CA2359145A1

CA2359145A1 - Exocytosis pathway proteins and methods of use

Info

Publication number: CA2359145A1
Application number: CA002359145A
Authority: CA
Inventors: Ying Luo
Original assignee: Individual
Current assignee: Rigel Pharmaceuticals Inc
Priority date: 1999-01-20
Filing date: 2000-01-20
Publication date: 2000-07-27
Also published as: JP2003521230A; EP1161442A2; WO2000043419A3; WO2000043419A2; AU2416600A

Abstract

(57) Abstract: The present invention is directed to novel polypeptides such as the Exo protein and related molecules which have an effect on or are related to exocytosis and to nucleic acid molecules encoding those polypeptides. Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides of the present invention and to methods for producing the polypeptides of the present invention. Further provided by the present invention are method for identifying novel compositions which mediated Exo bioactivity, and the use of such compositions in diagnosis and treatment of disease.

Description

DEMANDES OU BREVETS VOLUMINEUX
lA PRESENTS PARTIE DE CETTE DEMANDS OU CE BREVET
COMPREND PLUS D'UN TOME.
CECI EST LE TOME ~- DE Z
t~~T'E: Pour les tomes additionels, veuiilez cvntacter le Bureau canadien des brevets THAN ONE VOLUME
. THIS IS VOLUME OF
NOTE. For additivnai vviumes-phase contact the Canadian Patent Office -EXOCYTOSIS PATHWAY PROTEINS
AND METHODS OF USE
FIELD OF THE INVENTION
The invention relatesto moleculesinvolved in the exocytosispathway and more particularly to novelpolypeptideswhich associatewithexocytoticproteins,nucleicacids and antibodies.
The invention further relates fo the use of proteins associated with the exocytosis pathway in methods for identifying candidate agents which modulate exocytosis.
BACKGROUND OF THE INVENTION
In eukaryotic cells, proteins destined for the plasma membrane or the extracellular space are delivered along the secretory pathway. This comprises a series of sequential, vesicle-mediatedtransportsteps, each ofwhich requires the specific targeting oftransportvesicles to the appropriate acceptor membrane and the subsequent fusion of vesicle and acceptor membranes. In this way, proteins to be secreted by the cell are translocated into the endoplasmic reticulum and then travel through the Golgi complex. The proteins are sorted into secretory vesicles in the trans-Golgi network and these vesicles then fuse with the plasma membrane. This final membrane fusion event is known as exocytosis and results in the discharge ofvesicle contents into the extracellularspace as well as the incorporation of vesicle membrane lipids and proteins into the plasma membrane.
Exocytosis can be divided into two classes constitutive and regulated. In constitutive exocytosis, secretor vesicles fuse with the plasma membrane immediately after formation;
in regulated exocytosis, secretory vesicles accumulate in the cytoplasm and only undergo fusion upon receipt of an appropriate signal. All eukaryotic cells exhibit constitutive Although the fundamental purpose of exocytosis is to deliver lipids and proteins to the plasma membrane and to release vesicle contents from the cell, different cell types utilize this mechanism to fulfill their own particular physiological role. Some examples of the various functions of exocytosis in different cell types are listed in Table 1.
Table 1. Functions of exocytosis Constitutive All cells Insertion of plasma membrane proteins Liver cells Serum protein secretion Mammary cells Milk protein secretion Fibroblasts Connective tissue protein secretion Regulated Neurons Neurotransmitter release Adrenal chromaffln Adrenaline secretion cells Pancreatic acinar Digestive enzyme secretion cells (exocrine) Pancreatic (3-cells Insulin secretion (endocrine) Mast cells Histamine secretion Mammary cells Milk protein secretion Sperm Enzyme secretion Egg Creation of fertilization envelope Adipocytes Insertion of glucose transporters into plasma membrane It should be noted that exocytosis (.e., the fusion of vesicles with the plasma membrane) is not only the end point of the secretory pathway, but can also involve vesicles which did not originatefromtheendoplasmicreticulum. For instance,transcytosisoccursinpolarized cells and involves endocytic vesicle budding from one pole ofthe cell, transport to the other pole (often via endosomes) and subsequent exocyticfusion. In mammary cells, transcytosis is used in the uptake of antibodies from the blood and their subsequent secretion in milk, Similarly, some vesicles undergo cycles of exolendocytic fusion via endosomes without returning to the Golgi. Exocytosis of recycling vesicles may be either constitutive (e.g., transferrin receptor-containing vesicles) or regulated (e.g., synaptic vesicles).
3D Since all cells exhibit constitutive exocytosis, it follows that regulated secretory cells must possess two types of secretory vesicle: one constitutive and on regulated.
Morphological studies indicate this to be the Case, since constitutive secretory vesicles appear small and clear in the electron microscope, whereas regulated secretory vesicles typically appear SUBSTITUTE SHEET (RULE 26) larger and opaque. Furthermore, the two types of vesicle usually contain different substances (an exception is the mammary cell, where casein secretion occurs by both constitutive and regulated exocytosis).
It should be noted that cells may contain more than one type of regulated secretary vesicle.
The best example of this is seen in neurons, which may possess synaptic vesicles and large dense-core vesicles in addition to constitutive secretary vesicles. Some properties of the two type of neuronal regulated secretary vesicle are listed in Table 2.
Large dense-core vesicles contain pe~ide neurotransmitters and these are very similar to regulated secretary vesicles in endocrine cells. indeed, much ofthe information on large dense-core vesicle biogenesis and exocytosis has come from studies of adrenal chromaf5n cells and theirtumorcellderivatives,PCl2cells,bothpopufarneuronalcellmodels.
Synapticvesicles appear clear in the electron microscope, are much smalierthan large dense-core vesicles and contain fast neurotransmitters. Synaptic vesicles have evolved in animals to allow the extremely rapid point-to-point communication required forbrain function.
Recently synaptic-like vesicles have been found in endocrine cells, such as adrenal chromaffin cells and pancreatic (3-cells. These vesicles also appearto contain fast neurotransmitters, although their physiological role is undear.
Table 2. Characteristics of regulated secretary vesicles in neurons Synaptic vesiclesLarge dense-core vesicles Vesicle size50 nm 200 nm Speed of 200 Ns milliseconds-seconds transmissionFast neurotransrr>ittersPeptides (endorphins, VIP, Neurotransmitter(GABA, glutamate,etc.) ACh, etc.) Cells affectedPost-synaptic Cells in surrounding contact only area Duration Short-lived Longer lived of effect Stimulation Low-frequency High-frequency Caz' concentrationHundreds of micromolarTens of micromolar Vesicle recyclingYes No via endosome?

Abbreviations used: GABA, ~-arrunobutyric acid; ACh, acetylcholine:
VIP, vasoactive intestinal peptide.
The more infom~ationthatis gathered regarding exocytosis, the easier it will be to manipulate exocytosis. Moreover, the more information which is gathered, the easier it will be to diagnosis and treat disorders involving exocytosis. Far example, inflammatory mediator release from mast cells leads to a variety ofdisorders, including asthma.
Therapy for allergy SUBSTITUTE SHEET (RULE 26) .q_ remains limited to blocking the individual mediators released from mast cells (anti-histamines),non-specificanti-inflammatoryagentssuchassteroidsand mastcellstabilizers which are only marginally effective at limiting the symptomatology of allergies.
Similarly, the Chediak-Higashi Syndrome (CHS) is a rare autosomal recessive disease in which neutrophils, monocytes and lymphocytes contain giant cytoplasmic granules.
Similar disorders have been described in mice, mink, cattle, cats and killer whales, with the murine homolog of CHS (designated beige or bg) being the best characterized. See Perou etal., J. Biol. Chem.272(47):29790(1997) and Barbosa et al., Nature 382:262 ( 1996), both of which are hereby incorporated by reference.
There is a therefore a need to determine the proteins Involved In exocytosis.
Some insights into the process of regulated secretion at the molecular level have allowed the definition of G proteins as important regulators. Early experiments showed that non-hydrolyzable analogues of GTP could inducesecretionin peritoneal mast cells (Fernandes, et al., Nature 312: 453 (1984)). More recently, a large body of evidence has been accumulating implicatingsmaIIGproteinsoftherabfamllyasregulatorsinthefusionofsecretorygranul es with plasma membranes during exocytosis, The rab GTPases represent a diverse family of homologous proteins that are generally associated with the membrane of organelles in awidevariety ofcells, wherethey regulatedefined steps of intracellular membrane traffic (Zeriai, M. and Stenmark, H., Curr. Opin. Cell Biol. 5:613 ( 1993)). An example of this are the rab3 subfamily proteins which have been found to have limited expression in regulated secretion-competent cells, and to be associated with synaptic or secretory granules, suggesting that they are involved in stimulus-secretion coupling (Lledo, et al., Trends Neurobiol. Sci. 17:426 ( 1994)). Furthermore, overexpression of rabid or its GTP binding mutant form ( N 1351) in the rat basophilline RBL leads to significantinhibitionof IgE mediated :?5 exocytosis (Roa, J. lmmunol., 159:2815 (1997)).
Rab3a, Rab3b, Rab3c, and Rabid constitute a subgroup of the rab family implicated in regulated exocytosis. Rab3a has been detected in regulated secretory cells such as neurons, endocrine cells, and exocrine cells but not in constitutive secretory cells such as hepatocytes and lymphocytes (Fischer von Motlard, Proc. Natl Acad. Sci. 87:

',30 (1990), Takai, et al., Int. Rev. Cytol. 133: 187-230 (1992)). In neuromuscular synapses, rab3a has been localized at the synaptic vesicles (Mizoguchi, et al., Biochem.
Biophys.
Res. Commun. 202:1235-43 (1994)). Furthermore, Rab3a has also been detected atthe secretory granules of chromaffin cells and at the zymogen granules in the exocrine cells of pancreatic acini(Padfield,etal., Proc. Nad. Acad. Sci., USA 89:1656-60 (1992)). Rab3a has been shown to interact with a numberofother proteins including the exchange proteins GDI and GRF (Matsui, et al., Mol. Cell. Biol. 10' 4115-22(1990), Burstein, E.S. and Macara I.G., Proc. Natl. acad. Sci., USA 89:1154-8 (1992)) as well as GAP (Burstein, J. Biol.
Chem. 266: 2689-92 (1991)) and Rabphilin (Shirataki, et al., Mol. Cell Biol.
13: 2061-8 (1993)). It isbelievedthatRab3amoduiatesexocytosisinregulatorysecretorycells.
Rab3a is cloned and known in the art (see, i.e., Genbank accession number (no.) M28210).
Rab3d is thought to be involved in the modulation of regulated secretion in a number of cell types. Rab3dispredominantlyexpressedinfattissue but can also be found expressed, at lower levels, in a wide range of tissue types including lung, spleen, heart, and brain.
Baldini, G., et al., (1992) Proc. Natl. Acad. Sci. USA 89: 5049-52. Rab3d has also been implicated in the translocation of the GIut4 glucose transporter in adipocytes. Rab3d has been cloned and is known in the art, i.e., Genbank accession no.: AF081353.
Thus, rabs, particularly, tissue /cell specific isoforms of rabs and the proteins which they interact with are of great pharmaceutical interest. Rab7 is cloned and known in the art, (see, i.e., Genbank accession no. U44104).
Rab9 is localized to the surtace of lake endosomes where it appears to act to stimulate the transportofmannose6-phosphate receptors between late endosomes and the traps-Golgi network, both in vitro and in vivo. Recent studies suggest that this GTPase is a rate-limiting component for transport between late endosomes and the traps-Golgi network.
Rab9 has been cloned and is known in the art, (see i.e., U44103).
Rab11 was identified by screening a Madin-Darby canine kidney cell cDNA
library using degenerate oligonucleotides derived from conserved sequences of the Rab superfamily.
Chavrier, P., et al., (1990), Mol. Cell. Biol. 10:6578-85. The predicted amino acid sequences ofthe canine, human, rat and rabbit Rab11 are 100% identical. This high level of conservation between species might reflect a particular importance of this member of the Rab family. Rabl1 has been localized to both the constitutive and regulated secretory pathway in PC12 cells. Ora3, a homolog of Rabl1 (91 % identity at the amino acid level) has been found to be associated with cholinergicsynapticvesiclesderived from the electric organ of the marine ray. Although the function of Rab11 has yet to be definitively determined, a number of lines of evidence suggest that it may play a role in the targeting of transport vesicles of different origin to a common destination, the plasma membrane.
Northern blot analysis has shown that Rabl1 is ubiquitously expressed but is generally more abundant in tissues with a high level of secretion. Rab11 has been cloned and is known in the art, i.e., Genbank accession no. X56740.
RabS (a, b, and c) make up a subgroup of the Rab protein family. They are located at the cytoplasmic surface of the plasma membrane, on early endosomes and on plasma-s membrane derived clathrin coated vesicles. Antibodies directed against RabSa inhibit the fusion of early endosomes in vitro suggesting that its activity is required in this process.
In vivo, overexpression of wild type and mutant Rab5a leads to changes in the rate of internalizationofendocyticmarkersand in morphologicalalterationsofthe earlyendosomes.
These data suggest that RabSa is a rate-limiting factor that regulates the kinetics of both lateral fusion ofearly endosomesand fusionof plasma membranederivedendocyticvesicles with early endosomes. Some proteins have been identified to associate with RabS. For example, a 62 kDa coiled-coil protein that specifically interacts with the GTP-bound form of RabS has been identified. This protein shares 42% sequence identity with Rabaptin-5, a previously identified effector of RabS, and has been named it Rabaptin-5beta. Like Rabaptin-5, Rabaptin-5beta displays heptad repeats characteristic of coiled-coil proteins and is recruited on the endosomal membrane by Rab5 in a GTP-dependent manner, However, Rabaptin-5beta has features that distinguish it from Rabaptin-5. The relative expression levels of the two proteins varies in different cell types. Rabaptin-5beta does not heterodimerize with Rabaptin-5, and forms a distinct complex with Rabex-5, the GDPIGTP exchange factor for RabS. Immunodepietion of the Rabaptin-5beta complex from cytosol only partially inhibits early endosome fusion in vitro, whereas the additional depletion of the Rabaptin-5 complex has a stronger inhibitory effect. Fusion activity can mostly be recovered by addition of the Rabaptin-5 complex alone, but maximal fusion efficiency requires the presence of both Rabaptin-5 and Rabaptin-5beta complexes.
Gournier H., et al., (1998), EMBU J. 17(7):1930-1940. RabS is cloned and known in the art (see, i.e., Genbank accession no. M28215).
Moreover, key proteins that act in Ca2' -regulated exocytosis in neurons and endocrine cells includethevesicleproteinssynaptotagmin,VAMPlsynaptobrevin,the target membrane protein SYNTAXINs and SNAP-23/25 and, in addition, the soluble N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF-attachment proteins (a-, a-, y-SNAPs).
Functional evidence for the importance of the membrane proteins has come from their sensitivitytothe specific proteolyticactionsof clostridialneurotoxinsand I
orgeneticanalysis in mice and Drosophila. The soluble factors NSF and SNAP were found to interact, in a 20S complex, with the neurotoxin substrates leading to them being designated as SNAP-receptors (SNAREs). Many of the proteins that make up the SNARE complexes contain _7_ coiled coil domainswhich are thoughtto intereactprimarily th rough hydrophobicinteractions.
These proteins described function at some point in the exocytic pathway either as central membersofthe vesiclefusion complexoras accessory proteins involved in some regulatory step in the vesicle fusion cycle.
GS27isassociatedwiththeGolgiapparatusandisbelievedtobehavelikeaSNARE. GS27 (forGoIgiSNAREof27K), is identicaltomembrin,a protein implicated earlier in ER-to-Golgi transport. Regarding SNARES, these proteins are known to mediate vesicle transport by docking vesicles onto the target membrane. One study has have reported that the cytoplasmic domain of GS27 or antibodies raised against it quantitatively inhibit transport in vitro from the ER to the trans-GoIgiITGN, acting at a stage between the cis/medial- and the traps-GoIgi/TGN,indicatingthatprotein movement from medial-to the traps-Golgi/TGN
depends on SNARE-mediated vesicular transport and that GS27 plays a functional role (Lowe, S. L., et al., Nature, 389:881-884 (1997)). Therefore, GS27 is implicated in exocytosis by its relation to vesicular transport.
SNAP-23(usedinterchangeablywith snap-23)wasfirstidentifiedinahumanBlymphocyte cDNA library (Ravichandran, V., et al., (1996), J. Biol. Chem. 271:13300-03).
Subsequently, others have independently reported the identification of SNAP-23 in several cell types including mast cells. The primary structure of SNAP-23 is 59%
identical to SNAP-25; it contains a central clusterofcysteine residues that is a site of palmitoylation in SNAP-25 and predicted coiled-coil that are thought to serve in binding other SNAREs, especially syntaxins 1,3, and 4. SNAP-23, like SNAP-25, has been localized mainly to the plasma membrane. However, recent evidence suggests that SNAP-23 translocates to the surface of secretory granules upon cellular activation and forms a complex with SYNTAXIN-3 and VAMP-2 (Guo,Z.,etal.,(1998),Ce11.94:537-48).ThissamestudysuggeststhatSNAP-23 function iscrucialforoompoundexocytosisinmastcellsinthatSNAP-23specificantibodies can completely block secretion in permeabilized cells. SNAP-23 is cloned and known in the art, (see, i.e., Genbank accession no. U55936).
NSF and alpha-snap were originally detected as factors required for transport through the Golgi in in vitro assays and yeast homologues of these proteins, secl8 and secl7, are essential for secretion in vivo. In Golgi transport assays and in the formation of a Golgi membrane derived 20S complex, a and p-SNAP appear to be functionally redundant. In contrast more recent resultssuggestthataandp-SNAPhavedistinctfunctionsin regulated exocytosis based on the ability of alpha-snap to displace synaptotagmin from the SNARE

-8_ complex. Sollner, T., et al., Cell, 75: 409-18 (1993). Alpha-snap has been cloned and is known in the art, i.e., Genbank accession number (no.) 039412.
Sec1 is a hydrophilic protein that plays an essential role in exocytosis from the yeast Saccharomyces cerevisiae. Syntaxin (a T-SNARE), together with SNAP-25 and synaptobrevinNAMP (a T- and a V-SNARE, respectively), is thought to form the core of the docking-fusion complex in synaptic vesicle exocytosis. Proteins that exhibit similarity to Sec1 were identified in the nervous system of Drosophila melanogaster (Rop) and Caenorhabditiselegans(UNC18). Munc-18/n-SeclIrbSec1,a brain homoiogueoftheyeast Sec1p protein, is thought to participate in regulating the docking and fusion of synaptic vesicles. Munc-18/n-Sec1lrbSecl expression has been reported to be neural-speci6cand a number of non-neural isoforms have been identified which are more ubiquitously expressed. Shaywitz, D. A., et al , J. Cell. Biol. 128:769-777 (1995).
The role of Munc18c, previously identified as an n-Sec1/Munc18 homolog in 3T3-adipocytes, in insulin-regulated GLUT4 trafficking has been investigated in adipocytes. Lowe, S. L., et al, Nature. 389:881-884 (1997). In these cells, Munc18c predominantly associated with syntaxin4, although it bound both syntaxin2 and syntaxin4 to similar extents in vitro. In additian, SNAP-23, an adipocyte homolog of SNAP-25, associated with both syntaxins 2 and 4 in 3T3-L1 adipocytes. Overexpression of Munc18c in 3T3-L1 adipocytes by adenovirus-mediated gene transfer results in inhibition of insulin-stimulated glucose transport in a virus dose-dependent manner (maximal effect, approximately 50%) as well as in inhibition of sorbitol-induced glucose transport (by approximately 35%), which is mediated by a pathway different from that used by insulin.
In contrast, Munc18b, which is also expressed in adipocytes but which did not bind to syntaxin4, had no effect on glucose transport. These results suggest that MunclBc is involved in the insulin-dependent trafficking of GLUT4 from the intracellular storage compartment to the plasma membrane in 3T3-L1 adipocytes by modulating the formation of a SNARE complex that includes syntaxin4. Unc18-1 has been cloned and is known in the art, i.e., Genbank accession number (no.) D63851.
Tetanus toxin inhibits neurotransmitter release by selectively blocking fusion of synaptic vesicles. Tetanus toxin has been shown to proteolytically degrade synaptobrevin II (also named VAMP-2), a synaptic vesicle-specific protein, in vitro and in nerve terminals. As targetsoftetanustoxin, synaptobrevinsprobablyfunction in theexocytoticfusion of synaptic vesicles. A synaptobrevinhomologue, cellubrevin (VAMP-3), present in all cells and tissues tested, is a membrane trafficking protein of a constitutively recycling pathway. McMahon _g_ H.T., et al, Nature. 364(6435):346-52 (1993). Like synaptobrevin II, cellubrevin is proteolysed by tetanus toxin lightchain in vitro and aftertransfection.Theseresultsindicate that constitutive and regulated vesicu larpathways use homologousproteins for membrane trafficking, likely for membrane fusion at the plasma membrane, indicating a greater mechanistic and evolutionary similarity between these pathways than previously thought.
The homologueofVamp3, vamp2 (synaptobrevin), has been localizedto mast cell granules and may play a critical role in mast cell exocytosis (Guo, Z., et al., Cell, 94:537-48 (1998)).
Vamp3 has been cloned and is known in the art, i.e., Genbank accession no.:
AF26007.
Accordingly, the proteins involved in exocytosis, termed Exo proteins herein, particularly those associatedwith GS27, Rab3a, Rab7, Rab9, Rab11, Rabid, RabS, alpha snap, unc18-1, vamp3, and snap-23areof interest, and it is desirable to providesuch proteinsand related molecules. It is a further aspect of the invention to provide recombinant nucleic acids encoding Exo proteins and expression vectors and host cells containing the nucleic acid encoding the Exo protein. A further aspect of the invention is to provide methods for screening for antagonists and agonists of Exo proteins, particularly those which modulate exocytosis, secretion andlor vesicular transport.
SUMMARY OF THE INVENTION
Accordingly, the present invention provides recombinant nucleic acids encoding an Exo protein that has at least about 85% sequence identity, and mare preferably at least about 90% sequence identity, and most preferably about 95% sequence identity with an amino acid sequence encoded by a nucleic acid comprising the first 100 nucleic acid residues of a sequence selected from the group consisting of SEA ID NOS:15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 63, 64, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 147, 148, 150, 151, 152, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 210, 211.
Preferably, the Exo proteins bind to a protein selected from the group consisting of GS27, rab7, rab9, snap-23, ;30 rab3a,rab1l,rabid,rab5,alpha-snap,unc18-1,andvamp3. Also provided are recombinant nucleic acids which have at least about 75% sequence identity, more preferably, at least 85% sequence identity and most preferably at least about 95°l°
sequence identity with a nucleicacid sequencecomprisingthe first 100 nucleic acid residues of a sequenceselected from the group consisting of SEQ ID NOS: 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 63, 64, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142. 143, 147, 148, 150, 151, 152, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 210, 211 and their complements. Recombinant Exo proteins, expression vectors and host cells comprising the nucleic acids are also included.
In an additionalaspect, the invention provides recombinant Exo proteinsExo3, Exo4, ExoS, Exo6, Exo7, Exo8, Exo9, Exo10, Exo11, Exo12, Exo13, Exol4, Exo15, Exo16, Exo17a, Exo17b, ExolB, Exo19, Exo20, Exo2l, Exo22, Exo23. Exo24, Exo25, Exo26, Exo27, Exo28, Exo29, Exo30, Exo31, Exo32, Exo33, Exo34, Exo35, Exo36, Exo37, Exo38, Exo39, Exo40, Exo41, Exo42, Exo43, Exo44, Exo45, Exo46, Exo47, Exo48, Exo49, Exo50, Exo51, Exo52, Exo53, Exo54, Exo55, Exo56, Exo57, Exo58, Exo59, Exo60, Exo61, Exo62, Exo63, Exo64, Exo65, Exo66, Exo67, Exo68, Exo69., Exo70, Exo71, Exo72, Exo73, Exo74, Exo75, Exo76, Exo77, Exo78, Exo79, Exo80, Exo8l, Exo82, Exo83, Exo84, Exo85, Exo86, Exo87, ExoBB, Exo89, Exo90, Exo91, Exo92, Exo93, Exo94, Exo95, Exo96, Exo97, Exo98, Exo99,Exo100, Exo101, Exo102, Exo103, Exo104, Exo105, Exo106, Exo107, Exo108, Exo109, Exo110, Exo111, Exo112, Exo113, Exo114, Exo115, Exo116,Exo117,andExo118,andthenucleic acids encoding said Exo proteins.
In a further aspect, the invention provides methods of making Exo proteins, comprising providing a cell comprising an Exo nucleic acid and subjecting the cell to conditions which allow the expression of Exo proteins.
In a further aspect, the present invention provides methods for screening for a bioactive agent capable of binding to an Exo protein. The method comprises combining an Exo protein and a candidate bioactive agent, and determining the bindingofthecandidateagent to the Exo protein.
1n an additional aspect, the present invention provides methods for screening for agents capable of intertering with the binding of an Exo protein and GS27. The methods comprise combining an Exo protein, a candidate bioactiveagentand a GS27 protein, and determining the binding of the Exo protein and the GS27 protein.
In anotheraspect, the present invention provides methods for screeningforagentscapable of interferingwith the binding ofan Exo protein and rab7. The methods comprise combining an Exo protein,a candidatebioactive agentand a rab7 protein, and determining the binding of the Exo protein and the rab7 protein.
I n a furtheraspect, the presentinvention provides methods for screening foragentscapable of interfering with the binding ofan Exo proteinand rab9. The methodscomprise combining an Exo protein, a candidate bioactiveagentand a rab9 protein, and determining the binding of the Exo protein and the rab9 protein.
In yet another aspect, the present invention provides methods for screening for agents capable of interfering with the binding of an Exo protein and snap-23. The methods comprise combining an Exo protein, a candidate bioactive agent and a snap-23 protein, and determining the binding of the Exo protein and the snap-23 protein.
In an additional aspect, the present invention provides methods for screening for agents capable of interfering with the binding of an Exo protein and rab3a. The methods comprise combining an Exo protein, a candidatebioachveagentand a rab3a protein, and determining the binding of the Exo protein and the rab3a protein.
In anotheraspect, the present inventionprovidesmethodsforscreening for agents capable of interfering with the binding of an Exo protein and rab1 i. The methods comprise combiningan Exo protein, a candidate bioactive agent and a rabl 1 protein, and determining the binding of the Exo protein and the rabll protein.
In a further aspect, the present invention providesmethodsforscreeningforagentscapable of interfering with the binding of an Exo protein and rabid. The methods comprise combining an Exo protein, a candidatebioactiveagentand a rabid protein, and determining the binding of the Exo protein and the rabid protein.
In yet an additional aspect, the present invention providesmethodsforscreeningforagents capable of interfering with the binding of an Exo protein and rab5. The methods comprise combining an Exo protein, a candidate bioactive agent and a rab5 protein, and determining the binding of the Exo protein and the rab5 protein.
In a furtheraspect, the presentinvention provides methods for screeningforagentscapable of intertering with the binding of art Exo protein and alpha-snap. The methods comprise combining an Exo protein, a candidate bioactive agent and an alpha-snap protein, and determining the binding of the Exo protein and the alpha-snap protein.

In yet another aspect, the present invention provides methods for screening for agents capable of intertering with the binding of an Exo protein and unc18-1. The methods comprise combining an Exo protein, a candidate bioactive agent and an uncl8-1 protein, and determining the binding of the Exo protein and the uncl8-1 protein.
In an additional aspect, the present invention provides methods for screening for agents capable of interfering with thebindingofanExoproteinandvamp3. The methods comprise combining an Exo protein,a candidatebioactiveagentand a varnp3 protein, and determining the binding of the Exo protein and the vamp3 protein.
In another aspect, the invention provides methods for screening for an bioactive agent capable of modulating the activity of an Exo protein. The method comprises the steps of addinga candidatebioactiveagentto a cellcomprisinga recombinant nucleic acid encoding an Exo protein, and determining the effect of the candidate bioactive agent on cellular activity. In a preferred embodiment the cellular activity is exocytosis or vesicular transport.
In anotheraspect, the invention provides a methodoftreatingan exocytosisrelateddisorder comprising administering an agent that interferes with specific bindingofaproteinselected from those shown in the Sequence Listing with a protein selected from the group consisting of GS27, Rab3a, Rab7, Rab9, Rabll, Rab3d, RabS, alpha snap, uncl8-1, vamp3, and snap-23, expressed in a tissue such that said disorder is ameolerated.
Also provided herein is a method of treating an exocytosis related disorder comprising administering to a patient an agent that binds to a protein encoded by a sequence selected from the group consisting of those set forth in the Sequence Listing, such that exocytosis is altered.
Further provided herein is a method ofreducing or inhibiting exocytosis in a cell comprising administering an agent that interferes with specific binding of a protein selected from those encoded by a sequence selected from the group consisting of SEQ ID NOS:1-51 (odd numbers) and 53-211with a protein selected from the group consisting of GS27, Rab3a, Rab7, Rab9, Rab1l, Rab3d, Rab5, alpha snap, uncl8-1, vamp3, and snap-23, expressed in said cell such that exocytosis is inhibited.
In yet another aspect, the invention provides a method of neutralizing the effect of a protein encoded by a sequence selected from the group consisting of SEO ID NOS:1-51 (odd numbers) and 53-211 comprising contacting an agent specific for said protein with said protein in an amount sufficient to effect neutralization. Other aspects of the invention are set forth as described below.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1 A and 1 B showthe underlying mechanism ofyeasttwo-hybridand yeastone-hybrid systems. Figure 1A shows the two-hybrid system: GAL4A represent GAL4 transcription activation domain. cDNA represents cDNA library inserts. X represents any bait gene.
GAL4B represents GAL4 DNA binding domain. HIS/IacZ indicates that the reporter gene is either HIS or IacZ.
Figure 2 shows the outline of yeast two-hybrid screening. Solid black dots represent colonies on plates. Transformation steps of both bait plasmid and cDNA library plasmids are indicated.
Figure 3 shows the outline of yeast one-hybrid screening. Solid black dots represent colonies on plates.
Figures 4A-4D show vectors used in the yeast two-hybrid and one-hybrid screening, respectively. Figures4A-4Bshowthetwo-hybridvectors. Baitvectors can be pHybLex2eo (Invitrogen), pBD-GAL4 (Stratagene), pAS2-1 (Clontech), pGBT9 (Clontech), or pGilda (Origene, Clontech). Arrows indicatetranscription of fusion proteins on either bait orcDNA
vector. The binding domain can be either GAL4 or LexA. MCS underlined represents multiple cloning sites, where either bait gene or eDNA fragments should be cloned. 2 N
Orirepresentsyeast2micronreplicationorigin.cDNAvectorscanbepYESTrp2(Invitrogen) , pAD-GAL4 (Stratagene), pACT2 (Clontech), pGADGH (Clontech), pGAD424 (Clontech), or p,lG4-5 (Origene).Activationdomain can be GAL4, VP16, or other transcription activator.
Figures 4C-4D show the one-hybrid reporter vectors. DNA sequences of interest should be inserted into the multiple cloning sites (MCS) underlined. The enzyme used to Linearize :?5 reporter vector for integration is shown by solid arrow. The dashed arrow indicates the transcription of either HIS or IacZ gene.
DETAILED DESCRIPTION OF THE INVENTION
The present invention provides Exo proteins and nucleic acids involved in the exocytotic pathway. In a preferred embodiment, the Exo proteins are from vertebrates and more a0 preferably from mammals, including rodents (rats, mice, hamsters, guinea pigs, etc.), primates, farm animals (including sheep, goats, pigs, cows, horses, etc) and in the most preferred embodiment, from humans.
An Exo protein of the present invention may be identified in several ways.
"Protein" in this sense includes proteins, polypeptides, and peptides The Exo proteins of the invention fall into two general classes: proteins that are completely novel, i.e. are not part of a public database as of the time of discovery, although they may have homology to either known proteins or expressed sequence tags (ESTs). Alternatively, the Exo proteins are known proteins, but thatwere not known to be involved in exocytosis; i.e. they are identified herein as having a novel biological function. Accordingly, an Exo protein may be initially identified by its association with a protein known to be involved in exocytosis or vesicular transport, In one embodiment provided herein, Exo proteins bind to a protein selected from the group consisting of GS27, rab7, rab9, snap-23, rab3a, rab11, rabid, rab5, alpha-snap, unc18-1, and vamp3. Exo proteins may be novel or may have been known in the art to exist, but not known to bind to GS27, rab7, rab9, snap-23, rab3a, rabll, rabid, rab5, alpha-snap, uncl8-1, or vamp3. Wherein the Exo proteins and nucleic acids are novel, compositions and methodsof use are provided herein. In the case that the Exo proteins and nucleic acids were known but not known to bind toGS27, rab7, rab9, snap-23, rab3a, rabll, rabid, rab5, alpha-snap, unc18-1, or vamp3, methods of use, i.e. functional screens, are provided.
In one embodiment, Exo nucleic acids or Exo proteins are initially identified by substantial nucleic acid andlor amino acid sequence identity or similarity to the sequences provided herein. In a preferred embodiment, Exo nucleicacids or Exo proteinshave sequence identity orsimilarityto the sequencesprpvided herein as described below and bind to an exocytosis orvesiculartransportprotein.
Preferredexocytosisandvesiculartransportproteinsinclude GS27, rab7, rab9, snap-23, rab3a, rab11, rabid, rab5, alpha-snap, unc1B-1, and vamp3 (these proteins are known in the art are considered the same whether'=" is used within the name or capitols are used). Such sequence identity or similarity can be based upon the overall nucleic acid or amino acid sequence.
SEQ ID N0:1 shows nucleic acid sequence encoding at least a portionofmousesyntaxin4, Genbankaccessionno.: U76832, described in Hay JC, et al., J Cell Biol 1998,141 (7):1489-1502.
SEQ ID N0:3 shows nucleic acid sequence encoding at least a portion of mouse and LZIP-2, Genbankaccessionno.:AC003675,describedin BurbeloPD, etal. Gene 1994, 139(2 ):241-245.

SEQ ID N0:5 shows the nucleic acid sequence encoding at least a portion of mouse IL-3 receptor, Genbank accession no.: M29855, described in Tabira T, et al., Ann N
Y Acad Sci.
1998, 840: 107-116.
SEQ ID N0:7 shows nucleic acid sequence encoding at least a portion of mouse receptor, accession no.: M27959, described in Ryan JJ, et al., J I
mmunol.1998,161 (4):1811-1821.
SEQ ID N0:9 shows a second nucleic acid sequence encoding at least a portion of mouse IL-4 receptor, accession no.:M2T959, described in Ryan JJ, et al., J Immunol.
1998, 161 (4):1811-1821.
SEQ ID N0:11 shows nucleic acid sequence encoding at least a portion of mouse LDL
receptor-related protein 6 (Lrp6), accession no.:AF074265, described in Brown,SD, etal., Biochem. Biophys. Res. Commun. 248 (3):879-888 (1998).
SEQ ID N0:13 shows nucleic acid sequence encoding at least a portion of mouse abc2, accession no.:X75927, described in Illing M, etal., J Biol Chem 1997,11;272(15):10303-10310.
SEQ ID NOS:15,17,19, 21, 23, and 25 show nucleic acid sequences which encode Exo3-8, respectively.
SEQ ID N0:27 shows the nucleic acid sequence encoding Exo9, which may share some characteristicswith human syntaxinl6A, accession no.:AF008937, described in Hay JC, et al., J Cell Biol 1998, 141 (7):1489-1502.
SEQ ID N0:29 shows the nucleic acid sequence encoding Exo10, which which may share some characteristics with human putative RNA binding protein (RBP56), accession no.:U51334, described in Genomics 38:51-57 (1996}.
SE~IDN0:31showsthenucleicacidsequenceencodingExo11,whichhassomesimilarity with GenBank accession no.: AA144083.
SEQ ID N0:33 shows the nucleicacid sequenceencoding Exo12, which has some similarity with GenBank accession no.: AA103185.

SEA ID N0:35 shows the nuclelcacid sequenceencoding Exo13, which has some similarity with GenBank accession no.: AA919222.
SEQ ID N0:37showsthe nucleic acid sequenceencoding Exo14,which has some similarity with GenBank accession no.:AA276016 and human (xs99).
SEQ ID N0:39showsthe nucleicacid sequenceencoding Exo15, which has some similarity with GenBank accession no.:AA617266, and CREB-Rf~ (creb-rp), Genebank accession no.: 031903.
SEO I D N0:41 showsthe nucleicacid sequence encoding Exo 16, which has some similarity with Genl3ank accession no.:AA221293 and rat lamina-assocated peptide.
SEQ ID N0:43 shows the nucleic acid sequence encoding Exol7a, which has some similarity with GenBank accession no.:AA166109 and ratsyntaxin5, Genebankaccession no.:L20822, described in Rowe T, et al., Science 1998 279(5351):696-700.
SE4 ID N0:45 shows the nucleic acid sequence encoding Exol7b, which has some similarity with GenBank accession no.:AA166109 and rat syntaxin5, Genebank accession no.:L20822, described in Rowe 1', et al., Science 1998 279(5351):696-700.
SEQ ID N0:47showsthenucleicacid sequenceencodingExo18,which has some similarity withGenBankaccessionno.:AA166109andhassomesimilaritytoratsyntaxin5,Genebank accession no.:120822, described in Rowe T, et al., Science 1998 279(5351):696-700.
SEQ ID N0:49showsthe nucieicacid sequenceencoding Exo19, which has some similarity with GenBank accession no.:U76832 and mouse syntaxin4, described in Hay JC, J
Cell Biol 1998, 141 (7):1489-1502.
SEA ID N0:51 shows the nucleic acid sequence which encodes Exo20.
SEA ID N0:53 shows the nucleic acid sequence which encodes Exo21.
SEA ID N0:54 shows the nucleic acid sequence encoding a portion of human axonal transporter of synaptic vesicles, Genbank accession no.: X90840.

_17 SEQ ID N0:55 shows the nucleic acid sequence encoding a portion of human cargo selection protein TIP47 (TIP47), Genbank accession no.: AF057140.
SEA ID N0:56 shows the nucleic acid sequence encoding a portion of human cargo selection protein TIP47 (TIP47), Genbank accession no.: AF057140.
5EQ ID N0:57 shows the nucleic acid sequence encoding a portion of human cargo selection protein TIP47 (TIP47), Genbank accession no.: AF057140.
SEQ ID N0:58 shows the nucleic acid sequence encoding a portion of human cargo selection protein TIP47 (TIP47), Genbank accession no.: AF057140.
SEQ I D N0:59 shows the nucleicacid sequenceencodinga portion of human tax interaction protein, Genbank accession no.: AF028824.
SEQ ID N0:60shows the nucleic acid sequenceencodinga portionofhumantax interaction protein, Genbank accession no.: AF028824.
SEA ID N0:61 showsthe nucleic acid sequenceencodinga portion of humantax interaction protein, Genbank accession no.: AF028824.
SE~IDN0:62showsthenucleicacidsequenceencodingaportionofhumanhumaninositol polyphosphate 5-phosphatase, Genbank accession no.: M74161 SEQ ID N0:63 shows the nucleic acid sequence encoding Exo22.
SEf~ ID N0:64 shows the nucleic acid sequence encoding Exo23.
SEQ ID N0:65 shows the nucleic acid sequence encoding a portion of mouse Sec61 protein complex gamma subunit; Genbank accession no.: U11027.
SEQ ID N0:66 shows the nucleic acid sequence encoding a portion of mouse HMG-1;
GenBank accession no.: U00431.
SEQ ID N0:67 shows the nucleic acid sequence encoding a portion of mouse cyclin B2;
GenBank accession no.: X66032.

SEQ lD N0:68 shows the nucleic acid sequence encoding a portion of mouse cyclin B2;
GenBank accession no.: X66032.
SEQ ID N0:69 shows the nucleic acid sequence encoding a portion of mouse pancreatic beta-cell kinesin heavy chain; GenBank accession no. 086090.
SEQ ID N0:70 shows the nucleic acid sequence encoding a portion of mouse pancreatic beta-cell kinesin heavy chain; GenBank accession no.: 086090.
SEQ ID N0:71 shows the nucleic acid sequence encoding a portion of mouse syntaxin4;
GenBank accession no.:U76832.
SEQ ID N0:72 shows the nucleic acid sequence encoding a portion of mouse syntaxin4;
GenBank accession no.:U76832.
SEQ ID N0:73 shows the nucleic acEd sequence encoding a portion of mouse syntaxin4;
GenBank accession no.:U76832.
SEQ ID N0:74 shows thenucleicacidsequenceencodinga portion ofmouse stearoyl-CoA
desaturase (SCD2); GenBank accession no.: M26270.
SEQ ID N0:75 shows the nucleic acid sequence encoding a portion of mouse spermidine aminopropyltransferase (Mspmsy); GenBank accession no.: AF031486.
SEQ ID N0:76 shows the nucleic acid sequence encoding a portion of mouse prothymosin alpha; GenBank accession no.: X56135.
SEQ ID N0:77 shows the nucleic acid sequence encoding a portion of mouse protein cofactor; GenBank accession no.: 074079.
SEo ID N0:78 shows the nucleic acid sequence encoding a portion of mouse outer dense fiber protein 2 (Odf2); GenBank accession no.: AF000968.
SEQ ID N0:79 shows the nucleic acid sequence encoding a portion of mouse protein expressed in E12 brain (clone C2); GenBank accession no.: X83589.

_19_ SED ID N0:80 shows the nucleic acid sequence encoding a portion of mouse hnRNP
K
homologue;GenBank accession na.: L29769.
SEQ ID N0:81 shows the nucleic acid sequence encoding a portion of mouse NRF1 (NFE2-related factor 1 ); GenBank accession no.: X78709.
SEQ ID N0:82 shows the nucleic acid sequenceencodinga portion of mouse RNA-binding protein; GenBank accession no.: L~7076.
SEo iD N0:83 shows the nucleic acid sequence encoding a portion of mouse dynactinl;
GenBank accession no.: 060312.
SEQ ID N0:84 shows the nucleic acid sequence encoding a portion of mouse hormone-sensitive lipase; GenBank accession no.: 008188.
SED ID N0:85 shows the nucleic acid sequence encoding a portion of mouse mtprda (human TPRD homologue); GenBank accession no.: AB008516.
SEQ ID N0:86 shows the nucleic acid sequence encoding Exo24, having some similarity with GenBank accession no.: AA268561.
SEQ ID N0:87 shows the nucleic acid sequence encoding Exo25, having some similarity with GenBank accession no.: AA097037.
SEQ ID N0:88 shows the nucleic acid sequence encoding Exo26, having some similarity with GenBank accession no.: AA097037.
SEQ ID N0:89 shows the nucleic acid sequence encoding Exo27, having some similarity with GenBank accession no.: AA259474.
SEQ ID N0:90 shows the nucleic acid sequence encoding Exo28, having some similarity with GenBank accession no.: AA555886.
SEQ ID N0:91 shows the nucleic acid sequence encoding Exo29, having some similarity with GenBank accession no.: AA770839.

SEQ ID N0:92 shows the nucleic acid sequence encoding Exo30, having some similarity with GenBank accession no.: AA770839.
SEQ ID N0:93 shows the nucleic acid sequence encoding Exo3l, having some similarity with GenBank accession no.: AA415504.
SEQ ID N0:94 shows the nucleic acid sequence encoding Exo32, having some similarity with GenBank accession no.: AA415504.
SEQ ID N0:95 shows the nucleic acid sequence encoding Exo33, having some similarity with GenBank accession no.: AA415504.
SEQ ID N0:96 shows the nucleic acid sequence encoding Exo34, having some similarity with Gent3ank accession no.: AA415504.
SEQ ID N0:97 shows the nucleic acid sequence encoding Exo35, having some similarity with GenBank accession no.: AA415504.
SEQ ID N0:98 shows the nucleic acid sequence encoding Exo36, having some similarity with GenBank accession no.: AA415504.
SEQ ID N0:99 shows the nucleic acid sequence encoding Exo37, having some similarity with GenBank accession no.: AA415504, SEQ ID N0:100 shows the nucleic acid sequence encoding Exo38, having some similarity with GenBank accession no.: AA415504.
SEA ID N0:101 shows the nucleic acid sequence encoding Exo39, having some similarity with GenBank accession no.: AA415504.
SEG7 ID N0:102 shows the nucleic acid sequence encoding Exo40, having some similarity with GenBank accession no.: AA415504.
SEQ ID N0:103 shows the nucleic acid sequence encoding Exo41, having some similarity with GenBank accession no.: AA172925.

SEQ ID N0:104 shows the nucleic acid sequence encoding Exo42, having some similarity with GenBank accession no.: AA288130.
SEQ ID N0:105 shows the nucleic acid sequence encoding Exo43, having some similarity with GenBank accession no.: AI181639.
SEQ ID N0:106 shows the nucleic acid sequence encoding Exo44, having some similarity with GenBank accession no.: AA184709.
SEO ID N0:107 shows the nucleicacid sequence encoding Exa45, having some similarity with GenBank accession no.: AA266406.
SEQ ID N0:108 shows the nucleic acid sequence encoding Exo46, having some similarity with GenBank accession no.: AA563185.
SED ID N0:109 shows the nucleic acid sequence encoding Exo47, having some similarity with GenBank accession no.: AA519170.
SEQ ID N0:110 shows the nucleic acid sequence encoding Exo48, having some similarity with GenBank accession no.: AA519170.
SEQ I D N0:111 shows the nucleic acid sequence encoding Exo49, having some similarity with yeast ORMI, GenBank accession no.: AA175198.
SEQ ID N0:112 shows the nucleic acid sequence encoding Exo50, having same similarity with rat mt-GrpE no.1 precursor, GenBank accession no.: AA06D861.
SEQ ID N0:113 shows the nucleic acid sequence encoding Exo51, having some similarity with human CENP-F kinetochore protein, GenBank accession no.: A1034171.
SEQ ID N0:114 shows the nucleic acid sequence encoding Exo52, having some similarity with human arfaptin 2 (putative target of ADP-ribosylation factor), GenBank accession no.:AA543955.
SEQ ID N0:115 shows the nucleic acid sequence encoding Exo53, having some similarity with human brain and reproductive organ-expressed protein (BRE); GenBank accession no.: AA200608.

SEQ ID N0:116 shows the nucleic acid sequence encoding Exo54, having some similarity with human brain and reproductive organ-expressed protein (BRE); GenBank accession no.: AA200608.
SEQ ID N0:117 shows the nucleic acid sequence encoding Exo55, having some similarity with human cell cycle progression 2 protein (CPR2); GenBank accession no.:
W87077.
SEQ ID N0:118 shows the nucleic acid sequence encoding Exo56, having some similarity with humanspliceosomeassociatedprotein(SAP145); GenBankaccession no.:
A1119401.
SEQ ID N0:119 shows khe nucleic acid sequence encoding Exo57, having some similarity with mesocricetus auratus stearyl-CoA desaturase (FAR-17c); GenBank accession no.:
AA387696.
SEQ ID N0:120 shows the nucleic acid sequence encoding Exo58, having some similarity with rat inositol trisphosphate receptor subtype 3 (IP3R-3); GenBank accession no.:
AA823026.
SEQ ID N0:121 shows the nucleic acid sequence encoding Exo59, having some similarity with L-Asparaginase; GenBank accession no.: AI118730.
SEQ ID N0:122 shows the nucleic acid sequence encoding Exo60, having some similarity with L-Asparaginase; GenBank accession no.: AI118730.
SEQ ID N0:123 shows the nucleic acid sequence encoding Exo61, having some similarity with human RB-binding protein 2 (RBBP-2); GenBank accession no.: AA755315.
SEQ ID N0:124 shows the nucleic acid sequence encoding Exo62, having some similarity with human secreted apoptosis related protein 3 (SARP3); GenBank accession no.:
AU018890.
SEQ ID N0:125 shows the nucleic acid sequence encoding Exo63, having some similarity with myosin heavy chain; GenBank accession no.: AA237764.
SEQ ID N0:126 shows the nucleic acid sequence encoding Exo64, having some similarity with myosin heavy chain; GenBank accession no.: AA237764.

SEQ ID N0:127 shows the nucleic acid sequence encoding Exo65, having some similarity with myosin heavy chain; Gen6ank accession no.: AA237764.
SEa ID N0:128 shows the nucleic acid sequence encoding Exo66, having some similarity With myosin heavy chain; GenBank accession no.: AA237764.
SEQ ID NO:129 shows the nucleicacid sequence encoding Exo67, having some similarity with rat tomosyn; GenBank accession no.: AA437465.
SEQ ID N0:130 shows the nucleic acid sequence encoding Exo68, having some similarity with rat tomosyn; GenBank accession no.: AA437465.
SEQ ID N0:131 shows the nucleic acid sequence encoding Exo69, having some similarity with rat tomosyn; GenBank accession no.: AA437465.
SEQ ID N0:132 shows the nucleic acid sequence encoding Exo70, having some similarity with rat tomosyn; GenBank accession no.: AA437465.
SEQ ID N0:133 shows the nucleic acid sequence encoding Exo71, having some similarity with human mcag29 CTG repeat region; GenBank accession no.: AA089340.
SEO ID N0:134 shows the nucleic acid sequence encoding Exo72, having some similarity with rat G protein gamma-5 subunit; GenBank accession na.: AA021879.
SEQ ID N0:135 shows the nucleic acid sequence encoding Exo73, having some similarity with rat G protein gamma-5 subunit; GenBank accession no.: AA021879.
SEQ ID N0:136 shows the nucleic acid sequence encoding Exo74, having some similarity with rat G protein gamma-5 subunit; GenBank accession no.: AA021879.
5EG1 ID N0:137 shows the nucleic acid sequence encoding Exo75, having some similarity with rat G protein gamma-5 subunit; GenBank accession no.: AA021879.
SEQ ID N0:138 shows the nucleic acid sequence encoding Exo76, having some similarity with rat G protein gamma-5 subunit; GenBank accession no.: AA021879.

SEQ ID N0:139 shows the nucleic acid sequence encoding Exo77, having some similarity with rat G protein gamma-5 subunit; GenBank accession no.: AA021879.
SEQ ID N0:140 shows the nucleic acid sequence encoding Exo78.
SEQ ID N0:141 shows the nucleic acid sequence encoding Exo79.
SEQ ID N0:142 shows the nucleic acid sequence encoding Exo80.
SEQ ID N0:143 shows the nucleic acid sequence encoding Exo81.
SEQ ID N0:144 shows the nucleic acid sequence encoding a portion of human HLA-B-associated transcript 3; GenBank accession no.: M33519.
SEA ID N0:145 shows the nucleic acid sequence encoding a portion of human Hlh-B-associated transcript 3; GenBank accession no.: M33519.
SEQ ID N0:146 shows the nucleic acid sequence encoding a portion of human T
cell leukemia/lymphoma 1; GenBank accession no.: X82240.
SEA ID N0:147 shows the nucleic acid sequence encoding Exo82.
SEQ ID N0:148 shows the nucleic acid sequence encoding Exo83; may have some homology with rat rabin3, SEO ID N0:149 shows the nucleic acid sequence encoding at least a portion of human KIAA0665; GenBank accession no.: AB014565.
SEQ ID N0:150 shows the nucleic acid sequence encoding Exo84 which may have some similarity with Robin 3, GenBank accession no.: AA846576.
SEQ ID N0:151 shows the nucleic acid sequence encoding Exo85 which may have some similarity with Robin 3, GenBank accession no.: AA846576.
SEA ID N0:152 shows the nucleic acid sequence encoding Exo86 which may have some similarity with human KIAA0665, GenBank accession no.: AA757034.

SEQ ID N0:153 shows the nucleic acid sequence encoding a portion of mouse protein cofactor; GenBank accession no.: 074079.
SEQ ID N0:154 shows the nucleic acid sequence encoding a portion of mouse protein cofactor; GenBank accession no.: 074079.
SEQ ID N0:155 shows the nucleic acid sequence encoding Exo87, may have some similarity with calcium-dependent protein kinase, Genbank accession no.:
AA770736.
SEQ ID N0:156 shows the nucleic acid sequence encoding Exo88, may have some similarity with calcium-dependent protein kinase, Genbank accession no.:
AA770736.
SEQ ID N0:157 shows the nucleic acid sequence encoding Exo89, may have some similarity with calcium-dependent protein kinase, Genbank accession no.:
AA770736.
SEQ ID N0:158 shows the nucleic acid sequence encoding Exo90, may have some similarity with calcium-dependent protein kinase, Genbank accession no.:
AA770736.
SEQ ID N0:159 shows the nucleic acid sequence encoding Exo91, may have some similarity with human mcag29 CTG repeat region, Genbank accession no.:
AA473325.
SEA ID N0:160 shows the nucleic acid sequence encoding Exo92, may have some similarity with human mcag29 CTG repeat region, Genbank accession no.:
AA473325.
SEQ ID N0:161 shows the nucleic acid sequence encoding Exo93, may have some similarity with Genbank accession no.: AA138122.
SEQ ID N0:162 shows the nucleic acid sequence encoding Exo94, may have some similarity with Genbank accession na.: AA138122.
SEQ 1D N0:163 shows the nucleic acid sequence encoding Exo95, may have some similarity with Genbank accession no.; AA138122.
SEQ ID N0:164 shows the nucleic acid sequence encoding Exo96, may have some similarity with Genbank accession no.: AA138122.

SEQ ID N0:165 shows the nucleic acid sequence encoding Exo97, may have some similarity with Genbank accession no.: AA138122, SEA ID N0:166 shows the nucleic acid sequence encoding Exo98, may have some similarity with Genbank accession no.: AA060976.
SEQ ID N0:167 shows the nucleic acid sequence encoding Exo99, may have some similarity with Genbank accession no.: AA277208.
SE4 ID N0:168 shows the nucleic acid sequence encoding Exo100, may have some similarity with Genbank accession no.: AA277208.
SEQ ID N0:169 shows the nucleic acid sequence encoding Exo101, may have some similarity with Genbank accession no.: AA467477.
SEQ ID N0:170 shows the nucleic acid sequence encoding Exo102, may have some similarity with Genbank accession no.: AA467477.
SEQ ID N0:171 shows the nucleic acid sequence encoding Exo103, may have some similarity with Genbank accession no.: AA833213.
SEQ 1D N0:172 shows the nucleic acid sequence encoding a portion of Rab2;
GenBank accession no.: X95403.
SEA ID N0:173 shows the nucleic acid sequence encoding a portion of Rab2;
GenBank accession no.: X95403.
SEQ ID N0:174 shows the nucleic arid sequence encoding a portion of Rab2;
GenBank accession no.: X95403.
SEQ ID N0:175 shows the nucleic acid sequence encoding a portion of Rab2;
GenBank accession no.: X95403.
SEQ ID N0:176 shows the nucleic acid sequence encoding a portion of Rab2;
GenBank accession no.: X95403.

SEQ ID N0:177 shows the nucleic acid sequence encoding a portion of Rab2;
GenBank accession no.: X95403.
SEQ ID N0:178 shows the nucleic acid sequence encoding a portion of Rab2;
GenBank accession no.: X95403.
SEA ID N0:179 shows the nucleic acid sequence encoding a portion of Rab2;
GenBank accession no.: X95403.
SEQ ID N0:180 shows the nucleic acid sequence encoding a portion of Rab2;
GenBank accession no.: X95403.
SECT ID N0:1 B1 shows the nucleic acid sequence encoding a portion of Rab2;
GenBank accession no.: X95403.
SEQ ID N0:182 shows the nucleic acid sequence encoding a portion of Rab2;
GenBank accession no.: X95403.
SEQ ID N0:183 shows the nucleic acid sequence encoding a portion of Rab2;
GenBank accession no.: X95403.
SEQ ID N0:184 shows the nucleic acid sequence encoding a portion of Rab2;
GenBank accession no.: X95403.
SED ID N0:185 shows the nucleic acid sequence encoding a portion of Rab2;
GenBank accession no.: X95403.
SEC7 ID N0:186 shows the nucleic acid sequence encoding a portion of Rab2;
GenBank accession no.: X95403.
SEQ ID N0:187 shows the nucleic acid sequence encoding a portion of Rab2;
GenBank accession no.: X95403.
SEQ ID N0:188 shows the nucleic acid sequence encoding a portion of Rab2;
GenBank accession no.: X95403.

_28_ SEQ ID N0:189 shows the nucleic acid sequence encoding a portion of Rab2;
GenBank accession no.: X95403.
SEQ ID N0:190 shows the nucleic acid sequence encoding a portion of RabSC;
GenBank accession no.: AA230407.
SEQ ID N0:191 shows the nucleic acid sequence encoding a portion of Rab5C;
GenBank accession no.: AA230407.
SEQ ID N0:192 shows the nucleic acid sequence encoding a porticn of RabSC;
GenBank accession no.: AA230407.
SEQ ID N0:193 shows the nucleic acid sequence encoding Exo104.
SEO ID N0:194 shows the nucleic acid sequence encoding Exo105, a human gene similar to mouse testis-specific protein PBS13; may have some similarity to GenBank Accession no,: AA184365.
SEQ ID N0:195 shows the nucleic acid sequence encoding Exo106; may have some similarity to GenBank Accession no.: AA504490.
SEQ ID N0:196 shows the nucleic acid sequence encoding Exo107; may have some similarity to GenBank Accession no.: AA504490.
SEQ ID N0:197 shows the nucleic acid sequence encoding Exo108; may have some similarity to GenBank Accession no.: AI181750.
SEO ID N0:198 shows the nucleic acid sequence encoding Exo109; may have some similarity to GenBank Accession no.: AI181750.
SEQ ID N0:199 shows the nucleic acid sequence encoding Exo110; may have some similarity to GenBank Accession no.: AU043111.
SEQ ID N0:200 shows the nucleic acid sequence encoding Exo111, a human gene similar to rat alpha-soluble NSF attachment protein (SNAP) _29_ SEQ ID N0:201 shows the nucleic acid sequence encoding Exo112. shows the nucleic acid sequence encoding Exo105, a human gene similar to mouse testis-specific protein PBS13; may have some similarity to GenBank Accession no.: AA184365.
SEQ ID N0:202 shows the nucleic acid sequence encoding Exol13 which may be similar to a mouse zinc finger protein.
SEQ ID N0:203 shows the nucleic acid sequence encoding Exo114 which may be similar to a mouse zinc finger protein.
SEQ ID N0:204 shows the nucleic acid sequence encoding Exo115 which may be similar to chicken c-hairy 1; may have some similarity to GenBank Accession no.:
AA116067.
SEQ ID N0:205 shows the nucleic acid sequence encoding Exo116 which may be similar to chicken c-hairy 1; may have some similarity to Genl3ank Accession no.:
AA116067.
SEQ ID N0:206 shows the nucleic acid sequence encoding a portion of mouse syntaxin4;
GenBank accession no.: U76832.
SEQ ID N0:207 shows the nucleic acid sequence encoding a portion of mouse interleukin (IL) -3 receptor; GenBank accession no.: M29855.
SEQ ID N0:208 shows the nucleic acid sequence encoding a portion of mouse interleukin (IL) -3 receptor; GenBank accession no.: M29855.
SEQ ID N0:209 shows the nucleic acid sequence encoding a portion of mouse low density lipoprotein (LDL) receptor-related protein; GenBank accession no.: AF074265.
SEQ ID N0:210 shows the nucleic acid sequence encoding Exo117, similar to a human ANF126 zinc protein.
SEQ ID N0:211 shows the nucleic acid sequence encoding Exo118, similar to a rat isoprenylated 67 kDa protein.
As indicated above, Exo3-Exo118 are novel. The Exo proteinsencodedby SEQ I D
NOS:1-5i (odd numbers) and Sequence ID NOS:53-211 are each novel in the aspect that they are shown hereinto bind to an exocytosisorvesiculartransport protein, or fragment thereof, for the first time. in preferred embodiments, the proteins encoded by SEO ID
NOS:1-51 (odd numbers) bind to GS27; the proteins encoded by SEQ ID N0:53 bind to rab7;
the proteins encoded by SEQ ID NOS:54-64 bind to rab9; the proteins encoded by SEQ
ID
NOS:65-143bind to snap-23; the proteins encoded by SEO ID NOS:144-148bind to rab3a;
the proteins encoded by SEQ (D NOS:149-152 bind to rab11; the proteinsencoded by SEQ
I D NOS:153-171 bind tv rab3d; the proteins encoded by SEQ ID NOS:172-193 bind to rab5;
the proteins encoded by SEQ ID NOS:194-201 bind to alpha-snap; the proteins encoded by SE(~ ID NOS:202-205 bind to uncl8-1; and, the proteins encoded by SEQ ID
NOS:206-211 bind to vamp3.
In a preferred embodiment, a protein is a "Exo protein" if the overall sequence identity of the protein sequence to any one of the amino acid sequences encoded by SEQ ID
NOS:1-51, odd numbers, and SEQ ID NOS:53-211, preferably those sequences encoding Exo3-118, is preferably greater than about 75%, more preferably greater than about 80%, even more preferably greater than about 85% and most preferably greater than 90%.
In some embodiments the sequence identity will be as high as about 93 to 95 or 98%. As is known in the art, a number of different programs can be used to identify whether a nucleic acid has sequence identity or similarity to a known gene or expression sequence tag (EST).
Sequence identitywill be determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith & Waterman, Adv. Appl.
Math.2:482(1981),bythesequenceidentityalignmentalgorithmofNeedleman&Wunsch, J. Mol. Biool. 48:443 ( 1970), by the search for similarity method of Pearson & Lipman, PNAS
U SA 85:2444 ( 1988), by computerized irnplementationsof these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Drive, Madison, WI), the Best Fit sequence program described by Devereux et al., NucJ. Acid Res. 92:387-395 (1984), preferably using the default settings, or by inspection. Preferably, percent identity is calculated by FastDB based upon the following parameters: mismatch penalty of 1; gap penalty of 1; gap size penalty of 0.33;
and joining penalty of 30, "Current Methods in Sequence Comparison and Analysis,"
MacromoleculeSequencingand Synthesis, Selected Methods and Applications, pp (1988), Alan R. Liss, Inc.
AnexampleofausefulalgorithmisPILEUP, PILEUPcreatesamultiplesequencealignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment.
PILEUP uses a simpliflcationofthe progressivealignmentmethod of Feng & Doolittle, J. Mol.
Evol. 35:351-360 (1987); the method is similartothatdescribed by Higgins & Sharp CABIOS
5:151-153 WO 00/43419 PCTlUS00/01431 (1989). Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.
Another example of a useful algorithm is the BLAST algorithm, described in Altschul et al., J. Mol. Biol. 295, 403-410, (1990) and Karlin et al., PNAS USA 90:5873-5787 (1993). A
particularly useful BLAST program is the WU-BLAST-2 program which was obtained from Altschul et al., Methods in Enzvmolooy, 266: 460-480 (1996);
http:/lblast.wustlledulblast!
README.html]. WU-BLAST-2 uses several search parameters, most ofwhich are set to the default values. The adjustable parameters are set with the following values: overlap span =1, overlap fraction = 0.125, word threshold (T) = 11. The HSP S and HSP

parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity. A °ro amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "longer' sequence in the aligned region. The "longer" sequence is the one having the most actual residues in the aligned region (gaps introduced by WU-Blast-2 to maximi2e the alignment score are ignored).
In a similar manner, "percent (%) nucleic acid sequence identity" with respect to the coding sequence of the polypeptides identified herein is defined as the percentage of nucleotide residuesinacandidatesequencethatareidenticalwiththenucleotideresiduesinthecadin g sequence of the Exo protein. A preferred method utilizes the BLASTN module of WU-BLAST-2 set to the default parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively.
The alignment may include the introduction of gaps in the sequences to be aligned. In addition, for sequences which contain either more or fewer amino acids than the protein encoded by the sequences in the Sequence Listing, it is understood that the percentage of sequence identity will be determined based on the number of identical amino acids in relation to the total number of amino acids. Thus, for example, sequence identity of sequences shorter than that shown in the Sequence Listing, as discussed below, will be determined using the number of amino acids in the shorter sequence.
As an example, SED ID NOS:1-51 (odd numbers)and SEQ ID NOS:53-211 were identified as follows. A basic Blast search has been pertormed using program "Blastn" and database "nr". Blastn is a NCBI BLAST family of program used to compared a nucleotide query WO 00/43419 PCTlUS00/01431 sequence against a nucleotide sequence database, and nr is a nucleotide sequence database that includes all non-redundant GenBank CDS
translations+PDB+SwissProt+PIR+PRF. Two numbers, Score (bits) and E values, will be returned after search querry is submitted. In general sequences considered known had a Score > 100 and E < D.001. Using the same Blast search, the nucleic acid sequences encoding Exo3-118 had a Score < 100 or E > 0.001. These nucleic acid sequences encoding Exo3-118 were then further searched using program "Blastn" and database "dbest". Thedbestdatabaseisanucleotidesequencedatabasethatincludesnon-redundant Database ofGenBank+EMBL+DDBJESTDivisions. Usingthiscriteria,someofthenucleic acid sequences had a Score > 100 and E < 0.001, thus. these sequences are considered novel, yet have "some similarity"too known sequenceas indicated by the accession number provided. The sequences ofthe accession numbers provided herein are readily available to the skilled artisan.
As will be appreciated by those in the art, the nucleic acid sequences of the invention can be used to generate protein sequences. There are a variety of ways to do this, including cloning the entire gene and verifying its frame and amino acid sequence, or by comparing it to known sequences to search for homology to provide a frame, assuming the novel Exo protein has homology to some protein in the database being used. Generally, the nucleic acid sequences are input into a program that will search all three frames for homology.
This is done in a preferred embodiment using the following NCB/ Advanced BLAST
parameters. The program is blastx or blastn. The database is nr. The input data is as "Sequence in FASTA format". The organism list is "none". The "expect" is 10;
the filter is default. The "descriptions" is 500, the "alignments" is 500, and the "alignment view" is pairwise. The "Query Genetic Codes" is standard (1). The matrix is BLOSUM62;
gap existence cost is 11, per residue gap cost is 1; and the lambda ratio is .85 default. This results in the generation of a putative protein sequence. While this program can be used togeneratea preferredproteinsequence, it is understoodthatthe presentinventionprovides polypeptides encoded by each of the three frames of each nucleic acid provided herein.
Thus, when a protein encoded by the nucleic acid herein is described, the skilled artisan understands that the protein begins with the first amino acid encoded by the first colon of the coding region, which is not necessarily the first nucleotide in the sequence listing.
As will be appreciated by those skilled in the art, the sequences of the present invention may contain sequencing errors. That is, there may be incorrect nucleosides, frameshifts, unknown nucleosides,orothertypesofsequencingerrorsinanyofthe sequences;
however, the correct sequences will fall within the homology and stringency definitions herein. In WO 00!43419 PCT/US00/01431 addition, as will be appreciated by those in the art, in general, the first 200 bases or so of sequence contains the fewest errors. In a preferred embodiment, the Exo proteins are encoded by a nucleic acid comprising the first 100 nucleotides of the sequences set forth in the Sequence Listing and bind to an exocytosis orvesicu lartransport proteinorfragment thereof.
Exo proteins of the present invention may be shorter or longer than the amino acid sequences encoded by the nucleic acids shown in the Sequence Listing. Thus, in one embodiment, Exo proteins can be portions or fragments of the amino acid sequences encoded by the nucleic acid sequences provided herein. In one embodiment herein, fragmentsofExoproteinsareconsideredExoproteinsifa)theyshareatleastoneantigen~
epitope; b) have at least the indicated sequence identity; c) and preferably have Exo biologicalactivity, including binding to an exocytosis or vesicular transport protein. I n some cases, where the sequence is used diagnostically, that is, when the presence or absence of Exo protein nucleic acid is determined, only the indicated sequence identity is required.
The nucleic acidsofthepresentinventionmayalsobeshorterorlongerthanthesequences in the Sequence Listing.
Thenucleicacidfragmentsincludeanyportionofthenucleicacids provided herein whichhavea sequencenotexactly previously identified; fragments having sequences with the indicated sequence identity to that portion not previously identified are provided in an embodiment herein.
In addition, as is more fully outlined below, Exo proteins can be made that are longer than those depicted in the Sequence Listings; for example, by the addition of epitope or purification tags, the addition of other fusion sequences, or the elucidation of additional coding and non-coding sequences. As described below, the fusion of an Exo peptide to a fluorescent peptide, such as Green filuorescent Peptide (GFP), is particularly preferred.
Exo proteins may also be identified as encoded by Exo nucleic acids which hybridize to any one of the sequences depicted in SEQ ID NOS:1-51, odd numbers, and SEQ ID
NOS:53-211, preferably those encoding Exo3-118. Hybridization conditions are further described below.
In a preferred embodiment, when an Exo protein is to be used to generate antibodies, an Exo protein must share at least one epitope or determinant with the full length protein. By "epitope" or "determinant" herein is meant a portion of a protein which will generate and/or bind an antibody. Thus, in most instances, antibodies made to a smaller Exo protein will be able to bind to the full length protein. In a preferred embodiment, the epitope is unique;

that is, antibodies generated to a unique epitope show little or no cross-reactivity. The term "antibody" includes antibody fragments, as are known in the art, including Fab Fab2, single chain antibodies (Fv for example;/, chimeric antibodies, etc., either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA
technologies.
In a preferred embodiment, the antibodies to Exo are capable of reducing or eliminating the biological function of Exo, as is described below. That is, the addition of anti-Exo antibodies(eitherpolyclonal or preferably monoclonal) to Exo (or cells containing Exo) may reduce or eliminate the Exo activity. Generally, at least a 25% decrease in activity is preferred, with at least about 50% being particularly preferred and about a 95-100%
decrease being especially preferred.
The Exo antibodies of the invention specifically bind to Exo proteins. In a preferred embodiment, the antibodies specifically bind to Exo proteins. By "specifically bind" herein is meant that the antibodies bind to the protein with a binding constant in the range of at least 10'°- 10'° M'', with a preferred range being 10'' - 10'9 M''. Antibodies are further described below.
In the case of the nucleic acid, the overall sequence identity of the nucleic acid sequence is commensurate with amino acid sequence identity but takes into account the degeneracy in the genetic code and codon bias of different organisms. Accordingly, the nucleic acid sequence identity may be either lower or higher than that of the protein sequence. Thus the sequence identity of the nucleic acid sequence as compared to the nucleic acid sequences ofthe Sequence Listing, is preferablygreaterthan 75%, more preferablygreater than about80%, particularly greaterthan about85% and most preferablygreaterthan 90%.
In some embodiments the sequence identity will be as high as about 93 to 95 or 98%.
In a preferred embodiment, an Exo nucleic acid encodes an Exo protein. As will be appreciated by those in the art, due to the degeneracy of the genetic code, an extremely large number of nucleic acids may be made, all of which encode the Exo proteins of the present invention. Thus, having identified a particular amino acid sequence, those skilled in the art could make any number of different nucleic acids, by simply modifying the sequence of one or more codons in a way which does not change the amino acid sequence of the Exo.

In one embodiment, the nucleic acid is determined through hybridization studies. Thus, for example, nucleic acids which hybridize under high stringency to the nucleic acid sequences shown in the sequence listing, or its complement is considered an Exo gene.
High stringency conditions are known in the art; see for example Maniatis et al., Molecular Cloning: A Laboratory Manual, 2d Edition,1989, and Short Protocols in Molecular Biology, ed. Ausubel, et al., both ofwhich are hereby incorporatedby reference.
Stringentconditions are sequence-dependentand will be differentin differentcircumstances.
Longersequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in Tijssen, Techniques in Biochemistry and Molecular Biology--Hybridization with Nucleic Acid Probes, "Overview of principles of hybridization and the strategy of nucleic acid assays" (1993). Generally, stringent conditions are selected to be about 5-10°C lower than the thermal melting point (T,") for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium) Stringent conditions will be those in which the salt concentration is less than about 1.0 sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g. 10 to 50 nucleotides) and at least about 60°C
for long probes (e.g. greater than a0 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide.
In another embodiment, less stringent hybridization conditions are used; for example, moderate or low stringency conditions may be used, as are known in the art;
see Maniatis and Ausubel, supra, and Tijssen, supra.
The Exo proteins and nucleic acids tit the present invention are preferably recombinant.
As used herein, "nucleic acid" may refer to either DNA or RNA, or molecules which contain both deoxy- and ribonucleotides. The nucleic acids include genomic DNA, cDNA
and oligonucleotides including sense and anti-sense nucleic acids. Such nucleic acids may also contain modifications in the ribose-phosphate backbone to increase stability and half life of such molecules in physiological environments.
The nucleicacid may be doublestranded,singlestranded, or contain portionsofbothdouble stranded or single stranded sequence. As will be appreciated by those in the art, the depiction of a single strand ("Watson°) also defines the sequence of the other strand ("Crick"); thus the sequences depicted in the Sequence Listingalso includethecomplement of the sequence. By the term "recombinant nucleic acid" herein is meant nucleic acid, originally formed in vitro, in general, by the manipulation of nucleic acid by endonucleases, in a form not normally found in nature. Thus an isolated Exo nucleic acid, in a linear form, or an expression vector formed in vitro by (igating DNA molecules that are not normally joined, are both considered recombinantforthe purposes ofthis invention. It is understood that once a recombinant nucleicacid is made and reintroduced into a host cell or organism, it will replicate non-recombinantly, i.e. using the in vivo cellular machinery of the host cell rather than in vitro manipulations; however, SUCK nucleic acids, once produced recombinantly, although subsequently replicated nan-recombinantly, are still considered recombinant for the purposes of the invention.
Similarly, a "recombinant protein" is a protein made using recombinant techniques, i.e.
through the expression of a recombinant nucleic acid as depicted above. A
recombinant protein is distinguished from naturally occurring protein by at least one or more characteristics. For example, the protein may be isolated or purified away from some or all of the proteins and compounds with which it is normally associated in its wild type host, and thus may be substantially pure. For example, an isolated protein is unaccompanied by at least some of the material with which it is normally associated in its natural state, preferably constituting at least about 0.5%, more preferably at least about 5%
by weight of the total protein in a given sample. A substantially pure protein comprises at least about 75% by weight of the total protein, with at least about 80°~ being preferred, and at least about 90% being particularly preferred. The definition includes the production of an Exo protein from one organism in a different organism or host cell. Alternatively, the protein may be made at a significantly higher concentration than is normally seen, through the use of a inducible promoter or high expression promoter, such that the protein is made at increased concentration levels. Alternatively, the protein may be in a form not normally found in nature, as in the addition of an epitope tag or amino acid substitutions, insertions and deletions, as discussed below.
Also included within the definition of Exo proteins of the present invention are amino acid sequence variants. These variants fall into one or more of three classes:
substitutional, insertional or deletional variants. 'These variants ordinarily are prepared by site speck mutagenesis of nucleotides in the DNA encoding an Exo protein, using cassette or PCR
mutagenesisorothertechniqueswell known in the art, to produceDNA
encodingthevariant, andthereafterexpressingtheDNAinrecombinantcellcultureasoutlinedabove. However, variant Exo protein fragments having up to about 100-150 residues may be prepared by in vitro synthesis using established techniques. Amino acid sequence variants are WO 00!43419 PCT/US00/01431 characterized by the predetermined nature of the variation, a feature that sets them apart from naturally occurring allelic ar interspecies variation of the Exo protein amino acid sequence. The variants typically exhibit the same qualitative biological activity as the naturally occurring analogue, although variants can also be selected which have modified characteristics as will be more fully outlined below.
While the site or region for introducing an amino acid sequence variation is predetermined, the mutation per se need not be predetermined. Eor example, in order to optimize the performance of a mutation at a given site, random mutagenesis may be conducted at the targetcodon or region and the expressedExovariantsscreenedforthe optimal combination of desired activity. Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are welt known, for example, M13 primer mutagenesis and PCR mutagenesis. Screening of the mutants is done using assays of Exo protein activities.
Amino acid substitutions are typically of single residues; insertions usually will be on the order of from about 7 to 20 amino acids, although considerably larger insertions may be tolerated. Deletions range from about 1 to about 20 residues, although in some cases deletions may be much larger.
Substitutions, deletions, insertions or any combination thereof may be used to arrive at a final derivative. Generally these changes are done on a few amino acids to minimize the alteration of the molecule. However, larger changes may be tolerated in certain circumstances. When small alterations in the characteristics of the Exo protein are desired, substitutions are generally made in accordance with the following chart:
Chart I
Original Residue Exemplary Substitutions Ala Ser Arg Lys Asn Gln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gin Ile Leu, Val Leu Ile, Val Lys Arch, Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr Ser Thr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu Substa ntialchanges in fu nction or immunological identity are made by se lectingsubstitutions that are less conservative than those shown in Chart I. For example, substitutions may be made which more significantly affect: the structure of the polypeptide backbone in the area of the alteration, for example the alpha-helical or beta-sheet structure;
the charge or 5 hydrophobicity of the molecule at the target site; or the bulk of the side chain. The substitutions which in general are expected to produce the greatest changes in the polypeptide's properties are those in which (a) a hydrophilic residue, e.g.
seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substituted for (or by) any other residue; (c) a residue 10 having an electrop4sitive side chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by) an electronegative residue, e.g. glutamyl or aspartyl; or {d) a residue having a bulky side chain, e.g. phenylalanine, is substituted for (or by) one not having a side chain, e.g. glycine.
The variants typically exhibit the same qualitative biological activity and will elicit the same immune response as the naturally-occurringanalogue, although variants also are selected to modify the characteristics ofthe Exo proteins as needed. Alternatively, the variant may be designed such that the biological activity of the Exo protein is altered.
For example, glycosylationsitesmaybealteredorremoved.
Similarly,mutationswithinthekinasedomain and/or the cell death domain may be made.
Covalent modifications of Exo polypeptides are included within the scope of this invention.
One type of covalent modification includes reacting targeted aminoacid residues of an Exo polypeptide with an organic derivatizing agent that is capable of reacting with selected side chainsorthe N-orC-terminalresiduesofan Exo polypeptide. Derivatizationwith bifunctional agents is useful, for instance, for crosslinking Exo to a water-insoluble support matrix or surface for use in the method for purifying anti-Exo antibodies or screening assays, as is WO 00/43419 PCTIUS00l01431 more fullydescribedbelow. Commonly usedcrosslinkingagentsinclude,e.g.,1,1-bis(diazo-acetyl)-2-phenylethane, glutaratdehyde, N-hydroxysuccinimideesters,forexample,esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N-maleimido-1,8-octaneand agents such as methyl-3-((p-azidophenyl)dithio)propioimidate.
Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of hydroxyl groups ofseryl or threonyl residues, methylation ofthe "-amino groups of lysine, arginine, and histidine side chains [T.E. Creighton, Proteins:
Structureand MolecularProoerties, W. H. Freeman& Co., San Francisco, pp. 79-86 (1983)), acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group.
Another type of covalent mod~cation of the Exo polypeptide included within the scope of thisinventioncomprisesalteringthenativeglycosylationpatternofthepolypeptide."Al tering the native glycosylation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence Exo polypeptide, andJor adding one or more glycosylation sites that are not present in the native sequence Exo polypeptide.
Addition of glycosylation sites to Exa polypeptides may be accomplished by altering the amino acid sequence thereof. The alteration may be made, for example, by the addition of, or substitution by, one or more serine orthreonine residues to the native sequence Exo polypeptide(for0-linkedglycosylationsites).
TheExoaminoacidsequencemayoptionally be altered through changes at the DPJA level, particularly by mutating the DNA
encoding the Exo polypeptide at preselected bases such that codons aregeneratedthatwill translate into the desired amino acids.
Another means of increasing the number of carbohydrate moieties on the Exo polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide. Such methods are described in the art, e.g., in WO 87105330 published 11 September 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).
Removal of carbohydrate moieties present on the Exo polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residuesthatserveastargetsforglycosylation.
Chemicaideglycosylationtechniques are known in the art and described, for instance, by Ha'~cimuddin, et al., Arch. Biochem.
Bioehvs., 25:52 (1987) and by Edge et al., Anal. Biochem., 118:131 (1981 ).
Enzymatic wo ooi434>t9 rcrnrsooro>ta3i cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo-and exo-glycosidases as described by Thotakura et al., Meth. Enz~, 138:350 { 1987).
Another type of covalent modification of Exo comprises linking the Exo polypeptide to one ofavarietyofnonproteinaceouspolymers,e.g.,polyethyleneglycol,polypropyleneglyco l, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835;

4,496,689;
4,301,144; 4,670,417; 4,791,192 or 4,179,337.
Exo polypeptides of the present invention may also be modified in a way to form chimeric molecules comprising an Exo polypeptide fused to another, heterologous polypeptide or amino acid sequence. In one embodiment, such a chimeric molecule comprises a fusion ofan Exo polypeptidewith a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind. Theepitopetagisgenerallyplacedattheamino-orcarboxyl-terminus of the Exo polypeptide. ~1'he presence of such epitope-tagged forms of an Exo polypeptide can be detected using an antibody against the tag polypeptide.
Also, provis'ron of the epitope tag enables the Exo palypeptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag.
In an alternative embodiment, the chimeric molecule may comprise a fusion of an Exo polypeptide with an immunoglobulin or a particular region of an immunoglobulin. For a bivalent form of the chimeric malecuie, such a fusion could be to the Fc region of an IgG
molecule as discussed further below, Varioustagpolypeptidesandtheirrespectiveantibodiesareweilknownintheart.
Examples include poly-histidine (poly-his) or poly-histidine-glycine {poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al., Molecular and Cellular Biolo4y, 5:3610-3618 (1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein En ineering, 3(8):547-553 {1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al., BioTechnolo4y, 6:1204-1210 {1988)]; the KT3 epitopepeptide[Martin etal., Science, 255:192-194 (1992j];
tubulin epitope peptide [Skinner et al., J. Biol. Chem., 2_øfi:15163-15166 (1991 )]; and the T7 gene 10 proteinpeptidetag [Lutz-Freyermuthetal., Proc. Natl. Acad. Sci.
USA, 87;6393-6397 (1990)].
In an embodiment herein, Exo proteins of the Exo family and Exo proteins from other organisms are cloned and expressed as outlined below. Thus, probe or degenerate polymerase chain reaction (PCR) primer sequences may be used to find other related Exo proteins from humans or other organisms. As will be appreciated by those in the art, particularly useful probe and/or PCR primer sequences include the unique areas of the Exo nucleic acid sequence. As is generally known in the art, preferred PCR
primers are from about 15 to about 35 nucleotides in length, with from about 20 to about 30 being preferred, and may contain inosine as needed. The conditians for the PCR
reaction are well known in theart. It is therefore also understood that provided along with the sequences in the sequences listed herein are portions of those sequences, wherein unique portions of 15 nucleotides or mare are particularly preferred. The skilled artisan can routinely synthesize or cut a nucleotide sequence to the desired length.
Once the Exo nucleic acid is identified, it can be cloned and, if necessary, its constituent parts recombinedtoform the entire E.xo nucleic acid. Once isolated from its natural source, e.g., contained within a plasmid or ether vector or excised therefrom as a linear nucleic acid segment, the recombinant Exo nucleic acid can be further-used as a probe to identify and isolate other Exo nucleic acids.. It can also be used as a "precursor"
nucleic acid to make modified or variant Exo nucleic acids and proteins. The skilled artisan understands that wherein two or more nucleic acids overlap, the overlapping portions) of one of the overlapping nucleic acids can be omitted and the nucleic acids combined for example, by ligation, to form a longer linear Exo nucleic acid so as to, for example, encode the foil length or mature peptide. The same applies to the amino acid sequences of Exo polypeptides in that they can be combined so as to form one contiguous peptide.
Using the nucleic acids of the present invention which encode an Exo protein, a variety of expression vectors are made. The expression vectors may be either self-replicating extrachromosomalvectorsorvectorswhichintegrateintoahostgenome. Generally, these expressionvectorsincludetranscriptionalandtranslationalregulatorynucleicacidope rably linked to the nucleic acid encoding tare Exo protein. The term "control sequences" refers to DNA sequences necessary for khe expression of an aperably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleicacidsequence. For example,DNAforapresequenceorsecretoryleaderisoperably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretionofthe polypeptide;a promoterorenhanceris operablylinked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation atconvenient restriction sites. Ifsuch sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice. The transcriptional and translational regulatory nucleic acid will generally be appropriate to the host cell used to express the Exo protein; for example, transcriptional and translational regulatory nucleic acid sequences from Bacillus are preferably used to express the Exo protein in Bacillus. Numerous types of appropriate expression vectors, and suitable regulatory sequences are known in the art for a variety of host cells.
In general, the transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhanceroractivatorsequences.
In a preferredembodiment,the regulatory sequences include a promoterand transcriptional start and stop sequences.
Promoter sequences encode either constitutive or inducible promoters. The promoters may be either naturally occurring promoters or hybrid promoters. Hybrid promoters, which combine elements of more than one promoter, are also known in the art, and are useful in the present invention.
In addition, the expression vector may comprise additional elements. For example, the expression vector may have two replication systems, thus allowing it to be maintained in twoorganisms,forexampleinmammalianorinsectcellsforexpressionandinaprocaryotic host for cloning and amplification. Furthermore, for integrating expression vectors, the expression vector contains at least one sequence homologous to the host cell genome, and preferably two homologous sequences which flank the expression construct.
The integrating vector may be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector. Constructs for integrating vectors are well known in the art.

In addition, in a preferred embodiment, the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selection genes are welt known in the art and will vary with the host cell used.
A preferred expression vector system is a retroviral vector system such as is generally described in PCTIUS97101019 and NCT/US97/01048, both of which are hereby expressly incorporated by reference.
Exo proteins of the present invention are produced by culturing a host cell transformed with an expressionvectorcontainingnucleicacidencodingan Exo protein, under the appropriate conditions to induce or cause expression of the Exo protein. The conditions appropriate for Exo protein expression will vary 'with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation.
For example, the use of constitutive promoters in the expression vector will require optimizingthe growth and proliferationofthe hostcell, while the use of an inducible promoter requires the appropriate growthconclitionsforinduction, In addition, insomeembodiments, the timing ofthe harvest is important For example, the baculoviral systems used in insect cell expression are lytic viruses, and thus harvest time selection can be crucial for product yield.
Appropriate host cells include yeast, bacteria, archebacteria, fungi, and insect and animal cells, including mammalian cells. Of particular interest are Drosophila melangastercells, 5accharomycescerevisiaeandotheryeasts,E.coli,8acillussubtilis,SF9ceIIs,C129cell s, 293 cells, Neurospora,BHK, CHO, COS, and HeLacells, fibroblasts, Schwanoma cell lines, immortalized mammalian myeloid and lymphoidcell lines, Jurkatcells, livercells, mammary cells, sperm, egg, adipocytes, granulocytes,adrenalchromaffin cells, mast cells, basophils, endocrine and exocrine cells, muscle cells, eosinophils and neuronal cells.
Inapreferredembodiment,theExoproteinsareexpressedinmammaliancells. Mammalian expressionsystemsare also known in the art, and include retroviralsystems. A
mammalian promoter is any DNA sequence capable of binding mammalian RNA polymerase and initiating the downstream (3') transcription of a coding sequencefor Exo protein into mRNA.
A promoter will have a transcription initiating region, which is usually placed proximal to the 5' end of the coding sequence, and a TATA box, using a located 25-30 base pairs upstream of the transcription initiation site. The TATA box is thought to direct RNA
polymerase II to begin RNA synthesis at the correct site. A mammalian promoter will also contain an upstream promoter element (enhancer element), typically located within 100 to 200 base pairs upstream of the TATA box. An upstream promoter element determines the rate at which transcription is initiated and can act in either orientation. Of particular use as mammalian promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter.
Typically, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. The 3' terminus of the mature mRNA is formed by site-specific post-translational cleavage and polyadenylation.
Examplesoftranscription terminator and polyadenlytion signalsincludethosederivedform SV40.
The methods of introducing exogenous nucleic acid into mammalian hosts, as well as other hosts, is well known in the art, and will vary with the host cell used.
Techniques include dextrin-mediated transfection, calcium phosphate precipitation, polybrene mediated transfection, protoplast fusion, electroporation, viral infection, encapsulation of the polynucleotide(s) in liposomes, and direct microinjection of the DNA into nuclei.
In a preferred embodiment, Exo proteins are expressed in bacterial systems.
Bacterial expression systems are well known in the art.
AsuitablebacterialpromoterisanynucleicacidsequencecapableofbindingbacteriaIRNA
polymerise and initiating the downstream (3') transcription of the coding sequence of Exo protein into mRNA. A bacterial promoter has a transcription initiation region which is usually placed proximal to the 5' end of the coding sequence. This transcription initiation region typically includes an RNA polymerise binding site and a transcription initiation site.
Sequences encoding metabolic pathway enzymes provide particularly useful promoter sequences. Examples include promoter sequences derived from sugar metabolizing enzymes, such as galactose, lactose an d maltose, and seq uencesderived from biosynthetic enzymes such as tryptophan. Promoters from bacteriophage may also be used and are known in the art. In addition, synthetic promoters and hybrid promoters are also useful;
for example, the tic promoter is a hybrid of the trp and lac promoter sequences. .
Furthermore,a bacterialpromotercan include naturallyoccurringpromotersof non-bacterial origin that have the ability to bind bacterial RNA polymerise and initiate transcription.

in addition to a functioning promoter sequence, an efficient ribosome binding site is desirable. In E. coli, the ribosome binding site is called the Shine-Delgarno (SD) sequence and includes an initiation colon and a sequence 3-9 nucleotides in length located 3 - 11 nucleotides upstream of the initiation colon.
The expression vector may also include a signal peptide sequence that provides for secretion of the Exo protein in bacteria. The signal sequence typically encodes a signal peptide comprised of hydrophobic amino acids which direct the secretion of the protein from the cell, as is well known in the art. The protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria).
The bacterial expression vector may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed. Suitable selection genes include genes which render the bacteria resistant to drugs such as ampicillin, chloramphenicol,erythromycin,kanamycin,neomycinandtetracycline.
Selectablemarkers also include biosynthetic genes, such as those in the histidine, tryptophan and leucine biosynthetic pathways.
Thesecomponentsareassembledintoexpressionvectors. Expressionvectorsforbacteria are well known in the art, and include vectors for Bacillus subtilis, E. coli, Streptococcus cremoris, and Streptococcus lividans, among others.
The bacterialexpression vectors are transformed into bacterial host cells using techniques well known in the art. such as calcium chloride treatment, electroporation, and others.
In one embodiment, Exo proteins are produced in insect cells. Expression vectors for the transformation ofinsectcells, and in particular, baculovirus-based expression vectors, are well known in the art.
In a preferred embodiment, Exo protein is produced in yeast cells. Yeast expression systems are well known in the art, and include expression vectors for Saccharomyces cerevisiae, Candidaalbicansand C. maltosa, Hansenulapolymorpha, Kluyveromycesfragilis and K. lacfis, Pichia guillerimondii and P. pastoris, Sct~izosaccharomyces pombe, and Yarrowia lipolytica. Preferred promoter sequences for expression in yeast include the inducible GAL1,10 promoter, the promoters from alcohol dehydrogenase, enofase, glucokinase, glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase, hexokinase, phosphotructokinase, 3-phosphoglycerate mutase, pyruvate kinase, and the acidphosphatasegene. YeastselectablemarkersincludeADE2,HIS4,LEU2,TRP1,and ALG7, which confers resistance to tunicamycin; the neomycin phosphotransferase gene, which confers resistance to 6418; and the CUP1 gene, which allows yeast to grow in the presence of copper ions.
The Exo protein may also be made as a fusion protein, using techniques well known in the art. Thus, for example, for the creation of monoclonal antibodies, if the desired epitope is small, the Exo protein may be fused to a carrier protein to form an immunogen.
Alternatively, the Exo protein may be made as a fusion protein to increase expression, or for other reasons. For example, when the Exo protein is an Exo peptide, the nucleic acid encoding the peptide may be linked to othernucleicacid forexpressionpurposes.
Similarly, Exo proteins of the invention can be linked to protein labels, such as green fluorescent protein (GFP), red fluorescent protein (RFP), blue fluorescent protein (BFP), yellow fluorescent protein (YFP), etc.
In one embodiment, the Exo nucleic acids, proteins and antibodies of the invention are labeled. By "labeled" herein is meant that a compound has at least one element, isotope or chemical compound attached to enablethedetectionofthe compound. In general, labels fall into three classes: a) isotopic labels, which may be radioactive or heavy isotopes; b) immune labels, which may be antibodies or antigens; and c) colored or fluorescent dyes.
The labels may be incorporated into the compound at any position.
In a preferred embodiment, the Exo protein is purified or isolated after expression. Exo proteins may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample. Standard purification methods includeelectrophoretic,molecular, immunological and chromatographic techniques, including ion exchange, hydrophobic, affinity, and reverse-phase HPLC
chromatography, and chromatofocusing. For example, the Exo protein may be purified using a standard anti-Exo antibody column. Uftrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. For general guidance in suitable purification techniques, see Scopes, R., Protein Purification, Springer-Verlag, NY ( 1982).
The degree of purification necessary will vary depending on the use of the Exo protein.
In some instances no purification will be necessary.
Once expressed and purified if necessary, the Exo proteins and nucleic acids are useful in a number of applications.

The nucleotide sequences (or their complement) encoding Exo proteins have various applications in the art of molecular biology, including uses as hybridization probes, in chromosome and gene mapping and in the generation of anti-sense RNA and DNA.
Exo protein nucleic acid will also be useful for the preparation of Exo protein polypeptides by the recombinant techniques described herein.
The full-length native sequence Exo protein gene, or portions thereof, may be used as hybridizationprobesfora cDNAlibraryto isolate the full-length Exo proteingeneorto isolate still other genes (for instance, those encoding naturally-occurring variants of Exo protein or Exo proteinfrom other species) which have a desired sequence identity to the Exo protein coding sequence. Optionally, the length of the probes will be about 20 to about 50 bases.
The hybridization probes may be derived from the nucleotide sequences herein or from genomic sequences including promoters, enhancer elements and introns of native sequences as provided herein. By way of example, a screening method will comprise isolating the coding region of the Exo protein gene using the known DNA
sequence to synthesize a selected probe of about 40 bases. Hybridization probes may be labeled by a variety of labels, including radionucleotides such as 3?P or'SS, or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems. Labeled probes having a sequence complementary to that of the Exo protein gene of the present invention can be used to screen libraries of human cDNA, genomic DNA or mRNA
to determine which members of such libraries the probe hybridizes.
The probes may also be employed in PCR techniques to generate a pool of sequences for identification of closely related Exo protein coding sequences.
Nucleotide sequences encoding a Exo protein can also be used to construct hybridization probes for mapping the gene which encodes that Exo protein and for the genetic analysis of individuals with genetic disorders. The nucleotide sequences provided herein may be mapped to a chromosome and specific regions ofa chromosome using known techniques, such as in situ hybridization, linkage analysis against known chromosomal markers, and hybridization screening with libraries.
N ucleic acids which encode Exo protein or its modified forms can also be used to generate eithertransgenicanimalsor"knockout"animalswhich,in turn, are useful in the development and screening of therapeutically useful reagents. A transgenic animal (e.g., a mouse or rat) is an animal having cells that contain a transgene, which transgene was introduced into the animal or an ancestor of the animal at a prenatal, e.g., an embryonic stage. A

-48~
transgene is a DNA which is integrated into the genome of a cell from which a transgenic animal develops. In one embodiment, cDNA encoding an Exo protein can be used to clone genomic DNA encoding an Exo protein in accordance with established techniques and the genomic sequences used to generate transgenic animals that contain cells which express the desired DNA. Methods for generating transgenic animals, particularly animals such as mice or rats, have become canventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009. Typically, particular cells would be targeted for the Exo protein transgene incorporation with tissue-specific enhancers.
Transgenic animals that include a copy of a transgene encoding an Exo protein introduced into the germ line of the animal at an embryonic stage can be used to examine the effect of increased expression of the desired nucleic acid. Such animals can be used as tester animalsforreagentsthought to confer protection from, forexample, pathoiogicalconditions associated with its overexpression. In accordance with this facetof the invention, an animal is treated With the reagent and a reduced incidenceofthe pathologicalcondition, compared to untreated animals bearing the transgene, would indicate a potential therapeutic intervention for the pathological condition.
Alternatively, non-human homologues of the Exo protein can be used to construct a Exo protein "knock out" animal which has a defective or altered gene encoding an Exo protein as a result of homologous recombination between the endogenous gene encoding an Exo protein and altered genomic DNA encoding an Exo protein introduced into an embryonic cell of the animal. For example, cDNA encoding an Exo protein can be used to clone genomicDNAencodingan Exo pratein in accordancewithestablished techniques. A
portion ofthegenomicDNAencoding an Exo protein can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration.
Typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector [see e.g., Thomasand Capecchi, Cell, 51:503 (1987) for a description of homologous recombination vectors]. The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected [see e.g., Li et al., Cell, X9:915 ( 1992)].
The selected cells are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras [see e.g., Bradley, in Teratocarcinomasand Embryonic Sfem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152]. A
chimeric embryocanthen be implantedintoa suitablepseudopregnantfemalefosteranimal and the embryo brought to term to create a "knock out" animal. Progeny harboring the homologously recombined DNA in theirgermcellscan be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA. Knockout animals can be characterized for instance, for their ability to defend against certain pathological conditions and for their development of pathological conditions due to absence of the Exo protein polypeptide. It is understood that cell based knock-out or "knock-in" systems can also be made and utilized in accordance with the present disclosure.
It is understood that the models described herein can be varied. For example, "knock-in"
models can be formed, or the models can be cell-based rather than animal models.
Nucleic acid encoding the Exo polypeptides, antagonists or agonists may also be used in gene therapy. In gene therapy applications, genes are introduced into cells in order to achieve in vivo synthesis of a therapeutically effective genetic product, for example for replacement of a defective gene. "Gene therapy" includes both conventional gene therapy where a lasting effect is achieved by a single treatment, and the administration of gene therapeutic agents, which involves the one time or repeated administration of a therapeutically effective DNA or mRNA. Antisense RNAs and DNAs can be used as therapeutic agents for blocking the expression of certain genes in vivo. It has already been shown that short antisense oligonucleotides can be imported into cells where they act as inhibitors, despite their low intracellular concentrations caused by their restricted uptake by the cell membrane. (Zamecnik etal., Proc. Natl. Acad. Sci. tJSA >~3, 4143-4146 [1986]).
The oligonucleotides can be modified to enhance their uptake, e.g. by substituting their negatively charged phosphodiester groups by uncharged groups.
There are a variety of techniques available for introducing nucleic acids into viable cells.
The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, orin vivo in the cells ofthe intended host. Techniques suitable for the transfer of nucleic acid into mammalian cells un vitro include the use of liposomes, electroporation, microinjection,cellfusion,DEAE-dextran,thecalciumphasphateprecipitationmethod,etc.
The currently preferred in vivo gene transfer techniques include transfection with viral (typically retroviral) vectors and viral coat protein-liposome mediated transfection (Dzau et al., Trends in Biotechnologv 11, 205-210 [1993]). In some situations it is desirable to provide the nucleic acid source with an agent that targets the target cells, such as an antibodyspecificforacellsurfacemembraneproteinorthetargetcell,aligandforarecept or on the target cell, etc. Where liposomes are employed, proteins which bind to a cell surface membrane proteinassociatedwith endocytosismay be used fortargetingand/orto facilitate uptake, e.g. capsid proteins or frag ments thereof tropic for a particular cell type, antibodies for proteins which undergo internalization in cycling, proteins that target intracellular localization and enhance intracellular half-fife. The technique of receptor-mediated endocytosis is described, for example, by Wu etal., J. Biol. Chem. 262, 4429-4432 (9987);
and Wagner et al,, Proc. Natl. Acad. Sci. USA 87, 3410-3414 (1990). For review of gene marking and gene therapy protocols see Anderson et aL, i nce 2~ø, 808-813 (1992).
In a preferred embodiment, the Exn proteins, nuGeic acids, modified proteins and cells containing the native or modified Exo proteins are used in screening assays.
Identification of this important exocytosis protein permits the design of drug screening assays for compounds that modulate Exo activity.
Screens may be designed to first find candidate agents that can bind to Exo proteins, and then these agents may be used in assays that evaluate the ability of the candidate agent to modulate Exo activity. Thus, as will be appreciatedby those in the art, there are a number of different assays which may be run; binding assays and activity assays.
Thus, in a preferred embodiment, the methods comprise combining an Exo protein and a candidate bioactive agent, and determining the binding ofthe candidate agent to the Exo protein. Preferred embodiments utilize the human Exo protein, although other mammalian proteins may also be used, including rodents (mice, rats, hamsters, guinea pigs, etc.), farm animals (cows, sheep, pigs, horses, etc.) and primates. These fatter embodiments may be preferred in the developmentofanimal modelsofhuman disease. In some embodiments, as outlined herein, variant or derivative Exo proteins may be used, including deletion Exo proteins as outlined above.
The term "candidatebioactiveagent" ar"exogeneous compound' as used herein describes any molecule, e.g., protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc., with the capabilityofdirectlyor indirectly altering the bioactivity of Exo.
Generallya pluralityofassay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection.
Candidateagentsencompassnumerouschemicalclasses,thoughtypicallytheyareorganic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons. Candidate agents comprise functional groups necessaryforstructuralinteractionwith proteins, particularlyhydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures andlor aromatic or polyaromatic structures substituted with one or more ofthe abovefunctionalgroups. Candidateagentsarealsofoundamong biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides.
Candidate agents are obtained froma wide varietyofsourcesincludinglibrariesof synthetic ornaturalcompounds. Forexample, numerousmeansareavailableforrandomanddirected synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form ofbacterial,fungal,plantandanimalextractsareavailableorreadilyproduced.Addition ally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acyla6on, alkylation, esterification, amidification to produce structural analogs.
In a preferredembodiment,thecandidate bioactive agents are proteins. By "protein" herein is meantatleasttwocovalentlyattachedaminoacids,which includesproteins, polypeptides, oligopeptides and peptides. The proteinmay be madeup of naturallyoccurringamino acids and peptide bonds, or synthetic p~p~domimetic structures. Thus "amino acid", or "peptide residue", as used herein means both naturally occurring and synthetic amino acids. For example, homo-phenylalanine, citrulline and noreleucine are considered amino acids for the purposes of the invention. "Amino acid" also inGudes imino acid residues such as praline and hydroxyproline. The side chains may be in either the (R) or the (S) configuration. Inthepreferredembodiment,theaminoacidsare in the (S)orL-configuration.
If non-naturally occurring side chains are used, non-amino acid substituents may be used, for example to prevent or retard in vivo degradations.
In a preferred embodiment, the candidate bioactive agents are naturally occurring proteins or fragments of naturallyoccuringproteins.
Thus,forexample,cellularextractscontaining proteins, or random or directed digests of proteinaceous cellular extracts, may be used.
In this way libraries of procaryotic and eukaryotic proteins may be made for screening againstExo.
Particularlypreferredinthisembodimentarelibrariesofbacterial,fungal,viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred.

In a preferred embodiment, the candidate bioactive agents are peptides of from about 5 to about 30 amino acids, with from about 5 to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred. The peptides may be digests of naturally occuring proteins as is outlined above, random peptides, or "biased"
random peptides. By "randomized" or grammatical equivalents herein is meant that each nucleic acid and peptide consists ofessentially random nucieotidesand aminoacids, respectively.
Since generally these random peptides (or nucleic acids, discussed below) are chemically synthesized, they may incorporate any nucleotide or amino acid at any position. The synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible cornbinatians over the length of the sequence, thus forming a library of randomized candidate bioactive proteinaceousagents.
In one embodiment, the library i fully randomized, with no sequence preferences or constants at any position. In a preferred embodiment, the library is biased.
That is, some positions within the sequence areeitherheld constant, or are selectedfrom a limited number of possibilities. For example, in a preferred embodiment, the nucleotides or amino acid residues are randomized within a defined class, for example, of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc., or to purines, etc.
In a preferred embodiment, the candidate bioactive agents are nucleic acids.
By "nucleic acid"or"oligonucleotide"orgrammaticalequivalentsherein means atleasttwonucleotides covalently linked together. A nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramide (Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein;
Letsinger, J.
Org. Chem. 35:3800 (1970); Sprinal et al., Eur. J. Biochem. 81:579 (1977);
Letsinger et al., Nucl. Acids Res.14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsingeretal., J. Am. Chem. Soc. 110:4470 (1988)" and Pauwels etal., Chemica Scripta 26:141 91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437 (1991); and U.S.
Patent No.

5,644,048), phosphorodithioate (Briu et al., J. Am. Chem. Soc. 111:2321 (1989), O-methylphophoroamiditelinkages(see Eckstein, Oligonucleotides and Analogues: A
Practical Approach, Oxford UniversityPress), and peptide nucleic acid backbones and linkages (see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed. Engl.
31:1008 (1992); Nielsen, Nature, 365:566( 1993); Carlssonet al., Nature380:207(1996), all ofwhich are incorporated by reference). Other analog nucleic acids include those with positive WO 00/43419 PCTlIJS00/01431 backbones(Denpcyetal., Proc. Nati. Acad. Sci. USA92:6097 (1995); non-ionicbackbones (U.S. Patent Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863;
Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1991); Letsinger et al., J. Am.
Chem. Soc.
110:4470 (1988); Letsinger et al., Nucleoside & Nucleotide 13:1597 (1994);
Chapters 2 and 3, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed. Y.S. Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic 8~ Medicinal Chem. Lett.
4:395 (1994); Jeffs et al., J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 ( 1996)) and non-ribose backbones, includingthosedescribed in U. S. Patent Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, "Carbohydrate Modifications in Antisense Research", Ed. Y.S. Sanghui and P. Dan Cook.
Nucleic acids containing one or more carbocyclic sugars are also included within the definition of nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995) pp169-176). Several nucleic acid analogs are described in Rawls, C & E News June 2, 1997 page 35. All of these references are hereby expressly incorporated by reference. These modifications of the ribose-phosphate backbone may be done to facilitate the addition of additional moieties such as labels, or to increase the stability and half-life of such molecules in physiological environments. In addition, mixtures of naturally occurring nucleic acids and analogs can be made. Alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occuringnucleicacidsandanalogsmaybemade. Thenucleicacidsmaybesinglestranded or double stranded, as specified, or contain portions of bath double stranded or single stranded sequence. The nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid,wherethe nucleicacid contains any combination ofdeoxyribo-and ribo-nucleotides, and any combination of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xathanine hypoxathanine, isocytosine, isoguanine, etc.
As described above generally far proteins, nucleic acid candidate bioactive agents may be naturally occuring nucleicacids, random nucleicacids, or"biased" random nucleic acids.
For example,digestsofprocaryoticoreucaryoticgenomesmay be used as isoutlinedabove for proteins.
I n a preferred embodiment, the candidate bioactive agents are organic chemical moieties, a wide variety of which are available in the literature.
The assays provided utilize Exo proteins as defined herein. In one embodiment, portions of Exo proteins are utilized; in a preferred embodiment, portions having Exo activity are used. Exo activity is described further below and includes binding activity to GS27, rab7, rab9, snap-23, rab3a, rab11, rabid, rab5, alpha-snap, unc18-1, vamp3 or Exo protein modulatorsas furtherdescribed below. In addition, the assays described herein may utilize either isolated Exo proteins or cells comprising the Exo proteins.
Generally, in a preferred embodiment of the methods herein, the Exo protein or the candidate agent is non-diffusibly bound to an insoluble support having isolated sample receiving areas (e.g. a microtiterplate, an array, etc.}. The insolublesupports may be made of any composition to which the compositions can be bound. is readily separated from soluble material, and is otherwise compatible with the overall method of screening. The surface of such supports may be solid or porous and of any convenient shape.
Examples of suitable insoluble supports include microtiter plates, arrays, membranes and beads.
These are typically made of glass, plastic (e.g., polystyrene), polysaccharides, nylon or nitrocellulose,teflonT"',etc.
Microtiterplatesandarraysareespeciallyconvenientbecause a large number of assays can be carried out simultaneously, using small amounts of reagents and samples. In some cases magnetic beads and the like are included.
The particular manner of binding of the composition is not crucial so long as it is compatible with the reagents and overall methods of the invention, maintains the activity of the compositionand is nondiffusable. Preferredmethods of binding includethe useofantibodies (which do not sterically block either the ligand binding site or activation sequence when the protein is bound to the support), direct binding to "sticky" or ionic supports, chemical crosslinking,the synthesisofthe proteinoragenton the surtace, etc. In same embodiments, GS27 can be used. Other embodiments include using, rab7, rab3a, rabid, snap23, rabg, rab5, alpha snap, rab1l, unc18-1 or vamp3. Following binding of the protein or agent, excess unbound material is removed by washing. The sample receiving areas may then be blocked through incubationwith bovine serum albumin (BSA), casein or other innocuous protein or other moiety. Also included in this invention are screening assays wherein solid supports are not used.
In a preferred embodiment, the Exo protein is bound to the support, and a candidate bioactive agent is added to the assay. Alternatively, the candidate agent is bound to the support and the Exo protein is added. Novel binding agents include specific antibodies, non-natural binding agents identified in screens of chemical libraries, peptide analogs, etc.
Of particular interest are screening assays for agents that have a low toxicity for human cells. A wide variety of assays may be used for this purpose, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc.) and the like.

The determination of the binding of the candidate bioactive agent to the Exo protein may be done in a number of ways. In a preferred embodiment, the candidate bioactive agent is labelled, and binding determined directly. For example, this may be done by attaching all or a portion of the Exo protein to a solid support, adding a labelled candidate agent (for example a fluorescent label), washing off excess reagent, and determining whether the label is present on the solid support. Various blocking and washing steps may be utilized as is known in the art.
By "labeled" herein is meant that the compound is either directly or indirectly labeled with a label which provides a detectable signal, e.g. radioisotope, fluorescers, enzyme, antibodies, particles such as magnetic particles, chemiluminescers, or specific binding molecules, etc. Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin etc. Forthe specific binding members, the complementarymember would normally be labeled with a molecule which provides fordetection, in accordance with known procedures, as outlined above. The label can directly or indirectly provide a detectable signal.
In some embodiments, only one of the components is labeled. For example, the proteins (or proteinaceous candidate agents) may be labeled at tyrosine positions using '251, or with fluorophores.Alternatively, more than one component may be labeled with different labels;
using '251 for the proteins, for example, and a fluorophor for the candidate agents.
In a preferred embodiment, the binding of the candidate bioactive agent is determined through the use of competitive binding assays. In this embodiment, the competitor is a binding moiety known to bind to the targetmolecule{i.e. Exo), such as an antibody, peptide, binding partner, ligand, etc. In a preferred embodiment, the competitoris GS27, rab7, rab9, snap-23, rab3a, rab11, rabid, rab5, alpha-snap, uncl8-1, or vamp3. Under certain circumstances, there may be competitive binding as between the bioactive agent and the binding moiety, with the binding moiety displacing the bioactive agent. This assay can be used to determine candidate agents which interfere with binding between Exo proteins and GS27, rab7, rab9, snap-23, rab3a, rab11, rabid, rab5, alpha-snap, uncl8-1, or vamp3.
In oneembodiment,thecandidatebioactiveagentislabeled.
Eitherthecandidatebioactive agent, or the competitor, or both, is added first to the protein for a time sufficient to allow binding, if present. Incubations may be performed at any temperature which facilitates optimalactivity, typically between 4 and 40°C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high through put screening. Typically between 0.1 and 1 hourwill be sufficient. Excess reagent is generally removed or washed away. The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding.
In a preferred embodiment,thecompetitoris added first, followedby the candidate bioactive agent. Displacement of the competitor is an indication that the candidate bioactive agent is binding to the Exo protein and thus is capable of binding to, and potentially modulating, the activity of the Exo protein. In this embodiment, either component can be labeled. Thus, forexample, ifthe competitor is labeled, the presence oflabel in the wash solution indicates displacement by the agent. Alternatively, if the candidate bioactive agent is labeled, the presence of the label on the support indicates displacement.
In an alternative embodiment, the candidate bioactive agent is added first, with incubation and washing, followed by the competitor. The absence of binding by the competitor may indicate that the bioactive agent is bound to the Exo protein with a higher affinity. Thus, ifthe candidate bioactive agent is labeled, the presenceof the label on the support, coupled with a lackofcompetitorbinding, may indicatethat the candidate agent is capable of binding to the Exo protein.
In a preferredembodiment,the methodscomprisedifferential screening to identity bioactive agents that are capable of modulating the activity of the Exo proteins. In this embodiment, the methods comprise combining an Exo protein and a competitor in a first sample. A
second sample comprises a candidate bioactive agent, an Exo protein and a competitor.
The binding of the competitor is determined for both samples, and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding to the Exo protein and potentially modulating its activity. That is, if the binding of the competitor is different in the second sample relative to the first sample, the agent is capable of binding to the Exo protein.
Alternatively, a preferred embodiment utilizes differential screening to identify drug candidates that bind to the native Exo protein, but cannot bind to modified Exo proteins.
The structure of the Exo protein rnay be modeled, and used in rational drug design to synthesize agents that interact with that site. Drug candidates that affect Exo bioactivity are also identified by screening drugs for the ability to either enhance or reduce the activity of the protein.

WO 00/43419 PC'T/US00/01431 Positive controls and negative controls may be used in the assays. Preferably all control and test samples are performed in at least triplicate to obtain statistically significant results.
Incubation of all samples is for a time sufficient for the binding of the agent to the protein.
Following incubation, all samples are washed free of non-specifically bound material and the amount of bound, generally labeledagentdetermined.Forexample,wherearadiolabel is employed, the samples may be counted in a scintillation counterto determinethe amount of bound compound.
A variety of other reagents may be included in the screening assays. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc which may be used to facilitate optimal protein-protein binding andlor reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbialagents, etc., may be used. The mixture of components may be added in any order that provides for the requisite binding.
The components provided herein for the assays provided herein may also be combined to form kits. The kits can be based on the use ofthe proteinand/orthe nucleicacid encoding the Exo proteins. Assays regarding the use of nucleic acids are further described below.
Screening for agents that modulate the activity of Exo may also be done. In a preferred embodiment, methods forscreening fora bioactive agent capableof modulatingthe activity of Exo comprise the steps of adding a candidate bioactive agent to a sample of Exo, as above, and determiningan alteration in the biologicalactivity of Exo.
"Modulating the activity of Exo" includes an increase in activity, a decrease in activity, or a change in the type or kind of activity present. Thus, in this embodiment, the Candidate agent should both bind to Exo (although this may not be necessary), and alter its biological or biochemical activity as defined herein. The methods include both in vitro screening methods, as are generally outlined above, and in vivo screening of cells for alterations in the presence, distribution, activity or amount of Exo.
Thus, in this embodiment, the methods comprise combiningan Exo sampleand a candidate bioactive agent, and evaluating the effect on exocytosis. By "Exo activity' or grammatical equivalents herein is meant one of Exo's biological activities, including, but not limited to, its ability to affect exocytosis, secretion andlor vesicular transport.
Included within exocytosis, secretionandlorvesiculartransportactivitiesincluderegulatingorinvolvement in steps therein such as docking, fusion and targeting activities of proteins involved in the entire pathway of exocytosis, secretion andlor vesicular transport. In one embodiment, vesicular refers to any vesicle including synaptic or secretory granules and vesicles those involved in exocytosis, endocytosis or the traps-golgf network. In one embodiment, exo activity includes GTPase activity or regulation thereof. One exo activity herein is binding to at least one protein selected from the group consisting of GS27, rab7, rab9, snap-23, rab3a, rabll, rabid, rab5, alpha-snap, uncl8-1 and vamp3. Other exo activities include the activities and regulation thereof of GS27, rab7, rab9, snap-23, rab3a, rab1l, rabid, rab5, alpha-snap, unc18-1 and vamp3.
!n a preferred embodiment, the activity ofthe Exo protein is increased; in another preferred embodiment, the activity ofthe Exo protein is decreased. Thus, bioactive agents that are antagonists are preferred in some embodiments, and bioactive agents that are agonists may be preferred in other embodiments.
I n a preferred embodiment, the invention provides methods for screeningfor bioactiveagents capable of modulating the activity of an Exo protein. The methods comprise adding a candidate bioactive agent, as defined above, to a cell comprising Exo proteins. Preferred cell types include almost anycell.l'hecellscontainarecombinantnucleicacidthatencodes an Exo protein. In a preferred embodiment, a library of candidate agents are tested on a plurality of cells.
In some embodiments, the assays include exposing the cells to an exocytosis agent that will induce exocytosis in control cells, i.e. cells of the same type but that do not contain the exogeneous nucleic acid encoding an Exo. Suitable exocytosis agents are known in the art and include but are not limited to ionomycin and Ca", such as, but not limited to the Ca"ionophoreA23187. Alternatively,thecellsmaybeexposedtoconditionsthatnormally result in exocytosis, and changes in the normal exocytosis progression are determined.
Alternatively, the cells into which the Exo nucleic acids are introduced normally under exocytosis, and thus changes (far example, inhibition of exocytosis) are determined.
Optionally, the cells normally do not undergo exocytosis, and the introd uction of a candidate agent causes exocytosis.
Thus, the effect of the candidate agent on exocytosis is then evaluated.
Detection of exocytosis may be done as will be appreciated by those in the art. In one embodiment, indicators of exocytosis are used. Suitable exocytosis labels include, but are not limited to, annexin. Accordingly, these agents can be used as an affinity ligand, and attached to a solid support such as a bead, a surface. etc. and used to pull out cells _59-that are undergoing exocytosis. Similarly, these agents can be coupled to a fluorescent dye such as PerCP, and then used as the basis of a fluorescent-activated cell sorting (FACS) separation. Moreover, FAGS or other optical methods can be used to detect exocytosis activity and the modulation thereof based on light scattering, light absorption, dye uptake and release, granule enzyme activity and quantification of granule specific proteins.
In this way, bioactive agents are identified. Compounds with pharmacological activity are able to enhance or intertere with the activity of the Exo protein. The compounds having the desired pharmacological activity may be administered in a physiologically acceptable carrier to a host, as previously described. The agents may be administered in a variety of ways, orally, parenterally e.g., subcutaneously, intraperitoneally, intravascularly, etc.
Depending upon the mannerof introduction, the compounds may be formulated in a variety ofways. The concentration oftherapeutically active compound in the formulation may vary from about 0.1-100 wt.%.
The pharmaceutical compositions can be prepared in various forms, such as granules tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like.
Pharmaceutical grade organic or inorganic carriers and/or diluents suitable for oral and topical use can be used to make up compositions containing the therapeutically-active compounds. Diluents known to the art include aqueous media, vegetable and animal oils and fats. Stabilizing agents, wetting and emulsifying agents, salts for varying the osmotic pressure or buffers for securing an adequate pH value, and skin penetration enhancers can be used as auxiliary agents.
without being bound by theory, it appears that Exo is an important protein in exocytosis.
Accordingly, disorders based on mutant or variant Exo genes may be determined.
In one embodiment, the invention provides methods for identifying cells containing variant Exo genes comprising determining all or part of the sequence of at least one endogeneous Exo genes in a cell. As will be appreciated by those in the art, this may be done using any number of sequencing techniques. In a preferred embodiment, the invention provides methods of identifying the Exo genotype of an individual comprising determining all or part of the sequence of at least one Exo gene of the individual. This is generally done in at least one tissue of the individual, and may include the evaluation of a number of tissues or different samples of the same tissue The method may include comparing the sequence of the sequenced Exo gene to a known Exo gene, i_e. a wild-type gene.

The sequence of all or part of the Exo gene can then be compared to the sequence of a known Exo gene to determine if any differences exist. This can be done using any number of known sequence identity programs, such as Bestfit, etc. I n a preferred embodiment, the presenceofa difference in the sequencebetweenthe Exo geneofthe patientand the known Exo gene is indicative of a disease state or a propensity for a disease state, as outlined herein.
The present discovery relating to the role of Exo in exocytosis thus provides methods for inducing or preventing exocytosis in cells. In a preferred embodiment, the Exo proteins, and particularly Exo fragments, are useful in the study or treatment of conditions which are mediated byexocytosis,i.e.todiagnose,treatorpreventexocytosis-mediateddisorders.
Thus, "exocytosis mediated disorders" or"disease state" include conditions involving both insufficientorexcessive exocytosisa vesiculartransport, and/or secretion via the secretory pathway, including inflammatory mediator release from mast cells including asthma, allergies, and Chediak-Higashi Syndrome (CHS). Additionally, control of neurotransmitter release can be used to treat Alzheimer's disease, Parkinson's and Huntington's disease states as well as some skitzophrenia, thus these can also be included in exocytosis mediated disorders in some cases. In other cases, fertilization and lactation disorders can be included as disease states which can be treated with the compositions provided and/or identified herein. Additionally, some diabetes, digestion and wound healing disorders can be exocytosis mediated disorders.
Thus, in one embodiment, methods of modulating exocytosis in cells or organisms are provided. In one embodiment, the methods comprise administering to a cell an anti-Exo antibody that reduces or eliminates the biological activity of the endogeneous Exo protein.
Alternatively, the methods comprise administering to a cell or organism a recombinant nucleic acid encoding an Exo protein. As will be appreciated by those in the art, this may be accomplished in any number of ways. In a preferred embodiment, the activity of Exo is increased by increasing the amount of Exo in the cell, for example by overexpressing the endogeneous Exo or by administering a gene encoding an Exo, using known gene-therapy techniques, for example. In a preferred embodiment, the gene therapy techniques include the incorporation of the exogeneous gene using enhanced homologous recombination (EHR), for example as described in PCT/US93103868, hereby incorporated by reference in its entireity.
In one embodiment, the invention provides methods for diagnosing an exocytosis related condition in an individual. The methods comprise measuring the activity of Exo in a tissue from the individual or patient, which may include a measurement of the amount or specific activity of Exo. This activity is compared ko the activity of Exo from either a unaffected second individual or from anunaffectedtissuefromthefirstindividual. When these activities are different, the first individual may be at risk for an exocytosis mediated disorder.
The proteins and nucleic acids provided herein can also be used for screening purposes wherein the protein-protein interactions of the Exo proteins can be identified. Genetic systems have been described to detect protein-protein interactions. The first work was done in yeast systems, namely the "yeast two-hybrid" system. The basic system requires a protein-protein interaction in order to turn ontranscriptianofa reportergene. Subsequent work was done in mammalian cells. See Fields et al., Nature 340:245 (1989);
Vasavada et al., PNAS USA 88:10686 (1991); Fearon et al., PNAS USA 89:7958 (1992); Dang et al., Mol. Cell. Biol. 11:954 ( 1991 ); Chien etal., PNAS USA 88:9578 ( 1991 );
and U.S. Patent Nos. 5,283,173, 5,667,973, 5,468,614, 5,525,490, and 5,637,463 a preferred system is described in Serial No. 091050,863, fled March 30. 1998, entitled "Mammalian Protein InteractionCloningSystem". For use inconjunctionwiththesesystems,aparkicularlyuseful shuttle vector is described in Serial No. 091133,944, filed August 14, 1998, entitled "Shuttle Vectors".
In general, two nucleic acids are transformed into a cell, where one is a "bait" such as the geneencodingGS27, rab7, rab9, snap-23, rab3a, rabl1, rabid. rab5, alpha-snap, unc1 B-1, vamp3 or a portion thereof, and the other encodes a test candidate. Only if the two expression products bind to one another will an indicator, such as a fluorescent protein, be expressed. Expression of the indicator indicates when a test candidate binds to the GS27, rab7, rab9, snap-23, rab3a, rabll, rabid, rab5, alpha-snap, unc18-1 orvamp3 and can be identified as an Exo protein. Using the same system and the identified Exo proteins the reverse can be performed. Namely, the Exo proteins provided herein can be used to identify new baits, or agents which interact with Exo proteins. Additionally, the two-hybrid system can be used wherein a test candidate is added in addition to the bait and the Exo protein encoding nucleic acids to determine agents which interfere with the bait, such as GS27, rab7, rab9, snap-23, rab3a, rabll, rabid, rab5, alpha-snap, uncl8-1 orvamp3, and the Exo protein.
In one embodiment, a mammalian two-hybrid system is preferred. Mammalian systems provide post-translational modifications of proteins which may contribute significantly to their ability to interact. In addition, a mammalian two-hybrid system can be used in a wide varietyofmammaliancelltypestomimictheregulation,induction,processing,etc.ofspec ific WO 00/43419 PCTlUS00/01431 proteins within a particularcelltype. Forexample, proteins involved in a disease state such as those described above could be tested in the relevant disease cells.
Similarly, for testing of random proteins, assaying them underthe relevant cellular conditionswill give the highest positive results. Furthermore, the mammalian cells can be tested under a variety of experimental conditions that may affect intracellular protein-protein interactions, such as in the presence of hormones, drugs, growth factors and cytokines, cellular and chemical stimuli, etc., that may contribute to conditions which can effect protein-protein interactions, particularlythoseinvolved in exocytosis, the secretory pathway, and/or vesiculartransport.
Expression in various cell types, and assays for Exo activity are described above. The activity assays, such as having an affect on exocytosis, secretion and/orvesiculartransport can be performed to confirm the activity of Exo proteins which have already been ident~ed by their sequence identity/similarity or binding to GS27, rab7, rab9, snap-23, rab3a, rab11, rabid, rab5, alpha-snap, unc1&1 orvamp3 as well as to further confirm the activity of lead compounds identified as modulators of exocytosis, secretion and/or vesicular transport.
Assays involving binding such as the two-hybrid system may take into accountnon-specific binding proteins (NSB).
In one embodiment, the Exo proteins of the present invention may be used to generate polyclonaland monoclonalantibodiesto Exo proteins,whichare useful as described herein.
Similarly, the Exo proteins can be coupled, using standard technology, to affinity chromatography columns. These columns may then be used to purify Exo antibodies.
In a preferred embodiment, the antibodies are generated to epitopes unique to the Exo protein; that is, the antibodies show little or no cross-reactivity to other proteins. These antibodies find use in a number of applications. For example, the Exo antibodies may be coupled to standard affinity chromatography columns and used to purify Exo proteins as further described below. The antibodies may also be used as blocking polypeptides, as outlined above, since they will specifically bind to the Exo protein.
The anti-Exoproteinantibodiesmay comprisepolyclonalantibodies.
Methodsofpreparing polyclonal antibodies are known to the skilled artisan. Palyclonal antibodies can be raised in a mammal, for example, by one or more injections ofan immunizing agent and, ifdesired, an adjuvant. Typically, the immunizing agent and/oradjuvantwill be injected in the mammal by multiple subcutaneous or intraper7toneal injections. The immunizing agent may include the Exo protein polypeptide or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized.

Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin,serumalbumin,bovinethyroglobulin,andsoybeantrypsininhibitor, Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM
adjuvant(monophosphorylLipida,synthetictrehalosedicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.
The anti-Exo protein antibodies may, alternatively, be monoclonal antibodies.
Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Natur , 2;495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro.
The immunizing agent will typically include the Exo protein polypeptide or a fusion protein thereof. Generally, either peripheral blood lymphocytes ("PBLs") are used if cellsof human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [coding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103].
Immortalized cell lines are usually transformedmammaliancells, particularlymyelomacells ofrodent,bovineandhumanorigin. Usually,ratormousemyelomacelllinesareemployed.
The hybridoma cells may be cultured in a suitable culture medium that preferably contains oneor moresubstancesthat inhibit the growth or survivalofthe unfused, immortalizedcells.
For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase(HGPRTor HPRT), the culture medium forthe hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"),which substances prevent the growth of HGPRT-deficient cells.
Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines aremurinemyeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distribution Center, San Diego, California and the American Type Culture Collection, Rockville, Maryland.
Human myeloma and mouse-human heteromyeloma cell lines also have been described for the prod uction of human monoclonal antibodies [Kozbor, ,l. I
mmunol.,133:3001 (1984);
Brodeur et al., Monoclonal Antibody Production Technigues and Applicatiens, Marvel Dekker, Inc., New York, (1987) pp. 51-63].

WO 00/43419 PC'TIUS00/01431 The culture medium in which the hybridoma cells are cultured can then be assayed forthe presence of monoclonal antibodies directed against Exo protein. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Bioch~m., 107:220 (1980).
After the desired hybridoma cells are identified, the clones may be subcloned by limiting dilutionproceduresandgrownbystandardmethods[Goding,su ra. Suitableculturemedia forthis purpose include, forexample, Dulbecco'sModified Eagie's Medium and medium. Alternatively, the hybridorrma cells may be grown in vivo as ascites in a mammal.
The monoclonal antibodies secreted by the subclones may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, protein a-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.
Themonoclonalantibodiesmayalsobemadeby recombinantDNAmethods, such as those described in U.S. Patent No. 4,816,567. DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and lightchainsof murineantibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells, Chinese hamsterovary (CHO) cells, or myelomacellsthatdo nototherwiseproduceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells.
TheDNAalsomaybemodified,forexample,bysubstitutingthecodingsequenceforhuman heavy and light chain constant domains in place of the homologousmurine sequences[U.S.
Patent No. 4,816,567; Morrison etal., suaral or by covalentlyjoining to the immunoglobulin coding sequence all or part of the coding sequence fora non-immunoglobulin polypeptide.
Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody ofthe invention, or can be substituted for the variable domains of one antigen-combining site of an antibody of the invention to create a chimeric bivalent antibody.
The antibodies may be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art, For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain crosslinking.
Alternatively,the relevantcysteineresiduesaresubstitutedwith another amino acid residue or are deleted so as to prevent crosslinking.
In vitro methods are also suitable for preparing monovalent antibodies.
Digestion of antibodies to produce fragments thereof, particularly, Fabfragments,can be accomplished using routine techniques known in the art.
Theanti-Exoprotein antibodiesoftheinventionmayfurthercomprisehumanizedantibodies or human antibodies. Humanizedformsofnon-human(e.g., murine)antibodiesarechimeric immunoglobulins,immunoglobulinchainsorfragmentsthereof(suchasFv,Fab,Fab',F(ab') or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins(recipientantibody)in which residues from a complementary determining region (CDR) ofthe recipientare replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by correspondingnon-hurnanresidues.
Humanizedantibodiesmayalsocomprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that: of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op.
Struct. Biol., 2:593-596 (1992)].
Methods for humanizing non-human antibodies are well known in the art.
Generally, a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as "import"residues,whicharetypicallytakenfroman"import"variable domain.
Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988);
Verhoeyen et al., Science, 2239:1534-1536 (1988)], by substituting rodent CDRs or CDR
sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized"

antibodies are chimeric antibodies (ll.S. Patent No. 4,816,567), wherein substantially less than an intacthuman variable domain has been substituted by the correspondingsequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
Human antibodiescan also be produced usingvarioustechniquesknown in theart, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991);
Marks et al., J. Mol. Biol., 222:581 (1991 )]. The techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 ( 1985) and Boerneretal., J. Immunol., 147 1 :86-95 (1991 )]. Similarly, human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire.
This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806;
5,569,825;
5,625,126; 5,633,425; 5,661,016, and in the following scientific publications:
Marks et'aL, BioITechnoloay 10, 779-783 (1992); Lonberg et al., Nature 368 856-859 (1994);
Morrison, Nature 368, 812-13 {1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);
Neuberger, Nature Biotechnolopy '14, 826 (1996); Lonberg and Huszar, Intern.
Rev.
Immunol. 13 65-93 (1995).
Bispecific antibodies are monoclonal,preferablyhumanor humanized,antibodiesthathave binding specificitiesforat leasttwo differentantigens. In the present case, one ofthe binding speciflcities is for the Exo protein, the other one is for any other antigen, and preferably for a cell-surface protein or receptor' or receptor subunit.
Methodsformakingbispecificantibodiesareknownintheart.
Traditionally,therecombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chainllight-chain pairs, where the two heavy chains have different specificities [Milstein and Cuello, Nature, 305:537-539 (1983)]. Because of the random assortment of immunoglobulin heavy and light chains, these hybridamas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinitychromatographysteps.
SimilarproceduresaredisclosedinW093108829,published 13 May 1993, and in Traunecker et al., EMBO J., 10:3655-3659 (1991).

Antibodyvariabledomainswiththedesiredbindingspecificities(antibody-antigencombining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It is preferred to have the first heavy-chain constant region {CH1 ) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism. For further details of generating bispecific antibodies see, for example, Suresh et al., Methods in Enzvmoloav, 121:210 (1986).
Heteroconjugate antibodies are also within the scope of the present invention.
Heteroconjugate antibodies are composed of two covalently joined antibodies.
Such antibodies have, for example, been proposed to target immune system cells to unwanted cells [U.S. Patent No. 4,676,980], and for treatment of HIV infection [WO
91100360; WO
92/200373; EP 03089]. It is contemplated that the antibodies may be prepared in vitro using known methodsin syntheticprotein chemistry,includingthose involvingcrosslinkingagents.
For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolateand methyl-4-mercaptobutyrimidateand those disclosed, for example, in U.S.
Patent No. 4,676,980.
The anti-Exoproteinantibodies of the inventionhavevariousutilities.
Forexample,anti-Exo protein antibodies may be used in diagnostic assays for an Exo protein, e.g., detecting its expressioninspecificcells,tissues,orserum. Various diagnostic assaytechniquesknown in the art may be used, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted in eitherheterogeneousor homogeneous phases [Zola, Monoclonal Antibodies: a Manual of Techniaues, CRC Press, Inc.
(1987) pp.147-158J. Theantibodiesused in the diagnosticassays can be labeled with a detectable moiety. The detectable moiety should be capable of producing, either directly or indirectly, a detectable signal. For example, the detectable moiety may be a radioisotope, such as aH ,eC azP 35S or'251,afluorescentorchemiluminescentcompound,suchasfluorescein isothiocyanate, rhodamine, or luciferin, oran enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase. Any method known in the art for conjugating theantibodytothedetectablemoietymay be employed, including those methods described by Hunter etal., Nature, 144:945 (1982); David etal., Biochemistry, 13:1014 (1974); Pain etal., J. Immunol. Meth., 40:219 (1981 ); and Nygren, J. Histochem.and Cytochem., 3Q:407 (1982).

Anti-Exo protein antibodies also are useful for the affinity purification of Exo protein from recombinant cell culture or natural sources. !n this process, the antibodies against Exo protein are immobilized on a suitable support, such a Sephadex resin or filter paper, using methods well known in the art. The immobilized antibody then is contacted with a sample containingtheExoproteintobepurified,andthereafterthesupportiswashedwithasuitabl e solvent that will remove substantially all the material in the sample except the Exo protein, which is bound to the immobilized antibody. Finally, the support is washed with another suitable solvent that will release the Exo protein from the antibody.
The anti-Exo protein antibodies may also be used in treatment. In one embodiment, the genes encoding the antibodies are provided, such thatthe antibodies bind to and modulate the Exo protein within the cell.
In one embodiment,atherapeuticaltyeffectivedose ofan Exo protein, agonistorantagonist is administered to a patient.'By "therapeutically effective dose" herein is meanta dose that producestheeffectsforwhichitisadministered. The exactddsewilldependonthepurpose ofthe treatment, and will be ascertainable by one skilled in the art using known techniques.
As is known in the art, adjustments for Exo degradation, systemicversus localizeddelivery, and rate of new protease synthesis, as well as the age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.
A "patient" for the purposes of the present invention includes both humans and other animals, particularly mammals, and organisms. Thus the methods are applicable to both human therapy and veterinary applications. In the preferred embodiment the patient is a mammal, and in the most preferred embodiment the patient is human.
The administration of the Exo protein, agonist or antagonist of the present invention can be done in a variety of ways, including, but not limited to, orally, subcutaneously, intravenously,intranasally,transdermally,intraperitoneally,intramuscularly, intrapulmonary, vaginally, rectally, or intraoculariy. In some instances, for example, in the treatment of wounds and int7ammation, the Exo may be directly applied as a solution or spray.
ThepharmaceuticalcompositionsofthepresentinventioncompriseanExoprotein,agonist or antagonist in a form suitable for administration to a patient. In the preferred embodiment, the pharmaceutical compositions are in a water soluble form, such as being present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts. "Pharmaceutically acceptable acid addition salt" refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoricacidandthelike,andorganicacidssuchasaceticacid, propionic acid, glycolic acid, pyruvic: acid, oxalic acid, malefic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
"Pharmaceuticallyacceptablebaseadditionsalts"includethosederivedfrominorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum saltsandthelike.
Particularlypreferredaretheammonium, potassium, sodium, calcium, and magnesium salts. Salts derived from pharmaceutically acceptableorgan is non-toxicbases include salts of primary, secondary, and tertiaryamines, substitutedamines includingnaturallyoccurringsubstitutedamines, cyclic aminesand basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.
The pharmaceutical compositions may also include one or more of the following:
carrier proteins such as serum albumin; buffers; fillers such as microcrystalline cellulose, lactose, com and other starches; binding agents; sweeteners and other flavoring agents;
coloring agents; and polyethylene glycol. Additives are well known in the art, and are used in a variety of formulations.
All references cited herein are incorporated by reference in their entireity.
The following examples are merely for illustration.
EXAMPLES
The yeast two-hybrid cDNA cloning technology is a powerful in vivo protein-protein interaction assay first introduced in Fields S, Song 0 ( 1989) Nature 340:245-247 (Chevray PM, Nathans D (1992) Proc Natl Acad Sci USA 89:5789-5793; Chien CT, Bartel PL, Sternglanz R, Fields S (1991 ) Proc Natl Acad Sci USA 88:9578-9582; Durfee T, Becherer K, Chen PL, Yeh SN, Yang Y, Kilbum,AE, Lee UVH, Elledge S (1993) Genes Dev 7:555-569;
Fields S, Song O (1989) Nature 340:245-247; Mendelsohn AR, Brent R (1994) Biotechnology 5:482-486; Zervos A, Gyuris J, Brent R (1993) Cell 72:223-232).
It is based on co-expression of two proteins, X and Y, fused to GAL4 DNA binding domain (GAL4B) and GAL4 transcription activation domain (GAL4A) respectively (Figure 1 A). If the protein X interacts with the protein Y, the GAL4 transcription activation domain will be brought to the promoter containing the GAL4 DNA binding sites and will activate the transcription of reporter gene HIS3 or IacZ. The two-hybrid system can be used to clone cDNA
encoding a novel protein that interacts with a known protein (bait) in yeast. It can also be used to study protein-proteininteractionsbetweentwo known proteins. Theyeasttwo-hybridsystem has several advantages over other conventional methods to study protein-protein interactions: Protein-protein interactions are studied in eukaryotic cells (yeast); cDNA
clones are immediately available after screening.
Screening is very fast and convenient. No protein purification is necessary.
Both growth selection and IacZcolorselectionarevery sensitive. No radioisotope is needed.
However, this technology also presents a challenge to new users due to certain technical difficulties:
Depending on the bait protein used in screening, false positive rates vary significantly.
Yeast transformation efficiency is difficult to control.
High quality cDNA libraries can be very difficult to construct. The major difference between the yeast one-hybrid system and the yeast-two hybrid system is that the one-hybrid system is used to clone cDNA encoding DNA-bound proteins, rather than protein-bound proteins (Figure 1 B). DNA sequences of interest are inserted upstream of the minimal promoter controlling the expression of either HIS or IacZ gene. cDNA fragments are fused to the C-terminal of GAL4 transcription activation domain (GAL4A) to construct cDNA
library. If the protein encoded by the cDNA can bind to the specific DNA sequences of interest, the transcription ofHIS/IacZisactivated.Theyeastone-hybrid system is widely used in cloning new transcription factors (Lehming N, Thanos D, Brickman JM, Ma J, Maniatis T, Ptashne M (1994) Nature 371:175-179; Li JJ, Herskowitz I (1993) Science 262:1870-1873;
Luo Y, Stile J, Zhu L (1996) BioTechniques 20:564-568; Shang J, Luo Y, Clayton D
(1997) Developmen#al Dynamics 209:242-253; Strubin M., Newell JW, Matthias P (1995) Cell 80:497-506; Wilson TE, Fahmer TJ, Johnston M, Milbrandt J (1991) Science 252:1296-1300; WangMM,ReedRR(1993)Cel174:205-214). Experimental protocolsbetweenthese two methods are very similar except one notable exception. The one-hybrid system yeast reporterstrains need to be constructed by individual researcher. The expressionof reporter genes (HIS/IacZ) should be underthe control of specific DNA sequences of interest, rather than the GAL4 DNA-binding sites in the yeast tuvo-hybrid system. cDNA
libraries used for two-hybrid screening can also be used for one-hybrid screening.
The experimental flow-chart of a yeast two-hybrid cDNA screening experiment is outlined in Figure 2.

_71 The experimental flow-chart of a yeast one-hybrid cDNA screening experiment is outlined in Figure 3.
MATERIALS
Medium and Yeast Strains All yeast culture mediums, including YPD, YPD Agar, DOB, DOBA, CSM-TRP, CSM-LEU, CSM-HIS, CSM-URA, CSM-LYS, CSM-LEU-TRP, CSM-LEU-HIS, and CSM-LEU-TRP-HIS, are available from Bio101, Inc. 3AT (3-amino-1,2,4-triazol) is available from Sigma (Cat no.: A-8056, St. Louis, MO, USA).
Yeast two-hybrid system reporter strain Y190 (MATa, ura3-52, his3-200, lys2-801, ade2-101, trpl-901, leu2-3, 112, gal4A, ga180B, cyhr2, LYS2::GAL1~-HIS3Tnrn HIS3, URA3::GALI~AS-GALITnTA-IacZ) and yeast one-hybrid system reporter strain YM4271 (MATa, ura3-52, his3,200, lys2-8D1, ade2-101, trp1-903, leu2-3, 112, tyr1-501) are available from Clantech Laboratories, Inc. (Cat no.K1603-1, Clontech, Palo Alto, CA, USA).
Plasmids and cDNA Libraries pAS2 and pACT2 series were originally constructed by Elledge lab (Durfee et al.
1993) and are available from Clontech laboratories (Cat no.K1604-A, K1604-B).
Other GAL4 based two-hybrid vectors, such as pGBT9 and pGAD424 series, were originally published by Fields lab and are available from both Stratagene, Inca (Cat no.235700,235722) and Clontech Laboratories, Inc. (Cat no.K1605-A, K1605-B).
LexA based two-hybrid vectors are available from Origene Technologies, lnc.
(Cat no.DPL-100, DPL-102). cDNA libraries for two-hybrid and one-hybrid screening are available from Origene, Stratagene, Clontech, and Invitrogen. Rigel also makes its own two-hybrid cDNA libraries from various tissues. All of these two-hybrid vectors share basic structures as shown in Figure 4A.
The cDNA library is to be amplified before screening. It is recommended that at least 200 15cm-plates should be used Lo grow up 10 million independent cDNA
clones. High quality plasmid can be obtained with Qiagene DNA preparation kits.
Single-strand carrier DNA for yeast transformation is available from Origene or Clontech. Carrier DNA can also be made according to protocol by Ito et al.
(Ito H, Fukada Y, Murata K, Kimura A (1993) Journal of Bacteriology 153:163-168).
Special Buffers Z buffer oH7.0 (per liter) NazHP0~.7H20 16.1 g NaH2P04.Hz0 5.50 g MgS04.7H20 0.246 g Kcl 0.75 g Z buffer + X-Gal Z buffer 1 ml 20mglml X-Gal 40 ul (i-mercaptoethanol(optional) 2 ul PEG/LiAc (10 ml) 50°~ PEG(3350) 8 ml 10X TE (pH7.5) 1 ml 1 M LiAc 1 ml Equipment and Others 30°C incubators and liquid nitrogen containers are required. Nylon membrane and Whatman filter for IacZ color assay are available from Fisher Scientific.
X-Gal is from either Promega (Cat no.V3941, Madison, WI, USA) or Denville Scientific (Cat no.CX-3000-3, Metuchen, NJ, USA). All plastic wares are from Fisher Scientific or VWR.
PROCEDURE: Yesat Two-Hybrid System Screening Grow up Yeast reporter strains on YPD plates from frozen stock.
Since no antibiotics are added into the yeast medium, very stringent sterilization procedures are required during inoculation. It is also recommended that yeast reporter strain be streaked tin SD-W, SD-L, SD-H, SD-U, and SD-K plates to test other markers of the yeast before cDNA library screening. Reporter strain such as Y190 should be able to grow up on SD-K, SD-U and SD-H plates, but not on SD-W, and SD-L plates. Growth on SD-H plate is due to leaky expression of HIS
reporter gene.
There are many reporter strains available from different resources. In General, Y190 consistently showed higher sensitivity than other yeast strains such as HF7c.
Yeast reporter strains with both IacZ reporter gene and HIS3 reporter gene are strongly recommended. HIS selection will ensure that only interaction positive clones will grow, which makes colony picking much easier later.
Determine optimal 3AT concentration.

3AT can be used to suppress background expression from HIS reporter gene of Y190. 3AT concentration varies amang different reporter strains and ranges from 0 mM (HF7c) to 15 mM (Y190). To test the optimal concentration of 3AT, one yeast colony should be re-suspended in 1 D ml of TE. 100 ul of the re-suspended yeast is spread on SD-H+OmM3AT, SD-H+5mM3AT, SD-H+10mM3AT, SD-H+15mM3AT, SD-H+25mM3AT, and SD-H+40mM3AT plates. Although 15 mM 3AT is sufficient to suppress background HIS expression of Y190, higher concentrations of 3AT
(30-40 mM) are routinely used in our cDNA library screening.
Construct baitplasmid.
pAS2IpACT2 series plasmids showed higher level of sensitivity than pGAD424 /pGBT9 series plasmids (Estojak J, Brent R, Golemis EA (1995) Molecular and Cellular Biology 15:5820-5829; Legrain P, Dokhelar MC, Transy C (1994) Nucleic Acids Research 22:3241-3242). The disadvantage of using pAS2 is the large size of this plasmid (8 kb), which may present a challenge to cloning large cDNA fragments into the plasmid. cDNA fragments should fused to the C-terminal of Gal4 binding domain in frame (Figure 4A). The junction sequence between GAL4 and cDNA should have a GGG amino acid sequence to avoid any interruption of domain structure. Either full-length cDNA or partial fragments can used to generate bait plasmid.
Transform bait into yeast: 1st round.
1 Ng of bait plasmid is transformed into Y19D with small-scale yeast transformation protocol (see SUBPROTOCOL section). Transformants should be plated on 5D-W, SD-WH, and SD-WH+3AT(5-40mM) plates. LacZ color assay can also be done after colonies grow to a diameter of 1 mm. If colonies grow up on SD-WH+40mM3AT plates after 3 days incubation and/or LacZ color assay of these colonies show positive result after only 30 minutes incubation with X-Gal, the bait gene should be determined not suitable for two-hybrid screening without further modification. The bait gene itself may be able to activate transcription of reporter genes HISIIacZ.
Although co-transformation of bait plasmid and cDNA library can be done in a single step, co-transformation efficiency is at least 10 fold lower than single plasmid transformation. Mating approach may also be used to introduce cDNA
library into yeast cells containing the bait vector. Please refer to protocol published by Finley and Brent (Finley R, Brent R (1994) Proc Natl Acad Sci USA 91:12980-12984).
Transform cDNA library: 2nd round.

Y190 containing bait plasmid is grown up for second round of transformation by cDNA library plasmid (see SUBPROTOCOL section). Incubation time after transformation varies significantly from 4 days to 11 days.
Identify aositive clones.
Identification of positive clones needs experience. It should also be pointed out that background colonies at lightly populated area of the plate tend to grow bigger, occasionally reaching the size of a positive colony in a dense area on the same plate. The size of the positive colony should at least 4 times bigger than the neighboring background colonies, Positive colonies may also turn red faster.
Pertorm IacZ color assay.
Positive colonies should be re-streaked to another SD-LWH+3AT plate to isolated single colonies for color assay and plasmid retrieval. See SUBPROTOCOL section for the IacZ color assay protocol. If a colony does not turn blue after a 4-hour incubation, strong protein-protein interaction is highly unlikely. It is not recommended to pick positive clones after 12 hours incubation, except that you know the protein-protein interaction you are studying is very weak.
Retrieve alasmids.
There are several methods to retrieve plasmids from yeast, ranging from lyticase lysis to glass beads. The glass beads method is listed in SUBPROTOCOL section.
Electroporation method is by far the most efficient method to transform plasmids from yeast miniprep into E. coli. Bait and cDNA plasmid may carry different antibiotic selection markers to facilitate separation in E. coli. For example, Rigel's bait plasmid carries Kan~ gene and the cDNA plasmid carries Amp gene.
Verifv positive clones.
cDNA clones recovered from positive HIS/IacZ positive colonies should be re-transformed into yeast with other non-specific bait control to rule non-specific binding. In vitro protein binding assays and function assays should also be done to rule out false positive clones.
PROCEDURE: Yeast One-Hybrid System Screening Constrict HIS and IacZ reporter plasmids.
Selection of a very-well-defined DNA sequence is the most important step for one-hybrid screening. Many DNA sequences lead to significant elevation of the basal expression levels of the reporter genes in yeast even in the absence of the cDNA
library. Multiple copies (-3) of the DNA sequences of interest should be inserted into the multiple cloning sites of both HIS reporter plasmid pHISi-1 and pLacZi (Clontech Cat no.K1603-1" Figure 4B).

Grow up yeast reporter strains on YPD plates from frozen stock.
It is also recommended that the yeast reporter strain be streaked on SD-W, SD-L, SD-H, SD-U, and SD-K plates to test other markers of the yeast before cDNA
library screening. Reporter strain such as YM4271 should be able to grow up on SD-K, SD-U SD-H, SD-W, and SD-L plates.
Integrate HIS reporter into yeast.
To facilitate integration of pHISi reporter into yeast chromosome, pHISI-1 should be linearized at Xho I site. Since pHISi-1 has no yeast replication origin and can not survive in yeast without integration, no gel purification of digested plasmid is required. Transform 1 Ng of digested plasmid into YM4271 using the small-scale yeast transformation protocol (see SUBPROTOCOL section). Use more plasmids if integration efficiency is low. Transformants should be plated on 5D-H, and SD-H
plates with different concentration of 3AT(5-40 mM) and incubated at 30°C for at least 4 days.
Determine optimal 3AT concentrafron.
If more than 40 mM 3AT is needed to suppress transformants growth, the DNA
sequences inserted into pHISi are not suitable for one-hybrid screening.
Note: Integration efficiency of pHISi is very low. 20-100 colonies are expected on SD-H plate.
integrate LacZ reporter plasmid into yeast.
Pick a colony from a SD-H plate from step 3 and freeze as single HIS reporter strain YM4271/H. Linearize pLacZi at Nco I site. Transform 1 ug of linearized plasmid into yeast YM4271M and plate transformants on SD-U plates. This step of integration is very efficient. Several hundred to thousand colonies are expected to grow on each SD-U plates. Pick colonies and freeze as YM4271/HB.
Screen cDNA library for DNA binding protein.
Transform 100-200 ug of cDNA library into YM4271IHB with large-scale yeast transformation protocol (see SUBPROTOCOL section). Transformants should be plated on SD-LH+3AT ( concentration determined at step 4 ).
Identify positive clones.
Same as step 6 in two-hybrid screening procedure.
Perform IacZ color assays.
Same as step 7 in tuvo-hybrid screening procedure.
Retrieve cDNA plasmid.
Same as step 8 in two-hybrid screening procedure.
Verify aositive clones.

DNA gel retardation assay and other function assays are required to verify one-hybrid screening results.
SUBPROTOCOL
Small Scale Yeast Transformation (10S transformantslNg DNA) Inoculate one colony of yeast in 100 ml YPD (without plasmid) or corresponding selection medium (SD-W for Y190 with pAS2) at 240 rpm in a 30°C shaker overnight.
Check ODsoo the next day. If ODooo is between 0.6 and 1.0, the yeast can be used to prepare competent cells. Otherwise, dilute to ODsoo=0.4 and grow another 3 to hours.
Spin down cells in two 50 ml plastic tubes at 3000 rpm at room temperature for minutes. Remove medium.
Add 30 ml TE pH7.5 and re-suspend the cell pellet on vortex at high speed.
Combine cell pellet.
Spin down cells again in at 3000 rpm at room temperature for 5 minutes.
Remove TE.
Estimate the size of the cell pellet and add TE up to total volume of 0.9 ml.
Re-suspend cell completely by pipetting up and down.
Add 100 NI 1M LiAc and mix well by pipetting. Competent cells are ready.
Note: Competent cells can be kept on at room temperature for several hours without significant reduction of transformation efficiency, or at 4°C
overnight with a slight reduction of transformation efficiency.
In a clean eppendorf, mix 1 ug of plasmid with 10 NI 10 mg/ml carrier DNA.
Add 100 NI competent cells from step 8 to the eppendorf and mix well with the DNA.
Add 600 ul PEGILiAc and mix well.
Note: PEG/LiAc should be freshly made. Pre-mixed PEG/LiAc of up to 2 weeks old can also be used if transformation efficiency is not critical.
Incubate at 30°C for 30 minutes with or without shaking.
Add 70 ul DMSO and mix well.
Incubate in 42°C water bath for 15 minutes.
Put on ice for 2 minutes..
Spin dawn cells in an eppendorf centrifuge at 8000 rpm for 1 minute.
Remove supernatant.
Add 150 ul of TE to re-suspend cell pellet.
Plate on selection medium plate. (e.g. SD-W for Y190 transformed by pAS2).
Incubate in a 30°C incubator for 2 to 3 days.

WO 00/43419 PCTliJS00/01431 _77_ Large scale cDNA library transformation (1-10X106 transformants/100 ug cDNA) Inoculate one colony of yeast in 200 mi YPD (one-hybrid screening) or corresponding selection medium (SD-W for two-hybrid screening) at 24U rpm in a 30°C
shaker overnight.
1. Check ODsoo the next day. If ODD is between 0.8 and 1.0, the yeast can be used to prepare competent cells, Otherwise, dilute to ODsoo=0.6 and grow another 3 to 4 hours.
2. Spin down cells in a 250 ml bottles at 3000 rpm at room temperature for 5 minutes.
3. Remove medium.
4. Add 50 ml TE pH7.5 and re-suspend the cell pellet on vortex at high speed.
5. Spin down cells again in at 3000 rpm at room temperature for 5 minutes.

6. Remove TE.

7. Repeat steps 4 to 7 one more time.
Estimate the size of cell pellet and add TE up to total volume of 1.8 ml. Re-suspend cell pellet completely by vortex.
Add 200 NI 1 M LiAc and mix well by vortex.
In a clean eppendort, mix 100-200Ng of plasmid with 200 ul 10 mg/ml carrier DNA.
Add DNA to competent cells drop by drop on a vortex at 5000 rpm.
To ensure suffrcient mixture, vortex at the highest speed for 30 seconds.
Add 12 ml PEGILiAc and mix well.
Note: PEG/LiAc should be freshly made. Pre-mixed PEG/LiAc of up to 2 weeks old can also be used if transformation efficiency is not critical.
Incubate at 30°C for 30 minutes with shaking. Either an orbital shaker or rotator can be used.
Add 140 ul DMSO and mix well.
Incubate in 42°C water bath for 15 minutes. Invert several times during incubation.
Put on ice for 5 minutes to chill.
Spin down cells at 3000 rpm in a bench-top centrifuge for 1 minute.
Remove supernatant.
Add 20 ml TE to re-suspend cell pellet by vortex.
Plate 40o ul on each 15 cm selection medium plates (50 plates total). SD-LWH+40 mM3AT plates are used for Y190 strain two-hybrid screening; SD-LH+3AT plates are used for one-hybrid screening.
Plate 1 ul on a 10 cm plate of SD-LW for transformation efficiency control.
incubate at 30°C for up to 8 days until big colonies appear.

LacZ color assay.
Grow up fresh yeast colonies to a 1 mm diameter.
Fill a container (e.g. ice bucket) with liquid nitrogen.
Use a nylon membrane to lift colonies up from the plate. No special replica-plating device is needed. Simply press the nylon membrane to the plate.
Immerse the nylon membrane (Cat no. N04HY08250, N04HY13250, Fisher Scientific, PA, USA) colony side face down into the liquid nitrogen.
Wait for 20 seconds, remove the nylon membrane and allow to dry on a paper towel for 5 minutes.
In a 10 ml tube, add 40 NI X-Gal to each ml of Z buffer.
Add 1.5 ml Z bufferlX-Gal solution to'a clean 10 cm petri-dish. For 15 cm diameter petri-dish, add 4 ml Z bufferlX-Gal solution.
Add a Whatman circle to the petri-dish, ensuring it is evenly soaked any air bubbles are squeezed out.
Use forceps to transfer the dried nylon membrane with colony side facing up to lie over the soaked Whatman cin:le (Cat no.09-805C, Fisher Scientific, PA, USA). Make sure no air bubble in between the membrane and the circle.
Incubate the petri-dish with lid on in 37°C until blue color is visible.
Yeast plasmid mini-isolation.
Inoculate 3 ml of selection medium ( e.g. SD-L for eDNA library plasmid pACT ) with a yeast colony.
Incubate in a 30°C shaker or rotator overnight or until confluent.
Spin down yeast in a bench-top centrifuge at 3000 rpm at room temperature.
Remove medium and re-suspend pellet in 200 ul lysis buffer. Transfer to an eppendorf.
Add 200 ul volume glass beads.
Note: The lid of eppendorf can be used as scoop to collect 200 NI glass beads.
Add 200 NI phenollchloroformlisoamyl alcohol (25:24:1).
Vortex at the highest speed for 3 minutes.
Spin in micro-centrifuge at 14000 rpm for 10 minutes.
Transfer top water layer to another eppendorf, add 20 ul 3M NaAc and 500 NI
ethanol.
Precipitate should be visible immediately.
Put the eppendort into a dry ice bath for 15 minutes or until frozen.
Spin in a micro-centrifuge at 14000 rpm for 10 minutes.
Remove supernatant and dry pellet.
Wash pellet by 100 ul of 80% ethanol, and dry the pellet in air.
Re-suspend pellet in 30 ul H20 and use 1 ml for electroporation to transform E. coli.

_79_ TWO-HYBRID SCREENING RESULTS
Bait peptide was cloned into pAS2-1 to screen for proteins that can bind thereto.
F~esults are listed below.
cDNA Library Human Lymphocyte Bait Vector pAS2-1 Bait Protein Protein involved in exocytosis Yeast Strain Y190 Number of Transformants 15 million HIS'/IacZ' clones 2 Clone Identity PCNA (2) PCNA is a published binding protein of the bait peptide. Yeast one-hybrid screening results were previously published ( Luo Y, Stile J, Zhu L (1996) BioTechniques 20:564-568).

WO 00/43419 PCflUS00/01431 SEQUENCE LISTING
<110> Rigel Pharmaceuticals, Inc.
<120> Exocytosis Pathway Proteins And Methods Of Use <130> FP-67080-PC/RMS/DAV
<140> Not Yet Assigned <141> 2000-O1-20 <150> 60/116,534 <151> 1999-O1-20 <150> 60/117,308 <151> 1999-O1-26 <150> 60/117,312 <151> 1999-02-26 <150> 60/117,309 <151> 1999-O1-26 <150> 60/117,274 <151> 1999-O1-26 <150> 60/118,178 <151> 1999-02-O1 <150> 60/118,177 <151> 1999-02-O1 <150> 60/118,179 <151> 1999-02-O1 <150> 60/119,998 <151> 1999-02-11 <150> 60/119,759 <151> 1999-02-11 <150> 60/119,286 <151> 1999-02-09 <160> 211 <170> PatentIn Ver. 2.0 <210> 1 <211> 1219 <212> DNA
<213> Mouse <220>
<221> CDS
<222> (123)..(1217) <400> 1 cccggggtgt actggagttc gctgggtgag ggagggggtg tcaagtgtga gacgaggtgc 60 aggacttcca aatttagaac cttgaggccc tttgggctcg ggtccagggc actccgacca 120 cc atg cgc gac agg acc cac gag ttg agg cag ggg gat aac atc tca 167 Met Arg Asp Arg Thr H:is Glu Leu Arg Gln Gly Asp Asn Ile Ser gac gat gaa gat gag gtt cga gtc gcg ctg gtg gtg cac tca ggt get 215 Asp Asp Glu Asp Glu Val Arg Val Ala Leu Val Val His Ser Gly Ala gcc cgg ttg ggc agc ccg gac gac gag ttc ttc cag aag gtg cag aca 263 Ala Arg Leu Gly Ser Pro Asp Asp Glu Phe Phe Gln Lys Val Gln Thr att cgg cag act atg gcc aaa ctg gag agt aaa gtc cgg gag ttg gag 311 Ile Arg Gln Thr Met Ala hys Leu Glu Ser Lys Val Arg Glu Leu Glu aaa cag cag gtc acc atc ctg gcc acg cct ctt ccc gag gag agc atg 359 Lys Gln Gln Val Thr Ile Leu Ala Thr Pro Leu Pro Glu Glu Ser Met aag cag ggc ctg cag aac ctg cga gag gng nnn nna nag ctg ggg aga 407 Lys Gln Gly Leu Gln Asn Leu Arg Glu Xaa Xaa Xaa Xaa Leu Gly Arg a so s5 90 9s gag gtc cgg gcg cag cta aaa gcc ata gag ccc caq aag gag gaa get 455 Glu Val Arg Ala Gln Leu Lys Ala Tle Glu Pro Gln Lys Glu Glu Ala gat gag aat tac aac tca gtc aac aca agg atg aag aaa acc cag cat 503 Asp Glu Asn Tyr Asn Ser Val Asn Thr Arg Met Lys Lys Thr Gln His gga gtc ctg tcc cag caa ttt gtc gag ctc atc aac aag tgc aac tca 551 Gly Val Leu Ser Gln Gln Phe V;~1 Glu Leu Ile Asn Lys Cys Asn Ser atg cag tcc gaa tac cga gag aag aat gtg gag cgg atc cgg agg cag 599 Met Gln Ser Glu Tyr Arg Glu Lys Asn Val Glu Arg Ile Arg Arg Gln ctg aag atc acc aat get gga atg gtg tct gac gag gag ctg gaa cag 647 Leu Lys Ile Thr Asn Ala Gly Met Va1 Ser Asp Glu Glu Leu Glu Gln atg ctg gac agt ggg cag agt gag gtg ttt gtg tct aat atc ctg aag 695 Met Leu Asp Ser Gly Gln Ser Glu Val Phe Val Ser Asn Ile Leu Lys 1s0 185 190 gac acg cag gtg act cgg cag gcc ctg aat gag atc tct gcg cgg cac 743 Asp Thr Gln Val Thr Arg Gln Ala Leu Asn Glu Ile Ser Ala Arg His agt gag atc cag cag ttg gag cgc~ agt atc cga gag ct:c cat gag atc 791 Ser Glu Ile Gln Gln Leu Glu Arg Ser Ile Arg Glu Leu His Glu Ile ttt acg ttt cta get acg gag gtg gag atg cag ggg gag atg atc aac 839 Phe Thr Phe Leu Ala Thr Glu Val. Glu Met Gln Gly Glu Met Ile Asn cgc atc gag aag aac atc ctg agc tcg gcc gac tac gtg gaa cgt ggg 887 Arg Ile Glu Lys Asn Ile Leu Ser Ser Ala Asp Tyr Val Glu Arg Gly caa gag cac gtc aag ata gcc cta gag aat cag aag aag gcg agg aag 935 Gln Glu His Val Lys Ile Ala Leu Glu Asn Gln 'Lys Lys Ala Arg Lys aaa aaa gtc atg att gcc atc tgt gtc tct gtc act ggt ctc atc ttg 983 Lys Lys Val Met Ile Ala Ile Cys Val Ser Val 1'hr Gly Leu Ile Leu get gtc atc att ggc atc acc ata acc gtt gga r_aa tgt cac atg ttc 1031 Ala Val Ile Ile Gly Ile Thr Ile Thr Val Gly Cys His Met Phe ttg gtg aca nat tgg cga gag aca gag acc caa ccc ttg tgc agg tgg 1079 Leu Val Thr Xaa Trp Arg Glu Thr Glu Thr Gln Pro Leu Cys Arg Trp ggc aag cct ccc cgc tcc tgt cag acc cat tt.c ttt ctg ctt ccc ttg 1127 Gly Lys Pro Pro Arg Ser Cys Gln Thr His Phe Phe Leu Leu Pro Leu cag gca ttg gga gag aca gag acc cag ctt ttg tgg agg tgg ggc agg 1175 Gln Ala Leu Gly Glu Thr Glu Thr Gln Leu Leu Trp Arg Trp Gly Arg cct nca act gaa ccc gtc cgc aca ctg gac ctt gtg cag ang gc 1219 Pro Xaa Thr Glu Pro Val Arg Thr Leu Asp Leu Val Gln Xaa <210> 2 c211> 298 <212> PRT
<213> Mouse <400> 2 Met Arg Asp Arg Thr His Ga.u Leu Arg Gln Gly Asp Asn Ile Ser Asp Asp Glu Asp Glu Val Arg Val Ala Leu Val Val His Ser Gly Ala Ala .Arg Leu Gly Ser Pro Asp Asp Glu Phe Phe Gln Lys Val Gln Thr Ile Arg Gln Thr Met Ala Lys Leu Glu Ser Lys Val Arch Glu Leu Glu Lys Gln Gln Val Thr Ile Leu Ala Thr Pro Leu Pro Glu Glu Ser MeL Lys Gln Gly Leu Gln Asn Leu Arg Glu Xaa Xaa Xaa Xaa Leu Gly Arg Glu Val Arg Ala Gln Leu Lys Ala Lle Glu Pro Gln Lys Glu Glu Ala Asp Glu Asn Tyr Asn Ser Val Asn Thr Arg Met Lys Lys Thr Gln His Gly Val Leu Ser Gln Gln Phe Val Glu Leu Ile Asn Lys Cys Asn Ser Met Gln Ser Glu Tyr Arg Glu Lys Asn Val Glu Arg Ile Arg Arg Gln Leu Lys Ile Thr Asn Ala Gly Met Val Sex Asp Glu Glu Leu Glu Gln Met Leu Asp Ser Gly Gln Ser Glu Val Phe Val Ser Asn Ile Leu Lys Asp WO 00/43419 PCT'/US00I01431 Thr Gln Val Thr Arg Gln Ala Leu Asn Glu Ile Ser Ala Arg His 5er Glu Ile Gln Gln Leu Glu Arg Ser Ile Arg Glu Leu His Glu Ile Phe Thr Phe Leu Ala Thr Glu Val Glu Met Gln Gly Glu Met Ile Asn Arg 225 ~ 230 235 240 Ile Glu Lys Asn Ile Leu Ser Ser Ala Asp Tyr Val Glu Arg Gly Gln Glu His Val Lys Ile Ala Leu Glu Asn Gln Lys Lys Ala Arg Lys Lys Lys Val Met Ile Ala Ile Cys Val Ser Val Thr GIy Leu Ile Leu Ala Val Ile Ile Gly Ile Thr Ile Thr val Gly <210> 3 <211> 244 <212> DNA
<213> Mouse a <220>
<221> CDS
<222> (2)..(244) <400> 3 t atg get tan cca tat tnt gtt cta gan tac cct agc ttg ggt ggt can 49 Met Ala Xaa Pro Tyr Xaa Val Leu Xaa Tyr Pro Ser Leu Gly Gly Xaa ntn gcc ang gan gnc nag ggg atc tca att cgc ggt cgn gtc gag gag 97 Xaa Ala Xaa Xaa Xaa Xaa Gly ile Ser Ile Arg Gly Xaa Val Glu Glu aga gag aac tcc gtg naa gnc ctg anc nac tgg gat gtc gag gat nta 145 Arg Glu Asn Ser Val Xaa Xaa Leu Xaa Xaa Trp Asp Val Glu Asp Xaa ctg anc tct ctg cnc ant cnc tca gca tcg cgg gat ntt cnc ggc nct 193 Leu Xaa Ser Leu Xaa Xaa Xaa Ser Ala 5er Arg Asp Xaa Xaa Gly Xaa tcg anc tcg tcg not ctc cat gat cac aan nac nca ctt cca tcn gag 241 Ser Xaa Ser Ser Xaa Leu His Asp His Xaa Xaa Xaa Leu Pro Xaa Glu cag 244 Gln <210> 4 <211> B1 <212> PRT
<213> Mouse <400> 4 Met Ala Xaa Pro Tyr Xaa Val Leu Xaa Tyr Pro Ser Leu Gly Gly Xaa Xaa Ala Xaa Xaa Xaa Xaa Gly Ile Ser Ile Arg Gly Xaa Val Glu Glu Arg Glu Asn Ser Val Xaa Xaa Leu Xaa Xaa Trp Asp Val Glu Asp Xaa Leu Xaa Ser Leu Xaa Xaa Xaa Ser Ala Ser Arg Asp Xaa Xaa Gly Xaa Ser Xaa Ser Ser Xaa Leu His Asp His Xaa Xaa Xaa Leu Pro Xaa Glu Gln <210> 5 <211> 905 <212> DNA
c213> Mouse <220>
c221> CDS
<222> (19..(822) c400> 5 cgg cac gag gtg cct gag ccc agt gca cac agc cag tac aca gtc tct 48 Arg His Glu Val Pro Glu Pro Ser Ala His Ser Gln Tyr Thr Val Ser gtt aag cac ctg gaa caa ggg aag ttc atc atg agc tat aac cac atc 96 Val Lys His Leu Glu Gln Gly Lys Phe Ile Met Ser Tyr Asn His Ile cag atg gag cct cca acc ctc aac ctg acc aag aac aga gac agc tac 144 Gln Met Glu Pro Pro Thr Leu Asn Leu Thr Lys Asn Arg Asp Ser Tyr agc ctg cat tgg gaa act cag aag atg get taC tca ttc att gag cac 192 Ser Leu His Trp Glu Thr Gln Lys Met Ala Tyr Ser Phe Ile Glu His aca ttc cag gtc cag tac aag aag aaa tcg gac agc tgg gag gac agc 240 Thr Phe Gln Val Gln Tyr Lys Lys Lys Ser Asp Ser Trp Glu Asp Ser aag aca gag aac cta gat cga gcc cat agc atg gac ctc tcc cag ctg 288 Lys Thr Glu Asn Leu Asp Arg Ala His Ser Met Asp Leu Ser Gln Leu gag cca gac acc tca tac tgc gcc agg gtg agg gtc aag ccc atc tct 336 Glu Pro Asp Thr Ser Tyr Cys Ala Arg Val Arg Val Lys Pro Ile Ser aac tac gat ggg atc tgg agc aag tcg gca cga get cgt gcc get tgt 384 Asn Tyr Asp Gly Ile Trp Ser Lys Ser Ala Arg Ala Arg Ala Ala Cys gcc gat tga gcg tgt gat gcc cac get gtg gat agt cct cat cct ggt 432 Ala Asp Ala Cys Asp Ala His Ala Val Asp Ser Pro His Pro Gly ctt tct cat cct cac ctt get cct ggc tct tcg ctt: tgg ctg tgt ttc 480 Leu 5er His Pro His Leu Ala Pro Gly Ser Ser Leu Trp Leu Cys Phe tgg gta cag gac gta cag gaa gtg gaa gga aaa gat ccc caa ccc cag 528 Trp Val Gln Asp Val Gln Glu Val Glu Gly Lys Asp Pro Gln Pro Gln caa gag cct cct gtt cca gga tgg agg taa agg tct ctg gcc tcc tgg 576 Gln Glu Pro Pro Val Pro Gly Trp Arg Arg Ser Leu Ala Ser Trp lz cag cat ggc agc ctt cgc cac taa gaa ccc cgc tct cca ggg gcc aca 624 Gln His Gly Ser Leu Arg His Gl.u Pro Arg Ser Pro Gly Ala Thr gag cag get tct tgc tga gca aca ggg ggt gtc ata tga aca ttt gga 672 Glu Gln Ala Ser Cys Ala Thr Gly Gly Val Ile Thr Phe Gly 210 215 22 C) aga caa caa cgt gtc acc tct cac tat aga gga ccc taa tat aat tcg 720 Arg Gln Gln Arg Val Thr Ser His Tyr Arg Gly Pro Tyr.Asn Ser agt tcc acc atc cgg gcc tga tac aac ccc agc tgc ctc atc cga atc 768 Ser Ser Thr Ile Arg Ala Tyr Asn Pro Ser Cys Leu Ile Arg Ile cac aga gca act tcc caa tgt tca agt aga ggg acc aac tcc ttc tag 816 His Arg Ala Thr Ser Gln Cys Ser Ser Arg Gly Thr Asn Ser Phe ccg acc taggaagcaa ttacccagct ttgacttcaa tgggccctac ctggggcctt 872 Pro Thr cccaatccca ctctttggct gatctnccan acn g05 <210> 6 <211> 130 <212> PRT

WO 00/43419 PCTlUS00/01431 <213> Mouse <400> 6 Arg His Glu Val Pro Glu Pro Ser Ala His Ser Gln Tyr Thr Val Ser Val Lys His Leu Glu Gln Gly Lys Phe Ile Met Ser Tyr Asn His Ile Gln Met Glu Pro Pro Thr Leu Asn Leu Thr Lys Asn Arg Asp Ser Tyr Ser Leu His Trp Glu Thr Gln Lys Met Ala Tyr 5e~ Phe Ile Glu His Thr Phe Gln Val Gln Tyr :Lys Lys Lys Ser Asp Ser Trp Glu Asp Ser Lys Thr Glu Asn Leu Asp Arg Ala His Ser Met Asp Leu Ser Gln Leu Glu Pro Asp Thr Ser Tyr Cys Ala Arg Va1 Arg Val Lys Pro Ile Ser Asn Tyr Asp Gly Ile Trp Ser Lye Ser Ala Arg Ala Arg Ala Ala Cys 1.15 12 0 12 S
Ala Asp <210> 7 <211> 204 c212> DNA
<213> Mouse <220>
<221> CDS
<222> (1)..(204) <400> 7 cgg cac gag gac gaa tgg ctg ctg acc tgg aat anc ctg tac cca tcg 48 Arg His Glu Asp Glu Trp Leu Leu Thr Trp Asn Xaa Leu Tyr Pro Ser aac aac taa ctg tac aaa gac ctc atc tcc atg gtc aac atc tcc aga 96 Asn Asn Leu Leu Tyr Lys Asp Leu Ile Ser Met Val Asn Ile Ser Arg nag gac aac cct gca gaa ttc ata gtc tat aan gtg acn tac aag gaa 144 Xaa Asp Asn Pro Ala Glu Phe Ile Val Tyr Xaa Val Xaa Tyr Lys Glu ncc agg ctg anc ttc ccg atc aac atc ctg atg tca ggg gtc tac tat 192 Xaa Arg Leu Xaa Phe Pro Ile Asn Ile Leu Met Ser Gly Val Tyr Tyr tan ggc gng ttt Xaa Gly Xaa Phe <210> 8 <211> 68 <212> PRT
<213> Mouse <400> 8 Arg His Glu Asp Glu Trp Leu Leu Thr Trp Asn Xaa Leu Tyr Pro Ser Asn Asn Leu Leu Tyr Lys Asp Leu Ile Sex Met Val. Asn Ile Ser Arg Xaa Asp Asn Pro Ala Glu Phe Ile Val Tyr Xaa Val Xaa Tyr Lys Glu Xaa Arg Leu Xaa Phe Pro Ile Asn lle Leu Met Ser Gly Val Tyr Tyr Xaa Gly Xaa Phe <210> 9 <211> 254 <212> DNA
<213> Mouse <220>
<221> CDS
<222> (3) .. (254) <400> 9 gg cac gag agt gac ctt ggg ggc tgc ngt ggt gag gag agc tca cgg 47 His Gl.u Ser Asp Leu Gly Gly Cys Xaa Gly Glu Glu Ser Ser Arg gaa tcc tgg ngc agt gta act ggc gtg tca aaa gca gaa acg cag gat 95 Glu Ser Trp Xaa Ser Val Thr Gly Val Sex Lys Ala Glu Thr Gln Asp atg cgn ctt eca get gcc cct gat aca ncg cct tcc act ggg ggt cac 143 Met Xaa Leu Pro Ala Ala Pro Asp Thr Xaa Pro Ser Thr Gly Gly His cat ctc ctg cnt ctg cat cnc gtt gtt ttg cct ytt ctg tta ntt can 191 His Leu Leu Xaa Leu His Xaa Val Val Leu Pro Val Leu Leu Xaa Xaa cat tac cag gan taa gaa cat ntg gng gga cca gat tnc cac ccc agc 239 His Tyr Gln Xaa Glu His Xaa Xaa Gly Pro Asp Xaa His Pro Ser acg cag tnn ctt gnt 254 Thr Gln Xaa Leu Xaa BO
<210> 10 <211> 67 <212> PRT
<213> Mouse <400> 10 His Glu Ser Asp Leu Gly Gly Cys Xaa Gly Glu Glu Ser 5er Arg Glu Ser Trp Xaa Ser Val Thr Gly V'al. Ser Lys Ala Glw Thr Gln Asp Met Xaa Leu Pro Ala Ala Pro Asp Thr Xaa Pro Ser Thx Gly Gly His His Leu Leu Xaa Leu His Xaa Val Val. Leu Pro Val Leu Leu Xaa Xaa His Tyr Gln Xaa 1.8 <210> 11 <211> 526 <212> DNA
<213> Mouse <220>
<221> CDS
<222> (11..(525) <400> 11 cgg cac gag ggt ggc tgt tca cat atc tgc ctt gta aaa gga atg gta 48 Arg His Glu Gly Gly Cys Ser His Ile Cys Leu Va:L Lys Gly Met Val 1 5 :LO 15 cga caa gat get cct gce cca tga act tag ttc tgc ttc agg atg agc 96 Arg Gln Asp Ala Pro Ala Pro Cys. Thr Phe Cys Phe Arg Met Ser tgt cet gtg gag agc ctc ~~aa cgt gtt ctc ctc agc agt tta cct get 144 Cys Pro Val Glu Ser Leu Gln Arg Val Leu Leu Ser Ser Leu Pro Ala tca ctg ggg aca ttg act gca tcc ctg tgg ctt ggc ggt gtg atg ggt 192 Ser Leu Gly Thr Leu Thr Ala Ser Leu Trp Leu Gly Gly Val Met Gly tca ctg agt gcg aag acc aca gcg atg aac tca att gtc ceg tgt get 240 Ser Leu Ser Ala Lys Thr Thr Ala Met Asn Ser Ile Val Pro Cys Ala cag agt ctc agt tcc agt gtg cca gcg ggc agt gca ttg atg gtg ccc 288 Gln Ser Leu Ser Ser Ser Val Pro Ala G1y Ser Ala Leu Met Val Pro ttc gat gca atg gcg atg cga act: gcc agg aca aat cag atg aga aga 336 Phe Asp Ala Met Ala Met Arg Thr Ala Arg Thr Asn Gln Met Arg Arg act gtg aag tgc ttt gtt taa ttg atc agt tcc get gtg cca atg gtc 384 Thr Val Lys Cys Phe Val Leu. Tle Ser Ser Ala Val Pro Met Val agt gcg ttg gaa agc aca aga aat gtg acc aca gtg tgg act gca gtg 432 Ser Ala Leu Glu Ser Thr Arg Asn Val Thr Thr Val Trp Thr Ala Val aca gat ctg acg agc tgg act ggt atc caa ctg agg agc cag cac cac 480 Thr Asp Leu Thr Ser Trp Thr Gly Ile Gln Leu Arg Ser Gln His His aag cca cca aca cag ttg c~tt ncc gta ttg gag taa ttg nca cca t 526 Lys Pro Pro Thr Gln Leu Val Xaa Val Leu Glu Leu Xaa Pro 165 1'10 175 <210> 12 <211> 25 <212> PRT
<213> Mouse <400> 12 Arg His Glu Gly Gly Cys Ser His Ile Cys Leu Val Lys Gly Met Val Arg Gln Asp Ala Pro Ala Pro Cys Thr <210> 13 <211> 301 <212> DNA
<213> Mouse <220>
<221> CDS
<222> (1)..(300) <400> 13 cgg cac gag agc ctg gat gcg ctc cat ctg agc agt ttt ggg ctg atg 48 Arg His Glu Ser Leu Asp Ala Leu His Leu Ser Ser Phe Gly Leu Met gac aca act ctg gag gag gtg ttc ctc aag gtg tct gaa gaa gat cag 96 Asp Thr Thr Leu Glu Glu Val Phe Leu Lys Val. Ser Glu Glu Asp Gln tca ctg gag aac agc gag get gat gtg aag gag tcc cgg aag gat gtg 144 Ser Leu Glu Asn Ser Glu Ala Asp Val Lys Glu Ser Arg Lys Asp Val ctg cct ggg gca gag ggc ctg aca get gtg ggg ggt caa get ggc aac 192 Leu Pro Gly Ala Glu Gly Leu Thr Ala Val Gly Gly G1n Ala Gly Asn ctg get cgn tgc tca gag ctg gcn cag t:ca cag gca tcg ctt gca gtc 240 Leu Ala Xaa Cys Ser Glu Leu Xaa Gln Ser Gln Ala Ser Leu Ala Val tgc atc ctc tgt ggg ctc tgc ccg tgg gna gga ggg cac ccg nct act 288 Cys Ile Leu Cys Gly Leu Cys Pro Trp Xaa Gly Gly His Pro Xaa Thr ntg atg get acg g Xaa Met Ala Thr <210> 14 <211> 100 c212> PRT

<213> Mouse <400> 14 Arg His Glu Ser Leu Asp Ala Leu His Leu Ser Ser Phe Gly Leu Met Asp Thr Thr Leu Glu Glu Val Phe Leu Lys Val Ser Glu Glu Asp Gln Ser Leu Glu Asn Ser Glu Ala Asp Val Lys Glu Ser Arg Lys Asp Val Leu Pro Gly Ala Glu Gly Leu Thr Ala Val Gly Gly Gln Ala Gly Asn Leu Ala Xaa Cys Ser Glu Leu Xaa Gln Ser Gln Ala Ser Leu Ala Val Cys Ile Leu Cys Gly Leu Cys Pro Trp Xaa Gly Gly His Pro Xaa Thr Xaa Met Ala Thr <210> 15 <211> 687 <212> DNA

<213> Unknown <220>
<221> CDS
<222> (1)..(687) <220>
<223> Description of Unknown Organism: Novel <400> 15 ctt acc cat acg atg ttc cag att acg cta get tgg gtg gtc ata tgg 48 Leu Thr His Thr Met Phe G1n Ile Thr Leu Ala Trp Val Val Ile Trp 1 s io 15 cca tgg agg ccc cgg gga tcc gag tgg cac gag cag aag ctc tcc acc 96 Pro Trp Arg Pro Arg Gly Ser Glu Trp His Glu Gln Lys Leu Ser Thr ggc gac gac ggt gca ctg cct ggg ttg att tca gag ggt aaa tat tca 144 Gly Asp Asp Gly Ala Leu Pro Gly Leu Ile Ser Glu Gly Lys Tyr Ser cag gCa cac cga aaa gaa ctc tgc ctt cct ctg cag gac gca ttt gaa 192 Gln Ala His Arg Lys Glu Leu Cys Leu Pro Leu Gln Asp Ala Phe Glu gcg ctg ccc agg gac cag cct cac agc agt gag gta get gag ccg cgg 240 Ala Leu Pro Arg Asp Gln Pro His Ser Ser Glu Val Ala Glu Pro Arg cag cct gat gtc acg gcc agt gat: ggg aaa tca gca cag agt cag get 28B
Gln Pro Asp Val Thr Ala Ser Asp G1y hys Ser Ala Gln Ser Gln Ala ggc ttg gag act ggt cct gag agt gca ctc tgt ggt gac agg aaa gcc 336 Gly Leu Glu Thr Gly Pro Glu Ser Ala Leu Cys Gl;y Asp Arg Lys Ala tgt gac gta agc aca ctg tgt ctg gaa gtg tgt atg gcc cca gaa gaa 384 Cys Asp Val Ser Thr Leu Cys Leu Glu Val Cys Met Ala Pro Glu Glu agg cgg gac agt gaa gac cga gtg agt aag gaa acg gaa gac tat ctg 432 Arg Arg Asp Ser Glu Asp Arg Val Ser Lys Glu Thr Glu Asp Tyr Leu 130 :135 140 cac agc ctt ttg gag aga tgc ttg aag gat get gaa gat tcc ctg tcc 480 His Ser Leu Leu Glu Arg Cys Leu Lys Asp Ala Glu Asp Ser Leu ser tat gag gac atc cag gac gac gac tct gac ctc ctc caa gac ctc tct 528 Tyr Glu Asp Ile Gln Asp Asp Asp Ser Asp Leu Leu Gln Asp Leu Ser cct gaa gaa gcg tcc tac agt ctc cag gay gat. ctg cct cct gac gag 576 Pro Glu Glu Ala Ser Tyr Ser Leu Gln Glu Asp Leu Pro Pro Asp Glu WO 00/43419 PC'I'/US00/01431 lao ls5 190 agc acg ctc ttc cct tga tga tct tgg nca aga aga tag aga tcg can 624 Ser Thr Leu Phe Pro Ser Trp Xaa Arg Arg Arg Ser Xaa aag gca ttc ctg cag aaa ggc tgg tgt nca tac tga aga aga gga atg 672 Lys Ala Phe Leu Gln Lys Gly Trp Cys Xaa Tyr Arg Arg Gly Met aca cag tag gca gcc 687 Thr Gln Ala Ala <210> 16 <211> 19'7 <212> PRT
<213> Unknown <400> 16 Leu Thr His Thr Met Phe Gln Ile Thr Leu Ala Trp Val Val Ile Trp Pro Trp Arg Pro Arg Gly Ser Glu Trp His Glu G1:: Lys Leu Ser Thr Gly Asp Asp Gly Ala Leu Pro Gly Leu Ile Ser t:,lu Gly Lys Tyr Ser Gln Ala His Arg Lys Glu Leu Cys Leu Pro Leu Gln Asp Ala Phe Glu Ala Leu Pro Arg Asp Gln Pro His Ser Ser Glu Val Ala Glu Pro Arg Gln Pro Asp Val Thr Ala Ser Asp Gly Lys Ser Ala Gln Ser Gln Ala Gly Leu Glu Thr Gly Pro Glu Ser Ala Leu Cys Gly Asp Arg Lys Ala Cys Asp Val Ser Thr Leu Cys Leu Glu Val Cys Met Ala Pro Glu Glu Arg Arg Asp Ser Glu Asp Arg Val. Ser Lys Glu Thr Glu Asp Tyr Leu His Ser Leu Leu Glu Arg Cys Leu Lys Asp Ala Glu Asp Ser Leu Ser Tyr Glu Asp Ile Gln Asp Asp Asp Ser Asp Leu Leu Gln Asp Leu Ser Pro Glu Glu Ala Ser Tyr Ser Leu Gln Glu Asp Leu Pro Pro Asp Glu WO 00/43419 PC'T/US00/01431 Ser Thr Leu Phe Pro <210> 17 <211> 503 <212> DNA
<213> Unknown <220>
<221> CDS
<222> (1)..(501) <220>
<223> Description of Unknown Organism: Novel <400> 17 ccc agt ggc tgc get get cat cct tct att cat ggt tct cat tgc gac 48 Pro Ser Gly Cys Ala Ala His Pro Ser T_le His Gly Ser His Cys Asp cta ctt taa aag caa gag acc taa gca aga gcc ttc tag cca agg atc 96 Leu Leu Lys Gln Glu Thr Ala Arg Ala Phe Pro Arg Ile tca aag tgc ctt gca gac aca tga act tgg agg tga aac cct gaa agt 144 Ser Lys Cys Leu Ala Asp Thr Thr Trp Arg Asn Pro Glu Ser 35 4tJ 45 ccc tat ttt tga aga gga cac ac<: ctc tgt tat gga aat aga gat gga 192 Pro Tyr Phe Arg Gly His Thr Leu Cys Tyr Gly Asn Arg Asp Gly aga get tga taa atg gat gaa cag cat gaa cag aaa cgc cga cta tga 240 Arg Ala Met Asp Glu Gln His Glu Gln Lys Arg Arg Leu gtg ttt acc tac ttt gaa gga aga aaa gga acc aaa ccc cag ccc aag 288 Val Phe Thr Tyr Phe Glu Gly Arg Lys Gly Thr Lys Pro Gln Pro Lys tga caa tga atc cta agt cca aga ggc gtt gat gtr_ ttc caa gag cgt 336 Gln Ile Leu Ser Pro Arg Gly Val Asp Val Phe Gln Glu Arg act tcc gga ggg cac ctg cca gcc tgc cag ggg agg atg aag aag aga 384 Thr Ser Gly Gly His Leu Pro Ala Cys Gln Gly Arg Met Lys Lys Arg caa gtt taa tta att tca aat cag tgt aga cac aag agc att tta gtg 432 Gln Val Leu Ile Ser Asn Gln Cys Arg His Lys Ser Ile Leu Val 130 135 14C~
ntt ctt cca ctg tcc cct ctt cgg acg atg aca tcc ctg gac ttg gaa 480 Xaa Leu Pro Leu Ser Pro Leu Arg Thr Met Thr Ser Leu Asp Leu Glu gaa tga gga ctg gaa gtc ttg ga 503 Glu Gly Leu Glu Val Leu <210> 18 <211> 18 <212> PRT
<213> Unknown <400> 18 Pro Ser Gly Cys Ala Ala His Pro Ser Ile His Gly Ser His Cys Asp Leu Leu <210> 19 <211> 499 <212> DNA
<213> Unknown <220>
<221> CDS
<222> (2) .. (499) :30 <220>
<223> Description of Unknown Organism: Nove <400> 19 c ggc acg agg acc ctc tct tgt cca agc tta aga cag cgc ctg tgg aac 49 Gly Thr Arg Thr Leu Ser Cys Pro Ser Leu Arg Gln Arg Leu Trp Asn cgt cac cag aga gga aaa gcc ttg caa ggg cga taa tgt ctg aag agg 97 Arg His Gln Arg Gly Lys Ala Leu Gln Gly Arg Cys Leu Lys Arg cag tgg gga cag aag cag cag cca aag agc cag aga ttg aaa cct gtc 145 Gln Trp Gly Gln Lys Gln Gln Pro Lys Ser Gln Arg Leu Lys Pro Val cca gca cgg atc caa gtg gag acc ggc atg agg agg agc ccc agg aga 193 Pro Ala Arg Ile Gln Val Glu Thr Gly Met Arg Arg Ser Pro Arg Arg gca gcc ccg get gtc atc aga tgg agt ggc aga cag ctt ccc ccg agc 241 Ala Ala Pro Ala Val Ile Arg Trp Ser G1y Arg Gln Leu Pro Pro Ser ttc cag gga ctg cag gaa agg acc aca cgg agg agc tgc cca gca gca 289 Phe Gln Gly Leu Gln Glu Arg Thr Thr Arg Arg Ser Cys Pro Ala Ala cca atg cca cac tgg gac ctc cac acc cag tcc ctg gga gac agc cgg 337 Pro Met Pro His Trp Asp Leu His Thr Gln Ser Le~u Gly Asp Ser Arg gag caa gtc ang gcc agc tgc tgn ctc gaa tgc ctg caa aga cag can 385 Glu Gln Val Xaa Ala Ser Cys Cys Leu Glu Cys Leu Gln Arg Gln Xaa ctg cgt gcc ggc gcc tcc cac aga agg acc act. gcg gtg tgg cca aaa 433 Leu Arg Ala Gly Ala Ser His Arg Arg Thr Thr Ala Val Trp Pro Lys gac cct aag gtg ggc tcc tcc taa tga atc agt ggc aga gca gaa get 481 Asp Pro Lys Val Gly Ser Ser Ile Ser Gly Arg Ala Glu Ala ntc caa cgg gga cga cgg 499 Xaa Gln Arg Gly Arg Arg <210> 20 <211> 27 <212> PRT
<213> Unknown <400> 20 3 ?.

Gly Thr Arg Thr Leu Ser Cys Pro Ser Leu Arg Gln Arg Leu Trp Asn Arg His Gln Arg Gly Lys Ala Leu Gln Gly Arg <210> 21 <211> 157 <212> DNA
<213> Unknown <220>
<221> CDS
<222> (2)..(157) c220>
c223> Description of Unknown Organism: Novel <400> 21 g cat naa nat gga cca gtc act can aga ggt agg aac agc ata ttg aaa 49 His Xaa Xaa Gly Pro Val. Thr Xaa Arg Gly Arg Asn Ser Ile Leu Lys tgg aac gta tag tgc gca tca gcc gca ttc agc agn acc nna gag cat 97 Trp Asn Val Cys Ala Ser Ala Ala Phe Ser Xaa Thr Xaa Glu His tgg nng aaa tgn ata ngn agn ntg cga tnc ngc ngg aan aca ngg aan 145 Trp Xaa Lys Xaa Ile Xaa Xaa Xaa Arg Xaa Xaa Xaa Xaa Thr Xaa Xaa aga gtt gtc nag 157 Arg Val Val Xaa <210> 22 <211> 19 <212> PRT
<213> Unknown <400> 22 His Xaa Xaa Gly Pro Val Thr Xaa Arg Gly Arg Asn Ser Ile Leu Lys Trp Asn Val <210> 23 <211> 169 <212> DNA
<213> Unknown <220>

<221> CDS
<222> (1)..(168) <220>
<223> Description of Unknown Organism: Novel <400> 23 tng ctt anc cat ntn acn tnc cac act ang ctn gnt tgg ntg ntc gtn 48 Xaa Leu Xaa His Xaa Xaa Xaa His Thr Xaa Xaa Xaa Trp Xaa Xaa Xaa agg gca gcg ctg ccc gat ccg atc ctc ngt ncg agg acg cgn nct tgg 96 Arg Ala Ala Leu Pro Asp Pro Ile Leu Xaa Xaa Arg Thr Xaa Xaa Trp tcc ata ctc nag ncn gta tct gtg caa ncg tnt gca cag gag aga aaa 144 Ser Ile Leu Xaa Xaa Val Ser Val Gln Xaa Xaa Ala Gln Glu Arg Lys gcc ctn cat ggg gca ann atn tnt g 169 Ala Xaa His Gly Ala Xaa Xaa Xaa <210> 24 <211> 56 <212> PRT
<213> Unknown <400> 24 Xaa Leu Xaa His Xaa Xaa Xaa His 'fhr Xaa Xaa Xaa Trp Xaa Xaa Xaa Arg Ala Ala Leu Pro Asp Pro Ile Leu Xaa Xaa Arg Thr Xaa Xaa Trp Ser Ile Leu Xaa Xaa Val Ser Val Gln Xaa Xaa Ala Gln Glu Arg Lys Ala Xaa His Gly Ala Xaa Xaa Xaa <210> 25 <211> 201 <212> DNA
<213> Unknown <220>
<221> CDS
<222> (3) . . (200) <220>
<223> Description of Unknown Organism: Novel <400> 25 cg gnn cna ngt tgt ttt ctt agg not ttt tgn ttn ntc ana agg ctc 47 Xaa Xaa Xaa Cys Phe Leu Arg Xaa Phe Xaa Xaa Xaa Xaa Arg Leu tta tca ctt tgg gat atg gca tnn nat ngc ttg cct cta acc cta nca 95 Leu Ser Leu Trp Asp Met Ala Xaa Xaa Xaa Leu Pro Leu Thr Leu Xaa tgt aac aag gnc ggc cat gga cnc ata ggn ctt cct gcc ttc anc ttc 143 Cys Asn Lys Xaa Gly His Gly Xaa Ile Xaa Leu Prc Ala Phe Xaa Phe caa gtg ntg ant ctn ann gtn tgn acn gcc ana ctc anc ttc ccc att 191 Gln Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala Xaa Leu Xaa Phe Pro Ile gtt ttg att g 201 Va1 Leu Ile <210> 26 <211> 66 <212> PRT
<213~ Unknown <400> 26 Xaa Xaa Xaa Cys Phe Leu Arg Xaa Phe Xaa Xaa Xaa Xaa Arg Leu Leu WO 00/43419 PCTlU500/01431 Ser Leu Trp Asp Met Ala Xaa Xaa Xaa Leu Pro Leu Thr Leu Xaa Cys Asn Lys Xaa Gly His Gly Xaa Ile Xaa Leu Pro Ala Phe Xaa Phe Gln Val Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala Xaa Leu Xaa Phe Pro Ile Val Leu Ile <210> 27 <211> 538 <212> DNA
<213> Mouse <220>
<221> CDS
<222> (1)..(537) <400> 27 gcc aag tcc tat cac caa taa tcc aca cag tag cac: tct cgg gaa cct 48 Ala Lys Ser Tyr His Gln Ser Thr Gln I3is Ser Arg Glu Pro 1 5 1.0 15 ggt gcc cac tag cga gac ttt va~ gcn gac gaa ggg cca cga gga gga 96 Gly Ala His Arg Asp Phe 'Cy.r Xaa Asp Glu Gly Pro Arg Gly Gly cta aga tga cgg cca cca agg atc acg gat cac aag cat ctt ccg gtt 144 Leu Arg Arg Pro Pro Arg Tle Thr Asp His Lys His Leu Pro Val 35 44) 45 ett ett etg gta ctt get cag ect tgt gaa get gtt tca agc cat ctt 192 Leu Leu Leu Val Leu Ala Gln Pro Cys Glu Ala Va1 Ser Ser His Leu cgg tct tga cac agg act gtt caa cat tat agt caa ttc tat caa gga 240 Arg Ser His Arg Thr Val Gln His Tyr Ser Gln Phe Tyr Gln Gly ccg tte ect get cca caa tca tgg ctc eca agt ccc tga aga ttt cat 288 Pro Phe Pro Ala Pro Gln Ser Trp Leu Pro Ser Pro Arg Phe His tga ggt ctg aaa tgg act gca caa tct gac gga tct etc get cce tct 336 Gly Leu Lys Trp Thr Ala Gln Ser Asp Gly Ser Leu Ala Pro Ser cat cca cca tca gtg tgt ttt get cta cca gca cca get ggt cat ccg 384 His Pro Pro Ser Val Cys Phe A1a Leu Pro Ala Pro Ala Gly His Pro tga agc cct gac cat aca gag tag cat cat ctc cat cat cca tta gtg 432 Ser Pro Asp His Thr Glu His His Leu His His Pro Leu Val gta ctg ggg tgt cga aga aat get gcg atc tct cct ctc ggt ttt tca 480 Val Leu Gly Cys Arg Arg Asn Ala Ala Ile Sex' Pro Leu Gly Phe Ser tgc ctg tgt gac ttc ctg ggt ggt gat ctc gat ggc atg ttc cgt cga 528 Cys Leu Cys Asp Phe Leu Gly Gl.y Asp Leu Asp G1y Met Phe Arg Arg ngc ggc cgn g Xaa Gly Xaa <210> 28 <211> 6 <212> PRT
<213> Mouse <400> 28 Ala Lys Ser Tyr His Gln <210> 29 <211> 510 <212> DNA
<213> Mouse <220>
<221> CDS
<222> (2)..(508) <400> 29 c gag gtg atc gag gtg gat atg gag gag acc gca gcc ggg ggg cct atg 49 Glu Val Ile Glu Val Asp Met Glu Glu Thr Ala Ala Gly Gly Pro Met gag gag acc gtg gtg gta gtg gta gtg gca gtg gag get acg gtg gag 97 Glu Glu Thr Val Val Val Val Val Val Ala Va1 Glu Ala Thr Val Glu aca gaa gtg gag get acg gtg gag acc gaa gtg gtg get acg gag gag 145 Thr Glu Val Glu Ala Thr Val Glu Thr Glu Val Val Ala Thr Glu Glu acc gag gtg get acg gag gag acc gag gtg get atg gag gca aaa tgg 193 Thr Glu Val Ala Thr Glu Glu Thr Glu Val Ala Met Glu Ala Lys Trp gag gaa gaa acg act aca gaa atg atc agc gca acc gac cat act gat 241 Glu Glu Glu Thr Thr Thr Glu Met Ile Ser Ala Thr Asp His Thr Asp gac tgt ttc aga tac tcc ttt gtc tct gac atg atc cgt ggg gaa ttg 289 Asp Cys Phe Arg Tyr Ser Phe Val Ser Asp Met Ile Arg Gly Glu Leu tca gag tgt gcc tgc tgc ttt cct cat ggc ctc ttg ggt agt gaa att 337 Ser Glu Cys Ala Cys Cys Phe Prc> His Gly Leu Leu Gly Ser Glu Ile gac att tgg gtt ttt att tgg gtg gga ggg ttg ggg gca gtt ttc ttt 385 Asp Ile Trp Val Phe Ile Trp Val. Gly Gly Leu Gly Ala Val Phe Phe tta gaa atg tcc att aaa aaa atc ccc ttt tat ttt ccc acc ttt tct 433 Leu Glu Met Ser Ile Lys Lys Ile Pro Phe Tyr Phe Pro Thr Phe Ser ttc cta acc ctt aaa get aag tgc gtt gta aaa tat tgg cca aat gga 481 Phe Leu Thr Leu Lys Ala Lys Cys Val Val Lys Tyr Trp Pro Asn Gly aag tgt ttt gna ata ctg caa taa aag cc 510 Lys Cys Phe Xaa Ile Leu Gln Lys <210> 30 <211> 167 <212> PRT
<213> Mouse <400> 30 Glu Val Ile Glu Val Asp Met Glu Glu Thr Ala Ala Gly Gly Pro Met Glu Glu Thr Val Val Val Val Val Val Al.a Val Glu Al.a Thr Val Glu Thr Glu Val Glu Ala Thr Val Glu Thr Glu Val Val Ala Thr Glu Glu Thr Glu Val Ala Thr Glu Glu Thr Glu Val Ala Met Glu Ala Lys Trp Glu Glu Glu Thr Thr Thr Glu Met Ile Ser Ala Thr Asp His Thr Asp Asp Cys Phe Arg Tyr Ser Phe Val Ser Asp Met Ile Arg Gly Glu Leu Ser Glu Cys Ala Cys Cys Phe Pro His Gly Leu Leu Gly Ser Glu Ile Asp Ile Trp Val Phe Ile Trp Val Gly Gly Leu Gly Ala Val Phe Phe 1.15 120 125 WO 00/43419 PGTlUS00/01431 Leu Glu Met Ser Ile Lys Lys Il.e Pro Phe Tyz Phe Pro Thr Phe Ser Phe Leu Thr Leu Lys Ala Lys Cys Val Val Lys Tyr Trp Pro Asn Gly Lys Cys Phe Xaa Ile Leu Gln <210> 31 <211> 513 <212> DNA
<213> Unknown <220>
<221> CDS
<222> (2)..(511) <220>
<223> Description of Unknown Organism: Novel <40D> 31 c ctg gca ctg aaa aca cca cca cgc gtg ctc aca ctg agt gag aga cca 49 Leu Ala Leu Lys Thr Pro Pro hrg Val Leu Thr Leu Ser Glu Arg Pro cta gat ttc ctg gat tta gaa aga cca ctt cca act cct caa agc gaa 97 Leu Asp Phe Leu Asp Leu Glu Arg Pro Leu Pro Thr Pro Gln Ser Glu gag agc cga gca gtt ggc agg cta aaa aga gag cgt tct atg agt gaa 145 Glu Ser Arg Ala Val Gly Arg Leu Lys Arg Glu Arg Ser Met Ser Glu aat get gtt cgc caa aat gga cag ttg gtc aga aac gat tcc atg tat 193 Asn Ala Val Arg Gln Asn Gly Gln Leu Val Arg Asn Asp Ser Met Tyr ggc att tca aac cta gat gca gcc att gaa gga get tca gat gac atg 241 Gly Ile Ser Asn Leu Asp Ala Ala Ile Glu Gly Ala Ser Asp Asp Met act gtg gta gat gca get tca tt~a aga cgt cag ata atc aaa tta aat 289 Thr Val Val Asp Ala Ala Ser Leu Arg Arg Gln Ile Ile Lys Leu Asn aga cgt ctc caa ctt ctg gaa gag gag aat aaa gag cgt get aaa aga 337 Arg Arg Leu Gln Leu Leu Glu Glu Glu Asn Lys Glu Arg Ala Lys Arg gaa atg gtc atg tat tca atc act gta gcg ttc tgg ctg ctt aac agc 385 Glu Met Val Met Tyr Ser Ile Thr Val A1a Phe Trp Leu Leu Asn Ser 115 1.2D 125 tgg ctc tgg ttt cga cgc tag agg taa cct cag cat tca cat ata ttg 433 Trp Leu Trp Phe Arg Arg Arg Pro Gln His Ser His Ile Leu get caa cac ctg gaa ata taa agg att tgc aaa ctt cat tgg ttc tgg 481 Ala Gln His Leu Glu Ile Arg Ile Cys Lys Leu His Trp Phe Trp ctt tgc atc atc gna tga ccg gcc tga aaa tg 513 Leu Cys Ile Ile Xaa Pro Ala Lys 165 1'70 <210> 32 <211> 134 <212> PRT
<213> Unknown <400> 32 Leu Ala Leu Lys Thr Pro Pro Arg Val Leu Thr Leu Ser Glu Arg Pro Leu Asp Phe Leu Asp Leu Glu Arg Pro Leu Pro ~hr Pro Gln Ser Glu Glu Ser Arg Ala Val Gly Arg Leu Lys Arg G1u Arg Ser Met Ser Glu Asn Ala Val Arg Gln Asn Gly Gln Leu Val Arg Asn Asp Ser Met Tyr Gly Ile Ser Asn Leu Asp Ala Ala Ile Glu Gly Ala Ser Asp Asp Met 65 70 ?5 80 Thr Val Val Asp Ala Ala Ser Leu Arg Arg Gln Ile Ile Lys Leu Asn Arg Arg Leu Gln Leu Leu Glu Glu Glu Asn Lys Glu Arg Ala Lys Arg Glu Met Val Met Tyr Ser Ile Thr Val Ala Phe Trp Leu Leu Asn Ser Trp Leu Trp Phe Arg Arg <210> 33 <211> 507 <212> DNA
<213 > Unknown <220>
<221> CDS
<222> (2)..(505) <220>

<223> Description of Unknown Organism: Novel <400> 33 g gtc ggg agc gag agc gcc agc tat cct gag ggc tcg tgc cga tct ttc 49 Val Gly Ser Glu Ser Ala Ser Tyr Pro Glu G1y Ser Cys Arg Ser Phe agc ctt ctg gcc ccc atc cgc acc aag gac atc aga agc agg agc tat 97 Ser Leu Leu Ala Pro Ile Arg Thr Lys Asp Ile Arg Ser Arg Ser Tyr ctg gag gga agt ctt ctg gcc agt ggg gcc ctg cta gga gca gaa gag 145 Leu Glu Gly Ser Leu Leu Ala Ser Gly Ala Leu Leu Gly Ala Glu Glu ctg gcc agg tac ttc cca gac cga aac atg get ctc ttc gtg get acc 193 Leu Ala Arg Tyr Phe Pro Asp Arg Asn Met Ala Leu Phe Val Ala Thr tgg aac atg cag ggc cag aag gag ctc cca gcg agc ctg gat gag ttt 241 Trp Asn Met Gln Gly Gln Lys Glu Leu Pro Ala Ser Leu Asp Glu Phe ctg ctc ccc acc gag get gac tac: act cag gac ctg tat gtc att gga 289 Leu Leu Pro Thr Glu Ala Asp Tyr Thr Gln Asp Leu Tyr Val Ile Gly att cag gag ggc tgc tct gac agg cgg gag tgg gag aca cgc ctg cag 337 Ile Gln Glu Gly Cys Ser Asp Arg Arg Glu Trp Glu Thr Arg Leu Gln gag aca ctg ggc cct cag tat gta ctg ctg gca tca gca gca cat ggg 385 Glu Thr Leu Gly Pro Gln Tyr Val Leu Leu Ala Ser Ala Ala His Gly gtc ctg tac atg tcc ctg gtt atc cgt agg gac ctc atc tgg ttc tgc 433 Val Leu Tyr Met Ser Leu Val Ile Arg Arg Asp Leu Ile Trp Phe Cys tca nag gtc cga gta ctc cac agg taa cta cac cgc atn gtg nct cag 481 Ser Xaa Val Arg Val Leu His Arg Leu His Arg Xaa Val Xaa Gln atc aag acc aag ggg gcc ctg gga gt 507 Ile Lys Thr Lys Gly Ala Leu Gly <210> 34 <211> 152 <212> PRT
<213> Unknown <400> 34 Val Gly Ser Glu Ser Ala Ser Tyr Pro Glu Gly Ser Cys Arg Ser Phe WO 00/43419 PC'IYUS00/01431 Ser Leu Leu Ala Pro Ile Arg Thr L~ys Asp Ile Arg Ser Arg Ser Tyr Leu Glu Gly Ser Leu Leu Ala Ser Gly .Ala Leu Leu Gly Ala Glu Glu Leu Ala Arg Tyr Phe Pro Asp Arg Asn Met Ala Leu Phe Val Ala Thr Trp Asn Met Gln Gly Gln Lys Glu Leu Pro Ala Ser Leu Asp Glu Phe Leu Leu Pro Thr Glu Ala Asp Tyr Thr Gln Asp Leu Tyr Val Ile Gly Ile Gln Glu Gly Cys Ser Asp Arg Arg Glu Trp Glu Thr Arg Leu Gln Glu Thr Leu Gly Pro Gln Tyr Val Leu Leu Ala Ser Ala Ala His Gly Val Leu Tyr Met Ser Leu Val Ile:.Arg Arg Asp Leu Iie Trp Phe Cys Ser xaa Val Arg Val Leu His Arg WO 00/43419 PCTlUS00/01431 <210> 35 <211> 505 <212> DNA
<213> Unknown <220>
<221> CDS
<222> (2).,(502) <220>
<223> Description of Unknown Organism: Novel <400> 35 c caa aga tgg agc tca gac gac cct agc gaa ggt tgc ggt aga ctc ggc 49 Gln Arg Trp Ser Ser Asp Asp Pro Ser Glu Gly Cys Gly Arg Leu Gly gcg gga gga ccc ggg caa gca ccg gcg get ggg gag get gcc att agc 97 Ala Gly Gly Pro Gly Gln Ala Pro Ala Ala Gly Glu Ala Ala Ile Ser tgc ctt cat tct gcc gaa tgg tcc aag atg gaa agt cat aaa cca agt 145 Cys Leu His Ser Ala Glu Trp Ser Lys Met Glu Ser His Lys Pro Ser act agc aaa gat gat tta ata ctt aat atc ata tcc aga aaa att aaa 193 Thr Ser Lys Asp Asp Leu Ile Leu Asn Ile Ile Ser Arg Lys Ile Lys caa ctt cca gaa tca gac agg aat ctg ctt gaa tat gga tca gca tat z41 Gln Leu Pro Glu Ser Asp Arg Asn Leu Leu Glu Tyr Gly Ser Ala Tyr att gga ctt aat get get ttt ggg ggc cta ata gca aac agt cta ttt 289 Ile Gly Leu Asn Ala Ala Phe Gly Gly Leu Ile Ala Asn Ser Leu Phe cgg ega atc ttg aac gtg aca caa get ega tta get tct agc tta cca 337 Arg Arg Ile Leu Asn Val Thr Gln Ala Arg Leu Ala Ser Ser Leu Pro atg get gtg atc cca ttt ttg aea gca aac ctg tcc tac caa agt ett 385 Met Ala Val Ile Pro Phe Leu Thr Ala Asn Leu Ser Tyr Gln Ser Leu gta agt tta cct ttg agt aca ggt gat ttg aac tgt gaa acc tgt acc 433 Val Ser Leu Pro Leu Ser Thr Gly Asp Leu Asn Cys Glu Thr Cys Thr aca aca cgg ggt gca cta gtt ggt ttg gtt atg ggt ggc tgt acc cta 481 Thr Thr Arg Gly Ala Leu Val Gly Leu Val Met GIy Gly Cys Thr Leu tcc ttt tgg cta tcc cgc aat ggg 505 Ser Phe Trp Leu Ser Arg Asn <210> 36 <211> 167 <212> PRT
<213> Unknown <400> 36 Gln Arg Trp Ser Ser Asp Asp Pro Ser Glu Gly Cys Gly Arg Leu Gly Ala Gly Gly Pro Gly Gln Ala Pro Ala Ala Gly Glu Ala Ala Ile Ser Cys Leu Hie Ser Ala Glu Trp Sex Lys Met Glu Ser His Lys Pro 5er Thr Ser Lys Asp Asp Leu Ile Letn Asn Ile Ile Ser Arg Lys Ile Lys Gln Leu Pro Glu Ser Asp Arg Asn Leu Leu Glu Tyr Gly Ser Ala Tyr Ile Gly Leu Asn Ala Ala Phe Gly Gly Leu Ile Ala Asn Ser Leu Phe Arg Arg Ile Leu Asn Val Thr G1n Ala Arg Leu Ala Ser Ser Leu Pro Met Ala Val Ile Pro Phe Leu Thr Ala Asn Leu Ser Tyr Gln Ser Leu 115 1<?0 125 Val Ser Leu Pro Leu Ser Thr Gly Asp Leu Asn Cys Glu Thr Cys Thr Thr Thr Arg Gly Ala Leu Val Gly Leu Val Met Gly Gly Cys Thr Leu Ser Phe Trp Leu Ser Arg Asn <210> 37 c211> 355 <212> DNA
<213> Mouse <220>
<221> CD5 <222> (3) . . (353) <400> 37 cg gca cga gag tct cac aga tca gcg aag aca aac tac ccg ggt gac 47 Ala Arg Glu Ser His Arg Sex' Ala Lys Thr Asn Tyr Pro Gly Asp tcc tcc ggc cag ccg ggc ccc tca gat gag ggc tgc tcc ccg aag agc 95 Ser Ser Gly Gln Pro Gly Pro Ser Asp Glu Gly Cys Ser Pro Lys Ser acc tgc agc tca gcc tcc agc agc agc tgc gcc atc tgc agt tgg ctc 143 Thr Cys Ser Ser Ala Ser Ser SeY Ser Cys Ala Ile Cys Ser Trp Leu acc tgc cgc tgc gcc ccg gca gcc agg cct gat ggc cca gat ggc tac 191 Thr Cys Arg Cys Ala Pro Ala Ala Arg Pro Asp Gly Pro Asp Gly Tyr cac cgc agc cgg tgt ggc tgt ggg ctc tgc agt ggg aca cac cct ggc 239 His Arg Ser Arg Cys Gly Cys Gly Leu Cys Ser Gly Thr His Pro Gly tca tac tgt att aaa ttg tat aat tcc tta aaa ctc aac ttc aca ggc 287 Ser Tyr Cys Ile Lys Leu Tyr Asn Ser Leu Lys Leu Asn Phe Thr Gly tca ctc gtg ccg att cga get cga gag atc tat gaa tcg tag ata ctg 335 Ser Leu Val Pro Ile Arg Ala Arg Glu Ile Tyr Glu Ser Ile Leu aaa aac ccc gca agg nan as 355 Lys Asn Pro Ala Arg Xaa <210> 38 <211> 108 <212> PRT
<213> Mouse <400> 38 Ala Arg Glu Ser Hie Arg Ser Ala Lys Thr Asn Tyr Pro Gly Asp Ser Ser Gly Gln Pro Gly Pro Ser Asp Glu G1y Cys Ser Pro Lys Ser Thr Cys Ser 5er Ala Ser Ser Ser Ser Cys Ala Ile Cys Ser Trp Leu Thr 35 40 q,5 Cys Arg Cys Ala Pro Ala Ala Arr3 Pro Asp Gly Pro Asp Gly Tyr His Arg Ser Arg Cys Gly Cys Gly Leu Cys Ser G1y Thr His Pro Gly Ser Tyr Cys Ile Lys Leu Tyr Asn Ser Leu Lys Leu Asn Phe Thr Gly Ser Leu Val Pro Ile Arg Ala Arg Glu Ile Tyr Glu Ser <210> 39 <211> 381 <212> DNA
<213> Mouse <220>
<221> CDS
<222> (1)..(381) <400> 39 ccg gca gca gcg gat gat caa gaa tcg aga gtc ggc ctg cca gtc ccg 48 Pro Ala Ala Ala Asp Asp Gln Glu Ser Arg Val Gly Leu Pro Val Pro ccg caa gaa gaa aga gta cct gca agg cct gga ggc ccg get gca ggc 96 Pro Gln Glu Glu Arg Val Pro Ales Arg Pro Gly Gly Pro Ala Ala Gly tgt get gge ega eaa cca gca get gcg cag gga gaa cge tgc cct ccg 144 Cys Ala Gly Arg Gln Pro Ala Ala Ala Gln G1y Glu Arg Cys Pro Pro gcg gcg get gga ggc ect get ggc aga gaa cag cgg get caa get ggg 192 Ala Ala Ala Gly Gly Pro Ala Gly Arg Glu Gln Arg Ala Gln Ala Gly WO 00!43419 PCTIUS00/01431 gtc tgg aaa cag gaa ggt tgt ctg cat cat ggt ctt cct tct ctt cat 240 Val Trp Lys Gln Glu Gly Cys Leu His His Gly Leu Pro Ser Leu His tgc ctt caa ctt tgg gcc tgt gag cat cag cga gcc gcc ttc agc tcc 288 Cys Leu Gln Leu Trp Ala Cys Glu His Gln Arg Al.a Ala Phe Ser Ser cat gtc tcc tcg gat gag cag gga gga acc tcg acc cca nag gca cct 336 His Val Ser Ser Asp Glu Gln Gly Gly Thr Ser Thr Pro Xaa Ala Pro get ggg ctt ctt cag aac cag ggc cag ctt cat ggc atg gaa ccc 381 Ala Gly Leu Leu Gln Asn Gln Gly Gln Leu His Gly Met Glu Pro 115 12 c) 125 <210> 40 <211> 127 <212> PRT
<213> Mouse <400> 40 Pro Ala Ala Ala Asp Asp Gln Glu Ser Arg Val Gly Leu Pro Val Pro WO 00!43419 PCTIUS00/01431 Pro Gln Glu Glu Arg Val Pro Ala Arg Pro Gl.y Gl.y Pro Ala Ala Gly Cys Ala Gly Arg Gln Pro Ala Ala Ala Gln Gly Glu Arg Cys Pro Pro Ala Ala Ala Gly Gly Pro Ala Gly Arg Glu Gln Arg Ala Gln Ala Gly Val Trp Lys Gln Glu Gly Cys Leu His His Gly Leu Pro Ser Leu His Cys Leu Gln Leu Trp Ala Cys Glu His Gln Arg Ala Ala Phe Ser Ser His VaI Ser Ser Asp Glu Gln Gly Gly Thr Ser Thr. Pro Xaa Ala Pro loo los zlo Ala Gly Leu Leu Gln Asn Gln Gly Gln Leu His Gly Met Glu Pro <210> 41 <211> 546 <212> bNA
<213> Mouse <220>

<221> CDS
<222> (3)..(545) <400> 41 ct atg get tac cca tac gat gtt cca gat tac get agc ttg ggt ggt 47 Met Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Gly Gly 1 5 1.0 15 cat atg gcc atg gag gcc ccg ggg atc cga gcg gga gag gaa gtg aga 95 His Met Ala Met Glu Ala Pro Gly Ile Arg Ala Gly Glu Glu Val Arg gaa agc get tac aac ctt cgc tct cga cca cgg agg cag cgg cga gcc 143 Glu Ser Ala Tyr Asn Leu Arg Ser Arg Pro Arg Arg Gln Arg Arg Ala cag gaa gcc gag gag atg aag acg cgg agg tct get cgc ctt gag cag 191 Gln Glu Ala Glu Glu Met Lys Thr Arg Arg Ser Ala Arg Leu Glu Gln cac tca cag cag ccg cag ctg tct ccg gcc acc agc ggc cga ggg tta 239 His Ser Gln Gln Pro Gln Leu Sex' Pro Ala Thr Ser Gly Arg Gly Leu cgg gac tct ccg tcc tcc agt gag gac cgc gaa gag gat gaa cct tct 287 Arg Asp Ser Pro Ser Ser Ser Glu Asp Ai:g Glu Glu Asp Glu Pro Ser tcc cga cct gtt aca agc caa aca gcc tca aag aaa act ctt aga aca 335 Ser Arg Pro Val Thr Ser Gln Thr Ala Ser Lys Lys Thr Leu Arg Thr 100 :L05 110 cca gag get tca gtg atg aat gaa gac cct ata agc aac tta tgc aga 383 Pro Glu Ala Ser Val Met Asri Glu Asp Pro Ile Ser Asn Leu Cys Arg ccc cca tta aga agc cca nga ctt gac tca aca tac caa nca aat gga 431 Pro Pro Leu Arg Ser Pro Xaa Leu Asp Ser Thr Tyr Gln Xaa Asn Gly aat act aaa acg aat gaa aga gaa get acc att gtc aac agg tca ant 479 Asn Thr Lys Thr Asn Glu Arg Glu Ala Thr Ile Val Asn Arg Ser Xaa tct tcg aat aag gng aan ccg aan atg atc tcg aga get ctt aca gtg 527 Ser Ser Asri Lys Xaa Xaa Pro Xaa Met Ile Ser Arg Ala Leu Thr Val aca tca cta tca ggg gca g 546 Thr Ser Leu Ser Gly Ala <210> 42 <211> 181 <212> PRT

<213> Mouse <400> 42 Met Ala Tyr Pro Tyr Asp Val Pro Asp Tyr Ala Ser Leu Gly Gly His Met Ala Met Glu Ala Pro Gly Ile Arg Ala Gly Glu Glu Val Arg Glu Ser Ala Tyr Asn Leu Arg Ser Arg Pro Arg Arg Gln Arg Arg Ala Gln Glu Ala Glu Glu Met Lys Thr Arg Arg Ser Ala Arg Leu Glu Gln His 5er Gln Gln Pro Gln Leu Ser Pro Ala Thr Ser Gly Arg G1y Leu Arg Asp Ser Pro Ser Ser Ser Glu Asp Arg Glu Glu Asp Glu Pro Ser Ser Arg Pro Val Thr Ser Gln Thr Ala Ser Lys Lys Thr Leu Arg Thr Pro Glu Ala Ser Val Met Asn Glu Asp .Pro Ile Ser Asn Leu Cys Arg Pro Pro Leu Arg Ser Pro Xaa heu Asp Ser Thr Tyr Gln Xaa Asn Gly Asn WO 00/43419 PCTlUS00/01431 Thr Lys Thr Asri Glu Arg Glu Ala Thr Ile Val Asn Arg Ser Xaa Ser Ser Asn Lys Xaa Xaa Pro Xaa Met Ile Ser Arg A1a Leu Thr Val Thr Ser Leu Ser Gly Ala <210> 43 <211> 875 <212> DNA
<213> Mouse <220>
<221> CDS
<222> (3)..(875) <400> 43 ct aca ttt gcc aag cyg gag aag cta aca atc ttg gca aag cgc aag 47 Thr Phe Ala Lys Xaa Glu Lys Leu Thr Ile Leu Ala Lys Arg Lys tcc ctc ttt gat gat aaa gca gta gaa att gag gag cta aca tac atc 95 Ser Leu Phe Asp Asp Lys Ala Val Glu ile Glu Glu Leu Thr Tyr Ile ate aaa cag gat atc aat age ctc aac aaa caa att get cag ett caa 143 Ile Lys Gln Asp Ile Asn Ser Leu Asn Lys Gln Ile Ala Gln Leu Gln gat ttt gtg agg gcc aag ggc agc cag agt ggc cgg cat ctg cag acc 191 Asp Phe Val Arg Ala Lys Gly Ser Gln Ser Gly Arg H.is Leu Gln Thr cat tct aac acc att gtg gtt tag ttg cag tca aag ctg get tct atg 239 His Ser Asn Thr Ile Val Val Ser Leu Gln Ser Lys Leu Ala Ser Met tcc aat gac ttc aag tca gtt ct.a gaa gtg agg aca gag aat ctg aaa 287 Ser Asn Asp Phe Lys Ser Val Leu Glu Val Arg Thr Glu Asn Leu Lys cag cag agg aac cgt cgg gaa cag ttc tcc agg gca cca gtg tca gcc 335 Gln Gln Arg Asn Arg Arg Glu Glri Phe Ser Arg Ala Pro Val Ser Ala ctg cct ctt gcc ccc aac aac ctt gga ggt ggt ccc ata att ttg gga 383 Leu Pro Leu Ala Pro Asn Asn Leu Gly Gly Gly Pro Ile Ile Leu Gly gca gag tcc cga get tcc agg gac gtg get att gac atg atg gae ect 431 Ala Glu Ser Arg Ala Ser Arg Asp Val Ala Ile Asp Met Met Asp Pro f~ 4 WO 00/43419 PCTlUS00/01431 aga aca agc cag cag ctt cag ctc att gat gag cag gat tcc tac atc 479 Arg Thr Ser Gln Gln Leu Gln Leu Ile Asp Glu Gln Asp Ser Tyr Ile cag agt cgg gca gac acc atg cag aac att gag tct aca atc gtt gag 527 Gln Ser Arg Ala Asp Thr Met Gln Asn Ile Glu Ser Thr Ile Val Glu ctg ggc tcc att ttt cag caa ttg gca cac atg gtt aaa gaa cag gag 575 Leu Gly Ser Ile Phe Gln Gln Leu Ala His Met Val Lys Glu Gln Glu gaa acg att cag agg atc gac gag aat gtg ctt gga gcc cag ctg gat 623 Glu Thr Ile Gln Arg Ile Asp Glu Asn Val Leu Gly A1a Gln Leu Asp gtg gag gca gcc cat tca gag atc: ctc aag tac ttc cag tca gtt acc 671 Val Glu Ala Ala His Ser Glu Ile Leu Lys Tyr Phe Gln Ser Val Thr tcc aat cgg tgg ctc atg gtc aaa atc ttc ctc atr ctc att gtc ttc 719 Ser Asn Arg Trp Leu Met Val Lys IIe Phe Leu Ile Leu Ile Val Phe ttc atc atc ttt gtg gtc ttc ctt gcc tga acc rtc ctc cct cat tct 767 Phe Ile Ile Phe Val Val Phe Leu Ala Thr Leu Leu Pro His Ser gag cca ctc cat gga ggg ctt ggg atc ctc ctg gga gga cag gtg get 815 Glu Pro Leu His Gly Gly Leu G1y Ile Leu Leu Gly Gly Gln Val Ala act att gcc act gag cct gtg caa ggt ggt tgg gag aaa ggc cat ttc 863 Thr Ile Ala Thr Glu Pro Val Gln Gly Gly Trp Glu Lys Gly His Phe ctt gga act get Leu Gly Thr Ala <210> 44 <211> 24B
<212> PRT
<213> Mouse <400> 44 Thr Phe Ala Lys Xaa Glu Lys Leu Thr Ile Leu Ala Lys Arg Lys Ser Leu Phe Asp Asp Lys Ala Val Glu Ile Glu Glu Leu Thr Tyr Ile Ile Lys Gln Asp Ile Asn Ser Leu Asn Lys Gln Ile Ala Gln Leu Gln Asp WO 00!43419 PCT/US00l01431 Phe Val Arg Ala Lys Gly Ser Gln Ser Gly Arg His Leu Gln Thr His Ser Asn Thr Ile Val Val Ser Leu G1n Ser Lys Leu Ala Ser Met Ser Asn Asp Phe Lys Ser Val Leu Glu Val Arg Thr Glu Asn Leu Lys Gln Gln Arg Asn Arg Arg Glu Gln Phe Ser Arg Ala Pro Va:l Ser Ala Leu Pro Leu Ala Pro Asn Asn Leu Gly Gly Gly Pro Ile Ile Leu Gly Ala Glu Ser Arg Ala Ser Arg Asp Val Ala Ile Asp Met Met Asp Pro Arg Thr Ser Gln Gln Leu Gln Leu Ile Asp Glu Gln Asp Ser Tyr Ile Gln Ser Arg Ala Asp Thr Met Gln Asn Ile G1u Ser 'chr Ile Val Glu Leu Gly Ser Ile Phe Gln Gln Leu Ala His Met Val Lys Glu Gln Glu Glu Thr Ile Gln Arg Ile Asp Glu Asn Val Leu Gly Ala Gln Leu Asp Val Glu Ala Ala His Ser Glu Ile Leu Lys Tyr Phe Gln Ser Val Thr Ser Asn Arg Trp Leu Met Val Lys Ile Phe Leu Ile Leu Ile Val Phe Phe Ile Ile Phe Val Val Phe Leu Ala <210> 45 <211> 761 <212> DNA
<213> Mouse <220>
<221> CDS
<222> (1)..(759) <400> 45 agn gga tnc cna ngg tcn gaa gna ggc gcn gtg ggg ggc gcc tat tgt 48 Xaa Gly Xaa Xaa Xaa Xaa Glu Xaa Gly Xaa Val Gly Gly Ala Tyr Cys can gnn ggn tta gga att ccn nat cag get tat cna nnc cgn cna cct 96 Xaa Xaa Xaa Leu Gly Ile Xaa Xaa Gln Ala Tyr Xaa Xaa Xaa Xaa Pro tgn ggg ggg ncc cgg gac cca ttt tan cct not gng ngn ttt att nca 144 Xaa Gly Gly Xaa Arg Asp Pro Phe Xaa Pro Xaa Xaa Xaa Phe Ile Xaa gtt cac tgt ncg ttg tat gac cac gga ctn act ggg aaa acc ctg get 192 Val His Cys Xaa Leu Tyr Asp His Gly Xaa Thr Gly Lys Thr Leu Ala atg ant gag agc ggc asg agg mag agg aac cgt cgg gaa cag ttc tcc 240 Met Xaa Glu Ser Gly Xaa Arg Xaa Arg Asn Arg Arg Glu Gln Phe Ser agg gca cca gtg tca gcc ctg cct ctt gcc ccc aac aac ctt gga ggt 288 Arg Ala Pro Val Ser Ala Leu Pro Leu Ala Pro Asn Asn Leu Gly Gly ggt ccc ata att ttg gga gca gag tcc cga get tcc agg gac gtg get 336 Gly Pro Ile Ile Leu Gly Ala Glu Ser Arg Ala Ser Arg Asp Val Ala att gac atg atg gac cct aga aca agc cag cag ctt cag ctc att gat 384 Ile Asp Met Met Asp Pro Arg Thr Ser Gln Gln Leu Gln Leu Ile Asp gag cag gat tcc tac atc cag agt cgg gca gac acc atg cag aac att 432 Glu Gln Asp Ser Tyr Ile Gln Ser Arg Ala Asp Thr M.et Gln Asn Ile gag tct aca atc gtt gag ctg ggc tcc att ttt cag caa ttg gca cac 480 Glu Ser Thr Ile Val Glu Leu Gly Ser Ile Phe Gln Gln Leu Ala His atg gtt aaa gaa cag gag gaa acg att cag agg atc gac gag aat gtg 528 Met Val Lys Glu Gln Glu Glu Thr Ile Gln Arg Ile Asp Glu Asn Val ctt gga gcc cag ctg gat gtg gag gca gcc cat tca gag atc ctc aag 576 Leu Gly Ala Gln Leu Asp Val Glu Ala Ala His Ser Glu Ile Leu Lys tac ttc cag tca gtt acc tcc aat. cgg tgg ctc atg gtc aaa atc ttc 624 Tyr Phe Gln Ser Val Thr Ser Asn Arg Trp Leu Met Val Lys Ile Phe ctc atc ctc att ggc ttc ttc atc atc ttt gtg gtc ttc ctt gcc tga 672 Leu Ile Leu Ile Gly Phe Phe Ile Ile Phe Val Vai Phe Leu Ala acc cty ctt cct yat tct gag cca ctc cat gga ggg ctt ggg atc ctc 720 Thr Xaa Leu Pro Xaa Ser Glu Pro Leu His Gly Gly Leu Gly Ile Leu WO 00/43419 PC1'IUS00/01431 ctg gga gga cat gtg get act att ycc act gag cct gtg tt 761 L~eu Gly Gly His Val Ala Thr Ile Ala Thr Glu Pro Val <210> 46 <211> 223 <212> PRT
c213> Mouse c400> 46 X;~a Gly Xaa Xaa Xaa Xaa Glu Xaa Gly Xaa Val Gly Gly Ala Tyr Cys Xaa Xaa Xaa Leu Gly Ile Xaa Xaa Gln Ala Tyr Xaa Xaa Xaa xaa Pro Xaa Gly Gly Xaa Arg Asp Pro Phe Xaa Pro Xaa Xaa Xaa Phe Ile Xaa Val Hia Cys Xaa Leu Tyr Asp His Gly Xaa Thr Gly Lys Thr Leu Ala Met Xaa Glu Ser Gly Xaa Arg Xaa Arg Asn Arg Arg Glu Gln Phe Ser Arg Ala Pro Val Ser Ala Leu Pro Leu Ala Pro Asn Asn Leu Gly Gly wo ooi434i9 pc~r~sooma3i <rly Pro Ile Ile Leu Gly Ala Glu Ser Arg Ala Ser Arg Asp Va1 Ala 7:1e Asp Met Met Asp Pro Arg Thr Ser Gln Gln Leu ~31n Leu Ile Asp Glu Gln Asp Ser Tyr Ile Gln Ser Arg Ala Asp Thr Met Gln Asn Ile Glu Ser Thr Ile Val Glu Leu Gly ~~er Ile Phe Gln (31ri Leu Ala His Met Val Lys Glu Gln Glu Glu Thr Ile Glri Arg Ile Asp Glu Asn Val L~au Gly Ala Gln Leu Asp Val Glu Ala Ala His Ser Glu Ile Leu Lys T~,~r Phe Gln Ser Val Thr Ser Asri Arg Trp Leu Met Val Lys Ile Phe Ls:u Ile Leu Ile Gly Phe Phe I1e Ile Phe Val Val Phe Leu Ala <s:10> 47 <211> 453 <212> DNA
<213> Mouse <220>
<221> CDS
c222> (1)..(453) <400> 47 ;agn gga tnc cna ngg tcn gaa gna ggc gcn gtg ggg ggc gcc tat tgt 48 :Kaa Gly Xaa Xaa Xaa Xaa Glu Xaa Gly Xaa Val Gly Gly Ala Tyr Cys <:an gnn ggn tta gga att ccn nat cag get tat cna nnc cgn cna cct 96 :Caa Xaa Xaa Leu Gly Ile Xaa Xaa Gln Ala Tyx Xaa Xaa Xaa Xaa Pro t:gn ggg ggg ncc cgg gac cca ttt r_an cct not gng ngn ttt att nca 144 3;aa Gly Gly Xaa Arg Asp Pro Phe Xaa Pro Xaa Xaa Xaa Phe Ile Xaa crtt cac tgt ncg ttg tat gac cac gga ctn act ggg aaa acc ctg get 192 Val His Cys Xaa Leu Tyr Asp His Gly Xaa Thr Gly Lys 'rhr Leu Ala atg ant gag agt atc tgn aga acc cng aac ngt cac aaa cag ttc tct 240 Net Xaa Glu Ser Ile Xaa Arg Thr Ji:aa Asn Xaa His Lys Gln Phe Ser atg gca cca ntg tna ncc ctg cct ttt gcc ccc aac atc ctt gga ggt 288 Met Ala Pro Xaa Xaa Xaa Leu Pro Phe Ala Pro Asr Ile Leu Gly Gly gng tcc cat aat tct ngg agc atg gat ccg agc ttc cng gga cgn ggn 336 Xaa Ser His Asn Ser Xaa Ser Met Asp Pro Ser Phe Xaa Gly Xaa Xaa tat gtg aca tga tgg acc cta gaa caa gcc agc agc ttc agc tca ttg 384 Tyr Val Thr Trp Thr Leu Glu Gln Ala Ser Ser Phe Ser Ser Leu atg agc agg att cct aca tcc aga gtc ggg cag aca cca tgc aga aca 432 Met Ser Arg Ile Pro Thr Ser Arg Val Gly Gln Thr Pra Cys Arg Thr ttg agt cta caa tcg ttg agc 453 Leu Ser Leu Gln Ser Leu Ser c210> 48 ,:211> 115 .:212> PRT
.:213 > Mouse ~;400> 48 Xaa Gly Xaa Xaa Xaa Xaa Glu Xaa Gly Xaa Val Gly Gly Ala Tyr Cys Xaa Xaa Xaa Leu Gly Ile Xaa Xaa Gln Ala Tyr Xaa Xaa Xaa Xaa Pro Xaa Gly Gly Xaa Arg Asp Pro Phe Xaa Pro Xaa Xaa Xaa Phe Ile Xaa Val His Cys Xaa Leu Tyr Asp His Gly Xaa Thr Gly Lys Thr Leu Ala Met Xaa Glu Ser Ile Xaa Arg Thr Xaa Asn Xaa His Lys Gln Phe Ser Met Ala Pro Xaa Xaa Xaa Leu Pro Phe Ala Pro Asn Ile Leu Gly Gly Xaa Ser His Asn Ser Xaa Ser Met Asp Pro Ser Phe Xaa Gly Xaa Xaa 100 lU5 110 'Tyr Val Thr ~:210> 49 ~:211> 467 .; 212 > DNA

<213> Mouse <220>
<221> CDS
<222> (2)..(966) <400> 49 c ccg ggg tgt act gga gtt cgc tgg gtg agg gag ggg gtg tca agt gtg 49 Pro Gly Cys Thr Gly Val Arg Trp Val Arg Glu Gly Val Ser Ser Val aga gag gtg cag gac ttc caa att tag aac ctt gag gcc ctt tgg get 97 Arg Glu Val Gln Asp Phe Gln Ile Asn Leu Glu Ala Leu Trp Ala cgg gtc cag ggc act ccg acc acc atg cgc gac agg acc cac gag ttg 145 Arg Val Gln Gly Thr Pro Thr Thr Met Arg Asp Arg Thr His Glu Leu ~3gg cag ggg gat aac atc tca gac gat gaa gat gag gtt cga gtc gcg 193 :erg Gln Gly Asp Asn Ile Ser Asp Asp Glu Asp GLu Val Arg Val Ala ctg gtg gtg cac tca ggt get gcc cgg ttg ggc agc ccg gac gac gag 241 l~eu Val Val His Ser Gly Ala Ala Arg Leu Gly Ser Pro Asp Asp Glu tac ttc cag aag gtg cag aca att cgg cag act atg gcc aaa ctg gag 289 Phe Phe Gln Lys Val Gln Thr Ile Arg Gln Thr Met Ala Lys Leu Glu agt aaa gtc cgg gag ttg gag aaa cag cag gtc acc atc ctg gcc acg 337 Ser Lys Val Arg Glu Leu Glu Lys Gln Gln Val Thr Ile Leu Ala Thr cet ctt ccc gag gag aag gag gaa get gat gag aat tac aac tca gtc 385 Pro Leu Pro Glu Glu Lys Glu Glu Ala Asp Glu Asn Tyr Asn Ser Val aac aca agg atg aag aaa acc cag cat gga gtc ctg tcc can caa ttt 433 Asn Thr Arg Met Lys Lys Thr Gln His Gly Va1 Leu Ser Xaa Gln Phe gtc gag ctc atc aca agt gca act caa tgc agc c 467 Val Glu Leu Ile Thr Ser A1a Thr G1n Cys Ser c210> 50 c211> 24 ~c212> PRT
:213> Mouse ~:400> 50 I?ro Gly Cys Thr Gly Val Arg Trp Val Arg Glu G1y Val Ser Ser Val '17 Arg Glu Val Gln Asp Phe Gln Ile <210> 51 <211> 120 <212> DNA
<213> Mouse <220>
<221> CDS
<222> (1) .. (120) <400> 51 cgg cac gag aaa aaa aaa aaa aaa aaa aaa aaa aaa aaa aaa aaa aaa 48 T~rg His Glu Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys 1 5 10 1.5 ;aan ngg ggg ggg ggg naa aaa nnn naa nng ggg gnn aaa ann ggg gnc 96 :~Caa Xaa Gly Gly Gly Xaa Lys Xaa Xaa Xaa Gly Xaa Lys Xaa Gly Xaa ~:cn ccc cna aaa nnc ngg ggn aaa 120 Xaa Pro Xaa Lys Xaa Xaa Xaa Lys <:210> 52 <211> 40 <212> PRT
<213> Mouse <400> 52 A:rg His Glu Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys X~ia Xaa Gly Gly Gly Xaa Lys Xaa Xaa Xaa Gly Xaa i~ys Xaa Gly Xaa 20 2S~ 30 Xaa Pro Xaa Lys Xaa Xaa Xaa Lys <210> 53 <211> 216 <212> DNA
<213> Unknown <220>
<223> Description of Unknown Organism: Navel <400> 53 cgcggccgcg tcgaccaggg ccgaagcctg agacccgggt ctccccagct gcaggtgtcg 60 gcc~cagccgg cggagcagga gctgcggcgg ctgcnggagg agaacgagcg cctccgcang 120 WO 00/43419 PCT/t3S00/01431 gagctgcncg cggggccaca ngaggagcgc gcgcttgctg cngnanctca aggangtcac 180 ggaccgacan cggnaccaac ttcgggcnca caancn 216 <210> 54 <211> 280 <212> DNA
c213> Homo sapiens ~c400> 54 <~tgggggggt gtctttggtg cctgaagtct gcgcggcatg ganggtggtg tgagttcctc 60 t:ggtgggagg gagaacgcac atctcttntg ggcggccacc tgaggagtga ctncaagaag 120 agttccggca gctttcccca ngaaagggtg aggggtgaca ctnggctctg gctntganat 180 c~aggcagacg gcncccangc tgtgatctgt cctgggcgga gaccangagg gagcgggntn 240 g~ggatcacct gccagtgcca ngactctggg actgagtgcc 280 <210> 55 <211> 317 <212> DNA
<213> Homo Sapiens <400> 55 gccctaagcc tgatggaaac tgtcaagcaa ggcgttgatc agaagctggt ggaaggccag 60 gagaagctgc accagatgtg gctgctcacc atgttccggg acattgccca gcaactgcag 120 gccacctgta cctccctggg gtccagcatt cagggcctcc ccaccaatgt gaaggaccag 180 gtgcagcagg cccgccgcca ggtggaggac ctccaggcca cgttttccga ggccctggac 240 cacatggtgg aatatgtngc ccagaacaca cctgtcacgt ggctcgtggg acccttttnc 300 ccctggaatc actgaga 317 <210> 56 <211> 228 <212> DNA
c213> Homo sapieris <400> 56 ctgggacgtc cggttgaccg cgcgtctgct gcagagacca tgtctgccga cggggcagag 60 gctgatggca gcacccaggt gacaacatgg tgtccgcagc ctatgcctcc accaaggaga 120 gctacccgca cgtcaagact gtctgcgacg cagcagagaa tggagtgagg accctcacgg 180 cggctgctgt cagcggggct catcgatcct ctccaaagct gggagccc 228 <210>57 .c211>406 :212DNA
>

:213>Homo Sapiens .:400> 57 taacgctagc ttgggtggtc atatggccat ggaggccccg gggatccgat tcgcggccgc 60 gtcgacgtcg cggccgctgt tcctgggacg tccggttgac cgcgcgtctg ctgcagagac 120 catgtctgcc gacggggcag aggctgatgg cagcacccag gtgacagtgg aagaaccggt 180 n.cagcagccc agtgtggtgg accgtgtggc cagcatgcct ctgatcagct ccacctgcga 240 catggtgtcc gcagcctatg ccttcaccaa ggagagctac ccgcacgtca agactgtctg 300 cgacgcaanc agagaaggga gtgaggaccc tcacggnggc tgctgtcagc ggggcttagc 360 cgatcctctc caagctggag ccccatattg catcagccag cgaata 406 <210> 58 <211> 228 c?12> DNA
<13> Homo Sapiens <<E00> 58 ct:gggacgtc cggttgaccg cgcgtctgct gcagagacca tgtctgccgn cggggcagag 60 gctgatggca gcacccaggt gacagtggaa gaaccggtac agcagcccag tgtggtggac 120 cgtgtggcca gcatgcctct gatcagctcc acctgcnaca tggtgtcgca nnctatgcct 180 tnaccaanga gagctncccg cacatcaaga ctgtctgcga cgcancng 228 <210> 59 c211> 415 ~c212> DNA
~c213> Homo sapiens ~:400> 59 cacaaagtgg acatggacaa gctcctgggg ggccagatcg ggctggagga cttcatcttc 60 c~cccacgtga aggggcagcg caaggaggtg gaggtgttca agtcggagga tgcactcggg 120 cacaccatca cggacaacgg ggctggctac gccttcatca agcgcatcaa ggagggcagc 190 g~tgatcgacc acatccacct catcagcgtg ggcgacatga tcgaggccat taacgg~gcag 240 agcctgctgg gctgccggca ctacgaagtg gcccggcttg ctcaaggagc ttgccccgan 300 gccgtacctt cacgctgaag ctcacgggag cr_tcgcaagg cccacaactg gggcactggg 360 ccganggacc tgcggctccg atcccggggg ccccgccaan agtggaggat ctgcc 415 WO 00!43419 PCTlUS00/01431 <210> 60 <211> 199 <212> DNA
<213> Homo Sapiens e4D0> 60 ctggcccatg gcagtcccac tggccgcatc nagggcttca ccaacgtcaa ggagctgtat 60 ggcaanatcg ccgaggcctt ccgnctgcca actgccgatg tgatgttctg caccctnaan 120 acccacanag tggacatgga caatgctcct: ggggggccaa atcgggetgg angacttcat 180 cttcgcncac gtnaangng 199 <210> 61 <211> 243 c212> DNA
<213> Homo Sapiens c400> 61 ctggcccatg gcagtcccac tggccgcatc gagggcttca ccaacgtcaa ggagctgtat 60 <~gcaagatcg ccgaggcctt ccgcctgcca actgccgagg tgatgttctg caccctgaac 120 acccacaaag tggacatgga caagctcctg gggggccana tcgggctgga ggacttcatc 180 ttcgcccacg tgaaggggca nncaaggagg tggacgtgtt caagtcnngn ggatgcactc 240 ggg 243 <210> 62 <211> 309 <212> DNA
<213> Homo sapiens <400> 62 tggtgtgatc ggattctctg gaaagggaag aacatcactc agctgagtta ccagagccac 60 atggccctga agaccagtga ccacaagcct. gtcagctcag tgtttgacat cggggtgagg 120 ~3tcgtaaatg acgagcttta ccggaagaca ctggaggaaa ttgttcgctc cctggataag 1B0 ~~tggaaaatg ccaacattcc ttctgtgtcc ctgtccaagc gagagttctg ttttcaaaat 240 c~tgaagtaca tgcaataccc ctgtcatttt gaattcatca acaagcctga tgaagagtct 300 t:actgttng 309 <:210> 63 <211> 522 <212> DNA
<213> Unknown <220>
<223> Description of Unknown Organism: Novel <400> 63 tgncgacctg gnacnnccgg ctnaccgcgt gcctgcngnn gttatcangn ctnccgacng 60 ~~gtatnggnt gatngcagca cccatngcga cagtttgaag aatnggtatn ncatgncnat 120 ~3tgtgggctg gacctnnnnt ngntanntac anctctnnng antgattcnc natncnngct 180 ~latcanggnt gnnncgtcan gctcnntngn cctccnacnc nttngtatgn accgttgtct 240 ngcaacttgc anantatcgt tggtctggct gaantgttct gaattatcaa angggnaanc 300 ncttnngntt cccttcnatg tnntcncnct ctggtcttna tctatgcgtc ggggggtgnt 360 c:angttctgc atgtctgtcc tttcctaana tcctggtgct gtntttctca gnngatntgg 420 t:catatcnag nctcnctnct tnatngtcng atngnccnna atctattggg tntgtnctnc 480 nnnaagtanc gcgtcggttt cgtgtgtnan gactctnctn tt 522 <210> 64 <211> 530 <212> DNA
<213> Unknown WO 00/43419 PC1'NS00/01431 c220>
<223> Description of Unknown Organism: Novel <400> 64 gtcacanggc catcatgnct atgaggcact gcgngtcnac tactgcgcac ctnatgcgna 60 tnngnctcac nctctgcgag tattcgcnnt ataccacnct nacctcggtc cgctanantc 120 tcagcnccgc gggctgcgca cacntctatc nntagnngag cgcantntcn gtgnncagng 180 nnngttcntc tnatgngatn gtncgnctct gttcagnntc tcgaagganc gnntagcgtg 240 cnnaactntg nnntagnana ccncnggcag cnngnaatct nnntgacgcg catcnctgac 300 ccannntctg ncnagtccac ccgtccnncn ngacnnctna nngangccat nnatnnnncc 360 tctngntngn cntgnggcgt nacatagngt tntcngcntn tggcgntgca cctgncnngg 420 ~itgtctnncn ctngatntan ngntgnnnnn ggtnacgntc acgtnntngg tnncncngcn 480 l:annccnnnn ncgtcccnnn ctnggntgnn ngncnnnngn ntntnannnt 530 <;210> 65 <:211> 206 <:212 > DNA
<.213> Mouse <400> 65 ccattaataa cattattgtg ggtggctgag tccttctcat catgggacga gtgagccaga 60 gcgggggaaa gggcatgaag taaagcgttg cctgaatgct gtgtggtgtt ttgtttcttc 120 ctccttccta tgaggttttc tacttctcaa ttaaaataat ttcaaaataa aaaaaaaaaa 180 nnaaaanaaa annnnnanna nnnant 206 <210> 66 <211> 478 <212> DNA
<213> Mouse c400> 66 ~~caaaggcga gcatcctggc ttatccattg gtgatgttgc aaagaaacta ggagagatgt 60 ggaacaacac tgcagcagat gacaagcagc cctatgagaa gaaagctgcc aagctgaagg 120 :~gaagtatga gaaggatatt gctgcctaca gagctaaagg aaaacctgat gcagcgaaaa 180 <~gggggtggt caaggctgaa aagagcaaga aaaagaagga agaggaagat gatgaggagg 240 atgaagagga tgaggaagag gaggaagaag aggaagacga agatgaagaa gaagatgatg 300 atgatgaata agttggttct ancgcatttt tttttcctgt ctataaagca tttaaccccc 360 ctgttcacaa ctcactcctt ttaaagaaaa aatttgaaat gtaaggctgt gtaanatttg 420 tttttaaact gttcagtgtc cttttttgta nagttaacac actaccgaat gtgtcttt 478 <210> 67 <211> 386 c212> DNA
<213> Mouse c400> 67 caatttgaag cagcagtatt acacaggcta catggagagt gaagtcctgg aagtcatgca 60 gcacatggcc aagaacgttg tgaaagtcaa tgacaaccgt accaagttca tcgctgtcaa 120 gaacaagtat gccagcagca gactcctgaa gatcagcacg atccctcagc tgaactccaa 180 ~aatcatcaaa gacctggcct cccctctgct gggcagcccc taggcactgg actgccatcc 240 l:gcgcttctc agatcctgta tgtattttat tctagtttac atcacaaacc tcttctcaga 300 ca cattttct aattgtgtat tgatgaaaaa taaagctatt gattttctta taaaaaaaaa 360 aaaaaaaanc ncgnganntn tttgan 386 <210> 68 a9 <211> 392 <212> DNA
<213> Mouse <400> 68 catggagagt gaagtcctgg aagtcatgca gcacatggcc aagaacgttg tgaaagtcaa 60 tgacaaccgt accaagttca tcgctgtcaa gaacaagtat gccagcagca gactcctgaa 120 gatcagcacg atccctcagc tgaactccaa aatcatcaaa gacctggc:ct cccctctgct 180 ~gggcagcccc tagacactgg actgccatcc tgcgcttctc agatcctgta tgtattttat 240 '~ctagtttac atcacaaacc tcttctcaaa ctcattttct aattgtgtat tgatgaaaaa 300 t:aaagctatt gattttctta tactaaaaaa aaaaaaaaaa accccgnnaa anntnngaac 360 cntaaanncg gaaaancccc ccagnttttn as 392 <:210> 69 <.211> 502 <212> DNA
<213> Mouse <400> 69 cggactagct cctcaccaag ggaaagtcca tgaaatggaa aaagagcact tgaacaaggt 60 tcagactgca aatgaagtca agcaagctgt tgagcagcag atccagagtc acagagaaac 120 ccaccaaaaa caaatcagta gtttgcgaga tgaagtggag gcaaaggaaa agctaatcac 180 tgacctccaa gaccaaaacc agaagatggt gttggagcag gaacggctaa gggtggagca 240 r_gagaggctg aaggctacag accaagagaa gagcaggaag ctgcacgagc tcacggttat 300 c~caagacaga cgagaacaag caagacaaga cttgaagggt ttggaggaga ccgtggcaaa 360 <<gaacttcag actttacaca acctgcgtaa gctctttgtt caggacttgg ctaccaaggg 420 t.gaaaaagan cgccgaggtc gactctgacg acactggcgg cagtgctgca cagaancaga 480 aatctccttc ccttgaaaac as 502 <210> ?0 <211> 499 <212> DNA
<:213> Mause <400> 70 cc~gactagct cctcaccaag ggaaagtcca tgaaatggaa aaagagcact tgaacaaggt 60 tc:agactgca aatgaagtca agcaagctgt tgagcagcag atccagagtc acagagaaac 120 cc:accaaaaa caaatcagta gtttgcgaga t;gaagtggag gcaaaggaaa agctaatcac 180 WO 00/43419 PCTlUS00/01431 tgacctccaa gaccaaaacc agaagatggt gttggagcag gaacggctaa gggtggagca 240 tgagaggctg aaggctacag accaagagaa gagcaggaag ctgcacgagc tcacggttat 300 gcaagacaga cgagaacaag caagacaaga cttgaagggt ttggaggaga ccgtggcaaa 360 agaacttcag actttacaca acctgcgtaa gctctttgtt cangacttgg ctaccaaggg 420 tgaaaaagaa cgccgaggtc gactctgacg acactggcgg caattgctgc acagaagcag 480 aaaatctcct tccttgaaa 499 ~c210> 71 .211> 486 ~:212 > DNA
~:213 > Mouse <:400> 71 c:ctcagtcaa cacaaggatg aagaaaaccc agcatggagt cctgtcccag caatttgtcg 60 a.gctcatcaa caagtgcaac tcaatgcagt ccgaataccg agagaagaat gtggagcgga 120 tccggaggca gctgaagatc accaatgctg gaatggtgtc tgacgaggag ctggaacaga 180 tgctggacag tgggcagagt gaggtgtttg tgtctaatat cctgaaggac acgcaggtga 240 ctcggcaggc cctgaatgag atctctgcgc ggcacagtga gatccagcag ttggagcgca 300 WO 00!43419 PCTNS00/01431 gtatccgaga gctccatgag atctttacgt ttctagctac ggaggtggag atgcaggggg 360 agatgatcaa ccgcatcgag aagaacatcc tgagctcggc cgactacgtg gaacgtgggc 420 aagagcacgt caagatagcc ctagagaatc agaagaaggc gangaaggta ancctgggcc 480 ccaacc 486 <210> 72 <211> 486 <212> DNA
<213> Mouse <400> 72 cctcagtcaa cacaaggatg aagaaaaccc agcatggagt cctgtcccag caatttgtcg 60 ~agctcatcaa caagtgcaac tcaatgcagt ccgaataccg agagaagaat gtggagcgga 120 t:ccggaggca gctgaagatc accaatgctg gaatggtgtc tgacgaggag ctggaacaga 180 t:gctggacag tgggcagagt gaggtgtttg tgtctaatat cctgaaggac acgcaggtga 240 cacggcaggc cctgaatgag atctctgcgc ggcacagtga gatccagcag ttggagcgca 300 c~tatccgaga gctccatgag atctttacgt ttctagctac ggaggtggag atgcaggggg 360 WO 00!43419 PCT/US00/01431 agatgatcaa ccgcatcgag aagaacatcc~ tgagctcggc cgactacgtg gaacgtgggc 420 aagagcacgt caagatagcc ctagagaatc agaagaaggc gangaaggta ancctgggcc 480 ccaacc <210> 73 <211> 496 <212> DNA
<213> Mouse c400> 73 catcctgaag gacacgcagg tgactcggca ggccctgaat gagatctctg egcggcacag 60 vtgagatccag cagttggagc gcagtatccg agagctccat gagatcttta cgtttctagc 120 tacggaggtg gagatgcagg gggagatgat caaccgcatc gagaagaaca tcctgagctc 180 <lgccgactac gtggaacgtg ggcaagagca cgtcaagata. gccctagaga atcagaagaa 240 c~gcgaggaag aaaaaaagtc atgattgcca tctgtgtctc tgtcactgtt ctcatcttgg 300 <agtcatcat tggcatcacc ataaccgttg gataatgtca catgttcttg gtgagaaatg 360 cfcgggcctgc cccatccctg gcagcagcct ccccgcctcc tgtcagaccc atttctttct 420 ccttccctgc ganacagttt tcaatgtcct gtttgtctgg gatacatggn tttgttaaaa 480 aattttaaaa aaaaaa 496 c210> 74 <211> 453 <212> DNA
<213> Mouse <400> 74 cgggtgtacc agctcactaa gaaacactgc tatcaagatt tttaaagctc aaaccaagtg 60 aoacttttaa taagagacta agatctgatt gagggagttt tctaacagtc cttgcctcgt 120 ggacacgcta aagtttgact tgggtgcctt acctcttttc taaccagtgt tgctcgtgag 180 c:ctgtggcgc tcgcttgctc gctcgctcgc t.cgctccgtg gattccttaa aaccagagag 240 <:ctgtaagtg tggcaagatc aaaactggcc tggctggagg gttagctgga catggatctg 300 caggattgcc tctggggacc tcgttttggg aacttttctt ggggctgttt tgctcaagtg 360 c:ccgtaactg atggatgggt gaagtaattt tgaacgtacg cgatttgtag gttttgttat 420 ttgttttaaa gatttaaaag ccggttatag gaa 453 <210> 75 <211> 485 <212> DNA
c213> Mouse c400> 75 ctccacatgg gatttcctca gactgattct cgacctgtca atgaaggttc tgaaacagga 60 tggaaaatac ttcacacagg ggaactgtgt caacttgact gaagccctgt cgctctatga 120 agaacagctg gggcgtctgt attgtcctgt ggaattctca aaagagattg tctgtgtccc 180 ttcatacttg gaattgtggg tgttttacac tgtttggaag aaagctaagc cttaaagatg 240 agaatgacgt gtgctgcgag ccaccttcct gacctccata tgccgtatat gccatcaaat 300 gtgtcaggca actgctcatg aattccttct agtggttttc acttttaatt attattttta 360 atttaaaaaa gccaatggga aaaatgtata ttttgatgag ttaggatgtt aattttaaaa 420 agtcagccga aggacaatta gacagcccan caaagactgc agaatgcact gaccccctag 480 aatgt 485 <:210> 76 <211> 241 <212> DNA
<213> Mouse WO 00/43419 PCTNS00l01431 <400> 76 caaaaaacaa tcttattccg agcattccag taacttttgt gtatgtacct agctgtacta 60 taagtagttg gtttgtatga gatggttaaa aaggccaaag ataaaaagat tttttttttc 120 cttttctttc ngtctatgaa gttgctgttt attttttttt ggcctgtttg atgtatgtgt 180 gaaacaatgt ccaacaataa accggaattt tattttgctg anttgttcta aaaaaaaaaa 240 a 241 <210> 77 <211> 419 <212> DNA
<213> Mouse <400> 77 ccaggtggtg agccgcatcc gcgcagcgct gaacgccgtg cgcctgctgg tggtcgaccc 60 ~3gagaccgac gagcgtctca agaagctggg cgtctcgatc cgggaagagc tgctgc:gccc 120 ~acaggagaag tccgaacaag ccgagcctcc agcggccgcc gacactcatg aggctgggga 180 ccagaatgag gccgaaaaga gccacttgcg cgagctccgg ccccggct.ct gcaccatgaa 240 c~aaaggcccc aatggctatg gcttcaacct gcacagcgac aagtctaanc caggccagtt 300 catccgagca gtggacccag actcaccggc ggaggcgtct ggactccggg cccaaggacc 360 gaattgtgga ggtcaatggt gtctgcatgg agggcaagca gcacggggac gttgtgtcc 419 <210> 78 <211> 396 c212> DNA
<213> Mouse <400> 78 cctagatcgc tgcgacaaag agaacaagat gcttaaagac gagatgaaca aagagatcga 60 ggcggcccgt cggcagttcc agtcacagct ggctgaccta caacagctgc ctgacatcct 120 =aagatcacg gaggccaagc tggctgagtg ccaagaccag ctgcagggct acgagaggaa 1B0 c~aacatcgac ctcacagcca tcatttcaga cctgcgcagc cgggtaaggg actggcaaaa 240 c~ggatcccac gaactggccc gaacaagggc ccgcttaccg agatgagctg cacgcccccc 300 aagggaagac cacttcCttt ttcctggctg ccgatttctg aaaggatgag ctatcatcaa 360 tgctgtgaaa taaaagtctg gtgttccaaa aaaaaa 396 <210> 79 <211> 448 c212> DNA
<213> Mouse <400> 79 ccggcgttgt caggatcggc gcctccggag tactgaggct tccgactttg cggccggtcc 60 gagcagcttg ggtcttggcg cggcgcgatg tccgaaaaga agcagccggt cgacttgggt 120 ctcttggaag aggacgacga gttcgaggag tttcccgcgg aagactgggc tggcttagat 180 gaagatgaag atgcacatgt ctgggaggat aattgggatg atgacaatgt agaagatgac 240 ttctctaacc agttacgtgc tgagctggag aagcacggct acaagatgga gacatcatag 300 catctgggaa tgtcccagga acctcaatca tggactctac cacagtctag gacagagaaa 360 gcaggacggg atactttaaa gaacatgttt atttcattat ctgcttcaat ttatttttgt 420 tttatnacaa aaaaaataag taaataaa 448 c210> 80 <211> 498 <212> DNA
~:213> Mouse ~:400> 80 cgaacgagaa aggaggaggg cgctacaggc gacagcgctg cagacgccat tatcctctgc 60 ttctccgctg cacggacgtc gccgtcgtgc tcgtctccca ctcgattgcg gcctattggt 120 ggatccattt aattcaagaa aatggagacc gaacagccag aagaaacctt ccccaacacc 180 gaaaccaatg gtgaatttgg taaacgccct gcagaagata tggaagagga gcaagccttt 240 aaaagatcta gaaatactga tgagatggtt gaattgcgca ttttgcttca gagcaagaat 300 gctggagcag tgattggaaa aggaggcaag aatattaagg ctctccgtac agactacaat 360 ~~ccagtgttt cagtcccaga cagcagtggc cccgagcgca tactgagtat cagtgctgat 420 ;ittgagacat tggagaaatt ctgaagaaaa tcatccctac cttggaagaa ggcctncaag 480 ttgccatcac cactngan 498 <:210> 81 <:211> 470 <:212> DNA
<213> Mouse <400> 81 cgtcccgagg tctggtcttg atttcttttt tgggtttctt tctaggaaaa tgagaagtgc 60 atgcaagggg Caggagatga ccctccccta ggctttcagc ttcaggcagc ttcttcacag 120 cctgttcagc ctgggctcct ggaggacagc cctgggggag gcagtgaggg gcagcgccaa 180 gatagccagg tggttggttc caggaccaca gtgtcttttt tttgttgttg gtttttttgt 240 tgttgttgtt tgtttgtttg ttttttaact gccactaccg ccccctgacc cccaatcttg 300 gtcaactctg gagtactgcc tgccccagac gagcaggggt ggggggggga gcactgatcc 360 tcctccctgt gcanggcaaa gggctttcct aaccgancag tagggatana aancgtnagc 420 ctgggaatgc tttttaaaaa ttaatttcct tgtnnatttt aattttaatn 470 <210> 82 <211> 372 <212> DNA
<213> Mouse <400> 82 cagccttgca gtaagcagct taacaggagc attggccacc agcagaaggg catcactgtc 60 tcaggcctca agccaggcac ccatctctgg atgccagtct atagcgggta ccagaggaaa 120 gctggcagca gtaactctct ccccatgcat. cctagccagt gagtgctaca tcctttgcaa 180 gtggagttac tggcctaccc ttaccccatg cattcttcct gtctgcactg cctgggccaa 240 ggggcagaaa cactctgctc ttcttcccca ggacattccc aggcttgggg tttttctata 300 ggtttgaaag taaagggggg agggtgggaa gggtgggagg aacctgacaa taaagagatt 360 ggatccaaaa na 372 <210> 83 <211> 530 <212> DNA
<213> Mouse <400> 83 ocgggagacg gagctggagc tccgagagca gctggacatg gcgggcgccc gagtgaggga 60 agcgcataag cgagtggaag ccgcccagga gacagtcgcc gactaccagc agaccatcaa 120 <3aagtaccgc cagttgactg cccacctaca ggatgtcaat cgggagctga caaaccagca 180 clgaagcgtct gtagagaggc agcagcagcc gccgccagag acttttgatt tcaaaatcaa 240 c~tttgctgag acctaggaca gcgacaccgc ctggtgctga cccaggagca gctgcaccag 300 catcacagtc gcctcatctc ctaagcactc cttccctgcc ttccctgtgg tcccactgcg 360 cagggcacag ccacctcagc cactcccang ctggaccaca ggctaacctc ctgagtggtt 420 c:agctgcgtc tggtctgggt canggtcctg ctgcttccgt aacctgggtt tgcctgtcca 480 WO 00/43419 PCTlUS00/01431 accctggcct cttccgttat ttgcngttat cngctccctc cttccctgag 530 <210> 84 <21i> 217 <212> DNA
<213> Mouae <400> 84 caagnccttc atgtnncgtc agctggcgcn Ggatnncntn ctgnacacnt tnccgcctgt 60 gcaccntgtn gcttgcactn tngacnncat gctnnatgan tcggtcatnt tcgcgcggcg 120 antgcncgac ctgggcnacc cggtgangcn ganagtggta tancgatctg ccgcatggct 180 tcctgaacnt ggcggnactn tgtcnacnan aaccggg 217 <210> 85 <211> 188 <212> DNA
<213> Mouse ~c400> 85 c:ancntaaag agtnanaccc caagttggct ccctggatcc agagactgaa nanttttggc 60 taggntgcca taggaccctt tttcactcgg tacggagcat ctctcangga gcttgatttc 120 nacgtcgtga ctgtcctctg ggatgagaag tnCggccatn aactgggctc tanagaagga 180 nagcancc lg8 <210> 86 <211> 499 <212> DNA
<213> Unknown <220>
<223> Description of Unknown Organism: Novel <900> 86 cgaaaatgcc aataaagcac tggacaaggc aagagcgaaa aacaaagatg ttctacaggc 60 tgaaacttcc caacagttgt gctgtcagaa gtttgaaaaa atatctgaat ctgcaaaaca 120 agaactgata gattttaaga caagaagagt tgctgcattc agaaaaaatt tagtggaact 180 ~tgcagaacta gaactgaagc atgcaaaggg caaccttcag ctccttcaga actgccttgc 240 clgttttaaat ggagacacat aggcttcact ctgccttctg tcaaaaaggg ctgccttcaa 300 :~tccagtttg ttttcctgac gacctccctc cggcatctaa gctcactggg gaacaacaga 360 cracagcgggg cacagtgcat cagctcttct tctgagagac agtggagcag canggctcac 420 WO 00/43419 PCTlUS00/01431 cggctgcang gcaaacggnc tgccaacLCg cactccanct tccaagaagc ccaaaagttc 480 taacctctct tttctttgn 499 <210> 87 <211> 476 <212> DNA
<213> Unknown <220>
<223> Description of Unknown Organism: Novel <400> 87 caagaaagca gagctgcaac tgtctgcaga aaaggccaag gttgacagtc gccttcagaa 60 tatggacttc ctaaaagcga aggcagcgga gttccggttt ggaatcaaag ctgcagagga 120 <~caactttca gccagaggca tggatgcgtc tctgtctcat cggtcact.ag cggcactttc 180 agagaaactc tcggagttaa aagagcagac tatacccctg aagaagaagt tggagtccta 290 tatagattta atgccgagtc catctcttgc tcaactgaaa attgaagagg caaagagaga 300 attagacgcc atagaggctg aacttaccaa gaaagtagac atgatgggcc tgtgaggaca 360 g~ccatgtaga cgtggtcagg ggaaggaggt gactgttctt anagatgatg atgtctctat 420 tagtaaaaca acanggttcg cttctgcctt: taaactttgt ggcctgaata cagttn 476 <210> 88 <211> 484 <212> DNA
<213> Unknown <220>
~:223> Description of Unknown Oxganism: Novel ~:400> 88 c:ctggtgtta gaaaaatgtc tacgagaaga tctcaagaaa gcagagctgc aactgtctgc 60 aigaaaaggcc aaggttgaca gtcgccttca gaatatggac ttcctaaaag cgaaggcagc 120 c~gagttccgg tttggaatca aagctgcaga ggagcaactt tcagccagag gcatggatgc 180 c~tctctgtct catcggtcac tagcggcact ttcagagaaa ctctcggagt taaaagagca 240 c~actataccc ctgaagaaga agttggagtc ctatttagat ttaatgccga gtccatctct 300 tgctcaactg aaaattgaag aggcaaagag agaattagac gccatagagg ctgaacttac 360 caagaaagta gacatgatgg gcctgtgagg acagccatgt agacgtggtc aggggaagga 420 ggtgactgtt cttaaagatg atgatgtcnc attagtaaaa caacanggtt cgcttctgcc 480 ttta 484 <210> 89 <211> 214 <212> DNA
<213> Unknown <220>
<223> Description of Unknown Organism: Novel <400> 89 cattttaaag aagctcttct atataatcag gatggtgatc tttaataaaa aacatcctga 60 ttatatagaa gagcttcttt aaaatgtttg tgaatgtcat tttr_taatac tggaagaaaa 12D
gtattctgtt gtgtctgatg tgtttaggat gtcctttatg gagctaatta aaagatgaaa 180 cctgaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa 214 <210> 90 <211> 497 <212> DNA
c213> Unknown ~:220>

<223> Description of Unknown Organisms Novel <400> 90 cagaaagcag catgccctgt tgtgtcctgc agcttggaga catcatcgat ggctacaatg 60 cacagtataa agtatcagaa aagtctctag aacttgtcat gaacacattt cagatgctta 120 aagttccagt tcaccacacg tgggggaacc atgagttcta taacttcagc agagactact 180 tagcaagctc taaactgaac agcaagtttc tagaagacca gattgcacag catcctgaga 240 ccacgccatc agagaactat tacgcttat:~ actttgtacc attccctaag ttccggttca 300 ttttactcga ttcttatgac ctgagtgtct tgggcataga cccatcttct ccaaaatatg 360 agcagtgtat gaagatgctg agggagcaca acccaaatgt ggagttgaat aatccccaan 420 ggactttctg agccccanta tgtcagttta acggaggatc agccaagaac aagctgaact 480 ggctgaatga aattcnn 49~
<210> 91 <211> 499 <212> DNA
c213> Unknown <220>

WO 00!43419 PCTIUS00/01431 <223> Description of Unknown Organism: Novel.
<400> 91 ccgtgagggt gctgaaactt ttgctgacca ccgggagggt atcttaaaga cagcaaaggt 60 tcttgtggag gacaccaagg tcctagtgca gaatgcagct gggagccagg agaagttggc 120 acaagccgcc cagtcctccg tggccaccat tacccgcctc gctgatgtgg tcaagctcgg 180 tgcagccagc ctangagccg aagaccctga aactcangtg gtgctgatca atgcagtaaa 240 ggacgtagcc aaggccctgg gtgacctcat cagcgctacg aaggctgcag cgggcaaagt 300 tggggatgac cctgcantgt ggcagctcaa gaactctgcc aangtgatgg tgaccaatgt 360 gacatcattg ctcaagacag tgaangctgt ngaagatgag gccaccaaaa ggcacacggg 420 ncctaaaggc anncacaaga gcacaatacg tcaaggaact ggcggtcctc tgttccccan 480 aaccaactgc caaagaacn 4gg <210> 92 <211> 452 <212> DNA
<213> Unknown <220>

<223> Description of Unknown Organism: Novel <400> 92 cgaaactttt gctgaccacc gggagggtat cttaaagaca gcaaaggttc ttgtggagga 60 caccaaggtc ctagtgcaga atgcagctgg gagccaggag aagttggcac aagccgccca 120 gtcctccgtg gccaccatta cccgcctcgc tgatgtggtc aagctcggtg cagccagcct 180 aggagccgaa gaccctgaaa ctcaggtggt gctgatcaat gcagtaaagg acgtagccaa 240 ggccctgggt gacctcatca gcgctacgaa ggctgcagcg ggcaaaqttg gggatgaccc 300 tgcagtgtgg cagctcaaga actctgccaa ggtgatggtg accaatgtga catcattgct 360 caagacagtg aaggctgtgg aagatgangc caccaaaggc acacgggccc taaangcaac 420 cacaagagca catacgtcag gaactggcgg tc 452 <210> 93 <211> 91 <212> DNA
<213> Unknown <220>
<223> Description of Unknown Organism: Novel lla <400> 93 ccntcnangn tcncacngtc gtntctnccg cacggcntng cntcancaan ggantnannc 60 anngncnggn ttttngngnc cagnttnncg n gl <210> 94 <211> 447 <212> DNA
<213> Unknown <220>
<223> Description of Unknown Organism: Novel <400> 94 cgaggaatgt caactattca atatggaggc ggaggtcgat aagctggaac tgatgttcca 60 gaaagctgat tctgatttgg attaccttca atataggctg gaatatgaag tcaagactaa 120 tcacccacat tcagcaggag agaaaaatgc agttacagtt ttaaaggaat tatcagcgat 180 ~~aagtctcgc tatcaagctt tatgtgcacg ctttaaggca gtttctgttg agcaaaagga 240 claccaagagC tgcatttgtg ctactttgaa caagacaatg accatgatac aagaactaca 300 aaagcaaaca aacctggagc taactctact gactgaagaa gagaaagctg caacagagcc 360 attaaaatct catatgccgg actgataaaa aatgttaaag aggaaggctg gtggaaagaa 420 gancaactgc agagantggg cgagact 447 <210> 95 <211> 454 <212> DNA
<213> Unknown <220>
<223> Description of Unknown Organism: Novel <400> 95 ccaactattc aatatggagg cggaggtcga taagctggaa ctgatgttcc agaaagctga 60 ttctgatttg gattaccttc aatataggct ggaatatgaa gtcaagacta atcacccaca 120 ttcagcagga gagaaaaatg cagttacagt tttaaaggaa ttatcagcga taaagtctcg 180 ctatcaagct ttatgtgcac gctttaaggc agtttctgtt gagcaaaagg agaccaagag 240 ctgcatttgt gctactttga acaagacaat: gaccatgata caagaactac aaaagcaaac 300 aaacctggag ctaactctac tgactgaaga agagaaagct gcaacagagc cattaaaatc 360 tcatatgccg gactgataaa aaatgttaaa gaggaaggct ggtggaaaga agancaactg 420 cagagattgg gcgagacgtc taaaggcggg gctg 454 <210> 96 <211> 490 <212> DNA
<213> Unknown <220>
<223> Description of Unknown Organism: Novel <400> 96 ccaactattc aatatggagg cggaggtcga taagctggaa ctgatgttcc agaaagctga 60 ttctgatttg gattaccttc aatataggct ggaatatgaa gtcaagacta atcacccaca 120 ttcagcagga gagaaaaatg cagttacagt tttaaaggaa ttatcagcga taaagtctcg 180 ctatcaagct ttatgtgcac gctttaaggc agtttctgtt gagcaaaagg agaccaagag 240 ctgcatttgt gctactttga acaagacaat gaccatgata caagaactac aaaagcaaac 300 aaacctggag ctaactctac tgactgaaga agagaaagct gcaacagagc cattaaaatc 360 tcatatgccg gactgataaa aaatgttaaa gangaaggct ggtggaaaga agaccaactg 420 cagagattgg gcgagacgtc tanangcggg gctgatgaag aagcctgaca tttcccttga 480 ncgaaaagga 490 <210> 97 <211> 454 <212> DNA
<213> Unknown <220>
<223> Description of Unknown Organism: Novel <400> 97 cttcaatatg gaggcggagg tcgataagct: ggaactgatg ttccagaaag ctgattctga 60 tttggattac cttcaatata ggctggaata tgaagtcaag actaatcacc cacattcagc 120 aggagagaaa aatgcagtta cagttttaaa ggaattatca gcgataaagt ctcgctatca 180 agctttatgt gcacgcttta aggcagtttc tgttgagcaa aagyagacca agagctgcat 240 ttgtgctact ttgaacaaga caatgaccat gatacaagaa ctacaaaagc aaacaaacct 300 ggagctaact ctactgactg aagaagagaa agctgcaaca gagccattaa aatctcatat 360 gccggactga taaaaaatgt taaagangaa ggctgggtgg gaaagaagac caactgcaga 420 gattgggcga gacgtctana ngcggggctg atga 454 <210> 98 <211> 456 <212> DNA
<213> Unknown <220>
<223> Description of Unknown Organism: Novel <400> 98 cggcggaggt cgataagctg gaactgatgt tccagaaagc tgattctgat ttggattacc 60 ttcaatatag gctggaatat gaagtcaaga ctaatcaccc acattcagca ggagagaaaa 120 atgcagttac agttttaaag gaattatcag cgataaagtc tcgctatcaa gctttatgtg 180 cacgctttaa ggcagtttct gttgagcaaa aggagaccaa gagctgcatt tgtgctactt 240 tgaacaagac aatgaccatg atacaagaac tacaaaagca aacaaacctg gagctaactc 300 tactgactga agaagagaaa gctgcaacag agccattaaa atctcatatg ccggactgat 360 aaaaaatgtt aaagaggaag gctggtggaa agaagaccaa ctgcagagat tgggcgagac 420 gtctagangc ggggctgatg angaaacctg acattn 456 <210> 99 <211> 953 11~

<212> DNA
<213> Unknown <220>
<223> Description of Unknown Organism: Novel <400> 99 caatccaaat cacccacatt cagcaggaga gaaaaatgca gtt:acagttt taaaggaatt 60 atcagcgata aagtctcgct atcaagcttt atgtgcacgc tttaaggcag tttctgttga 120 gcaaaaggag accaagagct gcatttgtgc tactttgaac aagacaatga ccatgataca 180 agaactacaa aagcaaacaa acctggagct aactctactg actgaagaag agaaagctgc 240 aacagagcca ttaaaatctc atatgccgga ctgataaaaa atgttaaaga ggaaggctgg 300 tggaaagaag accaactgca gagattgggc gagacgtcta gaggcggggc tgatgaggaa 360 gcctgacatt tcccttgacc gagaggatcg tgatgcattt cagatgagga aattgtaaga 420 agcgtagatg tcctgtgact gaaaaccaaa agg 453 <210> 100 <211> 474 <212> DNA
<213> Unknown <220>
<223> Description of Unknown Organism: Novel <400> 100 catggaggcg gaggtcgata agctggaact gatgttccag aaagctgatt ctgatttgga 60 ttaccttcaa tataggctgg aatatgaagt caagactaat cacccacatt cagcaggaga 120 gaaaaatgca gttacagttt taaaggaatt atcagcgata aagtctcgct atcaagcttt 180 atgtgcacgc tttaaggcag tttctgttga gcaaaaggag accaagagct gcatttgtgc 240 tactttgaac aagacaatga ccatgataca agaactacaa aagcaaacaa acctggagct 300 aactctactg actgaagaag agaaagctgc aacagagcca ttaaaatctc atatgccgga 360 ctgataaaaa atgttaaaga ggaaggctgg tggaaagaag accaactgca gagattgggc 420 gagacgtcta aaggcggggc tgatgaggaa acctgacatt cccttgaccg aaag 474 <210> 101 <211> 473 <212> DNA
<213> Unknown <220>

WO 00/43419 PCTIUS00/0143i <223> Description of Unknown Organism: Novel <400> 101 ccaactattc aatatggagg cggaggtcga taagctggaa ctgatgttcc agaaagctga 60 ttctgatttg gattaccttc aatataggct ggaatatgaa gtcaagacta atcacccaca 120 ttcagcagga gagaaaaatg cagttacagt: tttaaaggaa ttatcagcga taaagtctcg 180 ctatcaagct ttatgtgcac gctttaaggc: agtttctgtt gagcaaaagg agaccaagag 290 ctgcatttgt gctactttga acaagacaat gaccatgata caayaactac aaaagcaaac 300 aaacctggag ctaactctac tgactgaaga agagaaagct gcaacagagc cattaaaatc 360 tcatatgccg gactgataaa aaatgttaaa gaggaaggct ggtggaaaga agaccaactg 420 cagagattgg gcgagacgtc tanangcggg gctgatgagg aaacctgaca ttt 473 <210> 102 <211> 519 <212> DNA
<213> Unknown ~:220>
~c223> Description of Unknown Organism: Novel WO 00143419 PCTNSOOl01431 <400> 102 ccagcggtct gaggaatgtc aactattcaa tatggaggcg gaggtcgata agctggaact 60 gatgttccag aaagctgatt ctgatttgga ttaccttcaa tataggctgg aatatgaagt 120 caagactaat cacccacatt cagcaggaga gaaaaatgca gttacagttt taaaggaatt 180 atcagcgata aagtctcgct atcaagcttt atgtgcacgc tttaaggcag tttctgttga 240 gcaaaaggag accaagagct gcatttgtgc tactttgaac aagacaatga ccatgataca 300 agaactacaa aagcaaacaa acctggagct aactctactg actgaagaag agaaagctgc 360 aacagagcca ttaaaatctc atatgccgga ctgataaaaa atgttaaaga ggaaggctgg 420 tggaaagaaa gaccaactgc agagattggg cgagacgtct agangcgggg ctgatgaaga 480 <~cctgacatt tcccttgacc gaaaaggatc gtgatgcat 519 ~:210> 103 <:211> 417 <:212 > DNA
<:213 > Unknown r220>
<223> Description of Unknown Organism: Novel <400> 103 caataaggta agtttacaaa agagatttga caaaacacaa tagtagttaa ttcaccatgg 60 tagttaaaaa tgtttacatc tttcttccaa agatacatca tccaagacac attcaagaca 120 attttctgct cacaaaattt tattactggt catcaagctt taactatatt gatgacaaga 180 aatccttgga ctttattagt aattcagaag atcactaggg gcaaaattta tttgaaaagt 240 accaagttct aaacagctct gtcaagaaat taggtgtggc tgctgccatg tttgcttact 300 aaaaagataa agactgagtt gttagaataa tgaaactgtg agcttgagtt actanggttt 360 ttggtttttg ttttgtcttt tttttttttt ttaaanattt gaanggactc caaancn 417 <210> 104 <211> 469 <212> DNA
<213> Unknown c220>
<223> Description of Unknown Organism: Novel ,:400> 104 c:agcatgtgc tctccctgtc ctcgcctcgt ctgccatccc catcccagtg acccacccaa 60 c~ggaggcctg gggtgcgctg ctgttgcttc ctgcgagcct ggccagctct tcccctcccc 120 gtggccaggg ttgggggtgg ctccagtagc taggggaggg tcaattgcca aaactcgatg 180 cctccagccc catctaggag gcagggtagg gtgcctcatt cctctgcctt ctctgtccct 240 tgtaggcagt actcggagcc cctgtcccca cagagagccc ctgagggaca aagccagccg 300 agccttactt gtctcacctg ggccacgtgg cctgaacctc gtgcctggcc tgtcagtatt 36D
taatgcctcc atttgggcca gctggnctga tgcctctgcc ctgggttcnt ggtgcaacat 420 ccctgggccg aacctccctg ggaccaacca aggacaaatt ttggaacaa 469 <210> 105 <211> 442 <212> DNA
<213> Unknown <220>
<223> Description of Unknown Organism: Novel <400> 105 caacgctgaa gaagacatag tcctccaaac caacaagcag atttactccc agcttttgag 60 agcgactgcc aacagaaact caactctgct ggaaagaatt gaagtcgtca tctgtttgct 120 ~3gagcagctt gcctccggta gcagccgatc gagtggcagt gctgtcccct gactgcagtg 180 gtactggagg actgtcttgt cagtgccttc ccgtcctgga gctgtctgtc tgtccactcg 240 gtgctgagac ttattaaata tgaggacata gcgtttaatc aggtccacag tggatacttg 300 cttcccatgg caggcagagg atgtggggtg gttcggcttc ttgtgaangt gagaaactat 360 aatggaaccg aaagctttgt aaatgtattt cctcgagaag tacatggttc cttaatgata 420 acacctcctg cgatgtcgcg an 442 <210> 106 <211> 444 <212> DNA
<213> Unknown <220>
<223> Description of Unknown Organism: Novel <400> 106 caataacagt gccattacca tatgtttgta tgttgcagtt ggatatagct gagactcctc 60 catcttggtt ttctcttcac tcatccattt cttcagctgc atgataagtc tgtcagtaaa 120 ~~catttaacc ccaacaaggt cagactatgg taaagttaga aaaacatatt taccgaagta 180 cacaattgat ctcattgaaa taagaagtga gaaccgctct gttgagcact gaggtgtaaa 240 cgtgcccaga tgcacagcgt gagtagcttc gagtacgcat ctgtttgcac atgttgtaga 300 agcttaaaat gggaacatga ccctgctgtg gagaaccttg gtagaattgt tgttagtact 360 aaaggagatt tccagcgcat acanggtggc actgaaaact gctgaagccc acaagccctc 420 aaccccgaag gctcancggg caat 444 <210> 10?
c211> 483 <212> DNA
c213> Unknown <220>
<223> Description of Unknown Organism: Novel <400> 107 cagcaaagtt ttatataaag gttcctataa tggcccagat agtaaatatt aatactgtct 60 ttggttttgc aactatgttg cttttgtgtg aaaaatagtg aacttggttt ttttgtgtca 120 tcaaataaat gaacttggtt atattccaat aaaacataac tgataaacac ttgacattgg 180 ~3atttcattg gcattttcag agtcaacatt cccaagtatt tgaaagtttg aaaatttccc 240 ~~aaggacatt gacaaagaga tagcagaata ggattagtaa catttctgac ttctggagtt 300 taggtcttga ggcttcaatg ttttatttgt tttgttgggg gaagttggag agacagttca 360 tcagttagaa ngcctggctg ctcttccaga ngacctaagt ggttcacaac catcnttang 420 ttcaaaccga agggaatctg atggcttctg tggtgcatan gcaaaacacc aattcacgtg 480 caa 483 <210> 108 <211> 456 c212> DNA
<213> Unknown <220>
<223> Description of Unknown Organism: Novel <400> 108 ctcactaacc ccacatccag taaagggcta atatccaaaa tatatgaaga agtcaagaag 60 ttaatcactg gccgggtgtg gtggctcacg cctttaatcc cagcactcgg gaggcagagg 120 caggcagatt tctgagtttg aggccagcct ggtctacaaa gtgagttcca ggacagccag 180 ggttatacag agaaaccctg tctcgaaaaa agcaaacaaa caaacaaagg aagttatcac 240 tgaaaaacca aacaacccaa acaaaaaatg gggtatagaa ctaaactgag aattcacaac 300 tgaggaatct cgaatgacgg agaaaaaact aaagaaatgt tccaagtcct tagtgataag 360 agaaatgcaa atcaaaatga ccctgagatt cccccttata ccagtcagaa tggctaagat 420 caaaacttca agtgacaaca catgttggag aggatg 456 <210> 109 <211> 485 <212> DNA
<213> Unknown <220>
<223> Description of Unknown Organism: Novel <900> 109 ccttgaaatg aaagatattg ctatcaacat cagcagaaac ctgaaggact taaaccagaa 60 atatgctgaa ctacagccat atctggacca gatcaacatg attgaggagc aggtggcagc 120 tctggagcag gcagcctaca agctcgatgc ttattcaaaa aaactggaag ccaagtacaa 180 ~~aagttggag aagcgatgag aatcgtgttc atgtggctca agtgtttgtt aatatggaag 240 atgttttata accactaagt cctgaggctg acacatcatc agagctcctc agaagaaaat 300 l3gctggttaa gcccaaggcc acgcaatact ggatgaactg gagggccgtg gctgctccgg 360 agtctcctct gaggtggtac ctctcagaaa ctcacacctg ggactcctca ccgtttactt 420 gaatgcttca tcatccatct aagatggaga ctgtanantt canggcatac ctgggtttgc 480 cagtn 485 <210> 110 <211> 450 c212> DNA
<213> Unknown <220>
<223> Description of Unknown Organism: Novel <400> 110 ccttgaaatg aaagatattg ctatcaacat cagcagaaac ctgaaggact taaaccagaa 60 atatgctgaa ctacagccat atctggacca gatcaacatg attgaggagc aggtggcagc 120 tctggagcag gcagcctaca agctcgatgc ttattcaaaa aaactggaag ccaagtacaa 180 gaagttggag aagcgatgag aatcgtgttc: atgtggctca agtgtttgtt aatatggaag 240 atgttttata accactaagt cctgaggctg acacatcatc agagctcctc agaagaaaat 300 ggctggttaa gcccaaggcc acgcaatact. ggatgaactg gagggccgtg gctgctccgg 360 WO 00/43419 PG"TlUS00/01431 agtctcctct gaggtggtac ctctcagaaa ctcacacctg ggactcctca ccgtttactt 420 gaatgcttca tcatccatct aagatggaga 450 <210> 111 <211> 552 <212> DNA
<213> Mouse <400> 111 cacaatttct ccaataattc tatactttct ggcaagtttc tatacaaagt atgacccgac 60 tcacttcatc ctaaacacag cttctctgct gagtgtactt attcccaaaa tgccacagct 12o gcatggtgtt cgcatctttg gaattaataa gtattgaaat gtctgtgcaa ctgaagagaa 180 ctgttaagag cttcctgtaa tgaaagagta gttaatgaac tgcactgttt ctgtgataat 240 gtgaaataag aggtatttac attggagggc caagtactgc tccttcacaa gctgtcttga 300 agtgctctac ttaaggatgc ctctgtctag tttgcttggt atatttctga gaaangcttt 360 gcangcaagc tgggcangcc cancttataa cttgatggta gtaggataag agtacagtaa 420 ctaaaatcca aggaaatgtg ggatttgtaa gaaactaggt ccagcttata gtgagcgagc 480 WO 00/43419 PC'TlUS00/01431 attatgttan ggaaaagagc attccaatca ttcgttcaaa accggttgaa naaaacttct 540 agaaaagcag an 552 <210> 112 <211> 467 <212> DNA
<213> Mouse <400> 112 cgcggtgcag cggctgcagc gcctcctggc ctcgggggct atgtctgaga gcaggggatg 60 gctccaccca ttcagcactg ccacccaaag aactgctggg gaagactgca gttccgagga 120 ccctcctgat ggactgggac cctcccttgc tgagcaggcc ttaaggctca aagctgttaa 180 actggaaaag gaagtccagg atttaaccct gagataccag agagctgtcg ctgattgtga 240 aaacataagg agaagaaccc agagatgtgt ggaagacgcg aagatatttg ggatccagag 300 cttctgcaag gacctggtgg aggtggcaga cattttggag aagactgcca agtctgttca 360 gaangggcgg aacctgagga ccacangcgr. actctggaaa aagtcntcca agggctgtcc 420 ctgctanaan ccanactgaa aagcgtgttt accaancatg gcctaaa 467 <210> 113 <211> 360 <212> DNA
<213> Mouse <400> 113 cgaattttgc agaagaaatg aaggctaaga atacctctca ggaaatcatg ttacgagatc 60 ttcaggaaaa actaaatcag caagaaaact cactaacttt agagaagctg aaacttgccc 120 tagctgatct ggaaagacag cgaaactgtt ctcaagatct cttgaagaaa agggaacatc 180 acattgatca actgaataat aagttaaata agatagagaa agagtttgaa actttgctga 240 gtgctttgga attaaaaaag aaagaatgtg aagaattgaa aaaaaaaaaa aaaaaccncn 300 ananatntan naancgtaaa tnnggaaaan. ccccnnagtt ttnnaannnn nnnnnnnnnt 360 <210> 114 <211> 488 <212> DNA
<213> Mouse <400> 114 ~~ctaagggag gaatttggct ataatgcaga gacacaaaag ctgctgtgca agaatgggga 60 <~acgctgcta ggggctgtga acttctttgt ctctagcatc aacacactgg tcaccaagac 120 catggaagac acactcatga ctgtcaaaca gtatgaggct gccaggctgg aatatgatgc 180 ctaccggaca gacttagaag agctgagcct agggccccgg gatgcaggga ctcgtggtcg 240 acttgagagt gcccaggcca ctttccagac ccatcgggac aaatatgaga agctgcgggg 300 agatgtggcc atcaagctga agttcctgga agaaaacaag atcaaggtga tgcacaagca 360 gctgctgctc ttccacaatg ccgtgtctgc ctacttcgct gggaaccaga aacagctgga 420 gcagactctt caacaagttc aacatcaagc tgcggcctcc aaggagccga naanccttcc 480 tggttaag 488 <210> 115 <211> 494 <212> DNA
<213> Mouse <400> 115 caagaaaaac aactggactg gtgaattttr. agctcgtttt ctattgaagt taccagtaga 60 tttcagcaac attcccacat accttctcaa ggatgtaaat gaagaccctg gagaagatgt 120 ggcccttctt tctgtcagtt ttgaggatac tgaagctacc caggtgtacc ccaagttgta 180 wo Qo~a34i9 rcTIUSOOioi43i cttgtcaccc cgaattgagc atgcactcgg aggctcctct gctcttcaca tccctgcttt 240 ccccggagga ggatgtctca ttgattatgt gcctcaagtg tgccacctgc tcaccaacaa 300 ggtacagtat gtgattcaag gctatcacaa aagaagagag tacatcgeag ctttcctcag 360 tcactttggc acaggtgtcg tggaatatga tgcagaaggc ttcacaaaac ttactctgct 420 gctgatgtgg aaagactttt gttttcctgt ccacattgac ctgcccctgt ttttccctcg 480 aagatcaacc tang 494 <210> 116 <211> 495 <212> DNA
<213> Mouse <900> 116 caagaaaaac aactggactg gtgaattttn agctcgtttt ctattgaagt taccagtaga 60 tttcagcaac attcccacat accttctcaa ggatgtaaat gaagaccctg gagaagatgt 120 ggcccttctt tctgtcagtt ttgaggatac tgaagctacc caggtgtacc ccaagttgta 180 cttgtcaccc cgaattgagc atgcactcgg aggctcctct gctcttcaca tccctgcttt 240 ccccggagga ggatgtctca ttgattatgt gcctcaagtg tgccacctgc tcaccaacaa 300 WO 00/43419 PCTlUS00/01431 ggtacagtat gtgattcaag gctatcacaa aagaagagag tacatcgcag ctttcctcag 360 tcactttggc acaggtgtcg tggaatatga tgcagaaggc ttcacaaaac ttactctgct 420 gctgatgtgg aaagactttt gttttcctgt ccacaatgac ctgcccctgt ttttccctcg 480 agatcagcta cgctc 4g5 <210> 117 <211> 485 <212> DNA
<213> Mouse <400> 117 ccgagacaga gctgcacacg gttctccatc ctggactcca cgctcggttt ctagagagca 60 agtctccaaa ggaccagagt accttccaga aattggtcca catcaacacc accgccctgc 120 tggagcaccc tgaatataaa ggccctttcc tgccagcctc agctgtagcc cccatcccct 180 caccctctaa taaaaagatg acacccctac agaaggagct gcaggagaca ctgaaggcac 240 tgctggggaa cactgacaag ggcagcctgg aggtggccac acagtatggc tgggtgctgg 300 ;atgcagaggt gctcctggat gccgacggcc acttcctgcc cctaagaaac ttcgttgctc 360 cccaccttgc ccagccagtg gggaaccagc cattgccccc angagctaaa aggatcgctt 420 tcctgcgctg ggagttccca acttcaacan cagaagcaag gacatgttgg gccgttttgt 480 cctgg 485 <210> 118 <211> 504 <212> DNA
<213> Mouse <400> 118 ccccgaacct cccaaaggag agttgcagtt gccaccgcct ccacctccag gccactatgg 60 agcctgggct gcgcaggagc ttcaggccag attggcagag ataggagcgc cgatccaggg 120 gagtcgcgag gagttagtag agcggctgca gacctacacc cgtcagacgg gcattgtcct 180 gaatcgacca gttttgagag gagaagatgg ggacaaagcg gctcctcctc ctatgtcagc 240 acagctgtct gggattccta tgccaccacc acctatggga cttccccctc tacagcctcc 300 tccaccacct ccaccacctc caccaggcct tggccttggc tttcctatgg ctcacccacc 360 aaatctgggg cccccaccac ctcttcgagt gggtgagcct gtggcgctgt cagaagaaga 420 ,acggctgaag ctggcacagc agcaggcaac tttgctgatc agcaaganga gcgttccaag 480 cangcaactg tgttaatgga ncag 504 <210> 119 <211> 482 <212> DNA
<213> Mouse <400> 119 cgaaattcgt tcctgtttct tttatagtaa aattgaagag ctggagaatg aaattgaaga 60 tgtaaaaagt aatattgaaa tgaaaagtct tgcactgagc aggatgaaac tttcagttgc 120 acttcgggat aacatggaga acatgggacc tgagaattgt ytgctcacag atgacatgaa 180 gcacatatta aagctacaaa agttaataat gaagtcccag gaggagtctt cagaactgga 240 gaaaaagctg cttgatgtca gaaagaagag actacggcct tattttggcg agtaaaacca 300 actgggcaga ggaccctgcc ctcagagaga cagtgctgca gctggagaag gactcaacac 360 gctatgagaa gaatcccgca gtttaaaatt ggctttgtga ncaatccgtc atttaaataa 420 ccgagttcaa ggggaaatgt aatggtgttt ttgggaacac tctgctgctt cagttttggg 48D
gg 482 <210> 120 <211> 473 <212> DNA
<213> Mouse <400> 120 caacaagaca gactacaccg gccccgagag ctatgtggct cagatgatca agaacaagaa 60 cctggactgg ttcccgcgga tgcgtgccat gtccctggtg agcggcgagg gggaggggga 120 gcagaatgag atccgcatcc tgcaggagaa gctcggctcc accatgaagc tggtgtcgca 180 cctcacctcc cagctcaatg aactcaagga gcagatgacg gagcagcgga aacggaggca 240 gcgtctgggc tttgtggatg tgcagaactg catgagccgc tgagcagagc caccgaggcc 300 ccctgcagga ggtccgctgc agatggcact gcccttcctt caggtcaggt ggcccaccac 360 cctctggaag ccangtgccc aacccacctc cagctctgct ggcaatctcc ctcccccaag 420 gcaaactggc caacangagt gcttcacaac aatcctgctt agtaccgttg aat 473 <210> 121 <211> 544 <z12> nNA
<213> Mouse <400> 121 caaaaactca ggaactgtgg gtgctgttgc cttggactgc agaggaaact tggcttacgc 60 aacctctact ggggggattg tcaataaaat ggttggccga gttggagact cgccttgcat 120 aggagctgga ggttacgcag ataataacct tggagccgtt tcaaccacag gacatgggga 180 aagtatcctg aaggtgaatc tggccagact tgccctcttc catgtagagc aaggaaagac 240 cgtagaggag gctgctcagt tggcattgga ttacatgaag tcaaaactca aaggtctagg 300 tggcctcatc ttggtcaaca aaacaggaga ctgggtggca aagtggacct ctgcctccat 360 gccctgggca gcggtgaaga atggcaagct gcangccggc attgacctct gtgagaccan 420 gacaagggac ctancttgct aacctgggac gtgaagctga actacctggg tgtnaagacc 480 taacttaatc aattanatgt nnacatggaa aacttaanan nnnnnnnnnn nnnnnnrinnc 540 <210> 122 <211> 485 <212> DNA
<213> Mouse <400> 122 cccacaggtc cctgtagaaa aactgataac tgagagaacc aagaagcacc tggagaaaga 60 gaagcttgag aagggagcac agaatgctga ctgccctaaa aactcaggaa ctgtgggtgc 120 tgttgccttg gactgcagag gaaacttggc ttacgcaacc tctactgggg ggattgtcaa 180 taaaatggtt ggccgagttg gagactcgcc ttgcatagga gctggaggtt acgcagataa 240 taaccttgga gccgtttcaa ccacaggaca tggggaaagt atcctgaagg tgaatctggc 300 cagacttgcc ctcttccatg tagagcaagg aaagaccgta naggaggctg ctcagttggc 360 attggattac atgaagtcaa aactcaaagg tctangtggc ctcatcttgg tcaacaaaac 920 aggagactgg gtggcaaant ggacctctgc ctccatgccc tggggcaacg gtgaanaatg 480 gcaan 485 <210> 123 <211> 479 <212> DNA
<213> Mouse <400> 123 cccaacatcc cccccatcca gtcatcccga ctcccctgaa aatgaaaaga cagagaccac 60 13'7 wo oomn9 pcTn~sooio~a3i attcactttc cctgcacctg ttcagcctgt gtctttgccc agccccacct ccacagacgg 120 tgatatccat gaggattttt gcagtgtttg cagaaaaagt ggccagttgc tgatgtgtga 180 cacgtgttcc cgtgtgtatc atctggactg cttagagccg cccctgaaaa caattcccaa 240 gggcatgtgg atctgtccca gatgtcagga ccagatgcta aagaaggaag aagcaattcc 300 ttggcctgga accttagcca ttgttcattc ctacatcgcc tacaaagcag caaaagaaga 360 ggagaaacag aagctactta agtggagttc agatttaaaa caggagcggg aacaactggg 420 agcanaaggt caaagagctc aacagttcta taaagtaaat gtatggagat gaagaacaa 479 <210> 124 <211> 430 <212> DNA
<213> Mouse <400> 124 cgggaagtga gtgctgggca cttgaggccc gtccctgcct gagcttctcc taccttctgc 60 tcattgtccc cttacaagta tcacttttag gataactagc cctggaaaaa cggtatctgt 120 ttgaagtgaa gggactggtt cttcttgctt tcagcccgtg taaatattta aataaaacag 180 tattaacatt tttgtgctgc ttctcgttag gttgtagatc tagccatgct ctggccgcct 240 ctgccatcct ggaggtagtt ttccttaact tccagaatag tgattttaaa actttaaaaa 300 aaaaaagggg gggggataac ctatccttac atacaagcat aacagattgt atctaacttt 360 atcacatatg atanagatgt atatgctgta aagtgagggg aaggagcttt ctaataaacc 420 antgctttgt 430 <210> 125 <211> 499 <212> DNA
<213> Mouse <400> 125 ccaagaggaa gggtatagcc agtaccaacg catgctgagc actctgtccc agtgtgaatt 60 ttcaatgggc aaaacattgc tggtatatga tatgaatctc agagaaatgg aaaattatga 120 aaaaatatac aaagaaatag aatgtagtat tgctggagca catgaaaaaa ttgctgagtg 180 taaaaagcag attcttcaag caaaacgaat acgaaaaaat cgacaagaat atgacgcttt 240 ~3gccaaagtg atccagcatc acccagacag gcatgagaca ctgaaggagc tagaggctct 300 c3ggcaaagaa ttagagcatc tctcacatat taaagaaagt. gttgaagata agctggaatt 360 gagacggaaa caatttcacg ttcttcttag taccatccat gaacttcaac agacattgga 420 gaatgatgac aagctgtcag aggtggatga agctcaagaa agcaccatgg aancagancc 480 taagccatag atggactgg 4g9 <210> 126 <211> 455 <212> DNA
<213> Mouse <400> 126 ccaagaggaa gggtatagcc agtaccaacg catgctgagc actctgtccc agtgtgaatt 60 ttcaatgggc aaaacattgc tggtatatga Catgaatctc agagaaatgg aaaattatga 120 aaaaatatac aaagaaatag aatgtagtat tgctggagca catgaaaaaa ttgctgagtg 180 taaaaagcag attcttcaag caaaacgaat. acgaaaaaat cgacaagaat atgacgcttt 240 ggccaaagtg atccagcatc acccagacag gcatgagaca ctgaaggagc tagaggctct 300 gggcaaagaa ttagagcatc tctcacatat taaagaaagt gttgaagata agctggaatt 360 gagacggaaa caatttcacg ttcttcctag t:accatccat gaacttcaac agacattgga 420 c3aatgatgac aagctgtcag aagtggatga aactn 455 WO 00/43419 PCT/US00l01431 <21D> 127 <211> 435 <212> DNA
<213> Mouse <400> 127 cgacgctttt ggccaaagtg atccagcatc acccagacag gcatgagaca ctgaaggagc 60 tagaggctct gggcaaagaa ttagagcatc~ tctcacatat taaagaaagt gttgaagata 120 agctggaatt gagacggaaa caatttcacg ttcttcttag taccatccat gaacttcaac 180 agacattgga gaatgatgac aagctgtcag aggtggatga agctcaagaa agcaccatgg 240 aagcagaccc taagccatag atggactgac cgtcgccatt tacagggaca gcgaataact 300 gcatgggctt cctgggttca gtgtgttgta cttttgggat atttcaactt cagcattgaa 360 gtactttgct ttcaagtatt catgtgtcgt tcatatttcc tcacanaacg gaaatgtntc 420 ccatatatat gtttn 435 <210> 128 ~:211> 457 ~c212> DNA

W O 00!43419 PCT/US00/01431 <213> Mouse <400> 128 caaaagcaga ttcttcaagc aaaacgaata cgaaaatcga caagaatatg acgctttggc 60 caaagtgatc cagcatcacc cagacaggca tgagacactg aaggagctag aggctctggg 120 caaagaatta gagcatctct cacatattaa agaaagtgtt gaagataagc tggaattgag 180 acggaaacaa tttcacgttc ttcttagtac catccatgaa cttcaacaga cattggagaa 240 tgatgacaag ctgtcagagg tggatgaagc tcaagaaagc accatggaag cagaccctaa 300 gccatagatg gactgaccgt cgccatttac agggacagcg aatagctgca tgggcttcct 360 gggttcagtg tgttgtactt ttgggatatt tcaacttcag cattgaagta ctttgcnttc 420 aagtattcat gtgtcgttca taattcctca caanaan 457 <210> 129 <211> 484 <212> DNA
<213> Mouse <400> 129 ccctacagaa atccagagac tcacctacag tcaggagaca tgtgaaaatc ttcaggagat 60 14z gctcggtgaa ctcttcacgc ctgtagagac accagaagca ccaaacagag gcttcttcaa 120 aggcttattt ggaggcggtg cacaatctct cgatagagaa gaactatttg gagagtcgtc 180 ctcgggaaag gcctccagga gcctcgcaca acacatcccg ggccctggtg ggatcgaagg 240 tgtgaaagga gccgcgtcag gagtggtggg tgaacttgcc cganccaggc tggccctcga 300 cgaaagagga cagaagctca gtgacttgga agaaaggacg gcagccatga tgtccagcgc 360 agactccttc tccaaacatg ctcatgagat gatgctgaaa tacanagata agaagtggta 420 ccagttctga caactagaac tcagtaagtc cagcttcaac cagaanggaa aagaatttcc 480 ttgt 484 <21D> 130 <211> 499 <212> DNA
<213> Mouse <400> 130 ccaggagaca tgtgaaaatc ttcaggagat gctcggtgaa ctcttcacgc ctgtagagac 60 accagaagca ccaaacagag gcttcttcaa aggcttattt ggaggcggtg cacaatctct 120 cgatagagaa gaactatttg gagagtcgtc ctcgggaaag gcctccagga gcctcgcaca 180 acacatcccg ggccctggtg ggatcgaagg tgtgaaagga gccgcgtcag gagtggtggg 240 tgaacttgcc cgagccaggc tggccctcga cgaaagagga cagaagctca gtgacttgga 300 agaaaggacg gcagccatga tgtccagcgc agactccttc tccaaacatg ctcatgagat 360 gatgctgaaa tacaaagata agaagtggta ccagttctga caactagaac tcagtaagtc 420 cagcttcaac cagaaggaaa aagaagtttc cttgttgata tcactgatgt atttggggaa 480 gataacgtaa aagggacgc 4gg <210> 131 <211> 474 <212> DNA
<213> Mouse <400> 131 caacagaggc ttcttcaaag gcttatttgg aggcggtgca caatctctcg atagagaaga 60 actatttgga gagtcgtcct cgggaaaggc: ctccaggagc ctcgcacaac acatcccggg 120 ccctggtggg atcgaaggtg tgaaaggagc cgcgtcagga gtggtgggtg aacttgcccg 180 agccaggctg gccctcgacg aaagaggaca gaagctcagt gacttggaag aaaggacggc 240 agccatgatg tccagcgcag actccttctc caaacatgct catgagatga tgctgaaata 300 caaagataag aagtggtacc agttctgaca actagaactc agtaagtcca gcttcaacca 360 agaaggaaaa agaagtttcc ttgttgatat cactgatgta tttggggaag ataacgtaaa 420 agggacgcac tttgctgana acttctttcc cagcacaatc atgcactgtt ttac 474 <210> 132 <211> 459 <212> DNA
<213> Mouse <400> 132 caacagaggc ttcttcaaag gcttatttgg aggcggtgca caatctctcg atagagaaga 60 actatttgga gagtcgtcct cgggaaaggc ctccaggagc ctcgcacaac acatcccggg 120 ~~cctggtggg atcgaaggtg tgaaaggagc egcgtcagga gtggtgggtg aacttgcccg 180 agccaggctg gccctcgacg aaagaggaca gaagctcagt gacttggaag aaaggacggc 240 ~~gccatgatg tccagcgcag actccttctc caaacatgct catgagatga tgctgaaata 300 c:aaagataag aagtggtacc agttctgaca actaagaact cagtaagtcc agcttcaacc 360 aagaaggaaa aagaaatttc cctgttgata tcactgatgt a~tttgggga anataacgtt 420 aaaaggggac ncactttgct gaaaacttct tcccancan 459 <210> 133 <211> 478 <212> DNA
<213> Mouse <400> 133 ccgcatcatg cagttctgcc acacactgag agagtttgcc cttgagtatc ggacttgtcg 60 ggaacgggta ctgcagcagc agcagaagcg ggctacatac cgtgagcgca acaagacccg 120 tggtcgcatg attaccgaga cagagaagtt ctcaggtgtg gctggggagg cccccaataa 180 cctgtctgtc ccagtggctg tgggcagcgg gccaggtcag ggtgatactg acaatcatgc 240 cagcatgaag agcctgctta ccagcaggcc ggaagatgcc acacacagcc gacgcagcag 300 ,~ggtatggtc cagagcagtt cccccgtctc acacacagca gtggggccct ccgctgcatc 360 ccctgaggag actgcagcct ccggcttacc caccgacacg tcagatgaga taatggacct 420 <lctggtgcag tcagttacca agagcggtcc taaancctta gctgctcggg aaaaggaa 478 ~:210> 134 <211> 476 <212> DNA
<213> Mouse <400> 134 cccgagcgcc atgtcgggtt cttctagcgt cgccgccatg aagaaggtgg tccagcagct 60 ccggctggag gccgggctca accgcgtgaa ggtttcccag gcagctgcag acttgaaaca 120 gttctgtctg cagaatgctc aacatgaccc tctgctgact ggagtgtctt caagtacgaa 180 tcccttcaga ccccagaaag tctgctcctt tttgtagtca tctatcttga ggtttctcaa 240 accacttttc atgaaccagt gaatattcaa gagaactaaa tttgaagtct gtacaaaagc 300 ttctctttaa cacgtgccat aatacactat: cttctgctcg tcagtcr_tta acatctacct 360 ctctgaattt catggatttc tgtctcacaa ggtttaacta ttttatatac actggctgta 420 gcatacaata aagcatcata caanananaa aaaaaaaann cccganagtt ntttnt 476 <210> 135 <211> 475 <212> DNA
<213> Mouse <400> 135 cccgagcgcc atgtcgggtt cttctagcgt cgccgccatg aagaaggtgg tccagcagct 60 ccggctggag gccgggctca accgcgtgaa ggtttcccag gcagctgcag acttgaaaca 120 gttctgtctg cagaatgctc aacatgaccc tctgctgact ggagtgtctt caagtacgaa 180 tcccttcaga ccccagaaag tctgctcctt tttgtagtca tctatcttga ggtttctcaa 240 accacttttc atgaaccagt gaatattcaa gagaactaaa tttgaagtct gtacaaaagc 300 ttctctttaa cacgtgccat aatacactat: cttctgctcg tcagtcctta acatctacct 360 ctctgaattt catggatttc tgtctcacaa ggtttaacta ttttatatac actggctgta 420 gcatacaata aagcatcata cannananaa aaaaaaaanc ccgnnaantn tttnt 475 <210> 136 <211> 458 c212> DNA
<213> Mouse <400> 136 cgtcccgagc gccatgtcgg gttcttctag cgtcgccgca atgaagaagg tggtccagca 60 gctccggctg gaggccgggc tcaaccgcgt gaaggtttcc caggcagctg. cagacttgaa 120 aicagttctgt ctgcagaatg ctcaacatga ccctctgctg actggagtgt cttcaagtac 180 gaatcccttc agaccccaga aagtctgctc ctttttgtag tcatctatct tgaggtttct 240 caaaccactt ttcatgaacc agtgaatatt caagagaact aaatttgaag tctgtacaaa 300 agcttctctt taacacgtgc cataatacac tatcttctgc tcgtcagtcc ttaacatcta 360 cctctctgaa tttcatggga tttctgtctc acaaggttta actaatttat atacactggc 420 tgtnncatac aataaagcat catacaaaac aanaaaaa 458 <210> 137 <211> 465 <212> DNA
<213> Mouse <400> 137 ccagccgggc gccatgtcgg gttcttctag cgtcgccgcc atgaagaagg tggtccagca 60 gctccggctg gaggccgggc tcaaccgcgt gaaggtttcc caggcagctg cagacttgaa 120 acagttctgt ctgcagaatg ctcaacatga ccctctgctg actggagtgt cttcaagtac 180 gaatcccttc agaccccaga aagtctgctc ctttttgtag tcat:ctatct tgaggtttct 240 caaaccactt ttcatgaacc agtgaatatt caagagaact aaatttgaag tctgtacaaa 300 agcttctctt taacacgtgc cataatacac tatcttctgc tcgtcagtcc ttaacatcta 360 cctctctgaa tttcatggat ttctgtctca caagggttaa ctattttata tacactggct 420 gtagcataca ataaagcatc atacaannrin nnnnaaaaaa annnn 465 <210> 138 <211> 472 <212> DNA
<213> Mouse <400> 138 cggcccggct gtcccgagcg ccatgtcggg ttcttctagc gtcgccgcca tgaagaaggt 60 ggtccagcag ctccggctgg aggccgggct aaaccgcgtg aaggtttccc aggcagctgc 120 agacttgaaa cagttctgtc tgcagaatgc tcaacatgac cctctgctga ctggagtgtc 180 ttcaagtacg aatcccttca gaccccagaa agtctgctcc tttttgtagt catctatctt 290 gaggtttctc aaaccacttt tcatgaacca gtgaatattc aagagaacta aatttgaagt 300 ~~tgtacaaaa gcttctcttt aacacgtgcc ataatacact atcttctgct cgtcagtcct 360 ~~aacatctac ctctctgaat ttcatgggat ttctgtctca caaggtttaa ctattttata 420 tacactggct gtagcataca ataaagcatc atacaanana naaaaaaaaa as 472 <210> 139 <211> 234 <212> DNA
<213> Unknown <220>
<223> Description of Unknown organism: Novel <40D> 139 cgttaggctt ccttttcttg ctgtaaactc ctctctggtg tgtccaacaa tgtattttaa 60 acttgtcttg atgtatgaat ttctttgttc tataaaatta tcaaattgct tccacgttgg 120 ~iatacaacat tttgcttaaa tctatgtccc tgtgataatg tatttacatc tccagaataa 180 actatctttt atttccacta aaaaaaaaaa aaaaaaannn naaananaaa aaaa 234 <:210> 140 <211> 79 <212> DNA
<213> Unknown <220>
<223> Description of Unknown organism: Novel <400> 140 cgagagagag aactagtctc gagttttttt tttttttttt tttgggaaaa aaaaaanggg 60 aaattttttn ngggggngg 79 <210> 141 <211> i34 <212> DNA
<213> Unknown <220>
<223> Description of Unknown Oxganism: Novel <400> 141 cgtcgacgcg gccgcgaatt cggggccnccfi qggnnnanat ggnnatacna tcaccaaggt 60 taagggacnt tngttgattg nannnnnaca tnnntnaggn nacttttang atttgntnaa 120 antgtcnntc atcn 134 <210> 142 <211> 359 c212> DNA
<213> Unknown ~:220>

<223> Description of Unknown Organism: Novel <400> 142 catgttttga tgtaaattcg aagtatgaga aactgtggga agtgctgcga gaacgacagg 60 aaagccttca gactgtcttc agcagaatgg aggaggttca aaaggaggcg agctctgtat 120 tacagtggtt agaatcaaaa gaagaagtcc tgaaagccat ggatgccact ttgtctccaa 180 ccaagacaga aacagtaaaa gcccaagcag aatctaacaa ggcttttctg gctgaatttg 240 gaacagaatt cccctaaaat tcagaaagta aaggaagccc tggctgggat tactgaagac 300 ctatcccaac tcacaagaag caaaanaaaa aaaaaaaacc ccnaaaattc tttganririt 359 <210> 143 <211> 176 <212> DNA
<213> Unknown <220>
<223> Description of Unknown Organism: Novel c400> 143 cgagagagag agagagagag agagagagag agagagagag agagagagag agagagagag 60 agagagagag tgtgngngng ngcgcgcgng nggggggggg gggggtgtgn gngngngngc 120 gcgcgcgcgc gcgcgcgcng tgggngagtg ngngggngtg agngcgcgcg cncttt 176 <210> 144 <211> 535 <212> DNA
<213> Homo sapiens <400> 144 ccatgccccc accaggctcc ccttctggtg gcgcagggag tcctggaggc ctgggtcttg 60 agagcctgtc accggagttt tttacctcag tggtgcaggg tgtgctcagc tccctgctgg 120 gctccctggg ggctcgggct ggcagcagtg aaagtattgc tgccttcata caacgcctca 180 gtggatccag caacatcttt gagcctggag ctgatggggc ccttggattc tttggggcct 240 tgctttctct tctgtgccag aacttctcta tggtggacgt agtgatgctt ctccatgggc 300 atttccagcc actacaacgg ctccagcccc: agctgcgatc cttcttccac cagcactacc 360 tgggtggtca ggagcccaca cccagtaaca t~ccggatggc aacccacaca ttgatcacgg 420 ggctagaaga gtatgtgcgg gagagttttt ccttggtgca ggttcagcca ggtgtggaca 480 tcatccggac aaacctggaa ttctccaaga gcagtttaat agcattgctg cgcaa 535 WO 00/43419 PCT/~3500101431 <210> 145 <211> 563 <212> DNA
<213> Homo sapiens <400> 145 cttctggtgg cgcagggagt cctggaggcc tgggtcttga gagcctgtca ccggagtttt 60 ttacctcagt ggtgcagggt gtgctcagct ccctgctggg ctccctgggg gctcgggctg 120 gcagcagtga aagtattgct gccttcatac aacgcctcag tggatccagc aacatctttg 180 agcctggagc tgatggggcc cttggattct ttggggcctt gctttctctt ctgtgccaga 240 acttctctat ggtggacgta gtgatgctt.r. tccatgggca tttccagcca ctacaacggc 300 tccagcccca gctgcgatcc ttcttccacc agcactacct gggtggtcag gagcccacac 360 ccagtaacat ccggatggca acccacaca;: tgatcacggg gctagaagag tatgtgcggg 420 agagtttttc cttggtgcag gttcagccag gtgtggacat catccgggac aaaacctggg 480 aatttctcca aagagcagtt taataagcaa ttgctgcgca tgttctgcaa ttgcacaaga 540 tagtgggatt tggggcccgg ttn 563 <210> 146 <211> 561 <212> DNA
<213> Homo sapiens <400> 146 cctgggagaa gttcgtgtat ttggacgaga agcagcacgc ctggctgccc ttaaccatcg 60 agataaagga taggttacag ttacgggtgc tcttgcgtcg ggaagacgtc gtcctgggga 120 ggcctatgac ccccacccag ataggcccaa gcctgctgoc tatcatgtgg cagctctacc 180 ctgatggacg ataccgatcc tcagactcca gtttctggcg cttagtgtac cacatcaaga 240 ttgacggcgt ggaggacatg cttctcgagc tgctgccaga tgactgatgt atggtcttgg 300 cagcacctgt ctcctttcac cccagggcct gagcctggcc agcctacaat ggggatgttg 360 tgtttctgtt caccttcgtt tactatgcct gtgtcttctc caccacgctg gggtctggga 420 ggaatggaca gacagaggat gagctctanc cagggcctgc aggacctgcc tgtagccact 480 ctgctcgcct tagcactaca ctcctgccaa ggaggatcca tttggcanan cttcttccag 54D
gtgccancta tanctgtgcc c 561 <210> 147 WO 00!43419 PCT/US00/01431 <211> 572 <212> DNA
<213> Unknown <220>
<223> Description of Unknown Organism: Novel <400> 147 tgaaactcga gcttcataaa ccagtgaaat gcctacccct atggagacgg ggagctggtc 60 atgggaccca ggtgcctgga ggacatccca gtggctttga aggctccagc cagtcaagac 120 ggtcaatatt agcccttctg ccaacatctg gcaatgtgag gctggggtgg acgttggcct 180 gatgttgcca ggagtaggat gctgatgctg ccagagagta ggtgggctcc aaaccccagg 240 cttctcactt gcttactaag cacagcagtc tgaagcttgg gacctggcag tgcgtctttg 300 gagaaggcaa aaaagccaca gcagcaacac ttaggagcaa gacccttccc gctctccacc 360 ctatttcctc ccctgaagaa gagcaacagc tcaagctcta gcatggcaca gaacgctagg 420 ttgggccagg caagcagcca tggtggggcc aggtgaagca atgtgggtct cagcaaggaa 480 ;3ccctctgaa ggtggcagtt ggctcccagg gctgctgcac actggacaac acaactttgg 540 ~:atcagctgc atggtgaact gcaaaagtgg tn 572 WO 00/43419 PCTlUS00/01431 <210> 148 <211> 569 <212> DNA
<213> Homo Sapiens <400> 148 ctataacaga ggcttgcgat ggcagtgatg atatttttgg gttgagtact gatagtctgt 60 ctcgtttacg aagcccatct gttttggaag ttagagaaaa gggctatgaa cgattaaaag 120 aagaactcgc aaaagctcag agggaactga agttaaaaga tgaagaatgt gagaggcttt 180 caaaagtgcg agatcaactt ggacaggaat tggaagaact cacagctagt ctatttgagg 240 aagctcataa aatggtgaga gaagcaaata tcaagcaggc aacagcagaa aaacagctaa 300 aagaagcaca aggaaaaatt gatgtacttc aagctgaagt agctgcattg aagacacttg 360 tattgtccag ttctccaaca tcacctacgc aggagccttt gccaggtgga aagacacctt 420 ttaaaaaggg gcatacaaga aataaaagca caagcagtgc tatgagtggc agtcatcagg 480 acctcagtgt gatacagcca attgttaaag actgcaaaga ggctgactta tccttgtata 540 atgaantccg attgtggaag gatganccc 569 WO 00/43419 PCT/USOOf01431 <210> 149 <211> 456 <212> DNA
<213> Homo Sapiens <400> 149 gacccttccc ccgagctcat ggagggccca gaggaggaca ttgctgacaa ggttgtcttc 60 ctggaaaggc gtgtgctgga gctggaaaag gacacggcag ccaccggtga gcaacacagc 120 cgcctgaggc aggagaacct gcagctggtg cacagagcaa acgccctgga ggagcagctg 180 aaggagcagg agctgagagc ctgcgagatg gtcctggaag agacccggcg tcagaaggag 240 ctcctgtgca agatggagag ggagaagagr. attgagatcg agaacct.gca gaccaggcta 300 cagcaactgg acgaggagaa cagtgaactc cggtcctgca cgccctgtct gaaggccaac 360 ~~ttgagcgtc tggaagagga gaacanaagc tgttggatga gatagagtcg ctgacgctgc 420 ~~gctcaatga anaacangag aacaagaaga gaattg 456 <:210> 150 <:211> 447 <212> DNA
<213> Homo Sapiens <400> 150 acacctttta aaaaggggca tacaagaaat aaaagcacaa gcagtgctat gagtggcagt 60 catcaggacc tcagtgtgat acagccaatt gtaaaagact gcaaagaggc tgacttatcc 120 ttgtataatg aattccgatt gtggaaggat gagcccacaa tggacaggac gtgtcctttc 180 ttagacaaaa tctaccagga agatatcttt ccatgtttaa cattctcaaa aagtgagttg 240 gcttcagctg ttctggaggc tgtggaaaac aatactctaa gcattgaacc agtgggatta 300 caacctatcc ggtttgtgaa agcttctgca gttgaatgcg gaggaccaaa aaaatgtgct 360 ctcactggcc agagtaagtc ctgtaaacac agaattaaat taggggactc aagcaactat 420 t:attatattt ctcctttttg cagaaac 447 <:210> 151 <:211> 590 <:212 > DNA
<213> Homo Sapiens <400> 151 gatctctatg gcttacccat acgatgttcc agattacgct agcttgggtg gtcatatggc 60 catggaggcc ccggcggcgc tgggaacgga cgcctgggcg ccccgaggcg ccgggatgtg 120 gagcggccca ccccagccag accagggcct cccgccgccc cttgcagctg tcccggtccc 180 ctggaagagc acggacccct gccaaggcca cagggagtcc ccaggagccc tggtggagac 240 ctctgcaggg gaggaggccc aaggccagga gggccccgca gccgcccagc tggacgtgtt 300 gcgcctgcgc agctcttcat ggagatccga gagaagggct ccnagttcct gaaggaggag 360 ctgcacagag cgcagaagga gctgaagcta aanggacnag gaatgtgagc ggctgtccaa 420 ngtgcgggag caagcttgaa caggagctgg aagagctgac ggccagcctg tttgaggaag 480 cttacaagat ggttcgagaa accaacatga aacangcggc attagaaaag cagctgaagg 540 aagctcgggg caagatcnac atgcttcang canangtgac acccttgaaa 590 <210> 152 <211> 551 <212> DNA
<213> Homo Sapiens <400> 152 agtgcctcct ctggcctagg cgagttcaat gccagggccc gcgaggtgga gctcgagcac 60 gaggtcaagc ggctcaagca ggagaattat aagctgcggg atcagaacga cgacttgaat 120 gggcagattt tgagcctcag cctctacgaa gcaaaaaacc tctttgctgc ccagactaaa 180 WO 00/43419 PCTlUS00/01431 gcccagtctc tggctgcaga gatagacacc gcctngcgcg atgagctaat ggaagccctg 240 aaggagcang aggagatcaa cttcggctga gcagtacatg gacaagatta tcctcgccat 300 cctggaccac aatccctcca tcctcgagat caaacactaa ggcacggggc ttggctgcan 360 aacaacctta ggaccctggg accaaggcan accttgccaa aggatgcagg cctaancccg 420 gccttacact tnnactggaa atgtctttnt tgccaccatg cntaacgtgt accccgtgta 4B0 tatgtgggga ggcttntncn cacnancgag gggtgaatgg ccntgggctg tggccagcat 540 tcacacngnt n 551 <210> 153 <211> 429 <212> DNA
<213> Mouse <400> 153 gcaaccgcgg ttcccgggac acctgttccc tgtgccccgt gcggtgctca gctcccaact 60 cgagctctgc ggctcgggga gaaaggtcgt gagtccccgg cctccgacgg cacccgcgct 120 gagccttcgc agaacaagat gagcgcggac gcagcggccg gggagcctct gccccggctc 180 tgctgcctgg agaagggtcc aaatggctac ggcttccacc tgcacgggga gaagggcaag 240 gtgggccagt ttatccgtct ggtagaacct ggctcgccgg ccgaaaagtc ngggttgttg 300 gctggagaac gattggtgga ggtgaacggt gagaatgtgg agaaggagac gcatcaacag 360 gtggtgagcc gcatccgcgc aacgctgaac gccgtgcgcc tgctggtggt cnacccggan 420 accgacgaa 429 <210> 154 <211> 431 <212> DNA
<213> Mouse <400> 154 gtgcgcctgc tggtggtcga cccggagacc gacgagcgtc tcaagaagct gggcgtctcg 60 atccgggaag agctgctgcg cccccaggag aagtccgaac aagccgagcc tccagcggcc 120 c~ccgacactc atgaggctgg ggaccagaat gaggccgaaa agagccactt gcgcgagctc 180 c:ggccccggc tctgcaccat gaagaaaggc cccaatggct atggcttcaa cctgcacagc 240 c~acaagtcta agccaggcca gttcatccga gcagtggacc cagactcacc ggcggangcg 300 tctggactcc gggcccanga ccgaattgtg gangtcaatg gtgtctgcat ggagggcaag 360 wo oo~a3ai9 Pcrmsooro~asi cancacgggg acgtggtgtc cgccatcaag ggtggangtg atgaggccaa gctgctggtg 420 gtanacaaag g 431 <210> 155 <211> 501 <212> DNA
<213> Mouse <400> 155 cgctgtctcc tccgcaagga accttcagag cgacttgtgg ccctgggaat cctcttgcat 60 ccctggttga gagaggatca cggccgagtc tctcctccac agtctgaccg aagggagatg 120 gaccaggtgg tcccagatgg gccacagctg gaggaggctg aggaagggga ggtggggctg 180 tacggctagg gccaccttac tggcccctca gctccaaggt gtgagttgag tacctgagtc 240 i:cagctttct cctgactttt tgggccaagc taaaccttaa gtgcctttct ggaggaagaa 300 acagccggcg tgccttattc gttcctgtgc ctagtggggt gatgctccct gagtgccacg 360 c:cctgctcta ggtgctgtga acagcaaagg aaaaagaggg agaaatatac cttgctggnc 420 a.agttgccac aantgccgca tgctctccgg gcaacccctg ccttgggcac gtttcctacc 480 ggggctgtct tctctggtgc n 501 <210> 156 <211> 407 <212> DNA
c213> Mouse <400> 156 cgctgtctcc tccgcaagga accttcagag cgacttgtgg ccctgggaat cctcttgcat 60 ccctggttga gagaggatca cggccgagtc tctcctccac agtctgaccg aagggagatg 120 gaccaggtgg tcccagatgg gccacagctg gaggaggctg aggaagggga ggtggggctg 180 tacggctagg gccaccttac tggcccctca gctccaaggt gtgagt.tgag tacctgagtc 240 tcagctttct cctgactttt tgggccaagc taaaccttaa gtgcctttct ggaggaagaa 300 acagccggng tgccttaatc gttcctgtgc ctaatggggt gatgctccct gagtgccacg 360 ccctgctcta agtgctgtga acaacaaagg aaaaanaggg anaaatn 407 <210> 157 <211> 424 <212> DNA
<213> Mouse <400> 157 cgctgtctcc tccgcaagga accttcagaa cgacttgtgg ccctgggaat cctcttgcat 60 ccctggttga gagaggatca cggccgagtc tctcctccac agtct.gaccg aagggagatg I20 gaccaggtgg tcccagatgg gccacagctg gaggaggctg aggaagggga ggtggggctg 180 tacggctagg gccaccttac tggcccctca gctccaaggt gtgagttgag tacctgagtc 240 tcagctttct cctgactttt tgggccaagc taaaccttaa gtgcctttct ggaggaagaa 300 acaaccggcg tgccntaatc gttcctgtgc ctaatggggt gatgctccct gaatgccacg 360 ccctgctcta ngtgctgtga acagcaaagg aaaaagaggg agaaatatac cttgctggcc 420 aunt 424 <210> 158 <211> 426 <212> DI3A
<213> Mouse <400> 158 cgctgtctcc tccgcaagga accttcagag cgacttgtgg ccctgggaat cctcttgcat 60 ccctggttga gagaggatca cggccgagtw tctcctccac agtctgaccg aagggagatg 120 WO 00/43419 PCT/US00l01431 gaccaggtgg tcccagatgg gccacagctg gaggaggctg aggaagggga ggtggggctg 180 tacggctagg gccaccttac tggcccctca gctccaaggt gtgagttgag tacctgagtc 240 tcagctttct cctgactttt tgggccaagc taaaccttaa gtgcctttct ggaggaagaa 300 acaaccggcg tgccttaatc gttcctgtgc ctaatggggt gatgctccct gaatgccacg 360 ccctgctcta ggtgctgtga acagcaaagg aaaaagangg agaaatatac cttgctggnc 420 aagttg 426 <210> 159 <211> 502 <212> DNA
<213> Mouse <400> 159 agcaggccgg aagatgccac acacagccga cgcagcagag gtatggtcca gagcagttcc 60 cccgtcccac aggcagcaag ggatgtacgc atcatgcagt tctgccacac actgagagag 120 tttgcccttg agtatcggac ttgtcgggaa cgggtactgc agcagcagca gaagcgggct 180 acataccgtg agcgcaacaa gacccgtggt cgcatgatta ccgagacaga gaagttctca 240 ggtgtggctg gggaggcccc caataacctg tctgtcccag tggctgtggg cagcgggcca 300 ggtcagggtg atactgacaa tcatgccagc atgaagagcc t_qcttaccag caggccggaa 360 gatgccacac acatcccctg aggagactgc agcctccggc ttacccaccg acacgtcaga 420 tgagataatg gacctgctgg tgcagtcagt taccaagagc ggtcctanan ccntanctgc 480 tccgganaag aaacnctctc tn 502 <210> 160 <211> 457 <212> DNA
<213> Mouse <400> 160 cacacactga gagagtttgc ccttgagtat cggacttgtc gggaacgggt actgcagcag 60 cagcagaagc gggctacata ccgtgagcgc aacaagaccc gtggtcgcat gattaccgag 120 acagagaagt tctcaggtgt ggctggggag gcccccaata acctgtctgt cccagtggct 180 gtgggcagcg ggccaggtca gggtgatact gacaatcatg ccagcatgaa gagcctgctt 240 accagcaggc cggaagatgc cacacacagc cgacgcagca gangtatggt ccagagcagt 300 tcccccgtct cacacacagc agtggggccc tccgctgcat cccctgagga gactgcagcc 360 tccggcttta ccaccgacac gtcagatgag ataatgggcc tgctggtgca gtcagttacc 420 aagagcggtc ctagagcctt anctgctegg ganaaga 457 <210> 161 <211> 498 <212> DNA
<213> Mouse <400> 161 atgaccaggt ggttgttaca aaaagaaatt. acaaccacgg gaaagaaatc ttaatgaaaa 60 aaagcatctc attgaggcat ttctgcctcg tgtgcactgg gccatgtttg ttttcttggt 120 actcattagt acacgtattt tttcccagat ctctttcccc gagttgctgt tatagagtac 180 tctgctgtgt gcagatgcat ttatacacat taaagcagat ctggagtctg gtgtggccaa 240 cgcagctaca aaaccgagaa acattcatag ctgcaaaaac catcataagt agaggacttt 300 tggtttttgt tttgtttttt ggtgttagaa attttaatta caaataaaaa tccagtgaat 360 tttgcagaaa tactggtttc tacaccatcc taaagaagtt gagtgtttgg gatagaaaac 420 gaaagtgggg aatattacat tgtaaaaata actgcagagt gaagagttct gaaaagcaga 480 DEMANDES OU BREVETS VOLUMINEUX
LA PRESENTF PARTIE DE CETTE DEMANDE OU CE BRE=VE'T
COMPREND PLUS D'UN TOME.
CECI EST LE TOME ~ DE Z
t'~OT'F: Pour les tomes additionels, veuillez contacter le Bureau canadien des brevets THiS SECTION OF THE APPUCATEONIPATENT CONTAINS MORE
THAN ONE VOLUME
THIS IS VOLUME OE=
' NOTE: For additional volumes-phase contact the Canadian Patent Ofific~ .

Claims

We claim:

1. A recombinant nucleic acid encoding an Exo protein comprising a nucleic acid that hybridizes under high stringency conditions to a sequence set forth in SEQ ID
NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ
ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID No:95, SEQ ID NO:96, SEQ ID
NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID
NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID
NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID
NO:112, SEQ ID NO:113, 5EQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID
NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID
NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID
NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO 130, SEQ ID NO:131, SEQ ID
NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID
NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID
NO:142, SEQ ID NO:143 and each complement thereof, respectively, wherein said Exa protein binds to SNAP-23.

2. A recombinant nucleic acid comprising a nucleic acid that is at least about 90%
identical to a nucleic acid sequence selected forth in SEQ ID NO:86, SEQ lD
NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ
ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID
NO:98, SEQ ID NO:99, SEQ IQ NO:100, SEQ ID NO:101, SEQ 1D NO:102, SEQ ID
NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID
NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID
NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID
NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID
NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID

NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID
NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID
NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID
NO:143 and complements, respectively, wherein said Exo protein binds to SNAP-23.

3. An expression vector comprising the recombinant nucleic acid according to claims 1 or 2 operably linked to regulatory sequences recognized by a host cell transformed with the nucleic acid.

4. A host cell comprising the recombinant nucleic acid according to claim 3.

5. A process for producing an Exo protein comprising culturing the host cell of claim 4 under conditions suitable for expression of an Exo protein.

6. A process according to claim 5 further comprising recovering said Exo protein.

7. A recombinant Exo protein encoded by the nucleic acid of claim 1 or 2.

8. A recombinant polypeptide comprising an amino acid sequence encoded by the first 100 nucleic acid residues of a sequence selected from the group consisting of the sequences set forth in SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ
ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID NO:98, SEO ID NO:99, SEQ ID
NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID
NO:105, SEQ ID NO:106, SEQ ID NO:107, SEG ID NO:108, SEG ID NO:109, SEQ ID
NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID
NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID
NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEG ID NO:124, SEQ ID

NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID
NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID
NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID
NO:140, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:143 and each complement thereof, wherein said polypeptide binds to SNAP-23.

9, A recombinant polypeptide comprising an amino acid sequence encoded by a nucleic acid sequence that will hybridize under high stringency to a nucleic acid selected from the group consisting of the sequences set forth SEQ ID NO:86, SEQ ID
NO:87, SEQ ID NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID
NO:92, SEQ ID NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID
NO:97, SEQ ID NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID
NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID
NO:107, SEQ ID NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID
NO:112, SEQ ID NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID
NO:117, SEQ ID NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID
NO:122, SEQ ID NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID
NO:127, SEQ ID NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID
NO:132, SEQ ID NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID
NO:137, SEQ ID NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID
NO:142, SEQ ID NO:143 and each complement thereof.

10. An isolated polypeptide which specifically binds to an Exo protein according to claim 9.

11. A polypeptide according to claim 10 that is an antibody.

12. A polypeptide according to claim 11 wherein said antibody is a monoclonal antibody.

13. The monoclonal antibody of claim 12 wherein said antibody reduces or eliminates the biological function of said Exo protein.

14. A method for screening for a bioactive agent capable of binding to an Exo protein, said method comprising combining an Exo protein and a candidate bioactive agent, and determining the binding of said candidate agent to said Exo protein.

15. A method for screening for agents capable of interfering with the binding of Exo and SNAP-23 comprising:

a) combining an Exo protein, a candidate bioactive agent and an SNAP-23 protein; and b) determining the binding of said Exo protein and said SNAP-23 protein.

16. A method according to claim 15 wherein said Exo protein and said SNAP-23 protein are combined first.

17. A method for screening for an bioactive agent capable of modulating the activity of an Exo protein, said method comprising the steps of:
a) adding a candidate bioactive agent to a cell comprising a recombinant nucleic acid encoding an Exo protein;
b) determining the effect of the candidate bioactive agent on said cell.

18. A method according to claim 17 wherein a library of candidate bioactive agents are added to a plurality of cells comprising a recombinant nucleic acid encoding an Exo protein.

19. A method according to claim 17 or 18 further comprising adding a labeling agent that will label exocytosing cells.

20. A method according to claim 19 further comprising separating the exocytosing cells from the non-exocytosing cells.

21. A method according to claim 20 wherein said separation is done by FACS.

22. A method of treating an exocytosis related disorder comprising administering an agent that interferes with specific binding of a protein selected from a protein encoded by the group consisting of SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID
NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID
NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID
NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:B1, SEQ ID NO:82, SEQ ID
NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID
NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID
NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID
NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID
NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID
NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID
NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID
NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID
NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID
NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID
NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO;136, SEQ ID NO:137, SEQ ID
NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, and SEQ ID NO:143, with SNAP23 expressed in a tissue such that said disorder is ameolerated.

23. A method of treating an exocytosis related disorder comprising administering to a patient an agent that binds to a protein encoded by a sequence selected from the group consisting of SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID NO:73, SEQ
ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID NO:78, SEQ ID
NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID
NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID NO:88, SEQ ID
NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID NO:93, SEQ ID
NO:94, SEQ ID NO:95, SEQ 1D NO:96, SEQ ID NO:97, SEQ ID NO:98, SEQ ID
NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID NO:103, SEQ ID
NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID NO:108, SEQ ID
NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO:113, SEQ ID
NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID
NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID NO:123, SEQ ID
NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID NO:128, SEQ ID
NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID NO:133, SEQ ID
NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID NO:138, SEQ ID
NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, and SEQ ID NO:143 such that exocytosis is altered.

24. A method of reducing or inhibiting exocytosis in a cell comprising administering an agent that interferes with specific binding of a protein encoded by a sequence selected from the group consisting of SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID
NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID
NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID
NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID
NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID

NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID
NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID
NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID
NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID
NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID
NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID
NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID
NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID
NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID
NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID
NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, and SEQ ID NO:143, with SNAP23 expressed in said cell such that exocytosis is inhibited.

25. A method of neutralizing the effect of a protein encoded by a sequence selected from the group consisting of SEQ ID NO:65, SEQ ID NO:66, SEQ ID NO:67, SEQ ID
NO:68, SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:71, SEQ ID NO:72, SEQ ID
NO:73, SEQ ID NO:74, SEQ ID NO:75, SEQ ID NO:76, SEQ ID NO:77, SEQ ID
NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID
NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86, SEQ ID NO:87, SEQ ID
NO:88, SEQ ID NO:89, SEQ ID NO:90, SEQ ID NO:91, SEQ ID NO:92, SEQ ID
NO:93, SEQ ID NO:94, SEQ ID NO:95, SEQ ID NO:96, SEQ ID NO:97, SEQ ID
NO:98, SEQ ID NO:99, SEQ ID NO:100, SEQ ID NO:101, SEQ ID NO:102, SEQ ID
NO:103, SEQ ID NO:104, SEQ ID NO:105, SEQ ID NO:106, SEQ ID NO:107, SEQ ID
NO:108, SEQ ID NO:109, SEQ ID NO:110, SEQ ID NO:111, SEQ ID NO:112, SEQ ID
NO:113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID
NO:118, SEQ ID NO:119, SEQ ID NO:120, SEQ ID NO:121, SEQ ID NO:122, SEQ ID
NO:123, SEQ ID NO:124, SEQ ID NO:125, SEQ ID NO:126, SEQ ID NO:127, SEQ ID
NO:128, SEQ ID NO:129, SEQ ID NO:130, SEQ ID NO:131, SEQ ID NO:132, SEQ ID

NO:133, SEQ ID NO:134, SEQ ID NO:135, SEQ ID NO:136, SEQ ID NO:137, SEQ ID
NO:138, SEQ ID NO:139, SEQ ID NO:140, SEQ ID NO:141, SEQ ID NO:142, and SEQ ID NO:143, comprising contacting an agent specific for said protein with said protein in an amount sufficient to effect neutralization.