AU2001249450A1 - Angiogenesis associated proteins, and nucleic acids encoding the same - Google Patents

Description

ANGIOGENISIS ASSOCIATES PROTEINS, AND NUCLEIC ACIDS ENCODING THE SAME

RELATED APPLICATIONS

This application claims priority to U.S. provisional application Serial No. 60/191,134 filed 03/22/2000.

BACKGROUND

Cities have roads and alleys, plants have xylem and phloem, and people have arteries, veins and lymphatics. Without these byways, the vertebrate animal cells would starve or drown in their metabolic refuse. Not only do blood vessels deliver food and oxygen and carry away metabolic wastes, but they also transport signaling substances that apprise cells of situations remote to them but to which they need to respond. Hormonal messages are a common signal.

All blood vessels are ensheathed by a basal lamina and a delicate monolayer of remarkably plastic endothelial cells lining the luminal walls. Depending on location and function, smooth muscle and connective tissue may also be present.

Not only do healthy cells depend on the blood resources transported by the circulatory system, but so, too, unwanted cells: tumorigenic and malignant cells. These cells colonize and proliferate if they are able to divert blood resources to themselves. Angiogenesis, the type of blood vessel formation where new vessels emerge from the proliferation of preexisting vessels (Risau, 1995; Risau and Flamme, 1995), is exploited not only by usual processes, such as in wound healing or myocardial infarction repair, but also by tumors themselves and in cancers, diabetic retinopathy, macular degeneration, psoriasis, and rheumatoid arthritis. Regardless of the process, whether pathological or usual physiological, endothelial cells mediate angiogenesis in a multi-step fashion: (1) endothelia receive an extracellular cue, (2) the signaled cells breach the basal lamina sheath, abetted by proteases they secrete, (3) the cells then migrate to the signal and proliferate, and finally, (4) the cells form a tube, a morphogenic event (Alberts et al., 1994). The complexity of this process indicates complex changes in cellular physiology and morphology, gene expression, and signaling. Angiogenic accomplices that are cues include basic fibroblast growth factors (bFGF), angiopoietins (such as ANG1) and various forms of vascular endothelial growth factor (VEGF).

The molecular events and the order in which they occur and the pathways that are required for this process are of fundamental importance to understand angiogenesis. In vitro models are useful for identifying alterations in gene expression that occur during angiogenesis. A particularly fruitful model systems involves the supspension in a three- dimensional type I collagen gel and various stimuli, such as phorbol myristate acetate (PMA), basic fibroblast growth factor (bFGF), and VEGF. The combination of the stimuli and the collagen gel results in the formation of a three-dimensional tubular network of endothelial cells with interconnecting lumenal structures. In this model, endothelial differentiation into tubelike structures is completely blocked by inhibitors of new mRNA or protein synthesis. Furthermore, the cells progress through differentiation in a coordinated and synchronized manner, thus optimizing the profile of gene expression (Kahn et al, 2000; Yang et al, 1999).

Tumor cells exploit angiogenesis to facilitate tumor growth. Controlling angiogenesis, by controlling the activity or expression of genes and proteins associated with angiogenesis, provides a way to prevent tumor growth, or even destoy tumors.

SUMMARY

The invention is based in part upon the discovery of novel nucleic acid sequences encoding novel polypeptides. Nucleic acids encoding the polypeptides disclosed in the invention, and derivatives and fragments thereof, will hereinafter be collectively designated as "AAP" nucleic acid or polypeptide sequences. AAP, or angiogenesis associated polypeptides (AAP) comprises kelch-like polypeptide (KXP), human ortholog of mouse BAZF (hBAZF), hmt-elongation factor G (hEF-G), human ortholog of rat TRG (hTRG), human myosin X (hMXl) and its splice variant (hMX2), nuclear hormone receptor (NHR), and human mitochondrial protein (hMP).

The invention is based in part upon the discovery of novel nucleic acid sequences encoding novel polypeptides. Nucleic acids encoding the polypeptides disclosed in the invention, and derivatives and fragments thereof, will hereinafter be collectively designated as "AAP" nucleic acid or polypeptide sequences." In a first aspect, the present invention is an isolated polypeptide having at least 80% sequence identity to the sequence SEQ ID NOS:2, 4, 6, 8, 10, 12, 14 or 16, polynucleotides encoding the same, and antibodies that specifically bind the same.

In a second aspect, the present invention is an isolated polynucleotide having at least 80% sequence identity to the sequence SEQ ID NOS:l, 3, 5, 7, 9, 11, 13 or 15, or a complement thereof.

In a third aspect, the present invention is a transgenic non-human animal, having a disrupted AAP gene or a transgenic non-human animal expressing an exogenous polynucleotide having at least 80% sequence identity to the sequence SEQ ID NOS:l, 3, 5, 7, 9, 11, 13 or 15, or a complement of said polynucleotide.

In a fourth aspect, the present invention is a method of screening a sample for a mutation in an AAP gene.

In a fifth aspect, the present invention is a method of modulating angiogenesis comprising modulating the activity of at least one AAP polypeptide.

In a sixth aspect, the present invention is a method of increasing, as well as decreasing angiogenesis, comprising modulating the activity of at least one AAP polypeptide. Activity modulation of AAP polypeptides may be over-expressing or eliminating expression of the gene, or impairing a AAP polypeptide' s function by contact with specific antagonists or agonists, such as antibodies or aptamers.

In a seventh aspect, the present invention is a method of treating various pathologies, including tumors, cancers, myocardial infarctions and the like.

In an eighth aspect, the present invention is a method of measuring a AAP transcriptional and translational up-regulation or down-regulation activity of a compound.

In a ninth aspect, the invention is a method of screening a tissue sample for tumorigenic potential.

In a tenth aspect, the invention is a method of determining the clinical stage of tumor which compares the expression of at least one AAP in a sample with expression of said at least one gene in control samples.

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

DETAILED DESCRIPTION

A model of angiogenesis-the suspension of endothelial cells in type I collagen gels with various stimuli- was used to identify a molecular fingerprint or transcriptional profile of endothelial differentiation into tubelike structures, using amplification and an imaging approach called GeneCalling (Shimkets et al, 1999). This method was previously shown to provide a comprehensive sampling of cDNA populations in conjunction with the sensitive detection of quantitative differences in mRNA abundance for both known and novel genes. Many differentially expressed cDNA fragments were found. The identification and differential expression of these gens was confirmed by a second independent method employing real-time quantitative polymerase chain reaction (PCR). Although some of the identified cDNA fragments were genes known to play some role in angiogenesis, many other differentially expressed genes were unexpected. The inventors have identified the unexpected genes and polypeptides that are expressed in response to this model of angiogenesis, collectively refered to as angiogenesis associated polypeptides (AAP). AAP are kelch-like polypeptide (KLP), human ortholog of mouse BAZF (hBAZF), hmt-elongation factor G (hEF-G), human ortholog of rat TRG (hTRG), human myosin X (hMXl) and its splice variant (hMX2), nuclear hormone receptor (NHR), and human mitochondrial protein (hMP).

Definitions

Unless defined otherwise, all technical and scientific terms have the same meaning as is commonly understood by one of skill in the art to which this invention belongs. The definitions below are presented for clarity.

The recommendations of (Demerec et al, 1966) where these are relevant to genetics are adapted herein. To distinguish between genes (and related nucleic acids) and the proteins that they encode, the abbreviations for genes are indicated by italicized (or underlined) text while abbreviations for the proteins start with a capital letter and are not italicized. Thus, AAP or AAP refers to the nucleotide sequence that encodes AAP. "Isolated," when referred to a molecule, refers to a molecule that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that interfere with diagnostic or therapeutic use.

"Container" is used broadly to mean any receptacle for holding material or reagent. Containers may be fabricated of glass, plastic, ceramic, metal, or any other material that can hold reagents. Acceptable materials will not react adversely with the contents.

1. Nucleic acid-related definitions

(a) control sequences

Control sequences are DNA sequences that enable the expression of an operably- linked coding sequence in a particular host organism. Prokaryotic control sequences include promoters, operator sequences, and ribosome binding sites. Eukaryotic cells utilize promoters, polyadenylation signals, and enhancers.

(b) operably-linked

Nucleic acid is operably-linked when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably- linked 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 positioned 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 conventional recombinant DNA methods.

(c) isolated nucleic acids

An isolated nucleic acid molecule is purified from the setting in which it is found in nature and is separated from at least one contaminant nucleic acid molecule. Isolated AAP molecules are distinguished from the specific AAP molecule, as it exists in cells. However, an isolated AAP molecule includes AAP molecules contained in cells that ordinarily express an AAP where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells. 2. Protein-related definitions

(a) purified polypeptide

When the molecule is a purified polypeptide, the polypeptide will be purified (1) to obtain at least 15 residues of N-terminal or internal amino acid sequence using a sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or silver stain. Isolated polypeptides include those expressed heterologously in genetically-engineered cells or expressed in vitro, since at least one component of an AAP natural environment will not be present. Ordinarily, isolated polypeptides are prepared by at least one purification step.

(b) active polypeptide

An active AAP or AAP fragment retains a biological and/or an immunological activity of the native or naturally-occurring AAP. Immunological activity refers to the ability to induce the production of an antibody against an antigenic epitope possessed by a native AAP; biological activity refers to a function, either inhibitory or stimulatory, caused by a native AAP that excludes immunological activity. A biological activity of AAP includes, for example, modulating angiogenesis.

(c) Abs

Antibody may be single anti-AAP monoclonal Abs (including agonist, antagonist, and neutralizing Abs), anti-AAP antibody compositions with polyepitopic specificity, single chain anti-AAP Abs, and fragments of anti-AAP Abs. A "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous Abs, i.e., the individual Abs comprising the population are identical except for naturally-occurring mutations that may be present in minor amounts

(d) epitope tags

An epitope tagged polypeptide refers to a chimeric polypeptide fused to a "tag polypeptide". Such tags provide epitopes against which Abs can be made or are available, but do not interfere with polypeptide activity. To reduce anti-tag antibody reactivity with endogenous epitopes, the tag polypeptide is preferably unique. Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8 and 50 amino acid residues, preferably between 8 and 20 amino acid residues). Examples of epitope tag sequences include HA from Influenza A virus and FLAG. The novel AAP of the invention include the nucleic acids whose sequences are provided in Tables 1, 3, 5, 7, 9, 11, 13 and 15, or a fragment thereof. The invention also includes a mutant or variant AAP, any of whose bases may be changed from the corresponding base shown in Tables 1, 3, 5, 7, 9, 11, 13 and 15 while still encoding a protein that maintains the activities and physiological functions of the AAP fragment, or a fragment of such a nucleic acid. The invention further includes nucleic acids whose sequences are complementary to those just described, including complementary nucleic acid fragments. The invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications. Such modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as anti-sense binding nucleic acids in therapeutic applications in a subject In the mutant or variant nucleic acids, and their complements, up to 20% or more of the bases may be so changed.

The novel AAP of the invention include the protein fragments whose sequences are provided in Tables 2, 4, 6, 8, 10, 12, 14 and 16. The invention also includes an AAP mutant or variant protein, any of whose residues may be changed from the corresponding residue shown in Tables 2, 4, 6, 8, 10, 12, 14 and 16 while still encoding a protein that maintains its native activities and physiological functions, or a functional fragment thereof. In the mutant or variant AAP, up to 20% or more of the residues may be so changed. The invention further encompasses Abs and antibody fragments, such as F_ab or (F_ab)'_2,that bind immunospecifically to any of the AAP of the invention.

The AAP nucleic acids are shown in Tables 1, 3, 5, 7, 9, 11, 13 and 15, and the corresponding polypeptides are shown in Tables 2, 4, 6, 8, 10, 12, 14 and 16, respectivly. Start and stop codons in the polynucleotide sequences are indicated in boldface and with underlining. SEQ ID NO:3 lacks a stop codon. The sequences of hMXl and hMX2 do not have start codons (see Table 17); consequently, hMXl and hMX2 polypeptides do not start with a Met. For any lacking polynucleotide sequence, one of skill in the art may retrieve the full length sequence by, for example, probing cDNA or genomic libraries with probes designed according to the sequences of the instant invention. Table 1 KLP nucleotide sequence (SEQ ID NO:l) ctggcctaga tactacaact gaactttttt tctttttagt tactccacag gatccgctga 60 acataggatg ttgccacaaa atctacctcg tgtatttttc tctttcactc atgagctgca 120 caattgcaga tttgagcaca atgtctgcag actgtgttga aaaactctga agaacctaat 180 taacacagga tgacctagga gtgattctaa gtctgtgtaa caagatatta ctcattagtg 240 aatgtgtcag tcttggtact gaatgctgca gataacagca agtaggttct cctttatttc 300 tgaagtattc acttgacctt ccatcagtaa gacggacttt tctaatctgt tcctggagat 360 attaatggaa tacagtcatg tccactcaag acgagaggca gatcaatact gaatatgctg 420 tgtcattgtt ggaacagttg aaactgtttt atgaacagca gttgtttact gacatagtgt 480 taattgttga gggcactgaa ttcccttgtc ataagatggt tcttgcaaca tgtagctctt 540 atttcagggc catgtttatg agtggactaa gtgaaagcaa acaaacccat gtacacctga 600 ggaatgtcga tgctgccacc ttacagataa taataactta tgcatacacg ggtaacttgg 660 caatgaatga cagcactgta gaacagcttt atgaaacagc ttgcttccta caggtagaag 720 atgtgttaca acgttgtcga gaatatttaa ttaaaaaaat aaatgcagag aattgtgtac 780 gattgttgag ttttgctgat ctcttcagtt gtgaggaatt aaaacagagt gctaaaagaa 840 tggtggagca caagttcact gctgtgtatc atcaggacgc gttcatgcag ctgtcacatg 900 acctactgat agatattctc agtagtgaca atttaaatgt agaaaaggaa gaaaccgttc 960 gagaagctgc tatgctgtgg ctagagtata acacagaatc acgatcccag tatttgtctt 1020 ctgttcttag ccaaatcaga attgatgcac tttcagaagt aacacagaga gcttggtttc 1080 aaggtctgcc acccaatgat aagtcagtgg tggttcaagg tctgtataag tccatgccca 1140 agtttttcaa accaagactt gggatgacta aagaggaaat gatgattttc attgaagcat 1200 cttcagaaaa tccttgtagt ctttactctt ctgtctgtta cagcccccaa gcagaaaaag 1260 tttacaagtt atgtagccca ccagctgatt tgcataaggt tgggaccgtt gtaactcctg 1320 ataatgatat ctacatagca gggggtcaag ttcctctgaa aaacacaaaa acaaatcaca 1380 gtaaaacaag caaacttcag actgccttca gaactgtgaa ttgcttttat tggtttgatg 1440 cacagcaaaa tacctggttt ccaaagaccc caatgctttt tgtccgcata aagccatctt 1500 tggtttgctg tgaaggctat atctatgcaa ttggaggaga tagcgtaggt ggagaactta 1560 atcggaggac cgtagaaaga tacgacactg agaaagatga gtggacgatg gtaagccctt 1620 taccttgtgc ttggcaatgg agtgcagcag ttgtggttca tgactgcatt tatgtgatga 1680 cactgaacct catgtactgt tattttccaa ggtctgactc atgggtagaa atggccatga 1740 gacagactag taggtccttt gcttcagctg cagcttttgg tgataaaatt ttctatattg 1800 gagggttgca tattgctacc aattccggca taagactccc ctctggcact gtagatgggt 1860 cttcagtaac tgtggaaatt tatgatgtga ataaaaatga gtggaaaatg gcagccaaca 1920 tccctgctaa gaggtactct gacccctgtg ttagagctgt tgtgatctca aattctctat 1980 gtgtgtttat gcgagaaacc cacttaaatg agcgagctaa atacgtcacc taccaatatg 2040 acctggaact tgaccggtgg tctctgcggc agcatatatc tgaacgtgta ctgtgggact 2100 tggggagaga ttttcgatgc actgtgggga aactctatcc atcctgcctt gaagagtctc 2160 catggaaacc accaacttat cttttttcaa cggatgggac agaagagttt gaactggatg 2220 gagaaatggt tgcactacca cctgtatagt ggggaagttc agggagtgca cgcctgagtt 2280 atgtgctttg tcattttctt tgctaaacaa aagaggctat gaaagaacta aatatgagta 2340 cataaaattc tatctttgat aaattttatt tttatgccct acttaatatt tgcatcagta 2400 taatatatat cagtgagtct tacagaaaga tatgcttcca taatatgaaa tagattattc 2460 aataattgag aaactttatg tgtaatcatg agagtataag aatctggatt atctaacatt 2520 gttagccctg tgtatgtaca gttcaaaaag ttcatttata aaagtagttt cctgttc 2577

Table 2 KLP polypeptide sequence (SEQ ID NO:2)

Met Ser Thr Gin Asp Glu Arg Gin He Asn Thr Glu Tyr Ala Val Ser 1 5 10 15

Leu Leu Glu Gin Leu Lys Leu Phe Tyr Glu Gin Gin Leu Phe Thr Asp 20 25 30

He Val Leu He Val Glu Gly Thr Glu Phe Pro Cys His Lys Met Val 35 40 45

Leu Ala Thr Cys Ser Ser Tyr Phe Arg Ala Met Phe Met Ser Gly Leu 50 55 60

Ser Glu Ser Lys Gin Thr His Val His Leu Arg Asn Val Asp Ala Ala 65 70 75 80

Thr Leu Gin He He He Thr Tyr Ala Tyr Thr Gly Asn Leu Ala Met 85 90 95

Asn Asp Ser Thr Val Glu Gin Leu Tyr Glu Thr Ala Cys Phe Leu Gin 100 105 110

Val Glu Asp Val Leu Gin Arg Cys Arg Glu Tyr Leu He Lys Lys He 115 120 125

Asn Ala Glu Asn Cys Val Arg Leu Leu Ser Phe Ala Asp Leu Phe Ser 130 135 140

Cys Glu Glu Leu Lys Gin Ser Ala Lys Arg Met Val Glu His Lys Phe 145 150 155 160

Thr Ala Val Tyr His Gin Asp Ala Phe Met Gin Leu Ser His Asp Leu 165 170 175

Leu He Asp He Leu Ser Ser Asp Asn Leu Asn Val Glu Lys Glu Glu 180 185 190

Thr Val Arg Glu Ala Ala Met Leu Trp Leu Glu Tyr Asn Thr Glu Ser 195 200 205

Arg Ser Gin Tyr Leu Ser Ser Val Leu Ser Gin He Arg He Asp Ala 210 215 220 Leu Ser Glu Val Thr Gin Arg Ala Trp Phe Gin Gly Leu Pro Pro Asn 225 230 235 240

Asp Lys Ser Val Val Val Gin Gly Leu Tyr Lys Ser Met Pro Lys Phe 245 250 255

Phe Lys Pro Arg Leu Gly Met Thr Lys Glu Glu Met Met He Phe He 260 265 270

Glu Ala Ser Ser Glu Asn Pro Cys Ser Leu Tyr Ser Ser Val Cys Tyr 275 280 285

Ser Pro Gin Ala Glu Lys Val Tyr Lys Leu Cys Ser Pro Pro Ala Asp 290 295 300

Leu His Lys Val Gly Thr Val Val Thr Pro Asp Asn Asp He Tyr He 305 310 315 320

Ala Gly Gly Gin Val Pro Leu Lys Asn Thr Lys Thr Asn His Ser Lys 325 330 335

Thr Ser Lys Leu Gin Thr Ala Phe Arg Thr Val Asn Cys Phe Tyr Trp 340 345 350

Phe Asp Ala Gin Gin Asn Thr Trp Phe Pro Lys Thr Pro Met Leu Phe 355 360 365

Val Arg He Lys Pro Ser Leu Val Cys Cys Glu Gly Tyr He Tyr Ala 370 375 380

He Gly Gly Asp Ser Val Gly Gly Glu Leu Asn Arg Arg Thr Val Glu 385 390 395 400

Arg Tyr Asp Thr Glu Lys Asp Glu Trp Thr Met Val Ser Pro Leu Pro 405 410 415

Cys Ala Trp Gin Trp Ser Ala Ala Val Val Val His Asp Cys He Tyr 420 425 430

Val Met Thr Leu Asn Leu Met Tyr Cys Tyr Phe Pro Arg Ser Asp Ser

435 440 445

Trp Val Glu Met Ala Met Arg Gin Thr Ser Arg Ser Phe Ala Ser Ala 450 455 460

Ala Ala Phe Gly Asp Lys He Phe Tyr He Gly Gly Leu His He Ala 465 470 475 480

Thr Asn Ser Gly He Arg Leu Pro Ser Gly Thr Val Asp Gly Ser Ser 485 490 495

Val Thr Val Glu He Tyr Asp Val Asn Lys Asn Glu Trp Lys Met Ala 500 505 510

Ala Asn He Pro Ala Lys Arg Tyr Ser Asp Pro Cys Val Arg Ala Val 515 520 525

Val He Ser Asn Ser Leu Cys Val Phe Met Arg Glu Thr His Leu Asn 530 535 540

Glu Arg Ala Lys Tyr Val Thr Tyr Gin Tyr Asp Leu Glu Leu Asp Arg 545 550 555 560

Trp Ser Leu Arg Gin His He Ser Glu Arg Val Leu Trp Asp Leu Gly 565 570 575

Arg Asp Phe Arg Cys Thr Val Gly Lys Leu Tyr Pro Ser Cys Leu Glu 580 585 590

Glu Ser Pro Trp Lys Pro Pro Thr Tyr Leu Phe Ser Thr Asp Gly Thr

595 600 605

Glu Glu Phe Glu Leu Asp Gly Glu Met Val Ala Leu Pro Pro Val 610 615 620

Table 3 hBAZF nucleotide sequence (SEQ ID NO:3) caagggagcg agggtgtcgt agagggcaga atgaacaaga agaattagga gggaggctgc 60 gtgtgccggg gctaggggct ggaagtcctg gctctagttg cacctcggaa ggaaaaggca 120 aacagaggag ggaaggcgtc ttaggactgc ctggatccag agcactttcc tcggcctcta 180 caggcctgtg tcgctatggg ttcccccgcc gccccggagg gagcgctggg ctacgtccgc 240 gagttcactc gccactcctc cgacgtgctg ggcaacctca acgagctgcg cctgcgcggg 300 atcctcactg acgtcacgct gctggttggc gggcaacccc tcagagcaca caaggcagtt 360 ctcatcgcct gcagtggctt cttctattca attttccggg gccgtgcggg agtcggggtg 420 gacgtgctct ctctgcccgg gggtcccgaa gcgagaggct tcgcccctct attggacttc 480 atgtacactt cgcgcctgcg cctctctcca gccactgcac cagcagtcct agcggccgcc 540 acctatttgc agatggagca cgtggtccag gcatgccacc gcttcatcca ggccagctat 600 gaacctctgg gcatctccct gcgccccctg gaagcagaac ccccaacacc cccaacggcc 660 cctccaccag gtagtcccag gcgctccgaa ggacacccag acccacctac tgaatctcga 720 agctgcagtc aaggcccccc cagtccagcc agccctgacc ccaaggcctg caactggaaa ^• 780 aagtacaagt acatcgtgct aaactctcag gcctcccaag cagggagcct ggtcggggag 840 agaagttctg gtcaaccttg cccccaagcc aggctcccca gtggagacga ggcctccagc 900 agcagcagca gcagcagcag cagcagcagt gaagaaggac ccattcctgg tccccagagc 960 aggctctctc caactgctgc cactgtgcag ttcaaatgtg gggctccagc cagtaccccc 1020 tacctcctca catcccaggc tcaagacacc tctggatcac cctctgaacg ggctcgtcca 1080 ctacccggga gtgaattttt cagctgccag aactgtgagg ctgtggcagg gtgctcatcg 1140 gggctggact ccttggttcc tggggacgaa gacaaaccct ataagtgtca gctgtgccgg 1200 tcttcgttcc gctacaaggg caaccttgcc agtcaccgta cagtgcacac aggggaaaag 1260 ccttaccact gctcaatctg cggagcccgt tttaaccggc cagcaaacct gaaaacgcac 1320 agccgcatcc attcgggaga gaagccgtat aagtgtgaga cgtgcggctc gcgctttgta 1380 caggtacgga gccagcctcc aagtggcttc caaggcaaac ctgcaagagg tggggtgggc 1440 caaaagggag ggttctgttc ctcccagagg caggacttga agtctcctcc ctcccaggtg 1500 gcacatctgc gggcgcacgt gctgatccac accggggaga agccctaccc ttgccctacc 1560 tgcggaaccc gcttccgcca cctgcagacc ctcaagagcc acgttcgcat ccacaccgga 1620 gagaagcctt accactgcga cccctgtggc ctgcatttcc ggcacaagag tcaactgcgg 1680 ctgcatctgc gccagaaaca cggagctgct accaacacca aagtgcacta ccacattctc 1740 ggggggccc 1749

Table 4 hBAZF polypeptide sequence (SEQ ID NO:4)

Met Gly Ser Pro Ala Ala Pro Glu Gly Ala Leu Gly Tyr Val Arg Glu 1 5 10 15

Phe Thr Arg His 'Ser Ser Asp Val Leu Gly Asn Leu Asn Glu Leu Arg 20 25 30

Leu Arg Gly He Leu Thr Asp Val Thr Leu Leu Val Gly Gly Gin Pro 35 40 45

Leu Arg Ala His Lys Ala Val Leu He Ala Cys Ser Gly Phe Phe Tyr 50 55 60

Ser He Phe Arg Gly Arg Ala Gly Val Gly Val Asp Val Leu Ser Leu 65 70 75 80

Pro Gly Gly Pro Glu Ala Arg Gly Phe Ala Pro Leu Leu Asp Phe Met 85 90 95

Tyr Thr Ser Arg Leu Arg Leu Ser Pro Ala Thr Ala Pro Ala Val Leu 100 105 110

Ala Ala Ala Thr Tyr Leu Gin Met Glu His Val Val Gin Ala Cys His 115 120 125

Arg Phe He Gin Ala Ser Tyr Glu Pro Leu Gly He Ser Leu Arg Pro 130 135 140

Leu Glu Ala Glu Pro Pro Thr Pro Pro Thr Ala Pro Pro Pro Gly Ser

145 150 155 160

Pro Arg Arg Ser Glu Gly His Pro Asp Pro Pro Thr Glu Ser Arg Ser 165 170 175

Cys Ser Gin Gly Pro Pro Ser Pro Ala Ser Pro Asp Pro Lys Ala Cys 180 185 190

Asn Trp Lys Lys Tyr Lys Tyr He Val Leu Asn Ser Gin Ala Ser Gin 195 200 205

Ala Gly Ser Leu Val Gly Glu Arg Ser Ser Gly Gin Pro Cys Pro Gin 210 215 220 Ala Arg Leu Pro Ser Gly Asp Glu Ala Ser Ser Ser Ser Ser Ser Ser 225 230 235 240

Ser Ser Ser Ser Ser Glu Glu Gly Pro He Pro Gly Pro Gin Ser Arg

245 250 255

Leu Ser Pro Thr Ala Ala Thr Val Gin Phe Lys Cys Gly Ala Pro Ala 260 265 270

Ser Thr Pro Tyr Leu Leu Thr Ser Gin Ala Gin Asp Thr Ser Gly Ser 275 280 285

Pro Ser Glu Arg Ala Arg Pro Leu Pro Gly Ser Glu Phe Phe Ser Cys 290 295 300

Gin Asn Cys Glu Ala Val Ala Gly Cys Ser Ser Gly Leu Asp Ser Leu 305 310 315 320

Val Pro Gly Asp Glu Asp Lys Pro Tyr Lys Cys Gin Leu Cys Arg Ser 325 330 335

Ser Phe Arg Tyr Lys Gly Asn Leu Ala Ser His Arg Thr Val His Thr 340 345 350

Gly Glu Lys Pro Tyr His Cys Ser He Cys Gly Ala Arg Phe Asn Arg 355 360 365

Pro Ala Asn Leu Lys Thr His Ser Arg He His Ser Gly Glu Lys Pro 370 375 380

Tyr Lys Cys Glu Thr Cys Gly Ser Arg Phe Val Gin Val Arg Ser Gin 385 390 395 400

Pro Pro Ser Gly Phe Gin Gly Lys Pro Ala Arg Gly Gly Val Gly Gin 405 410 415

Lys Gly Gly Phe Cys Ser Ser Gin Arg Gin Asp Leu Lys Ser Pro Pro 420 425 430

Ser Gin Val Ala His Leu Arg Ala His Val Leu He His Thr Gly Glu

435 440 445

Lys Pro Tyr Pro Cys Pro Thr Cys Gly Thr Arg Phe Arg His Leu Gin 450 455 460

Thr Leu Lys Ser His Val Arg He His Thr Gly Glu Lys Pro Tyr His 465 470 475 480

Cys Asp Pro Cys Gly Leu His Phe Arg His Lys Ser Gin Leu Arg Leu 485 490 495

His Leu Arg Gin Lys His Gly Ala Ala Thr Asn Thr Lys Val His Tyr 500 505 510

His He Leu Gly Gly Pro 515 Table 5 hEF-G nucleotide sequence (SEQ ID NO:5) tctttttcct cgcgtccttt gccccggaag tgctcttaca acattggctg ccggcgtgac 60 tttgaccgct tcccggtgcg ttaccggcag ctgaacccac ccggcgccac gggactttga 120 cgcgtgctct gcgcttgcca tgagactcct gggagctgca gccgtcgcgg ctctggggcg 180 cggaagggcc cccgcctccc taggctggca gaggaagcag gttaattgga aggcctgccg 240 atggtcttca tcaggggtga ttcctaatga aaaaatacga aatattggaa tctcagctca 300 cattgattct gggaaaacta cattaacaga acgagtcctt tactacactg gcagaattgc 360 aaagatgcat gaggtgaaag gtaaagatgg agttggtgct gtcatggatt ccatggaact 420 agagagacaa agaggaatca ctattcagtc agcagccact ttcaccatgt ggaaagatgt 480 caatattaac attatagata ctcctgggca tgtggacttc acaatagaag tggaaagggc 540 cctgagagtg ttggatggtg cagtccttgt tctctgtgct gttggagggg tacagtgcca 600 gaccatgact gtcaatcgtc agatgaagcg ctacaacgtt ccgtttctaa cttttattaa 660 caaattggac cgaatgggct ccaacccagc cagggccctg cagcaaatga ggtctaaact 720 aaatcataat acagcgttta tgcagatacc catgggtttg gagggtaatt ttaaaggtat 780 tgtagatctt attgaggaac gagccatcta ttttgatgga gactttagtc agattgttcg 840 atatggtgag attccagctg aattaagggc ggcggccact gaccaccggc aggagctaat 900 tgaatgtgtt gccaattcag atgaacagct tggtgagatg tttctggaag aaaaaatccc 960 ctcgatttct gatttaaagc tagcaattcg aagagctact ctgaaaagat catttactcc 1020 tgtatttttg ggaagcgcct tgaagaacaa aggagttcag cctcttttag atgctgtttt 1080 agaatacctc ccaaatccat ctgaagtcca gaactatgct attctcaata aaaaggatga 1140 ctcaaaagag aaaaccaaaa tcctaatgaa ctccagtaga cacaattccc acccatttgt 1200 aggcctggct tttcccctgg aggtaggtcg atttggacaa ttaacttatg ttcgcagtta 1260 tcagggagag ctaaagaagg gtgacaccat ctataacaca aggacaagaa agaaagtacg 1320 gttgcaacgg ctggctcgca tgcatgccga catgatggag gcaagtacag aggaagtata 1380 tgccggagac atctgtgcat tgtttggcat tgactgtgct agtggagaca cattcacaga 1440 caaagccaac agcggccttt ctatggagtc aattcatgtt cctgatcctg tcatttcaat 1500 agcaatgaag ccttctaaca agaacgatct ggaaaaattt tcaaaaggta ttggcaggtt 1560 tacaagagaa gatcccacat ttaaagtata ctttgacact gagaacaaag agacagttat 1620 atctggaatg ggagaattac acctggaaat ctatgctcag aggctggaaa gagagtatgg 1680 ctgtccttgt atcacaggaa agccaaaagt tgcctttcga gagaccatta ctgcccctgt 1740 cccgtttgac tttacacata aaaaacaatc aggtggtgca ggccagtatg gaaaagtaat 1800 aggtgtcctg gagcctctgg acccagagga ctacactaaa ttggaatttt cagatgaaac 1860 attcggatca aatattccaa agcagtttgt gcctgctgta gaaaaggggt ttttagatgc 1920 ctgcgagaag ggccctcttt ctggtcacaa gctctctggg ctccggtttg tcctgcaaga 1980 tggagcacac cacatggttg attctaatga aatctctttc atccgagcag gagaaggtgc 2040 tcttaaacaa gccttggcaa atgcaacatt atgtattctt gaacctatta tggctgtgga 2100 agttgtagct ccaaatgaat ttcagggaca agtaattgca ggaattaacc gacgccatgg 2160 ggtaatcact gggcaagatg gagttgagga ctattttaca ctgtatgcag atgtccctct 2220 aaatgatatg tttggttatt ccactgaact taggtcatgc acagagggaa agggagaata 2280 cacaatggag tatagcaggt atcagccatg tttaccatcc acacaagaag acgtcattaa 2340 taagtatttg gaagctacag gtcaacttcc tgttaaaaaa ggaaaagcca agaactaact 2400 ttgcttactg tgagttgact gactctaatt gaatctgcgt ggttttgata ctttgatgga 2460 ttccagtgga ataaattcag gctgctgaaa caagaaattc tgagcccagg aagcgggctc 2520 ttctttcttc aaaagaagcc cttcttgttc atattcagga gcttctgtta tattcaaagg 2580 taattctatg tctatctcaa ctctattgat tggttttata gttcattgaa aatcctcaaa 2640 taaaatataa ttattactga aatatgttta atatttaagg ggaaaagaga ctaatttcag 2700 ttatactttt aagcttagaa tgtatgttca tttccaaatt ttgtatcata agagttttca 2760 acatagagaa aagctgaaaa aatgcaaaga ataaccacat actttccatc taccttcctt 2820 tgttaacggg ttgtttatca tataataatt tgttttgtca tatttgcttt cactgtctat 2880 tatctgttta agtctcataa ctctattttt agtttgctga agacttgaaa gtgaatcgca 2940 tatatcatga cacttcttgg agtgtcatta atgggcaggc ttttctgttg aagagtggat 3000 tccgtatgtt cttcatagag agtgtttttc agattcttca ttgggatatt aaaatattag 3060 ccaaatttcn ctctgtttta tatatgncag tttatttcag tttgtggttt ctgcaaattt 3120 gtaactgcct ctgttttagg agtataagta ttacttcctt gtggtctatt gtgaagtaaa 3180 aagtagaccc ttgcatatac tattcttgtt tgtgttcatc ttaatgtttt tgtacagcta 3240 aatcaaatgt aatttataga gttagtttca tcaacctaat gaatgctagt taaatttgaa 3300 ttccttggaa tttatcgtat attgtattca ctgagattat gaagggacaa atgttaatct 3360 tttgtttcca gaaaaagttg ggctttccca agcagttcta ttacccggtt cagaattgct 3420 tcatccaaaa atcatctgat ggtatagatg gatcctagtc cttttcatta cctgatggta 3480 gaaataaaat aattgatttt a 3501

Table 6 hEF-G polypeptide (SEQ ID NO:6)

Met Arg Leu Leu Gly Ala Ala Ala Val Ala Ala Leu Gly Arg Gly Arg

1 5 10 15

Ala Pro Ala Ser Leu Gly Trp Gin Arg Lys Gin Val Asn Trp Lys Ala 20 25 30

Cys Arg Trp Ser Ser Ser Gly Val He Pro Asn Glu Lys He Arg Asn 35 40 45

He Gly He Ser Ala His He Asp Ser Gly Lys Thr Thr Leu Thr Glu 50 55 60

Arg Val Leu Tyr Tyr Thr Gly Arg He Ala Lys Met His Glu Val Lys 65 70 75 80

Gly Lys Asp Gly Val Gly Ala Val Met Asp Ser Met Glu Leu Glu Arg 85 90 95

Gin Arg Gly He Thr He Gin Ser Ala Ala Thr Phe Thr Met Trp Lys 100 105 110

Asp Val Asn He Asn He He Asp Thr Pro Gly His Val Asp Phe Thr 115 120 125

He Glu Val Glu Arg Ala Leu Arg Val Leu Asp Gly Ala Val Leu Val 130 135 140

Leu Cys Ala Val Gly Gly Val Gin Cys Gin Thr Met Thr Val Asn Arg 145 150 155 160

Gin Met Lys Arg Tyr Asn Val Pro Phe Leu Thr Phe He Asn Lys Leu 165 170 175

Asp Arg Met Gly Ser Asn Pro Ala Arg Ala Leu Gin Gin Met Arg Ser 180 185 190

Lys Leu Asn His Asn Thr Ala Phe Met Gin He Pro Met Gly Leu Glu 195 200 205

Gly Asn Phe Lys Gly He Val Asp Leu He Glu Glu Arg Ala He Tyr 210 215 220

Phe Asp Gly Asp Phe Ser Gin He Val Arg Tyr Gly Glu He Pro Ala 225 230 235 240

Glu Leu Arg Ala Ala Ala Thr Asp His Arg Gin Glu Leu He Glu Cys 245 250 255

Val Ala Asn Ser Asp Glu Gin Leu Gly Glu Met Phe Leu Glu Glu Lys 260 265 270

He Pro Ser He Ser Asp Leu Lys Leu Ala He Arg Arg Ala Thr Leu 275 280 285

Lys Arg Ser Phe Thr Pro Val Phe Leu Gly Ser Ala Leu Lys Asn Lys 290 295 300

Gly Val Gin Pro Leu Leu Asp Ala Val Leu Glu Tyr Leu Pro Asn Pro 305 310 315 320

Ser Glu Val Gin Asn Tyr Ala He Leu Asn Lys Lys Asp Asp Ser Lys 325 330 335

Glu Lys Thr Lys He Leu Met Asn Ser Ser Arg His Asn Ser His Pro 340 345 350

Phe Val Gly Leu Ala Phe Pro Leu Glu Val Gly Arg Phe Gly Gin Leu 355 360 365

Thr Tyr Val Arg Ser Tyr Gin Gly Glu Leu Lys Lys Gly Asp Thr He 370 375 380

Tyr Asn Thr Arg Thr Arg Lys Lys Val Arg Leu Gin Arg Leu Ala Arg 385 390 395 400

Met His Ala Asp Met Met Glu Ala Ser Thr Glu Glu Val Tyr Ala Gly 405 410 415 Asp He Cys Ala Leu Phe Gly He Asp Cys Ala Ser Gly Asp Thr Phe 420 425 430

Thr Asp Lys Ala Asn Ser Gly Leu Ser Met Glu Ser He His Val Pro 435 440 445

Asp Pro Val He Ser He Ala Met Lys Pro Ser Asn Lys Asn Asp Leu 450 455 460

Glu Lys Phe Ser Lys Gly He Gly Arg Phe Thr Arg Glu Asp Pro Thr 465 470 475 480

Phe Lys Val Tyr Phe Asp Thr Glu Asn Lys Glu Thr Val He Ser Gly 485 490 495

Met Gly Glu Leu His Leu Glu He Tyr Ala Gin Arg Leu Glu Arg Glu 500 505 510

Tyr Gly Cys Pro Cys He Thr Gly Lys Pro Lys Val Ala Phe Arg Glu 515 520 525

Thr He Thr Ala Pro Val Pro Phe Asp Phe Thr His Lys Lys Gin Ser 530 535 540

Gly Gly Ala Gly Gin Tyr Gly Lys Val He Gly Val Leu Glu Pro Leu

545 550 555 560

Asp Pro Glu Asp Tyr Thr Lys Leu Glu Phe Ser Asp Glu Thr Phe Gly 565 570 575

Ser Asn He Pro Lys Gin Phe Val Pro Ala Val Glu Lys Gly Phe Leu 580 585 590

Asp Ala Cys Glu Lys Gly Pro Leu Ser Gly His Lys Leu Ser Gly Leu 595 600 605

Arg Phe Val Leu Gin Asp Gly Ala His His Met Val Asp Ser Asn Glu 610 615 620

He Ser Phe He Arg Ala Gly Glu Gly Ala Leu Lys Gin Ala Leu Ala 625 630 635 640

Asn Ala Thr Leu Cys He Leu Glu Pro He Met Ala Val Glu Val Val 645 650 655

Ala Pro Asn Glu Phe Gin Gly Gin Val He Ala Gly He Asn Arg Arg 660 665 670

His Gly Val He Thr Gly Gin Asp Gly Val Glu Asp Tyr Phe Thr Leu 675 680 685

Tyr Ala Asp Val Pro Leu Asn Asp Met Phe Gly Tyr Ser Thr Glu Leu 690 695 700

Arg Ser Cys Thr Glu Gly Lys Gly Glu Tyr Thr Met Glu Tyr Ser Arg 705 710 715 720 Tyr Gin Pro Cys Leu Pro Ser Thr Gin Glu Asp Val He Asn Lys Tyr

725 730 735

Leu Glu Ala Thr Gly Gin Leu Pro Val Lys Lys Gly Lys Ala Lys Asn

740 745 750

Table 7 hTRG nucleotide sequence (SEQ ID NO:7) gccgcgggag caggcggagg cggaggcggc gggggcagga ggatgtcgca gccgccgctg 60 ctccccgcct cggcggagac tcggaagttc acccgggcgc tgagtaagcc gggcacggcg 120 gccgagctgc ggcagagcgt gtctgaggtg gtgcgcggct ccgtgctcct ggcaaagcca 180 aagctaattg agccactcga ctatgaaaat gtcatcgtcc agaagaagac tcagatcctg 240 aacgactgtt tacgggagat gctgctcttc ccttacgatg, actttcagac ggccatcctg 300 agacgacagg gtcgatacat atgctcaaca gtgcctgcga aggcggaaga ggaagcacag 360 agcttgtttg ttacagagtg catcaaaacc tataactctg actggcatct tgtgaactat 420 aaatatgaag attactcagg agagtttcga cagcttccga acaaagtggt caagttggat 480 aaacttccag ttcatgtcta tgaagttgac gaggaggtcg acaaagatga ggatgctgcc 540 tcccttggtt cccagaaagg tgggatcacc aagcatggct ggctgtacaa aggcaacatg 600 aacagtgcca tcagcgtgac catgaggtca tttaagagac gatttttcca cctgattcaa 660 cttggcgatg gatcctataa atttgaattt ttaaaagatc tccaaaagga accaaaagga 720 tcaatatttc tgggattcct gtatggggtg tcgttcagga acaacaaagt caggcgtttt 780 gcttttgagc tcaagatgca ggacaaaagt agttatctct tggcagcaga cagtgaagtg 840 gaaatggaag aatggatcac aattctaaat aagatcctcc agctcaactt tgaagctgca 900 atgcaagaaa agcgaaatgg cgactctcac gaagatgatg aacaaagcaa attggaaggt 960 tctggttccg gtttagatag ctacctgccg gaacttgcca agagtgcaag agaagcagaa 1020 atcaaactga aaagtgaaag cagagtcaaa cttttttatt tggacccaga tgcccagaag 1080 cttgacttct catcagctga gccagaagtg aagtcatttg aagagaagtt tggaaaaagg 1140 atccttgtca agtgcaatga tttatctttc aatttgcaat gctgtgttgc cgaaaatgaa 1200 gaaggaccca ctacaaatgt tgaacctttc tttgttactc tatccctgtt tgacataaaa 1260 tacaaccgga agatttctgc cgatttccac gtagacctga accatttctc agtgaggcaa 1320 atgatcgcca ccacgtcccc ggcgctgatg aatggcagtg ggccggaaac ccaatctgcc 1380 ctcaggggca tccttcatga agccgccatg cagtatccga agcagggaat attttcagtc 1440 acttgtcctc atccagatat atttcttgtg gccagaattg aaaaagtcct tcaggggagc 1500 atcacacatt gcgctgagcc atatatgaaa agttcagact cttctaaggt ggcccagaag 1560 gtgctgaaga atgccaagca ggcatgccaa agactaggac agtatagaat gccatttgct 1620 tgggcagcaa ggacattgtt taaggatgca tctggaaatc ttgacaaaaa tgccagattt 1680 tctgccatct acaggcaaga cagcaataag ctatccaatg atgacatgct caagttactt 1740 gcagactttc ggaaacctga gaagatggct aagctcccag tgattttagg caatctagac 1800 attacaattg ataatgtttc ctcagacttc cctaattatg ttaattcatc atacattccc 1860 acaaaacaat ttgaaacctg cagtaaaact cccatcacgt ttgaagtgga ggaatttgtg 1920 ccctgcatac caaaacacac tcagccttac accatctaca ccaatcacct ttacgtttat 1980 cctaagtact tgaaatacga cagtcagaag tcttttgcca aggctagaaa tattgcgatt 2040 tgcattgaat tcaaagattc agatgaggaa gactctcagc cccttaagtg catttatggc 2100 agacctggtg ggccagtttt cacaagaagc gcctttgctg cagttttaca ccatcaccaa 2160 aacccagaat tttatgatga gattaaaata gagttgccca ctcagctgca tgaaaagcac 2220 cacctgttgc tcacattctt ccatgtcagc tgtgacaact caagtaaagg aagcacgaag 2280 aagagggatg tcgttgaaac ccaagttggc tactcctggc ttcccctcct gaaagacgga 2340 agggtggtga caagcgagca gcacatcccg gtctcggcga accttccttc gggctatctt 2400 ggctaccagg agcttgggat gggcaggcat tatggtccgg aaattaaatg ggtagatgga 2460 ggcaagccac tgctgaaaat ttccactcat ctggtttcta cagtgtatac tcaggatcag 2520 catttacata attttttcca gtactgtcag aaaaccgaat ctggagccca agccttagga 2580 aacgaacttg taaagtacct taagagtctg catgcgatgg aaggccacgt gatgatcgcc 2640 ttcttgccca ctatcctaaa ccagctgttc cgagtcctca ccagagccac acaggaagaa 2700 gtcgcggtta acgtgactcg ggtcattatt catgtggttg cccagtgcca tgaggaagga . 2760 ttggagagcc acttgaggtc atatgttaag tacgcgtata aggctgagcc atatgttgcc 2820 tctgaataca agacagtgca tgaagaactg accaaatcca tgaccacgat tctcaagcct 2880 tctgccgatt tcctcaccag caacaaacta ctgaagtact catggttttt ctttgatgta 2940 ctgatcaaat ctatggctca gcatttgata gagaactcca aagttaagtt gctgcgaaac 3000 cagagatttc ctgcatccta tcatcatgca gtggaaaccg ttgtaaatat gctgatgcca 3060 cacatcactc agaagtttcg agataatcca gaggcatcta agaacgcgaa tcatagcctt 3120 gctgtcttca tcaagagatg tttcaccttc atggacaggg gctttgtctt caagcagatc 3180 aacaactaca ttagctgttt tgctcctgga gacccaaaga ccctctttga atacaagttt 3240 gaatttctcc gtgtagtgtg caaccatgaa cattatattc cgttgaactt accaatgcca 3300 tttggaaaag gcaggattca aagataccaa gacctccagc ttgactactc attaacagat 3360 gagttctgca gaaaccactt cttggtggga ctgttactga gggaggtggg gacagccctc 3420 caggagttcc gggaggtccg tctgatcgcc atcagtgtgc tcaagaacct gctgataaag 3480 cattcttttg atgacagata tgcttcaagg agccatcagg caaggatagc caccctctac 3540 ctgcctctgt ttggtctgct gattgaaaac gtccagcgga tcaatgtgag ggatgtgtca 3600 cccttccctg tgaacgcggg catgactgtg aaggatgaat ccctggctct accagctgtg 3660 aatccgctgg tgacgccgca gaagggaagc accctggaca acagcctgca caaggacctg 3720 ctgggcgcca tctccggcat tgcttctcca tatacaacct caactccaaa catcaacagt 3780 gtgagaaatg ctgattcgag aggatctctc ataagcacag attcgggtaa cagccttcca 3840 gaaaggaata gtgagaagag caattccctg gataagcacc aacaaagtag cacattggga 3900 aattccgtgg ttcgctgtga taaacttgac cagtctgaga ttaagagcct actgatgtgt 3960 ttcctctaca tcttaaagag catgtctgat gatgctttgt ttacatattg gaacaaggct 4020 tcaacatctg aacttatgga tttttttaca atatctgaag tctgcctgca ccagttccag 4080 tacatgggga agcgatacat agccagaaca ggaatgatgc atgccagatt gcagcagctg 4140 ggcagcctgg ataactctct cacttttaac cacagctatg gccactcgga cgcagatgtt 4200 ctgcaccagt cattacttga agccaacatt gctactgagg tttgcctgac agctctggac 4260 acgctttctc tatttacatt ggcgtttaag aaccagctcc tggccgacca tggacataat 4320 cctctcatga aaaaagtttt tgatgtctac ctgtgttttc ttcaaaaaca tcagtctgaa 4380 acggctttaa aaaatgtctt cactgcctta aggtccttaa tttataagtt tccctcaaca 4440 ttctatgaag ggagagcgga catgtgtgcg gctctgtgtt acgagattct caagtgctgt 4500 aactccaagc tgagctccat caggacggag gcctcccagc tgctctactt cctgatgagg 4560 aacaactttg attacactgg aaagaagtcc tttgtccgga cacatttgca agtcatcata 4620 tctgtcagcc agctgatagc agacgttgtt ggcattgggg gaaccagatt ccagcagtcc 4680 ctgtccatca tcaacaactg tgccaacagt gaccggctta ttaagcacac cagcttctcc 4740 tctgatgtga aggacttaac caaaaggata cgcacggtgc taatggccac cgcccagatg 4800 aaggagcatg agaacgaccc agagatgctg gtggacctcc agtacagcct ggccaaatcc 4860 tatgccagca cgcccgagct caggaagacg tggctcgaca gcatggccag gatccatgtc 4920 aaaaatggcg atctctcaga ggcagcaatg tgctatgtcc acgtaacagc cctagtggca 4980 gaatatctca cacggaaaga agcagtccag tgggagccgc cccttctccc ccacagccat 5040 agcgcctgcc tgaggaggag ccggggaggc gtgtttagac aaggatgcac cgccttcagg 5100 gtcattaccc caaacatcga cgaggaggcc tccatgatgg aagacgtggg gatgcaggat 5160 gtccatttca acgaggatgt gctgatggag ctccttgagc agtgcgcaga tggactctgg 5220 aaagccgagc gctacgagct cattgccgac atctacaaac ttatcatccc catttatgag 5280 aagcggaggg attttgagag gctggcccat ctgtatgaca cgctgcaccg ggcctacagc 5340 aaagtgaccg aggtcatgca ctcgggccgc aggcttctgg ggacctactt ccgggtagcc 5400 ttcttcgggc aggcagcgca ataccagttt acagacagtg aaacagatgt ggagggattc 5460 tttgaagatg aagatggaaa ggagtatatt tacaaggaac ccaaactcac accgctgtcg 5520 gaaatttctc agagactcct taaactgtac tcggataaat ttggttctga aaatgtcaaa 5580 atgatacagg attctggcaa ggtcaaccct aaggatctgg attctaagta tgcctacatc 5640 caggtgactc acgtcatccc cttctttgac gaaaaagagt tgcaagaaag gaaaacagag 5700 tttgagagat cccacaacat ccgccgcttc atgtttgaga tgccatttac gcagaccggg 5760 aagaggcagg gcggggtgga agagcagtgc aaacggcgca ccatcctgac agccatacac 5820 tgcttccctt atgtgaagaa gcgcatccct gtcatgtacc agcaccacac tgacctgaac 5880 cccatcgagg tggccattga cgagatgagt aagaaggtgg cggagctccg gcagctgtgc 5940 tcctcggccg aggtggacat gatcaaactg cagctcaaac tccagggcag cgtgagtgtt 6000 caggtcaatg ctggcccact agcatatgcg cgagctttct tagatgatac aaacacaaag 6060 cgatatcctg acaataaagt gaagctgctt aaggaagttt tcaggcaatt tgtggaagct 6120 tgcggtcaag ccttagcggt aaacgaacgt ctgattaaag aagaccagct cgagtatcag 6180 gaagaaatga aagccaacta cagggaaatg gcgaaggagc tttctgaaat catgcatgag 6240 cagatctgcc ccctggagga gaagacgagc gtcttaccga attcccttca catcttcaac 6300 gccatcagtg ggactccaac aagcacaatg gttcacggga tgaccagctc gtcttcggtc 6360 gtgtgattac atctcatggc ccgtgtgtgg ggacttgctt tgtcatttgc aaactcagga 6420 tgctttccaa agccaatcac tggggagacc gagcacaggg aggaccaagg ggaaggggag 6480 agaaaggaaa taaagaacaa cgttatttct taacagactt tctataggag ttgtaagaag 6540 gtgcacatat ttttttaaat ctcactggca atattcaaag ttttcattgt gtcttaacaa 6600 aggtgtggta gacactcttg agctggactt agattttatt cttccttgca gagtagtgtt 6660 agaatagatg gcctacagaa aaaaaaggtt ctgggatcta catggcaggg agggctgcac 6720 tgacattgat gcctggggga ccttttgcct cgaggctgag ctggaaaatc ttgaaaatat 6780 tttttttttc ctgtggcaca ttcaggttga atacaagaac tatttttgtg actagttttt 6840 gatgacctaa gggaactgac cattgtaatt tttgtaccag tgaaccagga gatttagtgc 6900 ttttatattc atttccttgc atttaagaaa atatgaaagc ttaaggaatt atgtgagctt 6960 aaaactagtc aagcagttta gaaccaaagg cctatattaa taaccgcaac tatgctgaaa 7020 agtacaaagt agtacagtat attgttatgt acatatcatt gttaatacag tcctggcatt 7080 ctgtacatat atgtattaca tttctacatt tttaatactc acatgggctt atgcattaag 7140 tttaattgtg ataaatttgt gctgttccag tatatgcaat acactttaat gttttattct 7200 tgtacataaa aatgtgcaat atggagatgt atacagtctt tactatatta ggtttataaa 7260 cagttttaag aatttcatcc ttttgccaaa atggtggagt atgtaattgg taaatcataa 7320 atcctgtggt gaatggtggt gtactttaaa gctgtcacca tgttatattt tcttttaaga 7380 ctttaattta gtaattttat atttgggaaa ataaaggttt ttaattttat ttaactggaa 7440 tcactgccct gctgtaatta aacattctgt accacatctg tattaaaaag acattgctga 7500 ccatta 7506

Table 8 hTRGpc )lype 3tide sequence (SEQ ID NO:8)

Met Ser Gin Pro Pro Leu Leu Pro Ala Ser Ala Glu Thr Arg Lys Phe

1 5 10 15

Thr Arg Ala Leu Ser Lys Pro Gly Thr Ala Ala Glu Leu Arg Gin Ser 20 25 30

Val Ser Glu Val Val Arg Gly Ser Val Leu Leu Ala Lys Pro Lys Leu 35 40 45

He Glu Pro Leu Asp Tyr Glu Asn Val He Val Gin Lys Lys Thr Gin 50 55 60

He Leu Asn Asp Cys Leu Arg Glu Met Leu Leu Phe Pro Tyr Asp Asp 65 70 75 80

Phe Gin Thr Ala He Leu Arg Arg Gin Gly Arg Tyr He Cys Ser Thr 85 90 95

Val Pro Ala Lys Ala Glu Glu Glu Ala Gin Ser Leu Phe Val Thr Glu 100 105 110

Cys He Lys Thr Tyr Asn Ser Asp Trp His Leu Val Asn Tyr Lys Tyr 115 120 125

Glu Asp Tyr Ser Gly Glu Phe Arg Gin Leu Pro Asn Lys Val Val Lys 130 135 140 Leu Asp Lys Leu Pro Val His Val Tyr Glu Val Asp Glu Glu Val Asp 145 150 155 160

Lys Asp Glu Asp Ala Ala Ser Leu Gly Ser Gin Lys Gly Gly He Thr 165 170 175

Lys His Gly Trp Leu Tyr Lys Gly Asn Met Asn Ser Ala He Ser Val 180 185 190

Thr Met Arg Ser Phe Lys Arg Arg Phe Phe His Leu He Gin Leu Gly 195 200 205

Asp Gly Ser Tyr Lys Phe Glu Phe Leu Lys Asp Leu Gin Lys Glu Pro 210 215 220

Lys Gly Ser He Phe Leu Gly Phe Leu Tyr Gly Val Ser Phe Arg Asn 225 230 235 240

Asn Lys Val Arg Arg Phe Ala Phe Glu Leu Lys Met Gin Asp Lys Ser 245 250 255

Ser Tyr Leu Leu Ala Ala Asp Ser Glu Val Glu Met Glu Glu Trp He 260 265 270

Thr He Leu Asn Lys He Leu Gin Leu Asn Phe Glu Ala Ala Met Gin 275 280 285

Glu Lys Arg Asn Gly Asp Ser His Glu Asp Asp Glu Gin Ser Lys Leu 290 295 300

Glu Gly Ser Gly Ser Gly Leu Asp Ser Tyr Leu Pro Glu Leu Ala Lys 305 310 315 320

Ser Ala Arg Glu Ala Glu He Lys Leu Lys Ser Glu Ser Arg Val Lys 325 330 335

Leu Phe Tyr Leu Asp Pro Asp Ala Gin Lys Leu Asp Phe Ser Ser Ala 340 345 350

Glu Pro Glu Val Lys Ser Phe Glu Glu Lys Phe Gly Lys Arg He Leu 355 360 365

Val Lys Cys Asn Asp Leu Ser Phe Asn Leu Gin Cys Cys Val Ala Glu 370 375 380

Asn Glu Glu Gly Pro Thr Thr Asn Val Glu Pro Phe Phe Val Thr Leu 385 390 395 400

Ser Leu Phe Asp He Lys Tyr Asn Arg Lys He Ser Ala Asp Phe His 405 410 415

Val Asp Leu Asn His Phe Ser Val Arg Gin Met He Ala Thr Thr Ser 420 425 430

Pro Ala Leu Met Asn Gly Ser Gly Pro Glu Thr Gin Ser Ala Leu Arg 435 440 445 Gly He Leu His Glu Ala Ala Met Gin Tyr Pro Lys Gin Gly He Phe 450 455 460

Ser Val Thr Cys Pro His Pro Asp He Phe Leu Val Ala Arg He Glu 465 470 475 480

Lys Val Leu Gin Gly Ser He Thr His Cys Ala Glu Pro Tyr Met Lys 485 490 495

Ser Ser Asp Ser Ser Lys Val Ala Gin Lys Val Leu Lys Asn Ala Lys 500 505 510

Gin Ala Cys Gin Arg Leu Gly Gin Tyr Arg Met Pro Phe Ala Trp Ala

515 520 525

Ala Arg Thr Leu Phe Lys Asp Ala Ser Gly Asn Leu Asp Lys Asn Ala 530 535 540

Arg Phe Ser Ala He Tyr Arg Gin Asp Ser Asn Lys Leu Ser Asn Asp 545 550 555 560

Asp Met Leu Lys Leu Leu Ala Asp Phe Arg Lys Pro Glu Lys Met Ala

565 570 575

Lys Leu Pro Val He Leu Gly Asn Leu Asp He Thr He Asp Asn Val 580 585 590

Ser Ser Asp Phe Pro Asn Tyr Val Asn Ser Ser Tyr He Pro Thr Lys 595 600 605

Gin Phe Glu Thr Cys Ser Lys Thr Pro He Thr Phe Glu Val Glu Glu 610 615 620

Phe Val Pro Cys He Pro Lys His Thr Gin Pro Tyr Thr He Tyr Thr 625 630 635 640

Asn His Leu Tyr Val Tyr Pro Lys Tyr Leu Lys Tyr Asp Ser Gin Lys 645 650 655

Ser Phe Ala Lys Ala Arg Asn He Ala He Cys He Glu Phe Lys Asp 660 665 670

Ser Asp Glu Glu Asp Ser Gin Pro Leu Lys Cys He Tyr Gly Arg Pro 675 680 685

Gly Gly Pro Val Phe Thr Arg Ser Ala Phe Ala Ala Val Leu His His 690 695 700

His Gin Asn Pro Glu Phe Tyr Asp Glu He Lys He Glu Leu Pro Thr 705 710 715 720

Gin Leu His Glu Lys His His Leu Leu Leu Thr Phe Phe His Val Ser 725 730 735

Cys Asp Asn Ser Ser Lys Gly Ser Thr Lys Lys Arg Asp Val Val Glu 740 745 750

Thr Gin Val Gly Tyr Ser Trp Leu Pro Leu Leu Lys Asp Gly Arg Val 755 760 765

Val Thr Ser Glu Gin His He Pro Val Ser Ala Asn Leu Pro Ser Gly 770 775 780

Tyr Leu Gly Tyr Gin Glu Leu Gly Met Gly Arg His Tyr Gly Pro Glu 785 790 795 800

He Lys Trp Val Asp Gly Gly Lys Pro Leu Leu Lys He Ser Thr His 805 810 815

Leu Val Ser Thr Val Tyr Thr Gin Asp Gin His Leu His Asn Phe Phe 820 825 830

Gin Tyr Cys Gin Lys Thr Glu Ser Gly Ala Gin Ala Leu Gly Asn Glu 835 840 845

Leu Val Lys Tyr Leu Lys Ser Leu His Ala Met Glu Gly His Val Met 850 855 860

He Ala Phe Leu Pro Thr He Leu Asn Gin Leu Phe Arg Val Leu Thr 865 870 875 880

Arg Ala Thr Gin Glu Glu Val Ala Val Asn Val Thr Arg Val He He 885 890 895

His Val Val Ala Gin Cys His Glu Glu Gly Leu Glu Ser His Leu Arg 900 905 910

Ser Tyr Val Lys Tyr Ala Tyr Lys Ala Glu Pro Tyr Val Ala Ser Glu 915 920 925

Tyr Lys Thr Val His Glu Glu Leu Thr Lys Ser Met Thr Thr He Leu 930 935 940

Lys Pro Ser Ala Asp Phe Leu Thr Ser Asn Lys Leu Leu Lys Tyr Ser 945 950 955 960

Trp Phe Phe Phe Asp Val Leu He Lys Ser Met Ala Gin His Leu He 965 970 975

Glu Asn Ser Lys Val Lys Leu Leu Arg Asn Gin Arg Phe Pro Ala Ser 980 985 990

Tyr His His Ala Val Glu Thr Val Val Asn Met Leu Met Pro His He 995 1000 1005

Thr Gin Lys Phe Arg Asp Asn Pro Glu Ala Ser Lys Asn Ala Asn 1010 1015 1020

His Ser Leu Ala Val Phe He Lys Arg Cys Phe Thr ] Phe Met Asp 1025 1030 1035

Arg Gly Phe Val Phe Lys Gin He Asn Asn Tyr He Ser Cys Phe 1040 1045 1050

| Ala Pro Gly Asp Pro Lys Thr Leu Phe Glu Tyr Lys ] Phe Glu Phe 1055 1060 1065

Leu Arg Val Val Cys Asn His Glu His Tyr He Pro Leu Asn Leu 1070 1075 1080

Pro Met Pro Phe Gly Lys Gly Arg He Gin Arg Tyr Gin Asp Leu 1085 1090 1095

Gin Leu Asp Tyr Ser Leu Thr Asp Glu Phe Cys Arg Asn His Phe 1100 1105 1110

Leu Val Gly Leu Leu Leu Arg Glu Val Gly Thr Ala Leu Gin Glu 1115 1120 1125

Phe Arg Glu Val Arg Leu He Ala He Ser Val Leu Lys Asn Leu 1130 1135 1140

Leu He Lys His Ser Phe Asp Asp Arg Tyr Ala Ser Arg Ser His 1145 1150 1155

Gin Ala Arg He Ala Thr Leu Tyr Leu Pro Leu Phe Gly Leu Leu 1160 1165 1170

He Glu Asn Val Gin Arg He Asn Val Arg Asp Val Ser Pro Phe 1175 1180 1185

Pro Val Asn Ala Gly Met Thr Val Lys Asp Glu Ser Leu Ala Leu 1190 1195 1200

Pro Ala Val Asn Pro Leu Val Thr Pro Gin Lys Gly Ser Thr Leu 1205 1210 1215

Asp Asn Ser Leu His Lys Asp Leu Leu Gly Ala He Ser Gly He 1220 1225 1230

Ala Ser Pro Tyr Thr Thr Ser Thr Pro Asn He Asn Ser Val Arg 1235 1240 1245

Asn Ala Asp Ser Arg Gly Ser Leu He Ser Thr Asp Ser Gly Asn 1250 1255 1260

Ser Leu Pro Glu Arg Asn Ser Glu Lys Ser Asn Ser Leu Asp Lys 1265 1270 1275

His Gin Gin Ser Ser Thr Leu Gly Asn Ser Val Val Arg Cys Asp 1280 1285 1290

Lys Leu Asp Gin Ser Glu He Lys Ser Leu Leu Met Cys Phe Leu 1295 1300 1305

Tyr He Leu Lys Ser Met Ser Asp Asp Ala Leu Phe Thr Tyr Trp 1310 1315 1320

Asn Lys Ala Ser Thr Ser Glu Leu Met Asp Phe Phe Thr He Ser 1325 1330 1335

Glu Val Cys Leu His Gin Phe Gin Tyr Met Gly Lys Arg Tyr He 1340 1345 1350 Ala Arg Thr Gly Met Met His Ala Arg Leu Gin Gin Leu Gly Ser 1355 1360 1365

Leu Asp Asn Ser Leu Thr Phe Asn His Ser Tyr Gly His Ser Asp 1370 1375 1380

Ala Asp Val Leu His Gin Ser Leu Leu Glu Ala Asn He Ala Thr 1385 1390 1395

Glu Val Cys Leu Thr Ala Leu Asp Thr Leu Ser Leu Phe Thr Leu 1400 1405 1410

Ala Phe Lys Asn Gin Leu Leu Ala Asp His Gly His Asn Pro Leu 1415 1420 1425

Met Lys Lys Val Phe Asp Val Tyr Leu Cys Phe Leu Gin Lys His 1430 1435 1440

Gin Ser Glu Thr Ala Leu Lys Asn Val Phe Thr Ala Leu Arg Ser

1445 1450 1455

Leu He Tyr Lys Phe Pro Ser Thr Phe Tyr Glu Gly Arg Ala Asp 1460 1465 1470

Met Cys Ala Ala Leu Cys Tyr Glu He Leu Lys Cys Cys Asn Ser 1475 1480 1485

Lys Leu Ser Ser He Arg Thr Glu Ala Ser Gin Leu Leu Tyr Phe 1490 1495 1500

Leu Met Arg Asn Asn Phe Asp Tyr Thr Gly Lys Lys Ser Phe Val 1505 1510 1515

Arg Thr His Leu Gin Val He He Ser Val Ser Gin Leu He Ala 1520 1525 1530

Asp Val Val Gly He Gly Gly Thr Arg Phe Gin Gin Ser Leu Ser 1535 1540 1545

He He Asn Asn Cys Ala Asn Ser Asp Arg Leu He Lys His Thr 1550 1555 1560

Ser Phe Ser Ser Asp Val Lys Asp Leu Thr Lys Arg He Arg Thr 1565 1570 1575

Val Leu Met Ala Thr Ala Gin Met Lys Glu His Glu Asn Asp Pro 1580 1585 1590

Glu Met Leu Val Asp Leu Gin Tyr Ser Leu Ala Lys Ser Tyr Ala 1595 1600 1605

Ser Thr Pro Glu Leu Arg Lys Thr Trp Leu Asp Ser Met Ala Arg 1610 1615 1620

He His Val Lys Asn Gly Asp Leu Ser Glu Ala Ala Met Cys Tyr 1625 1630 1635 Val His Val Thr Ala Leu Val Ala Glu Tyr Leu Thr Arg Lys Glu 1640 1645 1650

Ala Val Gin Trp Glu Pro Pro Leu Leu Pro His Ser His Ser Ala 1655 1660 1665

Cys Leu Arg Arg Ser Arg Gly Gly Val Phe Arg Gin Gly Cys Thr 1670 1675 1680

Ala Phe Arg Val He Thr Pro Asn He Asp Glu Glu Ala Ser Met 1685 1690 1695

Met Glu Asp Val Gly Met Gin Asp Val His Phe Asn Glu Asp Val 1700 1705 1710

Leu Met Glu Leu Leu Glu Gin Cys Ala Asp Gly Leu Trp Lys Ala 1715 1720 1725

Glu Arg Tyr Glu Leu He Ala Asp He Tyr Lys Leu He He Pro . 1730 1735 1740

He Tyr Glu Lys Arg Arg Asp Phe Glu Arg Leu Ala His Leu Tyr 1745 1750 1755

Asp Thr Leu His Arg Ala Tyr Ser Lys Val Thr Glu Val Met His 1760 1765 1770

Ser Gly Arg Arg Leu Leu Gly Thr Tyr Phe Arg Val Ala Phe Phe 1775 1780 1785

Gly Gin Ala Ala Gin Tyr Gin Phe Thr Asp Ser Glu Thr Asp Val 1790 1795 1800

Glu Gly Phe Phe Glu Asp Glu Asp Gly Lys Glu Tyr He Tyr Lys 1805 1810 1815

Glu Pro Lys Leu Thr Pro Leu Ser Glu He Ser Gin Arg Leu Leu 1820 1825 1830

Lys Leu Tyr Ser Asp Lys Phe Gly Ser Glu Asn Val Lys Met He 1835 1840 1845

Gin Asp Ser Gly Lys Val Asn Pro Lys Asp Leu Asp Ser Lys Tyr 1850 1855 1860

Ala Tyr He Gin Val Thr His Val He Pro Phe Phe Asp Glu Lys 1865 1870 1875

Glu Leu Gin Glu Arg Lys Thr Glu Phe Glu Arg Ser His Asn He 1880 1885 1890

Arg Arg Phe Met Phe Glu Met Pro Phe Thr Gin Thr Gly Lys Arg 1895 1900 1905

Gin Gly Gly Val Glu Glu Gin Cys Lys Arg Arg Thr He Leu Thr 1910 1915 1920

Ala He His Cys Phe Pro Tyr Val Lys Lys Arg He Pro Val Met 1925 1930 1935

Tyr Gin His His Thr Asp Leu Asn Pro He Glu Val Ala He Asp 1940 1945 1950

Glu Met Ser Lys Lys Val Ala Glu Leu Arg Gin Leu Cys Ser Ser 1955 1960 1965

Ala Glu Val Asp Met He Lys Leu Gin Leu Lys Leu Gin Gly Ser 1970 1975 1980

Val Ser Val Gin Val Asn Ala Gly Pro Leu Ala Tyr Ala Arg Ala 1985 1990 1995

Phe Leu Asp Asp Thr Asn Thr Lys Arg Tyr Pro Asp Asn Lys Val 2000 2005 2010

Lys Leu Leu Lys Glu Val Phe Arg Gin Phe Val Glu Ala Cys Gly 2015 2020 2025

Gin Ala Leu Ala Val Asn Glu Arg Leu He Lys Glu Asp Gin Leu 2030 2035 2040

Glu Tyr Gin Glu Glu Met Lys Ala Asn Tyr Arg Glu Met Ala Lys 2045 2050 2055

Glu Leu Ser Glu He Met His Glu Gin He Cys Pro Leu Glu Glu 2060 2065 2070

Lys Thr Ser Val Leu Pro Asn Ser Leu His He Phe Asn Ala^' He 2075 2080 2085

Ser Gly Thr Pro Thr Ser Thr Met Val His Gly Met Thr Ser Ser 2090 2095 2100

Ser Ser Val Val 2105 •

Table 9 hMXl nucleotide sequence (SEQ ID NO:9) ttttgtttac agggaacacg ggtctggctg agagaaaatg gccagcattt tccaagtact 60 gtaaattcct gtgcagaagg catcgtcgtc ttccggacag actatggtca ggtattcact 120 tacaagcaga gcacaattac ccaccagaag gtgactgcta tgcaccccac gaacgaggag 180 ggcgtggatg acatggcgtc cttgacagag ctccatggcg gctccatcat gtataactta 240 ttccagcggt ataagagaaa tcaaatatgg acctacatcg gctccatcct ggcctctgtg 300 aacccctacc agcccatcgc cgggctgtac gagcctgcca ccatggagca gtacagccgg 360 cgccacctgg gcgagctgcc cccgcacatc ttcgccatcg ccaacgagtg ctaccgctgc 420 ctgtggaagc gccacgacaa ccagtgcatc ctcatcaagg gtgaaagtgg ggcaggtaaa 480 accgaaagca ctaaattgat cctcaagttt ctgtcagtca tcagtcaaca gtctttggaa 540 ttgtccttaa aggagaagac atcctgtgtt gaacgagcta ttcttgaaag cagccccatc 600 atggaagctt tcggcaatgc gaagaccgtg tacaacaaca actctagtcg ctttgggaag 660 tttgttcagc tgaacatctg tcagaaagga aatattcagg gcgggagaat tgtagattgt 720 atcctctctt cccagaaccg agtagtaagg caaaatcccg gggaaaggaa ttatcacata 780 ttttatgcac tgctggcagg gctggaacat gaagaaagag aagaatttta tttatctacg 840 ccagaaaact accactactt gaatcagtct ggatgtgtag aagacaagac aatcagtgac 900 caggaatcct ttagggaagt tattacggca atggacgtga tgcagttcag caaggaggaa 960 gttcgggaag tgtcgaggct gcttgctggt atactgcatc ttgggaacat agaatttatc 1020 actgctggtg gggcacaggt ttccttcaaa acagctttgg gcagatctgc ggagttactt 1080 gggctggacc caacacagct cacagatgct ttgacccaga gatcaatgtt cctcagggga 1140 gaagagatcc tcacgcctct caatgttcaa caggcagtag acagcaggga ctccctggcc 1200 atggctctgt atgcgtgctg ctttgagtgg gtaatcaaga agatcaacag caggatcaaa 1260 ggcaatgagg acttcaagtc tattggcatc ctcgacatct ttggatttga aaactttgag 1320 gttaatcact ttgaacagtt caatataaac tatgcaaacg agaaacttca ggagtacttc 1380 aacaagcata ttttttcttt agaacaacta gaatatagca gggaaggatt agtgtgggaa 1440 gatattgact ggatagacaa tggagaatgc ctggacttga ttgagaagaa acttggcctc 1500 ctagccctta tcaatgaaga aagccatttt cctcaagcca cagacagcac cttattggag 1560 aagctacaca gtcagcatgc gaataaccac ttttatgtga agcccagagt tgcagttaac 1620 aattttggag tgaagcacta tgctggagag gtgcaatatg atgtccgagg tatcttggag 1680 aagaacagag atacatttcg agatgacctt ctcaatttgc taagagaaag ccggtttgac 1740 tttatctacg atctttttga acatgtttca agccgcaaca accaggatac cttgaaatgt 1800 ggaagcaaac atcggcggcc tacagtcagc tcacagttca aggttgactc actgcattcc 1860 ttaatggcaa cgctaagctc ctctaatcct ttctttgttc gctgtatcaa gccaaacatg 1920 cagaagatgc cagaccagtt tgaccaggcg gttgtgctga accagctgcg gtactcaggg 1980 atgctggaga ctgtgagaat ccgcaaagct gggtatgcgg tccgaagacc ctttcaggac 2040 ttttacaaaa ggtataaagt gctgatgagg aatctggctc tgcctgagga cgtccgaggg 2100 aagtgcacga gcctgctgca gctctatgat gcctccaaca gcgagtggca gctggggaag 2160 accaaggtat ttcttcgaga atccttggaa cagaaactgg agaagcggag ggaagaggaa 2220 gtgagccacg cggccatggt gattcgggcc catgtcttgg gcttcttagc acggaaacaa 2280 tacagaaagg tcctttattg tgtggtgata atacagaaga attacagagc attccttctg 2340 aggaggagat ttttgcacct gaaaaaggca gccatagttt tccagaagca actcagaggt 2400 cagattgctc ggagagttta cagacaattg ctggcagaga aaagggagca agaagaaaag 2460 aagaaacagg aagaggaaga aaagaagaaa cgggaggaag aagaaagaga aagagagaga 2520 gagcgaagag aagccgagct ccgcgcccag caggaagaag aaacgaggaa gcagcaagaa 2580 ctcgaagcct tgcagaagag ccagaaggaa gctgaactga cccgtgaact ggagaaacag 2640 aaggaaaata agcaggtgga agagatcctc cgtctggaga aagaaatcga ggacctgcag 2700 cgcatgaagg agcagcagga gctgtcgctg accgaggctt ccctgcagaa gctgcaggag 2760 cggcgggacc aggagctccg caggctggag gaggaagcgt gcagggcggc ccaggagttc 2820 ctcgagtccc tcaatttcga cgagatcgac gagtgtgtcc ggaatatcga gcggtccctg 2880 tcggggggaa gcgaattttc cagcgagctg gctgagagcg catgcgagga gaagcccaac 2940 ttcaacttca gccagcccta cccagaggag gaggtcgatg agggcttcga agccgacgac 3000 gacgccttca aggactcccc caaccccagc gagcacggcc actcagacca gcgaacaagt 3060 ggcatccgga ccagcgatga ctcttcagag gaggacccat acatgaacga cacggtggtg 3120 cccaccagcc ccagtgcgga cagcacggtg ctgctcgccc catcagtgca ggactccggg 3180 agcctacaca actcctccag cggcgagtcc acctactgca tgccccagaa cgctggggac 3240 ttgccctccc cagacggcga ctacgactac gaccaggatg actatgagga cggtgccatc 3300 acttccggca gcagcgtgac cttctccaac tcctacggca gccagtggtc ccccgactac 3360 cgctgctctg tggggaccta caacagctcg ggtgcctacc ggttcagctc tgagggggcg 3420 cagtcctcgt ttgaagatag tgaagaggac tttgattcca ggtttgatac agatgatgag 3480 ctttcatacc ggcgtgactc tgtgtacagc tgtgtcactc tgccgtattt ccacagcttt 3540 ctgtacatga aaggtggcct gatgaactct tggaaacgcc gctggtgcgt cctcaaggat 3600 gaaaccttct tgtggttccg ctccaagcag gaggccctca agcaaggctg gctccacaaa 3660 aaaggggggg gctcctccac gctgtccagg agaaattgga agaagcgctg gtttgtcctc 3720 cgccagtcca agctgatgta ctttgaaaac gacagcgagg agaagctcaa gggcaccgta 3780 gaagtgcgaa cggcaaaaga gatcatagat aacaccacca aggagaatgg gatcgacatc 3840 attatggccg ataggacttt ccacctgatt gcagagtccc cagaagatgc cagccagtgg 3900 ttcagcgtgc tgagtcaggt ccacgcgtcc acggaccagg agatccagga gatgcatgat 3960 gagcaggcaa acccacagaa tgctgtgggc accttggatg tggggctgat tgattctgtg 4020 tgtgcctctg acagccctga tagacccaac tcgtttgtga tcatcacggc caaccgggtg 4080 ctgcactgca acgccgacac gccggaggag atgcaccact ggataaccct gctgcagagg 4140 tccaaagggg acaccagagt ggagggccag gaattcatcg tgagaggatg gttgcacaaa 4200 gaggtgaaga acagtccaaa gatgtcttca ctgaaactga agaaacggtg gtttgtactc 4260 acccacaatt ccctggatta ctacaagagt tcagagaaga acgcgctcaa actggggacc 4320 ctggtcctca acagcctctg ctctgtcgtc cccccagatg agaagatatt caaagagaca 4380 ggctactgga acgtcaccgt gtacgggcgc aagcactgtt accggctcta caccaagctg 4440 ctcaacgagg ccacccggtg gtccagtgtc attcaaaacg tgactgacac caaggccccg 4500 atcgacaccc ccacccagca gctgattcaa gatatcaagg agaactgcct gaactcggat 4560 gtggtggaac agatttacaa gcggaacccg atccttcgat acacccatca ccccttgcac 4620 tccccgctcc tgccccttcc gtatggggac ataaatctca acttgctgaa agacaaaggc 4680 tataccaccc ttcaggatga ggccatcaag atattcaatt ccctgcagca actggagtcc 4740 atgtctgacc caattccaat aatccagggc atcctacaga cagggcatga cctgcgacct 4800 ctgcgggacg agctgtactg ccagcttatc aaacagacca acaaagtgcc ccaccccggc 4860 agtgtgggca acctgtacag ctggcagatc ctgacatgcc tgagctgcac cttcctgccg 4920 agtcgaggga ttctcaagta tctcaagttc catctgaaaa ggatacggga acagtttcca 4980 ggaaccgaga tggaaaaata cgctctcttc acttacgaat ctcttaagaa aaccaaatgc 5040 cgagagtttg tgccttcccg agatgaaata gaagctctga tccacaggca ggaaatgaca 5100 tccacggtct attgccatgg cggcggctcc tgcaagatca ccatcaactc ccacaccacc 5160 gctggggagg tggtggagaa gctgatccga ggcctggcca tggaggacag caggaacatg 5220 tttgctttgt ttgaatacaa cggccacgtc gacaaagcca ttgaaagtcg aaccgtcgta 5280 gctgatgtct tagccaagtt tgaaaagctg gctgccacat ccgaggttgg ggacctgcca 5340 tggaaattct acttcaaact ttactgcttc ctggacacag acaacgtgcc aaaagacagt 5400 gtggagtttg catttatgtt tgaacaggcc cacgaagcgg ttatccatgg ccaccatcca 5460 gccccggaag aaaacctcca ggttcttgct gccctgcgac tccagtatct gcagggggat 5520 tatactctgc acgctgccat cccacctctc gaagaggttt attccctgca gagactcaag 5580 gcccgcatca gccagtcaac caaaaccttc accccttgtg aacggctgga gaagaggcgg 5640 acgagcttcc tagaggggac cctgaggcgg agcttccgga caggatccgt ggtccggcag 5700 aaggtcgagg aggagcagat gctggacatg tggattaagg aagaagtctc ctctgctcga 5760 gccagtatca ttgacaagtg gaggaaattt cagggaatga accaggaaca ggccatggcc 5820 aagtacatgg ccttgatcaa ggagtggcct ggctatggct cgacgctgtt tgatgtggag 5880 tgcaaggaag gtggcttccc tcaggaactc tggttgggtg tcagcgcgga cgccgtctcc 5940 gtctacaagc gtggagaggg aagaccactg gaagtcttcc agtatgaaca catcctctct 6000 tttggggcac ccctggcgaa tacgtataag atcgtggtcg atgagaggga gctgctcttt 6060 gaaaccagtg aggtagtgga tgtggccaag ctcatgaaag cctacatcag catgatcgtg 6120 aagaagcgct acagcacgac acgctccgcc agcagccagg gcagctccag g 6171

Table 10 hMXl polypeptide sequence (SEQIDNO:10)

Phe Cys Leu Gin Gly Thr Arg Val Trp Leu Arg Glu Asn Gly Gin His 1 5 10 15

Phe Pro Ser Thr Val Asn Ser Cys Ala Glu Gly He Val Val Phe Arg 20 25 30

Thr Asp Tyr Gly Gin Val Phe Thr Tyr Lys Gin Ser Thr He Thr His

35 40 45

Gin Lys Val Thr Ala Met His Pro Thr Asn Glu Glu Gly Val Asp Asp 50 55 60

Met Ala Ser Leu Thr Glu Leu His Gly Gly Ser He Met Tyr Asn Leu 65 70 75 80

Phe Gin Arg Tyr Lys Arg Asn Gin He Trp Thr Tyr He Gly Ser He 85 90 95

Leu Ala Ser Val Asn Pro Tyr Gin Pro He Ala Gly Leu Tyr Glu Pro 100 105 110

Ala Thr Met Glu Gin Tyr Ser Arg Arg His Leu Gly Glu Leu Pro Pro 115 120 125 His He Phe Ala He Ala Asn Glu Cys Tyr Arg Cys Leu Trp Lys Arg 130 135 140

His Asp Asn Gin Cys He Leu He Lys Gly Glu Ser Gly Ala Gly Lys 145 150 155 160

Thr Glu Ser Thr Lys Leu He Leu Lys Phe Leu Ser Val He Ser Gin 165 170 175

Gin Ser Leu Glu Leu Ser Leu Lys Glu Lys Thr Ser Cys Val Glu Arg 180 185 190

Ala He Leu Glu Ser Ser Pro He Met Glu Ala Phe Gly Asn Ala Lys 195 200 205

Thr Val Tyr Asn Asn Asn Ser Ser Arg Phe Gly Lys Phe Val Gin Leu 210 215 220

Asn He Cys Gin Lys Gly Asn He Gin Gly Gly Arg He Val Asp Cys 225 230 235 240

He Leu Ser Ser Gin Asn Arg Val Val Arg Gin Asn Pro Gly Glu Arg

245 250 255

Asn Tyr His He Phe Tyr Ala Leu Leu Ala Gly Leu Glu His Glu Glu 260 265 270

Arg Glu Glu Phe Tyr Leu Ser Thr Pro Glu Asn Tyr His Tyr Leu Asn 275 280 285

Gin Ser Gly Cys Val Glu Asp Lys Thr He Ser Asp Gin Glu Ser Phe 290 295 300

Arg Glu Val He Thr Ala Met Asp Val Met Gin Phe Ser Lys Glu Glu 305 310 315 320

Val Arg Glu Val Ser Arg Leu Leu Ala Gly He Leu His Leu Gly Asn 325 330 335

He Glu Phe He Thr Ala Gly Gly Ala Gin Val Ser Phe Lys Thr Ala 340 345 350

Leu Gly Arg Ser Ala Glu Leu Leu Gly Leu Asp Pro Thr Gin Leu Thr 355 360 365

Asp Ala Leu Thr Gin Arg Ser Met Phe Leu Arg Gly Glu Glu He Leu 370 375 380

Thr Pro Leu Asn Val Gin Gin Ala Val Asp Ser Arg Asp Ser Leu Ala 385 390 395 400

Met Ala Leu Tyr Ala Cys Cys Phe Glu Trp Val He Lys Lys He Asn 405 410 415

Ser Arg He Lys Gly Asn Glu Asp Phe Lys Ser He Gly He Leu Asp 420 425 430

He Phe Gly Phe Glu Asn Phe Glu Val Asn His Phe Glu Gin Phe Asn 435 440 445

He Asn Tyr Ala Asn Glu Lys Leu Gin Glu Tyr Phe Asn Lys His He 450 455 460

Phe Ser Leu Glu Gin Leu Glu Tyr Ser Arg Glu Gly Leu Val Trp Glu 465 470 475 480

Asp He Asp Trp He Asp Asn Gly Glu Cys Leu Asp Leu He Glu Lys

485 490 495

Lys Leu Gly Leu Leu Ala Leu He Asn Glu Glu Ser His Phe Pro Gin 500 505 510

Ala Thr Asp Ser Thr Leu Leu Glu Lys Leu His Ser Gin His Ala Asn 515 520 525

Asn His Phe Tyr Val Lys Pro Arg Val Ala Val Asn Asn Phe Gly Val 530 535 540

Lys His Tyr Ala Gly Glu Val Gin Tyr Asp Val Arg Gly He Leu Glu 545 550 555 560

Lys Asn Arg Asp Thr Phe Arg Asp Asp Leu Leu Asn Leu Leu Arg Glu 565 570 575

Ser Arg Phe Asp Phe He Tyr Asp Leu Phe Glu His Val Ser Ser Arg 580 585 590

Asn Asn Gin Asp Thr Leu Lys Cys Gly Ser Lys His Arg Arg Pro Thr 595 600 605

Val Ser Ser Gin Phe Lys Val Asp Ser Leu His Ser Leu Met Ala Thr 610 615 620

Leu Ser Ser Ser Asn Pro Phe Phe Val Arg Cys He Lys Pro Asn Met 625 630 635 640

Gin Lys Met Pro Asp Gin Phe Asp Gin Ala Val Val Leu Asn Gin Leu 645 650 655

Arg Tyr Ser Gly Met Leu Glu Thr Val Arg He Arg Lys Ala Gly Tyr 660 665 670

Ala Val Arg Arg Pro Phe Gin Asp Phe Tyr Lys Arg Tyr Lys Val Leu 675 680 685

Met Arg Asn Leu Ala Leu Pro Glu Asp Val Arg Gly Lys Cys Thr Ser 690 695 700

Leu Leu Gin Leu Tyr Asp Ala Ser Asn Ser Glu Trp Gin Leu Gly Lys 705 710 715 720

Thr Lys Val Phe Leu Arg Glu Ser Leu Glu Gin Lys Leu Glu Lys Arg 725 730 735

Arg Glu Glu Glu Val Ser His Ala Ala Met Val He Arg Ala His Val 740 745 750 Leu Gly Phe Leu Ala Arg Lys Gin Tyr Arg Lys Val Leu Tyr Cys Val

755 760 765

Val He He Gin Lys Asn Tyr Arg Ala Phe Leu Leu Arg Arg Arg Phe

770 775 780

Leu His Leu Lys Lys Ala Ala He Val Phe Gin Lys Gin Leu Arg Gly

785 790 795 800

Gin He Ala Arg Arg Val Tyr Arg Gin Leu Leu Ala Glu Lys Arg Glu

805 810 815

Gin Glu Glu Lys Lys Lys Gin Glu Glu Glu Glu Lys Lys Lys Arg Glu 820 825 830

Glu Glu Glu Arg Glu Arg Glu Arg Glu Arg Arg Glu Ala Glu Leu Arg

835 840 845

Ala Gin Gin Glu Glu Glu Thr Arg Lys Gin Gin Glu Leu Glu Ala Leu 850 855 860

Gin Lys Ser Gin Lys Glu Ala Glu Leu Thr Arg Glu Leu Glu Lys Gin

865 870 875 880

Lys Glu Asn Lys Gin Val Glu Glu He Leu Arg Leu Glu Lys Glu He 885 890 895

Glu Asp Leu Gin Arg Met Lys Glu Gin Gin Glu Leu Ser Leu Thr Glu 900 905 910

Ala Ser Leu Gin Lys Leu Gin Glu Arg Arg Asp Gin Glu Leu Arg Arg

915 920 925

Leu Glu Glu Glu Ala Cys Arg Ala Ala Gin Glu Phe Leu Glu Ser Leu 930 935 940

Asn Phe Asp Glu He Asp Glu Cys Val Arg Asn He Glu Arg Ser Leu

945 950 955 960

Ser Gly Gly Ser Glu Phe Ser Ser Glu Leu Ala Glu Ser Ala Cys Glu

965 970 975

Glu Lys Pro Asn Phe Asn Phe Ser Gin Pro Tyr Pro Glu Glu Glu Val 980 985 990

Asp Glu Gly Phe Glu Ala Asp Asp Asp Ala Phe Lys Asp Ser Pro Asn

995 1000 1005

Pro Ser Glu His Gly His Ser Asp Gin Arg Thr Ser Gly He Arg

1010 1015 1020

Thr Ser Asp Asp Ser Ser Glu Glu Asp Pro Tyr Met Asn Asp Thr

1025 1030 1035

Val Val Pro Thr Ser Pro Ser Ala Asp Ser Thr Val Leu Leu Ala

1040 1045 1050 Pro Ser Val Gin Asp Ser Gly Ser Leu His Asn Ser Ser Ser Gly 1055 1060 1065

Glu Ser Thr Tyr Cys Met Pro Gin Asn Ala Gly Asp Leu Pro Ser 1070 1075 1080

Pro Asp Gly Asp Tyr Asp Tyr Asp Gin Asp Asp Tyr Glu Asp Gly 1085 1090 1095

Ala He Thr Ser Gly Ser Ser Val Thr Phe Ser Asn Ser Tyr Gly 1100 1105 1110

Ser Gin Trp Ser Pro Asp Tyr Arg Cys Ser Val Gly Thr Tyr Asn

1115 1120 1125

Ser Ser Gly Ala Tyr Arg Phe Ser Ser Glu Gly Ala Gin Ser Ser 1130 1135 1140

Phe Glu Asp Ser Glu Glu Asp Phe Asp Ser Arg Phe Asp Thr Asp 1145 1150 1155

Asp Glu Leu Ser Tyr Arg Arg Asp Ser Val Tyr Ser Cys Val Thr 1160 1Ϊ65 1170

Leu Pro Tyr Phe His Ser Phe Leu Tyr Met Lys Gly Gly Leu Met

1175 1180 1185

Asn Ser Trp Lys Arg Arg Trp Cys Val Leu Lys Asp Glu Thr Phe 1190 1195 1200

Leu Trp Phe Arg Ser Lys Gin Glu Ala Leu Lys Gin Gly Trp Leu 1205 1210 1215

His Lys Lys Gly Gly Gly Ser Ser Thr Leu Ser Arg Arg Asn Trp 1220 1225 1230

Lys Lys Arg Trp Phe Val Leu Arg Gin Ser Lys Leu Met Tyr Phe 1235 1240 1245

Glu Asn Asp Ser Glu Glu Lys Leu Lys Gly Thr Val Glu Val Arg 1250 1255 1260

Thr Ala Lys Glu He He Asp Asn Thr Thr Lys Glu Asn Gly He 1265 1270 1275

Asp He He Met Ala Asp Arg Thr Phe His Leu He Ala Glu Ser 1280 1285 1290

Pro Glu Asp Ala Ser Gin Trp Phe Ser Val Leu Ser Gin Val His 1295 1300 1305

Ala Ser Thr Asp Gin Glu He Gin Glu Met His Asp Glu Gin Ala 1310 1315 1320

Asn Pro Gin Asn Ala Val Gly Thr Leu Asp Val Gly Leu He Asp 1325 1330 1335

Ser Val Cys Ala Ser Asp Ser Pro Asp Arg Pro Asn Ser Phe Val 1340 1345 1350

He He Thr Ala Asn Arg Val Leu His Cys Asn Ala Asp Thr Pro 1355 1360 1365

Glu Glu Met His His Trp He Thr Leu Leu Gin Arg Ser Lys Gly 1370 1375 1380

Asp Thr Arg Val Glu Gly Gin Glu Phe He Val Arg Gly Trp Leu 1385 1390 1395

His Lys Glu Val Lys Asn Ser Pro Lys Met Ser Ser Leu Lys Leu 1400 1405 1410

Lys Lys Arg Trp Phe Val Leu Thr His Asn Ser Leu Asp Tyr Tyr 1415 1420 1425

Lys Ser Ser Glu Lys Asn Ala Leu Lys Leu Gly Thr Leu Val Leu 1430 1435 1440

Asn Ser Leu Cys Ser Val Val Pro Pro Asp Glu Lys He Phe Lys 1445 1450 1455

Glu Thr Gly Tyr Trp Asn Val Thr Val Tyr Gly Arg Lys His Cys 1460 1465 1470

Tyr Arg Leu Tyr Thr Lys Leu Leu Asn Glu Ala Thr Arg Trp Ser 1475 1480 1485

Ser Val He Gin Asn Val Thr Asp Thr Lys Ala Pro He Asp Thr 1490 1495 1500

Pro Thr Gin Gin Leu He Gin Asp He Lys Glu Asn Cys Leu Asn 1505 1510 1515

Ser Asp Val Val Glu Gin He Tyr Lys Arg Asn Pro He Leu Arg 1520 1525 1530

Tyr Thr His His Pro Leu His Ser Pro Leu Leu Pro Leu Pro Tyr 1535 1540 1545

Gly Asp He Asn Leu Asn Leu Leu Lys Asp Lys Gly Tyr Thr Thr 1550 1555 1560

Leu Gin Asp Glu Ala He Lys He Phe Asn Ser Leu Gin Gin Leu 1565 1570 1575

Glu Ser Met Ser Asp Pro He Pro He He Gin Gly He Leu Gin 1580 1585 1590

Thr Gly His Asp Leu Arg Pro Leu Arg Asp Glu Leu Tyr Cys Gin 1595 1600 1605

Leu He Lys Gin Thr Asn Lys Val Pro His Pro Gly Ser Val Gly 1610 1615 1620

Asn Leu Tyr Ser Trp Gin He Leu Thr Cys Leu Ser Cys Thr Phe 1625 1630 1635 Leu Pro Ser Arg Gly He Leu Lys Tyr Leu Lys Phe His Leu Lys 1640 1645 1650

Arg He Arg Glu Gin Phe Pro Gly Thr Glu Met Glu Lys Tyr Ala 1655 1660 1665

Leu Phe Thr Tyr Glu Ser Leu Lys Lys Thr Lys Cys Arg Glu Phe 1670 1675 1680

Val Pro Ser Arg Asp Glu He Glu Ala Leu He His Arg Gin Glu 1685 1690 1695

Met Thr Ser Thr Val Tyr Cys His Gly Gly Gly Ser Cys Lys He 1700 1705 1710

Thr He Asn Ser His Thr Thr Ala Gly Glu Val Val Glu Lys Leu 1715 1720 1725

He Arg Gly Leu Ala Met Glu Asp Ser Arg Asn Met Phe Ala Leu 1730 1735 1740

Phe Glu Tyr Asn Gly His Val Asp Lys Ala He Glu Ser Arg Thr 1745 1750 1755

Val Val Ala Asp Val Leu Ala Lys Phe Glu Lys Leu Ala Ala Thr 1760 1765 1770

Ser Glu Val Gly Asp Leu Pro Trp Lys Phe Tyr Phe Lys Leu Tyr 1775 1780 1785

Cys Phe Leu Asp Thr Asp Asn Val Pro Lys Asp Ser Val Glu Phe 1790 1795 1800

Ala Phe Met Phe Glu Gin Ala His Glu Ala Val He His Gly His 1805 1810 1815

His Pro Ala Pro Glu Glu Asn Leu Gin Val Leu Ala Ala Leu Arg 1820 1825 1830

Leu Gin Tyr Leu Gin Gly Asp Tyr Thr Leu His Ala Ala He Pro 1835 1840 1845

Pro Leu Glu Glu Val Tyr Ser Leu Gin Arg Leu Lys Ala Arg He 1850 1855 1860

Ser Gin Ser Thr Lys Thr Phe Thr Pro Cys Glu Arg Leu Glu Lys 1865 1870 1875

Arg Arg Thr Ser Phe Leu Glu Gly Thr Leu Arg Arg Ser Phe Arg 1880 1885 1890

Thr Gly Ser Val Val Arg Gin Lys Val Glu Glu Glu Gin Met Leu 1895 1900 1905

Asp Met Trp He Lys Glu Glu Val Ser Ser Ala Arg Ala Ser He 1910 1915 1920 He Asp Lys Trp Arg Lys Phe Gin Gly Met Asn Gin Glu Gin Ala 1925 1930 1935

Met Ala Lys Tyr Met Ala Leu He Lys Glu Trp Pro Gly Tyr Gly 1940 1945 1950

Ser Thr Leu Phe Asp Val Glu Cys Lys Glu Gly Gly Phe Pro Gin 1955 1960 1965

Glu Leu Trp Leu Gly Val Ser Ala Asp Ala Val Ser Val Tyr Lys 1970 1975 1980

Arg Gly Glu Gly Arg Pro Leu Glu Val Phe Gin Tyr Glu His He 1985 1990 1995

Leu Ser Phe Gly Ala Pro Leu Ala Asn Thr Tyr Lys He Val Val 2000 2005 2010

Asp Glu Arg Glu Leu Leu Phe Glu Thr Ser Glu Val Val Asp Val 2015 2020 2025

Ala Lys Leu Met Lys Ala Tyr He Ser Met He Val Lys Lys Arg 2030 2035 2040

Tyr Ser Thr Thr Arg Ser Ala Ser Ser Gin Gly Ser Ser Arg 2045 2050 2055

Table 11 hMX2 nucleotide sequence (SEQ ID NO:11) agctagtatc ttttattgtc agaacttctg tgagccaaca aacagttttg catggttgta 60 cacaaaggga caaggcaaat ttcttttttc gtgtgggtag acttagttgg cccaagtcct 120 taaaactttt ccatataaaa ataaaaagtc caagaccaga ttatttttct tctggtcata 180 aatgctgatt tatttacagg tgccttgttc agaccaccat tataaacttg ggataaaata 240 tgtgtgtatt aaagcctcag catttaatgt cagggtcctt tgaagattca ctcaagtgtt 300 aagacgtttc tggaatgcag cgtctctccc ccatagtcaa catggttatt atatctgtaa 360 tctatccaga atgatagaag ctaaccttcc aagtaacact ttgtttttaa cttaaatctt 420 ttagacatga aagactccaa aatgacttca ttcttgttct aaaaccagca ctggagccag 480 ctgttgaaga gtggtttata aatacagtta tcttgtaggc tgcttatctg tttataatac 540 agcagacaca gatggcagac tttgctacat gtaaaacaat ggagtcaaca cgtgtttttc 600 aaaatacagc aaagacagga aaatccagga tttgggtttg ttaataaaac caccttataa 660 agtaacaatt gagactatag ctctgcatta ttaaaatata cagactgtgt acaccattac 720 acatcctttt tccctttgct ttttaatgct catgaaacca tgattaaggt gttgagttta 780 tgaacacatg cacgaacagg caagcacgta cacttaaaag atgaaacaaa gaaaaaagtt 840 gattcatgtc attccatgag aaaggctgcc cgcagcactc cagctcaaac acactgtccc 900 ctcgagctct ccatccccct tcccactccc tcaccttccc tcagattcgg ggaaatcagg 960 ttgggaggtt agtgcatcat tgacagagaa tgcccccctt ccacgctctg ttaagtctcc 1020 cccagaaggg ggaaaggcag ttcccttcag tagcacagtt acggtcgatt agtgttggtt 1080 ccacaagtta aggcacttcc ggctgctttg gtggcagcgt ggttcctccc ctcctttttt 1140 aaggcatgtg tcctctaaga gtagtaaagc tttggaaact gtgcagactg ttaaagttga 1200 cagcttaata caggatcaat gaaggcggca ggcaaaagga tcctcggaga cacctccctc 1260 agaccagaag cttccagaaa gcctgggcag ctctgtgttt gttttggctg ggcatggcac 1320 actggagcca gcctaggcca gagggtggtg cgttcaggta gcaaagacag gtgggctctg 1380 tcccgccttc acctggagct gccctggctg ctggcggagc gtgtcgtgct gtagcgcttc 1440 ttcacgatca tgctgatgta ggctttcatg agcttggcca catccaccac ctcactggtt 1500 tcaaagagca gctccctctc atcgaccacg atcttatacg tattcgccag gggtgcccca 1560 aaagagagga tgtgttcata ctggaagact tccagtggtc ttccctctcc acgcttgtag 1620 acggagacgg cgtccgcgct gacacccaac cagagttcct gagggaagcc accttccttg 1680 cactggtggg caagagtcaa cagaagagtt aagtcatgaa gtggttggca acagaaagca 1740 tctaaaccat aagacaggct ttgagtgaag tcctctgtgc agaagattaa atatattcga 1800 tgtgcatgca tgcatggagg ggcctgaaat atgaaaaatg gcacctctct ggctatcttg 1860 atttctaact agttaatctc acgcttttgg gaaaacctca ctaactggca gagtctaaca 1920 tcttgctttg actctccact tctcagcatt attctactag ctgtttggat tagctacgtg 1980 gaagtggcct ggaaacgtac atgcttggcc gggggactta agaaagcttc cctgcaaccc 2040 aagccaagtc tactcttgta ttaatatctc cagttctgcc tccaatcctc tttgcggatg 2100 gttagtcttc aaatacaaaa tctaggatca cagaggaaaa ttctccaaat cacgcatgct 2160 gagcagttct ggctcctctt cacaaggagc agcaatggcc ttccatatgc agagtgggaa 2220 cagggacttt accagtttaa ctgtagactt tcctgtacag attggtggaa gaaaataaga 2280 ccccatatga aggggctaac aacacagggc tgatccaaac ctggacaagc aggagggcta 2340 taaattggag acgctgaaaa gagtctctag tttatatccc taataaccag acattctctg 2400 catcctccat gcaaaagcca gtagctttct ttttttcttt ttttttttga cggagtctca 2460 ctctgtcgcc caggctggag tgcagtggcg tgatcctggc tcactgcaac ctccacctcc 2520 tgagttcaag cgattctcct gtctcagcct ctcgagtagc tgggattaca ggtgcatgcc 2580 accacgccca gctaattttt tgcagagatg gggtttcacc gtgttagcca ggatggtctc 2640 gatctcctga cctcatgatc cgcccgcctt ggcctcccaa agcgctggga ttacaggcat 2700 gagccaccgc gcccggccaa gccagtagct ttctatgcta attcacagct cacgttttgc 2760 aggaagccaa gagtttaact gctattatct attccttgtc agggagaaat ggaattatgg 2820 ctttgtacaa agcacctgat ttttttatac ttaaaaacag gcataattga accaaccaaa 2880 ccaatcaaaa acatcaccta atgaaaagcc acccacggat tctagaattt ataatattta 2940 gaattttata cagcctcaat ataaagtcat cagatatacg ctgaattact gtgatcataa 3000 aaaatggaag ctaatctaga cgatgagctg gcacacttat ctgttaaggg ctgcatagta 3060 aagattttta gactttgtgg atcacatggt ctctgtcaca actactcaac tctgtacaaa 3120 aacagccagg gaatatatct aaaggaatga gcttggctgt gttccaataa aactttgttt 3180 agaaaaaaag gaggcaggca agatctgacc cacagaccag tttgccaaac tctcatctag 3240 acaattagta agatttcttt tcaataagcg gtctacttaa aacaaaacaa aaatcagtac 3300 tgggttgatg ccaatggcta aattccatta cgagatagac attcttcctt tcaaacagat 3360 ggctgtaaag aaaaaacaaa gtaaaatgca agtatatcca aagtttctaa tttgtatata 3420 cagctataac atttttt.taa atgtagattt ttatcagtgt ttaaaaaatt agatctatag 3480 cttccctaag gaagggtaga agaatagatg acatcttaat tttgcattca ttcctaatat 3540 tacagatgca tttactacac aggagaagag aaactgtgag gagaagggag gcgttaatgg 3600 tacaattttg ggggctcgaa aaaaagaggt tgagagagca aaatgctcca tcttgtcttc 3660 tctccacatg aacttggccg tgatccatgt tctcagatgc cagcacccag cccaccccaa 3720 cacatggcag ccagttctca cctccacatc aaacagcgtc gagccatagc caggccactc 3780 cttgatcaag gccatgtact tggccatggc ctgttcctgg ttcattccct gaaatttcct 3840 ccacttgtca atgatactgg ctcgagcaga ggagacttct tccttaatcc acatgtccag 3900 catctgctcc tcctcgacct tctgccggac cacggatcct gtccggaagc tccgcctcag 3960 ggtcccctct aggaagctcg tccgcctctt ctccagccgt tcacaagggg tgaaggtttt 4020 ggttgactgg ctgatgcggg ccttgagtct ctgcagggaa taaacctctt cgagaggtgg 4080 gatggcagcg tgcagagtat aatccccctg cagatactgg agtcgcaggg cagcaagaac 4140 ctggaggttt tcttccgggg ctggatggtg gccatggata accgcttcgt gggcctgttc 4200 aaacataaat gcaaactcca cactgtcttt tggcacgttg tctgtgtcca ggaagcagta 4260 aagtttgaag tagaatttcc atggcaggtc cccaacctcg gatgtggcag ccagcttttc 4320 aaacttggct aagacatcag ctacgacggt tcgactttca atggctttgt cgacgtggcc 4380 gttgtattca aacaaagcaa acatgttcct gctgtcctcc atggccaggc ctcggatcag 4440 cttctccacc acctccccag cggtggtgtg ggagttgatg gtgatcttgc aggagccgcc 4500 gccatggcaa tagaccgtgg atgtcatttc ctgcctgtgg atcagagctt ctatttcatc 4560 ■ tcgggaaggc acaaactctc ggcatttggt tttcttaaga gattcgtaag tgaagagagc 4620 gtatttttcc atctcggttc ctggaaactg ttcccgtatc cttttcagat ggaacttgag 4680 atacttgaga atccctcgac tcggcaggaa ggtgcagctc aggcatgtca ggatctgcca 4740 gctgtacagg ttgcccacac tgccggggtg gggcactttg ttggtctgtt tgataagctg 4800 gcagtacagc tcgtcccgca gaggtcgcag gtcatgccct gtctgtagga tgccctggat 4860 tattggaatt gggtcagaca tggactccag ttgctgcagg gaattgaata tcttgatggc 4920 ctcatcctga agggtggtat agcctttgtc tttcagcaag ttgagattta tgtccccata 4980 cggaaggggc aggagcgggg agtgcaaggg gtgatgggtg tatcgaagga tcgggttccg 5040 cttgtaaatc tgttccacca catccgagtt caggcagttc tccttgatat cttgaatcag 5100 ctgctgggtg ggggtgtcga tcggggcctt ggtgtcagtc acgttttgaa tgacactgga 5160 ccaccgggtg gcctcgttga gcagcttggt gtagagccgg taacagtgct tgcgcccgta 5220 cacggtgacg ttccagtagc ctgtctcttt gaatatcttc tcatctgggg ggacgacaga 5280 gcagaggctg ttgaggacca gggtccccag tttgagcgcg ttcttctctg aactcttgta 5340 gtaatccagg gaattgtggg tgagtacaaa ccaccgtttc ttcagtttca gtgaagacat 5400 ctttggactg ttcttcacct ctttgtgcaa ccatcctctc acgatgaatt cctggccctc 5460 cactctggtg tcccctttgg acctctgcag cagggttatc cagtggtgca tctcctccgg 5520 cgtgtcggcg ttgcagtgca gcacccggtt ggccgtgatg atcacaaacg agttgggtct 5580 atcagggctg tcagaggcac acacagaatc aatcagcccc acatccaagg tgcccacagc 5640 attctgtggg tttgcctgct catcatgcat ctcctggatc tcctggtccg tggacgcgtg 5700 gacctgactc agcacgctga accactggct ggcatcttct ggggactctg caatcaggtg 5760 gaaagtccta tcggccataa tgatgtcgat cccattctcc ttggtggtgt tatctatgat 5820 ctcttttgcc gttcgcactt ctacggtgcc cttgagcttc tcctcgctgt cgttttcaaa 5880 gtacatcagc ttggactggc ggaggacaaa ccagcgcttc ttccaatttc tcctggacag 5940 cgtggaggag cccccccctt ttttgtggag ccagccttgc ttgagggcct cctgcttgga 6000 gcggaaccac aagaaggttt catccttgag gacgcaccag cggcgtttcc aagagttcat 6060 caggccacct ttcatgtaca gaaagctgtg gaaatacggc agagtgacac agctgtacac 6120 agagtcacgc cggtatgaaa gctcatcatc tgtatcaaac ctggaatcaa agtcctcttc 6180 actatcttca aacgaggact gcgccccctc agagctgaac cggtaggcac ccgagctgtt 6240 gtaggtcccc acagagcagc ggtagtcggg ggaccactgg ctgccgtagg agttggagaa 6300 ggtcacgctg ctgccggaag tgatggcacc gtcctcatag tcatcctggt cgtagtcgta 6360 gtcgccgtct ggggagggca agtccccagc gttctggggc atgcagtagg tggactcgcc 6420 gctggaggag ttgtgtaggc tcccggagtc ctgcactgat ggggcgagca gcaccgtgct 6480 gtccgcactg gggctggtgg gcaccaccgt gtcgttcatg tatgggtcct cctctgaaga 6540 gtcatcgctg gtccggatgc cacttgttcg ctggtctgag tggccgtgct cgctggggtt 6600 gggggagtcc ttgaaggcgt cgtcgtcggc ttcgaagccc tcatcgacct cctcctctgg 6660 gtagggctgg ctgaagttga agttgggctt ctcctcgcat gcgctctcag ccagctcgct 6720 ggaaaattcg cttccccccg acagggaccg ctcgatattc cggacacact cgtcgatctc 6780 gtcgaaattg agggactcga ggaactcctg ggccgccctg cacgcttcct cctccagcct 6840 gcggagctcc tggtcccgcc gctcctgcag cttctgcagg gaagcctcgg tcagcgacag 6900 ctcctgctgc tccttcatgc gctgcaggtc ctcgatttct ttctccagac ggaggatctc 6960 ttccacctgc ttattttcct tctgtttctc cagttcacgg gtcagttcag cttccttctg 7020 gctcttctgc aaggcttcga gttcttgctg cttcctcgtt tcttcttcct gctgggcgcg 7080 gagctcggct tctcttcgct ctctctctct ttctctttct tcttcctccc gtttcttctt 7140 ttcttcctct tcctgtttct tcttttcttc ttgctccctt ttctctgcca gcaattgtct 7200 gtaaactctc cgagcaatct gacctctgag ttgcttctgg aaaactatgg ctgccttttt 7260 caggtgcaaa aatctcctcc tcagaaggaa tgctctgtaa ttcttctgta ttatcaccac 7320 acaataaagg acctttctgt attgtttccg tgctaagaag cccaagacat gggcccgaat 7380 caccatggcc gcgtggctca cttcctcttc cctccgcttc tccagtttct gttccaagga 7440 ttctcgaaga aataccttgg tcttccccag ctgccactcg ctgttggagg catcatagag 7500 ctgcagcagg ctcgtgcact tccctcggac gtcctcaggc agagccagat tcctcatcag 7560 cactttatac cttttgtaaa agtcctgaaa gggtcttcgg accgcatacc cagctttgcg 7620 gattctcaca gtctccagca tccctgagta ccgcagctgg ttcagcacaa ccgcctggtc 7680 aaactggtct ggcatcttct gcatgtttgg cttgatacag cgaacaaaga aaggattaga 7740 ggagcttagc gttgccatta aggaatgcag tgagtcaacc ttgaactgtg agctgactgt 7800 aggccgccga tgtttgcttc cacatttcaa ggtatcctgg ttgttgcggc ttgaaacatg 7860 ttcaaaaaga tcgtagataa agtcaaaccg gctttctctt agcaaattga gaaggtcatc 7920 tcgaaatgta tctctgttct tctccaagat acctcggaca tcatattgca cctctccagc 7980 atagtgcttc actccaaaat tgttaactgc aactctgggc ttcacataaa agtggttatt 8040 cgcatgctga ctgtgtagct tctccaataa ggtgctgtct gtggcttgag gaaaatggct 8100 ttcttcattg ataagggcta ggaggccaag tttcttctca atcaagtcca ggcattctcc 8160 attgtctatc cagtcaatat cttcccacac taatccttcc ctgctatatt ctagttgttc 8220 taaagaaaaa atatgcttgt tgaagtactc ctgaagtttc tcgtttgcat agtttatatt 8280 gaactgttca aagtgattaa cctcaaagtt ttcaaatcca aagatgtcga ggatgccaat 8340 agacttgaag tcctcattgc ctttgatcct gctgttgatc ttcttgatta cccactcaaa 8400 gcagcacgca tacagagcca tggccaggga gtccctgctg tctactgcct gttgaacatt 8460 gagaggcgtg aggatctctt ctcccctgag gaacattgat ctctgggtca aagcatctgt 8520 gagctgtgtt gggtccagcc caagtaactc cgcagatctg cccaaagctg ttttgaagga 8580 aacctgtgcc ccaccagcag tgataaattc tatgttccca agatgcagta taccagcaag 8640 cagcctcgac acttcccgaa cttcctcctt gctgaactgc atcacgtcca ttgccgtaat 8700 aacttcccta aaggattcct ggtcactgat tgtcttgtct tctacacatc cagactgatt 8760 caagtagtgg tagttttctg gcgtagataa ataaaattct tctctttctt catgttccag 8820 ccctgccagc agtgcataaa atatgtgata attcctttcc ccgggatttt gccttactac 8880 tcggttctgg gaagagagga tacaatctac aattctcccg ccctgaatat ttcctttctg 8940 acagatgttc agctgaacaa acttcccaaa gcgactagag ttgttgttgt acacggtctt 9000 cgcattgccg aaagcttcca tgatggggct gctttcaaga atagctcgtt caacacagga 9060 tgtcttctcc tttaaggaca attccaaaga ctgttgactg atgactgaca gaaacttgag 9120 gatcaattta gtgctttcgg ttttacctgc cccactttca cccttgatga ggatgcactg 9180 gttgtcgtgg cgcttccaca ggcagcggta gcactcgttg gcgatggcga agatgtgcgg 9240 gggcagctcg cccaggtggc gccggctgta ctgctccatg gtggcaggct cgtacagccc 9300 ggcgatgggc tggtaggggt tcacagaggc caggatggag ccgatgtagg tccatatttg 9360 atttctctta taccgctgga ataagttata catgatggag ccgccatgga gctctgtcaa 9420 ggacgccatg tcatccacgc cctcctcgtt cgtggggtgc atagcagtca ccttctggtg 9480 ggtaattgtg ctctgcttgt aagtgaatac ctgaccatag tctgtccgga agacgacgat 9540 gccttctgca caggaattta cagtacttgg aaaatgctgg ccattttctc tcagccagac 9600 ccgtgttccc tgtaaacaaa a 9621

Table 12 hMX2 polypeptide sequence (SEQ ID NO: 12)

Phe Cys Leu Gin Gly Thr Arg Val Trp Leu Arg Glu Asn Gly Gin His

1 5 10 15

Phe Pro Ser Thr Val Asn Ser Cys Ala Glu Gly He Val Val Phe Arg 20 25 30

Thr Asp Tyr Gly Gin Val Phe Thr Tyr Lys Gin Ser Thr He Thr His 35 40 45

Gin Lys Val Thr Ala Met His Pro Thr Asn Glu Glu Gly Val Asp Asp 50 55 60 Met Ala Ser Leu Thr Glu Leu His Gly Gly Ser He Met Tyr Asn Leu 65 70 75 80

Phe Gin Arg Tyr Lys Arg Asn Gin He Trp Thr Tyr He Gly Ser He 85 90 95

Leu Ala Ser Val Asn Pro Tyr Gin Pro He Ala Gly Leu Tyr Glu Pro 100 105 110

Ala Thr Met Glu Gin Tyr Ser Arg Arg His Leu Gly Glu Leu Pro Pro 115 120 125

His He Phe Ala He Ala Asn Glu Cys Tyr Arg Cys Leu Trp Lys Arg 130 135 140

His Asp Asn Gin Cys He Leu He Lys Gly Glu Ser Gly Ala Gly Lys 145 150 155 160

Thr Glu Ser Thr Lys Leu He Leu Lys Phe Leu Ser Val He Ser Gin 165 170 175

Gin Ser Leu Glu Leu Ser Leu Lys Glu Lys Thr Ser Cys Val Glu Arg 180 185 190

Ala He Leu Glu Ser Ser Pro He Met Glu Ala Phe Gly Asn Ala Lys 195 200 205

Thr Val Tyr Asn Asn Asn Ser Ser Arg Phe Gly Lys Phe Val Gin Leu 210 215 220

Asn He Cys Gin Lys Gly Asn He Gin Gly Gly Arg He Val Asp Cys 225 230 235 240

He Leu Ser Ser Gin Asn Arg Val Val Arg Gin Asn Pro Gly Glu Arg

245 250 255

Asn Tyr His He Phe Tyr Ala Leu Leu Ala Gly Leu Glu His Glu Glu 260 265 270

Arg Glu Glu Phe Tyr Leu Ser Thr Pro Glu Asn Tyr His Tyr Leu Asn 275 280 285

Gin Ser Gly Cys Val Glu Asp Lys Thr He Ser Asp Gin Glu Ser Phe 290 295 300

Arg Glu Val He Thr Ala Met Asp Val Met Gin Phe Ser Lys Glu Glu 305 310 315 320

Val Arg Glu Val Ser Arg Leu Leu Ala Gly He Leu His Leu Gly Asn 325 330 335

He Glu Phe He Thr Ala Gly Gly Ala Gin Val Ser Phe Lys Thr Ala 340 345 350

Leu Gly Arg Ser Ala Glu Leu Leu Gly Leu Asp Pro Thr Gin Leu Thr 355 360 365 Asp Ala Leu Thr Gin Arg Ser Met Phe Leu Arg Gly Glu Glu He Leu 370 375 380

Thr Pro Leu Asn Val Gin Gin Ala Val Asp Ser Arg Asp Ser Leu Ala 385 390 395 400

Met Ala Leu Tyr Ala Cys Cys Phe Glu Trp Val He Lys Lys He Asn 405 410 415

Ser Arg He Lys Gly Asn Glu Asp Phe Lys Ser He Gly He Leu Asp 420 425 430

He Phe Gly Phe Glu Asn Phe Glu Val Asn His Phe Glu Gin Phe Asn 435 440 445

He Asn Tyr Ala Asn Glu Lys Leu Gin Glu Tyr Phe Asn Lys His He 450 455 460

Phe Ser Leu Glu Gin Leu Glu Tyr Ser Arg Glu Gly Leu Val Trp Glu 465 470 475 480

Asp He Asp Trp He Asp Asn Gly Glu Cys Leu Asp Leu He Glu Lys 485 490 495

Lys Leu Gly Leu Leu Ala Leu He Asn Glu Glu Ser His Phe Pro Gin 500 505 510

Ala Thr Asp Ser Thr Leu Leu Glu Lys Leu His Ser Gin His Ala Asn 515 520 525

Asn His Phe Tyr Val Lys Pro Arg Val Ala Val Asn Asn Phe Gly Val 530 535 540

Lys His Tyr Ala Gly Glu Val Gin Tyr Asp Val Arg Gly He Leu Glu 545 550 555 560

Lys Asn Arg Asp Thr Phe Arg Asp Asp Leu Leu Asn Leu Leu Arg Glu 565 570 575

Ser Arg Phe Asp Phe He Tyr Asp Leu Phe Glu His Val Ser Ser Arg 580 585 590

Asn Asn Gin Asp Thr Leu Lys Cys Gly Ser Lys His Arg Arg Pro Thr 595 600 605

Val Ser Ser Gin Phe Lys Val Asp Ser Leu His Ser Leu Met Ala Thr 610 615 620

Leu Ser Ser Ser Asn Pro Phe Phe Val Arg Cys He Lys Pro Asn Met 625 630 635 640

Gin Lys Met Pro Asp Gin Phe Asp Gin Ala Val Val Leu Asn Gin Leu 645 650 655

Arg Tyr Ser Gly Met Leu Glu Thr Val Arg He Arg Lys Ala Gly Tyr 660 665 670

Ala Val Arg Arg Pro Phe Gin Asp Phe Tyr Lys Arg Tyr Lys Val Leu 675 680 685

Met Arg Asn Leu Ala Leu Pro Glu Asp Val Arg Gly Lys Cys Thr Ser 690 695 700

Leu Leu Gin Leu Tyr Asp Ala Ser Asn Ser Glu Trp Gin Leu Gly Lys 705 710 715 720

Thr Lys Val Phe Leu Arg Glu Ser Leu Glu Gin Lys Leu Glu Lys Arg 725 730 735

Arg Glu Glu Glu Val Ser His Ala Ala Met Val He Arg Ala His Val 740 745 750

Leu Gly Phe Leu Ala Arg Lys Gin Tyr Arg Lys Val Leu Tyr Cys Val 755 760 765

Val He He Gin Lys Asn Tyr Arg Ala Phe Leu Leu Arg Arg Arg Phe 770 775 780

Leu His Leu Lys Lys Ala Ala He Val Phe Gin Lys Gin Leu Arg Gly 785 790 795 800

Gin He Ala Arg Arg Val Tyr Arg Gin Leu Leu Ala Glu Lys Arg Glu 805 810 815

Gin Glu Glu Lys Lys Lys Gin Glu Glu Glu Glu Lys Lys Lys Arg Glu 820 825 830

Glu Glu Glu Arg Glu Arg Glu Arg Glύ Arg Arg Glu Ala Glu Leu Arg 835 840 845

Ala Gin Gin Glu Glu Glu Thr Arg Lys Gin Gin Glu Leu Glu Ala Leu 850 855 860

Gin Lys Ser Gin Lys Glu Ala Glu Leu Thr Arg Glu Leu Glu Lys Gin 865 870 875 880

Lys Glu Asn Lys Gin Val Glu Glu He Leu Arg Leu Glu Lys Glu He 885 890 895

Glu Asp Leu Gin Arg Met Lys Glu Gin Gin Glu Leu Ser Leu Thr Glu 900 905 910

Ala Ser Leu Gin Lys Leu Gin Glu Arg Arg Asp Gin Glu Leu Arg Arg 915 920 925

Leu Glu Glu Glu Ala Cys Arg Ala Ala Gin Glu Phe Leu Glu Ser Leu 930 935 940

Asn Phe Asp Glu He Asp Glu Cys Val Arg Asn He Glu Arg Ser Leu 945 950 955 960

Ser Gly Gly Ser Glu Phe Ser Ser Glu Leu Ala Glu Ser Ala Cys Glu 965 970 975

Glu Lys Pro Asn Phe Asn Phe Ser Gin Pro Tyr Pro Glu Glu Glu Val 980 985 990 Asp Glu Gly Phe Glu Ala Asp Asp Asp Ala Phe Lys Asp Ser Pro Asn 995 1000 1005

Pro Ser Glu His Gly His Ser Asp Gin Arg Thr Ser Gly He Arg 1010 1015 1020

Thr Ser Asp Asp Ser Ser Glu Glu Asp Pro Tyr Met Asn Asp Thr 1025 1030 1035

Val Val Pro Thr Ser Pro Ser Ala Asp Ser Thr Val Leu Leu Ala 1040 1045 1050

Pro Ser Val Gin Asp Ser Gly Ser Leu His Asn Ser Ser Ser Gly 1055 1060 1065

Glu Ser Thr Tyr Cys Met Pro Gin Asn Ala Gly Asp Leu Pro Ser 1070 1075 1080

Pro Asp Gly Asp Tyr Asp Tyr Asp Gin Asp Asp Tyr Glu Asp Gly 1085 1090 1095

Ala He Thr Ser Gly Ser Ser Val Thr Phe Ser Asn Ser Tyr Gly 1100 1105 1110

Ser Gin Trp Ser Pro Asp Tyr Arg Cys Ser Val Gly Thr Tyr Asn 1115 1120 1125

Ser Ser Gly Ala Tyr Arg Phe Ser Ser Glu Gly Ala Gin Ser Ser 1130 1135 1140

Phe Glu Asp Ser Glu Glu Asp Phe Asp Ser Arg Phe Asp Thr Asp 1145 1150 1155

Asp Glu Leu Ser Tyr Arg Arg Asp Ser Val Tyr Ser Cys Val Thr 1160 1165 1170

Leu Pro Tyr Phe His Ser Phe Leu Tyr Met Lys Gly Gly Leu Met 1175 1180 1185

Asn Ser Trp Lys Arg Arg Trp Cys Val Leu Lys Asp Glu Thr Phe 1190 1195 1200

Leu Trp Phe Arg Ser Lys Gin Glu Ala Leu Lys Gin Gly Trp Leu 1205 1210 1215

His Lys Lys Gly Gly Gly Ser Ser Thr Leu Ser Arg Arg Asn Trp 1220 1225 1230

Lys Lys Arg Trp Phe Val Leu Arg Gin Ser Lys Leu Met Tyr Phe 1235 1240 1245

Glu Asn Asp Ser Glu Glu Lys Leu Lys Gly Thr Val Glu Val Arg 1250 1255 1260

Thr Ala Lys Glu He He Asp Asn Thr Thr Lys Glu Asn Gly He 1265 1270 1275 Asp He He Met Ala Asp Arg Thr Phe His Leu He Ala Glu Ser 1280 1285 1290

Pro Glu Asp Ala Ser Gin Trp Phe Ser Val Leu Ser Gin Val His 1295 1300 1305

Ala Ser Thr Asp Gin Glu He Gin Glu Met His Asp Glu Gin Ala 1310 1315 1320

Asn Pro Gin Asn Ala Val Gly Thr Leu Asp Val Gly Leu He Asp 1325 1330 1335

Ser Val Cys Ala Ser Asp Ser Pro Asp Arg Pro Asn Ser Phe Val 1340 1345 1350

He He Thr Ala Asn Arg Val Leu His Cys Asn Ala Asp Thr Pro 1355 1360 1365

Glu Glu Met His His Trp He Thr Leu Leu Gin Arg Ser Lys Gly 1370 1375 1380

Asp Thr Arg Val Glu Gly Gin Glu Phe He Val Arg Gly Trp Leu 1385 1390 1395

His Lys Glu Val Lys Asn Ser Pro Lys Met Ser Ser Leu Lys Leu 1400 1405 1410

Lys Lys Arg Trp Phe Val Leu Thr His Asn Ser Leu Asp Tyr Tyr 1415 1420 1425

Lys Ser Ser Glu Lys Asn Ala Leu Lys Leu Gly Thr Leu Val Leu 1430 1435 1440

Asn Ser Leu Cys Ser Val Val Pro Pro Asp Glu Lys He Phe Lys 1445 1450 1455

Glu Thr Gly Tyr Trp Asn Val Thr Val Tyr Gly Arg Lys His Cys 1460 1465 1470

Tyr Arg Leu Tyr Thr Lys Leu Leu Asn Glu Ala Thr Arg Trp Ser 1475 1480 1485

Ser Val He Gin Asn Val Thr Asp Thr Lys Ala Pro He Asp Thr 1490 1495 1500

Pro Thr Gin Gin Leu He Gin Asp He Lys Glu Asn Cys Leu Asn 1505 1510 1515

Ser Asp Val Val Glu Gin He Tyr Lys Arg Asn Pro He Leu Arg 1520 1525 1530

Tyr Thr His His Pro Leu His Ser Pro Leu Leu Pro Leu Pro Tyr 1535 1540 1545

Gly Asp He Asn Leu Asn Leu Leu Lys Asp Lys Gly Tyr Thr Thr 1550 1555 1560

Leu Gin Asp Glu Ala He Lys He Phe Asn Ser Leu Gin Gin Leu 1565 1570 1575

Glu Ser Met Ser Asp Pro He Pro He He Gin Gly He Leu Gin 1580 1585 1590

Thr Gly His Asp Leu Arg Pro Leu Arg Asp Glu Leu Tyr Cys Gin 1595 1600 1605

Leu He Lys Gin Thr Asn Lys Val Pro His Pro Gly Ser Val Gly 1610 1615 1620

Asn Leu Tyr Ser Trp Gin He Leu Thr Cys Leu Ser Cys Thr Phe 1625 1630 1635

Leu Pro Ser Arg Gly He Leu Lys Tyr Leu Lys Phe His Leu Lys 1640 1645 1650

Arg He Arg Glu Gin Phe Pro Gly Thr Glu Met Glu Lys Tyr Ala 1655 1660 1665

Leu Phe Thr Tyr Glu Ser Leu Lys Lys Thr Lys Cys Arg Glu Phe 1670 1675 1680

Val Pro Ser Arg Asp Glu He Glu Ala Leu He His Arg Gin Glu 1685 1690 1695

Met Thr Ser Thr Val Tyr Cys His Gly Gly Gly Ser Cys Lys He 1700 1705 1710

Thr He Asn Ser His Thr Thr Ala Gly Glu Val Val Glu Lys Leu 1715 1720 1725

He Arg Gly Leu Ala Met Glu Asp Ser Arg Asn Met Phe Ala Leu 1730 1735 1740

Phe Glu Tyr Asn Gly His Val Asp Lys Ala He Glu Ser Arg Thr 1745 1750 1755

Val Val Ala Asp Val Leu Ala Lys Phe Glu Lys Leu Ala Ala Thr 1760 1765 1770

Ser Glu Val Gly Asp Leu Pro Trp Lys Phe Tyr Phe Lys Leu Tyr 1775 1780 1785

Cys Phe Leu Asp Thr Asp Asn Val Pro Lys Asp Ser Val Glu Phe 1790 1795 1800

Ala Phe Met Phe Glu Gin Ala His Glu Ala Val He His Gly His 1805 1810 1815

His Pro Ala Pro Glu Glu Asn Leu Gin Val Leu Ala Ala Leu Arg 1820 1825 1830

Leu Gin Tyr Leu Gin Gly Asp Tyr Thr Leu His Ala Ala He Pro 1835 1840 1845

Pro Leu Glu Glu Val Tyr Ser Leu Gin Arg Leu Lys Ala Arg He 1850 1855 1860 Ser Gin Ser Thr Lys Thr Phe Thr Pro Cys Glu Arg Leu Glu Lys 1865 1870 1875

Arg Arg Thr Ser Phe Leu Glu Gly Thr Leu Arg Arg Ser Phe Arg 1880 1885 1890

Thr Gly Ser Val Val Arg Gin Lys Val 'Glu Glu Glu Gin Met Leu 1895 1900 1905

Asp Met Trp He Lys Glu Glu Val Ser Ser Ala Arg Ala Ser He 1910 1915 1920

He Asp Lys Trp Arg Lys Phe Gin Gly Met Asn Gin Glu Gin Ala 1925 1930 1935

Met Ala Lys Tyr Met Ala Leu He Lys Glu Trp Pro Gly Tyr Gly 1940 1945 1950

Ser Thr Leu Phe Asp Val Glu Val Arg Thr Gly Cys His Val Leu 1955 1960 1965

Gly Trp Ala Gly Cys Trp His Leu Arg Thr Trp He Thr Ala Lys 1970 1975 1980

Phe Met Trp Arg Glu Asp Lys Met Glu His Phe Ala Leu Ser Thr 1985 1990 1995

Ser Phe Phe Arg Ala Pro Lys He Val Pro Leu Thr Pro Pro Phe 2000 2005 2010

Ser Ser Gin Phe Leu Phe Ser Cys Val Val Asn Ala Ser Val He 2015 2020 2025

Leu Gly Met Asn Ala Lys Leu Arg Cys His Leu Phe Phe Tyr Pro 2030 2035 2040

Ser Leu Gly Lys Leu 2045

Table 13 hMP nucleotide sequence (SEQ ID NO:13) ccaacttttg cagctccacc caggatgtgg cctcgctcca ccccagctgt gcgcctctct 60 ccacccctag gcgaaggcac tagaatttcc caaattaaga acgaagagga agtttggacc 120 ttttcggcca ccgctcgctt caatatggct gcccccaggg agagacgagg ctaccatgaa 180 ggagccgagc gcagaccctg agtccgtcac ccatggatcg cagcgcggag ttcaggaaat 240 ggaaggcgca atgtttgagc aaagcggacc tcagccggaa gggcagtgtt gacgaggatg 300 tggtagagct tgtgcagttt ctgaacatgc gagatcagtt tttcaccacc agctccttcg 360 ctggccgcat cctactcctt gaccggggta taaatggttt tgaggttcag aaacaaaact 420 gttgctggct actggttaca cacaaacttt gtgtaaaaga tgatgtgatt gtagctctga 480 agaaagcaaa tggtgatgcc actttgaaat ttgaaccatt tgttcttcat gtgcagtgtc 540 gacaattgca ggatgcacag attctgcatt ccatggcaat agattctggt ttcaggaact 600 ctggcataac ggtgggaaag agaggaaaaa ctatgttggc tgtccggagt acacatggct 660 tagaagttcc attaagccat aagggaaaac tgatggtgac agaggaatat attgacttcc 720 tgttaaatgt ggcaaatcaa aaaatggagg aaaacaagaa aagaattgag aggttttaca 780 actgcctaca gcatgctttg gaaagggaaa cgatgactaa cttacatccc aagatcaaag 840 agaaaaataa ctcatcatat attcataaga aaaaaagaaa cccagaaaaa acacgtgccc 900 agtgtattac taaagaaagt gatgaagaac ttgaaaatga tgatgatgat gatctaggaa 960 tcaatgttac catcttccct gaagattact aagctttggt tctgatgtgt cttggccgta 1020 atgtttctag taggttttat aaagctgctc ttcataagag tattttagtt tgttgagtgt 1080 atcagccatt cataagccag taatgacaag tgcagagctt caaactataa ctttgttgcc 1140 cagaggatgt gcagttgtca tctaagctct cagcagtacc cggcttatcc tacgacttca 1200 cctgaaatgc tatagttatc cctacttttt taccagtttc tcccagaagc acctgcttaa 1260 taaatcaaag atgtttgaaa aaaaaaaa 1288

Table 14 hMP polypeptide sequence (SEQ ID NO:14)

Met Asp Arg Ser Ala Glu Phe Arg Lys Trp Lys Ala Gin Cys Leu Ser 1 5 10 15

Lys Ala Asp Leu Ser Arg Lys Gly Ser Val Asp Glu Asp Val Val Glu 20 25 30

Leu Val Gin Phe Leu Asn Met Arg Asp Gin Phe Phe Thr Thr Ser Ser 35 40 45

Phe Ala Gly Arg He Leu Leu Leu Asp Arg Gly He Asn Gly Phe Glu 50 55 60

Val Gin Lys Gin Asn Cys Cys Trp Leu Leu Val Thr His Lys Leu Cys 65 70 75 80

Val Lys Asp Asp Val He Val Ala Leu Lys Lys Ala Asn Gly Asp Ala 85 90 95

Thr Leu Lys Phe Glu Pro Phe Val Leu His Val Gin Cys Arg Gin Leu 100 105 110

Gin Asp Ala Gin He Leu His Ser Met'Ala He Asp Ser Gly Phe Arg 115 120 125

Asn Ser Gly He Thr Val Gly Lys Arg Gly Lys Thr Met Leu Ala Val 130 135 140

Arg Ser Thr His Gly Leu Glu Val Pro Leu Ser His Lys Gly Lys Leu 145 150 155 160

Met Val Thr Glu Glu Tyr He Asp Phe Leu Leu Asn Val Ala Asn Gin 165 170 175

Lys Met Glu Glu Asn Lys Lys Arg He Glu Arg Phe Tyr Asn Cys Leu 180 185 190 Gin His Ala Leu Glu Arg Glu Thr Met Thr Asn Leu His Pro Lys He 195 200 205

Lys Glu Lys Asn Asn Ser Ser Tyr He His Lys Lys Lys Arg Asn Pro 210 215 220

Glu Lys Thr Arg Ala Gin Cys He Thr Lys Glu Ser Asp Glu Glu Leu 225 230 235 240

Glu Asn Asp Asp Asp Asp Asp Leu Gly He Asn Val Thr He Phe Pro 245 250 255

Glu Asp Tyr

Table 15 NHRnucleotide sequence (SEQ ID NO:15) acgcgtgcag gtggcgtggc gccagggatt tgaaccgcgc tgacgaagtt tggtgatcca 60 tcttccgagt atcgccggga tttcgaatcg cgatgatcat cccctctcta gaggagctgg 120 actccctcaa gtacagtgac ctgcagaact tagccaagag tctgggtctc cgggccaacc 180 tgagggcaac caagttgtta aaagccttga aaggctacat taaacatgag gcaagaaaag 240 gaaatgagaa tcaggatgaa agtcaaactt ctgcatcctc ttgtgatgag actgagatac 300 agatcagcaa ccaggaagag ctgagagaca gccacttggc catgtcacca aaacaaggag 360 aaggtgcaag actgtccgtg tggaccctga ctcacagaga atcattcaga gataaaaata 420 agtaatccca ctgaattcca gaatcatgaa aagcaggaaa gccaggatct cagagcactg 480 caaaagttcc ttctccacca gacgagcacc aagaagctga gaatgctgtt tcctcaggta 540 acagagattc aaaggtacct tcagaaggaa agaaatctct ctacacagat gagtcatcca 600 aacctggaaa aaataaaaga actgcaatca ctactccaaa ctttaagaag cttcatgaag 660 ctcattttaa ggaaatggag tccattgatc caatatatng aggagaaaaa aagaaacatt 720 ttgaagaaca caattccatg aatgaactga agcagccgcc catcaataag ggaggggtca 780 ggactccagt acctccaaga ggaagactct ctgtggcttc tactcccatc agccaacgac 840 gctcgcaagg ccggtcttgt ggccctgcaa gtcagagtac cttgggtctg aaggggtcac 900 tcaagcgctc tgctatctct gcagctaaaa cgggtgtcag gttttcagct gctactaaag 960 ataatgagca taagcgttca ctgaccaaga ctccagccag aaagtctgca catgtgaccg 1020 tgtctggggg cacccaaaaa ggcgaggctg tgcttgggac acacaaatta aagaccatca 1080 cggggaattc tgctgctgtt attaccccat tcaagttgac aactgaggca acgcagactc 1140 cagtctccaa taagaaacca gtgtttgatc ttaaagcaag tttgtctcgt cccctcaact 1200 atgaaccaca caaaggaaag ctaaaaccat gggggcaatc taaagaaaat aattatctaa 1260 atcaacatgt caacaaatta acttctacaa gaaaacttac aaacaacccc atctccagac 1320 aaaggaagag caacggaaga aacgcgagca agaagaaagg agaagaaagc aaaggttttg 1380 ggaatgcgaa ggggcctcat tttggctgaa gattaataat tttttaacat cttgtaaata 1440 ttcctgtatt ctcaactttt ttccttttgt aaattttttt tttttgctgt catccccact 1500 ttagtcacga gatctttttc tgctaactgt tcatagtctg tgtagtgtcc atgggttctt 1560 catgtgctat gatctctgaa aagacgttat caccttaaag ctcaaattct ttgggatggt 1620 ttttacttaa gtccattaac aattcaggtt tctaacgaga cccatcctaa aattctcttt 1680 ctagtttttt aatgtcacca tcccaaactc ccgtttctgg atttttaatc cccagctccc 1740 cagttccctc ttatcgtact aatattaaca gaactgcagt cttctgctag ccaatagcat 1800 ttacctgatg gcagctagtt atgcaagctt caggagaatt tgaacaataa caagaatagg 1860 gtaagctggg atagaaaggc cacctcttca ctctctatag aatatagtaa cctttatgaa 1920 acggggccat atagtttggt tatgacatca atattttacc taggtgaaat tgtttaggct 1980 tatgtacctt cgttcaaata tcctcatgta attgccatct gtcactcact atattcacaa 2040 aaataaaact ctacaactca ttctaacatt gcttacttaa aagctacata gccctatcga 2100 aatgcgagga ttaatgcttt aatgctttta gagacagggt ctcactgtgt tgcccaggct 2160 ggtctcaaac tccaccaaat gtacttctta ttcattttat ggaaaagact aggctttgct 2220 tagtatcatg tccatgtttc cttcacctca gtggagcttc tgagttttat actgctcaag 2280 atcgtcataa ataaaatttt ttctcattgt caaaaaaaaa aaaaaaaaaa aaaaaaaaaa 2340 aaaaaaaaaa aa 2352

Table 16 NHRpolypeptide sequence (SEQ ID NO:16)

Met He He Pro Ser Leu Glu Glu Leu Asp Ser Leu Lys Tyr Ser Asp 1 5 10 15

Leu Gin Asn Leu Ala Lys Ser Leu Gly Leu Arg Ala Asn Leu Arg Ala 20 25 30

Thr Lys Leu Leu Lys Ala Leu Lys Gly Tyr He Lys His Glu Ala Arg 35 40 45

Lys Gly Asn Glu Asn Gin Asp Glu Ser Gin Thr Ser Ala Ser Ser Cys 50 55 60

Asp Glu Thr Glu He Gin He Ser Asn Gin Glu Glu Ala Glu Arg Gin 65 70 75 80

Pro Leu Gly His Val Thr Lys Thr Arg Arg Arg Cys Lys Thr Val Arg 85 90 95

Val Asp Pro Asp Ser Gin Gin Asn His Ser Glu He Lys He Ser Asn 100 105 110

Pro Thr Glu Phe Gin Asn His Glu Lys Gin Glu Ser Gin Asp Leu Arg 115 120 125

Ala Thr Ala Lys Val Pro Ser Pro Pro Asp Glu His Gin Glu Ala Glu 130 135 140

Asn Ala Val Ser Ser Gly Asn Arg Asp Ser Lys Val Pro Ser Glu Gly 145 150 155 160

Lys Lys Ser Leu Tyr Thr Asp Glu Ser Ser Lys Pro Gly Lys Asn Lys 165 170 175

Arg Thr Ala He Thr Thr Pro Asn Phe Lys Lys Leu His Glu Ala His 180 185 190 Phe Lys Glu Met Glu Ser He Asp Pro He Tyr Xaa Gly Glu Lys Lys 195 200 205

Lys His Phe Glu Glu His Asn Ser Met Asn Glu Leu Lys Gin Pro Pro 210 215 220

He Asn Lys Gly Gly Val Arg Thr Pro Val Pro Pro Arg Gly Arg Leu 225 230 235 240

Ser Val Ala Ser Thr Pro He Ser Gin Arg Arg Ser Gin Gly Arg Ser 245 250 255

Cys Gly Pro Ala Ser Gin Ser Thr Leu Gly Leu Lys Gly Ser Leu Lys 260 265 270

Arg Ser Ala He Ser Ala Ala Lys Thr Gly Val Arg Phe Ser Ala Ala 275 280 285

Thr Lys Asp Asn Glu His Lys Arg Ser Leu Thr Lys Thr Pro Ala Arg 290 295 300

Lys Ser Ala His Val Thr Val Ser Gly Gly Thr Gin Lys Gly Glu Ala 305 310 315 320

Val Leu Gly Thr His Lys Leu Lys Thr He Thr Gly Asn Ser Ala Ala 325 330 335

Val He Thr Pro Phe Lys Leu Thr Thr Glu Ala Thr Gin Thr Pro Val 340 ^'345 350

Ser Asn Lys Lys Pro Val Phe Asp Leu Lys Ala Ser Leu Ser Arg Pro 355 360 365

Leu Asn Tyr Glu Pro His Lys Gly Lys Leu Lys Pro Trp Gly Gin Ser 370 375 380

Lys Glu Asn Asn Tyr Leu Asn Gin His Val Asn Arg He Asn Phe Tyr 385 390 395 400

Lys Lys Thr Tyr Lys Gin Pro His Leu Gin Thr Lys Glu Glu Gin Arg 405 410 415

Lys Lys Arg Glu Gin Glu Arg Lys Glu Lys Lys Ala Lys Val Leu Gly 420 425 430

Met Arg Arg Gly Leu He Leu Ala Glu Asp 435 440

Table 17 displays alignment of hMXl, hMX2 with human myosin (SEQ ID NO:31; GenBank AF247457) (Berg et al, 2000). As seen from the alignment, hMXl and hMX2 have a likely N-terminus of M N D residues. One of skill in the art can easily verify this observation by probing cDNA or genomic human libraries, or PCR techniques, to acquire the full length polynucleotide sequence.

Table 17 Alignment ofhMXl, hMX2 andhuman myosinX

10 1 FCLQGTRV LRENGQHFPSTVNSCAEGIWFRTDYGQVFTYKQSTIT

12 ^■ 1 ^■ FCLQGTRVWLRENGQHFPSTVNSCAEGIWFRTDYGQVFTYKQSTIT humX 1 MDNFFTEGTRVWLRENGQHFPSTVNSCAEGIWFRTDYGQVFTYKQSTIT

10 48 HQKVTAMHPTNEEGVDDMASLTELHGGSIMYNLFQRYKRNQI TYIGSIL

12 48 HQKVTAMHPTNEEGVDDMASLTELHGGSIMYNLFQRYKR QIWTYIGSIL humX 51 HQKVTAMHPTNEEGVDDMASLTELHGGSIMYNLFQRYKRNQIYTYIGSIL

10 98 ASVNPYQPIAGLYEPATMEQYSRRHLGELPPHIFAIANECYRCL KRHDN

12 98 ASV PYQPIAGLYEPATMEQYSRRHLGELPPHIFAIANECYRCL KRHDN humX 101 ASVNPYQPIAGLYEPATMEQYSRRHLGELPPHIFAIANECYRCL KRYDN

10 148 QCILIKGESGAGKTESTKLILKFLSVISQQSLELSLKEKTSCVERAILES

12 148 QCILI GESGAGKTESTKLILKFLSVISQQSLELSLKEKTSCVERAILES humX 151 QCILISGESGAGKTESTKLILKFLSVISQQSLELSLKEKTSCVERAILES

10 198 SPIMEAFGNAKTVYNNSSRFGKFVQLNICQKGNIQGGRIVDCILSSQNR

12 198 SPIMEAFGNAKTVYNNNSSRFG FVQLNICQKGNIQGGRIVDCILSSQ R humX 201 SPIMEAFGNAKTVYNNSSRFGKFVQLNICQKGNIQGGRIVDYLLE-KNR

10 248 WRQNPGERNYHIFYALLAGLEHEEREEFYLSTPE YHYLNQSGCVEDKT 12 248 WRQNPGERNYHIFYALLAGLEHEEREEFYLSTPENYHYLNQSGCVEDKT humX 250 WRQNPGERNYHIFYALLAGLEHEEREEFYLSTPENYHYLNQSGCVEDKT

10 298 ISDQESFREVITAMDVMQFSKEEVREVSRLLAGILHLGNIEFITAGGAQV

12 298 ISDQESFREVITAMDVMQFSKEEVREVSRLLAGILHLGNIEFITAGGAQV humX 300 ISDQESFREVITAMDVMQFSKEEVREVSRLLAGILHLGNIEFITAGGAQV

10 348 SFKTALGRSAELLGLDPTQLTDALTQRSMFLRGEEILTPLNVQQAVDSRD

12 348 SFKTALGRSAELLGLDPTQLTDALTQRSMFLRGEEILTPLNVQQAVDSRD humX 350 SFKTALGRSAELLGLDPTQLTDALTQRSMFLRGEEILTPL VQQAVDSRD

10 398 SLAMALYACCFE VIKKINSRIKGNEDFKSIGILDIFGFENFEVNHFEQF

12 398 SLAMALYACCFE VIKKINSRIKGNEDFKSIGILDIFGFENFEVNHFEQF humX 400 SLAMALYACCFE VIKKINSRIKGNEDFKSIGILDIFGFENFEV HFEQF

10 448 NI YANEKLQEYFNKHIFSLEQLEYSREGLV EDID IDNGECLDLIEKK

12 448 NIKTYANEKLQEYFW HIFSLEQLEYSREGLV EDID IDNGECLDLIEKK humX 450 NI YA EKLQEYFNKHIFSLEQLEYSREGLVWEDID IDNGΞCLDLIEKK

10 498 LGLLALINEESHFPQATDSTLLEKLHSQHANNHFYVKPRVAV NFGVKHY

12 498 LGLLALINEESHFPQATDSTLLE LHSQHANNHFYVKPRVAV NFGVKHY humX 500 LGLLALINEESHFPQATDSTLLEKϋHSQHANHFYV PRVAVNFGVKHY

10 548 AGEVQYDVRGILEK RDTFRDDLLNLLRESRFDFIYDLFEHVSSRN QDT

12 548 AGEVQYDVRGILEKNRDTFRDDLLNLLRESRFDFIYDLFEHVSSRNNQDT humX 550 AGEVQYDVRGILEKNRDTFRDDLLNLLRESRFDFIYDLFEHVSSR NQDT

10 598 LKCGSKHRRPTVSSQFKVDSLHΞLMATLSSSNPFFVRCIKPNMQKMPDQF

12 598 LKCGSiHRRPTVSSQFKVDSLHSLMATLSSSNPFFVRCIKP MQKMPDQF humX 600 LKCGSKHRRPTVSSQFKDS-LHSLMATLSSSNPFFVRCIKPNMQ MPDQF 10 648 DQAWLNQLRYSGMLETVRIRKAGYAVRRPFQDFYKRYKVLMRNLALPED

12 648 DQAWLNQLRYSGMLETVRIRKAGYAVRRPFQDFYKRYKVLMRNLALPED humX 649 DQAWLNQLRYSGMLETVRIRKAGYAVRRPFQDFYKRY VLMRNLALPED

10 698 VRGKCTSLLQLYDASNSE QLGKTKVFLRESLEQKLE RREEEVSHAAMV

12 698 VRGKCTSLLQLYDASNSE QLGKTKVFLRESLEQ LEKRREEEVSHAAMV humX 699 VRGKCTSLLQLYDASNSE QLGKTKVFLRESLEQKLEKRREEEVSHAAMV

10 748 IRAHVLGFLARKQYRKVLYCWIIQK YRAFLLRRRFLHLKKAAIVFQKQ

12 748 IRAHVLGFLARKQYRKVLYCWIIQK YRAFLLRRRFLHLKKAAIVFQKQ humX 749 IRAHVLGFLARKQYRKVLYCVVIIQKNYRAFLLRRRFLHLKKAAIVFQKQ

10 798 LRGQIARRVYRQLLAEiREQEEKKKQEEEEKKKREEEERERERERREAEL

12 798 LRGQIARRVYRQLLAEKREQEEKKKQEEEEKKKREEEERERERΞRREAEL humX 799 LRGQIARRVYRQLLAEKREQEEKKKQEEEEKPOCREEEERERERERREAEL

10 848 RAQQEEETRKQQELEALQ SQKEAELTRELEKQKENKQVEEILRLEKEIE

12 848 RAQQEEETRKQQELEALQ SQKΞAELTRELEKQKENKQVEEILRLEKEIE humX 849 RAQQEEETRKQQELEALQKSQKEAELTRELEKQKENKQVΞEILRLEKEIE

10 898 DLQRMKEQQELSLTEASLQKLQERRDQELRRLEEEACRAAQEFLESLNFD

12 898 DLQRMKEQQELSLTEASLQKLQERRDQELRRLEEEACRAAQEFLESLNFD humX 899 DLQRMKEQQELSLTEASLQKLQERRDQELRRLEEEACRAAQEFLESLNFD

10 948 EIDECVRNIERSLSGGSEFSSELAESACEE PNFNFSQPYPEEEVDEGFE

12 948 EIDECVRNIERSLSGGSEFSSELAESACEEKPNFNFSQPYPEEΞVDEGFE humX 949 EIDECVR IERSLSVGSEFSSELAESACEEKPNFNFSQPYPEEEVDEGFE

10 998 ADDDAFKDSPNPSEHGHSDQRTSGIRTSDDSSEEDPYMNDTWPTSPSAD

12 998 ADDDAFKDSPNPSEHGHSDQRTSGIRTSDDSSEEDPYMNDTWPTSPSAD humX 999 ADDDAF DSPNPSEHGHSDQRTSGIRTSDDSSEEDPYMNDTWPTSPSAD

10 1048 STVLLAPSVQDSGSLHNSSSGESTYCMPQNAGDLPSPDGDYDYDQDDYED

12 1048 STVLLAPSVQDSGSLH SSSGESTYCMPQNAGDLPSPDGDYDYDQDDYED humX 1049 STVLLAPSVQDSGSLHNSSSGESTYCMPQNAGDLPSPDGDYDYDQDDYED

10 1098 GAITSGSSVTFSNSYGSQWSPDYRCSVGTYNSSGAYRFSSEGAQSSFEDS

12 1098 GAITSGSSVTFSNSYGSQWSPDYRCSVGTYNSSGAYRFSSEGAQSSFEDS humX 1099 GAITSGSSVTFSNSYGSQWSPDYRCSVGTYNSSGAYRFSSEGAQSSFEDS

10 1148 EEDFDSRFDTDDELSYRRDSVYSCVTLPYFHSFLYMKGGLMMSWKRR CV

12 1148 EEDFDSRFDTDDELSYRRDSVYSCVTLPYFHSFLYMKGGLMNS KRR CV humX 1149 EEDFDSRFDTDDELSYRRDSVYSCVTLPYFHSFLYMKGGLMNS KRRWCV

10 1198 LKDETFL FRSKQEALKQG LHKKGGGSSTLSRR WKKRWFVLRQSKLMY

12 1198 LKDETFL FRSKQEALKQG LHKKGGGSSTLSRRN KKRWFVLRQSKLMY humX 1199 LKDETFLWFRSKQEALKQG LHKKGGGSSTLSRRNWKKR FVLRQSKLMY

10 1248 FENDSEEKLKGTVEVRTAKEIIDNTTKENGIDIIMADRTFHLIAESPEDA

12 1248 FENDSEEKLKGTVEVRTAKEIIDNTTKENGIDIIMADRTFHLIAESPEDA humX 1249 FENDSEEKLKGTVEVRTAKEIIDNTTKENGIDIIMADRTFHLIAESPEDA

10 1298 SQWFSVLSQVHASTDQEIQEMHDEQA PQNAVGTLDVGLIDSVCASDSPD

12 1298 SQ FSVLSQVHASTDQEIQEMHDEQANPQNAVGTLDVGLIDSVCASDSPD humX 1299 SQ FSVLSQVHASTDQEIQEMHDEQANPQNAVGTLDVGLIDSVCASDSPD

10 1348 RPNSFVIITANRVLHCNADTPEEMHH ITLLQRSKGDTRVEGQEFIVRGW 12 1348 RPNSFVIITANRVLHCNADTPEEMHHWITLLQRSKGDTRVEGQEFIVRG humX 1349 RPNSFVIITANRVLHCNADTPEEMHHWITLLQRSKGDTRVEGQEFIVRGW

10 1398 LHKEVKNSPKMSSL LKKR FVLTHNSLDYY SSΞKNALKLGTLVLNSLC

12 1398 LH EVKNSPi SSLKLKKR FVLTH SLDYYKSSEKNALKLGTLVLNSLC humX 1399 LHKEVKNSPKMSSLKLKKR FVLTH SLDYYKSSEKNALKLGTLVLNSLC

10 1448 SWPPDEKIFKETGYW VTVYGRKHCYRLYTKLLNEATR SSVIQ VTDT

12 1448 SWPPDEKIFKETGYWNVTVYGRKHCYRLYTKLLNEATRWSSVIQNVTDT humX 1449 SWPPDΞKIFKETGY NVTVYGRKHCYRLYTKLLNEATR SSAlQNVTDT

10 1498 KAPIDTPTQQLIQDIKENCLNSDWEQIYKRNPILRYTHHPLHSPLLPLP

12 1498 KAPIDTPTQQLIQDIKENCLNSDWEQIYKRNPILRYTHHPLHSPLLPLP humX 1499 KAPIDTPTQQLIQDIKENCLNSDWEQIYKRNPILRYTHHPLHSPLLPLP

10 1548 YGDINLNLLKDKGYTTLQDEAIKIFNSLQQLESMSDPIPIIQGILQTGHD

12 1548 YGDINLNLLKDKGYTTLQDEAIKIFNSLQQLESMSDPIPIIQGILQTGHD humX 1549 YGDINLNLLKDKGYTTLQDEAIKIFNSLQQLESMSDPIPIIQGILQTGHD

10 1598 LRPLRDEώYCQLIKQTNKVPHPGSVGNLYS QILTCLSCTFLPSRGILKY

12 1598 LRPLRDELYCQLIKQTNKVPHPGSVGNLYSWQILTCLSCTFLPSRGILKY humX 1599 LRPLRDELYCQLI QTNKVPHPGSVGNLYS QILTCLSCTFLPSRGILKY

10 1648 LKFHLKRIREQFPGTEMEKYALFTYESLKKTKCREFVPSRDEIEALIHRQ

12 1648 LKFHLKRIREQFPGTEME YALFTYESLKKTKCREFVPSRDEIEALIHRQ humX 1649 LKFHLKRIREQFPGTEMEKYALFTYESL KTKCREFVPSRDEIEALIHRQ

10 1698 EMTSTVYCHGGGSCKITINSHTTAGEWEKLIRGLAMEDSRNMFALFEYN ^'

12 1698 EMTSTVYCHGGGSCKITINSHTTAGEWEKLIRGLAMEDSRNMFALFEYN humX 1699 EMTSTVYCHGGGSCKITINSHTTAGEVVEKLIRGLAMEDSRNMFALFEYN

10 1748 GHVDKAIESRTVVADVLAKFEKLAATSEVGDLP KFYFKLYCFLDTDNVP

12 1748 GHVDKAIESRTVVADVLAKFEKLAATSEVGDLP KFYFKLYCFLDTDNVP humX 1749 GHVDKAIEΞRTWADVLAKFEKLAATSEVGDLPWKFYF LYCFLDTDNVP

10 1798 KDSVEFAFMFEQAHEAVIHGHHPAPEENLQVLAALRLQYLQGDYTLHAAI

12 1798 KDSVEFAFMFEQAHEAVIHGHHPAPEENLQVLAALRLQYLQGDYTLHAAI humX 1799 KDSVEFAFMFEQAHEAVIHGHHPAPEENLQVLAALRLQYLQGDYTLHAAI

10 1848 PPLEEVYSLQRLKARISQSTKTFTPCERLEKRTSFLEGTLRRSFRTGSV

12 1848 PPLEEVYSLQRLKARISQSTKTFTPCERLEKRRTSFLEGTLRRSFRTGSV humX 1849 PPLEEVYSLQRLKARISQSTKTFTPCERLEKRRTSFLEGTLRRSFRTGSV

10 1898 VRQKVEEEQMLDMWIKEEVSSARASIIDKWRKFQGMNQEQAMAKYMALIK

12 1898 VRQKVEEEQMLDM IKEEVSSARASIIDKWRKFQGMNQEQAMAKYMALIK humX 1899 VRQKVEEEQMLDM IKEEVSSARASIIDK RKFQGMNQEQAMAKYMALIK

10 1948 E PGYGSTLFDVECKEGGFPQEL LGVSADAVSVYKRGEGRPLEVFQYEH

12 1948 E PGYGSTLFDVEVRTG-CHVLG AGC HLRTWITAKFM REDKMEHFAL humX 1949 E PGYGSTLFDVECKEGGFPQEL LGVSADAVSVYKRGEGRPLEVFQYEH

10 1998 ILSFGAPLANTYKIWDERELLFETSEWDVAKLMKAYISMIVKKRYSTT

12 1997 STSFFRAPKIVPLTPPFSSQFLFSCWNASVILGMNAKLRCHLFFYPSLG humX 1999 ILSFGAPLANTYKIWDERELLFETSEWDVAKLMKAYISMIVKKRYSTT

10 2048 RSASSQGSSR

12 2047 KL humX 2049 RSASSQGSSR

The invention also includes polypeptides and nucleo tides having 80-100%, including 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98 and 99%, sequence identity to SEQ ID NOS:l-16, as well as nucleotides encoding any of these polypeptides, and compliments of any of these nucleotides. In an alternative embodiment, polypeptides and/or nucleotides (and compliments thereof) identical to any one of, or more than one of, SEQ ID NOS: 1-16 are excluded. In yet another embodiment, polypeptides and/or nucleotides (and compliments thereof) having 81-100% identical, including 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 91, 98 and 99%, sequence identity to SEQ ID NOS: 1-16 are excluded.

The nucleic acids and proteins of the invention are potentially useful in promoting wound healing, for example after organ transplantation, or in the treatment of myocardial infarction, but also in treating tumors, and in cancers, diabetic retinopathy, macular degeneration, psoriasis, and rheumatoid arthritis. For example, a cDNA encoding AAP may be useful in gene therapy, and AAP proteins may be useful when administered to a subject in need thereof. The novel nucleic acid encoding AAP, and the AAP proteins of the invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed. These materials are further useful in the generation of Abs that bind immunospecifically to the novel substances of the invention for use in therapeutic or diagnostic methods.

Kelch-like protein (KLP)

The putative protein encoded by KLP contains 1 putative BTB domain and 4 putative Kelch motifs. The BTB (broad complex, tramtrack, bric-a-brac)/POZ (poxvirus, zinc finger) domain is involved in protein protein interactions. The kelch motif is sixfold tandem element in the sequence of the Drosophila kelch ORF1 protein that also contains BTB. Kelch ORF1 localizes to the ring canals in the egg chamber and helps to organize the F-actin cytoskeleton (Adams et al, 2000). The repeated kelch motifs predict a conserved tertiary structure, a β-propeller. This module appears in many different polypeptide contexts and contains multiple potential protein-protein interaction sites. Members of this growing superfamily are present throughout the cell and extracellularly and have diverse activities (Adams et al, 2000). Such activities include cytoskeleton organization, as well as other morphological processes, gene expression, interactions with viruses, and various extracellular events, such as cell spreading.

Alignment with Drosophila kelch and other kelch-like proteins, human kelch-like protein (GenBank AAF20938 (SEQ ID NO:17)), hypothetical C. elegans (GenBank 061795 (SEQ ID NO:18) and the skeletal muscle-specific sarcosin (GenBank O60662 (SEQ ID NO: 19); (Taylor et al, 1998)) reveals that the disclosed protein (SEQ ID NO:2) is a member of a new subfamily

KLP is associated with tube formation and angiogenesis because it is upregulated in the in vitro model of angiogenesis of Example 1. Kelch mediates cytoskeletal associations, it is involved in morphogenetic processes, such as tube formation, that depend on cytoskeletal arrangements and signaling. KLP represents an attractive target for small molecule drug therapy.

Human ortholog of mouse BAZF (hBAZF) hBAZF (SEQ ID NO:4) is the human ortholog of mouse BAZF (GenBank ABOl 1665; SEQ ID NO:20), BAZF is a Bcl-6 (LAZ3) homolog, a transcription repressor that controls germinal center formation and the T cell-dependent immune response. Expression of Bcl-6 negatively correlates with cellular proliferation: Bcl-6 suppresses growth associated with impaired mitotic S phase progression and apoptosis (Albagli et al, 1999).

BAZF contains a BTB/POZ domain and five repeats of the Kruppel-like zinc finger motifs, instead of 6 in Bcl-6 (Okabe et al, 1998). Expression of BAZF mRNA is relegated to heart and lung, unlike Bcl-6 mRNA, but is induced in activated lymphocytes as an immediate-early gene, like Bcl-6 (Okabe et al, 1998).

The hBAZF sequence was derived by using tblastn (protein query -translated database) (Altschul et al, 1997), with the mouse protein sequence (GenBank 088282; SEQ ID NO:21) that has homology to GenBank AC015918 (SEQ ID NO:22), a clone of Homo sapiens chromosome 17. Human BAZF contains five Kruppel-like zinc finger motif repeats and a BTB/POZ domain.

The peptide sequence, "RSQ...PQV" that is present in the human sequence, might represent an alternative spliced form of the gene. Augment with mouse BAZF, and aligment with mouse and human Bcl-6 demonstrates that the four proteins are almost identical in this region, but only human BAZF has this inserted sequence. hBAZF is upregulated in HUVE cells grown embedded in collagen gels but not as a monolayer grown on collagen. When HUVE cells are suspended in collagen, they do not proliferate. Analagous to the role of mBAZF plays a role in regulating cell proliferation (Okabe et al, 1998), hBAZF plays a roll in cell proliferation in HUVE suspended in collagen. Because of its high expression during vessel morphogenesis, hBAZF represents an excellent molecular marker, as well as an attractive target for various therapies to inhibit angiogenesis.

hmt-Elongation Factor G (hEF-G)

The original isolation of hEF-G (SEQ ID NO: 6) is 84% identical and colinear with Rattus norvegicus nuclear encoded mitochondrial elongation factor G (GenBank LI 4684 (SEQ ID NO:23); (Barker et al, 1993). No human gene is described in GenBank. However, searching EST databases, the human gene is contained inside GenBank AC010936 (SEQ ID NO:24), a chromosome 3 clone. Augment of hEF-G with rat mtEF-G and yeast EF-G1 demonstrates that the novel sequence is the ortholog of rat nuclear-encoded mitochondrial elongation factor G.

Bacterial elongation factor G (EF-G) physically associates with translocation- competent ribosomes and facilitates transition to the subsequent codon through the coordinate binding and hydrolysis of GTP. The deduced amino acid sequence of hmt-EF- G reveals characteristic motifs shared by all GTP binding proteins. Therefore, similarly to other elongation factors, the enzymatic function of hmt-EF-G is predicted to depend on GTP binding and hydrolysis.

Hmt-EF-G is strongly induced (30-fold) in an in vitro model of angiogenesis (Example 1), and as such, hmt-EF-G represents an excellent molecular marker for vessel formation. Because of its putative localization to the mitochondrion, hmt-EF-G is also an attractive therapeutic target to treat disease states associated with mitochondrial dysfunction.

Human thyroid regulated transcript (hTRG) hTRG (SEQ ID NO:8) is the human ortholog of rat TRG, a novel thyroid transcript negatively regulated by TSH (GenBank KIAA1058 (SEQ ID NO:25); (Bonapace et al, 1990).

SEQ ID NO:25 appears to be a partial peptide since there are C. elegans homologous proteins of 2000 residues. Using tblastn (Altschul et al, 1997) against genomic sequences, the hTRG sequence (SEQ ID NO:8) was assembled.

In C. elegans, homologous proteins localize either to the plasma membrane or to the mitochondrial inner membrane. A partial sequence, KIAA0694 (SEQ ID NO:26) also localizes to the mitochondrial matrix. hTRG has a PH domain, and has weak homology to an extracellular fibronectin-binding protein precursor. SEQ ID NO:26 has homology to Drosophila DOS and mouse Gab-2 proteins; both of which are involved in signal transduction, acting as adapter proteins between receptors and kinases like Rasl (Hibi and Hirano, 2000).

Because of hTRG is upregulated during the in vitro model of angiogenesis (Example 1), and because of its homologies with adapter proteins, hTRG is likely to be involved in signal transduction between receptors and kinases. As such, hTRG represent an excellent candidate for small molecule drug therapy to modulate angiogenesis and treat angiogenesis-related diseases. In addition, because of its putative ability to respond to thyroid stimulating hormone (TSH), modulation of hTRG is useful to treat diseases related to TSH imbalance.

Human myosin X (hMXl (SEQ ID NO: 10) and hMX2 (SEQ ID NO:12) The hMX proteins represent the human ortholog of bovine myosin X, (GenBank AAB39486; SEQ ID NO:27). Using tblastn (Altschul et al, 1997) and the bovine sequence, a series of genomic clones from human chromosome 5 were identified; GenBank AC010310 (SEQ ID NO:28) appears to contain the entire sequence. Interestingly, a partial cDNA sequence from mouse (GenBank AF184153; SEQ ID NO:29) localizes to a 0.8 cM interval on the short arm of chromosome 5, between the polymorphic microsatellite markers D5S416 and D5S2114. In this region lies the gene for familial chondrocalcinosis (CCAL2) (Rojas et al, 1999).

Another GenBank entry, AB018342 (SEQ ID NO:30) that represents the 3' region of hMX, appears to encode an alternative splice form. Noteworthy, this variant (hMX2) has a very hydrophobic carboxy terminus, while the more prevalent form (hMXl) is hydrophilic and potentially interacts with DNA/RNA since it has homology to high mobility group box (HMG) and ribosomal proteins. Additionaly, a myosin head domain was found in the NH terminus, as well as a myosin talin domain, two calmodulin binding domains, four pleckstrin domains and a band 4.1 domain.

The band 4.1 domain represents a crossroads between cytoskeletal organization and signal transduction. The domain was first described in the red blood cell protein band 4.1. The ERM proteins ezrin, radixin, and moesin and the unconventional myosins Vila and X all possess the band 4.1 domain (Louvet-Vallee, 2000). The band 4.1 domain binds single transmembrane protein at the membrane-proximal region in the C-terminal cytoplasmic tail.

HMX is upregulated during angiogenesis in an in vitro model (Example 1). Because hMX contains the protein-protein interaction domains PH and band 4.1 domain, hMXl and hMX2 are involved in angiogenesis, likely transducing signals from angiogenic factors, perhaps modulating the cytoskeleton.

Human mitochondrial protein (hMP)

Analysis of hMP (SEQ ID NO: 14) reveals several subdomain that are homologous to proteins involved in transport across membranes, K⁺ATPase α and γ chains. Further analysis indicates that hMP may bind DNA and or RNA, since hMP is homologous to histones and transcription factors, especially those possessing basic region plus leucine zipper domains.

Although PSORT analysis (Nakai and Horton, 1999) predicts nuclear localization (P=.6), hMP may in fact be a nuclear-encoded mitochondrial protein. Homologies with mostly bacterial proteins and a PSORT prediction ofrnitochondrial matrix space localization (P=0.4478) strongly support this contention.

Because hMP is upregulated in an in vitro model of angiogenesis (Example 1), and because of its homologies with mitochondrial and nuclear-localized polypeptides, hMP is important in vascular morphogenesis, most likely through either powering the cellular differentiation-redifferentiation process, and/or affecting changes in the nuclear matrix that change global gene expression. Alternatively, hMP may be a transcription factor for either the nuclear or mitochondrial genomes. Nuclear hormone receptor (NHR)

NHR (SEQ ID NO: 16) has two domains: (1) the NH region is similar to Swi3 (yeast SWI/SNF complexes regulate transcription by chromatin remodeling), indicating a role in transcriptional regulation, and (2) the COOH region is similar to parathyroid hormone-related proteins that bind parathyroid hormones. PSORT (Nakai and Horton, 1999) predicts the protein to localize in the nucleus P=0.9600.

The identification of this new putative hormone receptor-transcriptional regulator and hBAZF suggest a novel human transcriptional pathway that resembles, to some extent, that of Bcl-6.

Bcl-6 suppresses transcription via the BTB domain, which recruits a complex containing SMRT, retinoid thyroid hormone receptor, nuclear receptor corepressor (N- CoR), mammalian Sin3 A, and histone deacetylase (HDAC). hBAZF, which also possesses a BTB domain, might recruit a similar complex containing deacetylase. Expression data indicate that hBAZF is up-regulated while NHR is down-regulated. These data agree with other evidence related to tube formation. Testosterone (a steroid) and dexamethasone (a steroid-like molecule) strongly inhibit vessel formation, and all- trans retinoic acid (at-RA) and 9-cis retinoic acid (9-cis RA) stimulate capillary-like tubular structures (Lansink et al, 1998).

Upon angiogenic stimulation, endothelial cells may become incompetent to respond to anti-angiogenic responses mediated by hormones using a dual mechanism, sequestering hormones and suppressing transcription. Because nΗR is down-regulated during in vitro angiogenesis (Example 1), this polypeptide is likely to be involved in non- angiogenesis-specific gene transcription. nΗR is an attractive therapeutic target, especially in therapies that are directed at preventing vascularization.

AAP polynucleotides

One aspect of the invention pertains to isolated nucleic acid molecules that encode AAP or biologically-active portions thereof. Also included in the invention are nucleic acid fragments sufficient for use as hybridization probes to identify AAP-encoding nucleic acids (e.g., AAP mRΝAs) and fragments for use as polymerase chain reaction (PCR) primers for the amplification and/or mutation of AAP molecules. A "nucleic acid molecule" includes DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs. The nucleic acid molecule may be single-stranded or double-stranded, but preferably comprises double-stranded DNA.

1. probes

Probes are nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt), 100 nt, or many (e.g., 6,000 nt) depending on the specific use. Probes are used to detect identical, similar, or complementary nucleic acid sequences. Longer length probes can be obtained from a natural or recombinant source, are highly specific, and much slower to hybridize than shorter-length oligomer probes. Probes may be single- or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies. Probes are substantially purified oligonucleotides that will hybridize under stringent conditions to at least optimally 12, 25, 50, 100, 150, 200, 250, 300, 350 or 400 consecutive sense strand nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, or 15; or an anti-sense strand nucleotide sequence of these sequences; or of a naturally occurring mutant of these sequences.

The full- or partial length native sequence AAP may be used to "pull out" similar (homologous) sequences (Ausubel et al, 1987; Sambrook, 1989), such as: (1) full-length or fragments of AAP cDNA from a cDNA library from any species (e.g. human, murine, feline, canine, bacterial, viral, retroviral, yeast), (2) from cells or tissues, (3) variants within a species, and (4) homologues and variants from other species. To find related sequences that may encode related genes, the probe may be designed to encode unique sequences or degenerate sequences. Sequences may also be genomic sequences including promoters, enhancer elements and introns of native sequence AAP.

For example, an AAP coding region in another species may be isolated using such probes. A probe of about 40 bases is designed, based on an AAP, and made. To detect hybridizations, probes are labeled using, for example, radionuclides such as ³²P or ³⁵S, or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin-biotin systems. Labeled probes are used to detect nucleic acids having a complementary sequence to that of an AAP in libraries of cDNA, genomic DNA or mRNA of a desired species. Such probes can be used as a part of a diagnostic test kit for identifying cells or tissues which mis-express an AAP, such as by measuring a level of an AAP in a sample of cells from a subject e.g., detecting AAP mRNA levels or determining whether a genomic AAP has been mutated or deleted.

2. isolated nucleic acid

An isolated nucleic acid molecule is separated from other nucleic acid molecules that are present in the natural source of the nucleic acid. Preferably, an isolated nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5'- and 3'-termini of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. For example, in various embodiments, isolated AAP molecules can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell/tissue from which the nucleic acid is derived (e.g., brain, heart, liver, spleen, etc.). Moreover, an isolated nucleic acid molecule, such as a cDNA molecule, can be substantially free of other cellular material or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized.

A nucleic acid molecule of the invention, e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13 or 15, or a complement of this aforementioned nucleotide sequence, can be isolated using standard molecular biology techniques and the provided sequence information. Using all or a portion of the nucleic acid sequence of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13 or 15 as a hybridization probe, AAP molecules can be isolated using standard hybridization and cloning techniques (Ausubel et al, 1987; Sambrook, 1989).

PCR amplification techniques can be used to amplify AAP using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers. Such nucleic acids can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to AAP sequences can be prepared by standard synthetic techniques, e.g., an automated DNA synthesizer.

3. oligonucleotide

An oligonucleotide comprises a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction or other application. A short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue. Oligonucleotides comprise portions of a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length. In one embodiment of the invention, an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at least 6 contiguous nucleotides of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13 or 15, or a complement thereof. Oligonucleotides may be chemically synthesized and may also be used as probes.

4. complementary nucleic acid sequences; binding

In another embodiment, an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence shown in SEQ ID NOS: 1, 3,-5, 7, 9, 11, 13 or 15, or a portion of this nucleotide sequence (e.g., a fragment that can be used as a probe or primer or a fragment encoding a biologically- active portion of an AAP). A nucleic acid molecule that is complementary to the nucleotide sequence shown in SEQ ID NOS:l, 3, 5, 7, 9, 11, 13 or 15, is one that is sufficiently complementary to the nucleotide sequence shown in SEQ ID NOS:l, 3, 5, 7, 9, 11, 13 or 15, that it can hydrogen bond with little or no mismatches to the nucleotide sequence shown in SEQ ID NOS:l, 3, 5, 7, 9, 11, 13 or 15, thereby forming a stable duplex.

"Complementary" refers to Watson-Crick or Hoogsteen base pairing between nucleotides units of a nucleic acid molecule, and the term "binding" means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, van der Waals, hydrophobic interactions, and the like. A physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.

Nucleic acid fragments are at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full-length sequence. Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice.

5. derivatives, and analogs

Derivatives are nucleic acid sequences or amino acid sequences formed from the native compounds either directly or by modification or partial substitution. Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but differ from it in respect to certain components or side chains. Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type. Homologs are nucleic acid sequences or amino acid sequences of a particular gene that are derived from different species.

Derivatives and analogs may be full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid, as described below. Derivatives or analogs of the nucleic acids or proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins of the invention, in various embodiments, by at least about 70%, 80%, or 95% identity (with a preferred identity of 80-95%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions (Ausubel et al, 1987).

6. homology

A "homologous nucleic acid sequence" or "homologous amino acid sequence," or variations thereof, refer to sequences characterized by homology at the nucleotide level or amino acid level as discussed above. Homologous nucleotide sequences encode those sequences coding for isoforms of AAP. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA Alternatively, different genes can encode isoforms. In the invention, homologous nucleotide sequences include nucleotide sequences encoding for an AAP of species other than humans, including, but not limited to: vertebrates, and thus can include, e.g., frog, mouse, rat, rabbit, dog, cat cow, horse, and other organisms. Homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein. A homologous nucleotide sequence does not, however, include the exact nucleotide sequence encoding human AAP. Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14 or 16, as well as a polypeptide possessing AAP biological activity. Various biological activities of the AAP are described below.

7. open reading frames

The open reading frame (ORF) of an AAP gene encodes an AAP. An ORF is a nucleotide sequence that has a start codon (ATG) and terminates with one of the three "stop" codons (TAA, TAG, or TGA). In this invention, however, an ORF may be any part of a coding sequence that-may or may not comprise a start codon and a stop codon. To achieve a unique sequence, preferable AAP ORFs encode at least 50 amino acids.

AAP polypeptides

1. mature

An AAP can encode a mature AAP. A "mature" form of a polypeptide or protein disclosed in the present invention is the product of a naturally occurring polypeptide or precursor form or proprotein. The naturally occurring polypeptide, precursor or proprotein includes, by way of nonlimiting example, the full-length gene product, encoded by the corresponding gene. Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an open reading frame described herein. The product "mature" form arises, again by way of nonlimiting example, as a result of one or more naturally occurring processing steps as they may take place within the cell, or host cell, in which the gene product arises. Examples of such processing steps leading to a "mature" form of a polypeptide or protein include the cleavage of the N-terminal methionine residue encoded by the initiation codon of an open reading frame, or the proteolytic cleavage of a signal peptide or leader sequence. Thus a mature form arising from a precursor polypeptide or protein that has residues 1 to N, where residue 1 is the N- terminal methionine, would have residues 2 through N remaining after removal of the N- terminal methionine. Alternatively, a mature form arising from a precursor polypeptide or protein having residues 1 to N, in which an N-terminal signal sequence from residue 1 to residue M is cleaved, would have the residues from residue M+l to residue N remaining. Further as used herein, a "mature" form of a polypeptide or protein may arise from a step of post-translational modification other than a proteolytic cleavage event. Such additional processes include, by way of non-limiting example, glycosylation, myristoylation or phosphorylation. In general, a mature polypeptide or protein may result from the operation of only one of these processes, or a combination of any of them.

2. active

An active AAP polypeptide or AAP polypeptide fragment retains a biological and/or an immunological activity similar, but not necessarily identical, to an activity of a naturally-occuring (wild-type) AAP polypeptide of the invention, including mature forms. A particular biological assay, with or without dose dependency, can be used to determine AAP activity. A nucleic acid fragment encoding a biologically-active portion of AAP can be prepared by isolating a portion of SEQ ID NOS: 1, 3, 5, 7, 9, 11, 13 or 15 that encodes a polypeptide having an AAP biological activity (the biological activities of the AAP are described below), expressing the encoded portion of AAP (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of AAP. Immunological activity refers to the ability to induce the production of an antibody against an antigenic epitope possessed by a native AAP; biological activity refers to a function, either inhibitory or stimulatory, caused by a native AAP that excludes immunological activity.

AAP nucleic acid variants and hybridization

1. variant polynucleotides, genes and recombinant genes The invention further encompasses nucleic acid molecules that differ from the nucleotide sequences shown in SEQ ID NOS:l, 3, 5, 7, 9, 11, 13 or 15 due to degeneracy of the genetic code and thus encode the same AAP as that encoded by the nucleotide sequences shown in SEQ ID NO NOS:l, 3, 5, 7, 9, 11, 13 or 15. An isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14 or 16.

In addition to the AAP sequences shown in SEQ ID NOS:l, 3, 5, 7, 9, 11, 13 or 15, DNA sequence polymoφhisms that change the amino acid sequences of the AAP may exist within a population. For example, allelic variation among individuals will exhibit genetic polymoφhism in an AAP. The terms "gene" and "recombinant gene" refer to nucleic acid molecules comprising an open reading frame (ORF) encoding an AAP, preferably a vertebrate AAP. Such natural allelic variations can typically result in 1-5% variance in an AAP. Any and all such nucleotide variations and resulting amino acid polymoφhisms in an AAP, which are the result of natural allelic variation and that do not alter the functional activity of an AAP are within the scope of the invention.

Moreover, AAP from other species that have a nucleotide sequence that differs from the human sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13 or 15, are contemplated. Nucleic acid molecules corresponding to natural allelic variants and homologues of an AAP cDNAs of the invention can be isolated based on their homology to an AAP of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13 or 15 using cDNA-derived probes to hybridize to homologous AAP sequences under stringent conditions.

"AAP variant polynucleotide" or "AAP variant nucleic acid sequence" means a nucleic acid molecule which encodes an active AAP that (1) has at least about 80% nucleic acid sequence identity with a nucleotide acid sequence encoding a full-length native AAP, (2) a full-length native AAP lacking the signal peptide, (3) an extracellular domain of an AAP, with or without the signal peptide, or (4) any other fragment of a full- length AAP. Ordinarily, an AAP variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% nucleic acid sequence identity and yet more preferably at least about 99% nucleic acid sequence identity with the nucleic acid sequence encoding a full-length native AAP. An AAP variant polynucleotide may encode a full-length native AAP lacking the signal peptide, an extracellular domain of an AAP, with or without the signal sequence, or any other fragment of a full-length AAP. Variants do not encompass the native nucleotide sequence.

Ordinarily, AAP variant polynucleotides are at least about 30 nucleotides in length, often at least about 60, 90, 120, 150, 180, 210, 240, 270, 300, 450, 600 nucleotides in length, more often at least about 900 nucleotides in length, or more.

"Percent (%) nucleic acid sequence identity" with respect to AAP-encoding nucleic acid sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the AAP sequence of interest, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for puφoses of determining % nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

When nucleotide sequences are aligned, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) can be calculated as follows:

%nucleic acid sequence identity = W/Z " 100 where

W is the number of nucleotides cored as identical matches by the sequence alignment program's or algorithm's alignment of C and D and

Z is the total number of nucleotides in D.

When the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C.

2. Stringency

Homologs (i.e., nucleic acids encoding an AAP derived from species other than human) or other related sequences (e.g., paralogs) can be obtained by low, moderate or high stringency hybridization with all or a portion of the particular human sequence as a probe using methods well known in the art for nucleic acid hybridization and cloning.

The specificity of single stranded DNA to hybridize complementary fragments is determined by the "stringency" of the reaction conditions. Hybridization stringency increases as the propensity to form DNA duplexes decreases. In nucleic acid hybridization reactions, the stringency can be chosen to either favor specific hybridizations (high stringency), which can be used to identify, for example, full-length clones from a library. Less-specific hybridizations (low stringency) can be used to identify related, but not exact, DNA molecules (homologous, but not identical) or segments.

DNA duplexes are stabilized by: (1) the number of complementary base pairs, (2) the type of base pairs, (3) salt concentration (ionic strength) of the reaction mixture, (4) the temperature of the reaction, and (5) the presence of certain organic solvents, such as formamide which decreases DNA duplex stability. In general, the longer the probe, the higher the temperature required for proper annealing. A common approach is to vary the temperature: higher relative temperatures result in more stringent reaction conditions. (Ausubel et al, 1987) provide an excellent explanation of stringency of hybridization reactions.

To hybridize under "stringent conditions" describes hybridization protocols in which nucleotide sequences at least 60% homologous to each other remain hybridized. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and >H. The Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium.

(a) high stringency

"Stringent hybridization conditions" conditions enable a probe, primer or oligonucleotide to hybridize only to its target sequence. Stringent conditions are sequence-dependent and will differ. Stringent conditions comprise: (1) low ionic strength and high temperature washes (e.g. 15 mM sodium chloride, 1.5 mM sodium citrate, 0.1 % sodium dodecyl sulfate at 50°C); (2) a denaturing agent during hybridization (e.g. 50% (v/v) formamide, 0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone, 50mM sodium phosphate buffer (pH 6.5; 750 mM sodium chloride, 75 mM sodium citrate at 42°C); or (3) 50% formamide. Washes typically also comprise 5X SSC (0.75 M NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2 x SSC (sodium chloride/sodium citrate) and 50% formamide at 55°C, followed by a high-stringency wash consisting of 0.1 x SSC containing EDTA at 55°C. Preferably, the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other. These conditions are presented as examples and are not meant to be limiting.

(b) moderate stringency

"Moderately stringent conditions" use washing solutions and hybridization conditions that are less stringent (Sambrook, 1989), such that a polynucleotide will hybridize to the entire, fragments, derivatives or analogs of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13 or 15. One example comprises hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55°C, followed by one or more washes in IX SSC, 0.1% SDS at 37°C. The temperature, ionic strength, etc., can be adjusted to accommodate experimental factors such as probe length. Other moderate stringency conditions are described in (Ausubel et al, 1987; Kriegler, 1990).

(c) low stringency

"Low stringent conditions" use washing solutions and hybridization conditions that are less stringent than those for moderate stringency (Sambrook, 1989), such that a polynucleotide will hybridize to the entire, fragments, derivatives or analogs of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13 or 15. A non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide, 5X SSC, 50 mM Tris-HCI (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10% (wt/vol) dextran sulfate at 40°C, followed by one or more washes in 2X SSC, 25 mM Tris-HCI (pH 7.4), 5 mM EDTA, and 0.1% SDS at 50°C. Other conditions of low stringency, such as those for cross-species hybridizations are described in (Ausubel et al, 1987; Kriegler, 1990; Shilo and Weinberg, 1981).

3. Conservative mutations

In addition to naturally-occurring allelic variants of AAP, changes can be introduced by mutation into SEQ ID NO NOS: 1, 3, 5, 7, 9, 11, 13 or 15 sequences that incur alterations in the amino acid sequences of the encoded AAP that do not alter the AAP function. For example, nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14 or 16. A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequences of the AAP without altering their biological activity, whereas an "essential" amino acid residue is required for such biological activity. For example, amino acid residues that are conserved among the AAP of the invention are predicted to be particularly non-amenable to alteration. Amino acids for which conservative substitutions can be made are well-known in the art.

Useful conservative substitutions are shown in Table A, "Preferred substitutions." Conservative substitutions whereby an amino acid of one class is replaced with another amino acid of the same type fall within the scope of the subject invention so long as the substitution does not materially alter the biological activity of the compound. If such substitutions result in a change in biological activity, then more substantial changes, indicated in Table B as exemplary are introduced and the products screened for an AAP polypeptide's biological activity.

Table A Preferred substitutions

Non-conservative substitutions that effect (1) the structure of the polypeptide backbone, such as a β-sheet or α-helical conformation, (2) the charge or (3) hydrophobicity, or (4) the bulk of the side chain of the target site can modify an AAP polypeptide's function or immunological identity. Residues are divided into groups based on common side-chain properties as denoted in Table B. Non-conservative substitutions entail exchanging a member of one of these classes for another class. Substitutions may be introduced into conservative substitution sites or more preferably into non-conserved sites.

Table B Amino acid classes

The variant polypeptides can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (Carter, 1986; Zoller and Smith, 1987), cassette mutagenesis, restriction selection mutagenesis (Wells et al, 1985) or other known techniques can be performed on the cloned DNA to produce the AAP variant DNA (Ausubel et al, 1987; Sambrook, 1989).

In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 45%, preferably 60%, more preferably 70%, 80%, 90%, and most preferably about 95% homologous to SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, or 15.

A mutant AAP can be assayed for blocking angiogenesis in vitro.

4. Anti-sense nucleic acids

Using antisense and sense AAP oligonucleotides can prevent AAP polypeptide expressioa These oligonucleotides bind to target nucleic acid sequences, forming duplexes that block transcription or translation of the target sequence by enhancing degradation of the duplexes, terminating prematurely transcription or translation, or by other means.

Antisense or sense oligonucleotides are singe-stranded nucleic acids, either RNA or DNA, which can bind a target AAP mRNA (sense) or an AAP DNA (antisense) sequences. Anti-sense nucleic acids can be designed according to Watson and Crick or Hoogsteen base pairing rules. The anti-sense nucleic acid molecule can be complementary to the entire coding region of an AAP mRNA, but more preferably, to only a portion of the coding or noncoding region of an AAP mRNA For example, the anti-sense oligonucleotide can be complementary to the region surrounding the translation start site of an AAP mRNA Antisense or sense oligonucleotides may comprise a fragment of the AAP DNA coding region of at least about 14 nucleotides, preferably from about 14 to 30 nucleotides. In general, antisense RNA or DNA molecules can comprise at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 bases in length or more. Among others, (Stein and Cohen, 1988; van der Krol et al, 1988a) describe methods to derive antisense or a sense oligonucleotides from a given cDNA sequence.

Examples of modified nucleotides that can be used to generate the anti-sense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1- methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2- methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5- methylaminomethyluracil, 5 -methoxyaminomethyl-2-thiouracil, beta-D- mannosylqueosine, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6- isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2- thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5- oxyacetic acid methylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3- amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine. Alternatively, the anti-sense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been sub-cloned in an anti-sense orientation such that the transcribed RNA will be complementary to a target nucleic acid of interest. To introduce antisense or sense oligonucleotides into target cells (cells containing the target nucleic acid sequence), any gene transfer method may be used. Examples of gene transfer methods include (1) biological, such as gene transfer vectors like Epstein- Barr virus or conjugating the exogenous DNA to a ligand-binding molecule, (2) physical, such as electroporation and injection, and (3) chemical, such as CaPO₄ precipitation and oligonucleotide-lipid complexes.

An antisense or sense oligonucleotide is inserted into a suitable gene transfer retroviral vector. A cell containing the target nucleic acid sequence is contacted with the recombinant retroviral vector, either in vivo or ex vivo. Examples of suitable retroviral vectors include those derived from the murine retrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the double copy vectors designated DCT5A, DCT5B and DCT5C (WO 90/13641 , 1990). To achieve sufficient nucleic acid molecule transcription, vector constructs in which the transcription of the anti-sense nucleic acid molecule is controlled by a strong pol II or pol III promoter are preferred.

To specify target cells in a mixed population of cells cell surface receptors that are specific to the target cells can be exploited. Antisense and sense oligonucleotides are conjugated to a ligand-binding molecule, as described in (WO 91/04753, 1991). Ligands are chosen for receptors that are specific to the target cells. Examples of suitable ligand- binding molecules include cell surface receptors, growth factors, cytokines, or other ligands that bind to cell surface receptors or molecules. Preferably, conjugation of the ligand-binding molecule does not substantially interfere with the ability of the receptors or molecule to bind the ligand-binding molecule conjugate, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell.

Liposomes efficiently transfer sense or an antisense oligonucleotide to cells (WO 90/10448, 1990). The sense or antisense oligonucleotide-lipid complex is preferably dissociated within the cell by an endogenous lipase.

The anti-sense nucleic acid molecule of the invention may be an -anomeric nucleic acid molecule. An α-anomeric nucleic acid molecule forms specific double- stranded hybrids with complementary RNA in which, contrary to the usual α-units, the strands run parallel to each other (Gautier et al, 1987). The anti-sense nucleic acid molecule can also comprise a 2'-o-methylribonucleotide (Inoue et al, 1987a) or a chimeric RNA-DNA analogue (Inoue et al, 1987b). In one embodiment, an anti-sense nucleic acid of the invention is a ribozyme. Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Thus, ribozymes, such as hammerhead ribozymes (Haseloff and Gerlach, 1988) can be used to catalytically cleave AAP mRNA transcripts and thus inhibit translation. A ribozyme specific for an AAP-encoding nucleic acid can be designed based on the nucleotide sequence of an AAP cDNA (i.e., SEQ ID NOS:l, 3, 5, 1, 9, 11, 13 or 15). For example, a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an AAP-encoding mRNA (Cech et al, U.S. Patent No. 5,116,742, 1992; Cech et al, U.S. Patent No. 4,987,071, 1991). An AAP mRNA can also be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules (Bartel and Szostak, 1993).

Alternatively, AAP expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of an AAP (e.g. , an AAP promoter and/or enhancers) to form triple helical structures that prevent transcription of an AAP in target cells (Helene, 1991; Helene et αl, 1992; Maher, 1992).

Modifications of antisense and sense oligonucleotides can augment their effectiveness. Modified sugar-phosphodiester bonds or other sugar linkages (WO 91/06629, 1991), increase in vivo stability by conferring resistance to endogenous nucleases without disrupting binding specificity to target sequences. Other modifications can increase the affinities of the oligonucleotides for their targets, such as covalently linked organic moieties (WO 90/10448, 1990) or poly-(L)-lysine. Other attachments modify binding specificities of the oligonucleotides for their targets, including metal complexes or intercalating (e.g. ellipticine) and alkylating agents.

For example, the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (Hyrup and Nielsen, 1996). "Peptide nucleic acids" or "PNAs" refer to nucleic acid mimics (e.g., DNA mimics) in that the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained. The neutral backbone of PNAs allows for specific hybridization to DNA and RNA under conditions of low ionic strength. The synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols (Hyrup and Nielsen, 1996; Perry-O'Keefe et al, 1996).

PNAs of an AAP can be used in therapeutic and diagnostic applications. For example, PNAs can be used as anti-sense or antigene agents for sequence-specific modulation of gene expression by inducing transcription or translation arrest or inhibiting replication. AAP PNAs may also be used in the analysis of single base pair mutations (e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., S_\ nucleases (Hyrup and Nielsen, 1996); or as probes or primers for DNA sequence and hybridization (Hyrup and Nielsen, 1996; Perry- O'Keefe et al, 1996).

PNAs of an AAP can be modified to enhance their stability or cellular uptake. Lipophilic or other helper groups may be attached to PNAs, PNA-DNA dimmers formed, or the use of liposomes or other drug delivery techniques. For example, PNA-DNA chimeras can be generated that may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes (e.g., RNase H and DNA polymerases) to interact with the DNA portion while the PNA portion provides high binding affinity and specificity. PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup and Nielsen, 1996). The synthesis of PNA-DNA chimeras can be performed (Finn et al, 1996; Hyrup and Nielsen, 1996). For example, a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'- deoxy-thymidine phosphoramidite, can be used between the PNA and the 5' end of DNA (Finn et al, 1996; Hyrup and Nielsen, 1996). PNA monomers are then coupled in a step wise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn et al, 1996). Alternatively, chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment (Petersen et al, 1976).

The oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (Lemaitre et al, 1987; Letsinger et al, 1989) or PCT Publication No. WO88/09810) or the blood-brain barrier (e.g., PCT Publication No. WO 89/10134). In addition, oligonucleotides can be modified with hybridization-triggered cleavage agents (van der Krol et al, 1988b) or intercalating agents (Zon, 1988). The oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, and the like.

AAP polypeptides

One aspect of the invention pertains to isolated AAP, and biologically-active portions derivatives, fragments, analogs or homologs thereof. Also provided are polypeptide fragments suitable for use as immunogens to raise anti-AAP Abs. In one embodiment, a native AAP can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques. In another embodiment, AAP are produced by recombinant DNA techniques. Alternative to recombinant expression, an AAP can be synthesized chemically using standard peptide synthesis techniques.

1. Polypeptides

An AAP polypeptide includes the amino acid sequence of an AAP whose sequences are provided in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14 or 16. The invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residues shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14 or 16, while still encoding a protein that maintains its AAP activities and physiological functions, or a functional fragment thereof.

2. Variant AAP polypeptides

In general, an AAP variant that preserves an AAP-like function and includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further includes the possibility of inserting an additional residue or residues between two residues of the parent protein as well as the possibility of deleting one or more residues from the parent sequence. Any amino acid substitution, insertion, or deletion is encompassed by the invention. In favorable circumstances, the substitution is a conservative substitution as defined above.

"AAP polypeptide variant" means an active AAP polypeptide having at least: (1) about 80% amino acid sequence identity with a full-length native sequence AAP polypeptide sequence, (2) an AAP polypeptide sequence lacking the signal peptide, (3) an extracellular domain of an AAP polypeptide, with or without the signal peptide, or (4) any other fragment of a full-length AAP polypeptide sequence. For example, AAP polypeptide variants include AAP polypeptides wherein one or more amino acid residues are added or deleted at the N- or C- terminus of the full-length native amino acid sequence. An AAP polypeptide variant will have at least about 80% amino acid sequence identity, preferably at least about 81% amino acid sequence identity, more preferably at least about 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% amino acid sequence identity and most preferably at least about 99% amino acid sequence identity with a full-length native sequence AAP polypeptide sequence. An AAP polypeptide variant may have a sequence lacking the signal peptide, an extracellular domain of an AAP polypeptide, with or without the signal peptide, or any other fragment of a full-length AAP polypeptide sequence. Ordinarily, AAP variant polypeptides are at least about 10 amino acids in length, often at least about 20 amino acids in length, more often at least about 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 300 amino acids in length, or more.

"Percent (%) amino acid sequence identity" is defined as the percentage of amino acid residues that are identical with amino acid residues in a disclosed AAJ' polypeptide sequence in a candidate sequence when the two sequences are aligned. To determine % amino acid identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum % sequence identity; conservative substitutions are not considered as part of the sequence identity. Amino acid sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) software is used to align peptide sequences. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

When amino acid sequences are aligned, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) can be calculated as:

%amino acid sequence identity = X/Y ' 100 where

X is the number of amino acid residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and

Y is the total number of amino acid residues in B.

If the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A.

3. Isolated/purified polypeptides

An "isolated" or "purified" polypeptide, protein or biologically active fragment is separated and/or recovered from a component of its natural environment. Contaminant components include materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous materials. Preferably, the polypeptide is purified to a sufficient degree to obtain at least 15 residues of N-terminal or internal amino acid sequence. To be substantially isolated, preparations having less than 30% by dry weight of non-AAP contaminating material (contaminants), more preferably less than 20%, 10% and most preferably less than 5% contaminants. An isolated, recombinantly-produced AAP or biologically active portion is preferably substantially free of culture medium, i.e., culture medium represents less than 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the AAP preparation. Examples of contaminants include cell debris, culture media, and substances used and produced during in vitro synthesis of an AAP.

4. Biologically active

Biologically active portions of an AAP include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequences of an AAP (SEQ ID NOS:2, 4, 6, 8, 10, 12, 14 or 16) that include fewer amino acids than a full-length AAP, and exhibit at least one activity of an AAP. Biologically active portions comprise a domain or motif with at least one activity of a native AAP. A biologically active portion of an AAP can be a polypeptide that is, for example, 10, 25, 50, 100 or more amino acid residues in length. Other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native AAP.

Biologically active portions of an AAP may have an amino acid sequence shown in SEQ ID NOS:2, 4, 6, 8, 10, 12, 14 or 16, or substantially homologous to SEQ ID NOS:2, 4, 6, 8, 10, 12, 14 or 16, and retains the functional activity of the protein of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14 or 16, yet differs in amino acid sequence due to natural allelic variation or mutagenesis. Other biologically active AAP may comprise an amino acid sequence at least 45% homologous to the amino acid sequence of SEQ ID NOS:2, 4, 6, 8, 10, 12, 14 or 16, and retains the functional activity of native AAP.

5. Determining homology between two or more sequences

"AAP variant" means an active AAP having at least: (1) about 80% amino acid sequence identity with a full-length native sequence AAP sequence, (2) an AAP sequence lacking the signal peptide, (3) an extracellular domain of an AAP, with or without the signal peptide, or (4) any other fragment of a full-length AAP sequence. For example, AAP variants include an AAP wherein one or more amino acid residues are added or deleted at the N- or C- terminus of the full-length native amino acid sequence. An AAP variant will have at least about 80% amino acid sequence identity, preferably at least about 81%) amino acid sequence identity, more preferably at least about 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% amino acid sequence identity and most preferably at least about 99% amino acid sequence identity with a full-length native sequence AAP sequence. An AAP variant may have a sequence lacking the signal peptide, an extracellular domain of an AAP, with or without the signal peptide, or any other fragment of a full-length AAP sequence. Ordinarily, AAP variant polypeptides are at least about 10 amino acids in length, often at least about 20 amino acids in length, more often at least about 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 300 amino acids in length, or more.

"Percent (%) amino acid sequence identity" is defined as the percentage of amino acid residues that are identical with amino acid residues in a disclosed AAP sequence in a candidate sequence when the two sequences are aligned. To determine % amino acid identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum % sequence identity; conservative substitutions are not considered as part of the sequence identity. Amino acid sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) software is used to align peptide sequences. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

When amino acid sequences are aligned, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) can be calculated as:

%amino acid sequence identity = X/Y ' 100 where

X is the number of amino acid residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and

Y is the total number of amino acid residues in B.

If the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A.

6. Chimeric and fusion proteins

Fusion polypeptides are useful in expression studies, cell-localization, bioassays, and AAP purification. An AAP "chimeric protein" or "fusion protein" comprises an AAP fused to a non-AAP polypeptide. A non-AAP polypeptide is not substantially homologous to an AAP (SEQ ID NOS:2, 4, 6, 8, 10, 12, 14 or 16). An AAP fusion protein may include any portion to an entire AAP, including any number of the biologically active portions. An AAP may be fused to the C-terminus of the GST (glutathione S-transferase) sequences. Such fusion proteins facilitate the purification of a recombinant AAP. In certain host cells, (e.g. mammalian), heterologous signal sequences fusions may ameliorate AAP expression and/or secretion. Additional exemplary fusions are presented in Table C.

Other fusion partners can adapt an AAP therapeutically. Fusions with members of the immunoglobulin (Ig) protein family are useful in therapies that inhibit an AAP ligand or substrate interactions, consequently suppressing an AAP-mediated signal transduction in vivo. Such fusions, incoφorated into pharmaceutical compositions, may be used to treat proliferative and differentiation disorders, as well as modulating cell survival. An AAP-Ig fusion polypeptides can also be used as immunogens to produce an anti-AAP Abs in a subject, to purify AAP ligands, and to screen for molecules that inhibit interactions of an AAP with other molecules.

Fusion proteins can be easily created using recombinant methods. A nucleic acid encoding an AAP can be fused in-frame with a non-AAP encoding nucleic acid, to an AAP NH₂- or COO- -terminus, or internally. Fusion genes may also be synthesized by conventional techniques, including automated DNA synthesizers. PCR amplification using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (Ausubel et al, 1987) is also useful. Many vectors are commercially available that facilitate sub-cloning an AAP in-frame to a fusion moiety.

Table C Useful non-AAP fusion polypeptides

Therapeutic applications of AAP

1. Agonists and antagonists

"Antagonist" includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of an endogenous AAP. Similarly, "agonist" includes any molecule that mimics a biological activity of an endogenous AAP. Molecules that can act as agonists or antagonists include Abs or antibody fragments, fragments or variants of an endogenous AAP, peptides, antisense oligonucleotides, small organic molecules, etc.

2. Identifying antagonists and agonists

To assay for antagonists, an AAP is added to, or expressed in, a cell along with the compound to be screened for a particular activity. If the compound inhibits the activity of interest in the presence of an AAP, that compound is an antagonist to the LλALP; if an AAP activity is enhanced, the compound is an agonist.

(a) Specific examples of potential antagonists and agonist

Any molecule that alters AAP cellular effects is a candidate antagonist or agonist. Screening techniques well known to those skilled in the art can identify these molecules. Examples of antagonists and agonists include: (1) small organic and inorganic compounds, (2) small peptides, (3) Abs and derivatives, (4) polypeptides closely related to an AAP, (5) antisense DNA and RNA, (6) ribozymes, (7) triple DNA helices and (8) nucleic acid aptamers.

Small molecules that bind to an AAP active site or other relevant part of the polypeptide and inhibit the biological activity of the AAP are antagonists. Examples of small molecule antagonists include small peptides, peptide-like molecules, preferably soluble, and synthetic non-peptidyl organic or inorganic compounds. These same molecules, if they enhance an AAP activity, are examples of agonists.

Almost any antibody that affects an AAP's function is a candidate antagonist, and occasionally, agonist. Examples of antibody antagonists include polyclonal, monoclonal, single-chain, anti-idiotypic, chimeric Abs, or humanized versions of such Abs or fragments. Abs may be from any species in which an immune response can be raised. Humanized Abs are also contemplated.

Alternatively, a potential antagonist or agonist may be a closely related protein, for example, a mutated form of an AAP that recognizes an AAP-interacting protein but imparts no effect, thereby competitively inhibiting AAP actioa Alternatively, a mutated AAP may be constitutively activated and may act as an agonist.

Antisense RNA or DNA constructs can be effective antagonists. Antisense RNA or DNA molecules block function by inhibiting translation by hybridizing to targeted mRNA. Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which depend on polynucleotide binding to DNA or RNA. For example, the 5' coding portion of an AAP sequence is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length A DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix) (Beal and Dervan, 1991; Cooney et al, 1988; Lee et al, 1979), thereby preventing transcription and the production of the AAP. The antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the AAP (antisense) (Cohen, 1989; Okano et al, 1991). These oligonucleotides can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of the AAP. When antisense DNA is used, oligodeoxyribonucleotides derived from the translation-initiation site, e.g., between about -10 and +10 positions of the target gene nucleotide sequence, are preferred.

Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. Ribozymes act by sequence-specific hybridization to the complementary target RNA, followed by endonucleolytic cleavage. Specific ribozyme cleavage sites within a potential RNA target can be identified by known techniques (WO 97/33551, 1997; Rossi, 1994).

To inhibit transcription, triple-helix nucleic acids that are single-stranded and comprise deoxynucleotides are useful antagonists. These oligonucleotides are designed such that triple-helix formation via Hoogsteen base-pairing rules is promoted, generally requiring stretches of purines or pyrimidines (WO 97/33551, 1997).

Aptamers are short oligonucleotide sequences that can be used to recognize and specifically bind almost any molecule. The systematic evolution of ligands by exponential enrichment (SELEX) process (Ausubel et al, 1987; Ellington and Szostak, 1990; Tuerk and Gold, 1990) is powerful and can be used to find such aptamers. Aptamers have many diagnostic and clinical uses; almost any use in which an antibody has been used clinically or diagnostically, aptamers too may be used. In addition, they are cheaper to make once they have been identified, and can be easily applied in a variety of formats, including administration in pharmaceutical compositions, inbioassays, and diagnostic tests (Jayasena, 1999).

Anti-AAP Abs

The invention encompasses Abs and antibody fragments, such as F_ab or (F_ab) , that bind immunospecifically to any AAP epitopes.

"Antibody" (Ab) comprises single Abs directed against an AAP (anti-AAP Ab; including agonist, antagonist, and neutralizing Abs), anti-AAP Ab compositions with poly-epitope specificity, single chain anti-AAP Abs, and fragments of anti-AAP Abs. A "monoclonal antibody" is obtained from a population of substantially homogeneous Abs, i.e., the individual Abs comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts. Exemplary Abs include polyclonal (pAb), monoclonal (mAb), humanized, bi-specific (bsAb), and heteroconjugate Abs.

1. Polyclonal Abs (pAbs)

Polyclonal Abs can be raised in a mammalian host, for example, by one or more injections of an immunogen and, if desired, an adjuvant. Typically, the immunogen and/or adjuvant are injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunogen may include an AAP or a fusion protein Examples of adjuvants include Freund's complete and monophosphoryl Lipid A synthetic-trehalose dicorynomycolate (MPL-TDM). To improve the immune response, an immunogen may be conjugated to a protein that is immunogenic in the host, such as keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Protocols for antibody production are described by (Ausubel et al, 1987; Harlow and Lane, 1988). Alternatively, pAbs may be made in chickens, producing IgY molecules (Schade et α/., 1996).

2. Monoclonal Abs (mAbs)

Anti-AAP mAbs may be prepared using hybridoma methods (Milstein and Cuello, 1983). Hybridoma methods comprise at least four steps: (1) immunizing a host, or lymphocytes from a host; (2) harvesting the mAb secreting (or potentially secreting) lymphocytes, (3) fusing the lymphocytes to immortalized cells, and (4) selecting those cells that secrete the desired (anti-AAP) mAb.

A mouse, rat, guinea pig, hamster, or other appropriate host is immunized to elicit lymphocytes that produce or are capable of producing Abs that will specifically bind to the immunogen. Alternatively, the lymphocytes may be immunized in vitro. If human cells are desired, peripheral blood lymphocytes (PBLs) are generally used; however, spleen cells or lymphocytes from other mammalian sources are preferred. The immunogen typically includes an AAP or a fusion protein.

The lymphocytes are then fused with an immortalized cell line to form hybridoma cells, facilitated by a fusing agent such as polyethylene glycol (Goding, 1996). Rodent, bovine, or human myeloma cells immortalized by transformation may be used, or rat or mouse myeloma cell lines. Because pure populations of hybridoma cells and not unfused immortalized cells are preferred, the cells after fusion are grown in a suitable medium that contains one or more substances that inhibit the growth or survival of unfused, immortalized cells. A common technique uses parental cells that lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT). In this case, hypoxanthine, aniinopterin and thymidine are added to the medium (HAT medium) to prevent the growth of HGPRT-deficient cells while permitting hybridomas to grow.

Preferred immortalized cells fuse efficiently, can be isolated from mixed populations by selecting in a medium such as HAT, and support stable and high-level expression of antibody after fusion. Preferred immortalized cell lines are murine myeloma lines, available from the American Type Culture Collection (Manassas, VA). Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human mAbs (Kozbor et al, 1984; Schook, 1987).

Because hybridoma cells secrete antibody extracellularly, the culture media can be assayed for the presence of mAbs directed against an AAP (anti-AAP mAbs). Immunoprecipitation or in vitro binding assays, such as radio immunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA), measure the binding specificity of mAbs (Harlow and Lane, 1988; Harlow and Lane, 1999), including Scatchard analysis (Munson and Rodbard, 1980).

Anti-AAP mAb secreting hybridoma cells may be isolated as single clones by limiting dilution procedures and sub-cultured (Goding, 1996). Suitable culture media include Dulbecco's Modified Eagle's Medium, RPMI-1640, or if desired, a protein-free or -reduced or serum-free medium (e.g., Ultra DOMA PF or HL-1; Biowhittaker; Walkersville, MD). The hybridoma cells may also be grown in vivo as ascites.

The mAbs may be isolated or purified from the culture medium or ascites fluid by conventional Ig purification procedures such as protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, ammonium sulfate precipitation or affinity chromatography (Harlow and Lane, 1988; Harlow and Lane, 1999).

The mAbs may also be made by recombinant methods (U.S. Patent No. 4166452, 1979). DNA encoding anti-AAP mAbs can be readily isolated and sequenced using conventional procedures, e.g., using oligonucleotide probes that specifically bind to murine heavy and light antibody chain genes, to probe preferably DNA isolated from anti-AAP-secreting mAb hybridoma cell lines. Once isolated, the isolated DNA fragments are sub-cloned into expression vectors that are then transfected into host cells such as simian COS-7 cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce Ig protein, to express mAbs. The isolated DNA fragments can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Patent No. 4816567, 1989; Morrison et al, 1987), or by fusing the Ig coding sequence to all or part of the coding sequence for a non-Ig polypeptide. Such a non-Ig polypeptide can be substituted for the constant domains of an antibody, or can be substituted for the variable domains of one antigen-combining site to create a chimeric bivalent antibody.

3. Monovalent Abs

The Abs may be monovalent Abs that consequently do not cross-link with each other. For example, one method involves recombinant expression of Ig light chain and modified heavy chain. Heavy chain truncations generally at any point in the F_c region will prevent heavy chain cross-linking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted, preventing crosslinking. In vitro methods are also suitable for preparing monovalent Abs. Abs can be digested to produce fragments, such as F_ab fragments (Harlow and Lane, 1988; Harlow and Lane, 1999).

4. Humanized and human Abs

Anti-AAP Abs may further comprise humanized or human Abs. Humanized forms of non-human Abs are chimeric Igs, Ig chains or fragments (such as F_v, F_ab, F_ab>, F(ab_')2 or other antigen-binding subsequences of Abs) that contain minimal sequence derived from non-human Ig.

Generally, a humanized antibody has one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization is accomplished by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody (Jones et al, 1986; Riechmann et al, 1988; Verhoeyen et al, 1988). Such "humanized" Abs are chimeric Abs (U.S. Patent No. 4816567, 1989), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized Abs are typically human Abs in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent Abs. Humanized Abs include human Igs (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are 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, corresponding non-human residues replace F_v framework residues of the human Ig. Humanized Abs may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which most if not all of the CDR regions correspond to those of a non-human Ig and most if not all of the FR regions are those of a human Ig consensus sequence. The humanized antibody optimally also comprises at least a portion of an Ig constant region (F_c), typically that of a human Ig (Jones et al, 1986; Presta, 1992; Riechmann et al, 1988).

Human Abs can also be produced using various techniques, including phage display libraries (Hoogenboom et al, 1991; Marks et al, 1991) and the preparation of human mAbs (Boerner et al, 1991; Reisfeld and Sell, 1985). Similarly, introducing human Ig genes into transgenic animals in which the endogenous Ig genes have been partially or completely inactivated can be exploited to synthesize human Abs. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire (U.S. Patent No. 5545807, 1996; U.S. Patent No. 5545806, 1996; U.S. Patent No. 5569825, 1996; U.S. Patent No. 5633425, 1997; U.S. Patent No. 5661016, 1997; U.S. Patent No. 5625126, 1997; Fishwild et al, 1996; Lonberg and Huszar, 1995; Lonberg et al, 1994; Marks et al, 1992).

5. Bi-specific mAbs

Bi-specific Abs are monoclonal, preferably human or humanized, that have binding specificities for at least two different antigens. For example, a binding specificity is an AAP; the other is for any antigen of choice, preferably a cell-surface protein or receptor or receptor subunit.

Traditionally, the recombinant production of bi-specific Abs is based on the co- expression of two Ig heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, 1983). Because of the random assortment of Ig heavy and light chains, the resulting hybridomas (quadromas) produce a potential mixture often different antibody molecules, of which only one has the desired bi-specific structure. The desired antibody can be purified using affinity chromatography or other techniques (WO 93/08829, 1993; Traunecker et al, 1991).

To manufacture a bi-specific antibody (Suresh et al, 1986), variable domains with the desired antibody-antigen combining sites are fused to Ig constant domain sequences. The fusion is preferably with an Ig heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. Preferably, the first heavy-chain constant region (CHI) containing the site necessary for light-chain binding is in at least one of the fusions. DNAs encoding the Ig heavy-chain fusions and, if desired, the Ig light chain, are inserted into separate expression vectors and are co-transfected into a suitable host organism.

The interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from recombinant cell culture (WO 96/27011, 1996). The preferred interface comprises at least part of the CH3 region of an antibody constant domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This mechanism increases the yield of the heterodimer over unwanted end products such as homodimers.

Bi-specific Abs can be prepared as full length Abs or antibody fragments (e.g. F(ab_')₂ bi-specific Abs). One technique to generate bi-specific Abs exploits chemical linkage. Intact Abs can be proteolytically cleaved to generate F_(ab')2 fragments (Brennan et al, 1985). Fragments are reduced with a dithiol complexing agent, such as sodium arsenite, to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The generated F_ab' fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the F_ab'-TNB derivatives is then reconverted to the F_ab>-thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other F_a ^TNB derivative to form the bi-specific antibody. The produced bi-specific Abs can be used as agents for the selective immobilization of enzymes. F_ab' fragments may be directly recovered from E. coli and chemically coupled to form bi-specific Abs. For example, fully humanized bi-specific F_(ab')₂ Abs can be produced (Shalaby et al, 1992). Each F_a fragment is separately secreted from E. coli and directly coupled chemically in vitro, forming the bi-specific antibody.

Various techniques for making and isolating bi-specific antibody fragments directly from recombinant cell culture have also been described. For example, leucine zipper motifs can be exploited (Kostelny et al, 1992). Peptides from the Fos and Jun proteins are linked to the F_ab' portions of two different Abs by gene fusion. The antibody homodimers are reduced at the hinge region to form monomers and then re-oxidized to form antibody heterodimers. This method can also produce antibody homodimers. The "diabody" technology (Holliger et al, 1993) provides an alternative method to generate bi-specific antibody fragments. The fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (V ) by a linker that is too short to allow pairing between the two domains on the same chain. The V_H and V_L domains of one fragment are forced to pair with the complementary V_L and VH domains of another fragment, forming two antigen-binding sites. Another strategy for making bi-specific antibody fragments is the use of single-chain F_v (sF_v) dimers (Gruber et al, 1994). Abs with more than two valencies are also contemplated, such as tri-specific Abs (Tutt et al, 1991).

Exemplary bi-specific Abs may bind to two different epitopes on a given AAP. Alternatively, cellular defense mechanisms can be restricted to a particular cell expressing the particular AAP: an anti-AAP arm may be combined with an arm that binds to a leukocyte triggering molecule, such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or to F_c receptors for IgG (F_cγR), such as F_cγRI (CD64), F_cγRII (CD32) and F_cγRIII (CD 16). Bi-specific Abs may also be used to target cytotoxic agents to cells that express a particular AAP. These Abs possess an AAP-binding arm and an arm that binds a cytotoxic agent or a radionuclide chelator.

6. Heteroconjugate Abs

Heteroconjugate Abs, consisting of two covalently joined Abs, have been proposed to target immune system cells to unwanted cells (4,676,980, 1987) and for treatment of human immunodeficiency virus (HIV) infection (WO 91/00360, 1991; WO 92/20373, 1992). Abs prepared in vitro using synthetic protein chemistry methods, including those involving cross-linking agents, are contemplated. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents include iminothiolate and methyl-4- mercaptobutyrimidate (4,676,980, 1987).

7. Immunoconjugates

Immunoconjugates may comprise an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin or fragment of bacterial, fungal, plant, or animal origin), or a radioactive isotope (i.e., a radioconjugate).

Useful enzymatically-active toxins and fragments include Diphtheria A chain, non-binding active fragments of Diphtheria toxin, exotoxin A chain from Pseudomonas aeruginosa, ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii proteins, Dianthin proteins, Phytolaca americana proteins, Momordica charantia inhibitor, curcin, crotin, Sapaonaria officinάlis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated Abs, such as ^2U Bi, ¹³¹1, ¹³¹In, ⁹⁰Y, and ¹⁸⁶Re.

Conjugates of the antibody and cytotoxic agent are made using a variety of bi- functional protein-coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bi-functional derivatives of imidoesters (such as dimethyl adipimidate HC1), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bts-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bts-diazonium derivatives (such as bis-φ-diazoniumbenzoyl)- ethylenediamine), diisocyanates (such as tolyene 2,6- diisocyanate), and bw-active fluorine compounds (such as l,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin can be prepared (Vitetta et al, 1987). ¹⁴C-labeled 1-isothiocyanatobenzyl- 3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionuclide to antibody (WO 94/11026, 1994).

In another embodiment, the antibody may be conjugated to a "receptor" (such as streptavidin) for utilization in tumor pre-targeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a streptavidin "ligand" (e.g., biotin) that is conjugated to a cytotoxic agent (e.g., a radionuclide). 8. Effector function engineering

The antibody can be modified to enhance its effectiveness in treating a disease, such as cancer. For example, cysteine residue(s) may be introduced into the F_c region, thereby allowing interchain disulfide bond formation in this region. Such homodimeric Abs may have improved intemalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC) (Caron et al, 1992; Shopes, 1992). Homodimeric Abs with enhanced anti-tumor activity can be prepared using hetero-bifunctional cross-linkers (Wolff et al, 1993). Alternatively, an antibody engineered with dual F_c regions may have enhanced complement lysis (Stevenson et al, 1989).

9. Immunoliposomes

Liposomes containing the antibody may also be formulated (U.S. Patent No. 4485045, 1984; U.S. Patent No. 4544545, 1985; U.S. Patent No. 5013556, 1991; Eppstein et al, 1985; Hwang et al, 1980). Useful liposomes can be generated by a reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG-derivatized phosphatidylethanolamine (PEG- PE). Such preparations are extruded through filters of defined pore size to yield liposomes with a desired diameter. F_ab- fragments of the antibody can be conjugated to the liposomes (Martin and Papahadjopoulos, 1982) via a disulfide-interchange reaction. A chemotherapeutic agent, such as Doxorubicin, may also be contained in the liposome (Gabizon et al, 1989). Other useful liposomes with different compositions are contemplated.

10. Diagnostic applications of Abs directed against an AAP

Anti-AAP Abs can be used to localize and/or quantitate anAAP (e.g., for use in measuring levels of an AAP within tissue samples or for use in diagnostic methods, etc.). Anti-AAP epitope Abs can be utilized as pharmacologically-active compounds.

Anti-AAP Abs can be used to isolate an AAP by standard techniques, such as immunoaffinity chromatography or immunoprecipitation. These approaches facilitate purifying an endogenous AAP antigen-containing polypeptides from cells and tissues. These approaches, as well as others, can be used to detect an AAJL in a sample to evaluate the abundance and pattern of expression of the antigenic protein. Anti-AAP Abs can be used to monitor protein levels in tissues as part of a clinical testing procedure; for example, to determine the efficacy of a given treatment regimen. Coupling the antibody to a detectable substance (label) allows detection of Ab-antigen complexes. Classes of labels include fluorescent, luminescent, bioluminescent, and radioactive materials, enzymes and prosthetic groups. Useful labels include horseradish peroxidase, alkaline phosphatase, β-galactosidase, acetylcholinesterase, streptavidin/biotin, avidin/biotin, umbelhferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazmylamine fluorescein, dansyl chloride, phycoerythrin, luminol, luciferase, luciferin, aequorin, and ¹²⁵I, ¹³¹I, ³⁵S or ³H

11. Antibody therapeutics

Abs of the invention, including polyclonal, monoclonal, humanized and fully human Abs, can be used therapeutically. Such agents will generally be employed to treat or prevent a disease or pathology in a subject. An antibody preparation, preferably one having high antigen specificity and affinity generally mediates an effect by binding the target epitope(s). Generally, administration of such Abs may mediate one of two effects: (1) the antibody may prevent ligand binding, eliminating endogenous ligand binding and subsequent signal transduction, or (2) the antibody elicits a physiological result by binding an effector site on the target molecule, initiating signal transduction.

A therapeutically effective amount of an antibody relates generally to the amount needed to achieve a therapeutic objective, epitope binding affinity, administration rate, and depletion rate of the antibody from a subject. Common ranges for therapeutically effective doses may be, as a nonlimiting example, from about 0.1 mg/kg body weight to about 50 mg/kg body weight. Dosing frequencies may range, for example, from twice daily to once a week.

12. Pharmaceutical compositions of Abs

Anti-AAP Abs, as well as other AAP interacting molecules (such as aptamers) identified in other assays, can be administered in pharmaceutical compositions to treat various disorders. Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components can be found in (de Boer, 1994; Gennaro, 2000; Lee, 1990).

Since some AAP are intracellular, Abs that are internalized are preferred used when whole Abs are used as inhibitors. Liposomes may also be used as a delivery vehicle for intracellular introduction. Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the epitope is preferred. For example, peptide molecules can be designed that bind a preferred epitope based on the variable- region sequences of a useful antibody. Such peptides can be synthesized chemically and/or produced by recombinant DNA technology (Marasco et al, 1993). Formulations may also contain more than one active compound for a particular treatment, preferably those with activities that do not adversely affect each other. The composition may comprise an agent that enhances function, such as a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent.

The active ingredients can also be entrapped in microcapsules prepared by coacervation techniques or by interfacial polymerization; for example, hydroxymethylcellulose or gelatin-microcapsules and poly-(methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions.

The formulations to be used for in vivo administration are highly preferred to be sterile. This is readily accomplished by filtration through sterile filtration membranes or any of a number of techniques.

Sustained-release preparations may also be prepared, such as semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (Boswell and Scribner, U.S. Patent No. 3,773,919, 1973), copolymers of L-glutamic acid and γ ethyl-L-glutamate, non-degradable ethylene- vinyl acetate, degradable lactic acid-glycolic acid copolymers such as injectable microspheres composed of lactic acid-glycolic acid copolymer, and poly-D-(-)-3- hydroxybutyric acid. While polymers such as ethylene- vinyl acetate and lactic acid- glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods and may be preferred.

AAP recombinant expression vectors and host cells

Vectors are tools used to shuttle DNA between host cells or as a means to express a nucleotide sequence. Some vectors function only in prokaryotes, while others function in both prokaryotes and eukaryotes, enabling large-scale DNA preparation from prokaryotes for expression in eukaryotes. Inserting the DNA of interest, such as an AAP nucleotide sequence or a fragment, is accomplished by ligation techniques and/or mating protocols well-known to the skilled artisan. Such DNA is inserted such that its integration does not disrupt any necessary components of the vector. In the case of vectors that are used to express the inserted DNA protein, the introduced DNA is operably-linked to the vector elements that govern its transcription and translation.

Vectors can be divided into two general classes: Cloning vectors are replicating plasmid or phage with regions that are non-essential for propagation in an appropriate host cell, and into which foreign DNA can be inserted; the foreign DNA is replicated and propagated as if it were a component of the vector. An expression vector (such as a plasmid, yeast, or animal virus genome) is used to introduce foreign genetic material into a host cell or tissue in order to transcribe and translate the foreign DNA In expression vectors, the introduced DNA is operably-linked to elements, such as promoters, that signal to the host cell to transcribe the inserted DNA. Some promoters are exceptionally useful, such as inducible promoters that control gene transcription in response to specific factors. Operably-linking an AAP or anti-sense construct to an inducible promoter can control the expression of an AAP or fragments, or anti-sense constructs. Examples of classic inducible promoters include those that are responsive to α-interferon, heat-shock, heavy metal ions, and steroids such as glucocorticoids (Kaufman, 1990) and tetracycline. Other desirable inducible promoters include those that are not endogenous to the cells in which the construct is being introduced, but, however, is responsive in those cells when the induction agent is exogenously supplied.

Vectors have many difference manifestations. A "plasmid" is a circular double stranded DNA molecule into which additional DNA segments can be introduced. Viral vectors can accept additional DNA segments into the viral genome. Certain vectors are capable of autonomous replication in a host cell (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. In general, useful expression vectors are often plasmids. However, other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses) are contemplated.

Recombinant expression vectors that comprise an AAP (or fragments) regulate an AAP transcription by exploiting one or more host cell-responsive (or that can be manipulated in vitro) regulatory sequences that is operably-linked to an AAP. "Operably- linked" indicates that a nucleotide sequence of interest is linked to regulatory sequences such that expression of the nucleotide sequence is achieved.

Vectors can be introduced in a variety of organisms and/or cells (Table D). Alternatively, the vectors can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.

Table D Examples of hosts for cloning or expression

Table D Examples of hosts for cloning or expression

Vector choice is dictated by the organism or cells being used and the desired fate of the vector. Vectors may replicate once in the target cells, or may be "suicide" vectors. In general, vectors comprise signal sequences, origins of replication, marker genes, enhancer elements, promoters, and transcription termination sequences. The choice of these elements depends on the organisms in which the vector will be used and are easily determined. Some of these elements may be conditional, such as an inducible or conditional promoter that is turned "on" when conditions are appropriate. Examples of inducible promoters include those that are tissue-specific, which relegate expression to certain cell types, steroid-responsive, or heat-shock reactive. Some bacterial repression systems, such as the lac operon, have been exploited in mammalian cells and transgenic animals (Fieck et al, 1992; Wyborski et al, 1996; Wyborski and Short, 1991). Vectors often use a selectable marker to facilitate identifying those cells that have incoφorated the vector. Many selectable markers are well known in the art for the use with prokaryotes, usually antibiotic-resistance genes or the use of autotrophy and auxotrophy mutants.

Using antisense and sense AAP oligonucleotides can prevent an AAP polypeptide expression. These oligonucleotides bind to target nucleic acid sequences, forming duplexes that block transcription or translation of the target sequence by enhancing degradation of the duplexes, terminating prematurely transcription or translation, or by other means.

Antisense or sense oligonucleotides are singe-stranded nucleic acids, either RNA or DNA, which can bind a target AAP mRNA (sense) or an AAP DNA (antisense) sequences. According to the present invention, antisense or sense oligonucleotides comprise a fragment of an AAP DNA coding region of at least about 14 nucleotides, preferably from about 14 to 30 nucleotides. In general, antisense RNA or DNA molecules can comprise at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 bases in length or more. Among others, (Stein and Cohen, 1988; van der Krol et al, 1988a) describe methods to derive antisense or a sense oligonucleotides from a given cDNA sequence.

Modifications of antisense and sense oligonucleotides can augment their effectiveness. Modified sugar-phosphodiester bonds or other sugar linkages (WO 91/06629, 1991), increase in vivo stability by conferring resistance to endogenous nucleases without disrupting binding specificity to target sequences. Other modifications can increase the affinities of the oligonucleotides for their targets, such as covalently linked organic moieties (WO 90/10448, 1990) or poly-(L)-lysine. Other attachments modify binding specificities of the oligonucleotides for their targets, including metal complexes or intercalating (e.g. ellipticine) and alkylating agents.

To introduce antisense or sense oligonucleotides into target cells (cells containing the target nucleic acid sequence), any gene transfer method may be used and are well known to those of skill in the art. Examples of gene transfer methods include 1) biological, such as gene transfer vectors like Epstein-Barr virus or conjugating the exogenous DNA to a ligand-binding molecule (WO 91/04753, 1991), 2) physical, such as electroporation, and 3) chemical, such as CaPO₄ precipitation and oligonucleotide-lipid complexes (WO 90/10448, 1990).

The terms "host cell" and "recombinant host cell" are used interchangeably. Such terms refer not only to a particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term.

Methods of eukaryotic cell transfection and prokaryotic cell transformation are well known in the art. The choice of host cell will dictate the preferred technique for introducing the nucleic acid of interest. Table E, which is not meant to be limiting, summarizes many of the known techniques in the art. Introduction of nucleic acids into an organism may also be done with ex vivo techniques that use an in vitro method of transfection, as well as established genetic techniques, if any, for that particular organism.

Table E Methods to introduce nucleic acid into cells

Table E Methods to introduce nucleic acid into cells

Table E Methods to introduce nucleic acid into cells

Vectors often use a selectable marker to facilitate identifying those cells that have incoφorated the vector. Many selectable markers are well known in the art for the use with prokaryotes, usually antibiotic-resistance genes or the use of autotrophy and auxofrophy mutants. Table F lists often-used selectable markers for mammalian cell transfection.

Table F Useful selectable markers for eukaryote cell transfection

Table F Useful selectable markers for eukaryote cell transfection

A host cell of the invention, such as a prokaryotic or eukaryotic host cell in culture, can be used to produce an AAP. Accordingly, the invention provides methods for producing an AAP using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of the invention (into which a recombinant expression vector encoding an AAP has been introduced) in a suitable medium, such that an AAP is produced. In another embodiment, the method further comprises isolating an AAP from the medium or the host cell. Transgenic AAP animals

Transgenic animals are useful for studying the function and/or activity of an AAP and for identifying and/or evaluating modulators of AAP activity. "Transgenic animals" are non-human animals, preferably mammals, more preferably a rodents such as rats or mice, in which one or more of the cells include a transgene. Other transgenic animals include primates, sheep, dogs, cows, goats, chickens, amphibians, etc. A "transgene" is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops, and that remains in the genome of the mature animal. Transgenes preferably direct the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal with the puφose of preventing expression of a naturally encoded gene product in one or more cell types or tissues (a "knockout" transgenic animal), or serving as a marker or indicator of an integration, chromosomal location, or region of recombination (e.g. cre/loxP mice). A "homologous recombinant animal" is a non-human animal, such as a rodent, in which an endogenous AAP has been altered by an exogenous DNA molecule that recombines homologously with an endogenous AAP in a. (e.g. embryonic) cell prior to development the animal. Host cells with an exogenous AAP can be used to produce non-human transgenic animals, such as fertilized oocytes or embryonic stem cells into which an y 4E-coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals or homologous recombinant animals.

1. Approaches to transgenic animal production

A transgenic animal can be created by introducing an AAP into the male pronuclei of a fertilized oocyte (e.g., by microinjection, retroviral infection) and allowing the oocyte to develop in a pseudopregnant female foster animal (pffa). n AAP cDNA sequences (SΕQ ID NO:l, 3, 5, 7, 9, 11, 13 or 15) can be introduced as a transgene into the genome of a non-human animal. Alternatively, a homologue of an AAP, such as the naturally- occuring variant of an AAP, can be used as a transgene. Intronic sequences and polyadenylation signals can also be included in the transgene to increase transgene expression. Tissue-specific regulatory sequences can be operably-linked to the AAP transgene to direct expression of the AAP to particular cells. Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art, e.g. (Evans et al, U.S. Patent No. 4,870,009, 1989; Hogan, 0879693843, 1994; Leder and Stewart, U.S. Patent No. 4,736,866, 1988; Wagner and Hoppe, US Patent No. 4,873,191, 1989). Other non-mice transgenic animals may be made by similar methods. A transgenic founder animal, which can be used to breed additional transgenic animals, can be identified based upon the presence of the transgene in its genome and/or expression of the transgene mRNA in tissues or cells of the animals. Transgenic animals can be bred to other transgenic animals carrying other transgenes.

2. Vectors for transgenic animal production

To create a homologous recombinant animal, a vector containing at least a portion of an. AAP into which a deletion, addition or substitution has been introduced to thereby alter, e.g., disrupt or alter the expression of, an AAP. An AAP can be a murine gene, or other AAP homologue, such as a naturally occurring variant. In one approach, a knockout vector functionally disrupts an endogenous AAP gene upon homologous recombination, and thus a non-functional AAP protein, if any, is expressed.

Alternatively, the vector can be designed such that, upon homologous recombination, an endogenous AAP is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of an endogenous AAP). In this type of homologous recombination vector, the altered portion of the AAP is flanked at its 5'- and 3'-termini by additional nucleic acid of the AAP to allow for homologous recombination to occur between the exogenous AAP carried by the vector and an endogenous AAP in an embryonic stem cell. The additional flanking AAP nucleic acid is sufficient to engender homologous recombination with the endogenous AAP. Typically, several kilobases of flanking DNA (both at the 5'- and 3'- termini) are included in the vector (Thomas and Capecchi, 1987). The vector is then introduced into an embryonic stem cell line (e.g., by electroporation), and cells in which the introduced AAP has homologously-recombined with an endogenous AAP are selected (Li et αl, 1992).

3. Introduction of an AAP transgene cells during development Selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (Bradley, 1987). A chimeric embryo can then be implanted into a suitable pffa and the embryo brought to term. Progeny harboring the homologously-recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously-recombined DNA by germline transmission of the transgene. Methods for constructing homologous recombination vectors and homologous recombinant animals are described (Berns et al, WO 93/04169, 1993; Bradley, 1991; Kucherlapati et al, WO 91/01140, 1991; Le Mouellic and Brullet, WO 90/11354, 1990).

Alternatively, transgenic animals that contain selected systems that allow for regulated expression of the transgene can be produced. An example of such a system is the cre/loxP recombinase system of bacteriophage PI (Lakso et al, 1992). Another recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al, 1991). If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be produced as "double" transgenic animals, by mating an animal containing a transgene encoding a selected protein to another containing a transgene encoding a recombinase.

Clones of transgenic animals can also be produced (Wilmut et al, 1997). In brief, a cell from a transgenic animal can be isolated and induced to exit the growth cycle and enter G₀ phase. The quiescent cell can then be fused to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated The reconstructed oocyte is then cultured to develop to a morula or blastocyte and then transferred to a pffa. The offspring borne of this female foster animal will be a clone of the "parent" transgenic animal. Pharmaceutical compositions

The AAP nucleic acid molecules, AAP polypeptides, and anti-AAP Abs (active compounds) of the invention, and derivatives, fragments, analogs and homologs thereof, can be incoφorated into pharmaceutical compositions. Such compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier. A "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absoφtion delaying agents, and the like, compatible with pharmaceutical administration (Gennaro, 2000). Preferred examples of such carriers or diluents include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used Except when a conventional media or agent is incompatible with an active compound, use of these compositions is contemplated. Supplementary active compounds can also be incoφorated into the compositions.

1. General considerations

A pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration, including intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose. ThepH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

2. Injectable formulations

Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid so as to be administered using a syringe. Such compositions should be stable during manufacture and storage and must be preserved against contamination from microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (such as glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures. Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by using surfactants. Various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal, can contain microorganism contamination. Isotonic agents, for example, sugars, polyalcohols such as manitol, sorbitol, and sodium chloride can be included in the composition. Compositions that can delay absoφtion include agents such as aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incoφorating the active compound (e.g., an AAP or anti-AAP antibody) in the required amount in an appropriate solvent with one or a combination of ingredients as required, followed by sterilization. Generally, dispersions are prepared by incoφorating the active compound into a sterile vehicle that contains a basic dispersion medium, and the other required ingredients as discussed. Sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and freeze-drying that yield a powder containing the active ingredient and any desired ingredient from a sterile solutions.

3. Oral compositions

Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the puφose of oral therapeutic administration, the active compound can be incoφorated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included. Tablets, pills, capsules, troches and the like can contain any of the , following ingredients, or compounds of a similar nature: a binder such as microcrystallme cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, PP MOGEL, or corn starch; a lubricant such as magnesium stearate or STEROTES; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

4. Compositions for inhalation

For administration by inhalation, the compounds are delivered as an aerosol spray from a nebulizer or a pressurized container that contains a suitable propellant, e.g., a gas such as carbon dioxide.

5. Systemic administration

Systemic administration can also be transmucosal or transdermal. For transmucosal or transdermal administration, penetrants that can permeate the target barrier(s) are selected. Transmucosal penetrants include, detergents, bile salts, and fusidic acid derivatives. Nasal sprays or suppositories can be used for transmucosal administration. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams.

The compounds can also be prepared in the form of suppositories (e.g., with bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

6. Carriers

In one embodiment, the active compounds are prepared with carriers that protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such materials can be obtained commercially from ALZA Coφoration (Mountain View, CA) and NOVA Pharmaceuticals, Inc. (Lake Elsinore, CA), or prepared by one of skill in the art. Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, such as in (Eppstein et al. , US Patent No. 4,522,811, 1985).

7. Unit dosage

Oral formulations or parenteral compositions in unit dosage form can be created to facilitate administration and dosage uniformity. Unit dosage form refers to physically discrete units suited as single dosages for the subject to be treated, containing a therapeutically effective quantity of active compound in association with the required pharmaceutical carrier. The specification for the unit dosage forms of the invention are dictated by, and directly dependent on, the unique characteristics of the active compound and the particular desired therapeutic effect, and the inherent limitations of compounding the active compound.

8. Gene therapy compositions

The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors. Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (Nabel and Nabel, US Patent No. 5,328,470, 1994), or by stereotactic injection (Chen et al, 1994). The pharmaceutical preparation of a gene therapy vector can include an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded. Alternatively, where the complete gene delivery vector can be produced intact from recombinant cells, e.g., retroviral vectors, the pharmaceutical preparation can include one or more cells that produce the gene delivery system.

9. Dosage

The pharmaceutical composition and method of the present invention may further comprise other therapeutically active compounds as noted herein that are usually applied in the treatment of the above mentioned pathological conditions.

In the treatment or prevention of conditions which require AAP modulation an appropriate dosage level will generally be about 0.01 to 500 mg per kg patient body weight per day which can be administered in single or multiple doses. Preferably, the dosage level will be about 0.1 to about 250 mg/kg per day; more preferably about 0.5 to about 100 mg/kg per day. A suitable dosage level may be about 0.01 to 250 mg/kg per day, about 0.05 to 100 mg/kg per day, or about 0.1 to 50 mg/kg per day. Within this range the dosage may be 0.05 to 0.5, 0.5 to 5 or 5 to 50 mg/kg per day. For oral administration, the compositions are preferably provided in the form of tablets containing 1.0 to 1000 milligrams of the active ingredient, particularly 1.0, 5.0, 10.0, 15.0. 20.0, 25.0, 50.0, 75.0, 100.0, 150.0, 200.0, 250.0, 300.0, 400.0, 500.0, 600.0, 750.0, 800.0, 900.0, and 1000.0 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the patient to be treated. The compounds may be administered on a regimen of 1 to 4 times per day, preferably once or twice per day.

It will be understood, however, that the specific dose level and frequency of dosage for any particular patient may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the host undergoing therapy.

10. Kits for pharmaceutical compositions

The pharmaceutical compositions can be included in a kit, container, pack, or dispenser together with instructions for administration. When the invention is supplied as a kit, the different components of the composition may be packaged in separate containers and admixed immediately before use. Such packaging of the components separately may permit long-term storage without losing the active components' functions.

Kits may also include reagents in separate containers that facilitate the execution of a specific test, such as diagnostic tests or tissue typing. For example, AAP DNA templates and suitable primers may be supplied for internal controls.

(a) Containers or vessels

The reagents included in the kits can be supplied in containers of any sort such that the life of the different components are preserved, and are not adsorbed or altered by the materials of the container. For example, sealed glass ampules may contain lyophihzed luciferase or buffer that have been packaged under a neutral, non-reacting gas, such as nitrogen. Ampoules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, etc., ceramic, metal or any other material typically employed to hold reagents. Other examples of suitable containers include simple bottles that may be fabricated from similar substances as ampules, and envelopes, that may consist of foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, or the like. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix. Removable membranes may be glass, plastic, rubber, etc.

(b) Instructional materials

Kits may also be supplied with instructional materials. Instructions may be printed on paper or other substrate, and/or may be supplied as an electronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM, Zip disc, videotape, audiotape, etc. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an internet web site specified by the manufacturer or distributor of the kit, or supplied as electronic mail.

Screening and detection methods

The isolated nucleic acid molecules of the invention can be used to express an AAP (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect an AAP mRNA (e.g., in a biological sample) or a genetic lesion in an AAP, and to modulate AAP activity, as described below. In addition, AAP polypeptides can be used to screen drugs or compounds that modulate the AAP activity or expression as well as to treat disorders characterized by insufficient or excessive production of an AAP or production of AAP forms that have decreased or aberrant activity compared to an AAP wild-type protein, or modulate biological function that involve an AAP. In addition, the anti-AAP Abs of the invention can be used to detect and isolate an AAP and modulate AAP activity.

1. Screening assays

The invention provides a method (screening assay) for identifying modalities, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs), foods, combinations thereof, etc., that effect an AAP, a stimulatory or inhibitory effect, inlcuding translation, transcription, activity or copies of the gene in cells. The invention also includes compounds identified in screening assays.

Testing for compounds that increase or decrease AAP activity are desirable. A compound may modulate an AAP activity by affecting: (1) the number of copies of the gene in the cell (amplifiers and deamplifiers); (2) increasing or decreasing transcription of an AAP (transcription up-regulators and down-regulators); (3) by increasing or decreasing the translation of an AAP mRNA into protein (translation up-regulators and down- regulators); or (4) by increasing or decreasing the activity of an AAP itself (agonists and antagonists).

(a) effects of compounds

To identify compounds that affect an AAP at the DNA, RNA and protein levels, cells or organisms are contacted with a candidate compound and the corresponding change in an AAP DNA, RNA or protein is assessed (Ausubel et αl, 1987). For DNA amplifiers and deamplifiers, the amount of an AAP DNA is measured, for those compounds that are transcription up-regulators and down-regulators the amount of an AAP mRNA is determined; for translational up- and down-regulators, the amount of an AAP polypeptide is measured. Compounds that are agonists or antagonists may be identified by contacting cells or organisms with the compound, and then examining, for example, the model of angiogenesis in vitro.

In one embodiment, many assays for screening candidate or test compounds that bind to or modulate the activity of an AAP or polypeptide or biologically-active portion are available. Ttest compounds can be obtained using any of the numerous approaches in combinatorial library methods, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach is limited to peptides, while the other four approaches encompass peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, 1997).

(b) small molecules

A "small molecule" refers to a composition that has a molecular weight of less than about 5 kD and more preferably less than about 4 kD, most preferably less than 0.6 kD. Small molecules can be, nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules. Libraries of chemical and/or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened with any of the assays of the invention. Examples of methods for the synthesis of molecular libraries can be found in: (Carell et al, 1994a; Carell et al, 1994b; Cho et al, 1993; DeWitt et al, 1993; Gallop et al, 199 ; Zuckermann et al, 1994).

Libraries of compounds may be presented in solution (Houghten et al, 1992) or on beads (Lam et al, 1991), on chips (Fodor et al, 1993), bacteria, spores (Ladner et al, US Patent No. 5,223,409, 1993), plasmids (Cull et al, 1992) or on phage (Cwirla et al, 1990; Devlin et al, 1990; Felici et al, 1991; Ladner et al, US Patent No. 5,223,409, 1993; Scott and Smith, 1990). A cell-free assay comprises contacting an AAP or biologically-active fragment with a known compound that binds the AAP to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the AAP, where determining the ability of the test compound to interact with the AAP comprises determining the ability of the AAP to preferentially bind to or modulate the activity of an AAP target molecule.

(c) cell-free assays

The cell-free assays of the invention may be used with both soluble or a membrane-bound forms of an AAP. In the case of cell-free assays comprising the membrane-bound form, a solubilizing agent to maintain the AAP in solution. Examples of such solubilizing agents include non-ionic detergents such as n-octylglucoside, n- dodecylglucoside, n-dodecylmaltoside, octanoyl-N-methylglucamide, decanoyl-N- methylglucamide, TRITON^® X-100 and others from the TRITON^® series, THESIT^®, Isotridecypoly(ethylene glycol ether)_n, N-dodecyl--N,N-dimethyl-3-ammonio-l-propane sulfonate, 3-(3-cholamidopropyl) dimethylamminiol-1 -propane sulfonate (CHAPS), or 3- (3-cholamidopropyl)dimethylamminiol-2-hydroxy-l-propane sulfonate (CHAPSO). (d) immobilization of target molecules to facilitate screening In more than one embodiment of the assay methods, immobilizing either an AAP or its partner molecules can facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate high throughput assays. Binding of a test compound to an AAP, or interaction of an AAP with a target molecule in the presence and absence of a candidate compound, can be accomplished in any vessel suitable for containing the reactants, such as microtiter plates, test tubes, and micro-centrifuge tubes. A fusion protein can be provided that adds a domain that allows one or both of the proteins to be bound to a matrix. For example, GST- AAP fusion proteins or GST-target fusion proteins can be adsorbed onto glutathione sepharose beads (SIGMA Chemical, St. Louis, MO) or glutathione derivatized microtiter plates that are then combined with the test compound or the test compound and either the non-adsorbed target protein or an AAP, and the mixture is incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described. Alternatively, the complexes can be dissociated from the matrix, and the level of AAP binding or activity determined using standard techniques.

Other techniques for immobilizing proteins on matrices can also be used in screening assays. Either an AAP or its target molecule can be immobilized using biotin- avidin or biotin-streptavidin systems. Biotinylation can be accomplished using many reagents, such as biotin-NHS (N-hydroxy-succinimide; PIERCE Chemicals, Rockford, IL), and immobilized in wells of streptavidin-coated 96 well plates (PIERCE Chemical). Alternatively, Abs reactive with an AAP or target molecules, but which do not interfere with binding of the AAP to its target molecule, can be derivatized to the wells of the plate, and unbound target or an AAJL trapped in the wells by antibody conjugation. Methods for detecting such complexes, in addition to those described for the GST-immobilized complexes, include immunodetection of complexes using Abs reactive with an AAP or its target, as well as enzyme-linked assays that rely on detecting an enzymatic activity associated with the AAP or target molecule.

(e) screens to identify modulators

Modulators of AAP expression can be identified in a method where a cell is contacted with a candidate compound and the expression of an AAP mRNA or protein in the cell is determined. The expression level of the AAP mRNA or protein in the presence of the candidate compound is compared to the AAP mRNA or protein levels in the absence of the candidate compound. The candidate compound can then be identified as a modulator of the AAP mRNA or protein expression based upon this comparison. For example, when expression of an AAP mRNA or protein is greater (i.e., statistically significant) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of the AAP mRNA or protein expression. Alternatively, when expression of the AAP mRNA or protein is less (statistically significant) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of the AAP mRNA or protein expression. The level of an AAP mRNA or protein expression in the cells can be determined by methods described for detecting an AAP mRNA or protein.

(i) hybrid assays

In yet another aspect of the invention, an AAP can be used as "bait" in two-hybrid or three hybrid assays (Bartel et al, 1993; Brent et al, WO94/10300, 1994; Iwabuchi et al, 1993; Madura et al, 1993; Saifer et al, US Patent No. 5,283,317, 1994; Zervos et al, 1993) to identify other proteins that bind or interact with the AAP and modulate AAP activity. Such AAP-bps are also likely to be involved in the propagation of signals by the AAP as, for example, upstream or downstream elements of an AAP pathway.

The two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for an AAP is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL4). The other construct, a DNA sequence from a library of DNA sequences that encodes an unidentified protein ("prey" or "sample") is fused to a gene that codes for the activation domain of the known transcription factor. If the "bait" and the "prey" proteins are able to interact in vivo, forming an AAP-dependent complex, the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) that is operably-linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the AAP-interacting protein.

The invention further pertains to novel agents identified by the aforementioned screening assays and uses thereof for treatments as described herein.

2. Detection assays

Portions or fragments of an AAP cDNA sequences identified herein (and the complete AAP gene sequences) are useful in themselves. By way of non-limiting example, these sequences can be used to: (1) identify an individual from a minute biological sample (tissue typing); and (2) aid in forensic identification of a biological sample.

(a) Tissue typing

The AAP sequences of the invention can be used to identify individuals from minute biological samples. In this technique, an individual's genomic DNA is digested with one or more restriction enzymes and probed on a Southern blot to yield unique bands. The sequences of the invention are useful as additional DNA markers for "restriction fragment length polymoφhisms" (RFLP; (Smulson et al, US Patent No. 5,272,057, 1993)).

Furthermore, the AAP sequences can be used to determine the actual base-by-base DNA sequence of targeted portions of an individual's genome. AAP sequences can be used to prepare two PCR primers from the 5'- and 3'-termini of the sequences that can then be used to amplify an the corresponding sequences from an individual's genome and then sequence the amplified fragment.

Panels of corresponding DNA sequences from individuals can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences. The sequences of the invention can be used to obtain such identification sequences from individuals and from tissue. The AAP sequences of the invention uniquely represent portions of an individual's genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. The allelic variation between individual humans occurs with a frequency of about once ever 500 bases. Much of the allelic variation is due to single nucleotide polymoφhisms (SNPs), which include RFLPs.

Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification puφoses. Because greater numbers of polymoφhisms occur in noncoding regions, fewer sequences are necessary to differentiate individuals. Noncoding sequences can positively identify individuals with a panel of 10 to 1,000 primers that each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NOS:l, 3, 5, 7, 9, 11, 13, and 15 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.

Predictive medicine

The invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trials are used for prognostic (predictive) puφoses to treat an individual prophylactically. Accordingly, one aspect of the invention relates to diagnostic assays for determining an AAP and/or nucleic acid expression as well as AAP activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant AAP expression or activity, including cancer. The invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with an AAP, nucleic acid expression or activity. For example, mutations in an AAP can be assayed in a biological sample. Such assays can be used for prognostic or predictive puφose to prophylactically treat an individual prior to the onset of a disorder characterized by or associated with the AAP, nucleic acid expression, or biological activity.

Another aspect of the invention provides methods for determining AAP activity, or nucleic acid expression, in an individual to select appropriate therapeutic or prophylactic agents for that individual (referred to herein as "pharmacogenomics"). Pharmacogenomics allows for the selection of modalities (e.g., drugs, foods) for therapeutic or prophylactic treatment of an individual based on the individual's genotype (e.g., the individual's genotype to determine the individual's ability to respond to a particular agent). Another aspect of the invention pertains to monitoring the influence of modalities (e.g., drugs, foods) on the expression or activity of an AAP in clinical trials.

1. Diagnostic assays

An exemplary method for detecting the presence or absence of an AAP in a biological sample involves obtaining a biological sample from a subject and contacting the biological sample with a compound or an agent capable of detecting the AAP or the AAP nucleic acid (e.g., mRNA, genomic DNA) such that the presence of the AAP is confirmed in the sample. An agent for detecting the AAP mRNA or genomic DNA is a labeled nucleic acid probe that can hybridize to the AAP mRNA or genomic DNA. The nucleic acid probe can be, for example, a full-length AAP nucleic acid, such as the nucleic acid of SEQ ID OS: 1, 3, 5, 7, 9, 11, 13 or 15, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to an AAP mRNA or genomic DNA.

An agent for detecting an AAP polypeptide is an antibody capable of binding to the AAP, preferably an antibody with a detectable label. Abs can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment (e.g., F_a or F(ab')₂) can be used. A labeled probe or antibody is coupled (i.e., physically linking) to a detectable substance, as well as indirect detection of the probe or antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin. The term "biological sample" includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. The detection method of the invention can be used to detect an AAP mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of an AAP mRNA include Northern hybridizations and in situ hybridizations. In vitro techniques for detection of an AAP polypeptide include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations, and immunofluorescence. In vitro techniques for detection of n AAP genomic DNA include Southern hybridizations and fluorescence in situ hybridization (FISH). Furthermore, in vivo techniques for detecting an AAP include introducing into a subject a labeled anti- AAP antibody. For example, the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.

In one embodiment, the biological sample from the subject contains protein molecules, and/or mRNA molecules, and/or genomic DNA molecules. A preferred biological sample is blood.

In another embodiment, the methods further involve obtaining a biological sample from a subject to provide a control, contacting the sample with a compound or agent to detect an AAP, mRNA, or genomic DNA, and comparing the presence of the AAP, mRNA or genomic DNA in the control sample with the presence of the AAP, mRNA or genomic DNA in the test sample.

The invention also encompasses kits for detecting an AAP in a biological sample. For example, the kit can comprise: a labeled compound or agent capable of detecting an AAP or an AAP mRNA in a sample; reagent and/or equipment for determining the amount of an AAP in the sample; and reagent and/or equipment for comparing the amount of an AAP in the sample with a standard. The compound or agent can be packaged in a suitable container. The kit can further comprise instructions for using the kit to detect the AAP or nucleic acid.

2. Prognostic assays

The diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with an aberrant AAP expression or activity. For example, the assays described herein, can be used to identify a subject having or at risk of developing a disorder associated with AAP, nucleic acid expression or activity. Alternatively, the prognostic assays can be used to identify a subject having or at risk for developing a disease or disorder. Tthe invention provides a method for identifying a disease or disorder associated with an aberrant AAP expression or activity in which a test sample is obtained from a subject and the AAP or nucleic acid (e.g., mRNA, genomic DNA) is detected. A test sample is a biological sample obtained from a subject. For example, a test sample can be a biological fluid (e.g., serum), cell sample, or tissue. Pognostic assays can be used to determine whether a subject can be administered a modality (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, food, etc.) to treat a disease or disorder associated with an aberrant AAP expression or activity. Such methods can be used to determine whether a subject can be effectively treated with an agent for a disorder. The invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with an aberrant AAP expression or activity in which a test sample is obtained and the AAP or nucleic acid is detected (e.g., where the presence of the AAP or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with the aberrant AAP expression or activity).

The methods of the invention can also be used to detect genetic lesions in an AAP to determine if a subject with the genetic lesion is at risk for a disorder characterized by aberrant angiogenesis. Methods include detecting, in a sample from the subject, the presence or absence of a genetic lesion characterized by at an alteration affecting the integrity of a gene encoding an AAP polypeptide, or the mis-expression of an AAP. Such genetic lesions can be detected by ascertaining: (1) a deletion of one or more nucleotides from an AAP; (2) an addition of one or more nucleotides to an AAP; (3) a substitution of one or more nucleotides in an AAP, (4) a chromosomal rearrangement of an AAP gene; (5) an alteration in the level of an AAP mRNA transcripts, (6) aberrant modification of an AAP, such as a change genomic DNA methylation, (7) the presence of a non-wild-type splicing pattern of an AAP mRNA transcript, (8) a non-wild-type level of an AAP, (9) allelic loss of an AAP, and/or (10) inappropriate post-translational modification of an AAP polypeptide. There are a large number of known assay techniques that can be used to detect lesions in an AAP. Any biological sample containing nucleated cells may be used.

In certain embodiments, lesion detection may use a probe/primer in a polymerase chain reaction (PCR) (e.g., (Mullis, US Patent No. 4,683,202, 1987; Mullis et αl, US Patent No. 4,683,195, 1987), such as anchor PCR or rapid amplification of cDNA ends (RACE) PCR, or, alternatively, in a ligation chain reaction (LCR) (e.g., (Landegren et αl., 1988; Nakazawa et αl, 1994), the latter is particularly useful for detecting point mutations in ^LP-genes (Abravaya et αl., 1995). This method may include collecting a sample from a patient, isolating nucleic acids from the sample, contacting the nucleic acids with one or more primers that specifically hybridize to an AAP under conditions such that hybridization and amplification of the AAP (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.

Alternative amplification methods include: self sustained sequence replication (Guatelli et al, 1990), transcriptional amplification system (Kwoh et al, 1989); Qβ Replicase (Lizardi et al, 1988), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules present in low abundance.

Mutations in an AAP from a sample can be identified by alterations in restriction enzyme cleavage patterns. For example, sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA Moreover, the use of sequence specific ribozymes can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.

Hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high- density arrays containing hundreds or thousands of oligonucleotides probes, can identify genetic mutations in an AAP (Cronin et al, 1996; Kozal et al, 1996). For example, genetic mutations in an AAP can be identified in two-dimensional arrays containing light-generated DNA probes as described in Cronin, et al, supra. Briefly, a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations. This is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene. In yet another embodiment, any of a variety of sequencing reactions known in the art can be used to directly sequence an AAP and detect mutations by comparing the sequence of the sample AAP-with the corresponding wild-type (control) sequence. Examples of sequencing reactions include those based on classic techniques (Maxam and Gilbert, 1977; Sanger et al, 1977). Any of a variety of automated sequencing procedures can be used when performing diagnostic assays (Naeve et al, 1995) including sequencing by mass spectrometry (Cohen et al, 1996; Griffin and Griffin, 1993; Koster, WO94/16101, 1994).

Other methods for detecting mutations in an AAP include those in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al, 1985). In general, the technique of "mismatch cleavage" starts by providing heteroduplexes formed by hybridizing (labeled) RNA or DNA containing a wild-type AAP sequence with potentially mutant RNA or DNA obtained from a sample. The double-stranded duplexes are treated with an agent that cleaves single-stranded regions of the duplex such as those that arise from base pair mismatches between the control and sample strands. For instance, RNA DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with S_t nuclease to enzymatically digest the mismatched regions. In other embodiments, either DNA DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. The digested material is then separated by size on denaturing polyacrylamide gels to determine the mutation site (Grompe et al, 1989; Saleeba and Cotton, 1993). The control DNA or RNA can be labeled for detection.

Mismatch cleavage reactions may employ one or more proteins that recognize mismatched base pairs in double-stranded DNA (DNA mismatch repair) in defined systems for detecting and mapping point mutations in an AAP cDNAs obtained from samples of cells. For example, the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al, 1994). According to an exemplary embodiment, a probe based on a wild-type AAP sequence is hybridized to a cDNA or other DNA product from a test cell(s). The duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like (Modrich et al, US Patent No. 5,459,039, 1995). Electrophoretic mobility alterations can be used to identify mutations in an AAP. For example, single strand conformation polymoφhism (SSCP) may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Cotton, 1993; Hayashi, 1992; Orita et al, 1989). Single-stranded DNA fragments of sample and control AAP nucleic acids are denatured and then renatured. The secondary structure of single-stranded nucleic acids varies according to sequence; the resulting alteration in electrophoretic mobility allows detection of even a single base change. The DNA fragments may be labeled or detected with labeled probes. The sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a sequence changes. The subject method may use heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al, 1991).

The migration of mutant or wild-type fragments can be assayed using denaturing gradient gel electrophoresis (DGGE; (Myers et al, 1985). In DGGE, DNA is modified to prevent complete denaturation, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR. A temperature gradient may also be used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rossiter and Caskey, 1990).

Examples of other techniques for detecting point mutations include, but are not limited to, selective oligonucleotide hybridization, selective amplification, or selective primer extension. For example, oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions that permit hybridization only if a perfect match is found (Saiki et al, 1986; Saiki et al, 1989). Such allele-specific oligonucleotides are hybridized to PCR-amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.

Alternatively, allele specific amplification technology that depends on selective PCR amplification may be used. Oligonucleotide primers for specific amplifications may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization (Gibbs et al, 1989)) or at the extreme 3'-terminus of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prosser, 1993). Novel restriction site in the region of the mutation may be introduced to create cleavage-based detection (Gasparini et al, 1992). Certain amplification may also be performed using Taq ligase for amplification (Barany, 1991). In such cases, ligation occurs only if there is a perfect match at the 3'-terminus of the 5' sequence, allowing detection of a known mutation by scoring for amplification

The described methods may be performed, for example, by using pre-packaged kits comprising at least one probe (nucleic acid or antibody) that may be conveniently used, for example, in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving an AAP.

Furthermore, any cell type or tissue in which an AAP is expressed may be utilized in the prognostic assays described herein.

3. Pharmacogenomics

Agents, or modulators that have a stimulatory or inhibitory effect on AAP activity or expression, as identified by a screening assay can be administered to individuals to treat, prophylactically or therapeutically, disorders, including those associated with angiogenesis. In conjunction with such treatment, the pharmacogenomics (i.e., the study of the relationship between a subject's genotype and the subject's response to a foreign modality, such as a food, compound or drug) may be considered. Metabolic differences of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug. Thus, the pharmacogenomics of the individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of an AAP, expression of an AAP nucleic acid, or an AAP mutation(s) in an individual can be determined to guide the selection of appropriate agent(s) for therapeutic or prophylactic treatment.

Pharmacogenomics deals with clinically significant hereditary variations in the response to modalities due to altered modality disposition and abnormal action in affected persons (Eichelbaum and Evert, 1996; Linder et αl., 1997). In general, two pharmacogenetic conditions can be differentiated: (1) genetic conditions transmitted as a single factor altering the interaction of a modality with the body (altered drug action) or (2) genetic conditions transmitted as single factors altering the way the body acts on a modality (altered drug metabolism). These pharmacogenetic conditions can occur either as rare defects or as nucleic acid polymoφhisms. For example, glucose-6-phosphate . dehydrogenase (G6PD) deficiency is a common inherited enzymopathy in which the main clinical complication is hemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

As an illustrative embodiment, the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action. The discovery of genetic polymoφhisms of drug metabolizing enzymes (e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19) explains the phenomena of some patients who show exaggerated drug response and/or serious toxicity after taking the standard and safe dose of a drug. These polymoφhisms are expressed in two phenotypes in the population, the extensive metabolizer (EM) and poor metabolizer (PM). The prevalence of PM is different among different populations. For example, the CYP2D6 gene is highly polymoφhic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers due to mutant CYP2D6 and CYP2C19 frequently experience exaggerated drug responses and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM shows no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite moφhine. At the other extreme are the so- called ultra-rapid metabolizers who are unresponsive to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification

The activity of an AAP, expression of an AAP nucleic acid, or mutation content of an AAP in an individual can be determined to select appropriate agent(s) for therapeutic or prophylactic treatment of the individual. In addition, pharmacogenetic studies can be used to apply genotyping of polymoφhic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with an AAP modulator, such as a modulator identified by one of the described exemplary screening assays.

4. Monitoring effects during clinical trials Monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of an AAP (e.g., the ability to modulate angiogenesis) can be applied not only in basic drug screening, but also in clinical trials. For example, the effectiveness of an agent determined by a screening assay to increase an AAP expression, protein levels, or up- regulate an AAP's activity can be monitored in clinical trails of subjects exhibiting decreased AAP expression, protein levels, or down-regulated AAP activity. Alternatively, the effectiveness of an agent determined to decrease an AAP expression, protein levels, or down-regulate an AAP's activity, can be monitored in clinical trails of subjects exhibiting increased the AAP expression, protein levels, or up-regulated AAP activity. In such clinical trials, the expression or activity of the AAP and, preferably, other genes that have been implicated in, for example, angiogenesis can be used as a "read out" or markers for a particular cell's responsiveness.

For example, genes, including an AAP, that are modulated in cells by treatment with a modality (e.g., food, compound, drug or small molecule) can be identified. To study the effect of agents on angiogenesis, for example, in a clinical trial, cells can be isolated and RNA prepared and analyzed for the levels of expression of an AAP and other genes implicated in the disorder. The gene expression pattern can be quantified by Northern blot analysis, nuclear run-on or RT-PCR experiments, or by measuring the amount of protein, or by measuring the activity level of the AAP or other gene products. In this manner, the gene expression pattern itself can serve as a marker, indicative of the cellular physiological response to the agent. Accordingly, this response state may be determined before, and at various points during, treatment of the individual with the agent.

The invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, protein, peptide, peptidomimetic, nucleic acid, small molecule, food or other drug candidate identified by the screening assays described herein) comprising the steps of (1) obtaining a pre-administration sample from a subject; (2) detecting the level of expression of an AAP, mRNA, or genomic DNA in the preadministration sample; (3) obtaining one or more post- administration samples from the subject; (4) detecting the level of expression or activity of the AAP, mRNA, or genomic DNA in the post-administration samples; (5) comparing the level of expression or activity of the AAP, mRNA, or genomic DNA in the pre- administration sample with the AAP, mRNA, or genomic DNA in the post administration sample or samples; and (6) altering the administration of the agent to the subject accordingly. For example, increased administration of the agent may be desirable to increase the expression or activity of the AAP to higher levels than detected, i.e., to increase the effectiveness of the agent. Alternatively, decreased administration of the agent may be desirable to decrease expression or activity of the AAP to lower levels than detected, i.e., to decrease the effectiveness of the agent.

5. Methods of treatment

The invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant AAP expression or activity. Furthermore, these same methods of treatment may be used to induce or inhibit angiogenesis, by changing the level of expression or activity of an AAP.

6. Disease and disorders

Diseases and disorders that are characterized by increased AAP levels or biological activity may be treated with therapeutics that antagonize (i.e., reduce or inhibit) activity. Antognists may be administered in a therapeutic or prophylactic manner. Therapeutics that maybe used include: (1) AAP peptides, or analogs, derivatives, fragments or homologs thereof; (2) Abs to an AAP peptide; (3) AAP nucleic acids; (4) administration of antisense nucleic acid and nucleic acids that are "dysfunctional" (i.e., due to a heterologous insertion within the coding sequences) that are used to eliminate endogenous function of by homologous recombination (Capecchi, 1989); or (5) modulators (i.e., inhibitors, agonists and antagonists, including additional peptide mimetic of the invention or Abs specific to an AAP) that alter the interaction between an AAP and its binding partner.

Diseases and disorders that are characterized by decreased AAP levels or biological activity may be treated with therapeutics that increase (i.e., are agonists to) activity. Therapeutics that upregulate activity may be administered therapeutically or prophylactically. Therapeutics that may be used include peptides, or analogs, derivatives, fragments or homologs thereof; or an agonist that increases bioavailability.

Increased or decreased levels can be readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying in vitro for RNA or peptide levels, structure and/or activity of the expressed peptides (or AAP mRNAs). Methods include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, and the like).

7. Prophylactic methods

The invention provides a method for preventing, in a subject, a disease or condition associated with an aberrant AAP expression or activity, by administering an agent that modulates an AAP expression or at least one AAP activity. Subjects at risk for a disease that is caused or contributed to by an aberrant AAP expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays. Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the AAP aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression. Depending on the type of AAP aberrancy, for example, an AAP agonist or AAP antagonist can be used to treat the subject. The appropriate agent can be determined based on screening assays.

8. Therapeutic methods

Another aspect of the invention pertains to methods of modulating an AAP expression or activity for therapeutic puφoses. The modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of AAP activity associated with the cell. An agent that modulates AAP activity can be a nucleic acid or a protein, a naturally occurring cognate ligand of an AAP, a peptide, an AAP peptidomimetic, or other small molecule. The agent may stimulate AAP activity. Examples of such stimulatory agents include an active AAP and an AAP nucleic acid molecule that has been introduced into the cell. In another embodiment, the agent inhibits AAP activity. Examples of inhibitory agents include antisense AAP nucleic acids and anti-AAP Abs. Modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject). As such, the invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of an AAP or nucleic acid molecule. In one embodiment, the method involves administering an agent (e.g., an agent identified by a screening assay), or combination of agents that modulates (e.g., up- regulates or down-regulates) AAP expression or activity. In another embodiment, the method involves administering an AAP or nucleic acid molecule as therapy to compensate for reduced or aberrant AAP expression or activity.

Stimulation of AAP activity is desirable in situations in which AAP is abnormally down-regulated and/or in which increased AAP activity is likely to have a beneficial effect. One example of such a situation is where a subject has a disorder characterized by aberrant angiogenesis (e.g., cancer).

9. Determination of the biological effect of the therapeutic

Suitable in vitro or in vivo assays can be performed to determine the effect of a specific therapeutic and whether its administration is indicated for treatment of the affected tissue.

In various specific embodiments, in vitro assays may be performed with representative cells of the type(s) involved in the patient's disorder, to determine if a given therapeutic exerts the desired effect upon the cell type(s). Modalities for use in therapy may be tested in suitable animal model systems including, but not limited to rats, mice, chicken, cows, monkeys, rabbits, and the like, prior to testing in human subjects. Similarly, for in vivo testing, any of the animal model system known in the art maybe used prior to administration to human subjects.

10. Prophylactic and therapeutic uses of the compositions of the invention AAP nucleic acids and proteins are useful in potential prophylactic and therapeutic applications implicated in a variety of disorders including, but not limited to those related to angiogenesis.

As an example, a cDNA encoding an AAP may be useful in gene therapy, and the protein may be useful when administered to a subject in need thereof.

AAP nucleic acids, or fragments thereof, may also be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein is to be assessed. A further use could be as an anti-bacterial molecule (i.e., some peptides have been found to possess anti-bacterial properties). These materials are further useful in the generation of Abs that immunospecifically bind to the novel substances of the invention for use in therapeutic or diagnostic methods.

The following example is meant to not be limiting. EXAMPLE

Identification of genes differentially-regulated

A comprehensive mRNA profiling technique (GeneCalling) was used to determine differential gene expression profiles of human endothelial cells undergoing differentiation into tube-like structures (Kahn et al, 2000). To confirm the expression data from GeneCalling, independent experiments were undertaken that used gene-specific PCR oligonucleotide primer pairs and an oligonucleotide probe labeled with a fluorescent dye at the 5' end and quencher fluorescent dye at the 3' end.. Total RNA (50 ng) was added to a 50 μl RT-PCR mixture and run. The following data were collected: hEF G collagen gel 24 hr versus 4h 4.5 fold upregulated hTRG collagen gel 24 hr versus 4h 3.5 fold upregulated

KLP collagen gel 24 hr versus 4h 3.5 fold upregulated myosin X collagen gel 24 hr versus 4h 3.5 fold upregulated

NHR collagen gel 24 hr versus 4h 7.3 fold downregulated

HBAZF collagen gel 24 hr versus 4h 2.1 fold upregulated

EQUIVALENTS

Although particular embodiments have been disclosed herein in detail, this has been done by way of example for puφoses of illustration only, and is not intended to be limiting with respect to the scope of the appended claims that follow. In particular, it is . contemplated by the inventors that various substitutions, alterations, and modifications may be made to the invention without departing from the spirit and scope of the invention as defined by the claims. The choice of nucleic acid starting material, clone of interest, or library type is believed to be a matter of routine for a person of ordinary skill in the art with knowledge of the embodiments described herein. Other aspects, advantages, and modifications considered within the scope of the following claims. REFERENCES

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Claims (41)

1. An isolated polypeptide comprising an amino acid sequence having at least 80% sequence identity to the sequence of SEQ ID NOS:2. 4, 6, 8, 10, 12, 14 or 16.

2. The polypeptide of claim 1 , wherein said polypeptide is an active AAP polypeptide.

3. The polypeptide of claim 2, wherein said amino acid sequence has at least 90% sequence identity to the sequence of SEQ ID NOS:2. 4, 6, 8, 10, 12, 14 or 16.

4. The polypeptide of claim 2, wherein said amino acid sequence has at least 98%o sequence identity to the sequence of SEQ ID NOS:2. 4, 6, 8, 10, 12, 14 or 16.

5. An isolated polynucleotide encoding the polypeptide of any one of claims 1-4, or a complement of said polynucleotide.

6. An isolated polynucleotide comprising a nucleotide sequence having at least 80% sequence identity to the sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13 or 15, or a complement of said polynucleotide.

7. The polynucleotide of claim 6, wherein said nucleotide sequence has at least 90% sequence identity to the sequence of NOS:l, 3, 5, 7, 9, 11, 13 or 15 , or a complement of said polynucleotide.

8. The polynucleotide of claim 6, wherein said nucleotide sequence has at least 98% sequence identity to the sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13 or 15, or a complement of said polynucleotide.

9. An antibody that specifically binds to the polypeptide of any one of claims 1-4.

10. A method of modulating angiogenesis comprising modulating the activity of at least one AAP.

11. The method of claim 10 wherein said modulating angiogenesis is increasing angiogenesis, and said modulating the activity comprises increasing the activity of at least one polypeptide selected from the group consisting of KLP, hBAZF, hTRG, hMXl, hMX2, hEF-G, and hMP.

12. The method of claim 10 wherein said modulating angiogenesis is decreasing angiogenesis, and said modulating the activity comprises increasing the activity of at least one polypeptide, wherein said at least one polypeptide comprises NHR.

13. The method of claim 10 wherein said modulating angiogenesis is decreasing angiogenesis, and said modulating the activity comprises decreasing the activity of at least one polypeptide selected from the group consisting of KLP, hBAZF, hTRG, hMXl , hMX2, hEF-G, and hMP.

14. The method of claim 10 wherein said modulating angiogenesis is increasing angiogenesis, and said modulating the activity comprises decreasing the activity of at least one polypeptide, wherein said at least one polypeptide comprises NHR.

15. The method of claim 11 or 12 wherein said increasing activity comprises increasing the expression of said at least one polypeptide.

16. The method of claim 13 or 14 wherein said decreasing activity comprises decreasing the expression of said at least one polypeptide.

17. The method of claim 15 wherein said increasing expression comprises transforming a cell to increase expression of a polynucleotide encoding said at least one polypeptide.

18. The method of claim 16 wherein said decreasing expression comprises transforming a cell to express a polynucleotide anti-sense to at least a portion of an endogenous polynucleotide encoding said at least one polypeptide.

19. The method of claim 13 or 14 wherein said decreasing activity comprises transforming a cell to express an aptamer to said at least one polypeptide.

20. The method of claim 13 or 14 wherein said decreasing activity comprises introducing into a cell an aptamer to said at least one polypeptide.

21. The method claim 13 or 14 wherein said decreasing activity comprises administering to a cell an antibody that selectively binds to said at least one polypeptide.

22. A method of treating tumors comprising decreasing angiogenesis by the method of claim 12 or 13.

23. A method of treating cancer comprising treating a cancerous tumor by the method of claim 22.

24. A method of treating myocardial infarction comprising increasing angiogenesis by the method of claim 11 or 14.

25. A method of promoting healing comprising increasing angiogenesis by the method of claim 11 or 14.

26. A method for determining whether a compound up-regulates or down- regulates the transcription of an AAP gene, comprising: contacting said compound with a composition comprising a RNA polymerase and said gene and measuring the amount of said AAP gene transcription.

27. The method of claim 26, wherein said composition is in a cell.

28. A method for determining whether a compound up-regulates or down- regulates the translation of an AAP gene, comprising: contacting said compound with a composition comprising a ribosome and a polynucleotide corresponding to a mRNA of said gene and measuring the amount of said AAP gene translation.

29. The method of claim 28, wherein said composition is in a cell.

30. A vector, comprising the polynucleotide of any one of claims 5-8.

31. A cell, comprising the vector of claim 30.

32. A method of screening a tissue sample for tumorigenic potential, comprising: measuring expression of at least one AAP gene in said tissue sample.

33. The method of claim 32, wherein said measuring is measuring an amount of a polypeptide encoded by said at least one AAP gene.

34. The method of claim 32, wherein said measuring expression is measuring an amount of mRNA corresponding to said at least one AAP gene.

35. A transgenic non-human animal, having at least one disrupted AAP gene.

36. The transgenic non-human animal of claim 35, wherein the non-human animal is a mouse.

37. A transgenic non-human animal, comprising an exogenous polynucleotide having at least 80% sequence identity to the sequence of SEQ TD NOS:l, 3, 5, 7, 9, 11, 13 or 15 , or a complement of said polynucleotide.

38. The transgenic non-human animal of claim 37, wherein said exogenous polynucleotide has at least 90% sequence identity to the sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13 or 15 , or a complement of said polynucleotide.

39. The transgenic non-human animal of claim 37, wherein said exogenous polynucleotide has at least 98% sequence identity to the sequence of SEQ ID NOS:l, 3, 5, 7, 9, 11, 13 or 15 , or a complement of said polynucleotide.

40. A method of screening a sample for an AAP gene mutation, comprising: comparing an AAP nucleotide sequence in the sample with SEQ ID NOS:l, 3, 5,

7, 9, 11, 13 or 15 .

41. A method of determining the clinical stage of tumor comprising comparing expression of at least one AAP gene in a sample with expression of said at least one gene in control samples.