatct gggagggaat 1140 ggagtcttcc ggagggagcc ctgggaaggg gccaggccag gcrccaggcca atggctttga 1200 cctgcatgag ccactcttca tcctggagga gcatgagctg ggragccatca cagaggagtc 1260 ggccactgcc tccccggaaa gctcctctcc cactgagggg cg;cagcccgg cccacctggc 1320 ccgggagctg aaagagctgg tgaaggagct gagcagcagt acccaggggg agctggtggc 1380 cccactgcac ccccgcatcg tgcagctctc ccacgtaatg ga.cagccacg tgagcgagcg 1440 cgtcaagaac aaggtctacc agctggcccg ccagtacagc ctccggatca agagcaacaa 1500 gccagtgatg gccaggccac cactgcagtg ggaaaaggtg gcccctgaga gggatgggaa 1560 gagccccact gtgccctgtc tacaggaaga ggctggagag ccattaggtg gcaaaggtaa 1620 gaggaagccg gtgctgtctc tatttgacta tgagcagctg atggcccagg agcacagccc 1680 tcccaagccc tcctcggctg gggagatgtc accacagcgt ttcttcttca acccgcctgc 1740 tgtcagccag aggaccacct cgcctggggg ccggccctcc gcccggagcc ccctcagccc 1800 cacagagacc ttcagctggc ccgacgtccg tgagctctgc tccaagtatg cctcccgcga 1860 tgaggcacgc cgagcagggg gcggccggcc ccgcggccca cccgtcaaca ggagccactc 1920 ggtgccggag aacatggtag agccacctct gtcgggcagg gtgggccgct gccgcagcct 1980 gagcaccaag aggggccggg gaggcggaga ggctgcccaa tcccctgggc ctctgcccca 2040 gagcaagccg gatggaggcg agaccctgta tgtcactgca gacctcaccc tggaggacaa 2100 ccggcgggtg attgtcatgg agaagggacc ccttcccagc cccactgcag ggctggagga 2160 gagcagtggc cagggaccaa gctcaccggt ggccctgctg gggcaggttc aggacttcca 2220 gcagtctgca gagtgccagc cgaaggaaga gggttccagg gacccggcag acccgagcca 2280 gcagggcaga gtgagaaacc ttagagagaa gttccaggcc ttgaactctg tcggttgatg 2340 ctgactcctg ggggagggag gagtcatgtt ggaggttggg gaagaacctg ggcatccttc 2400 ccctcaagcc tgggctcatg gagcccctgc ccagggccct caggtgggcg gaaagtccat 2460 cccctccgcc cttcaggaag gatgctcccg tgtgcagggg tct~cctgcct gtgccatcca 2520 ctggggctcg agacaatttc ccactcacct gtgaggccgg tgt:ggctgct tcccttgtaa 2580 atagttgttc tctggtaaga agccaaatat ttaagctcac ttcatcccag agagaggaag 2640 ctctgctcag gcctccagcg ttggctggcc atggccacag ccagatggag gagcccatcc 2700 ccaggagact caggcagtgg cctggagagg ctttgttctg taacggtgcc ttttcttagg 2760 gtccaggcag gaatgaagcc aataatttat tgctttccat tct:gtggtat gatgtgcgtg 2820 tgcgtgagtg tgtggcccct gtttattccc ctcctgtcaa gaatgaagtg gattcagttc 2880 aggtactttt gagggttgtt gtgctgaccc tgtggttgtc gct:gatgtac acacatttca 2940 ttatttgcca atggtgcaat aaccaatgct gacaaaccca aaa~aaaaaaa aaaa 2994 <210> 20 <211> 1298 <2I2> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 4094907081 <400> 20 cacgaccccc tgcctcttct tccagaagcc aatgggcaca gaagcaccaa ttctcccaca 60 atagtttcac ctgctattgt ttcccccacc caggacagtc ggcccaatat gtcaagacct 120 ctgatcacta gatcccctgc atctccactg aacaaccaag gcatccctac tccagcacaa 180 ctcacaaaat ccaatgcgcc tgtccacatt gatgtgggcg gccacatgta caccagcagc 240 ctggccaccc tcaccaaata ccctgaatcc agaatcggaa gac~tttttga tggtacagag 300 cccattgttt tggacagtct caaacagcac tatttcattg acagagatgg acagatgttc 360 agatatatct tgaattttct acgaacatcc aaactcetca ttcctgatga tttcaaggac 420 tacactttgt tatatgaaga ggcaaaatat tttcagcttc agcccatgtt gttggagatg 480 gaaagatgga 'agcaggacag agaaactggt cgattttcaa ggccctgtga gtgcctcgtc 540 gtgcgtgtgg ccccagacct cggagaaagg atcacgctaa gc:ggtgacaa atccttgata 600 gaagaagtat ttccagagat cggcgacgtg atgtgtaact ct:gtcaatgc aggctggaat 660 cacgactcga cgcacgtcat caggtttcca ctaaatggct acagtcacct caactcagtc 720 caggtcctcg agaggttgca gcaaagagga tttgaaatcg tgggctcctg tgggggagga 780 gtagactcgt cccagttcag cgaatacgtc cttcggcggg aactgaggcg gacgccccgt 840 gtaccctccg tcatccggat aaagcaagag cctctggact aaatggacat atttcttatg 900 caaaaaggaa aacacacaca accaataact caaacaaaaa ac~ggacattt atgtgcagtt 960 gggacagcaa accaagtcct ggacgtaaaa tcgaataaaa ga~cacattta tatccaatag 1020 agaccacacc tgtattcata tgggaacaat tggaatagtg at:atcctcaa ggtgtaaaaa 1080 atatataaat atatatatat atgtcaaaag gtaggaaatg ca.aaaaagaa aaaaaaaaaa 1140 aggtgacagc cgcagttggt gctgtgatgg ccgtgaagtg tcctgggcct cccgaggcct 1200 ctgacaaata aacaagccat gagtggtgag gacacagtct ccttacagtt tccattgcca 1260 acaacagcca tccatatttc ttttttcctt tgtctttc 1298 <210> 21 <2i1> 1877 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte TD No: 518158CB1 <400> 21 gcctggccgt caccactccc cagagggcac agggctctgc tgtgcctcag agcaaaagtc 60 ccagagccag-cagagcaggc tgacgacctg caagccacag tggctgccct gtgcgtgctg 120 cgaggtgggg gaccctgggc aggaagctgg ctgagcccca agaccccggg ggccatgggc 180 ggggatctgg tgcttggcct gggggccttg agacgccgaa agc:gcttgct ggagcaggag 240 aagtctctgg ccggctgggc actggtgctg gcaggaactg gcattggact catggtgctg 300 catgcagaga tgctgtggtt cggggggtgc tcgtgggcgc tct;acctgtt cctggttaaa 360 tgcacgatca gcatttccac cttcttactc ctctgcctca tcc~tggcctt tcatgccaaa 420 gaggtccagc tgttcatgac cgacaacggg ctgcgggact ggc;gcgtggc gctgaccggg 480 cggcaggcgg cgcagatcgt gctggagctg gtggtgtgtg ggc;tgcaccc ggcgcccgtg 540 cggggcccgc cgtgcgtgca ggatttaggg gcgccgctga cct:ccccgca gccctggccg 600 ggattcctgg gccaagggga agcgctgctg tccctggcca tgctgctgct cggcctcacg 660 cttggcctct ggctgaccac cgcctgggtg ctgtccgtgg ccg~agaggca ggctgttaat 720 gccactgggc acctttcaga cacactttgg ctgatcccca tca.cattcct gaccatcggc 780 tatggtgacg tggtgccggg caccatgtgg ggcaagatcg tctgcctgtg cactggagtc 840 atgggtgtct gctgcacagc cctgctggtg gccgtggtgg cccggaagct ggagtttaac 900 aaggcagaga agcacgtgca caacttcatg atggatatcc agtataccaa agagatgaag 960 gagtccgctg cccgagtgct acaagaagcc tggatgttct acaaacatac tcgcaggaag 1020 gagtctcatg ctgcccgcag gcatcagcgc aagctgctgg ccgccatcaa cgcgttccgc 1080 caggtgcggc tgaaacaccg gaagctccgg gaacaagtga actccatggt ggacatctcc 1140 aagatgcaca tgatcctgta tgacctgcag cagaatctga gca~gctcaca ccgggccctg 1200 gagaaacaga ttgacacgct ggcggggaag ctggatgccc tgactgagct gcttagcact 1260 gccctggggc cgaggcagct tccagaaccc agccagcagt cca~agtagct ggacccacga 1320 ggaggaacca ggctactttc cccagtactg aggtggtgga catcgtctct gccactcctg 1380 acccagccct gaacaaagca cctcaagtgc aaggaccaaa ggg<3gccctg gcttggagtg 1440 ggttggcttg 'ctgatggctg ctggagggga cgctggctaa agtgggtagg ccttggccca 1500 cctgaggccc caggtgggaa catggtcacc cccactctgc ata<:cctcat caaaaacact 1560 ctcactatgc tgctatggac gacctccagc tctcagttac aagt;gcaggc gactggaggc 1620 aggactcctg ggtccctggg aaagagggta ctaggggccc ggat:ccagga ttctgggagg 1680 WO 00/1271 I PCT/US99/2046$
cttcagttac cgctggccga gctgaagaac tgggtatgag gcaggggcgg ggctggaggt 1740 ggcgccccct ggtgggacaa caaagaggac accatttttc cagagctgca gagagcacct 1800 ggtggggagg aagaagtgta actcaccagc ctctgctctt at:ctttgtaa taaatgttaa 1860 agccagaaaa aaaaaaa 1877 <210> 22 c211> 2517 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 602926CB1 <400> 22 gccaccccgc tctcctcgcc gccgcggcgg caggcgcggg gccgcgccgc gaggcctgat 60 ccctgcagcg cgggcaggcg gcgtcgcaga gcggagctca gctggatgcg tccggactcc 120 tgcaggtgag ag'tgattttc cagtgattgc tttggcctgt acaaccagag aacaggattc 180 ttcccttctt tttggccacc aaatgcctat gtgcaccaca cattccagtg tgctgagaag 240 ggcagagctt cttggatgat gatggacgtc ccaccgggca gg<~tgaaggc agagcgtgtg 300 gcatctccac ctcaagggtg cagcctgatc ttcctcttct cccttgccag ccagcactct 360 gccttctgta tccaccatgg tgtttggtga gtttttccat cgccctggac aagacgagga 420 acttgtcaac ctgaatgtgg ggggctttaa gcagtctgtt gaccaaagca ccctcctgcg 480 gtttcctcac accagactgg ggaagctgct tacttgccat tct:gaagagg ccattctgga 540 gctgtgtgat gattacagtg tggccgataa ggaatactac ttt:gatcgga atccctcctc 600 gttcagatat gttttgaatt tttattacac ggggaagctg cat:gtcatgg aggagctgtg 660 cgtattctca ttctgccagg agatcgagta ctggggcatc aac:gagctct tcattgattc 720 ttgctgcagc aatcgctacc aggaacgcaa ggaggaaaac cac:gagaagg actgggacca 780 gaaaagccat gatgtgagta ccgactcctc gtttgaagag tcc~tctctgt ttgagaaaga B40 gctggagaag tttgacacac tgcgatttgg tcagctccgg aagraaaatct ggattagaat 900 ggagaatcca gcgtactgcc tgtccgctaa gcttatcgct atctcctcct tgagcgtggt 960 gctggcctcc atcgtggcca tgtgcgttca cagcatgtcg gag~ttccaga atgaggatgg 1020 agaagtggat gatccggtgc tggaaggagt ggagatcgcg tgcattgcct ggttcaccgg 1080 ggagcttgcc gtccgcctgg ctgccgctcc ttgrtcaaaag aaattctgga aaaaccctct 1140 gaacatcatt gactttgtct ctattattcc cttctatgcc acgttggctg tagacaccaa 1200 ggaggaagag agtgaggata ttgagaacat gggcaaggtg gtccagatcc tacggcttat 1260 gaggattttc cgaattctaa agcttgcccg gcactcggta ggacttcggt ctctaggtgc 1320 cacactgaga cacagctacc atgaagttgg gcttctgctt ctcttcctct ctgtgggcat 1380 ttccattttc tctgtgctta tctactccgt ggagaaagat gaccacacat ccagcctcac 1440 cagcatcccc atctgctggt ggtgggccac catcagcatg aca~actgtgg gctatggaga 1500 cacccacccg gtcaccttgg cgggaaagct catcgccagc acatgcatca tctgtggcat 1560 cttggtggtg gcccttccca tcaccatcat cttcaacaag ttttccaagt actaccagaa 1620 gcaaaaggac attgatgtgg accagtgcag tgaggatgca ccagagaagt gtcatgagct 1680 accttacttt aacattaggg atatatatgc acagcggatg cac<~ccttca ttaccagtct 1740 ctcttctgta 'ggcattgtgg tgagcgatcc tgactccaca gat<Icttcaa gcattgaaga 1800 caatgaggac atttgtaaca ccacctcctt ggagaattgc acadcaaaat gagcgggggt 1860 gtttgtgcct gtttctctta tcctttcccg acattaggtt aacacagctt tataaacctc 1920 agtgggttcg ttaaaatcat ttaattctca gggtgtacct ttcagccata gttggacatt 1980 cattgctgaa ttctgaaatg atagaattgt ctttattttt ctct:gtgagg tcaattaaat 2040 gccttgttct gaaatttatt ttttacaaga gagagttgtg atat:agtttg gaatataaga 2100 taaatggtat tgggtggggt ttgtggctac agcttatgca tcatactgtg tttgtcattt 2160 actcacattg agctaacttt aaattactga caagtagaat caaa~ggtgca gctgactgag 2220 acgacatgca tgtaagatcc acaaaatgag acaatgcatg taaa~tccatg ctcatgttct 2280 aaacatggaa actaggagcc taataaactt cctaattcag tatgrgtatac caaaaaatcc 2340 gggcggcctg cgactagcga gctcgtctga ccgggaatcc attccgcgac ggtacctgcc 2400 ggcgtaccag cctttcccat agtgagtcgg attagagctt ggcg~gaatca tggtcatagc 2460 cggttcccgt gggaaactgt tatccggcca caattccata caacactcga gccggga 2517 <210> 23 <211> 1154 <212> DNA
<213> Iiomo Sapiens <220>
<221> misc_feature <223> Tncyte ID No: 922119CB1 <400> 23 ttaactcagc tgggagttga agagccgatg ggcagcaggc ag~acttgagt ctcctttctg 60 tccatgggct cgggccactg tctcaggtcc acccgtggct ccaaaatggt ctcctggtcc 120 gtgatagcaa agatccagga aatactgcag aggaagatgg tgcgagagtt cctggccgag 180 ttcatgagca catatgtcat gatggtattc ggccttggtt ccgtggccca tatggttcta 240 aataaaaaat atgggagcta ccttggtgtc aacttgggtt ttggcttcgg agtcaccatg 300 ggagtgcacg tggcaggccg catctctgga gcccacatga acgcagctgt gacctttgct 360 aactgtgcgc tgggccgcgt gccctggagg aagtttccgg tctatgtgct ggggcagttc 420 ctgggctcct tcctggcggc tgccaccatc tacagtctct tctacacggc cattctccac 480 ttttcgggtg gacagctgat ggtgaccggt cccgtcgcta ca~gctggcat ttttgccacc 540 taccttcctg atcacatgac attgtggcgg ggcttcctga atgaggcgtg gctgaccggg 600 atgctccagc tgtgtctctt cgccatcacg gaccaggaga ac;aacccagc actgccagga 660 acagaggcgc tggtgatagg catcctcgtg gtcatcatcg gggtgtccct tggcatgaac 720 acaggatatg ccatcaaccc gtcccgggac ctgccccccc gc~atcttcac cttcattgct 780 ggttggggca aacaggtctt cagcaatggg gagaactggt ggtgggtgec agtggtggca 840 ccacttctgg gtgcctatct aggtggcatc atctacctgg tcttcattgg ctccaccatc 900 ccacgggagc ccctgaaatt ggaggattct gtggcgtatg aagaccacgg gataaccgta 960 ttgcccaaga tgggatctca tgaacccacg atctctcccc tcacccccgt ctctgtgagc 1020 cctgccaaca gatcttcagt ccaccctgcc ccacccttac at<~aatccat ggccctagag 1080 cacttctaag 'cagagattat ttgtgatccc atccattccc caataaagca aggcttgtec 1140 gacaaaaaaa aaaa 1154 <210> 24 <211> 1879 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2666782CB1 <400> 24 ggtaccgagg cccgagccgc gggagtcgag cgaaggcagc gccgaggccg cggtttcccc 60 ctgggcctcc ccagcagcag ccatgggcat caaattttta gaagttatca aaccattctg 120 tgcagttcta ccagaaattc agaaaccgga aaggaaaatc cagtttagag agaaggttct 180 gtggactgct ataacgctct tcattttctt agtgtgttgt cag~atcccac tgtttggaat 240 catgtcatca gattctgcag atcctttcta ctggatgaga gtt;attctgg cttccaatag 300 aggaacttta atggaattgg gtatctcccc aattgtaaca tctggtttga ttatgcagtt 360 gttagctgga gccaaaatca ttgaagttgg agatacaccg aaagatagag ctttattcaa 420 tggagcccag aaactgtttg gtatgatcat taccattggg caagccattg tgtatgtcat 480 gacggggatg tatggggacc ctgcagaaat gggtgccgga atc~tgtctcc tgatcatcat 540 tcagttgttt gttactagtt tgattgtgct actgttagat gag<~tactac agacaggtta 600 cagcttgggg tctgggattt ccctcgttat tgccaccaac atctgtgaga ccattgtctg 660 gaaggccttt agtcccacta ccattaacac tggcagaggt actgagtttg agggtgcagt 720 catagctctg ttccatttgt tggccaccag gacggacaaa gtcc:gagctt tacgggaggc 780 tttttatcgg cagaacttac ccaatctcat gaacctcatt gctacagttt ttgtgtttgc 840 tgttgttata tatttccaag gatttcgcgt tgatctgccc at taagtcgg cccgttaccg 900 aggacagtac agcagctacc ccatcaaact cttctacacc tccaacatcc ccatcatcct 960 ccagtcggcc ctggtgtcca acctgtatgt tatttcccag atgctgtctg ttcgatttag 1020 tggcaacttt ttagtaaatt tactaggaca gtgggccgat gt:cagtgggg gaggacccgc 1080 acgttcttac ccagttggag gcctttgtta ctatctttct ccacctgagt ccatgggcgc 1140 catctttgag gatcctgtcc atgtcgttgt ttatatcatc tt:catgttgg ggtcatgtgc 1200 attcttctct aagacatgga ttgaagtgtc tggttcctca gc:caaagatg tagctaaaca 1260 gctgaaagaa cagcagatgg taatgagggg ccaccgagat ac:ctctatgg ttcatgagct 1320 taataggtac atccccaccg cagctgagtt tggcggtttg tc~cattggcg ccctgtcagt 1380 gctggctgac ttcctggggg ccattggatc tggcactgga atactgctag cagtcactat 1440 tatttaccag tattttgaaa tatttgttaa agaacaggcc gaiagttggtg ggatgggtgc 1500 tttgtttttc taaatgttca aatatttcat tttgtgcgtg tg~aaagggaa aacactttga 1560 cggatcgttt ttgtcagatg acactggtgg ctccectttt ct.cccctcac agtttcttgt 1620 ttcgagtgct gactgacccg tttctgaaat gggcaccgag ctaagtctgt gtgcagcatt 1680 agtacccgct gccttaaaac tcaagtttac attattcatt aa.aaaaagta catctagtgt 1740 tgcctgtaat gctggaaacc agtgtatcta ccttgctgtg ttaaatcatg acagtgagac 1800 ggtgagatgg attcgttttg cacacaacat tcaaaacact tcatattgcc cccacttgtt 1860 gaaaaataaa tgtagttca 1879 <210> 25 <211> 1537 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2731369CB1 <400> 25 ctggcccagc cctggcagct gatgggagag cagatcgtcc aatacaagcc ttcctacggt 60 ctctttagaa gacatgggct gaaacttggg gtaatagagg ttc~gctggca tatccatgca 120 ggatgattgt cttcacatgt atctattacc ttgtagaata agc~tagaccc tgattttgga 180 acctgaagac 'caaagtgcaa gattagctct gctacttcca tct:gtggacc attgggcagg 240 tatctctggg ccttcactta ctctttgtga aatgaggaca ggc~gcaatcc ctaccctacc 300 aagtcattgg gagtgaagac atgatgacac ggtgattgtg aaa~agatttt gtcaatcgca 360 ccagcattaa gggtgcccat ctccaggttc ccccaggcct caaiggctccc aaggcctgag 420 tgggcaggta gcacccaggt atagaccttc cacgtgcagc acc:caggaca cagccagcat 480 gaactgggca tttctgcagg gcctgctgag tggcgtgaac aagtactcca cagtgctgag 540 ecgcatctgg ctgtctgtgg tgttcatctt tcgtgtgctg gtg~tacgtgg tggcagcgga 600 ggaagtgtgg gacgatgagc agaaggactt tgactgcaac accaagcagc ccggctgcac 660 caacgtctgc tacgacaact acttccccat ctecaacatc cgcctctggg ccctacagct 720 catcctggtc acgtgcccct cactgctcgt ggtcatgcac gtggcctacc gcgaggaacg 780 cgagcgcaag caccacctga aacacgggcc caatgccccg tccctgtacg acaacctgag 840 caagaagcgg ggcggactgt ggtggacgta cttgctgagc ctcatcttca aggccgccgt 900 ggatgctggc ttcctctata tcttccaccg cctctacaag gattatgaca tgccccgcgt 960 ggtggcctgc tccgtggagc cttgccccca cactgtggac tgttacatct cccggcccac 1020 ggagaagaag gtcttcacct acttcatggt gaccacagct gcc~atctgca tcctgctcaa 1080 cctcagtgaa gtcttctacc tggtgggcaa gaggtgcatg gag;atcttcg gccccaggca 1140 ccggcggcct cggtgccggg aatgcctacc cgatacgtgc ccaccatatg tcctctccca 1200 gggagggcac cctgaggatg ggaactctgt cctaatgaag get<~ggtcgg ccecagtgga 1260 tgcaggtggg tatccataac ctgcgagatc agcagataag atcaacaggt cccccccaca 1320 tgaggccacc caggaaaaaa ggcaggggca gtggcatcct tgccgtagca gggtggtgag 1380 gagggtggct gtgggggctc aggaagctcg cccaggggcc aatgtgggag gttgggggta 1440 gtttggtccc tgggtcctga gcctcagggg agggaggttg atagctactg gggattttgt 1500 atatggcaac 'agtatatgtc aaacctctta ataaatt 1537 <210> 26 <21I> 884 <212> DNA
<213> Homo Sapiens <220>
<221> unsure <222> 790, 827, 860 <223> a or g or c or t, unknown, or other <220>
<221> misc_feature <223> Incyte ID No: 1375415CB1 <400> 26 gcgcctggag ccacacaggg atccggagcc tgggggaaaa gcggcgcggg agccggcacc 60 caccgctgga ggggcggcga cggcggccgt agcgacctcg ggaggcaagc ggagccgcca 120 tggccgagtt cccgtcgaaa gttagcacgc ggaccagcag tcctgcgcag ggcgccgaag 180 cctcggtgtc ggcgctgcgc ccggacctgg gcttcgtgcg ctcccgcctc ggggcgctca 240 tgctgctgca gctggtgctg gggctgctgg tgtgggcgct gattgcggac accccgtacc 300 acctgtatcc ggcctatggc tgggtgatgt tcgtcgctgt cttcctctgg ctggtgacaa 360 tcgtcctctt caacctctac ctgtttcagc tgcacatgaa gttgtacatg gttccctggc 420 cactggtgtt aatgatcttt aacatcagcg ccaccgttct ct~acatcacc gccttcatcg 480 cctgctctgc ggcagttgac ctgacatccc tgaggggcac ccggccttat aaccagcgcg 540 cggctgcctc gttctttgcg tgtttggtga tgatcgccta tggagtgagt gccttcttca 600 gctaccaggc ctggcgagga gtaggcagca atgcggccac ca~3tcagatg gctggcggct 660 atgcctaaac cacctgtgcc acggccccct ctggggctga agccgccgct gggtcacaga 720 gcagggtcac cctgcaagcc tgaagctggg gagccctgcg tg<~agtcagc ccaacaggga 780 ctgcatttgn 'ctcctctctg cccgtcagac ataagctctc acagcgntaa ggaagcaggc 840 ccaggctggc agacatctcn gcttgcagga ggccaactgt ga<~a gg4 <210> 27 <211> 3156 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2733282CB1 <400> 27 gttcaggaag aaaccatctg catccatatt gaaaacctga cacaatgtat gcagcaggct 60 cagtgtgagt gaactggagg cttctctaca acatgaccca aaggagcatt gcaggtccta 120 tttgcaacct gaagtttgtg actctcctgg ttgccttaag ttcagaactc ccattcctgg 180 gagctggagt acagcttcaa gacaatgggt ataatggatt gctcattgca attaatcctc 240 aggtacctga gaatcagaac ctcatctcaa acattaagga aat~gataact gaagcttcat 300 tttacctatt taatgctacc aagagaagag tatttttcag aaatataaag attttaatac 360 ctgccacatg gaaagctaat aataacagca aaataaaaca aga;atcatat gaaaaggcaa 420 atgtcatagt gactgactgg tatggggcac atggagatga tccatacacc ctacaataca 480 gagggtgtgg aaaagaggga aaatacattc atttcacacc taaitttccta ctgaatgata 540 acttaacagc tggctacgga tcacgaggcc gagtgtttgt ccatgaatgg gcccacctcc 600 gttggggtgt gttcgatgag tataacaatg acaaaccttt cta<:ataaat gggcaaaatc 660 aaattaaagt gacaaggtgt tcatctgaca tcacaggcat ttttgtgtgt gaaaaaggtc 720 cttgccccca agaaaactgt attattagta agctttttaa agaaggatgc acctttatct 780 acaatagcac ccaaaatgca actgcatcaa taatgttcat gcaaagttat ctctgtggtg 840 aaatttgtaa tgccagtacc cacaaccaag aagcaccaaa cctacagaac cagatgtgca 900 gcctcagaag tgcatgggat gtaatcacag actctgctga ctttcaccac agctttccca 960 tgaacgggac tgagcttcca cctcctccca cattctcgct t.gtagaggct ggtgacaaag 1020 tggtctgttt agtgctggat gtgtccagca agatggcaga ggctgacaga ctccttcaac 1080 tacaacaagc cgcagaattt tatttgatgc agattgttga aattcatacc ttcgtgggca 1140 ttgccagttt cgacagcaaa ggagagatca gagcccagct acaccaaatt aacagcaatg 1200 atgatcgaaa gttgctggtt tcatatctgc ccaccactgt atcagctaaa acagacatca 1260 gcatttgttc agggcttaag aaaggatttg aggtggttga aaaactgaat ggaaaagctt 1320 atggctctgt gatgatatta gtgaccagcg gagatgataa gcttcttggc aattgcttac 1380 ccactgtgct cagcagtggt tcaacaattc actccattgc cctgggttca tctgcagccc 1440 caaatctgga ggaattatca cgtcttacag gaggttaaaa gttctttgtt ccagatatat 1500 caaactccaa tagcatgatt gatgctttca gtagaatttc ctctggaact ggagacattt 1560 tccagcaaca tattcagctt gaaagtacag gtgaaaatgt caaacctcac catcaattga 1620 aaaacacagt gactgtggat aatactgtgg gcaacgacac t<~tgtttcta gttacgtggc 1680 aggccagtgg tcctcctgag attatattat ttgatcctga tggacgaaaa tactacacaa 1740 ataattttat caccaatcta acttttcgga cagctagtct ttggattcca ggaacagcta 1800 agcctgggca ctggacttac accctgaaca atacccatca tt:ctctgcaa gccctgaaag 1860 tgacagtgac ctctcgcgcc tccaactcag ctgtgccccc ac~ccactgtg gaagcctttg 1920 tggaaagaga 'cagcctccat tttcctcatc ctgtgatgat tt:atgccaat gtgaaacagg 1980 gattttatcc cattcttaat gccactgtca ctgccacagt tgagccagag actggagatc 2040 ctgttacgct gagactcctt gatgatggag caggtgctga tcrttataaaa aatgatggaa 2100 tttactcgag gtattttttc tcctttgctg caaatggtag at:atagcttg aaagtgcatg 2160 tcaatcactc tcccagcata agcaccccag cccactctat tc:cagggagt catgctatgt 2220 atgtaccagg ttacacagca aacggtaata ttcagatgaa tgctecaagg aaatcagtag 2280 gcagaaatga ggaggagcga aagtggggct ttagccgagt ca,gctcagga ggctcctttt 2340 cagtgctggg agttccagct ggcccccacc ctgatgtgtt tccaccatgc aaaattattg 2400 acctggaagc tgtaaaagta gaagaggaat tgaccctatc ttggacagca cctggagaag 2460 actttgatca gggccaggct acaagctatg aaataagaat gagtaaaagt ctacagaata 2520 tccaagatga ctttaacaat gctattttag taaatacatc aaagcgaaat cctcagcaag 2580 ctggcatcag ggagatattt acgttctcac cccaaatttc cacgaatgga cctgaacatc 2640 agccaaatgg agaaacacat gaaagccaca gaatttatgt tgcaatacga gcaatggata 2700 ggaactcctt acagtctgct gtatctaaca ttgcccaggc gcctctgttt attcccccca 2760 attctgatcc tgtacctgcc agagattatc ttatattgaa aggagtttta acagcaatgg 2820 gtttgatagg aatcatttgc cttattatag ttgtgacaca tc~atacttta agcaggaaaa 2880 agagagcaga caagaaagag aatggaacaa aattattata aataaatatc caaagtgtct 2940 tccttcttag atataagacc catggccttc gactacaaaa acatactaac aaagtcaaat 3000 taacatcaaa actgtattaa aatgcattga gtttttgtac aatacagata agatttttac 3060 atggtagatc aacaaattct ttttgggggt agattagaaa acccttacac tttggctatg 3120 aacaaataat aaaaattatt ctttaaaaaa aaaaaa 3156 <210> 28 <211> 1774 <212> DNA
<213> Homo sapieas <220>
<221> misc_feature <223> Incyte ID No: 3148427081 <400> 28 cgctgctata ccgtgcaccc gcgtgctcgt gagggaactc gggctcgctt tggtggggag 60 aaaaatccat gcgctaatac tcggttccca gcttctgcaa aagaaataca aagagtatga 120 gaaagacgtt gccatagaag aacatcgatg gcctccaact ggtaaagaag ctggcaaaga 180 acatggaaga gatgtttcac aagaagtctg aggccgtcag gcgtctggtg gaggctgcag.240 aagaagcaca cctgaaacat gaatttgatg cagacttaca gtatgaatac ttcaatgctg 300 tgctgataaa tgaaagggac aaagacggga attttttgga gct~gggaaag gaattcatct 360 tagccccaaa tgaccatttt aataatttgc ctgtgaacat cagtctaagt gacgtccaag 420 taccaacgaa catgtacaac aaagaccctg caattgtcaa tc~gggtttat tggtctgaat 480 ctctaaacaa agtttttgta gataactttg accgtgaccc at:ctctcata tggcagtact 540 ttggaagtgc aaagggcttt tttaggcagt atccggggat taaatgggaa ccagatgaga 600 atggagtcat tgccttcgac tgcaggaacc gaaaatggta catccaggca gcaacttctc 660 cgaaagacgt ggtcatttta gttgacgtca gtggcagcat gs~aaggactc cgtctgacta 720 tcgcgaagca aacagtctca tccattttgg atacacttgg gg~atgatgac ttcttcaaca 780 taattgctta taatgaggag cttcactatg tggaaccttg cctgaatgga actttggtgc 840 aagccgacag gacaaacaaa gagcacttca gggagcatct gg~acaaactt ttcgccaaag 900 gaattggaat gttggatata gctctgaatg aggccttcaa ca.ttctgagt gatttcaacc 960 acacgggaca aggaagtatc tgcagtcagg ccatcatgct ca.taactgat ggggcggtgg 1020 acacctatga tacaatcttt gcaaaataca attggccaga tegaaaggtt cgcatcttca 1080 catacctcat tggacgagag gctgcgtttg cagacaatct aaagtggatg gcctgtgcca 1140 acaaaggatt ttttacccaa atctccacct tggctgatgt gcaggagaat gtcatggaat 1200 accttcacgt gcttagccgg cccaaagtca tcgaccagga gcatgatgtg gtgtggaccg 1260 aagcttacat tgacagcact ctccctcagg cacaaaagct gactgatgat cagggccccg 1320 tcctgatgac cactgtagcc atgcctgtgt ttagtaagca gaacgaaacc agatcgaagg 1380 gcattcttct gggagtggtt ggcacagatg tcccagtgaa agaacttctg aagaccatcc 1440 ccaaatacaa gttagggatt cacggttatg cctttgcaat cacaaataat ggatatatcc 1500 tgacgcatcc ggaactcagg ctgetgtacg aagaaggaaa aa~agcgaagg aaacctaact 1560 atagtagcgt tgacctctct gaggtggagt gggaagaccg ag~atgacgtg ttgagaaatg 1620 ctatggtgaa tcgaaagacg gggaagtttt ccatggaggt gaagaagaca gtggacaaag 1680 gggtacattt ttctcaaaca tttttgctgc ttaatttaaa ac~aaaccact gtgaaaaatt 1740 agctttgaaa gctatatctg gaataaatga cttc 1774 <210> 29 <211> 1505 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 3342358CB1 <400> 29 cgcggatcct gtgacacctc cgggcagccc ggcacttgtt gct:cccacga cctgttgtca 60 ttcccttaac ccggctttcc ccgtggcccc ccgcctcctc ccg~gcttcgc tccttttcat 120 gtgagcatct gggacactga tctctcagac cccgctgctc ggg~ctggaga atagatggtt 180 ttgtgaaaaa ttaaacaccg ccctgaagag gagccccgct gggcagcggc aggagcgcag 240 agtgctggcc caggtgctgc agaggtggcg cctccccggc ccg~ggacggt agccccgggc 300 gccaacggca tgacagactc ggcgacagct aacggggacg aca.gggaccc cgagatcgag 360 ctctttgtga aggctggaat cgatggagaa agcatcggca actgtccttt ctctcagcgc 420 ctcttcatga tcctctggct gaaaggagtc gtgttcaatg tcaccactgt ggatctgaaa 480 agaaagccag ctgacctgca caacctagcc cccggcacgc acccgccctt cctgaccttc 540 aacggggacg tgaagacaga cgtcaataag atcgaggagt tcctggagga gaccttgacc 600 cctgaaaagt accccaaact ggctgcaaaa caccgggaat ccaacacagc gggcatcgac 660 atcttttcca agttttctgc ctacatcaaa aataccaagc agcagaacaa tgctgctctt 720 gaaagaggcc taaccaaggc tctaaagaaa ttggatgact acctgaacac ccctctacca 780 gaggagattg acgccaacac ttgtggggaa gacaaggggt cccggcgcaa gttcctggat 840 gggrgatgagc tgaccctggc tgactgcaat ctgttgccca agctccatgt ggtcaagatt 900 gtggccaaga aataccgcaa ctatgatatc ccggctgaga tga~aaggcct gtggcggtac 960 ctcaagaacg cctatgcccg tgatgagttc accaacacct gtgcagctga cagtgagatc 1020 gagttggcct acgctgatgt cgccaaacgc ctcagccgat cctgagcaca gccattttgc 1080 cccatccccg ctgcagaagg actcaaccac tcccctaaga ctccagcttc atagactcct 1140 ctgtatcact gccttgaggc gcacttttta taatcaagcc tcatcttgct ggtatcatgg 1200 gaactccagc ctgctatctt tcatgaaggt cagcaccatc ctgggcctcc tcacataggg 1260 atctagcaga aatgatagac acagtccacc tttcggccgg ccacficctgat ctgggrgctca 1320 gcatgtttgg gggtcagtca gtgttgggag agcccacata tc~gggattgc cattaggttt 1380 tttttgccat tatcaaaaat tacttttcaa gaggctttag gc~aacatggc aacaacactt 1440 ctttttctaa cctccttttc ggcctatacc acaaggggca gc~ggcaaacg gcattttcat 1500 tcttt 1505 <210> 30 <211> 1478 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 1267774CB1 <400> 30 gaaactggag accagaattt tagaaaaaga gattaaggca tctcacttgg tggggtgggg 60 ggtgtctttt tatttttttt ttccttttct ttttaaaaaa aaaacactgc aactggaaca 120 gtttctgatc tcaaaaggca agcctcttcc cgtgtgatct ttataattta cactcttttc 180 cgtgagcttt cttacctccc tttbtttata actctccata ttctctattc atacatatat 240.
ccattatatt agtagtggaa taatttttat ttttatttat tttttttggc tttagtactt 300 gcaccctcac acacactctc ccgagaacca gaagtcggtt gggtgtttat ataatgaaga 360 attatggggc tgtttgatcg aggtgttcaa atgcttttaa cc~accgttgg tgctttcgct 420 gccttcagcc tgatgaccat agctgtggga accgactatt ggctctactc cagaggggtt 480 tgcaagacca aaagtgtcag tgagaatgaa accagcaaaa ag,aacgagga agttatgacc 540 cattccggat tatggagaac ctgctgccta gaagggaatt ca;aaaggtct gtgcaagcaa 600 attgatcact tcccagagga tgcagattac gaagctgaca cagcagaata tttcctccgg 660 gccgtgaggg cctccagcat tttcccaatc ctgagtgtga ttctgctttt catgggtggc 720 ctctgcatcg cagccagcga gttctacaaa actcgacaca acatcatcct gagtgccggc 780 atcttcttcg tgtctgcagg tctgagtaac atcattggca tcatagtgta catatctgcc 840 aatgccggag acccctccaa gagcgactcc aaaaagaata gttactcata cggctggtcc 900 ttctacttcg gggccctgtc cttcatcatc gccgagatgg tcggggtgct ggcggtgcac 960 atgtttatcg accggcacaa acagctgcgg gccacggccc gcc~ccacgga ctacctccag 1020 gcctctgcca tcacccgcat ccccagctac cgctaccgct accagcgccg cagccgctcc 1080 agctcgcgct ccacggagcc ctcacactcc agggacgcct cc<:ccgtggg catcaagggc 1140 ttcaacaccc tgccgtccac ggagatctcc atgtacacgc tcagcaggga ccccctgaag 1200 gccgccacca cgcccaccgc cacctacaac tccgacaggg ataacagctt cctccaggtt 1260 cacaactgta tccagaagga gaacaaggac tctctccact ccaacacagc caaccgccgg 1320 accacccccg tataaagacc gcgggcctcg ccagaagacc gcc~ggaggag ggcgcggtcc 1380 ccgggggcgg ggcggggcgg ggagacccag accctccgct ggc~agacctt ccaaaagcaa 1440 aaacaaaaaa caaaaaaaac aagtatacag gagagaga 1478 <210> 31 <211> 1971 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 1817329CB1 <400> 31 cgggcggggg agacctgcca gtcctcccag acttctcccg ggttgctcca gctggccctc 60 ctcgcccctt cccgggagag gcacatggag agacatgaat cag~gggagtg gactggacct 120 gctgaagatc tcatatggaa aaggagccag aaggaaaaac agatttaaag gatctgatgg 180 aagcacgtca tctgatacta cctcaaatag ttttgttcgc caggtaagag ttttaagttc 240 atggttttga taagtacctt aaaatgactt tagattttta aaggtgggtt tcactctttt 300 ccttaagatt tatgtagtat gattgtactt acttttaatt ga;agtgaaga cggtgctgtt 360 taatgtcata attaaaatat tttaatctta agtgagacta agtaataatt acactctttc 420 ctggcaagtt gaggaaagag aagtgtggca ttcattacag aggatttctt tgaaacagtg 480 agagaactcc agcagaaatg attatggatt tggggggatt gtitttttttt ttttttttca 540 gtgggacaga gagctgatgt gcatct:gtat ccctacctgt gacfiatactgg ggttcttcta 600 gttgagcttt cttcttctca cttgggcttg cattgaaaac ta<3aaaatca ttcttggtct 660 tgaaatggtt gaggctatcg aggttaaaca gaacatgcag tatcactgag acataattta 720 aaccatttct gcctttggag acatagcctt cgtctcattt aatgtgtagt atcttcctgc 780 caagcagcct ggaacagtcc cctaaacaga cagtgccagg ctccctaaca tataaacacg 840 attatatatg gtaacagaaa ggaattccat taccagagga agagcataga ggcagctgga 900 cctctgaggg ttgtggttat gtgttgagaa tgaagtctca tce~ggaatga gtgagcttgt 960 ttcttcctct aacccctctc catggggtgg aaagtagggg caclagcatgc agcagagaat 1020 ctgttcttgt ggcccaggga tgtccagtgt ctgatcagta ctca catcat ctcttgtcaa 1080 tgacagctca ctctacggaa cagtggtagt caactggaaa agcagcctcg tgtcatttgg 1140 atgccacttt ccccagtgcc gcttgagtcc atcatcatgg ccatagattc cacccctgcc 1200 ccctgtttgt gtggcaggac tgtttcctat aaatcctata tgaccttttg ggttttattt 1260 ttattgggga cagttacatt tccctagctg tctaccctta ttc~gctcctt gtggcactcc 1320 ccaagtgcct ccctcatgtt tccttcccac agttagctgc agtagaactt gaacttgtcc 1380 acctgcagca tcggtgggga ttttgatctt ggctggttgc tgca tctttg cactgtcctt 1440 agacgaagga tgatctgtcc ccagccatca gtccccgctt gcaacatttg agtatgccag 1500 tggtacttcc aagtaacttg gcaactggaa aaaaactggt cct:ggtccgt gccaattggg 1560 aattgttgtg gttgcaggaa gtgagaaaaa gagttactta aga~aggggca aagctttttt 1620 ggttagaaac atttagaaag aaaaaagatg aagccacgtg aggctcaacc ctagagctat 1680 gttctaatgg tgtcagcagg agctaggaag gctgtcagag gagtggaagg tgtatgcgtt 1740 ggcctcatgt tttcgtgggc agaatccagg catcatggtc ctgrctgatga agggagggcc 1800 ttgggacata gtgacttggg agagtcagtt aaaggaaagt tac~ccatcgc cttttccatt 1860 tcaagctatt tattctgcct ctgagacaaa gagattgaac ctg~gagagct agagttagaa 1920 tctacaactc tgagtcatca tgcaggacct gaaacagacc aaa.aaaaaaa a 1971 <210> 32 <211> 1424 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 3273307CB1 <400> 32 cccttctcat ttgtcccatc catttgaaat acagtgtact taaatttcca acaaaatcat 60 aacgtagact ctgtcagtta attaaagtat actaacaagc atacaaattg ctaagtcatt 120 ctgggaggct atcagtgctg gatgtactta tacattcagc act~ggaaaca tacccttcca 180 gtagtgcaca gccagaaata ccaagcaggc ttgcaaagga caa~gattaac cttggcctca 240 tggtattttg tctttctgca gggaaaggaa gatcatggag aca,acaattg caatacacgg 300 tggagaactc aacacccctt gattaaagaa aaaaaatagt tga~ataaaaa agaagtttcc 360 tgaaggagta agatagaatt taccacagaa agaaagtggt agtatctccc tatttcaatt 420 taagtgttgt tcccaggcag gtacaagtac ttctaaaaag gcatagatta aagaaaaatg 480 gtatttggag tcatggttac taaatgaagc tacagctcac catgatcctt gggatgaaga 540 ctatgacccc cagcaaagtt acaaaggatt ctgctctgga atttatcaac tgctttgttt 600 gttctcttaa caggtaaaaa atgcagctgc caatgtactc agggaaacat ggctaattta 660 caaaaataca aagctagtga aaaagataga tcatgcaaaa gta~agaaaac atcaacgaaa 720 attcctgcaa gctattcatc aattaagaag tgtaaaaatg gagcagagga aactgaatga 780 ccaagcaaac actttggtgg acttggcaaa gacccagaac atcatgtatg atatgatttc 840 tgacttaaac gaaaggagtg aagacttcga gaagaggatt gttaccctgg aaacaaaact 900 agagactttg attggtagca tccacgccct ccctgggctc ata<~gccaga ccatcaggca 960 gcagcagaga gatttcattg aggctcagat ggagagctac gacaagcacg tcacttacaa 1020 WO 00/12711 PCT/US99/z0468 tgctgagcgg tcccggtcct cgtccaggag gcggcggtcc tcttccacag caccaccaac 1080 ttcatcagag agtagctaga agagaataag ttaaccacaa aataagactt tttgccatca 1140 tatggtcaat attttagctt ttattgtaaa gcccctatgg ttctaatcag cgttatccgg 1200 gttctgatgt cagaatcctg ggaacctgaa cactaagttt t,aggccaaaa tgagtgaaaa 1260 ctcttttttt ttctttcaga tgcacaggga atgcacctat t;attgctata tagattgttc 1320 ctcctgtaat ttcactaact ttttattcat gcacttcaaa caaactttac tactacatta 1380 tatgatatat aataaaaaaa gttaatttct gcacaaaaaa aaaa 1424 <210> 33 <211> 1224 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 3824833CB1 <400> 33 cggccatatt agagagatgg aaataaagct tccttaatgt tc~tatatgtc tttgaagtac 60 atccgtgcat ttttttttag catccaacca ttcctccctt gt:agttctcg ccccctcaaa 120 tcaccctctc ccgtagccca cccgactaac atctcagtct ct:gaaaatgc acagagatgc 180 ctggctacct cgccctgcct tcagcctcac ggggctcagt ctctttttct ctttggtgcc 240 accaggacgg agcatggagg tcacagtacc tgccaccctc aa.cgtcctca atggctctga 300 cgcccgcctg ccctgcacct tcaactcctg ctacacagtg aa.ccacaaac agttctccct 360 gaactggact taccaggagt gcaacaactg ctctgaggag atgttcctcc agttccgcat 420 gaagatcatt aacctgaagc tggagcggtt tcaagaccgc gtggagttct cagggaaccc 480 cagcaagtac gatgtgtcgg tgatgctgag aaacgtgcag ccggaggatg aggggattta 540 caactgctac atcatgaacc cccctgaccg ccaccgtggt catggcaaga tccatctgca 600 ggtcctcatg gaagagcccc ctgagcggga ctccacggtg gccgtgattg tgggtgcctc 660 cgtcgggggc ttcctggctg tggtcatctt ggtgctgatg gtggtcaagt gtgtgaggag 720 aaaaaaagag cagaagctga gcacagatga cctgaagacc gaggaggagg gcaagacgga 780 cggtgaaggc aacccggatg atggcgccaa gtagtgggtg gccggcctgc agcctcctct 840 aggggttgca cccagcgctc cctcaggagg gccttggcct ggcacggctg tgctcctccc 900 ctgctcccag cccagagcag ccatcaggct ggaggtgacg at~gagttcct gaaacttgga 960 ggggcatgtt aaagggatga ctgtgcattc cagggcactg acggaaagcc agggctgcag 1020 gcaaagctgg acatgtgccc tggcccagga ggccatgttg gg~ccctcgtt tccattgcta 1080 gtggcctcct tggggctcct gttggctcct aatcccttag ga~~tgtggat gaggccagac 1140 tggaagagca gctccaggta gggggccatg tttcccagcg ggc~acccacc aacagaggcc 1200 agtttcaaag tcagctgagg gget 1224 <210> 34 <211> 1300 <212> DNA
<213> Homo sapiens <220>
<221> misc_feature <223> Incyte ID No: 2069907CB1 <400> 34 gctgtttacc ttgcatggtt gactgctctc ttctcacatt gtc~tgccagg aagggcactg 60 caccttggta taaatgctgc tgggcaccgt tctgttttct ttcatttctt aatcctatcc 120 aagtatgcag tacgctcttg ggtcgtctca tgagacccag gggcatgttg gaaagaactg 180 agagaaagag caacaaagcg gcgagtggtg tgagagggca gca,cgcgctg tggggccctt 240 ccagagaaat gtactgaaaa agtctacgca atgtctggga ttt.gctaaac aatacctgga 300 aagcagacag gtctttttgc cattcctcca ggacatccac cat.aaggaaa ggagaccctg 360 gaccaacatt ctctaagatg tttatatgga ccagtggccg gacctcttca tcttatagac 420 atgatgaaaa aagaaatatt taccagaaaa tcagggacca tgacctcctg gacaaaagga 480 aaacagtcac agcactgaag gcaggagagg accgagctat tcacctggga ctggctatga 540 tggtgtgctc catcatgatg tattttctgc tgggaatcac acacctgcgc tcatacatgc 600 agagcgtgtg gaccgaagag tctcaatgca ccttgctgaa tc~cgtccatc acggaaacat 660 ttaactgctc cttcagctgt ggtccagact gctggaaact tt;ctcagtac ccctgccccc 720 aggtgtacgt taacctgact tcttccgggg aaaagctcct cctctaccac acagaagaga 780 caataaaaat caatcagaag tgctcctata tacctaaatg tc~gaaaaaat tttgaagaat 840 ccatgtccct ggtgaatgtt gtcatggaaa acttcaggaa gt:atcaacac ttctcctgct 900 attctgaccc agaaggaaac cagaagagtg ttatcctaac ca~aactctac agttccaacg 960 tgctgttcca ttcactcttc tggccaacct gtatgatggc tg~ggggtgt:g gcaattgttg 1020 ccatggtgaa acttacacag tacctctccc tactatgtga ga.ggatccaa cggatcaata 1080 gataaatgca aaaatggata aaataatttt tgttaaagct ca.aatactgt tttctttcat 1140 tcttcaccaa agaaccttaa gtttgtaacg tgcagtctgt tatgagttcc ctaatatatt 1200 cttatatgta gagcaataat gcaaaagctg ttctatatgc aaacatgatg tctttattat 1260 tcaggagaat aaataactgt tttgtgttaa aaaaaaaaaa 1300 <210> 35 <211> 1060 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No; 2243917CB1 <400> 35 gcttgctggg atcatggcgg agaatcactg cgagctcctg tcgecggccc ggggcggcat 60 cggggcgggg ctggggggcg gcctgtgccg ccgctgcagc gctgggctcg gcgccctggc 120 ccagcgccct ggcagcgtgt ccaagtgggt ccgactcaac gtc:ggcggca cctacttcct 180 caccactcgg cagaccctgt gccgggaccc gaaatccttc ctdtaccgct tatgccaggc 240 cgatcccgac ctggactcag acaaggatga aacaggcgcc tatataatcg acagagaccc 300 cacctacttt gggcctgtgc tgaactacct gagacacggc aac~ctggtga ttaacaaaga 360 cctcgcggag gaaggagtgt tggaggaagc agaattttac aat:atcacct cattaataaa 420 acttgtaaag gacaaaatta gagaacgaga cagcaaaaca tcc~caggtgc ctgtgaagca 480 tgtgtaccgt gtgctgcagt gccaggagga ggagctcacg cac~atggtgt ccaccatgtc 540 cgacggctgg aagttcgagc agttggtcag catcggctcc tctaacaact atgggaacga 600 agaccaagcc gagttcctct gtgtggtgtc caaggagctg cac:aacaccc cgtacggtac 660 ggccagcgag cccagcgaga aggccaagat tttgcaagaa cga~ggctcaa ggatgtgagg 720 gacacagtat tgacagctga agaaatgatt tacgttttcc cga;gatgtaa tgaactgcca 780 tgtccaggaa gcttggctgt gagaagaaac ctgcttttga tca.tttttct agagatctgg 840 gtgtgaatcc ttttttgcct ctgaggtggg tggtgagaga cgg~gcccagc tgtccaaggc 900 cagacgtccc caagttgggg gagcacggcg gccgggtggg cgctgcctct tgggggggcc 960 tcgctctgtt ttttccaagt gccacgtggg actgaggcag acactcccag tcagcccgct 1020 cgatcctgaa gatcgtgtga aggaagcgtt cttggtgcta 1060 <210> 36 <211> 1815 <212> DNA
<213> Homo Sapiens <220>
<221> misc_feature <223> Incyte ID No: 2597476C'B1 <400> 36 WO ObII2711 PCT/US99I20468 gtgcctatgc agacagaggg agcagtgaat agcaataggg tgtttccacc atggtcttca 60 ctcaggcccc ggctgaaatc atgggccacc tccggatacg cagcctcctg gcccggcagt 120 gcctggcaga gtttctgggt gtgtttgtac tcatgctcct cacccaagga gctgtggccc 3.80 aggctgtcac cagtggagaa accaaaggca acttcttcac catgtttctg gctggctctc 240 tggccgttac gatagccatc tacgtgggtg gtaacgtctc a<~gggcccac ctgaatccag 300 ccttctccct ggccatgtgc atcgttggac gcctcccctg g<fitcaagctc cccatttaca 360 tcttggtgca gttgctgtct gctttctgtg cttcgggagc cacctatgtt ctctaccatg 420 atgccctaca gaactataca ggtgggaacc tgacagtgac t<;gccccaag gagacagcct 480 ccatttttgc cacctatcct gccccctatc tgtccctgaa caatggcttc ctggatcagg 540 ttctgggcac tgggatgctg attgtggggc tcttggccat ccaggacaga cggaacaagg 600 gagtccctgc gggtctggag cctgtggtgg tggggatgct gatcctggcc ctcgggttat 660 ccatgggtgc caactgcggg attccactca accctgcccg gc~acctgggc ccacgtctct 720 tcacctacgt ggctggctgg ggtcctgaag tcttcagtgc tc~gtaatggc tggtggtggg 780 tgcctgtggt ggcccctctg gtgggggcca ccgttggcac acfccacttac cagctgttgg 840 tggctctgca ccaccctgag ggcccagagc cagctcagga tcaggtgtct gctcaacaca 900 aagcctcaga gttggaaact cctgcctcag ctcagatgct gc~agtgtaag ctatgattag 960 gacaaccctc acttcactca tggaccctgg agccagccac ta~accccgcc tgggaacaac 1020 agtcattctt cctctttgtt aatgtgccag aacctgggag gcttctctgt ttatctgttt 1080 ggcatccctt cctcctaaac taagaaggat cctggacagg ga.gaagtgga ggaggataag 1140 gtaccaggac tcaggcttct catcccctcc tcccgcaaag cg~gttttctg accctcaggg 1200 cctctcggaa tgtagttgct cgaggtaacc gctagagggt gcgcacctgg atgctggatg 1260 gggacggctg cgggcatctg cagggtggag ggggccacca tccagtgtag ggcacaaccc 1320 tggggactgc cctccatagc ctgtcccgac tgccgactcc tagctctcat cgcctcggcg 1380 cctcccacct tcaccctctc ggggatgcct ccccaagagg gtagttaggg gtggggaagc 1440 cgcctccacc cagggggcgt ggtgggggcg gagggaagga gggcggcggg gcacagagac 1500 agagagcaag gctgtgaaac tgaggcaccg ttcctagaca tctcggtgct gtgtcgttca 1560 ttcaaggaga gttgagatac agtgaaatga gccagggcga ggagggaggg tgaaggaacg 1620 gagggcgggc ggctccgagg agcgagagtc gggctgaggg caacctggcg ccagggaaaa 1680 ttctggttat tcaccacttc tacagctctc ctgccgctcc cbgcagagga tgctcgtttt 1740 gcagagaagg cagtgttcct ctattccctt cttccgaatt aa~aaataccc cctcagagcg 1800 aaaaaaaaaa aaaaa 1815 <210> 37 <211> 315 <212> PRT
<213> Rattus norvegicus <220>
<221> misc_feature <223> GenBank ID No: 82924369 <400> 37 Met Leu Gly Trp Val Gln Arg Val Leu Pro Gln Pro Pro Gly Thr Pro Gln Lys Thr Glu GIu GIy Ala Gly Pro Gln Pro Glu Thr Glu Ser.Lys Pro GIu Ala Asn Pro Gln Pro Glu Pro Glu Val Gln Pro Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro Glu Pro G1u Pro Ala Pro Glu Glu Ala Ala Pro Glu Val Gln Thr Leu Pro Pro Glu Glu Pro Val Glu GIy Glu Asp Val Ala Glu Ala Gly Pro~ Ser Leu Gln Glu Thr Gln Glu Ala Asp Pro Pro Gln Pro Thr Ser Gln Ala Gln Val Ala Val Val Lys Val Asn Arg Pro Ser Ser Trp Met Leu Ser Trp Phe Trp Lys Giy Met Glu Lys Val Val Pro Gln Pro Val Tyr Ser Ser Ser Gly Gly Gln Asn Leu Ala Ala Gly G.lu Gly Gly Pro 140 . 145 150 Asp Gln Asp Gly Ala Gln Thr Leu Glu Pro Cys G:ly Thr Gly Asp Pro Gly Ser Giu Asp Gly Ser Asp Lys Thr Ser L~~rs Thr Gin Asp Thr Glu Pro Ser Leu Trp Leu Leu Arg Trp Leu Glu Leu Asn Leu Glu Lys Val Leu Pro Gln Pro Pro Thr Pro Ser G:Ln Ala Trp Lys Val Glu Pro Glu Gly Ala Val Leu Glu~Pro Asp Pro Pro Gly Thr Pro Met Glu Val Glu Pro Thr Glu Asn Pro Ser Gl.n Pro Asn Pro Gly Pro Val Glu Pro Glu Glu Glu Pro Ala Ala Gl.u Pro Gln Pro Gly Phe Gln.Ala Ser Ser Leu Pro Pro Pro Gly A~~p Pro Val Arg Leu Ile Glu Trp Leu Leu His Arg Leu Glu Met Ai.a Leu Pro Gln Pro Val Leu His Gly Lys Ala Ala Glu Gln Glu Pro Ser Cys Pro Gly Thr Cys Asp Val Gin Thr Arg Ala Thr Ala Ala Giy Giy Leu <210> 38 <211> 490 <212> PRT
<213> Drosophila melanogaster <220>
<221> misc_feature <223> GenBank ID No: g116443 <400> 38 Met Ala Ser Ala Trp Leu Pro Phe Ala Ala Val Ala Arch p~.7.a Aia ~

Ile Gly Trp Pro Ala Thr His Pro Leu Pro Val Ile Pro Pro Pro Met Pro Lys Arg Lys Thr Asp Asp Glu Leu Asp Arg Lys Leu Ile Asn Val Ser Arg Phe Glu Thr Txp Arg Leu Gly Arg Asn Thr Glu Lys Tyr Pro Thr Leu Gly Ser Asn Giu Phe Asp Leu Arch Glu Phe Tyr Asp Glu Cys Glu Tyr Phe Phe Asp Pro Asp Lys Arch Asp Asp Iie Phe Arg Ile Asn Tyr Tyr Arg Thr Leu His Leu Gly Lys His Tyr Pro Lys Giu Leu Thr Ser Tyr Asp Leu His Cys Glu Glu Ala WO 00/12711 PCT/US99/204b8 Phe Phe Gly Ile Met Pro Asp Cys Tyr Val Ile G1y Asp Cya Glu Asp Tyr Arg Asp Arg Lys Arg Leu Met Glu Asn Ala Glu Ar<1 Asp Asp Lys Leu Ser Glu Asn Gly Gln Leu Asp Gln Asn Leu Gln Thr Asn Met Arg Gln Lys Met Trp Pro His Arg Ala Phe Glu Asn Thr Ser Thr Ser Ala Leu Val Phe Phe Phe Tyr Tyr Val Thr Gly Ile Ala Val Ser Val Met Ala Asn Glu Thr Pro Cys Val Val VaI. Gly His Arg Pro Gly Arg Ala Gly Glu Arg Thr Leu Pro Cys Gly Tyr Lys Ile Val Phe Phe Cys Leu Ala Cys Met Ile Asp Thr Val Phe Thr Ala Glu Tyr Leu Leu Arg Ala Ala Asp Arg Leu Phe Pro Cys Lys Phe Val Arg Ser VaI Met Ile Asp Val Ala Ser Ile Val Ile Met Pro Tyr Tyr Ile Gly Leu Thr Asp Asp Asp Gly Ile Asn Val Ser Gly Ala Phe Val Thr Leu Phe Arg Phe Arg Arg Val Val Ile Phe Lys Phe Ser Arg His Ser Leu Arg Leu Gly Gln Gly Ile Tyr Thr Leu Lys Ser Cys Ala Ser Gly Phe Val Phe Glu Leu Leu Ser Leu Ala Met AIa Ile Ile Ile Thr Val Phe Tyr Phe Ala Met Ala Glu Lys Asn VaI Asn Gly Thr Thr Ser Pro Ala Asn Phe Ile Ala Phe Trp Tyr Thr Ile Val Thr Thr Leu Tyr Gly Met Thr Gly Asp Met Val Pro Glu Thr Ile Ala Ile Val Gly Val Gly Lys Gly Cys Ser Leu Ser Gly Val Leu Val Leu Pro Pro Val Ile Ala Val Ile Val Ser Asn Phe Ser Arg Ile Gln Asn Tyr His Gln Arg Ala Asp Lys Arg Lys Ala Gln Arg Lys Leu Ala Ile Arg Ala Arg Arg Ile AIa Lys Ala Ser Ser Gly Ala Val Ser Ala Phe Lys Lys Lys Ala Ala Glu Ala Arg Trp Ala Ala Ser Gly Gln Glu Ile Glu Leu Asp Asp Asn Tyr Arg Asp Glu Asp Glu Leu Ile Phe Gln His His His Leu Leu Arg Cys Leu Glu Lys Thr Thr Met <210> 39 <211> 478 <212> PRT
<213> Polyorchis penicillatus <220>
<221> misc_feature <223> GenBank ID No: g1763619 <400> 39 Met Asn Gly Asp Ile Gly Ala Trp Ile Ser Cys Ala Arg Thr Ala Gly Ile Gly Trp Val Glu Pro Ser Ala Pro Ile Ser Ser Lys Tyr Leu Asn Lys Gln Val Cys Asn Glu Asn GIu Lys Asn Asn Ala Lys Leu Thr Ile Asn Val Gln Th:r Tyr Ser Ser Gly Arg Arg Tyr His Thr Leu Arg Lys Phe Gly Ser Gln Glu Lys Glu Thr Leu Leu Arg Asp Tyr Phe Tyr Asp Leu Glu Tyr Ty~.~ Phe Glu Ser Glu Asp Arg Asp Pro Asp Leu Phe Ile Leu Tyr Tyr Arg Thr Arg His Asn Gly Lys Leu His Phe Pro Glu Cys Ser Ser Phe Glu Lys Asn Val Asp Glu Leu Thr Phe Phe Lys Gly Asn Ile: Asn Asn Gly Ile Phe Cys Cys Trp Asp Asp Tyr Lys Lys Glu Cye~ Thr Glu His Asp Arg Arg Leu Asn Glu Ser Asp Leu Thr Ser Glu Ile Asn Val Met Ser Glu Lys Ser Asp Thr Met Asp Val Met Asn Asn His Gly Ile Gln Gln Ala Lys Asn Phe Arg Val His Leu Phe. Glu Asn Gln Lys Gly Pro 185 ~ 190 195 Gln Ser Thr Phe Leu Ile Leu Tyr Ile Thr Gly Ala Arg Tyr Phe Phe Ile Ala Val Ser Ser Thr Ile Glu Thr Ile Val Gly Ile Asp Cys Ser Ala Asn Arg Gly Glu Tyr Asn Lys Ile Pro Cys Val Phe Phe Asn Ile Glu Ala Val Val Phe Thr Ile Glu Val Cys Val Tyr Leu Ala Arg Leu Tyr Pro Cys Phe Arg His Ala Ser Ala Arg Arg Ile Ser Leu Ser Ile Val Ile Ile Leu Pro Phe Ile Asp Ala Tyr Ile Gly Leu Ala Met Thr Ser Ser Gly Ala Phe Thr Lys Ile Val Ser Leu Arg Val Phe Phe Arg Phe Lys Phe Ser Arg Val Ile Arg His Ser Lys Gly Leu Leu Giy Thr Leu Thr Ser Arg Ile Ser Cys Ala Ser Glu Leu Gly Leu Phe Leu Ser Met Ala Phe Leu Ser Ile Ile Ile Phe Ala Thr Phe Tyr Val Val Val Glu Lys Asp Val Asn Asp Ser Asp Phe Thr Pro Ala Ser Ile Ser Phe Trp Tyr Thr Ile Val Thr Met Thr Thr Leu Gly Tyr Gly Asp Met Val Pro Lys Thr Ile Pro Gly Lys Leu Va1 Giy Ser Ile Cys Ser Le:u Ser Gly Val Leu Val Ile.Ala Leu Pro Val Pro Val Ile Val Ser Asn Phe Ser Arg Iie Tyr Leu GIn Asn Gln Arg Ala Asp Lys Arg Arg Ala Asn Gln Lys Leu Arg Asn Lys Cys Glu Glu Lys Giu Glu Lys Lys Lys Glu Ser Ser Ser Glu Thr Val Thr Arg Phe Ile Ile Ser Asn Gln Met Tyr Thr Ile Phe Ser Met Lys Phe Ala Leu Thr Arg <210> 40 <211> 732 <212> PRT
<213> Rattus norvegicus <220>
<221> misc_feature <223> GenBank ID No: 82564072 <400> 40 Met Asp Thr Ser Gly Phe His G1u Ser Gly Gly Asp His Va:L Leu Asp Glu Asp Pro Lys Pro Cys Pro Ser Ser Asp Glu Cys Gl;l Gln Gln Gln Gln Gln Gln Pro Pro Pro Ser Ala Pro Ala Pro Pro Val Pro Gln Gln Pro Pro Pro Leu Leu Gln Pro Pro Pro Gly Gln Gln Leu Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Gln Pro Leu His Pro Leu Gln Leu Ala Pro Ala Gln Leu Gln Ser Gln Val His Pro Gly Leu His Ser Val Leu Ser Pro Thr Ala Phe Arg Pro Asn Ser Ala Asn Thr Ala Ala Sex' Ile Leu His Pro Ser Ser Gln GIy Ser Gln Leu Leu Asn Arg Asn Asp His Leu Val Gly His Pro Ser Ser Thr Ala Ser Gly Ser Thr Pro 140 145 3.50 Gly Gly Gly Ser Arg Arg Gln Ala Ser Pro Val His His Val Arg Arg Asp Ser Asn Pro Thr Glu Ile Ala Met Ser Cys Phe Ser Lys Tyr Ser Gly Gly Val Lys Pro Leu Asn Arg Ser Ala Met Leu Ser Arg Arg Asn Leu Ile Ala Glu Pro Glu Gly Pro Leu Glu Gln Gln Leu Phe Ser Pro Ser Pro Pro Glu Ile ile Ser Ser Asn Ile Arg Glu Asp Asn His Ala Gln Thr Leu Leu His Pro Asn His His Ala Thr His Asn His.Gln His Ala Gly Thr Thr Ala G:Ly Ser Thr Thr Phe Pro Lys.Ala Asn Lys Arg Lys Asn Gln Asn I7Le Gly Tyr Lys Leu Gly His Arg Arg Ala Leu Phe Glu Lys Arg Lys Arg Leu Ser Asp Tyr Ala Leu Ile Phe Gly Met Phe Gly Ile Val Val Met Val Ile Glu Thr Glu Leu Ser Trp Gly Leu Tyr Ser Lys Asp Ser Met Phe Ser Leu Ala Leu Lys Cys Leu Ile Ser Leu Se:r Thr Ile Ile Leu Leu Gly Leu Ile Ile Ala Tyr His Thr Arg Glu Val Gln Leu Phe Val Ile Asp Asn Gly Ala Asp Asp Trp Arg Ile Ala Met Thr Tyr Glu Arg Ile Leu Tyr Ile Ser Leu Glu Met Leu Val Cys Ala Ile His Pro Ile Pro Gly Glu Tyr Lys Phe Phe Tr;p Thr Ala Arg Leu Ala Phe Ser Tyr Thr Pro Ser Arg Ala Glu Aha Asp Val Asp Ile Ile Leu Ser Ile Pro Met Phe Leu Arg Leu Ty:r Leu Ile Ala Arg Val Met Leu Leu His Ser Lys Leu Phe Thr Asp Ala Ser Ser Arg Ser Ile Gly Ala Leu Asn Lys Ile Asn Phe Asn Thr Arg Phe Val Met Lys Thr Leu Met Thr Ile Cys Pro Gly Thr Val Leu Leu Val Phe Ser Ile Ser Leu Trp Ile Ile Ala Ala Trl> Thr Val Arg Val Cys.Glu Arg Tyr His Asp Gln Gln Asp Val Thr Sex Asn Phe Leu Gly Ala Met Trp Leu Ile Ser IIe Thr Phe Leu Ser Ile Gly 500 505 51p Tyr Gly Asp Met Val Pro His Thr Tyr Cys Gly Lys~ Gly Val Cys Leu Leu Thr Gly Tle Met Gly A1a Gly Cys Thr Ala, Leu Val Val Ala Val Val Ala Arg Lys Leu Glu Leu Thr Lys Ala. Glu Lys His Val His Asn Phe Met Met Asp Thr Gln Leu Thr Lys Arg Ile Lys Asn Ala Ala Ala Asn VaI Leu Arg Glu Thr Trp Leu Ile Tyr Lys His Thr Lys Leu Leu Lyst Lys Ile Asp His Ala Lys Val Arg Lys His Gln Arg Lys Phe Leu Gln Ala Ile His Gln Leu Arg Gly Val Lys Met Glu Gln Arg Lys Leu Ser Asp Gln Ala Asn Thr Leu Val Asp Leu Ser Lys Met Gln Asn Val Met Tyr Asp Leu Ile Thr Glu Leu Asn Asp Arg Ser Glu Asp Leu Glu Lys Gln Ile Gly Ser Leu WO OO/i2713 PCT/US99/20468 Glu Ser Lys Leu Glu His Leu Ser A:an Leu Thr Ala Phe Ser Pro Leu Leu Ile Ala Asp Thr Leu Gln GAn Leu Arg Gln Gln Gln Leu Thr Ala Phe Val GIu Ala Arg Ser A7.a Gly Gly Ile VaI Val Thr Ser His Ala Pro Pro Ser Asp Ile Il.e Ser Ser Pro Gly Ser Thr Ser Phe Pro Thr Pro Tyr Thr Ser Cys Ser Ser Ser <210> 41 <211> 269 <212> PRT
<213> Rattus norvegicus <220>
<221> misc_feature <223> GenHank ID No: 82350843 <400> 41 Met Ala Gly Ser Val Leu Glu Asn Ile Gln Ser Va:1 Leu G1n Lys Thr Trp Val Arg Glu Phe Leu Ala Glu Phe Leu Asn Thr Tyr Val Leu Met Val Phe Gly Leu GIy Ser Val Ala His Met: Val Leu Gly Glu Arg Leu Gly Sex Tyr Leu Gly Val Asn Leu Gly Phe Gly Phe Gly VaI Thr Met Gly Ile His Val Ala Gly Gly Ile; Ser Gly Ala His Met Asn Pro Ala Val Thr Phe Thr Asn Cys Alai Leu Gly Arg Met Ala Gly Arg Lys Phe Pro Ile Tyr Val Leu Gly Gln Phe Leu Gly Ser Phe Leu Ala Ala Ala Thr Thr Tyr Leu Ile Phe Tyr Gly Ala IIe Asn His Tyr Ala Gly Glu Thr Leu Leu Val Thr Gly Pro Lys Ser Thr Ala Asn Ile Phe Ala Thr Tyr Leu Pro Glu His Met Thr Leu Tzp Arg Gly Phe Val Asp Glu Val Phe Val Thr Gly Met' Leu GIn Leu Cys Ile Phe Ala Ile Thr Asp Lys Leu Asn Ser Pro 1?0 I75 180 Ala Leu Gln Gly Thr Glu Pro Leu Met Ile Gly Ile Leu Val Cys Val Leu Gly VaI Ser Leu Gly Met Asn Thr Gly Tyr Ala Ile Asn Pro Ser Arg Asp Leu Pro Pro Arg Phe Phe Thr Phe Ile Ala Gly Trp Gly Lys Lys Val Phe Ser Ala Gly Asn Asn Trp Trp Trp Val Pro Val Val AIa Pro Leu Leu Gly Ala Tyr Leu Gly Gly Ile Val Tyr Leu Gly Leu Ile His Ala Gly Ile Pro Pro Gln Gly Ser <210> 42 <211> 266 <212> P12T
<213> Mus musculus <220>
<221> misc_feature <223> GenBank ID No: 8192647 <400> 42 Met Asn Trp Gly Phe Leu Ile Leu Gly Val Lys Gln Gly Ser Asn Tyr Ser Thr Ala Leu Gly Trp Leu Ile Arg Ile Ser Val Val Phe Phe Arg Val Leu Val Tyr Ala Ala GIu Val Asp Val Val Glu Trp Asp Asp Gln Lys Asp Phe Asn Thr Gln Pro Cys Ile Cys Lys Gly Pro Asn Vai Cys Tyr Asp Phe Pro Ser His Arg Glu Phe Val VaI

Leu Trp Ala Leu Gln Leu Val Thr Pro Ser Leu Ile Leu Cys Leu g5 90 Val Val Met His Val Ala Glu Glu Glu Arg His Tyr Arg Arg Lys Arg Leu Lys His Gly Pro Pro Ala Tyr Ser Leu Asn Ala Leu Asn Ser Lys Lys Arg Gly Gly Trp Thr Leu Leu Leu Leu Trp Tyr Ser Tle Phe Lys Ala Ala Val Gly Phe Tyr Ile His Asp Ser Leu Phe Cys Ile Tyr Lys Asp Tyr Pro Arg Val Ala Ser Asp Met Val Cys Val Thr Pro Cys Pro His Asp Cys Ile Ala Pro Thr Val Tyr Arg Thr Glu Lys Lys Val Phe Phe Met Val Thr Ala Thr Tyr Val Ala 1$5 190 195 Ile Cys Ile Leu Leu Asn Glu Va1 Tyr Leu Gly Leu Ser Val Val Lys Arg Cys Met Glu Val Pro Arg Arg Lys Ser Phe Arg Arg Ala Arg Arg His Gln Leu Pro Cys Pro Tyr Val Ser Asp Thr Pro Ile Lys Gly Gly His Pro Gln heu Thr Ala Asp Glu Ser Val Ile Lys Gly Met Ala Thr Val Asp Ala Gly Val Tyr Pro <210> 43 <21I> 191 < 212 > PitT

<213> Homo sapiens <220>
<221> misc_feature <223> GenBank ID No: 81055345 <400> 43 Met Val Lys Lys Leu Va1 Met Ala Gln Lys Arg Gl.y Glu Thr Arg Ala Leu Cys Leu Gly Val Thr Met Val Val Cys Ala Val Ile Thr Tyr Tyr Ile Leu VaI Thr Thr Val Leu Pro Leu Tyr Gln Lys Ser Val Trp Thr Gln Glu Ser Lys Cys His Leu Ile Glu Thr Asn Ile Arg Asp Gln Glu Glu Leu Lys Giy Lys Lys Val Pro Gln Tyr Pro Cys Leu Trp Val Asn Val Ser Ala Ala Gly Arg Trp Ala Val Leu 80 ' 85 90 Tyr His Thr Glu Asp Thr Arg Asp GIn Asn Gln Gln Cys Ser Tyr Ile Pro Gly Ser Val Asp Asn Tyr Gln Thr Ala Ar<3 Ala Asp Val 110 115 ' 120 Glu Lys Val Arg Ala Lys Phe Gln Glu Gln Gln Va:C Phe Tyr Cys Phe Ser Ala Pro Arg GIy Asn Glu Thr Ser Val Leu Phe Gln Arg Leu Tyr Gly Pro Gln Aia Leu Leu Phe Ser Leu Phe: Trp Pro Thr Phe Leu Leu Thr Gly Gly Leu Leu Ile Ile Ala Met: Val Lys Ser Asn Gln Tyr Leu Ser Ile Leu Ala Ala Gln Lys <210> 44 <211> 308 <212> PRT
<213> Caenorhabditis elegans <220>
<221> misc_feature <223> GenBank ID No: 83292929 <400> 44 Met Ser Thr Val Phe Ile Asn Ser Arg Lys Ser Pro Asn Val Leu Lys Lys Gln Gly Thr Asp Gln Trp Val Lys Leu Asn Val Gly Gly Thr Tyr Phe Leu Thr Thr Lys Thr Thr Leu Ser Arg Asp Pro Asn Ser Phe Leu Ser Arg Leu Ile Gln Glu Asp Cys Asp Leu Ile Ser Asp Arg Asp Glu Thr Gly Ala Tyr Leu ile Asp Arg Asp Pro Lys Tyr Phe Ala Pro Val Leu Asn Tyr Leu Arg His Gly Lys Leu Val Leu Asp Gly Val Ser Glu Glu Gly Val Leu Glu G~.u Ala Glu Phe Tyr Asn Val.Thr Gln Leu Ile Ala Leu Leu Lys G~.u Cys Ile Leu His Arg Asp Gln Arg Pro Gln Thr Asp Lys Lys Ax~g Val Tyr Arg Val Leu Gln Cys Arg Glu Gln Glu Leu Thr Gln Mea Ile Ser Thr Leu Ser Asp Gly Trp Arg Phe Glu Gln Leu Ile Ser Met Gln Tyr Thr Asn Tyr Gly Pro Phe Glu Asn Asn Glu Phe Leu Cys Val Val Ser Lys Glu Cys Gly Thr Thr Ala Gly Arg Glu Leu GIu Leu Asn Asp Arg Ala Lys Val Leu Gln Gln Lys G1y Ser Arg Ile Asn Thr Ile Ser His Ser Ala Thr Pro Thr Gln His Gln Leu Asp Ala Ala Lys Glu Ala Arg Ala Thr Ala Thr Ala Thr Ser Asn Thr Thr Asn His Thr Arg Ser Asp Gln Thr Gln Pro Gln Ala Gln Ile Thr His Gln Asp Gln Pro Glu Ser Pro Lys Gln Ser Pro Gln Gly Asp Tyr AIa Ser Phe Ala Phe Glu Thr Lys Leu Thr Gly Thi- Thr Ala Ile Arg Phe Ser Pro Leu Trp Pro Phe Cys Ala Leu Tyr Glu Val Cys Ala Gly Val His Val Phe Asn Leu <2I0> 45 <211> 295 <212> PRT
<213> Homo sapiens <220>
<221> misc_feature <223> GenBank ID No: 82887407 <400> 45 Met Gln Pro Glu Gly Ala Glu Lys Gly Lys Ser Phe Lys Gln Arg Leu Val Leu Lys Ser Ser Leu Ala Lys Glu Thr Leu Ser Glu Phe Leu GIy Thr Phe Ile Leu Ile Val Leu Gly Cys Gly Cys Val Ala GIn Ala Ile Leu Ser Arg Gly Arg Phe Gly Gly Val Ile Thr Ile Asn Val Gly Phe Ser Met Ala Val Ala Met Ala Ile Tyr VaI Ala Gly Gly Val Ser Gly Gly His Ile Asn Pro Ala Val Ser Leu Aia Met Cys Leu Phe Gly Arg Met Lys Trp Phe Lys Leu Pro Phe Tyr WO 00/12711 PC"T/US99120a68 ~5 100 105 Val Gly Ala Gln Phe Leu Gly Ala Phe Val Gly A7.a Ala Thr Val Phe Gly Ile Tyr Tyr Asp Gly Leu Met Ser Phe Al.a Gly Gly Lys Leu Leu Ile Val Gly Glu Asn AIa Thr Ala His Ile Phe Aia Thr Tyr Pro Ala Pro Tyr Leu Ser Leu Ala Asn Ala Ph.e Ala Asp Gln Val Val Ala Thr Met Ile Leu Leu Ile Ile Val Ph.e Ala Ile Phe Asp Ser Arg Asn Leu Gly Ala Pro Arg Gly Leu GIu Pro Ile Ala Ile Gly Leu Leu Ile IIe Val Ile Ala Ser Ser Leu Gly Leu Asn 5er Gly Cys Ala Met Asn Pro Ala Arg Asg Leu Ser Pra Arg Leu Phe Thr Ala Leu A1a Gly Trp Gly Phe Glu Val Ph~e Arg Ala Gly Asn Asn Phe Trp Trp Ile Pro VaI Val Gly Pro Leu VaI Gly Ala Val Ile Gly Gly Leu IIe Tyr Val Leu Val Ile Glu Ile His His Pro Glu Pro Asp Ser Val Phe Lys A1a Glu Gln Ser Glu Asp Lys 2?5 280 28S
Pro Glu Lys Tyr Glu Leu Ser Val Ile Met

Claims (20)

What is claimed is:

1. A substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID
NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID
NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:16, SEQ ID NO:17, and SEQ ID
NO:18, and fragments thereof.

2. A substantially purified variant having at least 95% amino acid sequence identity to the amino acid sequence of claim 1.

3. An isolated and purified polynucleotide encoding the polypeptide of claim 1.

4. An isolated and purified polynucleotide variant having at least 95%
polynucleotide sequence identity to the polynucleotide of claim 3.

5. An isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide of claim 3.

6. An isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide of claim 3.

7. A method for detecting a polynucleotide, the method comprising the steps of:
(a) hybridizing the polynucleotide of claim 6 to at least one nucleic acid in a sample, thereby forming a hybridization complex; and (b) detecting the hybridization complex, wherein the presence of the hybridization complex correlates with the presence of the polynucleotide in the sample.

8. The method of claim 7 further comprising amplifying the polynucleotide prior to hybridization.

9. An isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:19-36 and fragments thereof.

10. An isolated and purified polynucleotide variant having at least 95%
polynucleotide sequence identity to the polynucleotide of claim 9.

11, An isolated and purified polynucleotide having a sequence which is complementary to the polynucleotide of claim 9.

12. An expression vector comprising at least a fragment of the polynucleotide of claim 3.

13. A host cell comprising the expression vector of claim 12.

14. A method for producing a polypeptide, the method comprising the steps of:
(a) culturing the host cell of claim 13 under conditions suitable for the expression of the polypeptide; and (b) recovering the polypeptide from the host cell culture.

15. A pharmaceutical composition comprising the polypeptide of claim 1 in conjunction with a suitable pharmaceutical carrier.

16. A purified antibody which specifically binds to the polypeptide of claim 1.

17. A purified agonist of the polypeptide of claim 1.

18. A purified antagonist of the polypeptide of claim 1.

19. A method for treating or preventing a disorder associated with decreased expression or activity of MECHP, the method comprising administering to a subject in need of such treatment an effective amount of the pharmaceutical composition of claim 15.

20. A method for treating or preventing a disorder associated with increased expression or activity of MECHP, the method comprising administering to a subject in need of such treatment an effective amount of the antagonist of claim 18.