AU774591B2 - Novel molecules - Google Patents

Description

Regulation 3.2

AUSTRALIA

Patents Act 1990 DIVISIONAL APPLICATION Name of Applicant: Actual Inventor(s): Address for Service: Invention Title: The CouocQ or~ 'Ie &)ueehs1arlct Ynfi-#le f~ Mec! ,W ieee AAARAD Operations Pty T Ad ANTALIS, Toni Marie and HOOPER, John David DAVIES COLLISON CAVE, Patent Attorneys, Level 3, 303 Coronation Drive, Milton, Queensland, 4064, Australia "Novel molecules" Details of Parent Application No: 59734/98 The following statement is a full description of this invention, including the best method of performing it known to me/us: Q AOpcAVpa\23637o3 divisional filing.35 7 .doc 22/12/00 1A'- NOVEL MOLECULES FIELD OF THE INVENTION The present invention related generally to novel molecules and more particularly novel proteinaceous molecules involved in or associated with regulation of cell activities and/or viability. The present invention is particularly directed to novel serine proteinases and a novel kinase and to derivatives, agonists and antagonists thereof and to genetic sequences encoding same. In one embodiment, the present invention provides a novel serine proteinase, referred to herein as "HELA2" or "testisin", which has roles in spermatogenesis, in suppressing testicular cancer and as a marker for cancers. The present invention further provides cDNA and genomic sequences encoding the novel serine proteinases as well as promoters controlling expression of said sequence.

15 BACKGROUND OF THE INVENTION The rapidly increasing sophistication of recombinant DNA technology is greatly facilitating research and development in the medical and allied health fields. This is particularly the case in the area of cell regulation leading to a greater understanding of the events leading to or involved in cancer, development of acquired immunodeficiency disease syndrome (AIDS), neurological disorders, heart disease, tissue graft rejection and infertility amongst many other conditions.

Two particularly important classes of molecules are the proteinases and kinases.

25 Proteinases play important roles in a number of physiological and pathological processes such as proteolytic cascades involved in blood coagulation, fibrinolysis and complement activation as well as cleavage of growth factors, hormones and receptors, the release of bioactive molecules and processes involving cell proliferation and development, inflammation, tumour growth and metastasis. Of particular significance are the cellular proteinases, or those proteinases synthesized in cells and tissues which serve to activate or deactivate proteins responsible for performing specific functions. These proteinases may be found outside the cell, within the cell or may be present on the cell surface.

Serine proteinases are particularly important. These proteinases are characterised by a mechanism involving serine, histidine and aspartate amino acids in the serine proteinase active site. Members of the serine proteinase family which play important roles in a range of cellular functions and which have demonstrated causative roles in human diseases include tissue-type plasminogen activator and thrombin (thrombosis and blood clotting), urokinase-type plasminogen activator (cancer and metastasis), trypsin and elastase (emphysema and liver disease) and angiotensin converting enzyme (hypertension).

A serine proteinase is also implicated in TNFa degradation and soluble TNF-receptor release by THP1 cells (Vey et al. Eur. J. Imm. 26, 2404-2409, 1996). Serine proteinases have been implicated in the activation ofmacrophages (Nakabo et al. J. Leukocyte Biol. 60, 328-336, 1996), in nuclear laminin degradation in apoptosis (McConkey et al. J.Biol. Chem., 271, 22398- 15 22406, 1996), in prostaglandin-E2 induced release of soluble TNF receptor shedding (Choi et al. Cellular Immunology 170, 178-184, 1996), in PAF synthesis (Bussolino et al. Eur. J.

Immunol. 24, 3131-3139, 1994), and in the proteolysis of IkB, a regulatory molecule important in signal transduction and apoptosis. Release of serine proteinases known as granzymes is central to CTL killing and many of the substrates cleaved by granzymes are also cleaved by cellular proteinases (for example, IL-1 3 is a substrate for Granzyme B as well as the cysteine proteinase, interleukin 1 P-converting enzyme Granzyme A, a serine proteinase with Arg-amidolytic activity, has been reported to induce the production of 1L-6 and IL-8 in lung fibroblasts (Sower et al. Cellular Immunology 171, 159-163, 1996) and cleaves EL- 1 to a 17kD mature form that is biologically active.

Kinases are a large group of molecules, many of which regulate the response of cells to external stimuli. These molecules regulate proliferation and differentiation in eukaryotic cells frequently via signal transduction pathways.

The identification of new serine proteinases and kinases permits the development of a range of derivatives, agonists and antagonists at the nucleic acid and protein levels which in turn have applications in the treatment and diagnosis of a range of conditions such as cancer, inflammation, neurological disorders amongst many other conditions including conditions which initiate or promote apoptosis such as viral infection, old age and drug abuse. One particularly useful serine proteinase HELA2 (testisin) identified in accordance with the present invention is involved in spermatogenesis, testicular cancer and as a marker for cancer. Other useful molecules are referred to herein as ATC2 and BCON3. ATC2 is particularly useful in assessing and treating cardiac physiology.

SUMMARY OF THE INVENTION Sequence Identity Numbers (SEQ ID NOs.) for the nucleotide and amino acid sequences referred to in the specification are defined at the end of the subject specification.

Throughout this specification and the claims which follow, unless the context requires otherwise, 15 the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

One aspect of the present invention provides a novel molecule in isolated form involved in or associated with regulation of cell activity and/or viability.

I: Another aspect of the present invention contemplates an isolated proteinaceous molecule involved in or associated with regulation of cell activity and/or viability comprising a sequence of amino acids encoded by a nucleotide sequence, at least a portion of which, is capable of being 25 amplified by polymerase chain reaction (PCR) using the following primers: ACAGAATTCTGGGTIGTIACIGCIGCICAYTG3' [SEQ ID NO:1]; and ACAGAATTCAXIGGICCICCIC/GT/AXTCICC3' [SEQ ID NO:2]; or a complementary form of said primers.

The proteinaceous molecule of the present invention may be a serine proteinase or a kinase.

Yet another aspect of the present invention is directed to an isolated serine proteinase comprising the amino acid sequence substantially set forth in SEQ ID NO:4 or an amino acid sequence having at least about 50% similarity to all or part thereof. This serine proteinase is referred to herein as a short isoform of "HELA2" or "testisin".

Still another aspect of the present invention relates to an isolated serine proteinase comprising the amino acid sequence substantially as set forth in SEQ ID NO:6 or an amino acid sequence having at least about 50% similarity to all or part thereof. This serine proteinase is referred to herein as a long isoform of HELA2 (testisin).

Still yet another aspect of the present invention provides an isolated serine proteinase comprising an amino acid sequence substantially as set forth in SEQ ID NO:8 or an amino acid sequence *15 having at least about 50% similarity to all or part thereof. This serine proteinase is referred to herein as "ATC2".

Even yet another aspect of the present invention is directed to a serine proteinase in isolated form comprising a sequence of amino acids encoded by a nucleotide sequence substantially as set forth 20 in SEQ ID NO:3 or a nucleotide sequence having at least 50% similarity to all or part thereof or a nucleotide sequence capable of hybridising to the sequence set forth in SEQ ID NO:3 under low stringency conditions at 42 0

C.

SAnother aspect of the present invention relates to a serine proteinase in isolated form comprising 25 a sequence of amino acids encoded by a nucleotide sequence substantially as set forth in SEQ ID NO:5 or a nucleotide sequence having at least 50% similarity to all or part thereof or a nucleotide sequence capable of hybridising to the sequence set forth in SEQ ID NO:5 under low stringency conditions at 42 0

C.

Still another aspect of the present invention provides a serine proteinase in isolated form comprising a sequence of amino acids encoded by a nucleotide sequence substantially as set forth in SEQ ID NO:7 or a nucleotide sequence having at least 50% similarity to all or part thereof or a nucleotide sequence capable of hybridising to the sequence set forth in SEQ ID NO:7 under low stringency conditions at 420C.

Another embodiment of the present invention is directed to a kinase in isolated form comprising an amino acid sequence substantially as set forth in SEQ ID NO:10 or having 50% amino acid similarity to all or part thereof. This kinase is referred to herein as "BCON3".

In a related embodiment, the kinase comprises an amino acid sequence encoded by a nucleotide sequence substantially as set forth in SEQ ID NO:9 or a nucleotide sequence having at least similarity to all or part of the nucleotide sequence set forth in SEQ ID NO:9 or a nucleotide sequence capable of hybridising to the nucleotide sequence set forth in SEQ ID NO:9 under low stringency conditions at 42°C.

15 The present invention further provides an isolated nucleic acid molecule encoding a polypeptide .wherein at least a portion of said nucleic acid molecule is capable of being amplified by polymerase chain reaction (PCR) using the following primers: ACAGAATTCTGGGTIGTIACIGCIGCICAYTG3' [SEQ ID NO: and 5'ACAGAATTCAXIGGICCICCIC/GT/AXTCICC3' [SEQ ID NO:2]; o* or a complementary form of said primers.

.I 25 The present invention also provides an isolated nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:3 or having 50% similarity to all or part thereof or a nucleic acid molecule capable of hybridising to SEQ ID NO:3 under low stringency conditions at 42°C.

Another aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:5 or having 50% similarity to -6all or part thereof or a nucleic acid molecule capable of hybridising to SEQ ID NO:5 under low stringency conditions at 42°C.

Still another aspect of the present invention is directed to an isolated nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:7 or having similarity to all or part thereof or a nucleic acid molecule capable of hybridising to SEQ ID NO:7 under low stringency conditions at 42 0

C.

Even still another aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:9 or having similarity to all or part thereof or a nucleic acid molecule capable of hybridising to SEQ ID NO:9 under low stringency conditions at 42 0

C.

Another aspect of the present invention provides an isolated serine proteinase encoded by a gene 15 proximal to a cluster of genes on a mammalian chromosome.

More particularly, this aspect of the present invention is directed to a serine proteinase encoded by a gene proximal to a cluster of genes or human chromosome 16pl3.3 or its equivalent in a non-human species.

Still more particularly, the serine proteinase is encoded by a gene comprising a nucleotide sequence substantially as set forth in SEQ ID NO:3 or 5 or 28 or 29 or 30 or a nucleotide sequence having at least 50% similarity to any one thereof or a nucleotide sequence capable of hybridizing to any one of SEQ ID NO:3 or 5 or 28 or 29 or 30 under low stringency conditions S. 25 at 42°C or a nucleotide sequence encoding a serine proteinase having an amino acid sequence substantially as set forth in SEQ ID NO:4 or 6 or an amino acid sequence having at least about similarity to SEQ ID NO:4 or 6.

Another aspect of the present invention provides a nucleic acid molecule comprising a nucleotide sequence substantially as set forth in SEQ ID NO: 27 or a nucleotide sequence having at least similarity thereto or a nucleotide sequence capable of hybridizing thereto under low -7stringency conditions at 42 0 C which nucleic acid molecule includes a promoter or part thereof for testisin.

a a a.

a.

a a a.

a.

a a a.

a. a a a.

a a.

-8- BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a representation showing schematic and hydrophobicity plot of the HELA2 amino acid sequence.

Figure 2 is a diagrammatic representation showing: the amino acid sequence of HELA2 (testisin). The putative signal sequence, light chain, heavy chain and transmembrane domains are as indicated, the catalytic amino acids, His, Asp and Ser are as designated; insertion of Tyr-Ser (YS) 4 amino acids after the catalytic His is found in the long isoform of testisin and is due to alternative mRNA splicing; in vitro transcription/translation of HELA2 (testisin) showing the protein product.

Figure 3 is a diagrammatic representation of plasmid constructs pBluescriptHELA2(S) and pBluescriptHELA2(L) containing full length cDNAs for testisin (short isoform and testisin 15 (long isoform respectively.

Figure 4 is a diagrammatic representation of plasmid constructs pQET(20-295)N and 295)C, wherein the hydrophobic residues of testisin were removed and the remaining sequences cloned into pQE prokaryotic expression plasmids; plasmids pGEX-1 (90-279) comprising a carboxy terminal part of testisin fused to glutathione-S-transferase.

Figure 5 is a photographic representation of: silver stained gel showing purification of o* recombinant HELA2 (testisin) from E. coli. The purified HELA2 (testisin) is indicated by the arrow in the eluate fractions. Some HELA2 (testisin) is also found in the wash fractions as the 25 affinity matrix was not used in excess. His-N21 is one clone containing the amino-terminal His tag, and clones His-C21, His-C22 and His-C23 are three different clones with the carboxyterminal His tag. Western blot of native and denatured recombinant HELA2 (testisin) probed with Clontech anti-His tag-antibody. The 32kD band shown by the arrow is HELA2 (testisin). HELA2 (testisin) is not detected in the denatured samples as it appears that denaturation with urea destroys the His epitope recognised by the monoclonal antibody.

-9- Figure 6 is a representation of the amino acid sequence of HELA2 (testisin) showing the regions of the molecule selected for generation of peptide antigens.

Figure 7 is a photographic representation of a Western blot of HeLa cells and testis tissue under non-reducing and reducing conditions showing the native testisin protein.

Figure 8 is a diagrammatic representation ofeukaryotic expression constructs, pcDNA3-Test(SpcDNA3-Test(L-C) and pcDNA3-Test(1-297)L-C.

Figure 9 is a diagrammatic representation showing a histogram of the signal intensity from a Clontech Master RNA blot of the tissue distribution of HELA2 (testisin) in RNA from different normal tissues. Probed with HELA2 (testisin) specific probe; Probed with BCON3 specific prove which is ubiquitously expressed. The 8 tissues on the right hand side of the diagram are the control (negative) samples.

Figure 10 is a photographic representation of a multiple normal tissue Northern blot (Clonetech) S'. probed with: HELA2 (testisin) specific probe and BCON3 specific probe.

Figure 11 is a photographic representation of agarose gel of PCR products generated by 20 amplification of HELA2 (testisin) cDNA in prevasectomised and post-vasectomised ejaculate specimens. The HELA2 (testisin) PCR product is 464bp and the P2-macroglobulin product is 250 bp.

Figure 12 is a photographic representation of in situ hybridization of rat testis showing the 25 localisation of HELA2 (testisin) mRNA to the germ cells of the testis.

Figure 13 is a representation showing: spread of normal metaphase chromosomes showing bright dots where HELA2 (testisin) is expressed at 16p13.3; Diagrammatic representation of chromosome 16p13.3 showing location of HELA (testisin) and relationship to other disease causing genes.

Figure 14 is: a photographic representation of northern blot analysis of HELA2 (testisin) mRNA showing signals in normal testis of 4 patients and absence of signal in the tumours of these patients; a photographic representation of the localisation of HELA2 (testisin) protein in a human germ cell tumour section assessed by immunohistochemical staining using anti- HELA2 (testisin) peptide antibodies. Staining is only detected in the normal tissue and not present in the tumour tissue.

Figure 15 is a diagrammatic representation of the genomic map of HELA2 (testisin) showing experimentally determined intron/exon boundaries and relative sizes of the introns (marked with a letter) and exons (marked with a roman numeral).

Figure 16 is a representation of the HELA2 (testisin) nucleotide sequence. Nucleotides in introns are in lowercase and exons in uppercase. The putative transcription start site is marked by +1.

o Figure 17 is a representation of the DNA sequence of Intron C and flanking exons showing where alternative mRNA splicing occurs to generate the two isoforms of HELA2 (testisin).

Figure 18 is a representation of: the cDNA sequence of the mouse homologue of HELA2 (testisin). Catalytic residues are indicated by circles and cysteines likely involved in disulfide bonding are indicated by squares; Hydrophobicity plot of HELA2 (testisin) amino acid sequence.

Figure 19 is a diagrammatic representation of chromosome 16p13.3 showing the serine 25 proteinase gene cluster which includes HELA2 (testisin). Lines represent cosmids containing the respective serine proteinase genes.

Figure 20A is a representation of: the cDNA sequence of SP001LA (SEQ ID NO:34).

Catalytic residues are indicated by circles and cysteins likely involved in disulfide bonding are indicated by squares; hydrophobicity plots of SP001LA amino acid sequence.

-11- Figure 20B is a representation of: the cDNA sequence of SP002LA (SEQ ID Catalytic residues are indicated by circles and cysteines likely involved in disulfide bonding are indicated by squares. Hydrophobicity plot of SP002LA amino acid sequence.

Figure 20C is a representation of: the cDNA sequence of SP003LA (SEQ ID NO:36).

Catalytic residues are indicated by circles and cysteines likely involved in disulfide bonding are indicated by squares. Hydrophobicity plot of SP003LA amino acid sequence.

Figure 21 is a photographic representation of in vitro transcription/translation of BCON3 showing the protein products.

Figure 22 shows a photographic representation of northern blot analysis of polyA+ RNA obtained from 16 different normal tissues (Clontech Multiple Tissue Blots). Blots were probed with radiolabelled ATC2 DNA insert for 3h and washed to a final stringency of 0. lxSSC/0.1% O 15 w/v SDS at 60 0 C. a photographic representation of northern blot analysis of RNA obtained from different mouse tissues washed at lower stringency (0.5xSSC/0.1% w/v SDS at 55°C). (C) a diagrammic representation of ATC2 mRNA and the partial clones which span about 2.8kb (hatched box). The predicted structure of the catalytic region is shown at the top including the predicted disulfide linkages and the amino acid residues of the catalytic triad typical of serine proteinases.

-12- A summary of the SEQ ID NOs used throughout the specification is presented in Table 1.

TABLE 1

C

S

S..

*5

SS

S

S

S

S S

S

S.

S

00 SEQ ID NO 1 2 3 4 6 7 8 9 10 11 12 13 14 15 16 17 18 19 21

DESCRIPTION

PCR primer sequence PCR primer sequence Nucleotide sequence of short form of HELA2 Amino acid sequence of short form of HELA2 Nucleotide sequence of long form of HELA2 Amino acid sequence of long form of HELA2 Nucleotide acid sequence of ATC2 Amino acid sequence of ATC2 Nucleotide acid sequence of BCOM3 Amino acid sequence of BCOM3 Primers used to generate amino terminal tagged protein Primers used to generate amino terminal tagged protein Primers used to generated carboxy-linked terminal protein Primers used to generated carboxy-linked terminal protein Peptide antigen T20-33 Peptide antigen T46-63 Peptide antigen T175-190 Forward primer Reverse primer Forward primer Reverse primer 13- TABLE 1 (Continued) 22 Forward primer 23 Reverse primer 24 Serine proteinase activation motif 26 Mouse HELA2 cDNA sequence 27 Human HELA genomic DNA sequence 28 Amino acid sequence corresponding to human genomic nucleotide sequences of HELA2 29 Amino acid sequence corresponding to human genomic nucleotide sequences of HELA2 30 Amino acid sequence corresponding to human genomic nucleotide sequences of HELA2 31 Amino acid sequence corresponding to human genomic nucleotide sequences of HELA2 32 Amino acid sequence corresponding to human genomic nucleotide sequences of HELA2 33 Amino acid sequence corresponding to human genomic nucleotide sequences of HELA2 34 Clustered serine proteinase gene SP001LA *fe* 35 Clustered serine proteinase gene SP002LA 36 Clustered serine proteinase gene SP003LA 37 ATC2 peptide antigen A1A 38 ATC2 peptide antigen A1B 0 Abbreviations: X= A or G Y= C or T I= Inosine.

14 A list of single and three letter abbreviations for amino acid residues is presented in Table 2.

TABLE 2 Amino Acid Three-letter One-letter Abbreviation Symbol Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp

D

Cysteine Cys

C

Glutamnine Gin

Q

Glutamnic acid Glu E 15 Glycine Gly G *Histidine His

H

:Isoleucine Ile

I

Leucine Leu

L

Lysine Lys

K

ivieuuonine Met

M

Phenylalanine Phe

F

.:Proline Pro

P

Serine Ser

S

Threonine Thr

T

Tryptophan Trp,

W

Tyrosine Tyr

Y

Valine Val

V

Any residue Xaa

X

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS The present invention is predicated in part on a genetic engineering approach to identify nucleotide sequences encoding serine proteinases or kinases. The genetic engineering approach is based on the use of degenerate primers corresponding to conserved regions of serine proteinases (amino acids flanking His- and Ser- residues) to amplify gene fragments spanning these regions for cDNA, using low stringency reverse transcriptase-polymerase chain reaction

(RT-PCR).

This technique has been successfully used, in accordance with the present invention, to identify serine proteinases and kinases useful in modulating cell activity and viability including modulating spermatogenesis, acting as tumour suppressors and acting as a marker for non-testicular cancers.

Accordingly, one aspect of the present invention provides a novel molecule in isolated form 15 involved in or associated with regulation of cell activity and/or viability.

More particularly, the present invention contemplates a novel serine proteinase in isolated form comprising a sequence of amino acids encoded by a nucleotide sequence, at least a portion of which, is capable of being amplified by polymerase chain reaction (PCR) using the following 20 primers: 5' ACAGAATTCTGGGTIGTIACIGCIGCICAYTG3' [SEQ ID NO: and o• 5'ACAGAATTCAXIGGICCICCIC/GT/AXTCICC3' [SEQ ID NO:2]; or a complementary form of said primers.

Preferably, X is A or G, Y is C or T and I is inosine.

In a particularly preferred embodiment, the isolated serine proteinase comprises the amino acid sequence substantially set forth in SEQ ID NO:4 or an amino acid sequence having at least about -16similarity to all or part thereof. This serine proteinase is referred to herein as a short isoform of "HELA2" or "HELA2 (testisin)". The terms "HELA2" and "testisin" are used interchangedly throughout the subject specification to refer to the same molecule.

Genetic sequences encoding HELA2 (testisin) and other molecules are represented in italicized form.

In another preferred embodiment, the amino acid sequence of the serine proteinase is substantially as set forth in SEQ ID NO:6 or an amino acid sequence having at least about similarity to all or part thereof. This serine proteinase is the long isoform of HELA2 or HELA2 (testisin).

Yet another preferred embodiment of the present invention provides an amino acid sequence substantially as set forth in SEQ ID NO:8 or an amino acid sequence having at least about S 15 similarity to all or part thereof. This serine proteinase is referred to herein as "ATC2".

Another aspect of the present invention relates to a serine proteinase in isolated form comprising °a sequence of amino acids encoded by a nucleotide sequence substantially as set forth in SEQ ID NO:3 or a nucleotide sequence having at least 50% similarity to all or part thereof or a 20 nucleotide sequence capable of hybridising to the sequence set forth in SEQ ID NO:3 under low stringency conditions at 42"C.

Still another aspect of the present invention is directed to a serine proteinase in isolated form comprising a sequence of amino acids encoded by a nucleotide sequence substantially as set forth 25 in SEQ ID NO:5 or a nucleotide sequence having at least 50% similarity to all or part thereof or a nucleotide sequence capable of hybridising to the sequence set forth in SEQ ID NO:5 under low stringency conditions at 42"C.

In another aspect of the present invention, there is provided a serine proteinase in isolated form comprising a sequence of amino acids encoded by a nucleotide sequence substantially as set forth in SEQ ID NO:7 or a nucleotide sequence having at least 50% similarity to all or part thereof or 17a nucleotide sequence capable of hybridising to the sequence set forth in SEQ ID NO:7 under low stringency conditions at 42 0

C.

Another embodiment of the present invention is directed to a kinase in isolated form comprising an amino acid sequence substantially as set forth in SEQ ID NO: 10 or having 50% amino acid similarity to all or part thereof. This kinase is referred to herein as "BCON3".

In a related embodiment, the kinase comprises an amino acid sequence encoded by a nucleotide sequence substantially as set forth in SEQ ID NO:9 or a nucleotide sequence having at least similarity to all or part of the nucleotide sequence set forth in SEQ ID NO:9 or a nucleotide sequence capable of hybridising to the nucleotide sequence set forth in SEQ ID NO:9 under low stringency conditions at 42"C.

The present invention further provides an isolated nucleic acid molecule comprising a sequence 15 of nucleotides encoding or complementary to a sequence encoding a novel molecule involved in or associated with regulation of cell activity and/or viability. Preferably, the nucleic acid molecule is capable of being amplified by PCR using the primers set forth in SEQ ID NO:1 and/or SEQ ID NO:2.

More particularly, the present invention further provides an isolated nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:3 or having similarity to all or part thereof or a nucleic acid molecule capable of hybridising to SEQ ID NO:3 under low stringency conditions at 42°C.

25 Another aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:5 or having 50% similarity to all or part thereof or a nucleic acid molecule capable of hybridising to SEQ ID NO:5 under low stringency conditions at 42°C.

Another aspect of the present invention is directed to an isolated nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:7 or having 18similarity to all or part thereof or a nucleic acid molecule capable of hybridising to SEQ ID NO:7 under low stringency conditions at 42°C Still another aspect of the present invention provides an isolated nucleic acid molecule comprising a sequence of nucleotides substantially as set forth in SEQ ID NO:9 or having similarity to all or part thereof or a nucleic acid molecule capable of hybridising to SEQ ID NO:9 under low stringency conditions at 42*C.

Reference herein to a low stringency includes low stringency at 42 0 C includes and encompasses from at least about 1% v/v to at least about 15% v/v formamide and from at least about 1M to at least about 2M salt for hybridisation, and at least about 1M to at least about 2M salt for washing conditions. Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5M to at least about 0.9M salt for 15 hybridisation, and at least about 0.5M to at least about 0.9M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about v/v formamide and from at least about 0.01M to at least about 0.15M salt for hybridisation, and at least about 0.01M to at least about 0.15M salt for washing conditions. In general, washing is carried out Tm 69.3 0.41 (Marmur, J and Doty, However, the Tm of a duplex DNA decreases by 1lC with every increase of 1% in the number of mismatch base pairs (Bonner, W. M. and Laskey, R. Reference herein to similarity to "part" of a sequence means similarity to at least about 4 contiguous amino acids or at least about 12 contiguous nucleotide bases and more preferably at '25 least about 7 contiguous amino acids or at least about 21 contiguous nucleotide bases.

The term "similarity" includes exact identity between sequences or, where the sequence differs, different amino acids may be related to each other at the structural, functional, biochemical and/or conformational levels.

Preferably, comparisons of nucleotide and amino acid sequences are in terms of percentage P\OPEREJH\23942-C.US- 12/898 19identity and this includes the number of exact nucleotide or amino acid matches having regard to an appropriate alignment using a standard algorithm, such as but not limited to the Geneworks programme (Intelligenetics).

The term "isolated" includes biological purification and biological separation and encompasses molecules having undergone at least one purification, concentration or separation step relative to its natural environment. For example, a preparation may comprise at least about preferably at least about 20%, more preferably at least about 30%, still more preferably at least about 50% or greater of the molecule relative to at least one other component in a composition as determined by activity, mass, amino acid content, nucleotide content or other convenient means.

Hereinafter, the molecules of the present invention are referred to as a "proteinase/kinase". The term "proteinase/kinase" includes the serine proteinases HELA2 (testisin) and ATC2 and the 15 kinase BCON3. The proteinase/kinase of the present invention may be in isolated, naturally occurring form or recombinant or synthetic form or chemical analogues thereof.

The proteinase/kinase of the present invention is preferably of human origin but from non-human origins are also encompassed by the present invention. Non-human animals contemplated by the 20 present invention include primates, livestock animals sheep, cows, pigs, goats, horses, donkeys), laboratory test animals mice, rats, guinea pigs, hamsters, rabbits), domestic companion animals dogs, cats), birds chickens, geese, ducks and other poultry birds, game birds, emus, ostriches) and captive wild or tamed animals foxes, kangaroos, dingoes).

The present invention also encompasses a proteinase/kinase homologue from Xenopus and 25 plants.

The nucleic acid molecules encoding a proteinase/kinase may be genomic DNA, cDNA or RNA such as mRNA.

Yet another aspect of the present invention provides an isolated serine proteinase encoded by a gene proximal to a cluster of genes on a mammalian chromosome. The cluster of genes is P:OPERMEAP3942C.US 12/98 preferably on human chromosome 16pl3.3 or its equivalent in a non-human species. The cluster is made up of genes all encoding or having the potential to encode a serine proteinase or homologue, derivative or functional or evolutionary equivalent thereof. Preferably, the gene cluster comprises two or more of genes comprising a nucleotide sequence selected from SEQ ID NO:3 and 5 (HELA2, short and long forms, respectively) and SEQ ID NO:34 (SP001LA), SEQ ID NO:35 (SP002LA), SEQ ID NO:36 (SP003LA) and SP004LA (see Figure 19) or a nucleotide sequence having at least 50% similarity to any one of those sequences or capable of hybridizing to any one of those sequences under low stringency conditions at 42°C.

The term "proximal" is used in its broadest sense to mean a gene cluster and includes a gene within proximity to another gene.

Another aspect of the present invention contemplates a method for cloning a nucleotide sequence encoding a novel serine proteinase, said method comprising screening a nucleic acid library with 15 said one or more or oligonucleotides defined by SEQ ID NO:1 and/or SEQ ID NO:2 and obtaining a clone therefrom which encodes said novel serine proteinase or part thereof.

Preferably, the nucleic acid library is genomic DNA, cDNA, genomic or mRNA library.

20 Preferably, the nucleic acid library is a cDNA expression library.

Preferably, the nucleic acid library is of human origin such as from brain, liver, kidney, neo-natal tissue, embryonic tissue, tumour or cancer tissue.

25 With respect to HELA2 (HELA2 (testisin)), significant expression is generally only found in normal testis with respect to ATC2, expression is generally directed to cardiac tissue.

Accordingly, the present invention extends to nucleic acid molecules capable of tissue-specific or substantially tissue-specific expression.

Still another embodiment contemplates the promoter or a functional part thereof of the genomic gene encoding the subject proteinase/kinase of the present invention. The promoter may readily P\OPER\EJH023942-C.US 12/8/98 -21 be obtained by, for example, "chromosome walking". A particularly useful promoter is from HELA2 (testisin) which can be regarded as a testis specific promoter. This promoter can be used, for example, to direct testis specific expression of genetic sequences operably linked to the promoter and may be used inter alia gene therapy or modulation of fertility. The promoter for the HELA2 (testisin) gene comprises at least part of the nucleotide sequence set forth in SEQ ID NO:27.

Accordingly, another aspect of the present invention provides a nucleic acid molecule comprising a nucleotide sequence substantially as set forth in SEQ ID NO: 27 or a nucleotide sequence having at least 50% similarity thereto or a nucleotide sequence capable of hybridizing thereto under low stringency conditions at 42 0 C which nucleic acid molecule includes a promoter or part thereof for testisin.

The present invention further contemplates a range of derivatives of the subject S: 15 proteinase/kinase. Derivatives include fragments, parts, portions, mutants, homologues and S.analogues of the subject polypeptides and corresponding genetic sequences. Derivatives also include single or multiple amino acid substitutions, deletions and/or additions to the subject molecules or single or multiple nucleotide substitutions, deletions and/or additions to the genetic sequence encoding the molecules. "Additions" to amino acid sequences or nucleotide sequences include fusions with other peptides, polypeptides or proteins or fusions to nucleotide sequences.

Reference herein to the serine proteinase and kinase includes reference to all derivatives thereof including functional derivatives or immunologically interactive derivatives.

Analogues of the subject serine proteinase and kinase contemplated herein include, but are not 25 limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogues.

Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde P:\OPER\EH\023942-C.US 12/8/98 -22followed by reduction with NaBH 4 amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with phosphate followed by reduction with NaBH 4 The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides 15 with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted S. maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and mercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides.

Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.

25 Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4 -amino- 3 -hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of PA.OPER\ErnHn3942-C.US. I M89 23 amino acids. A list of unnatural amino acid, contemplated herein is shown in Table 3.

V

V.

V VV V P:%OPER\EJ1{I23942-C.US 12/089 24 TABLE 3 Non-conventional Code Non-conventional Code amino acid amino acid a.

a. a a a.

c-amninobutyric acid Abu c-amino-a-methylbutyrate Mgabu aminocyclopropane- Cpro carboxylate aminoisobutyric acid Aib aminonorbomyl- Norb carboxylate cyclohexylalanine cyclopentylalanine Cpen 15 D-alanine Dal D-arginine Darg D-aspartic acid Dasp D-cysteine Dcys D-glutamine Dgln D-glutamnic acid Dglu D-histidine Dis D-isoleucine Dile D-leucine Dleu D-lysine Dlys 25 D-methionine Dmet D-omnithine Dorn D-phenylalanine Dphe D-proline Dpro D-serine Dser D-threonine Dthr D-tryptophan Dtrp L-N-methylalanine L-N-methylarginine L-N-methylasparagine L-N-methylaspartic acid L-N-methylcysteine L-N-methylglutamine L-N-methylglutamic acid Chexa L-N-methylhistidine L-N-methylisolleucine L-N-methylleucine L-N-methyllysine L-N-methylmethionine L-N-methylnorleucine L-N-methylnorvaline L-N-methylomnithine L-N-methylphenylalanine L-N-methylproline L-N-methylserine L-N-methylthreonine L-N-methyltryptophan L-N-methyltyrosine L-N-methylvaline L-N-methylethylglycine L-N-methyl-t-butylglycine L-norleucine L-norvaline Nmala Nmarg Nmasn Nmasp Nmcys Nmgln Nmglu Nmhis Nrnile Nmleu Nmlys Nmmet Nmnle Nmnva Nmorn Nmphe Nmpro Nmser Nmthr Nmtrp Nmtyr Nmval Nmetg Nmtbug NMe Nva P.%OPER\FJHM3942-C.US IMS D-tyrosine D-valine D-a-methylalanine D-a-methylarginine D-a-methylasparagine D-a-methylaspartate D-a-methylcysteine D-a-methylglutamine D-a-methylWstidine D-a-methylisoleucine D-a-methylleucine D-a-methyllysine D-a-methylmethionine D-a-methylomitWne D-a-methylphenyWanine D-a-methylproline D-a-methylserine D-a-methylthreonine D-a-methyltryptophan D-a-methyltyrosine D-a-methylvaline D-N-methylalanine D-N-methylarginine D-N-methylasparagine D-N-methylaspartate D-N-methylcysteine D-N-methylglutarr ne D-N-methylglutamate D-N-methylhistidine D-N-methylisoleucine D-N-methylleucine Dtyr Dval Dmala Dmarg Dmasn Dmasp Dmcys Dmgln Dmhis Dmile Drudeu Dmlys Dmmet Dmom Dmphe Dmpro Dmser Dmthr Dmtrp Dmty Dmval Drimala Dnmarg D=asn Dninasp Druncys Dnmgln Dnmglu Dnrnhis Dnmile Dnn-deu a-methyl-arr-dnoisobutyrate a-methyl-y-aminobutyrate a-methylcyclohexyWanine a-methylcylcopentylalanine a-methyl-a-napthylalanine a-methylpenicillarnine N-(4-aminobutyl)glycine N-(2-arr noethyl)glycine N-(3-aminopropyl)glycine N-amino-a-methylbutyrate a-napthylalanine N-benzylglycine N-(2-carbamylethyl)glycine N-(carbamylmethyl)glycine N-(2-carboxyethyl)glycine N-(carboxymethyl)glycine N-cyclobutylglycine N-cycloheptylglycine N-cyclohexylglycine N-cyclodecylglycine N-cylcododecylglycine N-cyclooctylglycine N-cyclopropylglycine N-cycloundecylglycine N-(2,2-diphenylethyl)glycine N-(3,3-diphenylpropyl)glycine N-(3-guanidinopropyl)glycine N-(l -hydroxyethyl)glycine N-(hydroxyethyl))glycine N-(in-ddazolylethyl))glycine N-(3-indolylyethyl)glycine Maib Mgabu Mchexa Mcpen Manap Mpen Nglu Naeg Nom Nmaabu Anap Nphe Ngln Nasn Nglu Nasp Ncbut Nchep Nchex Ncdec Ncdod Ncoct Ncpro Ncund Nbhm Nbhe Narg Nthr Nser Nhis Nhtrp P-.OPER\EJfM3942-C.US 12/0~8 26 D-N-methyllysine N-methylcyclohexylalanine D-N-methylornithine N-methylglycine N-methylaminoisobutyrate 1 -methylpropyl)glycine N-(2-methylpropyl)glycine D-N-methylt-yptophan D-N-methyltyrosine D-N-methylvaline y-aminobutyric acid L-t-butylglycine L-ethylglycine L-homnophenylalanine L-a-methylarginine L-a-methylaspartate L-a-methylcysteine L-cz-methylglutamine L-a-methylhistidine 20 L-a-methylisoleucine L-a-methylleucine L-ac-methylmethionine L-a-methylnorvaline L-a-methylphenylalanine 25 L-a-methylserine L- a-methyltryptophan Dnmlys Nmchexa Dnmom Nala Nmaib Nile Nleu Dnmtrp Dnmtyr Dnmnval Gabu Thug Etg Hphe Marg Masp Mcys Mgln Mhis MWe Mleu Mmet Mnva Mphe Mser Mtrp N-methyl-y -aminobutyrate D-N-methylmethionine N-methylcyclopentylalanine D-N-methylphenylalanine D-N-methylproline D-N-methylserine D-N-methylthreonine I-methylethyl)glycine N-methyla-napthylalanine N-methylpenicillam-ine N-(p-hydroxyphenyl)glycine N-(thiomethyl)glycine penicillamnine L-a-methylalanine L-a-methylasparagine L-a-methyl-t-butylglycine L-methylethylglycine L-a-methylglutamnate L-a-methylhomophenylalanine N-(2-methylthioethyl)glycine L-a-methyllysine L-a-methylnorleucine L-a-methylomnithine L-cx-methylproline L-a-methylthreonine L-cx-methyltyrosine Nmgabu Dnmmet Nmcpen Dnmphe Dnmpro Dnimser Dnmthr NvaI Nmanap Nmpen Nhtyr Ncys Pen Mala Masn Mtbug Metg Mglu Mhphe Nmet Mys Mnle Momn Mpro Mthr Mtyr 6 66 S 6 0 S. S S S 0S

S.

5@SS

S

0 5656

S

S S 60 6 5.6655 0 5* 6 5 0S OS S

S.

S.

P:\OPER\EH023942-CUS 12/8/98 -27- L-a-methylvaline Mval L-N-methylhomophenylalanine Nmhphe N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycine carbamylmethyl)glycine 1-carboxy-l-(2,2-diphenyl- Nmbc ethylamino)cyclopropane Crosslinkers can be used, for example, to stabilise 3D conformations, using homo-bifunctional crosslinkers such as the bifunctional imido esters having (CH2)n spacer groups with n= 1 to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specificreactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COOH). In addition, peptides can be conformationally constrained by, for example, incorporation of Ca and N methylamino acids, introduction of double bonds between C, and Cp atoms of amino acids and 15 the formation of cyclic peptides or analogues by introducing covalent bonds such as forming *i an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.

These types of modifications may be important to stabilise the proteinase/kinase if administered to an individual or for use as a diagnostic reagent.

*i The present invention further contemplates chemical analogues of the proteinase/kinase capable of acting as antagonists or agonists of the native molecules or which can act as functional analogues of the native molecules. For example, an antagonist may be a proteinase inhibitor.

Chemical analogues may not necessarily be derived from the subject enzymes but may share certain conformational similarities. Alternatively, chemical analogues may be specifically designed to mimic certain physiochemical properties of the serine proteinases or kinases.

Chemical analogues may be chemically synthesised or may be detected following, for example, natural product screening.

The identification of the novel molecules of the present invention permits the generation of a P:\OPERXE 023942-C.US 12//98 -28range of therapeutic molecules capable of modulating expression of their native counterparts or modulating their activity. Modulators contemplated by the present invention includes agonists and antagonists of proteinase/kinase expression. Antagonists of proteinase/kinase expression include antisense molecules, ribozymes and co-suppression molecules. Agonists include molecules which increase promoter ability or interfere with negative regulatory mechanisms. Agonists of proteinase/kinase include molecules which overcome any negative regulatory mechanism. Antagonists of the proteinase/kinase include antibodies and inhibitor peptide fragments.

Other derivatives contemplated by the present invention include a range of glycosylation variants from a completely unglycosylated molecule to a modified glycosylated molecule.

Altered glycosylation patterns may result from expression of recombinant molecules in different host cells.

15 Another embodiment of the present invention contemplates a method for modulating expression of proteinase/kinase in a human, said method comprising contacting the proteinase/kinase gene encoding proteinase/kinase with an effective amount of a modulator of proteinase/kinase expression for a time and under conditions sufficient to up-regulate or downregulate or otherwise modulate expression of proteinase/kinase. For example, a nucleic acid molecule encoding proteinase/kinase or a derivative thereof may be introduced into a cell conversely, proteinase/kinase antisense sequences such as oligonucleotides may be introduced.

Another aspect of the present invention contemplates a method of modulating activity of proteinase/kinase in a human, said method comprising administering to said mammal a modulating effective amount of a molecule for a time and under conditions sufficient to increase or decrease proteinase/kinase activity. The molecule may be a proteinaceous molecule or a chemical entity and may also be a derivative of proteinase/kinase or its receptor or a chemical analogue or truncation mutant of proteinase/kinase or its receptor.

One particularly useful serine proteinase, HELA2 (testisin), is implicated in spermatogenesis and in testicular tumour development. It is proposed, in accordance with the present invention, P\OPER EM 23942-C.US I2/98 -29that HELA2 (testisin) is involved in fertility and infertility.

Northern blot analysis of Poly A+ RNA from normal tissue specimens showed a unique tissue distribution for HELA2 (testisin) with significant expression only in the testis. No signals are detected in any other tissue, with the exception of a minor signal in salivary gland. By RT-PCR, HELA2 (testisin) is detected in the ejaculate of normal males but not in the ejaculate of vasectomised males indicating that it is of germ cell origin. Hybridization data in situ indicated that HELA2 (testisin) is produced by immature germ cells in the testis, located near the basal epithelium and, hence, is an important factor for normal sperm maturation; defective expression or mutations would contribute to primary male infertility. Further, it is from the precursors of spermatocytes that 95% of testicular germ cell tumours, such as seminomas, embryonal carcinomas and teratocarcinomas arise. In the normal testis, germ cells undergo meiosis to become spermatocytes, but in individuals at risk, the germ cells continue to proliferate giving rise to germ cell tumours. Although not wishing to limit the present invention to any one theory 15 or mode of action, it is proposed, in accordance with present invention, that HELA2 (testisin) functions at this critical juncture cell growth versus maturation.

*o Familial forms of testicular cancer are rare, but linkage analysis of a large family with familial seminoma has demonstrated linkage to chromosome 16p, within a region adjacent to the HPKD1 (human polycystic kidney disease) gene at 16pl3.3. The HELA2 (testisin) gene localises to chromosome 16p13.3 which is near the telomere of chromosome 16 and is associated with high genetic instability. The HELA2 (testisin) gene is sandwiched between four genes which underlie other human genetic disorders; HPKDI and tuberous sclerosis (TSC2) on the one side, and familial mediterranean fever (MEF) and Rubenstein-Taybi syndrome (RSTS) on the other side. The question of whether HELA2 (testisin) may be a tumour suppressor for seminoma was determined by comparing HELA2 (testisin) mRNA expression in normal testes with corresponding germ cell tumours from patients with seminoma. HELA2 (testisin) was not detectable in the tumours of these patients, but was present in the corresponding normal testis specimens, indicative of a tumour suppressor role of HELA2 (testisin) in testicular germ cell cancers.

P:\OPERJH\023942-C.US 12/8/98 Although restricted in normal tissues to the testes, the HELA2 (testisin) gene is expressed in tumours of the colon, pancreas, prostate and ovary. This indicates that HELA2 (testisin) contributed to tumourigenesis and, therefore, has an application as a marker and also as a therapeutic anti-tumour target in these types of cancers.

These data point to a potentially very significant role for HELA2 (testisin) in testicular germ cell maturation (spermatogenesis) as well as in the genesis of testicular germ cell tumours. In accordance with the present invention, it is proposed that expression of the HELA2 (testisin) gene by immature germ cells may be essential for sperm cell development, such that loss of HELA2 (testisin) expression leads to continued and uncontrolled proliferation of immature germ cells leading to subsequent tumourigenesis. Germ cells wherein HELA2 (testisin) is mutated or absent may thus be prone to malignant transformation because of an inability to progress along the differentiation pathway.

HELA2 (testisin) is well-positioned to anchor on the surface of the germ cell where it would participate in a range of proteolytic activities, including cell migration, differentiation and/or activation of growth factors, receptors, or cytokines as well as initiate additional proteolytic cascades. Although not intending to limit the present invention to any one theory or mode of action, it is proposed, in accordance with the present invention, that the proteolytic target of HELA2 (testisin) is a cytokine, receptor or growth factor essential for either germ cell proliferation or differentiation ie. HELA2 (testisin) may either inactivate a factor important ofor proliferation, or activate a factor which promotes differentiation. Thus, HELA2 (testisin) may be critical in the regulation of specific cytokines, cytokine receptors or growth factors by .i means of post-translational proteolytic processing. That HELA2 (testisin) is not present in other normal tissues of the male urogenital tract, such as the prostate and kidney, also argues for such a role specific to the testis.

Diagnostic and therapeutic applications for HELA2 (testisin) have the potential to be wideranging both in the cancer and fertility/infertility markets. In tumours, other than the testis, it is desirable to block or inhibit HELA2 (testisin) activity. As HELA2 (testisin) is a member of the serine proteinase family, for which prototype crystal structures are known and the catalytic P:\OPER\IE023942-C.US 12/898 -31 mechanism reasonably well characterised, the design of drugs that target HELA2 (testisin) proteolytic activity as an anti-tumour therapy should be relatively straightforward. As HELA2 (testisin) is predicted to be anchored on the cell surface, there would not be difficulties associated with delivery of drugs to intracellular compartments. Further, it is very possible that some tumour-associated HELA2 (testisin) may be proteolytically cleaved from the surface of tumour cells, and the extracellular domain detectable in patient serum as a potential tumour associated marker.

Testicular cancer is the commonest malignancy in men aged 20-44 years. Early diagnosis correlates which an improved chance of cure and in a reduction in the severity of treatment.

If the cancer is not treated early, it becomes very aggressive. The incidence of testicular cancer is significant (9/100,000) and has been rising over the last 10 years. In testicular germ cell tumours, such as seminoma, delivery of recombinant HELA2 (testisin) using gene therapy techniques could lead to arrest of tumour growth and potentially allow commencement of 15 normal sperm cell maturation and differentiation, thereby reducing the need for surgical removal of the testis (orchidectomy). This may be particularly effective for patients who have already had one testicle removed because of testicular cancer. The risk of contralateral testicular cancer is increased in these patients and tumour development could be arrested through early treatment with HELA2 (testisin) to arrest growth and assist maturation of germ cells. The finding of mutant forms of HELA2 (testisin) may also lead to new markers for seminoma. Unlike other testicular non-seminoma cancers where a-fetoprotein and P-HCG are frequently elevated and can be used as tumour markers, the lack of an adequate marker for seminoma creates difficulties with staging and patient follow-up.

25 A demonstrated role for HELA2 (testisin) in sperm maturation and development would likely lead to improved diagnosis and new directed therapeutics for male primary infertility. Primary male infertility is responsible for conception problems in 5-10% of couples and the world market for a therapeutic in this area would be very substantial. Delivery of recombinant HELA2 (testisin) could assist sperm maturation and potentially trigger normal sperm development in some of these cases. The identification of mutant forms of HELA2 (testisin) could aid in diagnosis of infertility. If HELA2 (testisin) does not prove to be a tumour P:\OPEREIn23942-C.US- 12/898 -32suppressor, but is important for sperm maturation, it could provide a new target for the development of a male contraceptive. If hormonal regulation of HELA2 (testisin) can be demonstrated, HELA2 (testisin) may prove effective for the treatment of conditions arising from dysfunctional hormal responses, such as cryptorchidism, which is associated with both infertility and seminoma development.

Accordingly, the present invention contemplates a pharmaceutical composition comprising proteinase/kinase or a derivative thereof or a modulator of proteinase/kinase expression or proteinase/kinase activity and one or more pharmaceutically acceptable carriers and/or diluents.

These components are referred to as the "active ingredients" and include, for example, HELA2 (testisin).

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimersal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion P:\OPERUJH23942-C.US. 12/8/98 33 medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

When the active ingredients are suitably protected they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about S• 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained. Preferred compositions or -preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0./1 ug and 2000 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain the components as listed hereafter. A binder such as gum, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry *4flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of S° 25 the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound(s) may be incorporated into sustained-release P\OPERWJ Mo23942-C.US I2/19 34 preparations and formulations.

Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Parental compositions are generally suitable for administration by the intravenous, subcutaneous or intramuscular routes amongst other routes of administration. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association **with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on the unique characteristics of the .active material and the particular therapeutic effect to be achieved, and the limitations o..o inherent in the art of compounding such an active material for the treatment of disease in living 0 20 subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail. Other forms of administration include but are not limited to intranasal, buccal, rectal, suppository, inhalation, intracerebral and intraperitoneal.

oeo The principal active ingredient is compounded for convenient and effective administration in 25 effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as 0. 0. hereinbefore disclosed. A unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.5 lg to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 plg to about 2000 mg/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.

P\OPERIEJH\023942-CUS 12/898 The effective amounts include amounts calculated or predicted to have the desired effect and range from at least about 0.01 ng/kg body weight to about 10,000 mg/kg body weight.

Alternative amounts include 0.1 ng/kg body weight to about 1000 ng/kg body weight.

The pharmaceutical composition may also comprise genetic molecules such as a vector capable of transfecting target cells where the vector carries a nucleic acid molecule capable of modulating proteinase/kinase expression or proteinase/kinase activity. The vector may, for example, be a viral vector. This form of therapy is proposed to be particularly useful for gene replacement or enhancement therapy for HELA2 (testisin) especially for the modulation of fertility and/or treatment of testicular cancer.

This aspect of the present invention extends to pharmaceutical compositions comprising other molecules described herein such as but not limited to ATC2 and/or BCON3. Pharmaceutical compositions comprising ATC2, for example, are useful in the treatment and prophylaxis of cardiac physiology.

Still another aspect of the present invention is directed to antibodies to proteinase/kinase and its derivatives. Such antibodies may be monoclonal or polyclonal and may be selected from naturally occurring antibodies to proteinase/kinase or may be specifically raised to proteinase/kinase or derivatives thereof. In the case of the latter, proteinase/kinase or its derivatives may first need to be associated with a carrier molecule. The antibodies and/or recombinant proteinase/kinase or its derivatives of the present invention are particularly useful as therapeutic or diagnostic agents. For example, monitoring non-testicular cancer by measuring HELA2 (testisin) or screening for the presence of testicular cancer by an absence of 25 HELA2 (testisin).

Proteinase/kinase and its derivatives may also be used to screen for naturally occurring antibodies to proteinase/kinase. These may occur, for example in some autoimmune diseases.

Alternatively, specific antibodies can be used to screen for proteinase/kinase. Techniques for such assays are well known in the art and include, for example, sandwich assays and ELISA.

Knowledge of proteinase/kinase levels may be important for diagnosis of certain cancers or a P:\OPER\EUJ23942-C.US 12/898 -36predisposition to cancers or for monitoring certain therapeutic protocols.

Antibodies the proteinase/kinase of the present invention may be monoclonal or polyclonal.

Alternatively, fragments of antibodies may be used such as Fab fragments. Furthermore, the present invention extends to recombinant and synthetic antibodies and to antibody hybrids. A "synthetic antibody" is considered herein to include fragments and hybrids of antibodies. The antibodies of this aspect of the present invention are particularly useful for immunotherapy and may also be used as a diagnostic tool for assessing apoptosis or monitoring the program of a therapeutic regimen.

For example, specific antibodies can be used to screen for proteinase/kinase proteins. The latter would be important, for example, as a means for screening for levels of proteinase/kinase in a cell extract or other biological fluid or purifying proteinase/kinase made by recombinant means from culture supernatant fluid. Techniques for the assays contemplated herein are known in the art and include, for example, sandwich assays and ELISA.

It is within the scope of this invention to include any second antibodies (monoclonal, polyclonal or fragments of antibodies or synthetic antibodies) directed to the first mentioned antibodies discussed above. Both the first and second antibodies may be used in detection assays or a first antibody may be used with a commercially available anti-immunoglobulin antibody. An antibody as contemplated herein includes any antibody specific to any region of proteinase/kinase.

Both polyclonal and monoclonal antibodies are obtainable by immunization with the enzyme or protein and either type is utilizable for immunoassays. The methods of obtaining both types of sera are well known in the art. Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of proteinase/kinase, or antigenic parts thereof, collecting serum from the animal, and isolating specific sera by any of the known immunoadsorbent techniques. Although antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favoured because of the potential heterogeneity of the product.

PAOPER JHM023942-C.US- 12/898 37 The use of monoclonal antibodies in an immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product. The preparation ofhybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art.

Another aspect of the present invention contemplates a method for detecting proteinase/kinase in a biological sample from a subject said method comprising contacting said biological sample with an antibody specific for proteinase/kinase or its derivatives or homologues for a time and under conditions sufficient for an antibody-proteinase/kinase complex to form, and then detecting said complex.

The presence of proteinase/kinase may be accomplished in a number of ways such as by Western blotting and ELISA procedures. A wide range of immunoassay techniques are available as can be seen by reference to US Patent Nos. 4,016,043, 4, 424,279 and 4,018,653.

'.".These, of course, includes both single-site and two-site or "sandwich" assays of the noncompetitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labelled antibody to a target.

Sandwich assays are among the most useful and commonly used assays and are favoured for use in the present invention. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabelled antibody is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen complex, a second antibody specific to the antigen, labelled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibodyantigen-labelled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal produced by the reporter molecule. The results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of hapten. Variations on the P.\OPER\MEJf23942-C.US- 12/98 -38forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including any minor variations as will be readily apparent. In accordance with the present invention the sample is one which might contain proteinase/kinase including cell extract, tissue biopsy or possibly serum, saliva, mucosal secretions, lymph, tissue fluid and respiratory fluid. The sample is, therefore, generally a biological sample comprising biological fluid but also extends to fermentation fluid and supernatant fluid such as from a cell culture.

In the typical forward sandwich assay, a first antibody having specificity for the proteinase/kinase or antigenic parts thereof, is either covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay. The binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymerantibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient 2-40 minutes or overnight if more convenient) and under suitable conditions from about room temperature to about 37°C) to allow binding of any subunit present in the antibody.

Following the incubation period, the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of the hapten. The second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the hapten.

*o An alternative method involves immobilizing the target molecules in the biological sample and then exposing the immobilized target to specific antibody which may or may not be labelled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound target may be detectable by direct labelling with the antibody.

Alternatively, a second labelled antibody, specific to the first antibody is exposed to the targetfirst antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.

P:\OPER\EH\023942-C.US 12//98 -39- By "reporter molecule" as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative. The most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionucleotide containing molecules radioisotopes) and chemiluminescent molecules.

In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, betagalactosidase and alkaline phosphatase, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change. Examples of suitable enzymes include alkaline phosphatase and peroxidase. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labelled antibody is added to the first antibody hapten complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of hapten which was present in the sample. "Reporter molecule" also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.

Alternately, fluorescent compounds, such as fluorescein and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labelled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope. As in the EIA, the fluorescent labelled antibody is allowed to bind to the first antibody-hapten complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength the fluorescence observed indicates the presence of the hapten of interest.

Immunofluorescene and EIA techniques are both very well established in the art and are P:\OPEREJHD023942-C.US 2/8/98 particularly preferred for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.

The present invention also contemplates genetic assays such as involving PCR analysis to detect proteinase/kinase gene or its derivatives. Alternative methods or methods used in conjunction include direct nucleotide sequencing or mutation scanning such as single stranded conformation polymorphoms analysis (SSCP), specific oligonucleotide hybridisation, and methods such as direct protein truncation tests.

The nucleic acid molecules of the present invention may be DNA or RNA. When the nucleic acid molecule is in DNA form, it may be genomic DNA or cDNA. RNA forms of the nucleic acid molecules of the present invention are generally mRNA.

Although the nucleic acid molecules of the present invention are generally in isolated form, they may be integrated into or ligated to or otherwise fused or associated with other genetic o* molecules such as vector molecules and in particular expression vector molecules. Vectors and expression vectors are generally capable of replication and, if applicable, expression in one or both of a prokaryotic cell or a eukaryotic cell. Preferably, prokaryotic cells include E. coli, Bacillus sp and Pseudomonas sp. Preferred eukaryotic cells include yeast, fungal, mammalian and insect cells.

Accordingly, another aspect of the present invention contemplates a genetic construct comprising a vector portion and a mammalian and more particularly a human proteinase/kinase gene portion, which proteinase/kinase gene portion is capable of encoding an proteinase/kinase polypeptide or a functional or immunologically interactive derivative thereof.

Preferably, the proteinase/kinase gene portion of the genetic construct is operably linked to a promoter on the vector such that said promoter is capable of directing expression of said proteinase/kinase gene portion in an appropriate cell.

In addition, the proteinase/kinase gene portion of the genetic construct may comprise all or part P\OPER\EJHO23942-C.US 12/98 -41of the gene fused to another genetic sequence such as a nucleotide sequence encoding glutathione-S-transferase or part thereof.

The present invention extends to such genetic constructs and to prokaryotic or eukaryotic cells comprising same.

The present invention also extends to any or all derivatives of proteinase/kinase including mutants, part, fragments, portions, homologues and analogues or their encoding genetic sequence including single or multiple nucleotide or amino acid substitutions, additions and/or deletions to the naturally occurring nucleotide or amino acid sequence. The present invention further encompasses hybrids between the proteinase/kinases such as to broaden the spectrum of activity and to ligands and substrates of the proteinase/kinase.

The proteinase/kinase and its genetic sequence of the present invention will be useful in the generation of a range of therapeutic and diagnostic reagents.

Soluble proteinase/kinase polypeptides or other derivatives, agonists or antagonists are also contemplated to be useful in the treatment of disease, injury or abnormality in the nervous system, e.g. in relation to central or peripheral nervous system to treat Cerebral Palsy, trauma induced paralysis, vascular ischaemia associated with stroke, neuronal tumours, motoneurone disease, Parkinson's disease, Huntington's disease, Alzheimer's disease, Multiple Sclerosis, peripheral neuropathies associated with diabetes, heavy metal or alcohol toxicity, renal failure and infectious diseases such as herpes, rubella, measles, chicken pox, HIV or HTLV-1. Other conditions for which the proteinase/kinase are useful include cancer, metastasis and autoimmune disease amongst many others. Particular applications for HELA2 (testisin) include as a marker for non-testicular cancers, in the treatment of testicular cancer and in the treatment of infertility or in inducing infertility such for contraception.

A further aspect of the present invention contemplates the use of proteinase/kinase or its functional derivatives in the manufacture of a medicament for the treatment of proteinase/kinase mediated conditions defective or deficient.

P:\OPER\EJ023942C.US 12/8/98 -42- The present invention is further directed to the use of ATC2 or its derivatives or homologues in the treatment or prophylaxis of cardiac physiology and disease. Although not intending to limit the present invention to any one theory or mode of action, it is proposed that ATC2 functions through regulation of a coagulative or fibronolytic cascade. The state of cardiac physiology may also be determined using immunointeractive molecules such as antibodies to ATC2 or its derivatives and homologues.

The present invention is further described by the following non-limiting Examples.

P\AOPER\EJHD23942-C.US 12/98 -43- EXAMPLE 1 CLONING PROCEDURES In order to identify serine proteinases that may be involved in regulatory cellular functions, a genetic screening approach was applied using degenerate primers corresponding to conserved regions of serine proteinases (amino acids flanking His- and Ser- residues) to amplify gene fragments spanning these regions from cDNA, using a low stringency RT-PCR (Reverse Transcriptase-Polymerase Chain Reaction) approach.

By this technique, the aim was to isolate low abundance genes as well as those present in moderate to high abundance. The cDNA used for these experiments was isolated from a HeLa cell cytotoxicity model wherein PAI-2 expression inhibits TNF(-induced apoptosis (Dickinson et al J. Biol. Chem. 270: 27894-27904, 1995). These PAI-2 expressing cells provide a unique and viable system for investigating TNF(signalling pathways as they are protected from the cytotoxic effects of TNF).

cDNA was generated from RNA isolated from HeLa cells and PAI-2 expressing HeLa cells, both untreated and following treatment with TNF and cycloheximide. Amplification of each cDNA population using PCR and the following serine proteinase degenerate primers, His Primer: 5'ACAGAATTCTGGGTIGTIACIGCIGCICAYTG3' [SEQ ID NO: Ser Primer: 5'ACAGAATTCAXIGGICCICCIC/GT/AXTCICC3' [SEQ ID NO:2] (where X= A or G; Y= C or T; I= Inosine) produced DNA fragments in the range of 480bp, the approximate predicted size of the serine proteinase intergenic region. These amplified DNA fragments were cloned into E. coli generating a library containing approximately 150 independent clones. The inventors analysed 36 of these clones and found that 9 encoded previously identified serine proteinases or tissue-type or urokinase-type plasminogen activators, thereby demonstrating the efficacy of this approach. Of the other 36, two were found to encode novel open reading frames with high homology to serine proteinases and are referred to herein as "HELA2" (or "testisin") and P:\OPER\EJH\023942-C.US 12/898 -44- "ATC2". One additional clone designated herein, "BCON3", showed homology to a kinase.

Extension of the DNA fragments by RACE in both 5' and 3' directions using internally derived primers has verified the homology of HELA2 and ATC2 to the serine proteinase family. Each of the three DNA sequences are unique in that they are markedly different from any known DNA or protein sequence in the Genbank and NBRF databases.

EXAMPLE 2 HELA2 SERINE PROTEINASE (TESTISIN) The HELA2 mRNA transcript is approximately 1.5kb as determined from Northern blot analysis. Nucleic acid sequence was obtained for about 1.1kb of HELA2 which spans the entire coding region, the 3' noncoding region and part of the 5' noncoding region. The coding region starts with an ATG codon which is present in a motif analogous to the Kozak eukaryotic translation initiation consensus sequence. Alignment of the deduced amino acid sequence of HELA2 with homologous serine proteinases shows that the cDNA encodes a 314 amino acid (aa) polypeptide with a calculated molecular weight of 34.8kD (called Testisin), which is synthesized as a zymogen containing pre-, pro- and catalytic regions (Figure The pro- region (or light chain) and the catalytic region (heavy chain) are delineated by a classic serine proteinase activation motif Arg-Ile-Val-Gly-Gly [SEQ ID NO:24] with cleavage likely occurring between Arg and Ile. The catalytic region includes the catalytic triad of His, Asp and Ser in positions and motifs which are highly conserved among the serine proteinases. Ten Cys residues occur in conserved positions: by analogy to other serine proteinases, eight of these function to form disulfide bridges within the catalytic region and the remaining two link the proand catalytic regions.

Structural features conserved in the binding pockets of serine proteinases are present in HELA2 (testisin). An Asp residue at the bottom of the serine proteinase binding pocket six residues before the active site Ser in HELA2 (testisin) indicates that HELA2 (testisin) has trypsin-like specificity, with proteolytic cleavage after Arg or Lys in target substrates. HELA2 (testisin) also contains a conserved Ser-Trp-Gly motif at the top of the binding pocket which is likely involved in hydrogen bonding with target substrates in other serine proteinases.

P:OPER\EJH\23942-C.US 12/898 A hydrophobicity plot of the HELA2 (testisin) amino acid sequence (Figure 1) identifies two hydrophobic regions, one located at the amino terminus and the other at the carboxy terminus.

The 20 aa amino terminal hydrophobic region is likely to be a signal peptide, which would direct newly synthesized HELA2 to enter the endoplasmic reticulum. The 16 aa hydrophobic carboxy terminus of HELA2 (testisin) shows high homology to the transmembrane domain of prostasin (Figure suggesting that HELA2 (testisin) is likely to be a membrane-anchored serine proteinase. Immunostaining experiments performed on HeLa cells and on paraffin-fixed normal testis tissue specimens using anti-HELA2 (testisin) antibodies show strong staining on the plasma membrane, confirming the plasma membrane localisation of this protein. Thus HELA2 (testisin) anchors on the germ cell surface where it could participate in a range of proteolytic activities, including participation in cell migration, differentiation and/or activation of growth factors and proteolytic cascades. In prostasin, this protruding carboxy terminus may be cleaved, thus releasing the serine proteinase from the membrane. A similar cleavage event may also occur with Testasin.

Two isoforms of HELA2 were identified in a HeLa cell cDNA library (Stratagene UniZap HeLa Library) which differ by an insertion of 6 nucleotides which generates a Sfi 1 restriction enzyme site. At the protein level, there is a corresponding insertion of 2 aa's (Tyr-Ser) within the catalytic binding pocket (Figure 2A). The two isoforms of HELA2 cDNA are referred to as the short and long isoforms, respectively. The nucleotide and corresponding amino acid sequence for the short isoform of HELA2 is shown in SEQ ID NOs. 3 and 4, respectively. The long isoform is shown in SEQ ID NO:5 and 6, respectively.

EXAMPLE 3 GENERATION OF FULL LENGTH cDNA ENCODING HELA2 (TESTISIN) Partial cDNA fragments of the short and long isoforms of HELA2 were obtained using a combination of library screening techniques. Plasmids containing the full length cDNA of the two isoforms were then generated in pBluescriptSK(-) by ligating restriction enzyme-digested fragments of the partial cDNAs. A plasmid map of the two generated constructs, pBluescriptHELA2(S) and pBluescriptHELA2(L), and a restriction enzyme map of the long P\OPER\EIH023942-C.US- 12/8/98 -46isoform cDNA are diagrammed in Figure 3.

In vitro transcription/translation using HELA2 cDNA shows a major specific product of approximately 35kD (Fig. 2B), which is the same as size predicted from the open reading frame, demonstrating that HELA2 cDNA encodes a protein. The translation/transcription coupled rabbit reticulocyte lysate system (Promega) was used as per the manufacturer's instructions for labelling. Clones of HELA2 in pBluescript a PAI-2 positive control were used with T3-RNA polymerase (sense direction).

EXAMPLE 4 EXPRESSION OF RECOMBINANT HELA2 (TESTISIN) IN E.COLI Generation of expression constructs His(6)-tagged recombinant HELA2 (testisin) To reduce potential toxic effects on host cells, and therefore optimise expression, a strategy was employed to eliminate the hydrophobic residues of the secretory and membrane anchoring domains of HELA2 (testisin) (Testisin (20-295)). Testisin (20-295) fragments which were His6 tagged at either the amino or carboxy terminal were obtained by PCR and expression constructs were generated by inserting these into pQE vectors (Qiagen).

The primers used to generate the amino-terminal tagged protein were: forward: 5' GCACAGTCGACCAAGCCGGAGTCGCAGAG 3' [SEQ ID NO: 11] and reverse: 5' GCACAAAGCTTGCCAGGAGGGGTCTGGCTG 3' [SEQ ID NO: 12] The amplification product of 858bp was digested with Sail and HindIII and ligated into to give pQE-10(20-295)N (Figure 4).

The primers used to generate the carboxy-terminal tagged protein were: forward: 5' GCACAACCATGGCCAAGCCGGAGTCGCAGGAG 3' [SEQ ID NO:13] and reverse 5' GCACAAGATCTCCAGGAGGGGTCTGGCTG 3' [SEQ ID NO: 14].

The amplification product of 859 bp was digested with NcoI and BgIII and ligated into to give pQE-60(20-295)C (Figure 4).

P~AOPERIEMf23942-CUS 121IV98 -47- (ii) GST-tagged recombinant HELA2 (testisin) In order to generate a fusion of glutathione-S-transferase (GST) and HELA2 (testisin), pBluescriptHELA2(S) was digested with Sau3A releasing a 570bp DNA fragment encoding the 190 amino acids at the carboxy terminal end of HELA2 (testisin). This DNA fragment was cloned into the BamH1 site of pGEX-1 generating pGEX-1(90-279) (Figure 4) and subjected to DNA sequence analysis to confirm that the fusion was in frame.

Expression of His-tagged HELA2 (testisin) in E. coli pQE10(20-295)N and pQE60(20-295)C plasmids were electro-transformed into E. coli cells. Four different clones were selected for further analysis: His-N21 expressing amino terminal His6-tagged Testisin (20-295); and His-C21, His-C22, and His-C23 expressing carboxy terminal His6-tagged Testisin (20-295). To express recombinant HELA2 (testisin) protein, transformed cells were grown to log phase then induced for 4 hours in the presence of 2mM IPTG. Cells were lysed in a denaturing lysis buffer containing 8M urea, 0.1M NaH2PO4 ~and 0.01M Tris/HCI pH8. Alternatively the cells were lysed in a non-denaturing lysis buffer containing 0.1M NaH2PO4, 0.1M NaCI and 0.01 M Tris/HCI pH8. The His6 tagged protein was recovered by mixing the lysate with a metal affinity resin (Qiagen or Clontech). Purified testisin(L) was eluted with 100 mM EDTA in lysis buffer (pH A major band of approximately 32 kDa was obtained in the eluate as shown by the arrows in Figure Western blot analysis of a purification of the His-C23 clone using an anti-His6 antibody showed that the band at 32 kDa was His6 tagged HELA2 (testisin) (Figure EXAMPLE

IMMUNOLOGY

Rabbit Polyclonal Antibodies Directed Against HELA2 (testisin) Peptide Antigens Three peptides were selected from the HELA2 (testisin) amino acid sequence on the basis of predicted antigenicity, hydrophilicity and lack of identity with known proteins (Figure 6).

P\OPEREJHM023942-C.US 12/8/98 -48- Peptide antigen T20-33 KPESQEAAPLSGPC [SEQ ID Peptide antigen T46-63 EDAELGRWPWQGSLRLWDC [SEQ ID NO: 16] Peptide antigen T175-190 GYIKEDEALPSPHTLQC [SEQ ID NO: 17] These peptides were synthesized (Auspep) and coupled to keyhole limpet hemocyanin. The coupled peptide (500 in PBS (0.5 ml) was emulsified in an equal volume of Freund's complete adjuvant before injection into a rabbit. Booster injections of coupled peptide in Freund's incomplete adjuvant were made at intervals of 2 to 3 weeks. Each rabbit was bled (approximately 1 ml) before the initial injection and about 7 days after the second and subsequent boosters and the antibody titre assessed by direct ELISA assay. Immunoreactive antisera against the peptide antigens was demonstrated and when a sufficiently high titre was achieved (after 3 to 5 boosters), between 12 and 25 ml of blood was removed from each animal.

Rabbit antisera was affinity purified against the respective immunising peptides by chromatography using peptide-coupled affinity columns. Immunoreactivity of the affinity purified antibodies against HELA2 (testisin) was demonstrated by Western blot analysis of GST-tagged recombinant HELA2 (testisin). pGEX-1(90-279) plasmid DNA (described in Example 4) was electro-transformed into E. coli DH5a (cells and induced for 3 hours in the presence of 0.5mM IPTG. Cells were lysed in 1.5% v/v sarcosyl, 2% v/v Triton X100 and then sonicated. After removal of the insoluble fraction by centrifugation, the cell lysate was mixed with a 50% slurry of Glutathione Sepharose 4B, washed, and the purified GST-Testisin(90-279) was eluted by boiling with SDS-Sample buffer.

B) Rabbit Antibodies Directed Against HeLa Cell and Testis Tissue Lysates Purified rabbit antisera was used in SDS-PAGE-Western blot analysis of lysates prepared from HeLa cells and from testis tissues. Figure 7 shows an example of a Western blot using rabbit antipeptide T175-190 antibody, demonstrating an immunoreactive band at approximately 58kD in nonreduced samples and two bands of approximately 56kD and 48kD in reduced samples in both HeLa cells and testis tissue. Identical bands are obtained using antipeptide antibody T46- 63. The difference between the nonreduced and reduced testisin protein bands is likely due to RNOPMR Ef23942-CUS 12/89S -49the loss of the amino terminal light chain after proteolytic activation under reducing conditions.

These data suggest that the testisin protein may undergo post translational modification, (2) is bound to an inhibitor or other protein and/or exists in dimeric form under these conditions. Treatment of HeLa cells with the Trizol reagent (Gibco/BRL) which is a harsher extraction method, yields two bands at approximately 43kD and 40kD which is similar to the predicted molecular mass of testisin from the amino acid sequence.

Rabbit Polyclonal Antibodies Directed Against Purified Bacterially Expressed HELA2 (testisin) An SDS-PAGE gel slice containing purified His6 tagged HELA2 (testisin) (as described in Example 4, part is combined with adjuvant and rabbits immunized as described above.

Rabbit antisera are tested by Western blot analysis for immunoreactivity against purified recombinant HELA2 (testisin) and HELA2 (testisin) in cell extracts, as well as use in immunohistochemical analyses.

EXAMPLE 6 EXPRESSION OF HELA2 (TESTISIN) IN EUKARYOTIC CELLS 0@ Generation of expression constructs Eukaryotic expression constructs encoding testisin(s) and testisin(L) His6 tagged at the carboxy terminal were generated in the eukaryotic expression vector pcDNA3 (Invitrogen). DNA fragments encoding HELA2 (testisin) were generated by PCR from both pBluescriptHELA2(S) and pBluescriptHELA2(L) using the primers: forward: 5' GCACAGGTACCGAGGCCATGGGCGCGCGC 3' [SEQ ID NO:18] and reverse 5' GCACATCTAGATCAGTGGTGGTGGTGGTGGTGGACCGGCCCCAGGA GTGG 3' [SEQ ID NO:19] The PCR product of 985 bp obtained from amplification of HELA2 (testisin) from pBluescriptHELA2(S) as template was ligated into pGEM-T (Easy) vector (Promega).

P\OPER\EJH\023942-C.US- 12//98 Digestion of this shuttle construct with NotI released a 1025 bp fragment which was ligated into pcDNA3 generating the short isoform expression construct pcDNA3-Test(S-C) (Figure 8).

PCR amplification of the long isoform template gave a 991 bp product which was ligated into pGEM-T (easy) vector. NotI digestion of the shuttle construct released a 1031 bp fragment which was ligated into pcDNA3 giving pcDNA3-Test(L-C) (Figure 8).

Soluble testisin (1-295)-His6 in which the membrane anchoring sequence is deleted and the protein is carboxy-His6 tagged is to be obtained by PCR amplification of HELA2 (testisin) from pBluescriptHELA2(L) using the primers: forward: 5' GCACAGCGGCCGCGAGGCCATGGGCGCGCGC 3' [SEQ ID NO:20] and reverse: 5' GCACAGCGGCCGCTCAGTGGGTGGTGGTGGT

GCCAGGAGGGGTC

TGGCTG 3' [SEQ ID NO:21].

The PCR product will be digested with NotI and ligated into pcDNA3 generating the long isoform expression construct pcDNA3-Test(1-295)L-C (Figure 8).

Expression and cellular localisation of HELA2 (testisin) Each of the expression constructs is transiently transfected into a eukaryotic cell line (eg. HeLa, CHO or COS cells) by electroporation. Expression is confirmed by Northern blot and immunoblot. The His6 tag is a small, uncharged tag which reportedly does not interfere with cellular membrane interactions and is able to be detected with anti-His6 antibodies. HELA2 (testisin) cellular localisation is analysed by immunofluorescence using antibodies directed against the His6 tag and stained cells examined by confocal microscopy. Mock transfected cells is monitored as one of the controls in these experiments. Cells are examined under non-permeablised and permeabilised conditions to investigate intracellular and cell surface expression of HELA2 (testisin) tagged proteins. Possible release of HELA2 (testisin) into the supernatant is monitored by immunoblotting of conditioned media. Association of HELA2 (testisin) with a particular cellular compartment is confirmed by cellular fractionation studies.

Stable transfectants of the full length and truncated tagged HELA2 (testisin) is generated by selection in G418. Recombinant HELA2 (testisin) is purified from these stable transfectants using a metal affinity resin (eg. Qiagen or Clontech) for assay of its bioactivity and efficacy as P\OPEREH023942-C.US 12/8/98 -51a therapeutic reagent. Bulk production of HELA2 (testisin) from eukaryotic cells will be used to generate polyclonal and monoclonal antibodies for use as probes in diagnostic assays for the detection of HELA2 (testisin).

EXAMPLE 7 HELA2 (TESTISIN) IS SPECIFICALLY EXPRESSED IN THE NORMAL TESTIS, AND IS ASSOCIATED WITH SPERM DEVELOPMENT Normal Tissue Blot Dot blot analysis of PolyA+ RNA from 50 normal tissue specimens (strandardised to 8 different housekeeping genes) (Clontech) was performed using a 32P-labelled HELA2 (testisin) probe.

Hybridization of the radiolabelled probe was in ExpressHyb solution (Clontech) at 65C. The blots were washed to a final stringency of 0. lxSSC/0.5% w/v SDS. High level expression of 15 the HELA2 (testisin) gene was found only in the testis as shown by the histogram plot of the Signal Intensity in Figure 9. In contrast, probing of the same blot with BCON3 showed ubiquitous expression of BCON3 mRNA in a variety of tissues (Figure 9).

0# 0 00e Multiple Tissue Northern Blot *se* Northern blots displaying polyA+ mRNA from 16 different normal tissues (Clontech) were hybridised at 65C in ExpressHyb solution using a 400bp SacII/EcoRI 32P-labelled HELA2 probe for 3h and then washed to a final stringency of 0.1xSSC/0.1% w/v SDS at 60 0 C. After a 5h exposure, a strong band was observed only in the lane containing testis mRNA, 25 demonstrating the specificity of HELA2 (testisin) expression for the testis (Figure S0.: Prolonged exposure (4.5 days) of the blot revealed a very low level of HELA2 (testisin) mRNA expression in the prostate, lung and pancreas only. In contrast to HELA2, BCON3 is expressed in mRNA from most tissues present on the blot (Figure HELA2 (testisin) is Expressed in Sperm Cells, Demonstrating its Germ Cell Origin P:\OPEREJUf023942-C.US- 12/8/98 -52- To determine whether HELA2 (testisin) gene expression is associated with germ cells of the testis, ejaculate specimens from normal fertile males were compared with those of post-vasectomy males by RT-PCR analysis using HELA2 (testisin) specific primers. Sperm is the primary product from the testis that is found in ejaculate; other components of the ejaculate are derived from the prostate.

First strand cDNA was reverse transcribed from total RNA which has been isolated from frozen or fresh ejaculate specimens. PCR was performed on the cDNA templates using the primers: forward: 5' CTGACTTCCATGCCATCCTT 3' [SEQ ID NO:22] and reverse: 5' GCTCACGACTCCAATCTGAT 3' [SEQ ID NO:23].

As shown in Figure 11, strong signals of the expected size of 464 bp were detected in ejaculate from normal males (Patients #23 and while no HELA2 (testisin) was detected in Patient #153 (post-vasectomy). Patent #90 (post-vasectomy) showed a low level of amplification product which may reflect a small amount of residual sperm in the seminiferous tubules. PCR 15 using primers specific for (2-macroglobulin was performed on the same samples as a control for the presence of approximately equal amounts of cDNA in each sample.

HELA2 (testisin) is Expressed in Immature Germ Cells of the Testis In situ hybridization was performed on paraffin-embedded specimens of rat testis tissue using DIG- labelled HELA2 (testisin) RNA probes (T3 and T7 generated transcripts containing nucleotides 1-423 of HELA2 cDNA). The results using the antisense RNA probe showed strong positive staining near the basal lamina of the seminiferous tubules in the region associated with spermatocytes and spermatogonia (Figure 12, see arrows). HELA2 (testisin) 25 mRNA expression did not appear to be associated with Leydig cells and the pattern was not typical for Sertoli cell staining. The presence of HELA2 (testisin) mRNA in these cells indicates a role for HELA2 (testisin) in germ cell maturation and sperm development.

POPER.EJH\D23942-C.US- 12/8/98 -53- EXAMPLE 8 HELA2 (TESTISIN) EXPRESSION IS ASSOCIATED WITH TUMOURS IN NON-TESTIS CELL-TYPES The tissue and cell-type distribution of testisin mRNA transcripts in tumours were determined by Northern hybridization analyses of RNA extracted from in vitro cultured tumour cells lines derived from different cancerous tissues. HELA2 (testisin) was detected in the HeLa ovarian carcinoma, the U937 lymphoma, and the melanoma cell line 253-3D. HELA2 (testisin) is also associated with cDNA libraries derived from tumours of the colon, pancreas, prostate and ovary (NCBI-EST Database). The presence of HELA2 (testisin) in tumours where it is not expressed normally indicates that it likely plays a role. in tumourigenesis in several cell-types.

EXAMPLE 9 THE HELA2 (TESTISIN) GENE IS LOCATED 15 ON HUMAN CHROMOSOME 16pl3.3 The genetic location of testisin was mapped to the short arm of chromosome 16 at 16p13.3 by fluorescence in-situ hybridization to normal metaphase chromosomes (Figure 13A). Screening of a chromosome 16 hybrid panel then sub-localised HELA2 (testisin) to the cosmid 406D6 20 which has been mapped to this region (Sood, R. et al Genomics 42: 83-95, 1997; Doggett, N.A. et al. Nature 377 (Suppl.): 335-365, 1995. The cosmid lies between the markers D16S246 and D16S468 and the gene is located just centromeric to D16S246 (Figure 13B).

This region of the human genome is associated with high genetic instability and telomeric rearrangements underlie a variety of common human genetic disorders. Testisin is sandwiched 25 between the human disease genes PKD1 (polycystic kidney disease) and tuberous sclerosis (TSC2) on the on side, and MEF (familial mediterranean fever) and Rubenstein-Taybi syndrome (RSTS) on the other side as diagrammed in Figure 13B.

P:\OPER\EJHO23942-C.US 12/8/98 -54- EXAMPLE HELA2 (TESTISIN) mRNA AND PROTEIN EXPRESSION IS ABSENT IN TESTICULAR GERM CELL TUMOURS To determine whether HELA2 (testisin) may play a role in testicular tumourigenesis, HELA2 (testisin) mRNA expression in normal testes and testicular tumour tissue obtained from 4 patients diagnosed with seminoma were compared by Northern blot analysis. HELA2 (testisin) mRNA was detected in normal testes from all four patients but was not detectable in the corresponding tumours (Figure 14A). These data indicate a tumour suppressor role for HELA2 (testisin) in testicular germ cell tumours.

Expression of HELA2 (testisin) protein in testicular tissue was examined by immunohistochemistry. Paraffin-embedded tissue sections were fixed, treated, blocked, incubated with anti-peptide antibodies (1:10 dilution) and bound antibody detected with the 15 Vectastain Universal Elite ABC kit (Vector Laboratories). Negative controls were performed in the absence of antibody. Strong staining of HELA2 (testisin) was detected in the germ cells of normal testis but was absent in the adjacent tumour tissue (for example, see Figure 14B), providing further evidence of a tumour suppressor role for HELA2 (testisin) in testicular germ cell tumours.

In the seminiferous tubules of the testis, male germ cells differentiate from diploid spermatogonia to haploid spermatozoa through the process of spermatogenesis. Mitotic division of spermatogonia yields preleptotene primary spermatocytes which sequentially pass through leptotene, zygotene, pachytene, diplotene and diakeninesis stages during the first meiotic 25 prophase. Completion of this first meiotic division results in secondary spermatocytes which proceed through a second meiotic division giving rise to haploid round spermatids. These cells then undergo gross morphological changes during spermiogenesis to become spermatozoa. The immunostaining experiments show that testisin expression is localised to testicular germ cells undergoing meiosis, with strongest expression in pachytene spermatocytes. Significantly, it is the spermatocytes in this first meiotic midprophase (zygotene-pachytene-diplotene) that are believed to be the precursors for all male germ cell tumours. Transformation of these testicular P:\OPER.EIH\023942-C.US -12/8/98 germ cells gives rise to either a seminoma (an undifferentiated germ cell neoplasm) or an embryonal carcinoma (a neoplasm of totipotent cells). The latter tumours may undergo further somatic differentiation to become teratomas/ teratocarcinomas or may undergo extra-embryonic differentiation to yolk sac tumours (endodermal sinus tumour) or choriocarcinomas. The inventors have examined a number of these germ cell-derived tumours for testisin expression and find absence of testisin mRNA and protein expression in all testicular tumours examined to date. In addition, a screen of 8 human testicular tumour cell lines showed absence of testisin expression in both embryonal and yolk sac derived tumour cells. Thus, loss of testisin is likely to be a significant event in testicular germ cell tumour formation and progression.

In the germ cell, testisin would be well-positioned to anchor on the surface where it could proteolytic activate growth factors and cytokines, participate in cellular signalling events, activate other proteolytic cascades and/or participate in cell migration and matrix remodelling.

These activities may well impact on the processes of germ cell proliferation, apoptosis, and 15 differentiation such as occurs during spermatogenesis. Arrest of differentiation or apoptosis in germ cells could lead to uncontrolled growth and tumour progression due to an inability to progress to a differentiation stage with a more restricted cell growth.

EXAMPLE 11 20 GENOMIC ORGANISATION OF THE HELA2 (TESTISIN) GENE The HELA2 (testisin) gene is further characterised by determination of its genomic organisation. Intron-exon boundaries, promoter sequence and the full DNA sequence of the HELA2 (testisin) gene was determined from cosmid DNA by DNA sequencing. A genomic 25 map of HELA2 (testisin) is given in Figure 15. The testisin genomic DNA sequence is shown in Figure 16 and in SEQ ID NO 27. The intron/exon boundaries are highly conserved relative to prostasin, although the sizes of the introns show considerable variation. All intron/exon boundaries conform with the GT-AG rule. The HELA2 (testisin) promoter lacks a TATA box, but contains a putative CAAT box, and 3 potential Spl binding-GC boxes in the upstream region. The transcription initiation site was determined by primer extension. The results show that similar to other TATA-less promoters, the HELA2 (testisin) promoter has several P:OPER\EJH\023942-CUS- 12/8/98 -56transcription initiation sites located between nucleotides 500-700 in Figure 16. The promoter region also contains a number of elements associated with transcription of testis-specific genes.

It is possible that absence of testisin expression in tumours is due to mutations/deletions in the testisin gene or changes in the control of testisin gene expression in the tumour cells. Sequence of genomic DNA from matched tumour/normal tissue pairs is being compared to determine whether mutations in the gene may contribute to the HELA2 (testisin)'s predicted function as a tumour suppressor. Significant changes in methylation patterns of genes have also been found to occur during germ cell maturation. Methylation frequently occurs in regions of genes enriched for cytosine nucleotides linked on the 3' side to guanine (CpG islands). Methylation of CpG dinucleotides represses trancription, often to undetectable levels, and tumour suppressor genes are frequently inactivated by ectopic de novo methylation of promoter regions. Methylation inhibits gene expression by changing chromatin conformation and accessibility, and interfering with protein-DNA interactions required for transcription. The 15 HELA2 (testisin) gene contains an 800bp CpG island within the 5' region of the HELA2 (testisin) gene. Bisulfite genomic sequencing demonstrates that the methylation status of the 5' region of the testisin gene is a determinant of testisin gene expression; that is, in cells that express the testisin gene this gene is not methylated, while in cells that do not express testisin, the testisin gene is methylated. Thus the suppression of testisin gene expression in testicular 20 tumours is likely due to an effect of methylation on gene transcription. Thus, modulation of methylation status is a method of controlling testisin expression.

EXAMPLE 12 THE HELA2 (TESTISIN) SHORT AND LONG ISOFORMS 25 ARE GENERATED BY ALTERNATIVE mRNA SPLICING Two isoforms of HELA2 (testisin) were identified which differ by an insertion of 2 amino acids (Tyr-Ser) between the catalytic His and Asp residues. These constitute the long and short isoforms. At the DNA level there is a corresponding insertion of 6 nucleotides which generates a Sfcl restriction enzyme site. PCR amplification from single strand cDNA generated from HeLa cell total RNA followed by DNA sequence analysis of the amplified product P\OPER\EJ\023942.C.US. 12/898 -57demonstrated that the two isoforms are generated through the use of two alternative mRNA splice sites. The DNA sequence for the intron and the flanking exons are shown in Figure 17.

The resulting insertion of amino acids YS occurs 4 amino acids after the catalytic His residue of HELA2 (testisin). Preliminary molecular modelling shows the presence of this insertion is likely to alter the catalytic activity and/or specificity of HELA2 (testisin) for its substrates.

EXAMPLE 13 MUTATION ANALYSIS-HELA2 (TESTISIN) AS A TUMOUR SUPPRESSOR Intronic DNA sequence information generated above (see Example 11) is used to generate primers to amplify HELA2 (testisin) exons for SSCP analyses. Genomic DNA isolated from seminomas and corresponding normal testis as well as genomic DNA from wild-type and affected seminoma family members are analysed by SSCP for altered expression patterns indicative of genetic mutations. Evidence of genetic mutations are also being determined by 15 DNA sequence analysis.

EXAMPLE 14 HOMOLOGUES OF HUMAN HELA2 (TESTISIN) ARE PRESENT IN OTHER SPECIES Southern blot analysis of genomic DNA isolated from a range of species using a HELA2 (testisin) cDNA probe shows that homologues of HELA2 (testisin) are present in hamster, *oo mouse, marmoset and monkey. The mouse homologue of HELA2 (testisin) was identified and obtained as an EST clone. The cDNA sequence and corresponding amino acid sequence of 25 mouse HELA2 (testisin) was determined (Figure 18) and is given in SEQ ID NO 27. The mouse cDNA encodes a protein which contains the catalytic triad of His, Asp and Ser (circles) and 10 cysteine residues (small boxes), and an activation site (triangle) as found in HELA2 (testisin). The hydrophilicity plot shows the presence of a hydrophobic sequence at the carboxy terminus suggesting the presence of a putative membrane anchor. Comparison of the mouse and human sequences show 68.1% homology at the cDNA level and 69.1% homology at the amino acid level. The inventors have determined that the mouse testisin gene is contained within a P:AOPER\EH023942-C.US 2/8/98 -58- HindII restriction enzyme fragment and have isolated an approximately 20kb genomic clone containing the mouse gene.

EXAMPLE HELA2 (TESTISIN) IS PART OF A CLUSTER OF HOMOLOGOUS GENES ON CHROMOSOME 16pl3.3 Analysis of DNA sequences released to NCBI databases reveals the presence of homologues of HELA2 (testisin) in a cluster on Chromosome 16pl3.3. Figure 19 shows the positions of these genes, designated SP001LA, SP002LA, SP003LA, and SP004LA, relative to HELA2 (testisin) and the respective cosmids (Sood, R. et al (1997) Genomics 42: 83-95) in which they are located. Figure 20A, 20B and 20C show the partial cDNA and deduced amino acid sequences of SP001LA, SP002LA, and SP003LA respectively. Each cDNA encodes a protein which contains the catalytic triad of His, Asp and Ser (circles) and 10 cysteine residues (small 15 boxes), and an activation site (triangle) as found in HELA2 (testisin). Comparisons of the cDNA and amino acid sequences from the heavy chain region through to the poly A tail gives the identity with HELA2 (testisin) as follows: cDNA Protein 20 SP001LA 34.8% 47.3% S. SP002LA 41.0% 47.1% SP003LA 40.3% 51.3% Each of the serine proteinases encoded by these genes show that they have carboxy terminal extensions, and SP002LA is the only one with a hydrophobic carboxy terminal tail indicative of a membrane anchored protein. Identification of an expressed sequence tag (EST) from a human testis cDNA library demonstrates that this gene is expressed in the testis, like HELA2 (testisin). The location of this serine proteinase cluster on chromosome 16pl3.3 flanking HELA2 (testisin) suggests that these serine proteinases are also involved, like HELA2(testisin), PA\OPER\EJH\023942-C.US 12/8/98 -59in sperm maturation and development. Thus they may constitute a proteolytic cascade which is essential for these processes. Loss or mutation of these genes may lead to testicular germ cell tumours and to other testicular abnormalities, such as infertility.

EXAMPLE 16 ATC2 SERINE PROTEINASE ATC2 was isolated from the cDNA of PAI-2 expressing HeLa cells following treatment with TNF and cycloheximide. A partial DNA sequence for ATC2 cDNA has been obtained which encompasses the sequence encoding the serine proteinase catalytic region. Additional clones extending to both 5' and 3' directions have been obtained. The available nucleic acid sequence of ATC2 cDNA and its deduced amino acid sequence shows that it is a member of the serine proteinase family with homology to hepsin, prostasin, and acrosin. It thus belongs to the same family as HELA2. The catalytic region includes the His, Asp and Ser conserved motifs.

15 Preliminary Northern blot experiments have failed to detect ATC2 mRNA in total RNA isolated from resting HeLa cells, indicating it is not expressed in abundance in these cells, which may therefore be tightly regulated. As ATC2 was isolated from cells following treatment with TNF and cycloheximide, its expression may be induced by these agents in HeLa cells. These data have potential significance for a role for ATC2 in apoptosis and cell death. ATC2 may be intracellular, extracellular or found on the cell surface and is likely to be involved in regulating cell functions. Thus ATC2 may have potential significance in the treatment of cancer and diseases involving dysregulation of cell growth and survival. The nucleotide and corresponding amino acid sequence of ATC2 is shown in SEQ ID NOs: 7 and 8, respectively.

25 Northern blot analysis of mRNA from a range of normal human tissues shows that ATC2 mRNA is strongly expressed exclusively in the heart (Figure 22A). Further, homologues of ATC2 are present in other species. Northern blot analysis of mRNA from a range of normal mouse tissues shows the presence of ATC2 in mouse heart (Figure 22B). These data demonstrate that the ATC2 mRNA transcript is relatively large, approximately 5kb. The deduced amino acid sequence of the carboxy terminal 308 amino acids of ATC2 shows that it has closest homology to the serine proteinase, hepsin (31% identity). The catalytic region P\OPER\EIJH23942-C.US 12/98 includes the His, Asp and Ser conserved motifs and the nature of the binding pocket indicates that ATC2 has Arg specificity, as found with both hepsin and testisin (Figure 22C). The homology to hepsin and the size of the mRNA transcript may suggest that ATC2 has an extended amino-terminal pro-region linked to the catalytic region. The pro-region of hepsin contains an amino-terminal transmembrane domain which serves to anchor hepsin to the cell surface. Pro-regions in serine proteinases may be of variable size and frequently encode functional binding sites which confer specific biological activities and/or binding properties that can impact on the serine proteinase biological activity.

Antibodies (polyclonal and monoclonal) against ATC2 are used to detect ATC2 in biological fluids, cells and tissues. For example, rabbit polyclonal antibodies are generated against ATC2 peptide antigens. Two peptides are selected from the ATC2 amino acid sequence on the basis of predicted antigenicity, hydophilicity and lack of identity with known proteins.

15 Peptide antigen:A1A (amino) Ile-Gln-Glu-Gln-Glu-Lys-Glu-Pro-Arg-Trp-Leu-Thr-Leu-His- Ser-Asn-Trp-Glu-Cys [SEQ ID NO:37] Peptide antigen:A1B (catalytic) Gly-His-Met-Gly-Asn-Lys-Met-Pro-Phe-Lys-Leu-Gln-Glu- .Gly-Glu-Cys [SEQ ID NO:38] These peptides were synthesized (Auspep) and coupled to keyhole limpet hemocyanin. The coupled peptide (500micrograms) in PBS (0.5ml) was emulsified in an equal volume of S.:i Freund's complete adjuvant before injection into a rabbit. Booster injections of coupled peptide in Freund's incomplete adjuvant were made at intervals of 2 to 3 weeks. Each rabbit was bled (approximately 1 ml) before the initial injection and about 7 days after the second and 25 subsequent boosters and antibody titre assessed by direct ELISA assay. Rabbit antisera is affinity purified against the respective immunising peptides by chromatography using peptidecoupled affinity columns. Immunoreactivity of the respective peptides is demonstrated by immunostaining of tissue specimens..

The strong and specific expression pattern of ATC2 in heart would suggest that, in a similar way to testisin's role in the testis, ATC2 has a specific proteolytic function in the physiology P:\OPER\EJHM23942-C.US 12/8/98 -61of the heart. For example, ATC2 could play a role in the conduction system of the heart, such as in the regulation of cardiac sodium pump function. Other serine proteinases with Argspecificity similar to ATC2 (eg. urokinase, CAP-1) as well as serine proteinase inhibitors (eg.

aprotinin) have been implicated previously in the regulation of trans-epithelial sodium transport.

Hepsin is required for hepatoma cell growth and maintenance of normal cell morphology and has been demonstrated to activate the extrinsic blood coagulation pathway via Factor VII leading to thrombin formation. ATC2 could play a similar or related proteolytic function in the heart through regulation of a coagulative or fibrinolytic cascade.

ATC2 is likely to be important for the understanding of basic regulatory mechanisms in heart physiology and further may provide a new agent in the treatment of cardiac disease. The specificity of ATC2 suggests that it plays a unique and essential role in the heart, probably involving a critical function within the myocardium. ATC2 has potential as a marker of heart disease, a critical factor in cardiac organogenesis, and a new, rational target for 15 therapeutic design of inhibitors and other targeted drugs that can modify cardiac function by acting on ATC2 in the management of cardiovascular disease. If ATC2 proves to be myocyte specific, then release of this proteinase into the bloodstream as a result of myocardial infarction or other damage may provide a new and specific marker for heart attack.

EXAMPLE 17 BCON3 The deduced amino acid sequence ofBCON3 (SEQ ID NO: 10) reveals that it is novel. At both the DNA and protein level, BCON3 shows homology to members of the kinase family of 25 proteins. Although it cannot be classified as a member of any particular sub-family of kinases, alignments of the BCON3 protein with the conserved domains of thymidine kinases and tyrosine and serine/threonine protein kinases indicates possible ATP/GTP binding and phosphate transfer regions. Thus, it may be the first member of a new family of kinases. Analysis of the translation product using hydrophobicity plots and the Prosite protein analysis algorithms indicates BCON3 may lack an N-terminal signal sequence (that is, it is likely to encode an intracellular protein) and it possesses a nuclear localization signal. BCON3 mRNA is approximately 2300 P:\OPER IHE 23942-C.US 12/898 -62nucleotides in length. cDNA sequence (SEQ ID NO:9) has been obtained covering about of the transcript and including the 3' poly A tail. BCON3 mRNA is expressed in most normal tissues as demonstrated by dot blot analysis of 50 normal tissue specimens (standardised to 8 different housekeeping genes) (Clontech). (Figure Analysis of BCON3 mRNA expression using a multiple tissue Northern blot displaying polyA+ mRNA from 16 different normal tissues (Clontech) shows that BCON3 is expressed in most tissues (Figure 10B). Expression by in vitro transcription/translation expression using a partial BCON3 cDNA fragment shows BCON3 encodes a protein. Two major transcription/translation products are detected, one of 51kDa, the size predicted from the open reading frame, and a second product of about 43kDa, which may represent a partial translation product (Figure 21).

Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of 15 the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

9* 9. 9 9-- 9* °oe e P:OPEREJH\023942-C US 12//9 -63- SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT

(US only): ANTALIS Toni Marie and HOOPER John David (Other than US): AMRAD OPERATIONS PTY LTD (ii) TITLE OF INVENTION: NOVEL MOLECULES (iii) NUMBER OF SEQUENCES: 38 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: DAVIES COLLISON CAVE STREET: 1 LITTLE COLLINS STREET CITY: MELBOURNE STATE: VICTORIA COUNTRY: AUSTRALIA ZIP: 3000 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release Version #1.25 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: USSN S(B) FILING DATE: 13-AUG-1998

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o (vii) PRIOR APPLICATION DATA: S(A) APPLICATION NUMBER: PCT/AU98/00085 FILING DATE: 13-FEB-1998

CLASSIFICATION:

00 (vi) PRIOR APPLICATION DATA: APPLICATION NUMBER: USSN 09/023,942 FILING DATE: 13-FEB-1998

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: PO5101/97 FILING DATE: 13-FEB-1997

CLASSIFICATION:

P:AOPEMEf{W942-C.US. IDWS 64 (vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: PP0422/97 FILING DATE: 18-NOV- 1997

CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION: NAME: HUGHES, DR E JOHN L REFERENCE/DOCKET NUMB3ER: EJH-IAF (ix) TELECOMMUNICATION INFORMATION: TELEPHONE: +61 3 9254 2777 TELEFAX: +61 3 9254 2770 TELEX: AA 31787 P:\OPER\EJH\023942-C.US 12/8/98 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: ACAGAATTCT GGGTIGTIAC IGCIGCICAY TG 32 INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 1094 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: ACAGAATTCA XIGGICCICC IC/GT/AXTCICC 29 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 1094 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 17..965 PA~OPERW\023942-C.US 12/&98 66 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CGCGGGAGAG GAGGCC ATG GGC GCG CGC GGG GCG CTG CTG CTG GCG CTG Met Gly Ala Arg Gly Ala Leu Leu Leu Ala Leu 1 5 CTG CTG GCT CGG GCT GGA CTC AGG AAG CCG GAG TCG CAG GAG GCG C Leu Leu Ala Arg Ala Gly Leu Arg Lys Pro Glu Ser Gln Glu Ala Ala 20 CCG TTA TCA GGA CCA TGC GGC CGA COG OTC ATC ACO TCG CCC ATC OTO Pro Leu Ser Gly Pro Cys Oly Arg Arg Val Ile Thr Ser Arg Ile Vai 145 GGT GOA GAG Oly Gly Giu GAC GCC GAA Asp Ala Olu

CTC

Leu 50 GGG CGT TGG Gly Arg Trp CCG TGG Pro Trp CAG GGG AGC CTG Gin Oly Ser Leu 193

S.

S.

S.

CGC

Arg CTG TGG GAT TCC Leu Trp Asp Ser GTA TGC OGA Val Cys Oly GTG AGC Vai Ser 70 CTG CTC AOC CAC Leu Leu Ser His

CC

Arg TGO OCA CTC ACG Trp Ala Leu Thr

GCG

Ala GCG CAC TOC TTT Ala His Cys Phe

OAA

Olu 85 ACT GAC CTT AGT Thr Asp Leu Ser GAT CCC Asp Pro TCC GOG TOO Ser Gly Trp TGO AGC CTO Trp Ser Leu 110

ATG

Met GTC CAG TTT GGC Val Gin Phe Gly

CAG

Gin 100 CTG ACT TCC Leu Thr Ser CAG GCC TAC TAC Gin Ala Tyr Tyr

ACC

Thr 115 CGT TAC TTC OTA Arg Tyr Phe Val ATO CCA TCC TTC Met Pro Ser Phe 105 TCG AAT ATC TAT Ser Asn Ile Tyr 120 ATT GCC TTO GTG Ile Ala Leu Val CAG CCC ATC TOT Gin Pro Ile Cys 155 337 385 433 481 CTO AGC Leu Ser 125 CCT CGC TAC CTG Pro Arg Tyr Leu 000 Gly 130 AAT TCA CCC Asn Ser Pro TAT GAC Tyr Asp 135

AAG

Lys 140 CTG TCT OCA CCT Leu Ser Ala Pro GTC ACC Val Thr 145 TAC ACT AAA CAC ATC Tyr Thr Lys His Ile 150 CTC CAG GCC TCC Leu Gin Ala Ser ACA TTT GAG TTT GAG Thr Phe Giu Phe Olu 160 AAC COO ACA GAC TGC TGG GTG Asn Arg Thr Asp Cys Trp Val 165 170 PAOPER\FHfMD3942-C.US. IM89 67 ACT GGC Thr Gly TGG GGG TAC ATC AAA GAG Trp Giy Tyr Ile Lys Giu

GAT

Asp 180 GAG GCA CTG CCA TCT CCC CAC Giu Ala Leu Pro Ser Pro His 185 ACC CTC CAG Thr Leu Gin 190 GAA GTT CAG GTC Giu Val Gin Val

GCC

Ala 195 ATC ATA AAC AAC Ile Ile Asn Asn

TCT

Ser 200 ATG TGC AAC Met Cys Asn 625 CAC CTC His Leu 205 TTC CTC AAG TAC Phe Leu Lys Tyr

AGT

Ser 210 TTC CGC AAG GAC Phe Arg Lys Asp

ATC

Ile 215 TTT GGA GAC ATG Phe Gly Asp Met

GTT

Val1 220 TGT GCT GGC AAT Cys Ala Gly Asn

GCC

Ala 225 CAA GGC GGG AAG Gin Gly Gly Lys

GAT

Asp 230 GCC TGC TTC GGT Ala Cys Phe Gly

GAC

Asp 235 TCA GGT GGA CCC Ser Gly Gly Pro GGA GTC GTG AGC Gly Val Val Ser 255

TTG

Leu 240 GCC TGT AAC AAG Ala Cys Asn Lys

GAT

Asp 245 GGA CTG TGG TAT Gly Leu Trp Tyr CAG ATT Gin Ile 250 CCC GGT Pro Gly 769 TGG GGA GTG GGC Trp Giy Val Gly

TGT

Cys 260 GGT CGG CCC AAT Gly Arg Pro Asn

CGG

Arg 265 817 GTC TAC ACC Val Tyr Thr 270 AAT ATC AGC CAC Asn Ile Ser His

CAC

His 275 TTT GAG TGG ATC Phe Giu Trp Ile

CAG

Gin 280 AAG CTG ATG Lys Leu Met 865 913 GCC CAG Ala Gin 285 AGT GGC ATG TCC Ser Gly Met Ser

CAG

Gin 290 CCA GAC CCC TCC Pro Asp Pro Ser

TGG

Trp 295 CCG CTA CTC TTT Pro Leu Leu Phe

TTC

Phe 300 CCT CTT CTC TGG Pro Leu Leu Trp

GCT

Ala 305 CTC CCA CTC CTG Leu Pro Leu Leu

GGG

Gly 310 CCG GTC TGA Pro Val GCCTACCTGA GCCCATGCAG CCTGGGGCCA CTGCCAAGTC AGGCCCTGGT TCTCTTCTGT CTTGTTTGGT AATAAACACA TTCCAGTTGA TGCCTTGCAG GGCATTTTTC AAAAAA AAAAAAAA AAAAAA- 10i5 1075 1094 INFORMATION FOR SEQ ID NO:4: P:OPEMOEHMD3942-C.US. 12/81 68 SEQUENCE CHARACTERISTICS: LENGTH: 313 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: Met 1 Gly Ala Arg Gly Ala Leu Leu 5 Leu Ala Leu Leu Leu Ala Arg Ala 10 Giu Ala Ala Pro Leu Ser Gly Pro Gly Leu Arg Lys Pro Cys Gly Arg Arg Val Giu Leu Gly Arg Trp Glu Ser Gin Ile Thr Ser 40 Gin Arg Ile Val Gly Ser Leu Gly Gly Ara Leu Glu Asp Ala Trp Asp Ser Pro

S

*S

*5

S

His Val Trp 55 Leu Trp Cys Gly Vai 65 Ala Ser 70 Leu Ser His Arg 75 Pro Ala Leu Thr Ala His Cys Phe Giu Leu Thr Asp Leu Ser Asp 90 Ser Ser Gly Trp Met Val Gin Phe Gly Tyr Tyr Thr 115 Leu Gly Asn Gin 100 Arg Thr Ser Met Pro 105 Asn Phe Trp Ser Tyr Phe Val Ser 120 Ile Ile Tyr Leu Ser 125 Leu Leu Gin Ala 110 Pro Arg Tyr Ser Ala Pro Ser Pro Tyr 130 Val Thr Asp 135 Ile Ala Leu Val Lys 140 Leu Tyr Thr Lys 145 Phe His 150 Arg Gin Pro Ile Cys 155 Val1 Gin Ala Ser Thr 160 Tyr Glu Phe Glu Asn 165 Thr Asp Cys Trp 170 Thr Gly Trp Gly 175 Ile Lys Giu Asp Giu Ala Leu Pro Ser Pro His Thr Leu Gin Giu Val 180 185 190 P:\OPEREIHO23942-C.US 12/8/98 -69- Gln Val Ala 195 Ile Ile Asn Asn Ser Met Cys Asn His Leu Phe Leu Lys 200 205 Tyr Ser 210 Phe Arg Lys Asp Ile 215 Phe Gly Asp Met Val 220 Cys Ala Gly Asn Ala 225 Gin Gly Gly Lys Asp 230 Ala Cys Phe Gly Asp 235 Ser Gly Gly Pro Leu 240 Ala Cys Asn Lys Asp 245 Gly Leu Trp Tyr Gin 250 Ile Gly Val Val Ser Trp 255 Thr Asn Ile 270 Gly Val Gly Ser His His 275 Cys 260 Gly Arg Pro Asn Arg 265 Pro Gly Val Tyr Phe Glu Trp Ile Gin 280 Lys Leu Met Ala Gin Ser Gly Met 285 Ser Gin 290 Pro Asp Pro Ser Trp 295 Pro Leu Leu Phe Phe Pro Leu Leu Trp 300 r r Ala 305 Leu Pro Leu Leu Gly 310 Pro Val ee INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1100 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 17..961 (xi) SEQUENCE DESCRIPTION: SEQ ID CGCGGGAGAG GAGGCC ATG GGC GCG CGC GGG Met Gly Ala Arg Gly 1 GCG CTG CTG CTG GCG CTG Ala Leu Leu Leu Ala Leu P:%OPER\E1I-23942-C.US 12A/U9 CTG CTG GCT CGG GCT GGA CTC AGG AAG CCG GAG TCG CAG GAG GCG GCG Leu Leu Ala Arg Ala Gly Leu Arg Lys Pro Giu Ser Gin Glu Ala Ala CCG TTA TCA GGA CCA TGC GGC CGA CGG GTC ATC Gly Arg Arg Val Ile Pro Leu Ser Gly Pro Cys ACG TCG CGC ATC GTG Thr Ser Arg Ile Val 145 GGT GGA Gly Gly GAG GAC GCC GAA Glu Asp Ala Glu

CTC

Leu 50 GGG CGT TGG Gly Arg Trp CCG TGG Pro Trp CAG GGG AGC CTG Gin Gly Ser Leu 193 241

CGC

Arg CTG TGG GAT TCC Leu Trp Asp Ser

CAC

His 65 GTA TGC GGA GTG Vai Cys Gly Val

AGC

Ser 70 CTG CTC AGC CAC Leu Leu Ser His

CGC

Arg TGG GCA CTC ACG Trp Ala Leu Thr

GCG

Ala GCG CAC TGC TTT Ala His Cys Phe

GAA

Glu 85 ACC TAT AGT GAC Thr Tyr Ser Asp CTT AGT Leu Ser 289 r r r GAT CCC TCC Asp Pro Ser TCC TTC TGG Ser Phe Trp 110

GGG

Gly TGG ATG GTC CAG Trp Met Val Gin

TTT

Phe 100 GGC CAG CTG ACT Gly Gin Leu Thr TCC ATG CCA Ser Met Pro 105 GTA TCG AAT Val Ser Asn 337 AGC CTG CAG GCC Ser Leu Gin Ala TAC ACC CGT TAC Tyr Thr Arg Tyr

TTC

Phe 120 ATC TAT Ile Tyr 125 CTG AGC CCT CGC Leu Ser Pro Arg

TAC

Tyr 130 CTG GGG AAT TCA Leu Gly Asn Ser CCC TAT GAC ATT GCC Pro Tyr Asp Ile Ala 135 385 433 481 GTG AAG CTG TCT Val Lys Leu Ser

GCA

Ala 145 CCT GTC ACC TAC Pro Val Thr Tyr ACT AAA Thr Lys 150 CAC ATC CAG His Ile Gin

CCC

Pro 155 ATC TGT CTC CAG Ile Cys Leu Gin TGG GTG ACT GGC Trp Val Thr Gly 175

GCC

Ala 160 TCC ACA TTT GAG Ser Thr Phe Glu

TTT

Phe 165 GAG AAC CGG Glu Asn Arg ACA GAC TGC Thr Asp Cys 170 529 TGG GGG TAC ATC Trp Gly Tyr Ile AAA GAG Lys Glu 180 GAT GAG GCA CTG CCA TCT Asp Giu Ala Leu Pro Ser 185 CCC CAC ACC CTC CAG GAA GTT CAG GTC GCC ATC ATA AAC AAC TCT ATO 625 PAPEREH 23942-CUS I /98 -71 Pro His Thr 190 Leu Gin Giu Val Gin Val 195 Ala Ile Ile Asn Asn Ser Met 200 TGC AAG Gys Asn 205 CAC GTC TTC CTC His Leu Phe Leu

AAG

Lys 210

TAG

Tyr AGT TTC CGC AAG Ser Phe Arg Lys 215 GAG ATG TTT GGA Asp Ile Phe Gly 673 721

GAC

Asp 220 ATG GTT TGT GCT Met Val Cys Ala GGC AAT GGC CAA GGC GGG Gly Asn Ala Gin Giy Gly 225 230 AAG GAT GCC TGC TTC Lys Asp Ala Gys Phe 235 GGT GAG TCA GGT Gly Asp Ser Gly

GGA

Gly 240 CCC TTG GCC TGT Pro Leu Aia Gys AAG GAT GGA GTG Lys Asp Gly Leu TGG TAT Trp Tyr 250 CAG ATT GGA Gin Ile Gly CCC GGT GTG Pro Giy Val 270

GTC

Val 255 GTG AGC TGG GGA Val Ser Trp Gly

GTG

Val 260 GGG TGT GGT CGG Gly Cys Gly Arg CCC AAT CGG Pro Asn Arg 265 ATG CAG AAG Ile Gin Lys r r r r r r r TAG ACC AAT ATG Tyr Thr Asn Ile

AGC

Ser 275 CAC CAC TTT GAG His His Phe Glu

TGG

Trp 280 817 865 913 GTG ATG Leu Met 285 GCC CAG AGT GGG Ala Gin Ser Gly

ATG

Met 290 TCC CAG CCA GAG Ser Gin Pro Asp

CCC

Pro 295 TCC TGG CCG GTA Ser Trp Pro Leu CTC TTT TTC CCT 968 GTT CTC TGG GGT CTC CCA CTC CTG GGG CCG GTG TGAGCCTACC Leu Phe Phe Pro Leu Leu Trp Ala Leu Pro Leu Leu Gly Pro Val 300 305 310 315 TGAGCCCATG GAGCCTGGGG CCACTGCCAA GTGAGGCCCT GGTTCTCTTC TGTGTTGTTT GGTAATAAAG AGATTCCAGT TGATGCCTTG GAGGGGATTT TTGAAAAAAA AAAAAAAAAA AAAAAAAAAA AA 1028 1088 1100 INFORMATION FOR SEQ ID NO:6: SEQUENGE GHARAGTERISTIGS: LENGTH: 314 amino acids TYPE: amino acid TOPOLOGY: linear P AOPERTJH=~O3942-C.US M0199 72 (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met Gly Ala Arg Gly Ala Leu Leu Leu Ala Leu Leu Leu Ala Arg Ala Giy Leu Arg Lys Cys Giy Arg Arg Giu Leu Giy Arg Pro Glu Ser Gin Val Ile Thr Ser Giu Ala Ala 25 Arg Ile Val Pro Leu Ser Gly Pro Gly Gly Giu Asp Ala Arg Leu Trp Asp Ser Trp Pro His Val Trp 55 Leu Gin Giy Ser Leu Trp Cys Gly Vai Ala Ser 70 Thr Leu Ser His Arg 75 Ser Aia Leu Thr Ala S. .5.5 S S. S S

S.

His Cys Phe Giu Gly Tyr Ser Asp Leu 90 Met Asp Pro Ser Giy Trp Met Vai Gin Gin Ala Tyr 115 Arg Tyr Leu Phe 100 Tyr Gin Leu Thr Ser 105 Val1 Pro Ser Phe Trp Ser Leu 110 Leu Ser Pro Thr Arg Tyr Phe 120 Tyr Ser Asn lie Tyr 125 Vali Giy Asn Ser 130 Ala Pro Pro 135 Lys Asp Ile Ala Leu 140 Ile Lys Leu Ser Val Thr Tyr 145 Ser Thr 150 Glu His Ile Gin Pro 155 Cys Cys Leu Gin Thr Phe Glu Phe 165 Glu Asn Arg Thr Asp 170 Pro Trp Val Thr Gly Trp 175 Gly Tyr Ile Giu Val Gin 195 Lys 180 Val1 Asp Giu Ala Leu 185 Asn Ser Pro His Thr Leu Gin 190 His Leu Phe Ala Ile Ile Asn 200 Ser Met Cys Asn 205 Leu Lys 210 Tyr Ser Phe Arg Lys Asp 215 Ile Phe Gly Asp Met Val Cys Ala 220 P:\OPER\EfJ023942-C.US 12/898 -73- Gly Asn 225 Ala Gin Gly Gly Lys Asp Ala 230 Cys Phe Gly Asp Ser Gly Gly 235 240 Pro Leu Ala Cys Asn 245 Lys Asp Gly Ser Trp Gly Asn Ile Ser 275 Gly Cys Gly Arg Leu Trp Tyr Gin Ile Gly Val Val 250 255 Pro Asn Arg Pro Gly Val Tyr Thr 265 270 Ile Gin Lys Leu Met Ala Gin Ser 285 His His Phe Glu Trp 280 Gly Met 290 Ser Gin Pro Asp Pro 295 Ser Trp Pro Leu Leu Phe Phe Pro Leu 300 a a Leu Trp Ala Leu Pro Leu Leu Gly Pro Val 305 310 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 976 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 2..928 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: A GAA CCA TCT GTG ACC AAA TTG ATA CAG Glu Pro Ser Val Thr Lys Leu Ile Gin GAA CAG GAG AAA GAG CCG Glu Gin Glu Lys Glu Pro CGG TGG CTG ACA TTA CAC TCC AAC TGG GAG AGC CTC AAT GGG ACC ACT Arg Trp Leu Thr Leu His Ser Asn Trp Glu Ser Leu Asn Gly Thr Thr 25 PAOPEREJ{W3942-C.US I2/V8 74 TTA CAT GAA CTT CTA GTA AAT GGG CAG TCT TGT GAG AGC AGA AGT AAA 142 Leu His Giu Leu Leu Val Asn Gly Gin Ser Cys Giu Ser Arg Ser Lys ATT TCT CTT Ile Ser Leu CTG TGT ACT AAA Leu Cys Thr Lys CAA GAC TGT GGG CGC CGC CCT GCT GCC Gin Asp Cys Giy Arg Arg 55 Pro Aia Ala CGA ATG Arg Met AAC AAA AGG ATC Asn Lys Arg Ile

CTT

Leu 70 GGA GGT CGG ACG Gly Gly Arg Thr

AGT

Ser CGC CCT GGA AGG Arg Pro Giy Arg

TGG

Trp CCA TGG GAG TGT Pro Trp Gin Cys

TCT

Ser 85 CTG GAG AGT GAA Leu Gin Ser Giu

CCC

Pro 90 AGT GGA CAT ATC Ser Gly His Ile

TGT

Cys

TGG

Cys 286 GGC TGT GTG CTG Giy Cys Vai Leu

ATT

Ile 100 GGG AAG AAG TGG Aia Lys Lys Trp

GTT

Val1 105 GTG ACA GTT CC Leu Thr Val Ala

GAG

His 110 334 382 0000 o 0 0*1 TTG GAG GGG Phe Giu Giy AAG AAT CTA Asn Asn Leu 130

AGA

Arg 115 GAG AAT GCT GCA Giu Asn Ala Ala

GTT

Val1 120 GG AAA GTG GTG GTT GGG ATC Arg Lys Val Val Leu Giy Ile 125 GAG CAT CCA TGA Asp His Pro Ser GTG TTC ATG Val Phe Met 135 TAG AGT GGA Tyr Ser Arg GAG ACA GGC TTT GTG AGG Gin Thr Arg Phe Val Arg 140 GCA GTG GTG GAG TAT GAG Ala Val Vai Asp Tyr Asp 155 ACC ATC Thr Ile 145 ATG GTG CAT CCC Ile Leu His Pro

CGG

Arg 150 478 526

ATG

Ile 160 AGC ATC GTT GAG Ser Ile Val Giu

CTG

Leu 165 AGT GAA GAG Ser Glu Asp ATG AGT Ile Ser 170 GAG ACT GGG TAG Giu Thr Giy Tyr

GTC

Vali 175 CGG CCT GTC Arg Pro Val TAG TGC TAT Tyr Gys Tyr

TGG

Gys

TTG

Leu 180 CCC AAG CCG Pro Asn Pro GAG GAG Giu Gin 185 TGG GTA GAG CGT Trp Leu Giu Pro GAG ACG Asp Thr 190 ATG ACA GGC TGG GGC GAG ATG GGC AAT Ile Thr Gly Trp Gly His Met Giy Asn 195 200

AAA

Lys ATG CCA TTT Met Pro Phe 205 AAG CTG CAA GAG GGA GAG GTC CGC ATT ATT TGT CTG GAA CAT TGT GAG PA~OPERTEJH023942-C.US.- 12/SM 75 Lys Leu Gin Glu Gly Glu Val Arg Ile 210 215 Ile Ser Leu Glu His Cys Gin 220 TCG TAC Ser Tyr 225 TTT GAC ATG AAG Phe Asp Met Lys

ACC

Thr 230 ATC ACC ACT Ile Thr Thr CGG ATG Arg Met 235 ATA TGT GCT GGC Ile Cys Ala Gly 718 766

TAT

Tyr 240 GAG TCT GGC ACA Glu Ser Gly Thr

GTT

Val1 245 GAT TCA TGC ATG Asp Ser Cys Met

GGT

Gly 250 GAC AGC GGT GGG Asp Ser Giy Gly

CCT

Pro 255 CTT GTT TGT GAG Leu Val Cys Giu

AAG

Lys 260 CCT GGA GGA CGG Pro Giy Gly Arg

TGG

Trp 265 ACA TTA TTT GGA Thr Leu Phe Gly TTA ACT Leu Thr 270 GTT TAT Val Tyr 814 862 TCA TGG GGC Ser Trp Gly AGT AAT GTG Ser Asn Val 290

TCC

Ser 275 GTC TGC TTT TCC Val Cys Phe Ser

AAA

Lys 280 GTC CTG GGG CCT Val Leu Gly Pro

GGC

Giy 285 TCA TAT TTC GTC Ser Tyr Phe Val

GAA

Giu 295 TGG ATT AAA AGA Trp Ile Lys Arg

CAG

Gin 300 ATT TAG ATC Ile Tyr Ile 910 958 GAG AGG TTT GTG GTA AAG TAATTATAAG GATGATGAGA GAGTTTTGGC Gin Thr 305 Phe Leu Leu Asn AGCTAGAGTA AAAGAAAA INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 309 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Glu Pro Ser Val Thr Lys Leu Ile Gin Giu Gin Giu Lys Giu Pro Arg 1 5 10 Trp Leu Thr Leu His Ser Asn Trp Giu Ser Leu Asn Gly Thr Thr Leu 976 P*.OPERXEJH\023942-C.US IM/I8 76 Hius Giu Leu Leu Val Asn Gly Gin 40 Ser Cys Giu Ser Arg Ser Lys Ile Ser Leu Leu Cys Thr Lys Gin 55 Asp Cys Gly Arg Arg Pro Aia Ala Arg Asn Lys Arg Ile Leu Giy Gly Arg Thr Ser 75 Arg Pro Giy Arg Trp Pro Trp Gin Cys Ser Leu Gin Ser Giu Ser Gly His Ile Cys Gly Cys Vai Leu Giu Giy Arg 115 Ile 100 Aia Lys Lys Trp Val1 105 Leu Thr Val Ala His Cys Phe 110 Gly Ile Asn Giu Asn Ala Ala Val1 120 Arg Lys Val Val Leu 125 Asn Leu 130 Asp His Pro Ser Val1 135 Phe Met Gin Thr Phe Val Arg Thr Ile Leu His Pro Arg 150 Tyr Ser Arg Ala Val Asp Tyr Asp Ile 160 Ser Ile Vai Glu Leu Ser Glu Asp Ile 165 Ser 170 Giu Thr Gly Tyr Val Arg 175 Pro Val Cys Cys Tyr Ile 195 Leu 180 Pro Asn Pro Glu Gin 185 Trp Leu Glu Pro Asp Thr Tyr 190 Pro Phe Lys Thr Gly Trp, Giy Met Gly Asn Lys Met 205 Leu Gin 210 Glu Gly Glu Val Arg 215 Ile Ile Ser Leu Giu 220 His Cys Gin Ser Tyr 225 Phe Asp Met Lys Thr 230 Ile Thr Thr Arg Met 235 Ile Cys Ala Giy Tyr 240 Giu Ser Gly Thr Val1 245 Asp Ser Cys Met Gly 250 Asp Ser Gly Gly Pro Leu 255 Val Cys Giu Lys Pro Gly Gly Arg Trp Thr Leu Phe Gly Leu Thr Ser P:\OPER\EXH023942-C.US 12/898 -77- 265 270 Trp Gly Ser 275 Val Cys Phe Ser Lys 280 Glu Trp 295 Val Leu Gly Pro Gly Val Tyr Ser 285 Ile Lys Arg Gin Ile Tyr Ile Gin 300 Asn Val 290 Ser Tyr Phe Val Thr Phe Leu Leu Asn 305 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 2241 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 166..1773 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: ATTTAATACG ACTCACTATA GGGAATTTGG CCCTCGAGGA AGAATTCGGC ACGAGGCTGC GGCGCACTGT GAGGGAGTCG CTGTGATCCG GGGCCCCGAA CCCGACTGGA GCTGAAGCGC 120 AGGCTGCGGG GCGCGGAGTC GGGAGGCCTG AGTGTTCCTT CCAGC ATG TCG GAG Met Ser Glu GGG GAG TCC CAG ACA GTA CTT AGC AGT GGC TCA GAC CCA AAG GTA GAA Gly Glu Ser Gin Thr Val Leu Ser Ser Gly Ser Asp Pro Lys Val Glu 10 TCT TCA TCT TCA GCT CCT GGC CTG ACA TCA GTG TCA CCT CCT GTG ACC Ser Ser Ser Ser Ala Pro Gly Leu Thr Ser Val Ser Pro Pro Val Thr 25 30 270 PAOPER\FiH\O23942-C.US I M89 78 TGG AGA ACC TGA GCT GCT TGG CGA Ser Thr Thr Ser Ala Ala Ser Pro GAG GAA GAA GAA GAA AGT GAA GAT Giu Giu Giu Giu Glu Ser Glu Asp 45 CCC TGT GGG CGG TGG CAG AAG AGG Pro Gys Gly Arg Trp Gin Lys Arg 60 GAG TGT GAG ATT Giu Ser Giu Ile TTG GAA GAG TG Leu Giu Giu Ser 366 CGA GAA GAG Arg Giu Giu GTG AAT CAA CGG Val Asn Gin Arg AAT GTA GCA GGT ATT GAG Asn Val Pro Gly Ile Asp 75 AGT GCA TAC Ser Ala Tyr AAT GAG GTA Asn Giu Val 414 CTG GGG Leu Ala ATG GAT ACA GAG Met Asp Thr Giu

GAA

Giu 90 GGT GTA GAG GTT Gly Val Giu Vai

GTG

Val1

TGG

Trp

GAG

Gin 100 TTC TGT GAA CGG Phe Ser Giu Arg

AAG

Lys 105 AAC TAG AAG GTG Asn Tyr Lys Leu

GAG

Gin 110 GAG GAA AAG GTT Giu Giu Lys Val

TGT

Gys 115

AAG

Lys GGT GTG TTT GAT Ala Vai Phe Asp

AAT

Asn 120 TTG ATT GAA Leu Ile Gin TTG GAG Leu Giu 125 GAT GTT AAG ATT His Leu Asn Ile

GTT

Val1 130 510 558 606 654 TTT GAG AAA Phe His Lys TTT ATG AGA Phe Ile Thr 150

TAT

Tyr 135 TGG GGT GAG ATT Trp Ala Asp Ile

AAA

Lys 140 GAG AAG AAG GGG Giu Asn Lys Ala AGG GTG ATT Arg Val Ile 145 TTT GTG AAG Phe Leu Lys GGA TAG ATG TGA Gly Tyr Met Ser

TGT

Ser 155 GGG AGT GTG AAG Gly Ser Leu Lys

GAA

Gin 160 AAG AGG Lys Thr 165 GAA AAG AAG GAG Gin Lys Asn His

GAG

Gin 170 AGG ATG AAT GAA Thr Met Asn Glu

AAG

Lys 175 GGA TGG AAG GGT Ala Trp Lys Arg

TGG

Trp 180 TGG AGA GAA ATG Gys Thr Gin Ile

GTG

Leu 185 TGT GGG GTA AGG Ser Ala Leu Ser

TAG

Tyr 190 GTG GAG TGG TGT LeU His Ser Gys

GAG

Asp 195 GGG GGG ATG ATG Pro Pro Ile Ile GAT GGG His Giy 200 AAG GTG AGG TGT GAG AGG ATG TTG Asn Leu Thr Gys Asp Thr Ile Phe 205 ATG GAG Ile Gin 210 GAG AAG GGA GTG ATG AAG ATT GGG TGT GTG GGT GGT GAG AGT ATG AAG P-.OPR\EJH23942-C.US I2/MS9 -79- His Asn Gly Leu Ile Lys Ile Gly Ser Vai Ala Pro Asp Thr Ile Asn 225 215 220 AAT CAT GTG Asn His Val 230 AAG ACT TGT CGA Lys Thr Cys Arg

GAA

Glu 235 GAG CAG AAG Glu Gin Lys AAT CTA CAC TTC TTT Asn Leu His Phe Phe 240 GCA CCA Ala Pro 245 GAG TAT GGA GAA Glu Tyr Gly Glu

GTC

Val 250 ACT AAT GTG ACA ACA GCA GTG GAC ATC Thr Asn Val Thr Thr Ala Val Asp Ile 255

TAC

Tyr 260 TCC TTT GGC ATG Ser Phe Gly Met

TGT

Cys 265 GCA CTG GGG ATG Ala Leu Giy Met

GCA

Ala 270 GTG CTG GAG ATT Val Leu Glu Ile

CAG

Gin 275 990 GGC AAT GGA GAG Gly Asn Giy Glu

TCC

Ser 280 TCA TAT GTG CCA Ser Tyr Val Pro

CAG

Gin 285 GAA GCC ATC AGC Glu Ala Ile Ser AGT GCC Ser Ala 290 1038 4 .4 4 4 4 *r 444 ATC CAG CTT Ile Gin Leu CTG CAG TCT Leu Gin Ser 310

CTA

Leu 295 GAA GAC CCA TTA Glu Asp Pro Leu

CAG

Gin 300 AGG GAG TTC ATT Arg Giu Phe Ile CAA AAG TGC Gin Lys Cys 305 CTT CTG TTC Leu Leu Phe 1086 1134 GAG CCT GCT CGC Glu Pro Ala Arg

AGA

Arg 315 CCA ACA GCC AGA Pro Thr Ala Arg

GAA

Glu 320 CAC CCA His Pro 325 GCA TTG TTT GAA Ala Leu Phe Glu

GTG

Vai 330 CCC TCG CTC AAA Pro Ser Leu Lys

CTC

Leu 335 CTT GCG GCC CAC Leu Ala Ala His 4 44 4 4 44 4

C

4* 1182 1230

TGC

Cys 340 ATT GTG OGA CAC Ile Val Gly His

CAA

Gin 345 CAC ATG ATC CCA His Met Ile Pro

GAG

Glu 350 AAC GCT CTA GAG Asn Ala Leu Glu

GAG

Glu 355 ATC ACC AAA AAC Ile Thr Lys Asn

ATG

Met 360 GAT ACT AGT Asp Thr Ser GCC GTA Ala Val 365 CTG GCT GAA ATC Leu Ala Giu Ile CCT GCA Pro Ala 370 1278

GGA

Gly CCA GGA Pro Gly AGA GAA Arg Glu 375 CCA GTT CAG ACT TTG Pro Val Gin Thr Leu 380 TAC TCT CAG TCA CCA GCT Tyr Ser Gin Ser Pro Ala 385 1326 CTG GAA TTA GAT AAA Leu Giu Leu Asp Lys TTC CTT GAA GAT GTC AGG Phe Leu Giu Asp Val Arg AAT GGG ATC TAT CCT Asn Gly Ile Tyr Pro 1374 P-.%OPER\EIH\23942-C.US IM~/8 80 400 CTG ACA Leu Thr 405 GCC TTT GGG CTG CCT Ala Phe Gly Leu Pro 410 CGG CCC CAG CAG CCA CAG CAG GAG GAG Arg Pro Gin Gin Pro Gin Gin Giu Giu 415 1422

GTG

Val1 420 ACA TCA CCT GTC Thr Ser Pro Val

GTG

Val1 425

CCC

Pro CCC TCT GTC AAG ACT Pro Ser Val Lys Thr 430 CCG ACA CCT GAA Pro Thr Pro Giu 435 CCA GCT GAG GTG Pro Ala Glu Val

GAG

Glu 440 ACT CGC AAG GTG Thr Arg Lys Val GTG CTG Val Leu 445 ATG CAG TGC Met Gin Cys AAC ATT Asn Ile 450 CTG AAG Leu Lys GAG TCG GTG Giu Ser Val TTG GAG GAC Leu Glu Asp 470

GAG

Giu 455 GAG GGA GTC AAA Giu Gly Val Lys

CAC

His 460 CAC CTG ACA His Leu Thr CTT CTG Leu Leu 465 1470 1518 1566 1614 1662 1710

C

C. C C. CC

C

C*

C

CC. C C. C C C

CC

C

C

C. C C C

C.

CCC...

C

C.

C C

C.

CC C C C

C.

AAA CTG AAC CGG Lys Leu Asn Arg

CAC

His 475 CTG AGC TGT GAG CTG ATG CCA AAT Leu Ser Cys Asp Leu Met Pro Asn 480 GAG AAT Glu Asn 485 ATC CCC GAG TTG Ile Pro Glu Leu

GCG

Ala 490 GCT GAG CTG GTG Ala Giu Leu Val

GAG

Gin 495 CTG GGC TTC ATT Leu Gly Phe Ile

AGT

Ser 500 GAG GCT GAG Giu Ala Asp GAG AGC Gin Ser 505 CGG TTG ACT TCT Arg Leu Thr Ser

CTG

Leu 510 CTA GAA GAG ACC Leu Giu Giu Thr

TTG

Leu 515 AAC AAG TTC AAT TTT GCC AGG AAC AGT ACC CTC AAC TCA GCC GCT GTC Asn Lys Phe Asn Phe Ala Arg Asn Ser Thr Leu Asn Ser Ala Ala Val 520 525 530 ACC GTC TCC TCT TAGAGCTCAC TCGGGCCAGG CCCTGATCTG CGCTGTGGCT Thr Val Ser Ser 535 GTCCCTGGAC GTGCTGCAGC CCTCCTGTCC CTTCCCCCCA GTCAGTATTA CCCTGTGAAG CCCCTTCCCT CCTTTATTAT TCAGGAGGGC TGGGGGGGCT CCCTGGTTCT GAGCATCATC CTTTCCCCTC CCCTCTCTTC CTCCCCTCTG CACTTTGTTT ACTTGTTTTG CACAGACGTG GGCCTGGGCC TTCTCAGCAG CCGCCTTCTA GTTGGGGGCT AGTCGCTGAT CTGCCGGCTC 1758 1810 1870 1930 1990 2050 PAOPEM0JI23942-CUS 12A8/98 -81 CCGCCCAGCC TGTGTGGAAA GGAGGCCCAC GGGCACTAGG GGAGCCGAAT TCTACAATCC CGCTGGGGCG GCCGGGGCGG GAGAGAAAGG TGGTGCTGCA GTGGTGGCCC TGGGGGGCCA TTCGATTCGC CTCAGTTGCT GCTGTAATAA AAGTCTACTT TTTGCTAAAA AAAAAA AAAAAAAAA A INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 535 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein 2110 2170 2230 2241 (xi) SEQUENCE DESCRIPTION: SEQ ID Ser Gin Thr Val Leu NO: Ser Ser

S

0 *5 S S S. S

S

S.

*flS

S

0

S

~0 *5 SeeS

S

55 S. 55 50 0 0O Met Ser Giu Gly Glu Gly Ser Asp Pro Ser Val Ser Pro Val Glu Ser Ser Val Thr Ser Thr Ser Ser Ala Pro Gly 25 Ala Ser Leu Thr Glu Glu Glu Pro Thr Ser 35 Ser Glu Asp Ala 40 Leu Pro Glu Glu Glu Ser Glu 50 Gin Lys Ile 55 Val1 Glu Glu Ser Pro Gin Arg Asn Val 75 Cys Gly Arg Trp Arg Arg Glu Asn Pro Gly Ile 65 Ser Asp Ala Tyr Leu Ala Phe Met Asp Thr Ser Glu Arg Asn Glu Val Lys Val Cys 115 Gin 100 Ala Giu Glu 90 Lys Asn 105 Leu Ile Gly Val Glu Val Val Trp Tyr Lys Leu Gin Leu Glu 125 Gin Glu Glu 110 His Leu Asn Val Phe Asp Asn 120 Ile Val Lys Phe His Lys Tyr Trp Ala Asp Ile Lys Glu Asn Lys Ala PAOPERMMH023942-C.US -2/S98 -82- Val Ile Phe Ile Thr Gly Tyr Met Ser Ser Gly Ser Leu Lys Gin 150 155 Phe Leu Lys Lys Thr 165 Gin Lys Asn His Gin 170 Thr Met Asn Glu Lys Ala 175 Trp Lys Arg Ser Cys Asp 195 Trp 180 Cys Thr Gin Ile Leu 185 Ser Ala Leu Ser Tyr Leu His 190 Asp Thr Ile Pro Pro Ile Ile His 200 Gly Asn Leu Thr Cys 205 Phe Ile 210 Gin His Asn Gly Leu 215 Ile Lys Ile Gly Ser 220 Vai Ala Pro Asp Thr 225 Ile Asn Asn His Va1 230 Lys Thr Cys Arg Glu 235 Glu Gin Lys Asn e g.

S

*5

S

e g C 0S S

SO

@0 5 0 Oe C Leu 240 Ala His Phe Phe Ala Pro 245 Glu Tyr Gly Glu Va1 250 Thr Asn Val Thr Thr 255 Vai Asp Ile Glu Ile Gin 275 Tyr 260 Ser Phe Giy Met Cys 265 Ala Leu Giy Met Ala Val Leu 270 Glu Ala Ile Gly Asn Giy Glu Ser 280 Ser Tyr Vai Pro Gin 285 Ser Ser 290 Ala Ile Gin Leu Glu Asp Pro Leu Gin 300 Arg Giu Phe Ile 0e 0O 0 *0 0e S S go 0@ Gln 305 Lys Cys Leu Gin Ser 310 Glu Pro Ala Arg Arg 315 Pro Thr Ala Arg Glu 320 Leu Leu Phe His Pro 325 Ala Leu Phe Glu Val 330 Pro Ser Leu Lys Leu Leu 335 Ala Ala His Leu Giu Glu 355 Cys 340 Ile Val Gly His Gin 345 His Met Ile Pro Glu Asn Ala 350 Ile Thr Lys Asn Met 360 Asp Thr Ser Ala Val Leu Ala Glu 365 Ile Pro Ala Gly Pro Gly Arg Glu Pro Val Gin Thr Leu Tyr Ser Gin PAOPERUH0f23942-CUS 12/8/98 83 370 375 380 Pro Ala Leu Giu Leu 390 Asp Lys Phe Leu Glu 395 Asp Vai Arg Asn Gly 400 Ile Tyr Pro Leu Thr 405 Ala Phe Gly Leu Arg Pro Gin Gin Giu Giu Thr Pro Giu 435 Vali 420 Thr Ser Pro Val Val1 425 Pro Pro Ser Val Gin Pro Gin 415 Lys Thr Pro 430 Leu Met Gin Pro Aia Glu Val Giu 440 Thr Arg Lys Val Vali 445 Cys Asn 450 Ile Giu Ser Val Giu 455 Giu Gly Val Lys His 460 His Leu Thr Leu Leu 465 Leu Lys Leu Glu Asp 470 Lys Leu Asn Arg His 475 Leu Ser Cys Asp Leu 480 Met Pro Asn Glu Asn 485 Ile Pro Glu Leu Ala 490 Ala Giu Leu Val Gin Leu 495 Gly Phe Ile Glu Thr Leu 515 Glu Ala Asp Gin Ser Arg Leu Thr 505 Ser Leu Leu Giu 510 Asn Lys Phe Asn Phe 520 Ala Arg Asn Ser Thr Leu Asn Ser 525 Ala Ala Val Thr Val Ser Ser 535 INFORMATION FOR SEQ ID NO:ii: SEQUENCE CHARACTERISTICS: LENGTH: 39 base pairs TYPE: nucleic acid STRANDEDNESS: single P:\OPERIE1H\023942-C.US 12/8/98 -84- TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GCACAGTCGA CCAAGCCGGA GTCGCAGAG 39 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 30 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GCACAAAGCT TGCCAGGAGG GGTCTGGCTG INFORMATION FOR SEQ ID NO:13: S SEQUENCE CHARACTERISTICS: LENGTH: 32 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GCACAACCAT GGCCAAGCCG GAGTCGCAGG AG 32 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single P:\OPEREJHM023942-C.US -12/8/98 TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: GCACAAGATC TCCAGGAGGG GTCTGGCTG INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 14 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Lys Pro Glu Ser Gin Glu Ala Ala Pro Leu Ser Gly Pro Cys INFORMATION FOR SEQ ID NO:16: SEQUENCE CHARACTERISTICS: LENGTH: 19 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: Glu Asp Ala Glu Leu Gly Arg Trp Pro Trp Gin Gly Ser Leu Arg Leu Trp Asp 10 Cys INFORMATION FOR SEQ ID NO:17: SEQUENCE CHARACTERISTICS: LENGTH: 17 amino acids TYPE: amino acid TOPOLOGY: linear P:\OPEREJH023942-C.US 12/8/98 -86- (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: Gly Tyr Ile Lys Glu Asp Glu Ala Leu Pro Ser Pro His Thr Leu Gin Cys 10 INFORMATION FOR SEQ ID NO:18: SEQUENCE CHARACTERISTICS: LENGTH: 29 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: GCACAGGTAC CGAGGCCATG GGCGCGCGC 29 S. INFORMATION FOR SEQ ID NO:19: SEQUENCE CHARACTERISTICS: LENGTH: 50 base pairs TYPE: nucleic acid o' STRANDEDNESS: single TOPOLOGY: linear S(ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: S* GCACATCTAG ATCAGTGGTG GTGGTGGTGG TGGACCGGCC CCAGGAGTGG INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 31 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA P:\OPER\EJH023942-C.US 12/8/98 -87- (xi) SEQUENCE DESCRIPTION: SEQ ID GCACAGCGGC CGCGAGGCCA TGGGCGCGCG C 31 INFORMATION FOR SEQ ID NO:21: SEQUENCE CHARACTERISTICS: LENGTH: 52 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: GCACAGCGGC CGCTCAGTGG TGGTGGTGGT GGTGCCAGGA GGGGTCTGGC TG 52 INFORMATION FOR SEQ ID NO:22: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: *CTGACTTCCA TGCCATCCTT INFORMATION FOR SEQ ID NO:23: SEQUENCE CHARACTERISTICS: LENGTH: 20 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: GCTCACGACT CCAATCTGAT on PAOPEMMJHO3942CUS 128 88 INFORMATION FOR SEQ ID NO:24: SEQUENCE CHARACTERISTICS: LENGTH: 5 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: Arg Ile Val Gly Gly INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 959 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 2. .856 (xi) SEQUENCE DESCRIPTION: SEQ ID 0@ 0 0 00 0 0S *0 *0 0..e 4 0000 00.0 00 0#

SP

0**S*0 0 0* 0 4* 00 0* 00 C GAC CTA TTG TCA GGG CCC TGC GGT CAC Asp Leu Leu Ser Gly Pro Cys Gly His AGG ACC ATC CCT TCC CGT Arg Thr Ile Pro Ser Arg ATA GTG GGT GOC GAT GAT GCT GAG CTT GGC CGC TGG CCG TGG CAA GGG Ile Val Gly Gly Asp Asp Ala Glu Leu Gly Arg Trp Pro Trp Gln Gly 25 AGC CTG CdT GTA TGG GGC AAC CAC TTA TGT GGC GCA ACC TTG CTC AAC Ser Leu Arg Val Trp Gly Asn His Leu Cys Gly Ala Thr Leu Leu Asn 40 94 142 P:%OPER\WJI{3942-C.US 2/98 -89- CGC CGC TGG GTG CTT ACA GCT GCC CAC TGC TTC CAA AAG GAT AAC GAT Arg Arg Trp Val CCT TTT GAC TGG Pro Phe Asp Trp Leu Thr Ala Ala His Cys Phe Gin Lys Asp Asn Asp 55 ACA GTC CAG TTT GGT GAG CTG ACT TCC AGG CCA TCT Thr Val Gin Phe 70 Cly Giu Leu Thr Ser Arg Pro Ser

CTC

Leu TGG AAC CTA CAG Trp Asn Leu Gin GCC TAT Ala Tyr 85 TCC AAC CGT Ser Asn Arg

TAC

Tyr 90 CAA ATA CAA CAT ATT Gin Ile Glu Asp Ile 286 TTC CTG AGC CCC Phe Leu Ser Pro

AAG

Lys 100 TAC TCG GAG CAG Tyr Ser Glu Gin

TAT

Tyr 105 CCC AAT GAC ATA Pro Asn Asp Ile GCC CTG Ala Leu 110 334 CTG AAG CTG Leu Lys Leu TGC CTC CTG Cys Leu Leu 130

TCA

Ser 115 TCT CCA CTC ACC Ser Pro Val Thr TAC AAT Tyr Asn 120 AAC TTC ATC Asn Phe Ile CAC CCC ATC Gin Pro Ile 125 GAC TGC TGG Asp Cys Trp AAC TCC ACC Asn Ser Thr TAC AAG Tyr Lys 135 TTT GAG AAC Phe Giu Asn CGA ACT Arg Thr 140 430 GTG ACC Val Thr 145 GGC TGG CGG GCT Cly Trp Gly Ala

ATT

Ile 150 GGA GAA GAT GAG Gly Glu Asp Glu

AGT

Ser 155 CTG CCA TCT CCC Leu Pro Ser Pro

AAC

Asn 160 ACT CTC CAG CAA Thr Leu Gin Glu

GTG

Val 165 CAG GTA GCT ATT Gin Val Ala Ile

ATC

Ile 170 AAC AAC AGC ATG Asn Asn Ser Met

TGT

Cys 175 478 526 574 622 AAC CAT ATG TAC Asn His Met Tyr

AAA

Lys 180 AAC CCA CAC TTC Lys Pro Asp Phe

CGC

Arg 185 ACG AAC ATC TGG Thr Asn Ile Trp GGA GAC Gly Asp 190 TTT CGT Phe Gly ATG GTT TGC Met Val Cys GAC TCG GGA Asp Ser Gly 210

GCT

Ala 195 CGC ACT CCT GAA Gly Thr Pro Glu

GGT

Gly 200 GGC AAG GAT GCC Gly Lys Asp Ala

TGC

Cys 205 GGA CCC TTG GCC Gly Pro Leu Ala

TGC

Cys 215 GAC CAG GAT ACG Asp Gin Asp Thr GTG TGG TAT CAG Val Trp Tyr Gin 220 GTT GGA GTT GTG AGC TCG GGA ATA GGC TGT GGT CGC CCC AAT CGC CCT 718 P:\OPER\IEM023942-CUS 12/98 Val Gly Val 225 Val Ser Trp Gly Ile Gly Cys Gly Arg Pro Asn Arg Pro 230 235

GGA

Gly 240 GTC TAT ACC AAC Val Tyr Thr Asn

ATC

Ile 245 AGT CAT CAC TAC Ser His His Tyr

AAC

Asn 250 TGG ATC CAG TCA Trp Ile Gin Ser

ACC

Thr 255 ATG ATC CGC AAT Met Ile Arg Asn

GGG

Gly 260 CTG CTC AGG CCT Leu Leu Arg Pro

GAC

Asp 265 CCA GTC CCC TTG Pro Val Pro Leu CTA CTG Leu Leu 270 814 856 TTT CTT ACT Phe Leu Thr

CTG

Leu 275 GCC TGG GCT TCC Ala Trp Ala Ser

TCT

Ser 280 TTG CTG AGG CCT GCC Leu Leu Arg Pro Ala 285 TGAGCCCACA CGTGTACGTC ACACCTGTGA GGTCAGGGTG TGTCTCTTTT GTATCTTGCT TGCTAATAAA CCTGTTAATA TTTAAAAAAA AAAAAAAAAA AAA INFORMATION FOR SEQ ID NO:26: SEQUENCE CHARACTERISTICS: LENGTH: 285 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 916 959 Asp Leu Leu Ser Gly Pro Cys Gly His Arg Thr Ile Pro Ser Arg Ile Val Gly Gly Asp Asp Ala Glu Leu Gly Arg Trp Pro Trp Gin Gly Ser 25 Leu Arg Val Arg Trp Val Trp Gly Asn His Leu 40 Cys Gly Ala Thr Leu Leu Asn Arg Leu Thr Ala Ala His Cys Phe Gin Asp Asn Asp Pro Phe Asp Trp Thr Val Gin 70 Phe Gly Glu Leu Thr Ser Arg Pro Ser Leu P:XOPER'EJKMO3942-C.US M218 91 Trp Asn Leu Gin Ala Tyr Ser Asn Arg Tyr Gin Ile Giu Asp Ile Phe Leu Ser Pro Lys 100 Tyr Ser Giu Gin Tyr 105 Pro Asn Asp Ile Ala Leu Leu 110 Lys Leu Ser 115 Ser Pro Val Thr Tyr 120 Asn Asn Phe Ile Gin 125 Pro Ile Cys Leu Leu 130 Asn Ser Thr Tyr Lys 135 Phe Giu Asn Arg Thr 140 Asp Cys Trp Vai Thr 145 Gly Trp Gly Ala Ile 150 Gly Glu Asp Glu Ser 155 Leu Pro Ser Pro Asn 160 Thr Leu Gin Giu Val1 165 Gin Val Ala Ile Ile 170 Asn Asn Ser Met Cys Asn 179 0 4* His Met Tyr Val Cys Ala 195 Lys 180 Lys Pro Asp Phe Arg 185 Thr Asn Ile Trp Gly Asp Met 190 Phe Gly Asp Gly Thr Pro Glu Gly 200 Gly Lys Asp Ala Cys 205 Ser Gly 210 Gly Pro Leu Ala Cys 215 Asp Gin Asp Thr Val1 220 Trp Tyr Gin Val Gly 225 Val Val Ser Trp Gly 230 Ile Gly Cys Gly Arg 235 Pro Asn Arg Pro Gly 240 Val Tyr Thr Asn Ile Arg Asn Gly 260 Leu Thr Leu Ala 275 Ile 245 Ser His His Tyr Asn 250 Trp Ile Gin Ser Thr Met 255 Leu Leu Arg Trp Ala Ser Pro Asp 265 Ser Leu 280 Pro Val Pro Leu Leu Leu Phe 270 Leu Arg Pro Ala 285 INFORMATION FOR SEQ ID NO:27: SEQUENCE CHARACTERISTICS: LENGTH: 5318 base pairs TYPE: nucleic acid STRANDEDNESS: single P:\OPEREJHU023942-C.US 12/898 -92- TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 703..765 (ix) FEATURE: NAME/KEY: CDS LOCATION: 863..890 (ix) FEATURE: NAME/KEY: CDS LOCATION: 1237..140 (ix) FEATURE: NAME/KEY: CDS LOCATION: 2112..240 (ix) FEATURE: NAME/KEY: CDS LOCATION: 4388..454 (ix) FEATURE: NAME/KEY: CDS LOCATION: 4803..503 14 2 r

D

4 9 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: AAAATACAAA AATGAGCCAG GTGTGCTGGC GCGCGCCTGT AACCCCAGCT ACTTGGGAGG CTGAGGCAGG AGAATCGCTT GAACCCAGGA GGTGGAGGTT GCAGTGAGCT GGGATCACGC CAATGCACTC CAGCCTGGGC GACAAGAGCG AAACTCCATC TCAAAAAAAA AAAAAATTGT TCCAGAGAAG TGAAGAGAGC CCAGCGGGAG CCCGCAGTGG GGACAGCCTG GGCAGAGACT TGGGGAGATC CCCATTCTGG GTGGAACGGG CCGAGGACCA CTGTTTTCCC AAGAAGGTCC CATGAAGGAG CAGCCATGCC TCTAGGCCCC ACCCGGCCTT CAGGGGGTCC CCAGGCTTTA AGGGGACTCC TGGCTCAGGG CCAGGCCCTT GGTGCTGGAG GAGGTTGTAG GTGGAGGACG 120 180 240 300 360 420 P:\OPER\FEflfl23942-CUS- l2/8 93

GGGTCACCAA

CACCGGGTCT

CAGGGCGCTG

GGAGTAGAGG

GAGAGGAGGC

CTGCTGCTGG

GAGGGCAGCC

GGGCCACATC

CCAGGCATGA

GCAGAGCTCC

AGAGGGGGCG

CGCTGCTGCT

GGGACCCCTG

AAGAAGTGTG

GAGGCCCCAA

CACCCCGCCC

TCAGGCCGCG

GGCTCGGGCT

GGCTGCAGAC

GTTGAAGACC

ACAGCCCTTG

CGCCCCCAGG

GGAGAGGAGG

GGACTCAGGA

AAGAAAGGGA

CGCCCCTAGG

GCCCAGGAG

GGGCGCCCCG

CCATGGGCGC

AGCCGGGTGA

CTGTGGGGTC

GGCTGAAAGC

GGTGAAGCTG

GGCCCGGCGC

GCGCGGGGCG

GCTCGGGGCG

480 540 600 660 720 780 CTGCTGGCGG GATGGGGAGG CGGGGGAGCG GTGGGGAGGA CGGGAGGTGG AGGCCGCCGGG

*SSSSS

S.

S. S

GAGTCACTTC

CAGGACGCGC

TAGAGGGGGG

TGGAAAGTAA

ATCTCCAGTG

CGTGCTTTCC

TCCCGCGGCT

TCGCGCATCG

CGCCTGTGGG

GCGGCGCACT

GAACAGGGCT

TGAAAGTTGT

GTTACACCCA

TTGTCTCCCG

GATTCCTGCC

CCTTTACTGC

CTAACGGACG

TCACCTACTT

CGGACGGGGT

CAGCAGTTCC

TGGGTGGAGA

ATTCCCACGT

GCTTTGAAAC

GGAGGGAGTG

GCGTGGATGC

CTCCAGTTCC

TCTCTCGCCC

CTGGAGGGGG

CCTGCTGCAC

TGAAGGGGAG

TCTGACCATC

GGACGCCGAA

ATGCGGAGTG

GTGAGTGGGG

CCACCGAACT

GGCCTGGTGT

CTTTGGGTCT

CCGCCCCCGG GATCGAGAAC TCTGTTGGCG ATGGGCGGGC CCTGCAGAGC CAGAGTCGCA GGAGGCGGCG CCGTTATCAG GTAGGGCGCC AGGGCCGTTG GGCCGAGGTG GACGGGGGGC GGTGAGGGGG

ACACGCGAG

AAAGGGAGAG

CGAGGACCAT

CTCGGGCGTT

AGCCTGCTCA

GTGCGAACG

TTACCTCTGG

TCTCCTGAGC

GGACCCTGGG

GTCGGGCTTG

GCGGCCGACG

GGCCGTGGCA

GCCACCGCTG

AGGGGTGCGG

TCTGATGCCA

CCCAGGCTGT

ACGTGGGAGG

TGGGCAAAAA

GGGGGCTGCC

GGTCATCACG

GGGGAGCCTG

GGCACTCACG

GGACGGGCAG

GACTTGGGCG

GCTGCAGCCG

GGTTATTCCA

TTCTTTCAAC

GTGCGGTACA

840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 GAATGTTCTT CCAGAACATT TCCACACACT CCTGGAGGGA ACCCTGTTCA TTTGGGTATT CTCTCCCTTT CCAAAGTTCA CCACTGACCA TCCCACCCTC ATCCCCCCTC CTGGTGGACG GTGTGGGGCA CTGAGCCAAG GCCAGCACCC CCGGGCCGCT GTGTGGACTC CATCCTGCCA 1800 PR.OPFRTEH\023942-C.US i2/I'98 94

ATCCCACATT

GAGGCCTTGG

CTTCTGCTGC

CTGCAGGGGA

GGACCCCAGT

CTTCTGCCAG

TGACTTCCAT

GGCGTGGTGC

CTCAATGCAA

AGGAGCAGGT

CCCTGGGTGT

TGTGCTCAGG

CTATAGTGAC

GCCATCCTTC

ATCTCCCCAT

GGCTCCTTGG

CCTAGGACTT

TAGCAAGTAG

TAGCCAGCCC

CTTAGTGATC

TGGAGCCTGC

TCCTCCTTGG

GACAGCTCTG

TGGTTGTGGT

CAGCAACACC

TCCATCCAGG

CCTCCGGGTG

AGGCCTACTA

GCTGCATGGG

GGAGGTGACA

CTGTCTGGGC

ACAGTTTCCC

GCCCCTGACT

GATGGTCCAG

CACCCGTTAC

GGTGCCCCTG

AGACCCCACC

TCCTTCATTT

CTCCTGCACT

GCTCTCTTCT

TTTGGCCAGC

TTCGTATCGA

ATATCTATCT GAGCCCTCGC TACCTGGGGA ATTCACCCTA TGACATTGCC TTGGTGAAGC

TGTCTGCACC

TTGAGTTTGA

AGGGTGAGGC

CATAGGCACA

GTCTCTCCTC

TGGGGGCCAG

GGTGGTGGAG

CATCTTTCCA

CTCCATGCCT

CCTCTCCCCA

GGTTCCTCTG

AAGTGGGCCC

TGTCACCTAC

GAACCGGACA

TGGGGACAGG

ATAGCCCCCT

ACCTGCCAGG

AAGGAGAGTG

GGGTTCTGGA

CCTCCCCCAG

CCCACACCCA

GAAACTTCCA

GTGGGCCTGC

1GCATCCGGA

ACTAAACACA

GACTGCTGGG

TCCAGCCCAT

TGACTGGCTG

CTGTCTCCAG

GGGGTACATC

GCCTCCACAT

AAAGAGGATG

1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 CGGGTCAGGG AGGAACTGTC TTTGTTCACC TGTTCCCCTG GCTTGGTCTG GGGGTGCAGG

GCAGGGACCA

TGAGAGGGAG

GGGCTTGGCG

TGCTCACCAA

CCCTAGCTCT

AGCCATGCTC

CCTGGCACCT

GGAGCCCCGG

AACACCCAGT

GCCAGTTTGG

ACATAAACCT

TGCCCCAGGC

GGCCGATTCT

AACCGCAGCT

GCGTGTCCCC

CTATGCCCCT

TCTCTCCCTT

CGCAGGCCTG

CATACTTGGA

ATCAGGCTCC

CCTGCAGCAC

CCCACGGAAA

CAACACACAT

CT'rGCTTGCA

CCAGGGGCTG

TGGGTGGTGC

TTTATTCCTG

TGGGCTGCCT

TGAGCCCATT

CCCCTCTGGG

GCCCTGAAAG

CAGCCCCAGA CTGGGCGTGT TCCCTGTATC AGGAATCCCT TCCCTCTGCT CCCCTGTCTG GCCCGTCCCT GCATCATCCC ACAACAGTAG

TAACAATAAC

GGGGGCCCAG

AAACGTGGTT

CAGTGAGCCA

CCTTGAGCAC

GGCGCGGCCT

ACGCTGTGTG

CTCCTCCTGC

CCTGGCACTG

TGGAGCTCAC

TTCTAGCCAT

CTAGGAATGA

3060 3120 3180 P:%OPERTEflN023942-C.US 1218/98 95

AGTTGGTAAC

GCCAAGAACT

CCCAAGTCAC

GAGGAGGGAT

GCCAGGGGCA

CACACCAACC

CTGGAGCCCC

GCAAAGGTGT

TGCCACATCA

GACGGGAAGC(

GGTGCTGGCT(

GTTCTTATAC

AGTAAATGGA

ACCCCGGCAG

CCTTTGCCTT

CGGCCAGGCT

CCAGGCTGTG

CTGCCTGGTG

TCATCTTACA

GAGGCTAGTT

GCTGCCTGCA(

3GCACTCTGA

CCATCTTACP

GAAGTCAGGG

TTGGGAGTGG

CCCTGAGAAG

GCCTCTGCCA

CCTCAATTCA

TGCACCTTTG

GTGCCTCAGT

CCCAGGTTGT

3GTTGGGTCT

"GCAGGAATT

GATGAGAAAA CTGAGAGGGT CCCCTTGCCT TAGAGAGGGT GGAACTCAGC ACTGGAGCCG GACCCTTGCT GCAAACACGA CCCTCTCTCC CCAGCCCAGG CAGTGCCGGG GAGGCAGGGG CTGTGCCCTG CGCGGGCTGG TTCCTCCCCT GTGAAATAGG TAGTCCCGAG GGCTTAGGAG CATTGTCTTT TGTCATCCTC GGACAGGTGT GGGCTCCTGTC

TATGGAAATT

AAGGGATGTC

CCTCCCTCTG

GTGGATATGA

GTAGACAGGT

ACAGGCACTT

GAGGCCTTGG

GGAATGATGG

AGGCCCCGCT

rGCAGGACAA

'AGGCTCTGA

3240 3300 3360 3420 3480 3540 3600 3660 3720 3780 3840 3900 3960 4020 4080 4140 4200 4260 4320 4380 AACTTCCCCA GTGCCTTGCT GGGGGTCAGC GGGGCAATAT CCCCCTGACT CTCCTTATCT

GGGAGTAGGT

AACAAGCTTG

AGGCTGCCCC

TCGCCCCTCC

TGTTGCCATT

CCGGGGCCTT

GGGGTAATGG

CCTATGCCAT

CCTGATGACT

TGTCCAGGCC

TCCCCCAGGT

CTCGGCTCCT

CTACAGATAG

CTGTGATGCT

AGATGAACCT

CCCTCCATAG

GCCGCCTCCC AGGGGTGCTG GGGGTCCCTA

TGGAAAGCGC

CTGGCTTTGG

TTTGGCTGAG

GGAAAGTGCG

GCTGAGGGCC

GCCAGGCACA

AGGGGCCTCA

CCAGCTGCTA

ATGCTCATCT

GAATCTCTCC

GCTGGGGGAG

TCTGTTGTGC

GTGGGTGCCC

GGTTGCTGTC

CTGAGGCCAG

GAACACCGTC

ATGGCTGCAG

CTCTGACAGC

TGGGGTCTGG

CAGGGCCCCC

TCTCTCCTTC

AGAGTGCTGC

GGCTGCAGCC

TCAGCTGCCT

GCAGGGCCAT

CTGTCCCTCC

GTTGGAGCTG

ACCCCCGCAG

CCACTATCGT

CCGCACAGCA CTGCCATCTC CCCACACCCT CCAGGAAGTT CAGGTCGCCA TCATAAACAA CTCTATGTGC AACCACCTCT TCCTCAAGTA CAGTTTCCGC AAGCACATCT TTGGAGACAT GGTTTGTGCT GGCAATGCCC AAGGCGGGAA GGATGCCTGC TTCGTGAGTG TCCTTGCCAC 4440 4500 4560 P*.%0PER'EJHKf3942-C.US 120898 96

CACTCCCAGC

CCGGGAGGTG

CAGGGGGAGG

GCCCCTCCCC

GTGACTCAGG

TGAGCTGGGG

ACCACTTTGA

CCTGGCCGCT

CCTACCTGAG

TTGTTTGGTA

CTTCATGGAC

CACATCTGTT

TGCCCAGGCT

*CCAGGAAAGC ATCCTGTGTC GAGACTGTTG CCCCACTCTG AGGAGGATGT GCACCCAGTC TGCCCCACTC ACTCTGCCCC TGGACCCTTG GCCTGTAACA AGTGGGCTGT GGTCGGCCCA GTGGATCCAG AAGCTGATGG ACTCTTTTTC CCTCTTCTCT CCCATGCAGC CTGGGGCCAC ATAAACACAT TCCAGTTGAT AGCTCATTCT CTCTTGTGCA CTGCACCATA GAACCATCTGC GGTCTTGAAC GCCTAGGGTG 9

CCTGTGCCT'I

CAGATGCAGA

TACCCAGCCC

AGGCTGACCT

AGAATGGACT

ATCGGCCCGG

CCCAGAGTGG

GGGCTCTCCC

TGCCAAGTCA

GCCTTGCAGG

3ACAGCCTGT 3TTATTTCGA

[CTCGATC

ATTTGACCCI

AACGGAGGCT

CATAGCCCTT

CAGCCCCGCT

GTGGTATCAG

TGTCTACACC

CATGTCCCAG

ACTCCTGGGG

GGCCCTGGTT

GCATTCTTCA

CTGTGCCCCT

TCAGAAAGAG

CATGCCAACC

TGGCTGCTGC

CCCACTCTCA

GCTCCCCAGG

ATTGGAGTCG

AATATCAGCC

CCAGACCCCT

CCGGTCTGAG

CTCTTCTGTC

AAAGCAGTGG

GGCTCACACC

AATTGTGTGT

4620 4680 4740 4800 4860 4920 4980 5040 5100 5160 5220 5280 5318 C INFORMATION FOR SEQ ID NO:28: SEQUENCE CHARACTERISTICS: LENGTH: 21 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: Met Gly Ala Arg Gly Ala Leu Leu Leu Ala Leu Leu Leu Ala Arg Ala 1 5 10 Gly Leu Arg Lys Pro P:\OPERE)H\023942-CUS -12/898 -97- INFORMATION FOR SEQ ID NO:29: SEQUENCE CHARACTERISTICS: LENGTH: 10 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: Glu Ser Gin Glu Ala Ala Pro Leu Ser Gly 1 5 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 55 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID Pro Cys Gly Arg Arg Val Ile Thr Ser Arg Ile Val Gly Gly Glu Asp 1 5 10 Ala Glu Leu Gly Arg Trp Pro Trp Gin Gly Ser Leu Arg Leu Trp Asp 20 25 Ser His Val Cys Gly Val Ser Leu Leu Ser His Arg Trp Ala Leu Thr 40 Ala Ala His Cys Phe Glu Thr INFORMATION FOR SEQ ID NO:31: SEQUENCE CHARACTERISTICS: LENGTH: 97 amino acids P'.%OPER\EJHWD3942-C.US 12MS98 98 TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: Ser Asp Leu Ser Asp Pro Ser Gly Trp Met Val Gin Phe Gly Gin 5 10 Thr Ser Met Pro Ser Phe Trp Ser Leu Gin Ala Tyr Tyr Thr Arg 25 Phe Vai Ser Asn Ile Tyr Leu Ser Pro Arg Tyr Leu Gly Asn Ser Tyr 1 Leu Tyr Pro Tyr Asp 40 Vali Ile Ala Leu Lys His Val1 55 Cys Lys Leu Ile Gin Pro

C

C.

C

C. C C

C

C

C.

Asn Glu Ilie 70 Trp Leu Ser Ala Pro Gin Ala Ser Thr 75 Gly Trp Gly Tyr 90 Thr Tyr Thr Phe Giu Phe Giu Ile Lys Giu Asp Arg Thr Asp Cys 85 Val Thr INFORMATION FOR SEQ ID NO:32: SEQUENCE CHARACTERISTICS: LENGTH: 53 amino acids TYPE: amino acid TOPOLOGY: iinear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: Ala Leu Pro Ser Pro His Thr Leu Gin Giu Val Gin Val Ala Ile Ile 1 5 10 Asn Asn Ser Met Cys Asn His Leu Phe Leu Lys Tyr Ser Phe Arg Lys 25 P:\OPER\EJ{O23942-C.US 12/98 -99- Asp Ile Phe Gly Asp Met Val Cys Ala Gly Asn Ala Gin Gly Gly Lys 40 Asp Ala Cys Phe Gly INFORMATION FOR SEQ ID NO:33: SEQUENCE CHARACTERISTICS: LENGTH: 78 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: Asp Ser Gly Gly Pro Leu Ala Cys Asn Lys Asn Gly Leu Trp 1 5 10 Ile Gly Val Val Ser Trp Gly Val Gly Cys Gly Arg Pro Asn 25 Gly Val Tyr Thr Asn Ile Ser His His Phe Glu Trp Ile Gin 40 Met Ala Gin Ser Gly Met Ser Gin Pro Asp Pro Ser Trp Pro 55 Phe Phe Pro Leu Leu Trp Ala Leu Pro Leu Leu Gly Pro Val 70 Tyr Gin Arg Pro Lys Leu Leu Leu INFORMATION FOR SEQ ID NO:34: SEQUENCE CHARACTERISTICS: LENGTH: 1165 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA P-.\PER\EIFJHW23942-C.US I2J8i98 100 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: CTGAACCGGG TTGTGGGCGG CGAGGACAGC ACTGACACCG AGTGGCCCTG GATCGTGAGC ATCCAGAAGA ATGGGACCCA CCACTGCGCA GGTTCTCTGC TCACCAGCCG CTGGGTGATC ACTGCTGCCC ACTGTTTCA.A GGACAACCTG AACAAACCAT ACCTGTTCTC TGTGCTGCTG GGGGCCTGGC AGCTGOGGAA CCCTGGCTCT CGGTCCCAGA AGGTGGGTGT TGCCTGGGTG GAGCCCCACC CTGTGTATTC CTGGAAGGAA GGTGCCTGTG CAGACATTGC CCTGGTGCGT CTCGAGCGCT CCATACAGTT CTCAGAGCGG GTCCTGCCCA TCTGCCTACC TGATGCCTCT ATCCACCTCC CTCCAAACAC CCACTGCTGG ATCTCAGGCT GGGGGAGCAT CCAAGATGCA GTTCCCTTGC CCCACCCTCA GACCCTGCAG AAGCTGAAGG TTCCTATCAT CGACTCGGAA GTCTGCAGCC ATCTGTACTG GCGGGGAGCA GGACAGGGAC CCATCACTGA GGACATGCTG TGTGCCGGCT ACTTGGAGGG GGAGCGGGAT GCTTGTCTGG GCGACTCCGG GGGCCCCCTC ATGTGCCAGG TGGACGGCGC CTGGCTGCTG GCCGGCATCA TCAGCTGGGG CGAGGGCTGT GCCGAGCGCA ACAGGCCCGG GGTCTACATC AGCCTCTCTG CGCACCGCTC CTGGGTGGAG AAGATCGTGC AAGGGGTGCA GCTCCGCGGG CGCGCTCAGG GGGGTGGGGC CCTCAGGGCA CCGAGCCAGG GCTCTGGGGC CGCCGCGCGC TCCTAGGGCG CAGCGGGACG CGGGGCTCGG ATCTGAAAGG CGGCCAGATC CACATCTGGA TCTGGATCTG CGGCGGCCTC GGGCGGTTTC CCCCGCCGTA AATAGGCTCA TCTACCTCTA CCTCTGGGGG CCCGGACGGC TGCTGCGGAA AGGAAACCCC CTCCCCGACC CGCCCGACGG CCTCAGGCCC CGCCTCCAAG GCATCAGGCC CCGCCCAACG GCCTCATGTC CCCGCCCCCA CGACTTCCGC CCCCGCCCCG GGCCCCAGCG CTTTTGTGTA TATAAATGTT AATGATTTTT ATAGGTATTT GTAACCCTGC CCACATATCT TATTTATTCC TCCAATTTCA ATAAA 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1165 INFORMATION FOR SEQ ID PAOPEREM023942-C.US 12AU98 -101- SEQUENCE CHARACTERISTICS: LENGTH: 933 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA 0 0 0 00 0 00 0 00 0 00 0 (xi) SEQUENCE DESCRIPTION: SEQ ID AATGCGGCCA CTCCAAGGAG GCCGGGAGGA TTGTGGGAGG CCAAGACACC CAGGAAGGAC GCTGGCCGTG GCAGGTTGGC CTGTGGTTGA CCTCAGTGGG GCATGTATGT GGGGGCTCCC TCATCCACCC ACGCTGGGTG CTCACAGCCG CCCACTGCTT CCTGAGGTCT GAGGATCCCG GGCTCTACCA TGTTAAAGTC GGAGGGCTGA CACCCTCACT TTCAGAGCCC CACTCGGCCT TGGTGGCTGT GAGGAGGCTC CTGGTCCACT CCTCATACCA TGGGACCACC ACCAGCGGGG ACATTGCCCT GATGGAGCTG GACTCCCCCT TGCAGGCCTC CCAGTTCAGC CCCATCTGCC TCCCAGGACC CCAGACCCCC CTCGCCATTG GGACCGTGTG CTGGGTAAAC GGGCTGGGGG TCCACTCAGG AGAGGCCCTG GCGAGTGTCC TTCAGGAGGT GGCTGTGCCC CTCCTGGACT CGAACATGTG TGAGCTGATG TACCACCTAG GAGAGCCCAG CCTGGCTGGC CAGCGCCTCA TCCAGGACGA CATGCTCTGT GCTGGCTCTG TCCAGGGCAA GAAAGACTCC TGCCAGGGTG ACTCCGGGGG GCCGCTGGTC TGCCCCATCA ATGATACGTG GATCCAGGCC GGCATTGTGA GCTGGGGATT CGGCTGTGCC CGGCCTTTCC GGCCTGGTGT CTACACCCAG GTGCTAAGCT ACACAGACTG GATTCAGAGA ACCCTGGCTG AATCTCACTC AGGCATGTCT GGGGCCCGCC CAGGTGCCCC AGGATCCCAC TCAGGCACCT CCAGATCCCA CCCAGTGCTG CTGCTTGAGC TGTTGACCGT ATGCTTGCTT GGGTCCCTGT GAACCATGAG CCATGGAGTC CGGGATCCCC TTTCTGGTAG GATTGATGGA ATCTAATAAT AAA INFORMATION FOR SEQ ID NO:36: 120 180 240 300 360 420 480 540 600 660 720 780 840 900 933 P:OPERIOM023942-C.US 12V98 -102- SEQUENCE CHARACTERISTICS: LENGTH: 980 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: DNA S (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: CCTGTGGTCG CCCCAGGATG CTGAACCGAA TGGTGGGCGG GCAGGACACG CAGGAGGGCG AGTGGCCCTG GCAAGTCAGC ATCCAGCGCA ACGGAAGCCA CTTCTGCGGG GGCAGCCTCA TCGCGGAGCA GTGGGTCCTG ACGGCTGCGC ACTGCTTCCG CAACACCTCT GAGACGTCCC TGTACCAGGT CCTGCTGGGG GCAAGGCAGC TAGTGCAGCC GGGACCACAC GCTATGTATG CCCGGGTGAG GCAGGTGGAG AGCAACCCCC TGTACCAGGG CACGGCCTCC AGCGCTGACG TGGCCCTGGT GGAGCTGGAG GCACCAGTGC CCTTCACCAA TTACATCCTC CCCGTGTGCC TGCCTGACCC CTCGGTGATC TTTGAGACGG GCATGAACTG CTGGGTCACT GGCTGGGGCA GCCCCAGTGA GGAAGACCTC CTGCCCGAAC CGCGGATCCT GCAGAAACTC GCTGTGCCCA TCATCGACAC ACCCAAGTGC AACCTGCTCT ACAGCAAAGA CACCGAGTTT GGCTACCAAC CCAAAACCAT CAAGAATGAC ATGCTGTGCG CCGGCTTCGA GGAGGGCAAG AAGGATGCCT GCAAGGGCGA CTCGGGCGGC CCCCTGGTGT GCCTCGTGGG TCAGTCGTGG CTGCAGGCGG GGGTGATCAG CTGGGGTGAG GGCTGTGCCC GCCAGAACCG CCCAGGTGTC TACATCCGTG TCACCGCCCA CCACAACTGG ATCCATCGGA TCATCCCCAA ACTGCAGTTC CAGCCAGCGA GGTTGGGCGG CCAGAAGTGA GACCCCCGGG GCCAGGAGCC CCTTGAGCAG AGCTCTGCAC CCAGCCTGCC CGCCCACACC ATCCTGCTGG TCCTCCCAGC GCTGCTGTTG CACCTGTGAG CCCCACCAGA CTCATTTGTA AATAGCGCTC CTTCCTCCCC TCTCAAATAC CCTTATTTTA TTTATGTTTC TCCCAATAAA 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 980 PAOPEM0J~t23942-CUS 121S 103 INFORMATION FOR SEQ ID NO:37: SEQUENCE CHARACTERISTICS: LENGTH: 19 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: Ile Gin Glu Gin Glu Lys Glu Pro Arg Trp Leu Thr Leu His Ser Asn Trp Glu Cys 10 INFORMATION FOR SEQ ID NO:38: SEQUENCE CHARACTERISTICS: LENGTH: 16 amino acids TYPE: amino acid se:* TOPOLOGY: linear MOLECULE TYPE: protein 55 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38: Gly His Met Gly Asn Lys Met Pro Phe Lys Leu Gin Glu Gly Glu Cys 05 10

Claims (39)

1. 4-04:14:56 ;DAVIES COLLISON CAVE Pat.&Trad ;61 7 3358 2262 6/ 29 P.Oa&VrV1HA folginar3d70) teN'l d -as s< -SWGb a -104- THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. An isolated proteinaceous molecule having serine proteinase activity comprising an amino acid sequence encoded by a nucleotide sequence substantially as set forth in any one of SEQ ID NO:3, 5, 34, 35 and 36, or a nucleotide sequence having at least 50% similarity to any one of SEQ ID NO: NO:3 and 5, or its complementary form, or a nucleotide sequence capable of hybridizing to any one of SEQ ID NO:3, 5, 34, 35 and 36, or its complementary form, under medium stringency conditions at 42 0 C. 2. An isolated proteinaceous molecule having serine proteinase activity comprising an amino acid sequence substantially as set forth in any one of SEQ ID NO:4 and 6, or an amino acid sequence having at least 50% similarity to any one of SEQ ID NO:4 and 6. 3. An isolated derivative or homologue of a proteinaccous molecule having serine proteinase activity, wherein said derivative or homologue is encoded by a nucleotide sequence having at least 50% similarity to the nucleotide sequence set forth in any one of SEQ ID NO: NO:3 and 5, or its complementary form, or a nucleotide sequence capable of hybridizing to any one of SEQ ID NO: NO:3, 5, 34, 35 and 36, or its complementary form, under medium stringency conditions at 42 0 C. 4. A composition comprising a proteinaccous molecule according to Claim 1 or 2 and one or more pharmaceutically acceptable carriers and/or diluents. A composition comprising a proteinaceous molecule according to Claim 3 and one or more pharmaceutically acceptable carriers and/or diluents.
2. 6. An isolated immunointeractive molecule specific to a proteinaceous molecule according to any one of Claims 1 to 3.
3. 7. An isolated immunointeractive according to Claim 6, wherein the COMS ID No: SMBI-00728947 Received by IP Australia: Time 15:12 Date 2004-04-30 4-04:14:56 :DAVIES COLLISON CAVE Pat.&Trad :61 7 3368 2262 7/ 29 ttO.4pVaiFA houl tiamtD2i7 un2 (in -105- immunointeractive molecule is an antibody.
4. 8. An isolated immunointeractive molecule according to Claim 6, wherein the immunointeractive molecule is a monoclonal antibody.
5. 9. An isolated nucleic acid molecule comprising a sequence of nucleotides encoding, or complementary to, a sequence encoding a proteinaceous molecule having scrine proteinase activity and wherein said proteinaceous molecule comprises a sequence of amino acids substantially as set forth in any one of SEQ ID NO:4 and 6, or an amino acid sequence having at least about 50% similarity to any one of SEQ ID NO:4 and 6.
6. 10. An isolated nucleic acid molecule comprising a sequence of nucleotides S. substantially as set forth in any one of SEQ ID NO: NO:3, 5, 34, 35 and 36, or a nucleotide sequence having at least about 50% similarity to any one of SEQ ID NO: NO:3 and 5, or its complementary form, or a nucleotide sequence capable of hybridizing to any one of S lSEQ ID NO: NO:3, 5, 34, 35 and 36, or its complementary form, under medium stringency conditions at 42 0 C.
7. 11. An isolated derivative or homologue of a nucleic acid molecule according to Claim 9 or 10 wherein said derivative or homologue comprises a sequence of nucleotides o: which encodes a proteinaceous molecule having at least about 50% similarity to any one of O SEQ ID NO:4 and 6 P
8. 12. An isolated derivative or homologue of a nucleic acid molecule according to Claim 9 or 10 comprising a sequence of nucleotides having at least 50% similarity to any one of SEQ ID NO: NO:3 and 5, or its complementary form, or a nucleotide sequence capable of hybridizing to any one of SEQ ID NO: NO:3, 5, 34, 35 and 36, or its complementary form, under medium stringency conditions at 42 0 C.
9. 13. A vector comprising a nucleic acid molecule according to any one of Claims 9 to 12. COMS ID No: SMBI-00728947 Received by IP Australia: Time 15:12 Date 2004-04-30 4-04;14!56 :DAVIES COLLISON CAVE Pat.&Trad :61 7 3368 2262 8/ 29 P.OWVpVYA F ftMw m is. fctA a -106-
10. 14. An expression vector comprising a nucleic acid molecule according to any one of Claims 9 to 12. A vector or expression vector comprising a nucleic acid molecule according to any one of Claims 9 to 12, when used for gene therapy.
11. 16. A gene targeting vector comprising a nucleic acid molecule according to any one of Claims 9 to 12.
12. 17. A genetic composition comprising a nucleic acid molecule according to any one of Claims 9 to 12. 0* 9 9
13. 18. A method of treatment of a mammal, said method comprising administering 09 to said mammal an activity-modulating effective amount of a proteinaceous molecule 9. ""according to any one of Claims 1 to 3 or an antagonist thereof selected from an immunointeractive molecule specific to the proteinaceous molecule, or an antisense molecule, ribozyme or co-suppression molecule that is specifically interactive with a nucleic acid molecule according to any one of Claims 9-12. 9999
14. 19. A method according to Claim 18, wherein said antagonist is an immunointeractive molecule specific to a proteinaceous molecule according to any one of Claims 1 to 3. 9 9 A method according to Claim 18, wherein said antagonist is an antibody specific to a proteinaceous molecule according to any one of Claims I to 3.
15. 21. A method according to Claim 18, wherein said antagonist is a monoclonal antibody specific to a proteinaceous molecule according to any one of Claims I to 3.
16. 22. A method according to Claim 18, wherein said antagonist is an antisense COMS ID No: SMBI-00728947 Received by IP Australia: Time 15:12 Date 2004-04-30 4-04:14:56 :DAVIES COLLISON CAVE Pat.&Trad :61 7 3368 2262 S/ 29 KOXPAWpVI A nIOul«793e IA dtllisO Cdjta1uae4dL6 -107- molecule that is specifically interactive with a nucleic acid molecule according to any one of Claims 9-12.
17. 23. A method according to Claim 18, wherein said antagonist is a ribozyme that is specifically interactive with a nucleic acid molecule according to any one of Claims 9- 12.
18. 24. A method according to Claim 18, wherein said antagonist is a co- suppression molecule that is specifically interactive with a nucleic acid molecule according to any one of Claims 9-12.
19. 25. A method of immunotherapy, said method comprising administering to a 00 00*mammal an immunointeractive molecule specific to the proteinaceous molecule according to any one of Claims 1 to 3 or an antigen capable of inducing formation of immunointeractive molecules specific to said proteinaceous molecule, said administration being for a time and under conditions sufficient to reduce the activity or presence of the proteinaceous molecule.
20. 26. A method according to Claim 25 wherein the immunointeractive molecule is an antibody.
21. 27. A method according to Claim 26 wherein the immunointeractive molecule 0*b* is a monoclonal antibody.
22. 28. A method of treatment of a mammal, comprising administering to said mammal an expression-modulating effective amount of a nucleic acid molecule according to any one of Claims 9 to 12.
23. 29. A method according to any one of Claims 18 to 24, wherein the treatment is of a disease, injury or abnormality involving the nervous system, peripheral neuropathies, infectious disease, cancer, infertility, autoimmune disease, asthma or inflammation. COMS ID No: SMBI-00728947 Received by IP Australia: Time 15:12 Date 2004-04-30 4-04:14:56 :DAVIES COLLISON CAVE Pat.&Trad ;61 7 3358 2262 1 0/ 29 108 A method according to any one of Claims 18 to 24, wherein the treatment is of a condition involving infertility.
24. 31. of a cancer. A method according to any one of Claims 18 to 24, wherein the treatment is 0@ 0 0**0 0*0* 0* 0
25. 32. A method according to any one of Claims 18 to 24, wherein the treatment is of a cancer of the squamous cell carcinoma type.
26. 33. A method according to any one of Claims 18 to 24, wherein the treatment is of a cancer selected from testicular cancer, colon cancer, pancreatic cancer, oesophageal cancer, prostate cancer and ovarian cancer.
27. 34. A method according to any one of Claims 18 to 24, wherein the treatment is of a testicular cancer. A method of producing infertility in a mammal, comprising administering to said mammal an activity-modulating effective amount of an antagonist of a proteinaceous molecule according to any one of Claims 1 to 3.
28. 36. A method according to Claim 35, wherein said antagonist is an immunointeractive molecule specific to a proteinaceous molecule according to any one of Claims I to 3.
29. 37. A method according to Claim 35, wherein said antagonist is an antibody specific to a proteinaceous molecule according to any one of Claims I to 3.
30. 38. A method according to Claim 35, wherein said antagonist is a monoelonal antibody specific to a proteinaceous molecule according to any one of Claims 1 to 3.
31. 39. Use of a proteinaceous molecule according to any one of Claims I to 3 or a COMS ID No: SMBI-00728947 Received by IP Australia: Time 15:12 Date 2004-04-30 4-04;14:56 ;DAVIES COLLISON CAVE Pat.&Trad :61 7 3368 2262 ll/ 29 P-'O.WDoWA Pr tMlW7tuW a imdodaJdoIW -109- nucleic acid molecule according to any one of Claims 9 to 10 in the manufacture of a medicament for the treatment of a disease or condition in a mammal. Use of an immunointeractive molecule specific to the proteinaceous molecule according to any one of Claims 1 to 3 or an antigen comprising said proteinaceous molecule or part thereof in the manufacture of a medicament for the immunotherapy of a mammal.
32. 41. Use according to Claim 39 for the treatment of infertility. S
33. 42. Use according to Claim 40 for the treatment of a cancer.
34. 43. Use according to Claim 40 for the treatment of a cancer of the squamous cell carcinoma type.
35. 44. Use according to Claim 42, wherein the cancer is selected from testicular cancer, colon cancer, pancreatic cancer, oesophageal cancer, prostate cancer and ovarian 1 cancer.
36. 45. Use according to Claim 42, wherein the cancer is a testicular cancer.
37. 46. Use of an immunointeractive molecule specific to the proteinaceous molecule according to any one of Claims 1 to 3 in the manufacture of a medicament for producing infertility in a mammal.
38. 47. A method of detecting a proteinaceous molecule according to any one of Claims 1 to 3 in a biological sample, comprising contacting the biological sample with an immunointeractive molecule specific to the proteinaceous molecule, and detecting the presence of a complex comprising the immunointeractive molecule and the proteinaceous molecule in the contacted sample. COMS ID No: SMBI-00728947 Received by IP Australia: Time 15:12 Date 2004-04-30 4-04;14:56 :DAVIES COLLISON CAVE Pat.&Trad :61 7 3358 2262 12/ 29 ?NpP3*VTA Ituoa'fl6702 ashimt~ aS0O4
39. 110- 48. A method of detecting a proteinaceous molecule according to any one of Claims 1 to 3 in a biological sample, comprising detecting in said biological sample an expression product of a nucleic acid molecule encoding said proteinaceous molecule. 49. A method of diagnosis of a disease or condition in a mammal, comprising detecting absence in a biological sample obtained from the mammal of a proteinaceous molecule according to any one of Claims I to 3 or of an expression product of a nucleic acid molecule encoding said proteinaceous molecule. 50. A method of diagnosis of a disease or condition in a mammal, comprising detecting in a biological sample obtained from the mammal aberrant expression of a nucleic acid molecule according to any one of Claims 9 to 51. A method according to Claim 49 or 50, wherein the biological sample comprises a cell or tissue relating to the testes, colon, pancreas, oesophagus, prostate and ovaries. 0000 0@ 00 0@ 0 0 0 00 0 0 @0 0 000 0 00 0 00 00 0 00 0000 0 0000 0@0~ 0 0000 *000 0 @0 00 0 0 52. A method according comprises a testicular cell or tissue. 53. A method according comprises a biological fluid. to Claim 49 or 50, wherein the biological sample to Claim 49 or 50, wherein the biological sample 54. A method according to Claim 49 or 50, wherein the biological sample comprises blood. A method according comprises serum. to Claim 49 or 50, wherein the biological sample 56. A method according to Claim 49 or 50, wherein the disease or condition is selected from any one or more of a disease, injury or abnormality involving the nervous COMS ID No: SMBI-00728947 Received by IP Australia: Time 15:12 Date 2004-04-30 4-04:14:56 ;DAVIES COLLISON CAVE Pat.&Trad :61 7 3368 2262 13/ 29 -111- system, peripheral neuropathies, infectious disease, cancer, infertility, autoimmune disease, asthma or inflammation. 57. A method according to Claim 49 or 50, wherein the disease or condition relates to infertility. 58. A method according to Claim 49 or 50, wherein the disease is a cancer. 59. A method according to Claim 49 or 50, wherein the disease is a cancer of the squamous cell carcinoma type. 60. A method according to Claim 49 or 50, wherein the cancer is selected from testicular cancer, colon cancer, pancreatic cancer, oesophageal cancer, prostate cancer and ovarian cancer. 4e 61. A method according to Claim 49 or 50, wherein the cancer is testicular cancer. ex s p c a aeOs 62. A method according to Claim 50, comprising detecting expression of an aberrant or mutant form of a nucleic acid molecule according to any one of Claims 9 to or an expression product thereof. geeS 63. A method according to Claim 50, comprising detecting expression of an aberrant or mutant form of a proteinacous molecule according to any one of Claims 1 to 3. 64. A method of diagnosis of a disease or condition in a mammal, comprising detecting in a biological sample obtained from the mammal an immunointeractive molecule or imniunointeractive cell specific to a proteinaceous molecule according to any one of Claims i to 3 or to an aberrant or mutant form of said proteinaccous molecule. COMS ID No: SMBI-00728947 Received by IP Australia: Time 15:12 Date 2004-04-30 4-04:14:56 ;DAVIES COLLISON CAVE Pat.&Trad :61 7 3368 2262 1 4/ 29 -112- A method according to Claim 64, wherein the immunointeractive molecule is an antibody. 66. A method according to Claim 64, wherein the immunointeractive cell is a T cell. 67. A method according to Claim 64, wherein the biological sample comprises a biological fluid. 0e* 0*O* 0**e 68. blood. 69. serum. 70. tissue biopsy. A method according to Claim 64, wherein the biological sample comprises A method according to Claim 64, wherein the biological sample comprises A method according to Claim 64, wherein the biological sample comprises a 71. A method of diagnosis in a mammal of a disease or condition associated with an aberrant concentration of a proteinaceous molecule according to any one of Claims 1 to 3, comprising contacting a biological sample obtained from said mammal with an immunointeractive molecule specific to said proteinaccous molecule, measuring the concentration of a complex comprising said immunointeractive and the proteinaceous molecule in said contacted sample, and relating said measured complex concentration to the concentration of proteinaceous molecule in said sample, wherein the presence of said aberrant concentration is indicative of said disease or condition. 72. A method according to Claim 71, wherein the biological sample comprises a cell or tissue relating to the testes, colon, pancreas, prostate and ovaries. 73. A method according to Claim 71, wherein the biological sample comprises a COMS ID No: SMBI-00728947 Received by IP Australia: Time 15:12 Date 2004-04-30 4-04;14:56 :DAVIES COLLISON CAVE Pat.&Trad :61 7 3368 2262 1 5/ 29 -113- testicular cell or tissue. 74. A method according to Claim 71, wherein the disease or condition is selected from any one or more of a disease, injury or abnormality involving the nervous system, peripheral neuropathies, infectious disease, cancer, infertility, autoimmune disease, asthma or inflammation. A method according to claim 71, wherein the disease or condition relates to infertility. 76. A method according to Claim 71, wherein the disease is a cancer. 77. A method according to Claim 71, wherein the disease is a cancer of the squamous cell carcinoma type. 78. A method according to Claim 71, wherein the cancer is selected from testicular cancer, colon cancer, pancreatic cancer, ocsophageal cancer, prostate cancer and ovarian cancer. 79. A method according to Claim 71, wherein the cancer is testicular cancer. A method of detecting normal spermatogenesis, comprising detecting in a testicular cell or tissue a proteinaceous molecule according to any one of Claims 1 to 3 or an expression product of a nucleic acid molecule according to any one of Claims 9 to 12. 81. A method of detecting abnormal spermatogenesis, comprising detecting in a testicular cell or tissue an aberrant or mutant form of proteinaceous molecule according to any one of Claims I to 3 or an expression product of an aberrant or mutant form of a nucleic acid molecule according to any one of Claims 9 to 12. 82. A method of diagnosis of a disease or condition in a mammal, comprising COMS ID No: SMBI-00728947 Received by IP Australia: Time 15:12 Date 2004-04-30 4-04:14:56 :DAVIES COLLISON CAVE Pat.&Trad :61 7 3368 2262 16/ 29 P-.OVpA VpPatim\Tv3ln73eh.da1.1 m -114- detecting in a biological sample obtained from the mammal aberrant expression of an immunointeractive molecule or immunointeractive cell specific to a proteinaceous molecule according to any one of Claims 1 to 3. 83. A method according to Claim 82, wherein the immunointeractive molecule is an antibody. 84. A method according to Claim 82, wherein the immunointeractive cell is a T cell. A method according to Claim 82, wherein the disease or condition is selected from any one or more of a disease, injury or abnormality involving the nervous system, peripheral neuropathies, infectious disease, cancer, infertility, autoimmune disease, asthma or inflammation. 86. A method according to claim 82, wherein the disease or condition relates to infertility. 87. A method according to Claim 82, wherein the disease is a cancer. 88. A method according to Claim 82, wherein the disease is a cancer of the squamous cell carcinoma type. 89. A method according to Claim 82, wherein the cancer is selected from testicular cancer, colon cancer, pancreatic cancer, oesophageal cancer, prostate cancer and ovarian cancer. A method according to Claim 82, wherein the cancer is testicular cancer. 91. The isolated proteinaceous molecule according to Claim 1 or Claim 2, or the derivative or homologue according to Claim 3, or the composition according to Claim 4 or COMS ID No: SMBI-00728947 Received by IP Australia: Time 15:12 Date 2004-04-30 4-04:14!56. :DAVIES COLLISON CAVE Pat.V&rad :61 7 3368 2262 17/ 29 -115 Claim 5, or the isolated immunointeractive molecule according to any one of Claims 6-8, or the isolated nucleic acid molecule according to Claim 9 or Claim 10, or the derivative or homologue according to Claim 11 or Claim 12, or the vector according to any one of Claims 13-16, or the genetic composition according to Claim 17, or the method according to any one of Claims 18-38, or the use according to any one of Claims 39-46, or the method according to any one of Claims 47-90, substantially as herein before described with reference to the figures and/or examples. DATED this 30th day of April 2004 T>T~ COtJOUCLQ OF( rt-o~C~~~k J'Msh~t of HedbcA oANMAD EOe, .ations Pty DAVIES COLLISON CAVE RA4/ Patent Attorneys for the applicant .SEC S113 :0,0 00S COMS ID No: SMBI-00728947 Received by IP Australia: Time 15:12 Date 2004-04-30