MXPA98010005A - Similar gene to the toloid and protein of mamif - Google Patents

Abstract

A mammalian gene encoding a toloid-like protein, other than human or murine BMP-1 / mTld, is described. The gene is similar in structure to members of the BMP-1 gene family, but maps to a different site and encodes a different protein. The protein encoded by the gene can be used to select putative therapeutic agents in a constant effort to inhibit the activity of the BMP-1 gene family, to prevent scarring, fibrosis and the like

Description

GENE SIMILAR TO THE TOLOID AND PROTEIN OF MAMIFER BACKGROUND OF THE INVENTION The present invention relates to the field of bone morphogenetic proteins and more particularly to a gene in the BMP-1 / Tld gene family. Bone formation in mammals such as mice and humans is governed by a group of bone morphogenetic proteins (BMPs). Of the seven known BMPs involved in osteogenesis, six (designated BMP-2 to BMP-7) belong to the TGF-β superfamily. The seventh BMP (designated BMP-1) is not similar to TGF-β, but rather appears to be derived from a different gene family. The members of the BMP-1 gene family typically contain the following domains: a metalloprotease domain similar to astazin, one or more EGF-like portions which are thought to bind calcium to other proteins (Ca **), and a number of CUB domains. A CUB domain is a portion that mediates the protein-protein interactions in the components of the Clr / Cls complement, which has also been identified in various proteins involved in the processes of development. BMP-1 was described, at the level of the sequence REF: 28895 of nucleotides, by Wozney, J.M., et al., Science 242: 1528-1534 (1988). The structure of the mammalian BMP-1 domain is shared by proteins found in other species other than mammals. These proteins include Drosophila toloid (Tld) (Shimell, MJ, Cell 67: 469-481 (1991)), a genetic product of Drosophila similar to toloid (Tlr-1 or tolkin) (Nguyen, T., Dev. Biol. 166: 569-586 (1994) and Finelli, AL, et al., Genetics 141: 271-281 (1995)), a homologue of sea urchin BMP-1 (suBMP-1) (Hwang, S.-P. et al., Development 120: 559-568 (1994)), two related genetic products of sea urchin development, SpaN and BP10 (Reynolds, SD et al., Development 114: 769-786 (1992 and Lepage, T. et al., Development 114: 147-164 (1992)), a BMP-1 from Xenopus (xBMP-1) (Maeno, M. et al., Gene 134: 257-261 (1993) and a mammalian toloid (mTld) (Takahara, K. et al, J. Biol. Chem. 269: 32572-32578 (1994).) A gene similar to the toloid (xoloid) obtained from Xenopus has been briefly mentioned in the past - in an article reviewing the. family of metalloproteases of astacin, Bond, JS and RJ Benynon, Protein Science 4: 1 247-12-61 to 1249 (1995), but the data related to the gene itself has not been published Some of the nucleic acid sequences of the genes encoding these proteins are known. The mammalian BMP1 gene codes for the BMP-1 protein and for the mTld protein, although on two distinct mRNA molecules alternately spliced. The documents mentioned in this paragraph are incorporated herein by reference.

BRIEF DESCRIPTION OF THE INVENTION The present invention is summarized in that a new gene product similar to the mammalian toloid (mTll) and its related or cognate gene, which is distinct from mTld and the other known proteins and genes related to BMP-1, are also described. The murine and human versions of the gene are reported. An objective of the present invention is to provide a gene and a gene product involved in the deposition of the extracellular matrix in vertebrates (for example, in osteogenesis). Yet another objective of the present invention is to provide a target molecule for the rational development of a. drug to inhibit the activity of genes similar to the toloid, to treat fibrosis, scarring, keloids, surgical adhesions and the like. Yet another objective of the present invention is to provide a recombinant DNA construct, and a protein encoded by the construct for use in accelerated healing of wounds and fractures. Another additional objective of the present invention is to provide a marker gene that makes the map for the central portion of the chromosome 8 of the mouse. Yet another objective of the present invention is to provide a marker gene that maps the 4q32-4q33 region of human chromosome 4. Yet another objective of the present invention is to provide a nucleotide sequence that functions as a probe for a non-BMP-1 bone morphogenetic protein gene in mammalian cells.A characteristic of the present invention is that the described murine gene contains a novel repeat sequential simple in the 3 'untranslated region of the gene., features, and advantages of the present invention will become apparent upon consideration of the following detailed description, considered in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DIVERSE VIEWS OF THE DRAWINGS Figure 1 depicts a cDNA map of murine mTll including the 5 'and 3' untranslated portions thereof. Also aligned below the mTll cDNA for comparison, are the schematic representations of the cDNA clones of the related genes, drawn to the same scale as mTII. The portions of the cDNA corresponding to the domains of the gene product are highlighted. The dotted, darkly shaded, withdrawn, slightly shaded, and black boxes represent the domains of signal peptide, pro-region, metalloprotease, CUB, and EGF, respectively. The white boxes represent the domains unique to the various proteins. The wavy lines represent the 5 'and 3' untranslated regions. Abbreviations: mTld, mammalian toloid; mBMP-1, mammalian BMP-1; xBMP-1, xenopus BMP-1; suBMP-1, BMP-1 sea urchin; Tld, Drosophila toloid; Tlr-1, a gene related to the Drosophila toloid; SpaN and BP10, genes of the development of sea urchin, related. Restriction enzymes include: Bg, BglII; C, Clal; E, EcoRI; H, HincII; N, Ncol; S, Smal; St, Stul.

Figure 2 aligns the amino acid sequence of the described mTll gene to that of the mTld gene. The domain structure common to both proteins is shown schematically. The domains are represented as in Figure 1. The alignment was performed using the GAP program (Genetics Computer Group, Madison, Wisconsin), with a GAP weight of 3.0 and a GAP length weight of 0.1, with some additional manual alignment of putative signal peptide sequences. The cysteines are framed, the glycosylation sites linked to Asn are underlined, and the H? XXH portion of the active site of the metalloendopeptidase, is enclosed by a shaded box - with lines.

Figure 3 shows a schematic map of the central portion of mouse chromosome 8. The Map Manager program (Manly K., "A Macintosh Program for Storage and Analysis of Experimental Genetic Mapping Data", Mammalian Genome 4: 301-313 (1993) compared the segregation data for Til and for other loci from the cross-panel TJL BSS, performed the link analysis and generated the map.TJL BSS cross panel data is available in Jackson's public database Laboratories (http: // www. Jax.org / Resources / documents / cmdata).

DETAILED DESCRIPTION OF THE INVENTION A substantially pure preparation of the (mTll) cDNA similar to the mammalian toloid was isolated from mice by probing a cDNA library prepared from mouse embryo fibroblasts of the BMP-lt ,, lltl strain with a Aatll-Drdl restriction fragment of approximately 330 base pairs of the mouse BMP-1 gene and selecting at low demand, as described in more detail in the following examples. BMP-1, nlbln is a mouse from repeated crosses between siblings (knockout) (KO) of BMP-1 that is homozygous for a null allele of the BMP-1 gene. The probe, shown in SEQ ID NO. 1 corresponds to a segment of the 360 pass pair portion of the BMP-1 gene that is absent from the BMP-ltmlblh knockout strain. Since BMP-1 is absent from the cDNA library, the selection only found sequences related to but different from BMP-1 at the DNA sequence level. A simple region of the genome was discovered in the selection (see Figure 1). Clones KO 3 and KO 7-2 of cDNA that overlap, obtained in the selection initial as substantially pure preparations, they covered much, if not all, of the coding sequence. The selection was made under a low requirement using standard protocols (Ausubel, F.M., and collaborators, Current Protocols in Molecular Biology, Wiley, New York (1987) CA). The rest of the coding sequence was obtained by re-screening the embryo fibroblast cDNA library to a high requirement to reveal an additional clone (Ko 8-2), which extended into the 5 'untranslated portion of the gene. Clone KO 8-2 was also obtained as a substantially pure preparation. One of ordinary skill in the art can join together the cloned, separate sequences, as the inventors have done, to produce the full-length cDNA, shown in SEQ ID NO. 2. Presented herein as SEQ ID NO. 2 is an open reading frame of 3039 base pairs flanked by the 5 'and 3' untranslated sequences. The open reading structure encodes a protein similar to the mammalian toloid, called mTll. The sequence presented herein represents the combined nucleic acid sequences of three cDNA clones (KO 3, KO 7-2 and KO 8-2). In the 3 'untranslated region of the mTll gene is a single sequence repeat not previously reported (SSR). The SSR has the following sequence: (GT) 20GC (GT) 7GC (GT) 7GCAT (GT) 3GCAT (GT) 3 (shown at nucleotides 4148 to 4239 in SEQ ID NO.2). The sequential data presented in SEQ ID NO. 2 will be available in Access Number U34042 of Genbank. A preparation of the DNA molecules containing a sequence of the mTll gene from any source is considered substantially pure if more than 90% of any cellular material of any host cell in which it has received the DNA has been removed from the DNA preparation. The cellular material can be removed from a nucleic acid preparation, for example, by the use of commercial purification equipment such as that available from Qiagen (Chatsworth, CA). It is preferred that more than 10% of the nucleic acid molecules in a nucleic acid preparation comprise the complete or partial mTll gene or a portion thereof. More preferably, more than 50% and still more preferably more than 90%, of the nucleic acid molecules comprise the complete or partial sequence. It is noted that the additional genes that have some relation to mTll and other members of the family BMP-1 can be isolated from the embryo, null, double cDNA libraries lacking BMP-1 / mTld and mTll, using a comparable selection strategy. Such mutant, null, double animals could be produced by the copulation of heterozygous animals at each of the two loci. The murine mTll gene maps to central chromosome 8 near D8Bir22, which was placed at position 31 in the committee report of chromosome 8 of 1994 (Ceci, JD, "Mouse Chromosome 8", Mammalian Genome 5: S124- S138 (1994)). This same general regional region is the site of the map of four genetic defects that lead to apparent abnormalities in development: Hook (Hk), adrenocortical dysplasia (Acd), Quinky (Q), and proportional dwarfism (pdw). BMP-1 in contrast, maps the mouse chromosome 14 (Ceci, JD et al., "An interspecific backcross linkage map of the proximal half of mouse chromosome 14", Genomics 6: 673-678 (1990). The proposed murine mTll protein domain, predicted from the sequential similarities to the product of the m-toloid protein, is shown in Figure 2. In view of the similarity to other proteins similar to the toloid, it is expected that the encoded product for the mTll gene described will be a protease that has a key role in the development and homeostatic processes such as wound healing. It is likely that the protein is involved in the maturation of precursors of the extracellular matrix in macromolecular structures. Protein may also have a role in the activation of growth factors in vi and in vi tro, and may accelerate homeostatic and developmental processes when an effective amount of the protein is administered to a tissue. On the other hand, if the function of the mTII protein is inhibited, such processes can by themselves be inhibited, whose property can be exploited advantageously after the distribution of an effective amount of an inhibitor to prevent fibrosis and excessive scarring. other abnormalities of wound healing. An effective amount of the protein that is to be administered to a target site for the activation of the homeostatic and developmental properties, can be easily determined by testing a range of amounts of the protein on a selected veterinary species or on a species model that has biochemical or physiological similarity to humans, recognized. In the case of skin wound healing, for example, porcine skin is an appropriate model for human skin. Similarly, an effective amount of a Til protein inhibitor can also be determined. An effective amount is an effective amount after administration to a wound, which reduces the appearance of fibrosis, scarring or keloids compared to an untreated wound, where the evaluation of fibrosis, scarring or keloids is performed according to accepted clinical or veterinary standards. Such a test is preferably performed in a model system generally accepted as having relevance to human skin. The ability to bind with proteins of the BMP system has been hampered by the fact that proteins are typically present in very small amounts in animal tissues. It is demonstrated here (see below) that mTII, a previously known gene, can be cloned into a suitable expression vector containing an effective transcriptional promoter in a suitable host cell, introduced into and expressed in the appropriate host cells, and purified in a native configuration, always using conventional methods. The protein expressed in this way can remain inside the host cells or can be secreted into the medium of extracellular growth, if an adequate signal sequence is provided on the construction. The protein can be purified from the cell or from the growth medium by conventional methods. A suitable transcription promoter is the very late promoter of the baculovirus found on the pFASTBacl vector, which vector is commercially available from Gibco-BRL. Another suitable promoter is the immediate early promoter of the baculovirus as it is found on the pAcPIEl vector (Novagen, Madison, Wl). Any other advantageous expression elements such as enhancers, terminators, and the like, as are known in the art, can be included on the appropriate expression vector. A suitable host could be the insect tissue culture cells, such as the cell line Sf21, Sf9, or High Five. { Invitrogen, San Diego, CA). Suitable portions of the gene that comprise less than the entire coding sequence can also advantageously be cloned into the appropriate expression vector to form a recombinant genetic construct. It is understood that a construct prepared according to the invention does not necessarily need to contain the complete mTII locus or the complete coding region, but could contain one or more portions thereof encoding a desired function, or containing a portion of the gene having other useful properties, for example, complementarity to a desired genomic sequence. It is understood by those of skill in the art that some variation in the size or sequence of the mTll protein (and in the corresponding genetic material encoding the mTll protein) will not interfere with the mTll functions. Such modified forms can be engineered using known methods that can be advantageously employed when carrying out genetic constructs containing the complete or partial mTll gene, and on proteins encoded by it.

It is contemplated that such changes, modifications, additions and deletions fall within the scope of the present invention, as long as the protein retains a desired function that is known to be associated with other members of this protein family. The protein is competent if it retains an ability to cleave laminin 5 in a standard assay for such cleavage. It is also desired that the protein retain a C-proteinase activity against procollagen, as described by BMP-1 by Kessler, E., and collaborators, Science 271: 360-362 (1996), incorporated by reference herein. Someone with ordinary experience in the art is familiar with the necessary controls that must accompany any such test. Alternatively, it may be desired that the protein lose a certain function as a result of such a change, and such a situation is also considered within the scope of the present invention. A substantially pure preparation of the protein produced in this way is defined as a preparation wherein the laminin 5 cleaving activity of the mTll protein is not affected by the presence of other proteins or molecules in the preparation. Depending on the use to which the protein is to be put, it may be that the mTll protein represents at least 10%, preferably at least 50%, more preferably at least 75%, and still more preferably at least 95% of the protein in the preparation of substantially pure protein. The protein preparation can be improved for the protein of interest by labeling the protein with an affinity tag or label, and passing the preparation on a column having an affinity for the tag or label. It is also possible to employ a processing tag or label such that a Properly processed form of the protein (lacking the cleaved pro-region) can be eluted from a column loaded with a crude preparation. The translation product of mTl (SEQ ID NO 3), predicted from the DNA sequence, has a predicted molecular weight of 114.532 (pl 6.15). If the translation product is cleaved between the proregion and the protease domain in the limit shown in Figure 2, the predicted molecular weight for the mature protease could be 98.007 (pl 6.18). When the sequence of the murine mTll protein is compared to other genes similar to the toloid, there is no obvious homology between the pro-region of either of the two mammalian proteins (Tld and mTll) and the pro-region of either of the two proteins. of Drosophila (Tld or Tlr-1). The mTll protease domain was 66% similar (47% identical) to Tld and was 69% similar (52% identical) to Tlr-1. mTll is slightly more similar in sequence to both Drosophila proteins than what is mTld, and there is no obvious correlation between a particular member of the pair of mammalian proteins and a particular member of the pair of Drosophila proteins. An aligned pair of amino acids are "similar" if they have a similarity threshold above 0.5 using the rating system of Schwartz and Dayhoff, Atlas of Protein Sequence and Structure, Dayhoff, M.O., ed., Natinal Biomedical Research Foundation, Washington, D.C., p. 353-358 (1975). The mTII mRNA transcript does not appear to be alternately spliced, since only a single transcript was detected using a fragment of the KO3 clone internal to the coding region, such as a probe (SEQ ID NO.2, nucleotides 1113 to 2745) and because only a single mTll cDNA was isolated during selections from the cDNA library. An expression of relatively strong mTII mRNA was observed in brain and adult kidney, with somewhat less expression in the lung and skeletal muscle RNA, and a very low expression in the A-RN from the heart and testes. No signal for the spleen or liver was apparent. After a 60-hour exposure to Northern Spotting, a very weak signal could be detected for the liver, although no signal from the spleen was detected. The expression pattern of mTII mRNA differs from that previously reported for BMP-1 transcripts and mTld transcripts. Low levels of expression are observed even in the total embryo RNA seven days after intercourse. The level of mRNA increases slightly at eleven days of development, there are peaks at relatively high levels at fifteen days, and then decreases at seventeen-day embryos. In contrast, transcripts of BMP-1 and mTld were observed at higher levels in seven-day embryos than in eleven-day-old embryos. The same spotting was used to periodically check the transcript levels of mTll, BMP-1 and mTld. The mTII mRNA transcripts were detected throughout the embryonic development in the period of 9.5 to 15.5 days after intercourse. As previously observed with mTld RNA, mTll signals were observed throughout the mesenchyme, with higher levels of overlapping areas, of the future bone and the ventral portion of the neural tube. A strong signal, observed in the same portion of the anterior ventral brain in which the signal for mTld was previously observed, is consistent with the expression of mTll in the basal plate. A regular pattern of strong expression was observed overlapping the connective tissue between the developing vertebrae. The high signal of mTll observed in the mesenchyme of the developing lung contrasts with the absence, or very low level, of liver expression, which reflects the relative amounts of mTll mRNA found in the lung and adult mouse liver, through analysis of Spotted from Northern. In a parasagittal section of an embryo 13.5 days after intercourse, expression was observed in the mesenchymal elements of the developing tongue, the nasal process, and the jaws, and in the submucosal layer in the developing bowel. We observed the expression of mTll that overlaps an atrioventricular valve in development of the heart. A greater difference between the distribution of mTII and mTld mRNA in developing mouse tissues is observed by overlapping the neuroepithelium in the vestibular area of the base of the fourth ventricle of the developing brain, where a strong mTII expression was consistently observed, in various sections, and where no expression of mTld or BMP has been observed. The expression of mTll RNA was observed, in a number of sections, to overlap the neuroepithelial lining of the ventricles and the neonatal brain aqueduct. The expression of mTll was also observed by overlapping the specific nuclei within the thalamus and the neuroepithelial lining of the lateral ventricles. In the adult brain, strong expression of mTll was observed in the granular layer of the cerebellum. It was also observed weaker mTll expression overlapping other brain structures of neonate and adult mouse. The Northern blot analysis of RNA from various portions of the human brain, has also detected a relatively strong signal for mTll in the human cerebellum. Another perceived difference between the distribution of mTII mRNA and that previously described for mTld was in a developing spinal cord where the expression of mTII was more extensive than what was previously perceived for Tld, extending beyond the basal plate to portions more dorsals of the spinal cord. In other developing tissues, the distribution of mTII and mTLD transcripts seems to overlap. It is specifically considered that the mTll gene equivalents can be isolated from other species, by probing a cDNA library from the cells of an appropriate species with a probe selected to include a specific portion of mTll of the described mouse gene. A specific mTll portion of the mouse mTll gene can be obtained by comparing the nucleic acid sequence of the mouse mTll coding region to that of BMP-1 / mTld and selecting a portion of the mTll gene that does not have equivalent in BMP-1. To be an effective probe, the selected sequence it should not contain repeated sequences that could cross-hybridize to the numerous genomic sites. The probe should be at least about 200 base pairs in length. It is recognized that the genes of the BMP-1 family are more variable in the regions that encode the pro-region in the 17 C-terminal amino acids of the proteins, and it is anticipated that the appropriate probes can be isolated from those regions of the mTll gene. . In SEQ ID No. 2, this region corresponds to the sequence shown between-approximately bases 3599 and 3650 for the C-terminal portion and approximately 701-1051 for the pro-region. Such fragment can be converted into a probe by translating by cutting, and end-labeling or other suitable technique known in the art. It is also understood that a desired fragment (or of course a complete gene) can be synthesized in vi tro using well-known techniques available to the molecular biologist. This has been achieved using human source DNA. To obtain human mTll sequences, a 677 base pair NdeI-Eco7 I fragment of mouse mTll cDNA clone KO 3, corresponding to a portion of CUB4 and all of CUB5 and the carboxyl terminus, was used to select a library from Genetic DNA of human placenta. Genomic clone 151-2 was isolated, which contained the final three exons of the human TLL gene. A Taql fragment of 339 base pairs of mouse mTll KO 7-2 cDNA, corresponding to a part of the pro-region, was then used to select the same human DNA library, resulting in the isolation of the genomic clones 5-2 and 8, each of which contained the first exon (more towards the 5 'end of the Til gene.) Oligonucleotide primers were synthesized corresponding to the sequences in the 5' and 3 'untranslated regions, and were used with cDNA synthesized from human fetal cartilage RNA for long-distance PCR amplification of the remainder of the Til coding sequence.The forward primer was 5'-TCTTGCAGTCAGTTGCTTTGCTGG-3 '(SEQ ID NO: 10). The reverse primer was 5 '-TAGTGCGGCCGCACATTCCTTTGTGTTC-3' (SEQ ID NO.11) The nucleic acid sequence of human mTll is shown in SEQ ID NO 4. The protein encoded by the gene is shown in SEQ ID NO. 5. The gene (or portions thereof) can be used in the same ways as the murine gene, but with the additional benefit for genetic therapies, diagnostics and the like / since there is no need to adapt the gene for use in humans, as might be the case for the mouse mTll gene. Because defects in mTll can lead to genetic abnormalities in people, the chromosomal position of the human TLL gene was established. A cDNA PCR product of 527 base pairs, corresponding to the last 3 exons of the human TLL gene, was hybridized to the Southern Spotted genomic DNA digested with EcoRI from hybrid panels of human-mouse cells. Strong hybridization to the human bands of approximately 5.1 and 9.5 kb was observed, and DNA examination from the 30 hybrid lines, derived from 17 unrelated human cell lines and 4 mouse cell lines (Takahara, K. et al. , J. Biol. Chem. 269: 26280-26285 (1994)), showed that the segregation of TLL correlated with the distribution of human chromosome 4. Of the cell hybrids examined, one that retained a translocation of human chromosome 4 also located TLL on the long arm of chromosome 4. The 55R16 cell hybrid does not have "intact chromosome 4 but retains the 11/4 translocation, llqter-llpl3:: 4q25 These results placed TLL in the 4q25-4qter region.The TLL gene was independently mapped by fluorescence hybridization ip if you (FISH) on smear of human chromosomes in metaphase by the method of Trask, B., Methods Cell Biol. 35: 1-35 (1992). Clone 8 of human genomic DNA, which contains the first exon of TLL and has an insert size of approximately 16 kb, was labeled with digoxigenin-11-dUTP (Boehringer Mannheim) by random priming (Feinberg, AP, and B. Vogelstein, B. Anal. Biochem 132: 6-13 (1983)) and used as a probe for FISH analysis. The images were obtained and analyzed as described (Takahara, K. et al., Supra). Double fluorescent signals were found only in 4q32-4q33 in 16/18 of the smears "in metaphase examined (88.8%), with double fluorescent signals found on both chromosomes of the smears in metaphase 10/18 and on no other chromosome, pug TLL in this region It should also be possible to use PCR to amplify a portion of a genome corresponding to the mTll region, by selecting specific primers that are expected to flank the mTll gene (or any portion of the gene). mTll of the gene can serve as suitable primers It can not be effective to select primers outside the coding portion of the gene, because the selective pressure reduced on the non-coding portions of the gene Coding results in greater divergence between mice and other species in those regions. It is specifically noted that the genes of the BMP family from human species and model such as the mouse are particularly sought after their relationship to human deformities (see, for example, "The Chlcken With a Duck 's Feet: It's All in the Biochemical Signal ", The New York Times, National Edition, page B6 (May 21, 1996)). It is also specifically considered that large amounts of the protein encoded by the mTII gene can be expressed in (or secreted from) host cells, purified to a substantially pure preparation and used in subsequent functional assays. this type, the functional attributes of the expressed protein will be described.The functions of the protein are expected to include a metalloprotease activity, C-proteinase activity and laminin 5 processing activity, and an activation activity for proteins similar to TGF-β, such predictions being reasonable in view of the gross structural similarity for proteins known at the domain level In yet another assay, the protein can be used to select putative agents having inhibitory activity against the protein. Since mTll is capable of rescuing BMP-1 knockout mice, it will be important for any therapeutic system that it modifies or eliminates the function of BMP-1 protein to similarly alter the function of the mTll protein. "Thus, any panel of such agents must be selected against the mTll protein In such an assay, all the components of an assay that support the mTll function can be aggregated together, under appropriate salt and pH conditions, and combined with a panel of putative inhibitors of protein function Using the established assays of protein function (described in the documents incorporated elsewhere herein by reference), it will be possible to determine whether any agent tested can inhibit the activity of the protein, which makes it a likely candidate for use in a therapeutic amount to inhibit flbrosis, reduce scarring, and reduce keloids.Such selection efforts are under way using related proteins from the BMP-1 family of genes. See Kessler, described above It is now also possible to venture into a rational strategy of drug design ut illizing the described protein or the fragments thereof. To the In doing so, the protein or fragments will be subjected to X-ray crystallographic analyzes to determine their active sites and sites that are available for interaction with a putative therapeutic agent. It was recently shown that the protein encoded by BMP-1 breaks the procollagen near the C-terminus. It had been thought for a long time that this activity of C-proteinase, which is essential for the production of collagen, lies in a protein that had remained elusive There is great commercial interest in harnessing the activity of C-proteinase as a therapeutic agent in collagen-related diseases. Since mTll appears to be the only other mammalian gene closely related to BMP-1 (based on the results of cDNA library selection), it is also specifically contemplated that the protein encoded by mTll will be an alternative C-proteinase and, Furthermore, that the mTII gene can be used in an effort to produce an alternative C-proteinase, either by incorporating the gene into a recombinant vector for the ex vi ve production of the therapeutic protein, and for direct administration in a gene therapy The human gene has particular utility for these applications.

The invention will be better understood by considering the following non-limiting examples.

EXAMPLES BMP-1 / mTld Null Mouse Embryo cDNA library Mouse embryo fibroblasts (MEFs) were prepared as described (Hogan et al., "Manipulating the Mouse Embryo: A Laboratory Manual, 2nd Edition", pp. 260-261, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1994 )). Ten 150 mm plates of MEFs prepared from embryos made homozygous for the null alleles of the BMP-1 / mTld gene (do not express BMP-1 / mTld proteins) were developed to confluence (in DMEM, 10% fetal serum of beef), and 3 days later were treated with 50 μg / ml of ascorbate for 18 hours, harvested and 42 μg of poly (A *) mRNA were isolated using a FastTrack equipment (Invitrogen). An aliquot of 5 μg of poly (A *) was then used for the synthesis of double-stranded cDNA with EcoRI ends using the SuperScript Choice System (Gibco-BRL). This cDNA was then ligated to the gtlO arms cut with EcoRI and packaged using the Gigapak II Gold packaging extract (Stratagene). The 5 μg of poly (A *) provided an unamplified library of approximately 2.2 x 10 6 pFU plate-forming units). The randomly picked clones had an average insert size of approximately 2.9 kb.

Analysis of DNA Sequence Restriction fragments were subcloned into pBluescript II KS 'and the sequences were obtained from the double-stranded templates by dideoxy chain termination, as described in Lee ST et al., "Construction of a full-length cDNA encoding human pro -alpha 2 (1) collagen and its expression in pro-alpha 2 (1) - deficient W8 rat cells, "J. Biol. Chem: 263: 13414-13418 (1988). The ends of the subclones were sequenced using primers T3 and T7 with internal portions of subclones made accessible to sequencing by introducing deletions or by using primers complementary to the insert sequences. The mTll sequences reported herein were confirmed by sequencing both strands.

Polymerase Chain Reaction PCR was performed with 0.2 μM of each primer in a 480 thermal cycler (Perkin-Elmer Corp.) with denaturation at 94 ° C for 3 minutes. Followed by 35 cycles of 94 ° C / 1 minute, 57 ° C / 1 minute, 72 ° C / 1.5 minute, and a final incubation at 72 ° C / 8 minutes. The final volumes were 100 μl of 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 1.5 mM magnesium chloride, 0.01% (w / v) of gelatin, 0.2 mM of each dNTP, and 2.5 units of Taq polymerase ( Perkin Elmer Corporation).

Tissue Sections for In Situ Hybridization Sections of tissue mounted on slides for hybridization in if you were kindly provided by G.E. Lyons (University of Wisconsin-Madison). The mouse tissues were fixed and embedded, as in Lyons et al., "The expression of yosin genes in developin skeletal muscle", J. Cell Biol. 111: 14654-1476 (1990). Briefly, tissues were fixed in 4% paraformaldehyde in phosphate buffered saline, dehydrated and paraffin infiltrated. Sections were mounted in series, 5 to 7 μ thick, on gelatinized slides. One to three sections were mounted on the slide, deparaffinized in xylene, and rehydrated. The sections were digested with proteinase K, post-fixed, treated with tri-ethanolamine / acetic anhydride, washed and dehydrated.

Probes for Hybridization in if you Specific mTll probes corresponding to the portions of the 3 'untranslated region of mTll of 1104 base pairs were used for hybridization m si tu. Since the 3 'untranslated region has no similarity to the BMP-1 or mTld sequences, the probes did not hybridize cross-over with the BMP-1 or Tld RNA. To ensure that the probes did not hybridize to other RNA transcripts possessing repeat sequences similar to the long SSR identified in the central portion of the 3 'untranslated region of mTII (nucleotides 4148 to 4239), two separate riboprobes were prepared, corresponding to sequences 3 '-TU with upward direction or with downward direction of the SSR, according to the conditions of the manufacturer (Stratagene), marked with 35S-UTP (greater than 1000 Ci / mmol / Amersham Corporation) and combined to reinforce the hybridization signal in si tu. The probes were hydrolyzed with alkali at an average size of 70 bases. For the 3 'untranslated sequences downstream of the SSR, a PCR product of 399 base pairs (SEQ ID NO-2 nucleotides 4283 to 4681) was prepared using the forward primer 5'-CCAGCTTAACCTGTTCACAC-3' (SEQ ID No. 6) and the reverse primer 5 '-AACTCTACTTCCACTTCATC-3' (SEQ TD No. 7). The PCR product was ligated into the cloning site of the vector -pCRII T-A (Invitrogen). The uniformly labeled antisense riboprobe was generated by linearizing the template at the HindIII site in the pCRII polylinker and transcribing with the T7 RNA polymerase. The sense control riboprobe was generated by linearization of the Xhol site in the pCRII polylinker and transcribing with the SP6 RNA polymerase. For the 3 'untranslated sequences upstream of the SSR, a PCR product of 420 base pairs was prepared (SEQ ID NO: 2, nucleotides 3666 to 4085), using the forward primer 5'-TCAGAACAGAAAGGAATGTG-3' ( SEQ ID No. 8) and the reverse primer 5 '-GACCACTATTCCACATCACC-3' (SEQ ID NO 9) and bound within the cloning site of pCRII T-A. The antisense riboprobe was prepared by linearization at the Xhol site in the pCRII polylinker and transcribing with the RNA SP6 polymerase, while the sense riboprobe was prepared by linearization at the HindIII site in the pCRIT polylinker and transcribing with the polymerase. RNA T7.

In Hybridization and Washing Procedures Sections were hybridized overnight at 52 ° C in 50% deionized formamide, 0.3 M NaCl, 20 mM Tris-HCl, pH 7.4, 5 mM EDTA, 10 mM NaP04, 10% dextran sulfate, 1 x Denhardt's solution , 50 μg / ml of total yeast RNA, 25 μmol / l of thio-ATP (Boehringer-Mannheim), and 50-75,000 cpm / μl of 35S-labeled cRNA probe. The tissue was strictly washed at 65 ° C in 50% formamide, 2 x SSC, 10 mM dithiothreitol; rinsed in phosphate buffered saline; and treated with 20 μg / ml RNase A at 37 ° C for 30 minutes. After the washes in 2 x SSC and 0.1 x SSC for 15 minutes at 37 ° C, the slides were dehydrated, immersed in Kodak nuclear NTB-2 tracing emulsion, and exposed for 1 week in airtight boxes to the light with desiccator at 4 ° C. The photographic development was in Kodak D-19. The slides were analyzed using the optical field and dark field optical elements of a Zeiss Axiophot microscope.

Northern and Southern Spotting Analysis An EcoRI fragment of 1,633 base pairs (SEQ ID NO 2, nucleotides 1113 to 2745) corresponding to the 5 'end of the cDNA clone KO 3 (Figure 1) was purified and used as a probe for the Northern blot analysis. This fragment contains the sequences corresponding to most of the protease domain; all domains CUB1, CUB2 and EGFl; and most of the CUB3 domain. The 399 base pair PCR product described above for use in? N hybridization experiments if you were gel purified and used as a probe in Southern blot analysis. Both probes were radiolabeled at a specific activity of 4-6 x 109 cmp / μg by random priming (Feinberg and Vogelstem, "A technique for radiolabelmg DNA restriction endonuclease fragments to high specific activity; Addendum", Anal. Biochem. 137: 266- 267 (1984)) and hybridized to the spots in QulckHyb (Stratagene) at 68 ° C for 1 hour. The Northern blots (obtained from Clontech) were washed twice in 2 x SSC, 0.1% SDS at 68 ° C for 10 minutes and then twice in 0.1 x SSC, 0.1% SDS at 68 ° C for 15 minutes . Southern stains were washed twice in 2 x SSC, 0.1% SDS at 68CC for 10 minutes and then twice in 0.1 x SSC, 0.1% SDS at 68 ° C for 20 minutes.

Subcloning and expression of the mTll gene The mature active forms of BMP-1, mTld and mTll are all similar in their amino acid sequences. An exception to this is the C-terminus of each protein, where no homology is observed. This unique feature of the C-terminal sequences has been developed to be used in the production of a group of polyclonal antibodies capable of discriminating between the three protein forms. In the case of mouse mTll, the synthetic peptide Ac-CYIRYKSIRYPETMHAKN-OH, which corresponds to the final 17 amino acids of mTII, was ligated to the protein carrier Hemocyanin of Lapa in the form of Lock, suspended in saline and emulsified by mixing with an equal volume of Freund's adjuvant and injected in three to four subcutaneous dorsal doses in each of two rabbits. The bleeding for the sera was at 12 and 16 weeks after immunization and reinforcements. Contrary to BMP-1 and mTld, for which the C-terminal amino acid sequences are perfectly conserved between the mouse and the human, the C-terminal amino acid sequences of mouse and human mTll are different. It is perhaps because of this divergence across species that the peptide for the C-terminal peptide of mouse mTll has produced 3-fold higher titers of antibodies in rabbits than those that have produced the C-terminal peptides of BMP-1 and mTld. In order to produce antibodies specific for the C-terminus of human mTll, the Ac-CHIRYK? IRYPDTTHTKK-OH peptide will be used. These antibodies have commercial utility in an assay to visualize the production and localization of the mTll protein in mammalian cells, tissues and organisms, including, but not limited to model systems (eg, rodents, primates, and the like) as well as humans. In view of the rapid pace at which the understanding of bone morphogenetic proteins is advancing, the ability to distinguish individual components from one another is important, not merely from a research perspective, but in the verification Periodic evaluation of the level and distribution of BMP system components in patients with disorders of the BMP system. Such disorders could include, for example, in mice and in humans, fibrotic conditions. In addition, hereditary developmental abnormalities may be due to defects in the TLL gene. The determination of the role of mTll in such genetic abnormalities will be made possible by the antibody and the nucleic acid probes described herein. The mTll protein is very clearly important in the BMP system, since it apparently perfectly replaces BMP-1 in mice that have null BMP-1 alleles on both chromosomes. Such mice survive the full course of gestation but develop a persistent hernia of the intestine in the umbilical region. These mice die shortly after birth, presumably due to the loss of the BMP-1 / mTld gene. However, these do not show serious derangements of the formation of the pattern, the formation of collagen fibrils, or development in general. Clearly, the development of this order or even fibrinogenesis of collagen could not be possible without some activity similar to BMP-1 / mTld. Such activity has been found in mouse embryo fibroblasts from these null mice of BMP-1, in the form of C-proteinase activity. Such activity seems to be provided by mTII and there do not appear to be other closely related genes.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANT: (A) NAME: Greenspan, Daniel S Takahara, Kazuhiko Hoffman, Guy G (ii) TITLE OF THE INVENTION: Protein Similar to Mammalian Toloid (iii) NUMBER OF SEQUENCES: 13 (iv) ADDRESS FOR CORRESPONDENCE: (A) RECIPIENT: Quarles & Brady (B) STREET: 1 South Pinckney Street (C) CITY: Madison (D) STATE: Wl (E) COUNTRY: US (F) ZIP CODE: 53703 (v) COMPUTER LEGIBLE FORM: (A) TYPE OF MEDIUM: Diskette - - (B) COMPUTER: IBM compatible PC (C) OPERATING SYSTEM: PC-DOS / MS-DOS (D) SOFTWARE: Patentln Reléase # 1.0. Version # 1.30 (vi) DATA OF THE CURRENT APPLICATION: (A) APPLICATION NUMBER: (B) DATE OF PRESENTATION: (C) CLASSIFICATION: (viii) ATTORNEY / AGENT INFORMATION: (A) NAME: Berson, Bennett J (B) REGISTRATION NUMBER: 37094 (C) REFERENCE NUMBER / CASE: 960296.93839 (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 608-251-5000 (B) TELEFAX: 608-251-9166 (2) INFORMATION FOR SEQ ID NO. 1: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 330 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide probe" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 1: ACGTCCASAC CsGAGCsOß OTGßCCCGAT GGOßTCATCC CsTTTGTOAT TOGAGOGAAT 60 TTCACAGGCA GCCAGAGGGC AGTCTTCCßß CAGGCCATSA GACACTOOOft GAAGCATACC 120 TGTGTCACCT tctaa - ßca CACAGATGAG a - aw - ctATA TTSTATTCAC CTACCOACCC íßo TGCOsGTSCT OCTCCTACßt GCOTCOCCGA Gt-TQGQGaCC CCCASßOCAT CTCCATCGOC 240 AAßAACTQTs ACAAOTTTSa CAT GTQCTC CATSA3CT < 3Q GCCATGTCAX TaßCTTCTGG 300 CACOAÍ-CACA CGCßßCCCÍ-A CCGCÍ-ACCGC 330 (2) INFORMATION FOR SEQ ID NO. 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 4771 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: double (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: DNA (genomic) (ix) FEATURES: (A) NAME / KEY: CDS (B) LOCATION: 611..3652 (C) OTHER INFORMATION: / product »" murine mTll protein (Xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 2: CACACCCCTT TGCTCTCCGG GCAGTCGOGA GCTTCCCTAG CTTCGGCAGG CTTTTAAGGT «0 CTGGCGGCGT AGAAATGCCT ATCCCCCAOC CCCTTCCTCG GTCTCCCCTT TCAGTTCAGA 2T TGTGCTGATG TGCAGACCGG ATTCATCTTC CCCGAGCAGC GGCGGTGGCA GCGGCGGGCa 180 CAGGCGGCTG CAGCTCGCTC TCGGCCGCGG GGTCCTGACA GCGGCGGGGG CGCGGCGCGG 240 GAGCCGGAGC TCCGGTGGCA OCTGAGCCCG CCGTGCGCCT CTCGCCGCGO CCGGTCGTGA 300 TCGCsGGAAG TTCGACCGCT GGAAGGACGA CCTAGACCGA GCCGGGTTGG CTGCGGCTGC 360 CCTGCGCCGA GCTCCTCACC TGCCTTCCGC CCACCCGCGG GCCCCCGGCC AAGTTCCCCA 420 GCATCCGGGG GAGACAGGGA GACATTTGCC CTCTCTAGCG TCCTGAAGAC ATCCGCATGT 480 CTCCGGACAC CTGAACATTC AGGTCTTTCC GAGGAGCTTC CCAGTCGGGA TAAGAACACT 540 GTCCCTAGAG CCCCGCATAT CCACGCGGCC CTCCGGGTC GGTCCCCTCC TTTTCCTCTA 600 GGGGAGGAGG ATG GGT TG CAA OCG CTC TCC CCG AGG ATG CTC CTG TGG 649 Met Gly Leu Gln Ala Leu Ser Pro Arg Met Leu Leu Trp 1 S 10 TTG GTG GTC TCG GGT ATT GTT TTC TCC CGG GTG CTG TOG GTC TGC GCT 637 Leu Val Ser oly lie Val phe Ser Arg Val Leu Trp val Cys Wing 15 20 25 GGC CTC GAT TAT GAT TAC ACT TTT GAT GGG AAC GAA GAG GAC AAA ACO 745 Giy Leu Asp Tyr Asp Tyr Thr Phe Asp Gly Asn Glu Glu Asp Lys Thr 30 35 40 45 GAG CCT ATA GAT TAC AAG GAC CCG TGC AAA GCT GCT GTG TTT TGG GGT 793 Glu Pro l e Asp Tyr Lyß Asp Pro Cys Lys Ala Ala Val Phe Trp Gly 50 55. 60 GAC ATC GCC TTA GAT GAT GAA GAC TTA AAT ATC TTC CAA ATA GAC AGG 841 Asp le Ala Leu Asp Asp Glu Asp Leu Asn lie Phe Gln Xle Asp Arg 65 70 75 ACA ATT GAC CTG ACC CAG AGC CCC TTT GOA AAA CTT GGA CAT ATT ACA 8B9 Thr He Asp Leu Thr Gln Ser Pro Fhe Gly Lys Leu Gly His He Thr 30 85 90 ssc ssc 937 Gly Gly Phe Gly Asp His Gly Met Pro Lys Lyß Arg Gly Ala Leu Tyr 95 100 105 CAÁ CTT ATA GAG AGG ATC AGA AGA ATT GGC TCT GGC TTG GAG CAA AAT Gln Leu He Glu Arg He Arg Arg He Gly Ser Gly Leu Glu Gln Asn -10 115 120 12S AAC ACG ATG AAG OGA AAA GCA CCT CCA AAA TTG TCA GAG CA AGT GAG Asn Thr Met Lys Gly Lys Pro Pro Wing Lys Leu Ser Glu Gln Ser Glu 130 135 140 AAA AAT CGA GTT CCC AGA GCT GCT ACC TCA AGA ACG GAA AGG ATA TGG 1 Lys Asn Arg Val Pro Arg Wing Wing Thr Ser Arg Thr Glu Arg He Trp 145 150 155 CCT GGG GGT GTC ATT CCT TAT GTC ATA OGA GGA AAC TTT ACT GGC AGC 3 Pro Gly Gly Val Ha Pro Tyr Val He Gly Gly Asn Phe Thr Gly Ser 160 165 170 CAG AGA GCC ATG TTC AAG CAG GCC ATG AGA CAC TGG GAA AAG CAC ACC -Gln Arg Ala Met Phe Lys Glr. Ala Met Arg His Trp Glu Lys His Thr 175 180 185 TGT GTG ACG TTC ACT GAO AGA AGT GAT GAA GAA AGT TAT ATT GTG TTC: Cys Val Thr Phe Thr Glu Arg Ser Asp Glu Glu Ser Tyr He Val Pha 190 195 200 205 ACC TAC AGG CCT TGT GGA TGC TGC TCC TAT GTT GGT CGG CGG GGA AAT Thr Tyr Arg Pro Cys Gly cys Cys Ser Tyr Val Gly Arg Arg Gly Asn 210 215 220 GGC CCT CAG GCC ATC TCT ATT GGC AAG AAC TGT GAC AAG TTT GGA ATT Gly Pro Gln Wing He Ser He Gly Lys Asn Cys Asp Lyß Phe Gly He 225 230 235 GTT GTT CAT GAA CTG GGC CAC GTG ATA GGC TTC TGG CAT GAA CAT ACC Val Val His Glu Leu Gly His Val He Gly phe Trp Hls Glu His Thr 240 245 250 CGC CCA GAC CGA GAC AAC CAT GTC ACC ATC AGA GAG AAC ATC CAG Arg Pro Asp Arg Asp Asn Hs Val Thr He He Arg Glu Asn He Gln 255 260 265 CCA GGT CAA GAG TAC AAT TTT CTA AAG ATG GAG CCT GGA GAA GTG AAC Pro Gly Gli. Giu Tyr Asn Pha Leu Lys Met Glu Pro Gly Glu Val Asn 270 275 280 285 TCT CTT GOG GAA AGA TAT GAT TTT GAC AGT ATC ATG CAC TAC GCC AGG Ser Leu Gly Giu Arg Tyr Asp Phe Asp Ser He Met His Tyr Ala Arg 290 295 300 AAC ACC TTC TCA AGA GGG ATG TTT TTA GAC ACA ATA .CTC CCC TCC CGT Asn Thr Phe be Arg Gly Met Phe Leu Asp Thr He Leu Pro Ser Arg 305 310 315 GAT GAT AAT GGC ATT CGT CCT GCA ATT GGT CAA CGG ACC CGG TTA AGC Asp Asp Asn Gly Ha Arg Pro Wing He Gly Gln Arg Thr Arg Leu Ser 320 325 330 AAA GGA GAC ATT GCA CAA GCA AGA AAG CTG TAT CGA TGC CCA GCA TGT Lys Glv Asp He Wing Gln Wing Arg Lys Leu Tyr Arg Cys Pro Wing Cys 335 340 345 GGA GAA ACC CTG CAA GAA TCC AGT GGC AAC CTT TCT TCC CCA GGA TTC Gly Glu Thr Leu Gln Glu Be Ser Gly Asn Leu Ser Ser Pro Gly Phe 350 355 360 365 CCA AAT GGC TAC CCT TCC TAC ACA CAC TGC ATC TG3 ASA GTG TCT GTG Pro Asn Gly Tyr Pro Ser Tyr Thr His Cys He Trp Arg Val Ser Val 370 375 380 ACC CCG GGA GAA AAG ATT GTC TTG AAT TTT ACC ACA ATG GAC CTT TAC 1801 Thr Pro Gly Glu Lys He Val Leu Asn Phe Thr thr Met Asp Leu Tyr 335 390 39S AAA AGT AGT TTG TGC TGG TAT GAT TAC ATT GAA GTA AGA GAT GGT TAC 1849 Lys Ser Ser Leu Cys Trp Tyr Asp Tyr He Glu val Arg Asp Gly Tyr 400 405 410 TGG AGG AAG TCA CCT CTC CTT GOT AGA TTC TGT GGG GAC AAA GTG GCT 1897 Trp Arg Lys Ser Pro Leu Leu Gly Arg Phe Cys Gly Asp Lys Val Wing 415 420 425 GGA GTT CTT ACA TCT ACG GAC AGC AGA ATG TGG ATT GAG TTT CGT AGC 1945 Gly Val Leu Thr Ser Thr Asp Ser Arg Met Trp lie Glu Phe Arg Ser 430 435 440 445 AGC AGT AAC TGG GTA GGA AAA GGG TTT GCA GCT GTC TAT GAA GCG ATT 1993 Ser Ser Asn Trp Vai Gly Lys Gly Phe Ala Ala Val Tyr Glu Ala He 450 455 460 TGT GGA GOG GAG ATA AGG AAA AAC GAA GGG CAG ATT CAG TCT CCC AAT 2OH. Cys Gly Gly Glu He Arg Lys Asn Glu Gly Gln He Gln Ser Pro Asn 465 470 475 TAC CCC GAT GAC TAC CGA CGA ATG AAG GAG TGT GTA TGG AAA ATA ATG 2089 Tyr Pro Asp Asp Tyr Arg Pro Met Lys Glu Cys Val Trp Lyß He Met 480 48S 490 GTG TCC GAG GGC TAC CAT GTT GGA CTG ACC TTT CAG GCC TTT GAO ATC 2137 Val Ser Glu Gly Tyr? A Val Gly Leu Thr Phe Gln Wing Phe Glu He 495 500 505 GAA AGA CAT GAC AGC TGT GCC TAT GAC CAC CTA GAA GTT CGA GAT GGA 2185 Glu Arg Hrs Asp Ser Cys Ala Tyr Asp Kis Leu alu Val Arg Asp Gly 510 515 520 525 GCC AGT GAG AAC AGC CCT TTG ATA GGA CGG TTC TGT GGT TAT GAC AAA 2233 Wing Ser Glu Asn Ser pro Leu He Gly Arg Phe Cys Gly Tyr Asp Lys 530 '535' 540 CCT GAA GAT ATA AGG TCT ACT TCC AAC ACC CTG TOG ATG.AAG TTT GTC 2281 Pro Glu Asp He Arg Ser Thr Ser Asn Thr Leu Trp Met Lys Phe Val 545 550 555 TCT GAC GGG ACT GTß AAC AAG GCA GGG TTT GCT GCG AAC TTT TTT AAA 2329 Ser Asp Giy Thr Val Asn Lys Wing Gly Phe Wing Wing Asn Phe Phe Lys 5S0 565 570 GAG GAA GAT GAG TGT GCC AAA CCT GAC CGA GGA GGC GAA CAG AGG 2377 Glu Glu Asp Glu Cyß Ala Lys Pro Asp Arg Gly Gly Cys Glu Gln Arg 575 580 585 TGT CTT AAC ACA CTA GGC AGC TAC CAG TOT GCC TGT GAG CCT GGC TAT 242S Cys Leu Asn Thr Leu Gly Ser Tyr Gln Cyß Ala Cys Glu Pro Gly Tyr 590 59S 600 605 GAA CTG GGG CCA GAC AGA AGA AGC TGT GAA GCT GCT TGC GGA GGA CTT 2473 Glu Leu Gly Pro Asp Arg Arg Ser Cys Glu Ala Wing Cys Gly Gly Leu 610 615 620 CTG ACG AAG CTC AAT GGC ACC ATA ACC ACC CCC GGC TGG CCC AAA GAG 2521 Leu Thr Lys Leu Asn Gly Thr He Thr Thr Pro Gly Trp Pro Lys Glu 625 630 635 TAC CCT CCA AAC AAA AAC TGT GTG TGG CAA GTG ATC GCG CCA AGC CAG 2S69 Tyr Pro Pro Asn Lys Asn cys Val Trp Gln Val He Pro Pro Wing Gln 640 645 650 TAC AGA ATC TCT GTG AAG TTT GAG TTT TTT GAA TTG GAA GGC AAT GAA 2 Tyr Arg He Sar Val Lys Phe Glu Phe Phe Glu Leu Glu Gly Asn Glu 655 660 665 GTT TGC AAA TAC GAT TAC GTG GAG ATC TGG AGC GGC CCT TCC TCT GAG 2 Val Cys Lys Tyr Asp Tyr Val Glu He Trp Ser Gly Pro Ser Ser Glu 670 675 680 685 TCT AAA CTG CAT GGC AAG TTC TGT GGC GCT GAC ATA CCT GAA GTG ATG 2 Ser Lys Leu His Gly Lys Phe Cys Gly Wing Asp He Pro Glu Val Met 690 695 700 ACT TCC CAT TTC AAC AAC ATG AGG ATT GAA TTC AAG TCA GAC AAC ACT 2 Thr Ser His Phe Asn Asn Met Arg lie Glu Phe Lys Ser Asp Asn Thr 705 710 715 OTA TCC AAG AAG GTC TTC AAA OCA CAT TTT TTC TCA GAT AAG GAT GAG 2 Val Ser Lys Lys Gly Phe Lys Wing His Phe Phe Ser Asp Lys Asp Glu 720 72S 730 TOT TCA AAG GAT AAT GGT GGC TGT CAG CAT GAO TGT GTC AAC ACG ATG; Cys Ser Lys Asp Asn Gly Gly Cys Gln His Glu Cys Val Asn Thr Met 735 740 74S GGA AGT TAC ACG TGT CAG TGC CGG AAT GGA TTC GTG TTG CAT GAG AAC Gly Ser Tyr Thr Cys Gln Cys Arg Asn Gly Phe Val Leu His Glu Asn 750 755 760 765 AAG CAT GAT TGC AAO GAA GCC GAO TGT GAA CAG AAG ATA CAC AGC CCA: Lys His Asp Cys Lys Glu Ala Glu Cyß Glu Gln Lyß He His Ser Pro 770 775 780 AGT GGT CTC ATC ACC AGT CCC AAC TGG CCA GAC AAG TAT CCA AGC AGG Ser Gly Leu He Thr Ser Pro Asn Trp Pro Asp Lys Tyr Pro Ser Arg 785 790 7S5 AAA GAG TGC ACG TGG GTG ATC AGT GCC ATT CCT GGC CAC CGC ATC ACA Lys Glu Cys Thr Trp Val He Ser Wing He Pro Gly His Arg He Thr 800 805 810 TTA GCC TTC AAT GAG TTT GAG GTT GAA CAA CAT CA GAA TGT GCT TAT Leu Wing Phe Asn Glu Phe Glu Val Glu Gin His Gln Glu Cys Wing Tyr 815 820 825 GAT CAC TTG GAA ATT TTT OAT GGA GAA ACO GAG AAG TCA CCA ATA CTT Asp His Leu Glu He Phe Asp Gly Glu Thr Glu Lyß Ser Pro He Leu 830 835 340 845 GGC CGA CTA TGT GOC AGC AAG ATA CCA GAT CCC CTC 'ATO GCT ACT GGG Gly Arg Leu Cyß sly Ser Lys He Pro Asp Pro Leu Met Wing Thr Gly 850 855 860 AAT GAA ATO TTT ATT CGG TTT ATT TCT GAT GCC TCT GTT. CAA AGA AAA Asn Glu Met Phe He Arg Phe He Ser Asp Ala Ser Val Gln Arg Lyß 365 870 875 ssc TTT CAÁ CCT ACÁ CAT TCC ACÁ GAG TGT GGT GGT COA TTO AAA GCA Gly Phe Gln Ala Thr His Ser Thr Glu Cys Gly sly Arg Leu Lys Wing 880 885 890 GAG TCA AAG CCT AGA GAC CTG TAC TCC CAT GCT CAG TTT OOT GAT AAT Glu Ser Lys Pro Arg Asp Leu Tyr Ser His Wing Gln Phe Gly Asp Asn 895 900 905 AAC TAC CCA GOA CAÁ CTG OAC TGT GAA TGG TTG TTG OTG TCA GAA CGA Asn Tyr Pro Gly Gln Leu Asp Cys Giu Trp Leu Leu Val Ser Olu Arg 910 915 920 925 GGA TCT CGA CTT OAA TTG TCC TTC CAG ACA TTC GAA OTA GAA GAA GAA 3433 Gly Ser Arg Leu Glu Leu Ser Phe Gln Thr phe Giu Vai slu Glu Glu 930 935 940 GCT GAC TGT GGC TAT GAC TAT GTT GAA GTC TTT OAT GGT CTC AGT TCA 3481 Wing Asp Cys Gly Tyr Asp Tyr Val ßlu Val Phe Asp ßly Leu Ser Ser 945 950 955 AAA GCT OTO GGT CTT OGT AGA TTC TGT GGG TCA OGG CCA CCA GAA GAA 3529 Lys Wing Val Gly Leu Gly Arg Phe Cyß sly ser Gly Pro Pro Glu Olu 960 965 970 ATC TAT TCA ATT GGA GAT GTG GCT TTG ATT CAT TTC CAC ACA OAT GAC 3577 He Tyr Ser He sly Asp Val Aia Leu He Kis Phe His Thr Asp Asp 975 980 985 ACT ATC AAC AAG AAA GGA TTT TAT ATA AGA TAT AAA AGT ATA AGA TAC 3625 Thr He Asn Lys Lys Giy Phe Tyr He Arg Tyr Lys Ser He Arg Tyr 990 995 1000 1005 CCG GAA ACG ATG CAT GCC AAG AAC TAA TGCCGACCCT CCCTCAGAAC 367 --- Pro Glu Thr Met His Wing Lys Asn * 1010 AGAAAGOAAT GTGCATATGG AAAGAAGACA TTTTTAAAAT AGACAATATT AATACAATTO 3732 TTTTATATAA TGAATTTOAG CAAAAGAAAC CTOCAAGATT AOAGTTATCT CTGAAGTGAA 3792 AGAGAACTTT CCAGAAAACC TGATTGGCAT TGCAAGOATG TTTGAAAQTC ATGCTTGTTC 3852 AAGGAAGATT AACAGCTTGA AATAGATGCT TCACATTTTG OACAGTTCAT TTAATOAGCT 3912 GTGATTCTCT GGAGTGATTT CTTGACTACT TTTCCAAGAT CTOGGGACOT AGAAATGATG 3972 GGACGGATCA TAOCTTOGAA ACCCACTTCT TGGOTCTTAO CATGTTTGCT TAOACTCTOT 4032 AGGTCAGACA CAOTGTAAAC CAAATTCATO TAAGGTGATO TGOAATAGTG GTCTTTGGAA 4092 GGTGGTTCAT CATTTAAATG TAGGTTTOTG CTTGTGTGTA TGTTCACATA TGCAAGTGTG 4152 TGTGTGTGTG TGTGTGTGTG TGTGTOTGTG TGTGTGCGTG TOTGTGTGTO TGCGTGTGTG 4212 TGTGTGTGCA TGTGTGTGCA TGTGTGTTTO GAAACTGGAA TATTTCATCT TCATTATTTT 4272 CAAATGCAGO CCAGCTTAAC CTGTTCACAC AAATOATTTT GtGACCACTT CATTGTATCT 4332 GTATCTTGAG AAOTTTGAAA TATCTATAGT GTCTACAATO CAGTTAATCC CTAGATATCG 4392 GATAATACCC AGTTCACTAG TAAACTCATT TCTCTCTGOG GAAGTßCTGA ATAGTCTCCA 4452 AATTCAAGAA ACTCACCATG TCTTATAAAC CTTTAAGATA AAATTCCAAC GAGGTGTGTG 4512 CAGCCATCTT CCAAATGACT GCCTGGATGT TTCTTAGTCC AGTTACTACT GCTGCTOCTA 4572 TTGGTCTTTC TTTTATTGTT AATOTGTTGA TATGTTGTTA TTATTATGGT TATTATTATT 4632 GATGTTGTTA CTATATTTAA AAATGATGAG ATOAAGTGGA AGTAGAGTTT GGOAGAAATG 4692 AAATCTCTCT TTTTTGTTCT CTTCTTGAAA TTCAGTTTCA AAAAATACAA TATTGGGTGG 4752 CAAAAAAAAA AAAAAAAAA 4771 (2) INFORMATION FOR SEQ ID NO. 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 1014 amino acids - (B) TYPE: amino acid (D) TOPOLOGY: l ineal (ll) T MOLECULE IPO: protein (XI) DESCRIPTION OF SEQUENCE: SEQ ID NO. 3 : Met Gly Leu sln Ala Leu Ser Pro Arg Met Leu Leu Trp Leu Val Val 1 5 10 15 Ser Giy He Val Phe Ser Arg Val Leu Trp Val Cys Ala Oly Leu Asp 25 30 Tyr Asp Tyr Thr Phe Asp Gly Asn Glu Glu Asp Lyss Thr Glu Pro He 35 40 45 Asp Tyr Lys Asp Pro CYSS Lys Ala Aia Val Phe Trp Gly Asp He Ala 50 55 60 Leu Asp Asp Giu Asp Leu Asn He Phe Gln I Asp Arg Thr lie Asp 65 70 75 80 Leu Thr Gln Ser Pro Phe Gly Lys Leu Gly His He Thr Oly sly Phe 85 90 95 Gly Asp Hxs Gly Met Pro Lys Lyß Arg sly Ala Leu Tyr Gln Leu He 100 105 110 Glu Arg He Arg Arg He Gly Be Gly Leu Glu Gln Asn Asn Thr Met 115 120 125 Lys sly Lys Wing Pro Pro Lys Leu Ser Olu Gln Ser Glu Lyß Asn Arg 130 135 140 Val Pro Arg Wing Aia Thr Ser Arg Thr Olu Arg He Trp Pro 'Gly Gly 145 • 150 155 160 Val He Pro Tyr Val He Oly Gly Asn Phß Thr Gly Ser sln Arg Ala 165 170 175 Met Phß Lys Gin Ala Met Arg His Trp Glu Lys Hiß Ti-r Cyß Val Thr 180 185 190 Phe Thr olu Arg Ser Asp Glu Glu Ser Tyr He Val Phß Thr Tyr Arg 195 200 205 Pro Cys sly Cyß Cys Ser Tyr Val Gly Arg Arg sly Asn Gly Pro aln 210 215 220 Wing He Ser He Gly Lys Aßn Cy »Asp Lys Phe ßly He Val Val His 225 225 235 240 Giu Leu Gly His Val He Gly Phe Trp His Glu His T r Arg Pro Asp 245 250 255 Arg Asp Asn -lis Val Thr He He Arg Glu Asn He Oln Pro ßly sln 260 265 270 Glu Tyr Asn Phß Leu Lys Met Glu Pro Gly Glu Val Asn Ser Leu sly 275 280 28S Glu Arg Tyr Asp Phe Asp Ser Met Met Hiß Tyr Ala Arg Asn Thr Phß 290 295 300 Be Arg Gly Met Phß Leu Asp Thr He Leu pro Ser Arg Asp Aßp Asn 3-5 310 31S 320 Gly He Arg Pro Wing He sly Gin Arg Thr Arg Leu Ser Lys Gly Asp 325 330 335 He Aia sln Wing Arg Lys Leu Tyr Arg Cys Pro Wing Cys Gly Glu Thr 340 345 350 Leu Gin slu Ser Ser siy Asn Leu Ser Ser Pro Gly Phe pro Asn Gly 355 360 365 Tyr Pro be Tyr Thr Hxs Cys He Trp Arg Val Ser Val Thr Pro Oiy 370 375 380 Glu Lys He Val Leu Asn Phe Thr Met Asp Leu Tyr Lys Ser Ser 385 390 395 400 Leu Cys Trp Tyr Asp Tyr He Glu Val Arg Asp Gly Tyr Trp Arg Lys 405 410 415 Ser Pro Leu Leu Gly Arg Phe Cyß sly Asp Lys Val Wing Gly Val Leu 420 425 430 Thr Ser Thr Asp Ser Arg Met Trp He Glu Phe Arg Ser Ser Ser Asn 435 440 445 Trp Vai sly Lys Gly Phe Ala Ala Val Tyr Glu Ala He Cys sly Gly 450 455 460 slu He Arg Lys Asn Giu Gly Gln I sln Ser Pro Asn Tyr Pro Asp 465 470 475 480 Asp Tyr Arg Pro Met Lys Glu Cys Val Trp Lys He Met Val Ser Glu 485 490 495 Gly Tyr Hxs Val Gly Leu Thr Phß Gln Aia Phß Glu lie Glu Arg His 500 505 510 Asp Ser Cys Ala Tyr Asp His Leu Glu Val Arg Asp sly Ala Ser Olu SIS 520 525 Asn Ser Pro Leu He sly Arg Phe Cys Gly Tyr Asp Lys Pro slu Asp 530 535 540 He Arg Ser Thr Ser Asn Thr Leu Trp Met Lys PHSS Val Ser Asp sly 545 550 555 360 Thr Vai Asn Lys Ala sly Phß Ala Ala Asn Phß Phe Lyß Oiu Glu Aßp 56S 570 575 Glu Cys Ala Lys Pro Asp Arg sly Gly Cyß Glu Gln Arg Cys Leu Asn 580 585 590 Thr Leu Gly be Tyr Gln Cys Ala Cys Glu Pro Gly Tyr Glu Leu Gly 595 600 605 Pro Asp Arg Arg Ser Cys Glu Ala Ala Cys sly Gly Leu Leu Thr Lya 610 615 620 Leu Asn Gly Thr He Thr Thr Pro sly Trp Pro Lys Glu Tyr Pro Pro 625 630 635 640 Asn Lys Asn Cys Val Trp Gln Vai He Wing Pro Ser Gln Tyr Arg He 645 650 655 Ser Val Lys Phe olu Phe Phe Giu Leu Giu Gly Asn Glu Val Cys Lys 660 665 670 Tyr ASSP Tyr Vai olu He Trp Ser Gly Pro Ser Ser Glu Ser Lys Leu 675 680 685 HxS Gly Lys Phe Cys Gly Ala Asp He Pro Glu Val Met Thr Ser His 690 695 700 Phe Asn Asn Met Arg He slu Phe Lyss Ser Asp Asn Thr Val Ser Lys 705 710 715 720 Lys Gly Phe Lys Wing Hxs Phe Phe Ser Asp Lys Asp Glu Cyß Ser Lys 725 730 735 Asp Asn Gly Gly Cys Gln Hís slu Cys Val Asn Thr Met Oly Ser Tyr 740 745 750 Thr Cys sln Cys Arg Asn sly Phe Val Leu His Olu Asn Lyß Hiß Asp 7S5 760 765 Cys Lys Glu Wing Glu Cyn Glu Gln Lys He His Ser Ser Gly Leu 770 775 780 He Thr Ser Pro Asn Trp Pro Asp Lys Tyr Pro Ser Arg Lys Glu Cys 785 790 795 800 Thr Trp Val He Ser Wing He Pro Gly His Arg He Thr Leu Wing Phe 805 810 815 Asn slu Phe slu Val slu Gln Hxs sln Glu Cyß Wing Tyr Asp His Leu 820 825 830 slu He Phe Asp Oly slu Thr slu Lys Ser Pro He Leu sly Arg Leu 835 840 845 Cys Giy Ser Lys He Pro Asp Pro Leu Met Wing Thr Gly Asn Glu Met 850 855 860 Phe He Arg Phß He Ser Asp Ala Ser Val Gln Arg Lyß Gly Phe Gln 865 870 875. '880 Wing Thr Hxs Ser Thr Glu Cyß Gly Gly Arg Leu Lys Wing Glu Ser Lys 885 890 895 Pro Arg Asp Leu Tyr Ser Hxs Ala sln Phe Gly Asp Asn Asn Tyr Pro 900 905 910 Gly Gln Leu Asp Cys Olu Trp Leu Leu Val Ser Glu Arg Gly Ser Arg 915 920 925 Leu slu Leu Ser Phß Gin Thr Phß Giu Val Glu Olu Glu Wing Asp Cys 930 935 940 sly Tyr Asp Tyr Val Olu Val Phe Asp Gly Leu Ser Ser Lys Ala Val 945 950 955 960 Oly Leu sly Arg Phe Cys sly Ser Gly Pro Pro Olu Glu He Tyr Ser 965 970 975 He Giy Asp Vai Ala Leu He Hxs Phß His Thr Asp Aßp Thr He Aßn 980 985 990 Lys Lys Gly Phe Tyr He Arg Tyr Lys He He Arg Tyr Pro Glu Thr 995 1000 1005 Met His Ala Lys Asn 1010 (2) INFORMATION FOR SEQ ID NO. 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 3919 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: double (D) TOPOLOGY: linear (ll) TYPE OF MOLECULE: DNA (gendmico) (VI) ORIGINAL SOURCE: (A) ORGANISM: Homo Sapiens (IX) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 648..3689 (D) OTHER INFORMATION: / product »" human mTll protein " (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 4: CTCACACTTT TGCTCTCTTG CAGTCAGTTs CTTTsCTGOC TTCTGCAOGC TTTTAAGGTC 60 TCocsscoTA 0AAAtsccts 120 GTGCTGATGT GCAGACCGGA TTCATCTTCT CGGAOCTGCO GCOGCOGCTT TGGGCTCAGG 180 CGGCsscGGC TCGCOCTCGG CCGCsGAGTC CTOGCAGCAG CGGGGACGCs GCGCGGGAGT 240 CCGAGCTCTO GTGGCAGCTG AGCCCGCGGG GCGCCGCTCO CCGAGCCOCO GCCGCOOOAA 300 GTTCGGCAGC CAGAAOOACO ACCTGGCAGO CTGCOAßCGC CAGCGCCGCC AGAGCCCAOT 360 TTGCCTGCOC CCTCCCCOCC TCCGAOTOCA GAOTTCCTTA CCTOCCCTCC GCCCACCCGT 420 GGGCCCCTAO CCAACTTCTC CCTGCGACTG GGOOTAACAs OCAOTOCTTG CCCTCTCTAC 480 TGTCCCGGCG GCATCCACAT GTTTCCGGAC ACCTGAGCAC CCCGGTCCCG CCGAGGAGCC 540 TCCasGTGGG sAGAAsAGCA CCGaTGCCCC TAGCCCCGCA CATCAOCGCG OACCGCOGCT 600 OCCTAACCTC TGGGTCCCGT CCCCTCCTTT TCCTCCGGGG GAGOAOO ATO GOO TTG - 656 Met Gly Leu 1015 GGA ACG CTT TCC CCG AGG ATO CTC GTG TOG CTG GTG GCC TCO OGG ATT 704 sly Thr Leu be Pro Arg Mßt Leu Val Trp Leu Val Wing Ser Gly He 1020 1025 1030 'GTT TTC TAC GGO GAG CTA TGG GTC TGC GCT GTC CTC GAT TAT OAT TAC 752 Val Phe Tyr sly Olu Leu Trp Val Cys Wing Gly Leu Asp Tyr Asp Tyr 1035 1040 1045 ACT TTT GAT GOG AAC GAA GAG GAT AAA ACA GAG ACT ATA GAT TAC AAß 800 Thr Phe Asp Gly Asn Glu Glu Asp Lys Thr slu Thr He Asp Tyr Lys' 1050 105S 1060 1065 GAC CCG TGT AAA GCC OCT GTA TTT TOO OGC OAT ATT GCC TTA OAT GAT 848 Asp Pro Cys Lyß Wing Ala val Phß Trp aly Asp He Ala Leu Asp Asp 1070 1075 1080 OAA GAC TTA AAT ATC TTT CAA ATA OAT AOG ACA ATT GAC CTT ACO CAG 1 Glu Asp Leu Aßn He phß Gin He Asp Arg Thr He Asp Leu Thr Gln 1085 1090 1095 AAC CCC TTT GOA AAC CTT GGA CAT ACC ACA OGT GGA CTT OCA GAC CAT Asn Pro Phe sly Asn Leu Giy His Thr thr sly sly Leu Oly Asp His 1100 us mo GCT ATG TCA AAO AAO CGA GGG GCC CTC TAC CA CTT ATA GAC AGO ATA: Ala Mat Ser Lys Lys Arg oly Wing Leu Tyr Oln Leu He Asp Arg He 1115 1120 1125 AGA AGA ATT GOC TTT GGC TTG OAO CAA AAC AAC ACA OTT AAG GGA AAA 1 'Arg Arg He Gly Phe sly Leu Glu sln Asn Asn Thr Val Lys sly Lys 1130 1135 1140 1145 GTA CCT CTA CAÁ TTC TCA GOO CAA AAT GAG AAA AAT COA GTT CCC AGA 1 Val Ro Leu Gln Phe Ser sly Gln Asn Glu Lys Asn Arg Val Pro Arg 1150 1155 1160 GCC GCT ACA TCA AGA ACG GAA AGA ATA TOG CCT GGA GGC GTT ATT CCT 1 Wing Wing Thr Ser Arg Thr slu Arg He Trp Pro Oly Oly Val He Pro 1165 1170 1175 TAT GTT ATA GGA GOA AAC TTC ACT GGC AOC CAs ACA OCC ATO TTC AAO 1 Tyr Val He Gly Oly Asn Phe Thr Oiy Ser Gln Arg Aia Met Phe Lys 1180 1185 1190 CAG GCC ATO AOG CAC TGG GAA AAG CAC ACA TGT GTG ACT TTC ATA GAA 1 Gln Aia Met Arg Kis Trp Olu Lys Hxs Thr Cys Val Thr Phe He Glu 1195 1200 1205 AGA AGT GAT GAA GAG AGT TAC ATT OTA TTC ACC TAT AOG CCT TGT GOA 2 Arg Ser Asp Glu Olu Ser Tyr He Val Phe Thr Tyr Arg Pro Cys Gly 1210 1215 1220 1225 TGC TGC TCC TAT OTA GGT CGG COA OGA AAT GGA CCT CAO OCA ATC TCT 3 Cys Cys Ser Tyr Val aly Arg Arg Oly Asp Gly Pro Gln Ala He Ser 1230 1235 1240 ATC OGC AAG AAC TGT GAT AAA TTT GGG ATT GTT GTT CAT GAA TTG GGT: He sly Lys Asn Cyß Asp Lys phß sly He Val Val Hxß slu Leu Gly 1245 1250 1255 CAT GTG ATA OGC TTT TOG CAT GAA CAC ACA AGA CCA GAT CGA GAT AAC Hxs Val He oly Phß Trp His Glu Hia Thr Arg Pro Aßp Arg Asp Asn 1260 1265 1270 CAC OTA ACT ATC ATA AGA GAA AAC ATC CAG CCA GOT CAÁ GAG TAC AAT Hxs Val Thr He He Arg Glu Asn He Gin Pro Gly Gln ßlu Tyr Asn • 1275 1280 128S TTT CTG AAG ATG OAG CCT OGA GAA OTA AAC TCA CTT GOA GAA AGA TAT Phe Leu Lys Met Glu Pro Gly slu Val Asn Ser Leu sly Glu Arg Tyr 1290 1295 1300 1305 GAT TTC GAC AOT ATC ATG CAC TAT GCC AGO AAC ACC TTC TCA AGO GOO Asp Phe Asp Ser Met Met Hxs Tyr Ala Arg Asn Thr Phe Ser Arg Oly 1310 1315 1320 ATG TTT CTG OAT ACC ATT CTC CCC TCC CGT GAT GAT AAT GGC ATA COT Met Phe Leu Asp Thr He Leu ro Ser Arg Asp Asp Asn aly He Arg 1325 1330 1335 CCT GCA ATT GGT CAO CGA ACC CGT CTA AOC AAA GOA GAT ATC GCA CAG pro Wing He sly Gln Arg Thr Arg Leu Ser Lys Gly Asp He Aia Gln 1340 1345 1350 GCA AGA AAG CTG TAT AGA TGT CCA GCA TGT GGA GAA ACT CTA CAA GAA 1713 Wing Arg Lys Leu Tyr Arg Cys Pro Wing Cys Gly Glu Thr Leu sln slu 1355 1360 1365 tcc AAT soc AAC CTT TCC TCT CCA OGA TTT CCC AAT ssc TAC CCT TCT 1760 Ser Asn Oly Asn Leu Ser Ser Pro sly Phe Pro Asn sly Tyr pro Ser 1370 1375 1380 1385 TAC ACA CAC TCC ATC TGG AGA GTT TCT GTG ACC CCA GOG OAO AAG ATT 1808 Tyr Thr Hxs Cys He Trp Arg Val Ser Val Thr Pro Gly Glu Lys He 1390 1395 1400 OTT TTA AAT TTT ACA ACO ATG GAT CTA TAC AAO AGT AOT TTO TGC TOO 1856 Val Leu Asn Phe Thr Met Asp Leu Tyr Lys Ser Sar Leu Cys Trp 1405 1410 1415 TAT OAC TAT ATT OAA OTA AOA GAC GGG TAC TGG AGA AAA TCA CCT CTC 1904 Tyr Asp Tyr He slu Val Arg Asp Gly Tyr Trp Arg Lys Ser Pro Leu 1420 1425 1430 CTT OOT AOA TTC TOT GGO GAC AAA TTß CCT GAA GTT CTT ACT TCT ACA 1952 .. Leu Gly Arg Phß Cyß Gly Asp Lys Leu Pro slu Val Leu Thr Ser Thr 1435 1440 1445 GAC AOC AGA ATG TGO ATT GAO TTT CGT AGC AGC AGT AAT TGO OTA OGA 2000 Asp Ser Arg Met Trp He Giu Phe Arg Ser Ser As As Trp Val Giy 1450 1455 1460 1465 AAA GGC TTT GCA GCT GTC TAT GAA GCG ATC TGT GGA GGT GAO ATA CGT 2043 Lys sly Phe Ala Ala Val Tyr slu Ala He Cys sly aly Glu lie Arg 1470 1475 1480 AAA AAT GAA CCC 2096 Lys Asn slu Arg CCG ATG AAA GAA TGT GTO TGO AAA ATA ACA GTG TCT OAG AGC TAC CAC 2144 Pro Met Lys Glu Cys Val Trp Lys He Thr Val Ser slu Ser Tyr hxs 1500 1505 1510 OTC OGG CTO ACC TTT CAO TCC TTT GAG ATT GAA AGA CAT GAC AAT TGT 2192 Val sly Leu Thr Phe Oln be Phe Glu He slu Arg Hxs Asp Asn Cys 1515 1520 1525 GCT TAT GAC TAC CTG OAA sTT AOA GAT OOA ACC AOT ßAA AAT AßC CCT 2240 Wing Tyr Asp Tyr Leu Clu Val Arg Asp Gly Thr Ser Glu Asn Ser Pro 1530 1535 1540 1545 TTO ATA OGs COT TTC TOT sGT TAT GAO AAA CCT OAA OAC ATA ASA TCt 2288 Leu He Oly Arg Phe Cyß Oly Tyr Asp Lyß Pro slu Asp He Arg Ser 1SS0 1555 1560 ACC TCC AAT ACT TTO TOG ATO AAG TTT GTT TCT GAC GOA ACT GTO AAC 2336 Thr Ser Asn Thr Leu Trp Met Lys Phe Val Ser Asp ßly 'Thr Vai Asn 1565 1570 1575 AAA OCA GOG TTT GCT AAC TTT TTT AAA OAG OAA OAT OAO TGT GCC 2384 Lys Ala Gly Phe Ala Aia Asn Phe Phe Lys Glu Olu Asp Olu Cys Wing 1580 1585 1590 AAA CCT CAC CGT GGA GGC TOT GAG CAG CGA TGT CTG AAC ACT CTO GOC 2432 Lys Pro Asp Arg Oly Oly Cys Glu Gln Arg Cys Leu Asn Thr Leu Giy 1595 1600 1605 AGT TAC CAO TGT GCC TGT GAG CCT GGC TAT GAO CTG OGC CCA GAC AOA 2480 Ser Tyr Gin Cys Aia Cys Glu Pro Gly Tyr slu Leu Oly Pro Asp Arg 1610 1615 1620 1625 AOG AGC TGT OAA OCT GCT TGT GOT GGA CTT CTT ACC AAA CTT AAC GGC 2528 Arg Ser Cys Olu Ala Wing Cys Oly Gly Leu Leu Thr Lyß Leu Asn aly 1630 1635 1640 ACC ATA ACC ACC CCT GGC TOG CCC AAO GAG TAC CCT CCT AAT AAG AAC 2576 Thr Thr Thr Pro Pro Gly Trp Pro Lys slu Tyr Pro Pro Asn Lys Asn 1645 1650 1655 TOT GTO TGG CA GTG GTT OCA CCA ACC CAO TAC AOA ATT TCT OTO AAO 2624 Cys Val Trp Gln Vai Vai Wing Pro Thr Oln Tyr Arg He Ser Vai Lyß 1660 1665 1670 TTT GAO TTT TTT GAA TTG OAA OGC AAT GAA GTT TGC AAA TAT GAT TAT 2672 Phe Glu he Phe Glu Leu Glu Gly Aßn slu Val Cys Lyß Tyr Asp Tyr 1675 1680 1685 GTG GAG ATC TGG AOT GOT CTT TCC TCT GAG TCT AAA CTß CAT GGC AAA 2720 Val Glu He Trp Ser Gly Leu Ser Ser Glu Ser Lys Leu His Gly Lyß 1690 1695 1700 1705 TTC TGT GGC GCT GAA GTG CCT OAA GTG ATC ACA TCC CASE TTC AAC AAT 276J_ Phe Cys Gly Wing Glu Val Pro Glu Val He Thr Ser Qln Phß Asn Asn 1710 1715 1720 ATO AGA ATT GAA TTC AAA TCT OAC AAT ACT OTA TCC AAO AAO GOC TTC 2816 Met Arg lie Ghe Phe Lys Ser Asp Asn Thr Val Ser Lys Oly Phs 1725 1730 1735 AAA OCA CAT TTT TTC TAC GAC AAA GAT GAA TGC TCT AAO GAT AAT OGT 2864 Lys Ala His Phß Phe Ser Asp Lys Asp Glu Cys be Lys Asp Asn sly 1740 1745 1750 OGA TGT CAG CAC GAA TOT GTC AAC ACG ATO GGG AOC TAC ATG TGT CAA 2912 aly Cys sln Hxs Glu Cys Val Asn Thr Met Gly Ser Tyr Met Cyß aip 1755 1760 1765 TGC COT AAT GOA TTT GTG CTA CAT GAC AAT AAA CAT GAT TGC AAG GAA 2960 Cys Arg Asp Oly Phe Val Leu Hxs Asp Asn Lys Hxs Asp Cys Lys Glu 1770 1775 1780 '1785 GCT GAG TGT GAA CAG AAG ATC CAC AOT CCA AOT GOC CTC ATC ACC AGT 3008 Aia Glu Cys Glu sln Lys He Hxs Ser Pro Ser Gly Leu He Thr Ser 1790 1795 1800 CCC AAC TOO CCA GAC AAG TAC CCA AOC AGG AAA GAA TGC ACT TGO OAA 3056 Pro Asn Trp Pro Asp Lys Tyr Pro Ser Arg Lys Glu Cys Thr Trp Glu 1805 1810 1815 ATC AOC OCC ACT CCC GOC CAC CGA ATC AAA TTA GCC TTT AGT GAA TTT 3104 He Ser Ala Thr Pro Oly Hlß Arg lie Lys Leu Ala Phß Ser slu Phe 1820 1825 1830 OAG ATT GAG CAG CAT CAA GAA TOT GCT TAT GAC CAC TTA OAA OTA TTT 3152 Giu He Glu Gin Hxs Gln Glu Cys Wing Tyr Asp Hxs Leu Glu Val Phe 1835 1840 1845 GAT GGA OAA ACA GAA AAG TCA CCO ATT CTT OGA COA CTA TGT GOC AAC 3200 Asp Gly Glu Thru Glu Lys Ser Pro He Leu Gly Arg Leu Cy sly Asn 1850 1855 1860 1865 AAO ATA CCA GAT CCC CTT GTO GCT ACT OGA AAT AAA ATG TTT OTT CGG 3248 Lys He Pro Asp Pro Leu Vai Aia Thr Gly Asn Lys Met Phß Vai Arg 1870 1875 1880 TTT GTT TCT OAT GCA TCT GTT CAA AGA AAA GOC TTT CAA GCC ACA CAT 3296 Phe Val be Asp Ala Ser Vai Gln Arg Lys Giy Phßn Oln Ala Thr His 1885 1890 1895 TCT ACA GAG TGT GOC OGA COA TTG AAA OCA GAA TCA AAA CCA AOA GAT 3344 Ser Thr slu Cyß Giy Giy Arg Lau Lys Aia Glu Ser Lys Pro Arg Asp 1-CC 1.05 1910 CTG TAC TCA CAT GCT CAG TTT GGT GAT AAC AAC TAC CCA GOA CAO GTT 3392 Leu Tyr Ser Kxß Wing Gin Phe Giy Asp Asr. Asn Tyr Pro Gly Gln Val 1-15 192C 1925 GAC TGT GAA TGO CTA TTA GTA TCA GAA CGG GGC TCT COA CTT GAA TTA 3440 Asp Cys Glu Trp Leu Leu Val Ser Glu Arg Giy Ser Arg Leu Glu Leu 1935 1935 1940 1945 TCC TTC CAO ACA TTT GAA GTG GAG GAA GAA GAC GAC TGT GGC TAT GAC 3488 Being Phe Gin Thr Phe Glu Vai Glu Glu Glu Aia Asp Cys Oly Tyr Asp 1550 1955 1960 TAT OTO OAO CTC TTT GAT OOT CTT GAT TCA ACA GCT GTO GOT CTT GOT 3536 Tyr Val Glu Leu Phe Asp Siy Leu Asp Ser Thr Wing Vai Gly Leu Giy 1965 1970 1975 CGA TTC TOT GGA TCC GGG CCA CCA GAA GAG ATT TAT TCA ATT GGA OAT SÜ Arg Phe Cys to Ser Gly Pro Pro Giu Glu He Tyr Ser He Gly Asp 193C 1385 1990 TCA GTT TTA ATT CAT TTC CAC ACT GAT GAC ACA ATC AAC AAO AAG GGA 3632 Ser Val Leu He Hxs Phe ^ s Tr.r Asp Asp Thr He Asn Lys Lys Giy 1995 2000 2005 TTT CAT ATA AGA TAC AAA AGC ATA AGA TAT CCA GAT ACC ACA CAT ACC 3680 Phe Hxs He Arg Tyr Lys Ser He Arg Tyr Pro Asp Thr Thr His Thr 2C10 2015 2020 2025 AAA AAA TAA CACCAAAACC TCTOTC? GAA CACAAAGGAA TGTGCATAAT 3729 Lys Lys * GGAGAGAAGA CATATTTTTT TTAAAACTGA AOATATTOGC ACAAATGTTT TATACAAAGA 3789 GTTTGAACAA AAAATCCCTG TAAGACCAGA ATTATCTTTG TACTAAAAGA OAAGTTTCCA 3849 GCAAAACCCT CATCAGCATT ACAAGGATAT TTOAACTCCA TGCTTGATGG TATTAATAAA 3909 GCTGOTGAAA '.3919 (2) INFORMATION FOR SEQ ID NO. 5: (i) CHARACTERISTICS OF THE SEQUENCE! (A) LENGTH: 1014 amino acids (B) TYPE: amino acid (P) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO. 5: Met Gly Leu Gly Thr Leu Ser Pro Arg Met Leu Vai Trp Leu Val Wing 1 5 10 15 Be Gly He Val Phe Tyr Gly Clu Leu Trp Vai C? S Wing Gly Leu Asp 20 25 30 Tyr Asp Tyr Thr Phe Asp Ory Asu Olu Giu Asp Lys Thr Giu Thr He 35 40 45 Aso Tyr Lys Asp Pro Cys Lys Ala Wing Val Phe Trp Gly Asp He Aia Leu Asp Asp slu Asp Leu Asn He Phe Olí »He Asp Arg Thr He Asp 65 70 75 80 Leu Thr Gln Asn Pro Phe Giy Asn Leu Gly Hxs Thr Thr Gly Oiy Leu 85 90 95 Gly Asp Hxs Wing Met Ser Lys Lys Arg Gly Wing Leu Tyr Gln Leu He 100 105 lio Asp Arg He Arg Arg He sly Phe Oiy Leu slu oln Asn Asn Thr Vai 115 120 125 Lys Oiy Lys Val Pro Leu Gln Phe Ser Gly Gln Asn Olu Lys Asn Arg 130 135 140 Val Pro Arg Ala Wing Thr Ser Arg Thr Glu Arg He Trp Pro sly sly 145 150 155 150 Val He Pro Tyr Val He sly Gly Asn Pha Thr Gly Ser G n Arg Wing 165 170 175 Met Phe Lys oln Wing Met Arg Kxs Trp slu Lys His Thr Cys Val Thr 130 185 190 Phe He Olu Arg Being Asp Giu Olu Being Tyr He Val Phe Thr Tyr Arg 195 200 205 Pro Cys Gly Cys Cys Ser Tyr Val Gly Arg Arg Gly Asn Gly Pro Gln 210 215 220 Wing He Ser He Gly Lyß Asn Cys Asp Lys Phe Gly He Val Val Hxs 22S 230 235 240 Glu Leu Gly Hxs Val He sly Phß Trp His Olu Hiß Thr Arg Pro Asp 245 250 25S Arg Asp Asn Hxs Vai Thr He He Arg Oiu Asn He Gln Pro Gly Gln 260 265 270 Glu Tyr Asn Phe Leu Lys Met Giu Pro Oly Glu Val Asn Ser Leu sly 27S 280 285 Oiu Arg Tyr Asp Phe Asp Ser Met Met Hxs Tyr Ala Arg Asn Thr Phe 290 295 300 Ser Arg Gly Met Phe Leu Asp Thr He Leu Pro Ser Arg Asp Asp Asn 305 310 315 320 Gly lie Arg Pro Aia He Gly sln Arg Thr Arg Leu Ser Lyß Gly Asp 325 330 335 He Wing shan Wing Arg Lyß Leu Tyr Arg Cys Pro Wing Cys Gly Glu Thr 340 345 350 Leu sln Glu Ser Asn sly Asn Leu Ser Ser Pro sly Phe Pro Asn Gly 355 360 365 Tyr Pro Ser Tyr Thr Hxs Cys He Trp Arg Val Ser Val Thr Pro Gly 370 375 380 Giu Lys He Val Leu Asn Phe Thr Met Asp Leu Tyr Lys Ser Ser 385 390 395 400 Leu Cys Trp Tyr Aso Tyr He Glu Val Arg Asp Gly Tyr Trp Arg Lys 405 410 415 Ser Pro Leu Leu oly Arg Phe C / s Oly Asp Lys Leu Pro Glu Val Leu 420 425 430 Thr Ser Thr Asp Ser Arg Met Trp He ßlu Phe Arg Ser Ser Ser Asn 435 440 445 Trp Val Gly Lys Gly Phe Wing Wing Val Tyr Glu Wing He Cys Gly Gly 450 455 460 Glu He Arg Lys Asn Giu Gly sln He sln Ser Pro Asn Tyr Pro Asp 465 470 475 480 Asp Tyr Arg Pro Met Lys slu Cys Val Trp Lys He Thr Val Ser Glu 485 490 495 Ser Tyr Hxs Val Gly Leu Thr Phe Gln Ser Phe Olu He Glu Arg Hxs 500 505 510 Asp Asn Cys Wing Tyr Asp Tyr Leu Glu Val Arg Asp Gly Thr Ser Oiu 515 520 525 Asn Ser Pro Leu He Gly Arg Phe Cys Gly Tyr Asp Lys Pro Olu Asp 530 S35 540 He Arg Ser Tr.r Ser Asn Thr Leu Trp Met Lys Phe Val Ser Aßp Gly 545 550 555 560 Thr Val Asn Lys Wing Gly Phe Wing Wing Asn Phß Phe Lys Glu slu Asp 565 570 575 Glu cys Ala Lys Pro Asp Arg Oly sly Cys slu Gln Arg cys Leu Asn 580 585 590 Thr Leu Gly Ser Tyr Gln Cys Aia cys Glu Pro aly Tyr Glu Leu Gly 595 600 605 Pro Asp Arg Arg Ser Cys Glu Aia Wing Cys Oly sly Leu Leu Thr Lys 610 615 620 Leu Asn Gly T * r He Thr Thr Pro Gly Trp Pro Lys slu Tyr Pro Pro 625 630 635 640 Asn Lys Asn Cys Val Trp Oln Val Val Wing Pro Thr Oln Tyr Arg He 645 650 65S Ser Val Lys Phe slu Phß Phe Glu Leu Glu Gly Asn ßlu Val Cys Lys 660 665 670 Tyr Asp Tyr val slu He Trp Ser sly Leu Ser Ser slu Ser Lyß Leu 675 680 685 Hxs sly Lys Phe Cys sly Ala slu Val Pro Glu Val He Thr Ser sln 690 695 700 Phe Asn Asn et Arg He olu Phe Lys Ser Asp Asp Thr Vai be Lys 705 710 715 720 Lys Gly Phe Lys Aia Hxs Phe Pwe Ser Asp Lys Asp slu Cys Ser Lys 725 730 735 Asp Asn sly Gly Cys Gln Hls Glu Cys Val Asn Thr Met siy Ser Tyr "40 745 750 Met Cys Oln Cys Arg Asn Oiy P > -e Vai Leu His Asp Asn Lys Hxs Asp 755 760 765 Cys Lys slu Ala Glu Cys ßlu Gin Lys He Kis Ser Pro Ser aly Leu 770 775 780 He Thr Ser Pro Asn Trp Pro Asp Lys Tyr Pro Ser Arg Lys Glu Cys 785 790 795 800 Thr Trp Olu He Ser Wing Thr Pro Gly Hxs Arg He Lys Leu Wing Phe 805 810 815 Ser Glu Phe slu He Glu Gln Hxs Gln Glu Cys Ala Tyr Asp His Leu 820 825 830 Glu Vai Phe Asp Siy Oiu Thr Oiu Lys Ser Pro He Leu Gly Arg Leu 83S 840 845 Cys Gly Asn Lys He Pro Asp Pro Leu Val Wing Thr Gly Asn Lys Met 850 855 860 Phe Val Arg Phe Val Ser Asp Ala Ser Val Oln Arg Lys Oiy Phe Oin 865 870 875 880 Wing Thr His Ser Thr slu Cyß Gly Gly Arg Leu Lys Aia aiu Ser Lys 885 890 895 Pro Arg Asp Leu Tyr Ser Hxs Wing Oln Phe sly Asp Asn Asn Tyr Pro 900 905 910 Gly Gln Val Asp Cys slu Trp Leu Leu Val Ser Glu Arg Gly Ser Arg 915 920 92S Leu Glu Leu be Phe sln Thr Phe Glu Val Olu Olu Glu Wing Aso Cys 930 935 940 Gly Tyr Asp Tyr Val Glu Leu Phe Asp sly Leu Asp Be Thr Wing Val 945 950 955 9S0 sly Leu oly Arg Phe Cys Gly Ser Gly Pro Pro Glu Glu He Tyr Ser 965 970 97S I have 0? Asp Ser Val Leu He Hxß Phe His Thr Asp Asp Thr He Asn 980 985 990 Lys Lys sly Phe Kis He Arg Tyr Lys Ser He Arg Tyr Pro Asp Thr 995 1000 1005 (2) INFORMATION FOR SEQ ID NO. 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (11) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide primer" (x) DESCRIPTION OF THE SEQUENCE : SEQ ID NO. 6: CCAGCTTAAC CTGTTCACAC 20 (2) INFORMATION FOR SEQ ID NO. 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc - "oligonucleotide primer" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 7: AACTCTACTT CCACTTCATC 20 (2) INFORMATION FOR SEQ ID NO. 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (n) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide primer (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 8: TCAGAACAGA AAGGAATGTG 20 (2) INFORMATION FOR SEQ ID NO. 9: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 20 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide primer" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 9: GACCACTATT CCACATCACC - 20 (2) INFORMATION FOR SEQ ID NO. 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: other nucleic acid (A) DESCRIPTION: / desc - "oligonucleotide primer" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 10: TCTTGCAGTC AGTTGCTTTG CTGG 24 (2) INFORMATION FOR SEQ ID NO. 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 28 base pairs (B) TYPE: nucleic acid (C) TYPE OF HEBRA: simple (D) TOPOLOGY: linear (i i) TI PO OF MOLECULE: another nucleic acid (A) DESCRIPTION: / desc = "oligonucleotide primer" (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 11: TAGTGCGGCC GCACATTCCT TTGTGTTC "28 (2) INFORMATION FOR SEQ ID NO. 12: (1) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: (D) TOPOLOGY: linear (i i) TYPE OF MOLECULE: peptide (XI) DESCRIPTION OF SEQUENCE IA: SEQ ID NO. 12: Cys Tyr lie Arg Tyr Lys Ser lie Arg Tyr Pro Glu Thr Met His Wing 1 5 10 15 Lys Asn (2) INFORMATION FOR SEQ ID NO. 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 18 amino acids (B) TYPE: amino acid (C) TYPE OF HEBRA: (D) TOPOLOGY: linear (il) TYPE OF MOLECULE: peptide (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO. 13: Cys His lie Arg Tyr Lys Ser lie Arg Tyr Pro Asp Thr Thr Hls Thr 1 5 10 15 Lys Lys It is noted that in relation to this date, the least known method for the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Having described the invention as above, property is claimed as contained in the following:

Claims (8)

1. A substantially purified preparation of a DNA molecule, characterized in that it comprises the nucleotide sequence of SEQ ID NO. 2 between nucleotides 1052 and 3649.

2. A genetic construct, characterized in that it comprises in the order 5 'to 3': an active transcrinal promoter in a host cell; and a DNA fragment comprising a nucleotide sequence of SEQ ID NO. 2 between the nucleotides 1052 and 3649.

3. A method for the treatment of a wound, characterized in that it comprises the steps of: provision, in a suitable host cell, of a genetic construct comprising an active transcrinal promoter in the host cell, and a DNA fragment comprising a nucleotide sequence of SEQ ID NO. 2 between nucleotides 1052 and 3649, such that a protein is encoded by the nucleotide sequence; Y the administration of the protein to a wound in an amount effective to accelerate the healing of the wound in relation to an untreated wound.

4. A substantially pure preparation of a protein having the amino acid sequence - of SEQ ID NO. 3 between amino acids 1 and 1013.

5. A substantially purified preparation of a DNA molecule, characterized in that it comprises the nucleotide sequence of SEQ ID NO. 4 between nucleotides 1089 and 3686.

6. A genetic construct, characterized in that it comprises in the order 5 'to 3': an active transcrinal promoter in a host cell; and a DNA fragment comprising a nucleotide sequence of SEQ ID NO. 4 between nucleotides 1089 and 3686.

7. A method for the treatment of a wound, characterized by comprising the steps of: providing, in a suitable host cell, a genetic construct comprising a promoter transcrinal active in the host cell, and a DNA fragment comprising a nucleotide sequence of SEQ ID NO. 4 between nucleotides 1089 and 3686, so that a protein is encoded by the nucleotide sequence; and the administration of the protein to a wound, in an amount effective to accelerate the healing of the wound relative to an untreated wound.

8. A substantially pure preparation of a protein, characterized in that it has the amino acid sequence -of SEQ ID NO. 5 between amino acids 1 and 1013.