PL185293B1 - Novel growth factor and genetic sequence encoding same - Google Patents

Abstract

1. An isolated polypeptide having at least one of the following characteristics: (i) the ability to induce proliferation of vascular epithelial cells; (ii) the ability to interact with the receptor family, and (iii) the ability to induce cell migration, cell survival and / or growth intracellular levels of alkaline phosphatase; wherein the polypeptide comprises: a. Amino acid sequence encoded by the nucleotide sequence shown on SEQ ID No. 3 or a nucleotide sequence capable of hybridizing to SEQ ID No. 3 or not complementary under stringent hybridization conditions in the presence of 0.1-1 x SSC / 0.7% weight / weight SDS at 60 ° C for 1-3 hours, or b. the amino acid sequence shown in SEQ ID No. 4 or the amino acid sequence having at least 70% similarity to her; or a derivative of such a polypeptide, showing at least one of the features (i) - (iii).

Description

The invention relates to an isolated polypeptide having at least one of the following characteristics:

(i) the ability to induce proliferation of vascular epithelial cells;

(ii) the ability to interact with the flt-1 / flk-1 receptor family, and (iii) the ability to induce cell migration, cell survival and / or an increase in intracellular levels of alkaline phosphatase;

wherein the polypeptide comprises:

a) the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID No. 3 or a nucleotide sequence capable of hybridizing to SEQ ID No. 3 or complementary to it under stringent hybridization conditions in the presence of 0.1-1 x SSC / 0.1-1% w / w / wt. SDS at 60 ° C for 1-3 hours, or

b) the amino acid sequence shown in SEQ ID No. 4 or an amino acid sequence having at least 70% similarity thereto; or a derivative of such a polypeptide, showing at least one of the features (i) - (iii).

Preferably, an isolated polypeptide according to the invention comprises the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID No. 3.

Preferably, an isolated polypeptide according to the invention comprises the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID No. 5.

Preferably, an isolated polypeptide according to the invention comprises the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID No. 7.

Preferably, an isolated polypeptide according to the invention comprises the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID No. 9.

Preferably, an isolated polypeptide according to the invention comprises the amino acid sequence shown in SEQ ID No. 4.

Preferably, an isolated polypeptide according to the invention comprises the amino acid sequence shown in SEQ ID No. 6.

Preferably, an isolated polypeptide according to the invention comprises the amino acid sequence shown in SEQ ID No. 8.

Preferably, an isolated polypeptide according to the invention comprises the amino acid sequence shown in SEQ ID No. 10.

Preferably, an isolated polypeptide according to the invention is of human origin.

Preferably, an isolated polypeptide of the invention has at least one of the following characteristics: ability to induce astroglial proliferation, ability to promote neuronal viability and / or proliferation in mammals.

Preferably, an isolated polypeptide of the invention is a mature form of the polypeptide with a cleaved leader sequence. The invention also relates to an isolated nucleic acid molecule having the nucleotide sequence shown in SEQ ID No. 3 or

Nucleotide sequence capable of hybridizing to SEQ ID No. 3 or the complement thereof under stringent hybridization conditions in the presence of 0.1-1 x SSC / 0.1% w / w. SDS at 60 ° C for 1-3 hours.

Preferably, an isolated nucleic acid molecule according to the invention comprises the nucleotide sequence shown in SEQ ID No. 3.

Preferably, an isolated nucleic acid molecule according to the invention comprises the nucleotide sequence shown in SEQ ID No. 5.

Preferably, an isolated nucleic acid molecule according to the invention comprises the nucleotide sequence shown in SEQ ID No. 7.

Preferably, an isolated nucleic acid molecule according to the invention comprises the nucleotide sequence shown in SEQ ID No. 9.

Preferably, an isolated nucleic acid molecule according to the invention encodes a polypeptide having the amino acid sequence shown in SEQ ID No. 4 or an amino acid sequence having at least 70% similarity thereto.

Preferably, an isolated nucleic acid molecule according to the invention encodes a polypeptide comprising the amino acid sequence shown in SEQ ID No. 4.

Preferably, an isolated nucleic acid molecule according to the invention encodes a polypeptide comprising the amino acid sequence shown in SEQ ID No. 6.

Preferably, an isolated nucleic acid molecule according to the invention encodes a polypeptide comprising the amino acid sequence shown in SEQ ID No. 8.

Preferably, an isolated nucleic acid molecule according to the invention encodes a polypeptide comprising the amino acid sequence shown in SEQ ID No. 10.

Preferably, an isolated nucleic acid molecule of the invention encodes a polypeptide having at least one of the following characteristics:

(i) the ability to induce proliferation of vascular epithelial cells;

(ii) the ability to interact with the flt-1 / flk-1 receptor family, and (iii) the ability to induce cell migration, cell survival and / or an increase in intracellular levels of alkaline phosphatase;

or a derivative of such a polypeptide, showing at least one of the features (i) - (iii).

Preferably, an isolated nucleic acid molecule according to the invention encodes a polypeptide additionally having the ability to induce astroglial proliferation.

Preferably, an isolated nucleic acid molecule of the invention encodes a polypeptide additionally having the ability to promote neuronal viability and / or proliferation in mammals. .

Preferably, the isolated nucleic acid molecule according to the invention or the polypeptide encoded by it is of human origin.

The invention also relates to a method of obtaining a polypeptide having at least one of the following characteristics:

(i) the ability to induce proliferation of vascular epithelial cells;

(ii) the ability to interact with the flt-1 / flk-1 receptor family, and (iii) the ability to induce cell migration, cell survival, and / or an increase in intracellular levels of alkaline phosphatase, characterized by the expression of a nucleic acid molecule encoding the polypeptide containing:

a) the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID No. 3 or a nucleotide sequence capable of hybridizing to SEQ ID No. 3 or complementary to it under stringent conditions hybi ^ r ^ y ^ ^ / z ^ cci in the presence of 0.1-1 x SSC / 0.1% w / w SdS at 60 ° C for 1-3 hours, or

b) the amino acid sequence shown in SEQ ID No. 4 or the amino acid sequence. having at least 70% similarity to her;

or a derivative of such a polypeptide in a suitable host grown under conditions suitable for polypeptide synthesis.

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Preferably, the method of the invention is characterized in that the polypeptide comprises an amino acid sequence encoded by a nucleotide sequence selected from SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7 and SEQ ID No. 9, or an amino acid sequence selected from SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, wherein preferably the isolated polypeptide is of human origin.

The invention also provides an antisense molecule that hybridizes to a nucleic acid molecule having or complementary to the nucleotide sequence shown in SEQ ID No. 3, or to a nucleic acid molecule having a nucleotide sequence capable of hybridizing to SEQ ID No. 3 or complementary to it under stringent hybridization conditions under stringent hybridization conditions. 0.1-1 x SSC / 0.1% w / w SDS at 60 ° C for 1-3 hours.

Preferably, the antisense molecule of the invention is characterized in that it hybridizes with a nucleic acid molecule comprising a nucleotide sequence selected from SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, or a nucleic acid molecule that encodes a polypeptide containing the sequence an amino acid selected from SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, or an amino acid sequence having at least 70% similarity to SEQ ID No. 4.

The invention also relates to a pharmaceutical for inducing astroglial proliferation in a mammal and / or enhancing survival and / or proliferation of mammalian neurons comprising an active agent and one or more pharmaceutically acceptable carriers and / or diluents, characterized in that the active agent comprises an isolated polypeptide having at least one of the following characteristics:

(i) the ability to induce proliferation of vascular epithelial cells;

(ii) the ability to interact with the flt-1 / flk-1 receptor family, and (iii) the ability to induce cell migration, cell survival and / or an increase in intracellular levels of alkaline phosphatase;

wherein the polypeptide comprises:

a) the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID No. 3 or a nucleotide sequence capable of hybridizing to SEQ ID No. 3 or complementary to it under stringent hybridization conditions in the presence of 0.1-1 x SSC / 0.1% w / w / wt. SDS at 60176C for 1-3 hours, or

b) the amino acid sequence shown in SEQ ID No. 4 or an amino acid sequence having at least 70% similarity thereto;

or a derivative of such a polypeptide, showing at least one of the features (i) - (iii).

Preferably the pharmaceutical composition according to the invention is characterized in that the isolated polypeptide comprises an amino acid sequence encoded by a nucleotide sequence selected from SEQ ID No. 3, SeQ ID No. 5, sEq ID No. 7 and SEQ ID No. 9, or an amino acid sequence selected from SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, wherein the isolated polypeptide is preferably of human origin or the mature form of the polypeptide with a cleaved leader sequence.

The invention also relates to a host cell which contains a nucleic acid molecule having the nucleotide sequence shown in SEQ ID No. 3 or a nucleotide sequence capable of hybridizing to or complementary to SEQ ID No. 3 under stringent hybridization conditions in the presence of 0.1-1 × SSC / 0.1% w / w SDS at 60 ° C for 1-3 hours.

Preferably, the host cell according to the invention is characterized in that the nucleic acid molecule comprises a nucleotide sequence selected from SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9 or encodes a polypeptide comprising an amino acid sequence selected from SEQ ID No. 4. SEQ ID No. 6, SeQ ID No. 8, SEQ ID No. 10, or an amino acid sequence having at least 70% similarity to SEQ ID No. 4.

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The invention also relates to a vector which comprises a nucleic acid molecule, a nucleic acid molecule having the sequence shown in SEQ ID No. 3 or a ncklaocide sequence capable of hybridizing to SEQ ID No. 3 or complementary to it under stringent hybridization conditions of 0.1-1. x SSC / 0.1% w / w SDS at 60 ° C for 1-3 hours.

Preferably, the vector according to the invention is characterized in that the nucleic acid molecule comprises a nckleotide sequence selected from SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9 or encodes a polypeptide comprising an amino acid sequence selected from SEQ ID No. 4, SEQ ID NO: 6, SEq ID NO: 8, SEQ ID NO: 10, or an amino acid sequence having at least 70% similarity to SEQ ID NO: 4.

The invention will be more fully illustrated with the aid of the following non-limiting examples and figures:

Figure 1. Cucleotide sequence [SEQ ID No. 1] and the corresponding amino acid sequence [SEQ ID No. 2] of VEGF1 ₆₅ .

Figure 2. The nUclaotide sequence [SEQ ID No. 3] and the corresponding amino acid sequence [SEQ ID No. 4] of SOM175.

Figure 3. Results of a BLAST sequence search of the SOM175 protein.

Figure 4. Alignment of VEGF cDNA and SOM175 cDNA using BESTFIT.

Figure 5. Multiple matching VEGF 6 ₅ with SOM175 and its variants folding at kckleotydó> w.

Figure 6 Multiple matching _VEGF) 6 ₅ with SOM175 and its folding variants at the amino acid level.

Figure 7. Representation of SOM175 and its variants folded as diagrams.

Figure 8 (a). Presentation of the gecko structure of the human SOM175 gecko in the form of a diagram showing the agzók / iktron map.

Figure 8 (b). Diagrammatic representation of the genomic structure of the human ganomic SOM175 showing agsone / ictrok boundaries.

Figure 9. Cucleotide sequences and predicted peptide sequences derived from the mVRF clone cDNA. The nucleotide numbering is given to the left, starting with the A initiation codon. Amino acid numbering is given on the right hand side starting with the first residue of the predicted mature protein after removal of the putative signal peptide. The alternately folded area is double underlined and the resulting peptide sequence from each mRNA has been included. A potential polyiadanylation signal is shown in bold letters. The start and stop codons of mVRFj6 ₇ and mYRF ^ are underlined and the polymorphic repeat in the 3 'UTR is indicated by a dotted box. The positions of the iktrok / exon boundaries are marked with arrows.

Figure 10. Alignment of human and murine VRF protein isoforms. A: mVRFj67 and hYRF ^. B: mVRF _{] 8} 6 and hVRF _{] g} 6, from the point where the sequences begin to differ from the corresponding 167 amino acid isoforms. The identity of the amino acids is marked with vertical lines and the preserved amino acids with a colon. The arrow marks the predicted site for cleavage of the signal peptide from human and mouse VRF.

Figure 11. Alignment of the mVRF167 and mVEGFl _gg peptide sequences (Breier et al., 1992) using BESTFIT. The arrow marks the mVEGF signal peptide cleavage site. Identical amino acids are marked with vertical lines and conservative substitutions with colons. The amino acid numbering used is described in the legend of Figure 9.

Figure 12. Comparison of VRF gene structures (generic VRF gene shown as the ictroc / agsone organization in mouse and human homologs is nearly identical) with other members of the human VEGF / PIGF / PDGF gene family. The exons are framed. Protein coding and untranslated regions are shown as filled and empty, respectively.

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The hatched area in VRF represents an additional 3 'UTR sequence resulting from alternating splicing of the VRF1 ₈₆ isoform. The potential products of the alternating splicing of each gene are shown.

Figure 13. Northern blot autoradiogram of full mRNA from different tissues (marked) of adult mice hybridized with mVRF clone cDNA. The 11 kb main transcript was detected in all samples.

Figure 14. Photographs of autoradiography (A-C) and dark-field micrographs (D-E) illustrating the mVRF and mRNA pattern in mice, in the case of the E14 (A) mouse embryo, positive signals are seen on the developing heart (Ha) and cerebral cortex (Cx). A low background signal is also seen over the other tissues in this section, in the case of the E17 (B) mouse embryo, the heart (Ha) is clearly visible due to the strong hybridization signal. An equally strong signal is found over the brown adipose tissue (Fa) behind and around the chest. A moderate hybridization signal occurs above the spinal cord (SC) and the tongue (T). The background signal is attenuated compared to the E14 embryo. in the case of young adult mice (C-D), positive signals are seen above the heart (Ha) and the adipose tissue (Fa) around the chest, while for example in the lung (Lu) no significance was observed. The hybridization signal over the heart is evenly distributed over the entire left ventricle, including the papillary muscles (D). no positive signals are present for the E17 heart hybridized with an excess of the cold probe (D). Scale bars: 0.5mm (A), 1.2mm (B), 1mm (C), 0.3mm (D), 0.1mm (E).

Figure 15. Dark (A and C) and light field (B and D) micrographs showing mRNA expression of mVFR in mouse adipose tissue (A-B) and spinal cord (C-D). A strong hybridization signal is visible above the fat (A), confirming the strong importance in the Sudanese black stained sections (B). Weak signal is also present in skeletal muscle (M to A-B). in the spinal cord of an adult mouse (C), mVRF probes visualize the neural staining pattern above the gray matter. Toluidine counterstaining showed that the motor neurons in the ventral horn, the insertion neurons in the deep dorsal horn and around the central canal (not shown) were strongly positive for mVRF mRNA. Scale bars: 0.1mm (A), 0.1mm (B), 0.25mm (C), 0.015mm (D).

Figure 16. Effect of VEGF on the sensory neurons of an 8-day-old chicken embryo (E8), defined as% survival,% neurite outgrowth and mean neurite length (pm).

Figure 17. Effect of VEGF and SOM175 on hatchling glial. Central nervous system (CNS) glia, peripheral glia and CNS oligodendrocytes were examined.

Figure 18. Effect of various SOM175 proteins on murine astroglial cells. ³ H (signals / minute)

1. FGF-2 (10 ng / ml) - positive control

2. SOM? C6 * 1 ng / ml

3. SOMAX6 10 ng / ml

4. SOMAX6 100 ng / ml

5. SOMAX6 1000 ng / ml

6. SOMAX6 1000 ng / ml, no heparin

7. SOMX6 ** 1 ng / ml

8. SOMX6 10 ng / ml

9. SOMX6 100 ng / ml

10. SOMX6 1000 ng / ml

11. SOMX6 1000 ng / ml, no heparin * Doyyczy SOM17P absent exon 6 ** Applies to SOM175

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Figure 19. Effect of various SOM175 proteins on murine oligodendroglial cells. ³ H (signals / minute)

1. FGF-2 (10 ng / ml) - positive control

2. SOMAX6 * 1 ng / ml

3. SOMAX6 10 ng / ml

4. SOMAX6 100 ng / ml

5. SOMAX6 1000 ng / ml

6. SOMA 1000 ng / ml, no heparin

7. SOMX6 ** 1 ng / ml

8. SOMX6 10 ng / ml

9. SOMX6 100 ng / ml

10. SOMX6 1000 ng / ml

11. SOMX6 1000 ng / ml, without heparin * Applies to SOM175 with exon 6 absent ** Applies to SOM175

Figure 20. Effect of various SOM175 proteins on mouse forebrain neurons. % survival

1. FGF-2 (10 ng / ml) - positive control

2. SOMAX6 * 1 ng / ml

3. SOMAX6 10 ng / ml

4. SOMAX6 100 ng / ml

5. SOMAX6 1000 ng / ml

6. SOMAX6 1000 ng / ml, no heparin

7. SOMX6 ** 1 ng / ml

8. SOMX6 10 ng / ml

9. SOMX6 100 ng / ml

10. SOMX6 1000 ng / ml

11. SOMX6 1000 ng / ml, without heparin * Applies to SOM175 with exon 6 absent ** Applies to SOM175

Table 1

List of sequence identification numbers

SEQ ID No. 1 VEGF165 nucleotide sequence SEQ ID No. 2 VEGFi65 amino acid sequence SEQ ID No. 3 SOM175 nucleotide sequence (VEGF-like molecules SEQ ID No. 4 SOM175 amino acid sequence SEQ ID No. 5 Nucleotide sequence of SOM175 with exon 6 absent SEQ ID No. 6 Amino acid sequence of SOM175 with exon 6 absent SEQ ID No. 7 Nucleotide sequence of SOM175 with exon 6 and exon 7 absent SEQ ID No. 8 Amino acid sequence of SOM175 with exon 6 and exon 7 absent SEQ ID No. 9 Nucleotide sequence of SOM175 with exon 4 absent SEQ ID No. 10 Amino acid sequence of SOM175 with exon 4 absent SEQ ID No. 11 Oligonucleotide SEQ ID No. 12 Oligonucleotide SEQ ID No. 13 Oligonucleotide SEQ ID No. 14 Oligonucleotide

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Example 1. Human cDNA clones

The original SOM175 cDNA was isolated by selecting the human fetal brain library (/. Zapli, Stratagene) with cosmid D11S750 (Larsson et al., 1992). The plasmid was excised "in v / vo" to yield a single cDNA of 1.1 KB. Three independent SOM175 cDNA clones were also isolated from the human fetal spleen library (Stratagene, Unizap) using the above-mentioned SOM175 insert as probe. 3 clones were obtained: SOM175-4A, -5A and - 6A. SOM 175-4A is an alternating complex clone with exon 4 absent (SOM175-e4). Library selection was performed by performing hybridization under the conditions recommended by the library manufacturer (Stratagene) and randomly labeled SOM175 insert.

Two partial human SOM175 cDNAs were also isolated from the A2058 library of the human λ GT11 melanoma cell line (Clontech). Library selection was performed using the hybridization conditions described by Church and Gilbert (1994). in each case, the probe was generated by random spotting of the PCR product derived from SOM175 (18f-700r).

Mouse cDNA clones

Human SOM175 was also used to select a whole brain cDNA library from newborn mice (Unizap, Stratagene). 4 nonchimeric clones were isolated: M175-A, B, C and D. All clones were partial cDNAs and M175-C contained several introns. 3 of these cDNAs did not contain exon 6.

Another clone, designated M1, was fully sequenced and found to contain a complete open reading frame and a 5'utr part and all 3'utr.

Example 2. DNA sequence analysis

The complete cDNA sequence of the clone (SOM175) was assembled and shown in Figure 2 along with the corresponding amino acid sequence. This sequence was searched for open reading frames using the MAP program (GCG, University of Wisconsin). A single open reading frame of 672 bp was observed (Fig. 2). It turned out to contain small 5'-untranslated sequences (2 bp). The 3'-untranslated region was found to be complete as it contains the polyadenylation signal and the poly-A tail.

Database homology searches were performed using the BLAST algorithm (performed at NCBI, USA). The analysis confirmed homology with a number of human VEGF forms (see Figure 3). The degree of homology between SOM175 and human VEGF ₁₆₅ was determined using the BESTFIT program (CGC, University of Wisconsin, see Figures 4 and 5). The nucleotide homology was estimated to be 69.7%, and the protein homology was assessed as 33.3% identity and 52.5% behavior using BESTFIT analysis. BLAst analysis of the nucleotide sequences confirmed an almost complete alignment with the human expressed EST06302 tag sequence (Adams et al., 1993).

These results indicate that SOM175 codes for a growth factor structurally similar to VEGF. Both genes contain start and stop codons at similar positions and show discrete blocks of homology. All 8 cysteines are preserved as well as a number of other VEGF residues believed to play a role in dimerization. These residues include cysteine-47, proline-70, cysteine-72, valine-74, arginine-77, cysteine-78, glycine-80, cysteine-81, cysteine-82, cysteine-89, proline-91, cysteine- 122 and cysteine-124, as shown in Figure 6, due to the retention of structure in VEGF and the SOM175 gene products, it is also possible that they show similar effects. SOM175 is assumed to encode a VEGF-like molecule that shares some properties with VEGF, but also exhibits specific unique properties. The nucleotide sequence and the corresponding amino acid sequence of VEGF1 _{65 are} shown in Figure 1.

Example 3. Percentage similarity and difference between the VEGF1 ₆₅ family and the SOM175 family (proteins) was analyzed using the Clustal method, MegAlign software, DNSTAR, Wisconsin. The results are presented in Tables 2.1 and 2.2. The alternatively folded forms of SOM175 were shortened to SOM715-e6 in which the entirety of exon 6 was excised; SOM715-e6 and 7,

185,293 in which all exons 6 and 7 were cut out; and SOM175-e4, in which the entirety of exon 4 was cut out. The complex form of SOM175 is shown in Fig. 7. Ganomew maps of SOM175 with the int-on / exon boundaries marked are shown in Figs. 8a and 8b.

Table 2.1

A. Percentage of nucleotide similarity between composite SOM175 variants and human VEGF _{] 65}

vegf ₁₆ 5 SOM175 SOM175-e6 SOM175-e & 7 SOM175-e4 VEGF, 65 * * * 34.9 39.7 41.4 37.0 SOM175 * * * 98.9 95.6 99.2 SOM175-e6 98.8 84.0 SOM175-e6 & 7 * * * 80.3 SOM175-e4 ***

B. Percentage of differences in kuklaetydanh between complex SOM175 variants and human VEGF _I6 5

VEGF, 65 SOM175 SOM175-e6 SOM175-e & 7 SOM175-e4 VEGF, 65 * * * 46.7 41.6 41.7 41.8 SOM175 * * * 0.2 0.2 0.0 SOM175-e6 *** 0.0 0.2 SOM175-e6 & 7 * ** 0.3 SOM175-e4 * * *

Table 2.2

A. Percentage of amino acid identity between composite SOM175 variants and human VEGF ₁₆ 5

VEGF, 65 SOM175 SOM175-e6 SOM175-e & 7 SOM175-e4 VEGF "5 * * * 31.4 42.3 33.5 40.6 SOM175 * * * 74.7 73.7 99.1 SOM675-e6 *** 76.8 99.1 SOM175-e6 & 7 * * * 99.1 SOM175-e4 * * *

B. Percentage of amino acid differences between composite SOM175 variants and human VEGF _I6 5

VEGF, 65 SOM175 SOM175-e6 SOM175-e & 7 SOM175-e4 VEGFj65 * * * 65.7 55.4 54.6 57.4 SOM175 * * * 19.9 4.2 0.0 SOM175-e6 *** 0.0 0.0 SOM175-e6 & 7 * * * 0.0 SOM175-e4 * * *

Example 4. Bioassays to determine the activity of SOM175

Assays were performed to evaluate whether SOM175 exhibited similar activity to VEGF with respect to epithelial cell function, angiogenesis and wound healing. Other tests were performed based on the results of receptor binding degradation studies.

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Test of epithelial cells

Epithelial cell proliferation. Epithelial cell growth tests are described by Ferrara and Henzel (1989) and Gospodarowicz et al. (1989).

Vascular Permeability Test. This test, which uses the Miles guinea pig test, was performed as described by Miles and Miles (1952).

Cell adhesion test. The effect of SOM175 on adhesion of polymorphs to epithelial cells was analyzed.

Chemitaxy. It was performed using a standard Boyden chamber for chemotaxis assay.

Plasminogen activator test. in epithelial cells the plasminogen activator and the production of the plasminogen activator inhibitor were determined after the addition of SOM175 (Pepper et al (1991)).

Epithelial cell migration test. The ability of SOM175 to promote epithelial cell migration and tubular formation was investigated as described by Montesano et al (1986).

Angiogenesis test

The induction of an angiogenic reaction by SOM175 in a hatchling chorionic membrane was investigated as described by Leung et al. (1989).

The possible neurotrophic effects of SOM175 were assessed using the following tests:

Assessment of neurite hypertrophy and gene induction (C12 cells)

P12 cells (phaeochromocytoma cell line) respond to NGF and other neurotrophic factors by developing characteristic sympathetic neurons including early and late gene induction and neurite extension. The cells were exposed to SOM175 and their reaction was followed (Drinkwater et al. (1991) and Drinkwater et al. (1993)).

Cultured neurons from the peripheral nervous system (PNS)

Primary cultures of the following PNS neurons were exposed to SOM175 and monitored for possible reactions:

- sensory neurons from the ganglia of the crest and the posterior root

- sympathetic neurons from sympathetic chain ganglia

- posterior sensory neurons from the lower vagal ganglion

- motor neurons from the spinal cord.

These tests are described by Suter et al. (1992) and Marinou et al. (1992).

When an in vitro reaction is observed, in vivo tests are performed to determine properties such as uptake and retrograde transport as described by Hendry et al. (1992).

Nerve regeneration (PNS)

When the neutrotrophic effect of SOM175 is observed, its possible role in the regeneration of axonotrophied sensory neurons, sympathetic neurons, and motor neurons are analyzed by the methods described by Otto et al. (1989), Yip et al. (1984) and Hendry et al. (1976).

Action of SOM175 on CNS neurons

The ability of SOM175 to support the survival of central nervous system neurons is analyzed by the methods described by Hagg et al. (1992), Williams et al. (1986), Hefti (1986), and Kromer (1987).

Wound healing

The ability of SOM175 to support wound healing is tested with the most reliable models available as described by Schilling et al (1959) and used by Hunt et al (1967).

The hemopoietic system

Various in vitro and in vivo tests are available on specific populations of the haemopoietic system, which are listed below:

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Hematopoietic stem cell test

Mice test. A series of in vitro tests were developed for murine haematopoietic stem cells using FACS purified cells:

(a) Repopulation of mouse haematopoietic progenitor cells

These are cells capable of repopulation in the bone marrow of mice irradiated with a lethal dose, showing the Lin ', Rh ^hl , Ly-6A / E ⁺ , c-kit + phenotype. It is estimated effects on the cells of the test substance itself, or by incubation with multiple factors, and cell proliferation is measured by incorporation of ³ H thymidine.

(b) Late stage hematopoietic stem cells

These are cells that show a relatively low ability to repopulate in the bone marrow, but can produce D13 CFU-S. These are cells with the Lin ', Rh ^hl , Ly6A / E + c-kit + phenotype. The substance to be tested is incubated with these cells for a certain period, after which it is injected into recipients irradiated with a lethal dose and the number of spleen D13 colonies is determined.

(c) Cells enriched in direct ancestry

These are cells that react in vitro to single growth factors, with the phenotype Lin-, Rh ¹¹¹ , Ly-6A / E +, c-kit +. This test shows whether SOM175 can act directly on the haemopoietic parental cells. The substance to be tested is incubated with the cells in the agar and the number of colonies is determined after 7-14 days.

Atherosclerosis. Smooth muscle cells play a key role in the development or initiation of atherosclerosis, requiring their phenotype to be changed from contractile to synthetic. Macrophages, epithelial cells, T cells, and platelets all play a role in the development of atherosclerotic plaques by increasing the modulation of the smooth muscle cell phenotype. An in vitro assay that measures the proliferation rate and modulation of the smooth muscle cell phenotype in a multicellular environment is used to evaluate the effect of SOM175 on smooth muscle cells. The system uses a modified Rose chamber in which different types of cells are seeded onto opposing coverslips.

Effect of SOM175 on bones. The ability of SOM175 to regulate osteoblast proliferation is assessed as described by Lowe et al. (1991). Any effect on bone resorption is assessed as described by Lowe et al. (1991). The effects on osteoblast migration and changes in intracellular molecules (e.g., cAMP accumulation. Alkaline phosphatase levels) are analyzed as described by Midy et al. (1994).

Effect on the cells of the skeletal muscle The effect of SOM175 on myoblast proliferation and myotubule development can be determined as described by Ewton et al (1980) and Gospodarowicz et al (1976).

Example 5. Cloning of murine VEGF DNA

Secretion of cDNA. Mouse VRF clones (mVRF) were selected from whole brain neonatal cDNA libraries / .zap (Stratagene). Primary phage from the high-density filters (5x10 ⁴ pfu / plate) were identified by hybridization with a ³² P-labeled 628 bp probe made by PCR with the hVRF cDNA (pSOM175) as described above. Hybridization and stringency washing of the polyamide (Hybond-N) membranes were performed at 65 ° C under the conditions described by Church and Gilbert (1984). Positive plaques were isolated, purified and excised in vivo to yield bacterial colonies containing the cDNA clones in pBluescript SK-.

Secretion of genomic clones. Genomic clones were isolated from the murine szjZbpuSV / 129 library cloned in the X Fix II vector (Stratagene). High density filters (5 x 10 ⁴ pfu / filter) were selected ³² P-labeled 563 bp probe made by PCR amplification of the nucleotide 233-798 region of the cDNA mVRF (see Fig. 9). Positive clones were harvested with a tampon and re-selected using filters containing 400-800 pfu. Large scale phage preparations were obtained using the QUIAGEN X kit or by purification with ZnCl ₂ (Santos, 1991).

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Sequencing and nucleotide analysis. cDNA was sequenced in both strands with a variety of vector-based and internal primers using dye terminator sequencing kits from Applied Biosystems, Incorporated (ABI) according to the manufacturer's specifications. Sequences were analyzed on a tequeneee Model 373A robotic DNA apparatus from ABI. Peptide homology matching was performed using the BESTFIT program (GCG, Wisconsm).

Identification of ictroc / exot boundaries. Identification of exon boundaries and flanking areas was performed by PCR with mouse genomic DNA or with genomic XX mVFR clones as templates. The primers used in the PCR to identify introns were derived from the hVRF sequence, and to allow for potential hybridization of mismatched human-mouse sequences, the reaction was run at 5-10 ° C lower than the estimated T All PCR products were size separated by agarose gel electrophoresis and were gel purified using QIAquick fast spin columns (Quiagen) and mtron / exon boundaries were sequenced directly from these products. In addition, some splicing junctions were sequenced from tubcloned genomic MVRF fragments. Intron / exon boundaries were identified by comparing the cDNA with genomic cDNA sequences.

Northern analysis. Total cellular RNA was obtained from a set of fresh tissues of normal adult mice (brain, kidney, liver, muscle) using the method of Chomezyctky and SacchRego (1987). 20 µg of total RNA was electrophoresed, transferred to a polyamide membrane (Hybond N, Amersham) and hybridized under standard conditions (Church and Gilbert, 1984). Filters were washed at 65 ° C in 0.1 × SsC (20 × SSC in 3M NaCl / O, 3M trisodium citrate), 0.1% SDS, followed by exposure to X-ray film with intensifying screens at -70 ° C for 1-3 days.

Characterization of the mVRF cDNA. Mouse mVRF homologues were isolated by selecting the murine cDNA library with the hVRF cDNA clone. 5 clones ranging in size from 0.8-1.5 kb were recovered and subsequently sequenced. The cDNA sequences were assembled to yield a full-length 1041 bp cDNA sequence spanning the entire open reading frame (621 bp or 564 bp, depending on assembly, see below) and a 3 'UTR (379 bp), and 163 bp of the 5' UTR ( Fig. 9).

The predicted initiation codon matched the position of the start codon in the hVRF. Another codon from the ATG frame was located at position -47, and two stop codons were observed upstream (at positions -9 and -33, respectively) and in-frame with the putative initiation codon.

It turned out that the predicted N-terminal signal peptide from hVRF is present in mVRF with 81% identity (amino acids 17/21). The peptide cleavage in mVRF is expected to occur after residue 21 (Figure 10). These data suggest that mature mVRF is excreted and therefore it is not excluded that it may act as a growth factor.

As in hVRF, two open reading frames (ORFs) were also detected in cDNAs isolated by library selection. 4 or 5 clones were found to be alternating clones lacking the 101 bp fragment homologous to exon 6 in hVRF. The predicted peptide sequences of the two mVRF isoforms were determined and then aligned with the corresponding human isoforms (Figure 10).

The mVRF coding information ₈₆ contains an ORF of 621 bp with the coding sequences ending at position +622, towards the end of exon 7 (Fig. 9). The smaller mVR ^ 167 coding information actually terminates downstream of the +622 TAG site due to the frame shift due to exon 6 ^^ emments by 101 bp and the introduction of a stop codon at position +666 near the start of exon 8 (Figure 9) .

The VRFj86 protein shows strong homology to the amine and middle parts of VEGF, while the carboxyl terminus is quite different and is enriched in alanine. mVRF _{] 8} 6 shares these similarities, and also retains homology from mVEGF to the right to the C-terminus (Figure 11). The overall homology of mYRF ^ with hVRF167 was 85%

185,293 identity and 92% similarity (Fig. 10). In contrast, the homology between mVRF1 ₆₇ and VEGF (Breier et al., 1992) comprised 49% identity and 71% conservative amino acid substitutions, respectively (Fig. 11).

The canonical vertebrate polyadenylation signal (AATAAA) (Bimstiel et al., 1986) was not present in the mVRF cDNA, while the very similar sequence of GATAAA is found at similar positions in both the mouse and human VRF cDNA (Figure 9). In contrast to hVRF, mVRF was found to have an AC dinucleotide repeat at the very 3 'end of the 3' UTR (nucleotide positions 998 to 1011, Figure 9). Polymorphism of this repeat region has been observed in certain mVRF cDNAs with dinucleotide numbers ranging from 7 to 11.

Genomic characterization of mVRF. Intron / exon boundaries (Table 3) were mapped using primers that flank sequences homologous with the corresponding hVRF boundaries. The introns I, III, IV and VI of the mVRF (Table 3, Figure 12) were smaller than the hVRF intron sequences. The complete genomic sequence was assembled from the 5 'UTR from the mVRF to intron VI, the largest intron region (2.2 kb), by sequencing the amplified introns and cloned genomic portions of the mVRF. There was only one significant difference between mVRF and hVRF in the genomic structure that the exon 7 / intron VI boundary in mVRF was located 10 bp downstream of the cDNA sequence such that exon 7 in mVRF was 10 bp further downstream. bp longer than the corresponding exon in the hVRF.

Exons 6 and 7 are adjacent to the mVRF, similar to the human homologue. The strong homology of the sequence of exon 6 and mVRF hVRF (Fig. 10) suggests that this sequence is not a retained intronic sequence but rather encodes a functional part VRF ₁₈ 6 isoforms.

The overall intron / exon structure is conserved in the various members of the VEGF gene family (VEGF, PIGH, hVRF) and therefore it is not surprising that the overall genomic organization of the mVRF gene is very similar to the organization of these genes (Figure 12).

Previously performed comparative mappings have shown that the area surrounding the human disease locus of numerous type 1 endocrine system tumors on chromosome 19 is synthetic with a close segment of the mouse chromosome 19 (Rochelle et al., 1992). since the inventors mapped the hVRF gene at the 1 kb human MEN1 locus (see above), it is very likely that the mouse VRF gene maps to the centromere of chromosome 19.

Study of mVRF expression. Northem RNA analysis from adult mouse tissue (muscle, heart, lung and liver) showed that the expression is very widespread and manifests itself primarily as a major band of approximately 1.3 kb (Figure 14). This differs somewhat from that observed in hVRF, where two major bands of 2.0 and 5.5 kb were identified in all tissues examined. The 1.3 kb mouse information probably corresponds to the shorter of the human transcripts and the different size is most likely due to the difference in length of the respective 5 'UTRs.

Example 6. Expression of mouse VEGF in unborn and unborn mice

Animals. Pregnant (n = 4) and young adult (n-2) mice (C57 inbred, ALAB, Sweden) were sacrificed with carbon dioxide, then the appropriate tissues were harvested and frozen in medium. Tissues were kept at 70 ° C before further use, mice of two different gestational ages were used in the studies: embryonic day 8 * E8), 14 and E17.

Histochemistry of in situ hybridization

In situ hybridization was performed as previously described (Dagerlind et al., 1992). briefly, transverse sections (14 µm) were cut in a cryostat (Mierom, Germany), thawed on Probe-On slides (Fisher Scientific, USA) and stored in closed black boxes at -70 ° C until use. The sequences of the synthetic 42-mer oligonucleotides complementary to the mRNA encoding the mVRF were as follows:

ACCACCACCTCCCTGGGCTGGCATGTGGCACGTGCATAAACG [SEQ ID No. 11] (complementary to nt 120-161) and

AGTTGTTTGACCACATTGCCCATGAGTTCCATGCTCAGAGGC [SEQ ID No. 12]

185 293 (complementary to nt 162-203). An oligonucleotide was used to detect the two variant folding forms

GATCCTGGGGCTGGAGTGGGATGGATGATGTCAGCTGG [SEQ ID No.13] (complementary to ny xxx-xxx) and

GCGGGCAGAGGATCCTGGGGCTGTCTGGCCTCACAGCACT [SEQ ID No. 14]. The probes were labeled at the 3 'end [thio] triphosphate, deoxyadenosine ^[35 S] (NEN, USA) using terminal deoxynucleotidyl transferase (IBI, USA) to a specific activity of 7-10 x 10 ⁸ pulses per second / pg, and then hybridized with sections without pretreatment for 16-18 hours at 42 ° C. The hybridization mixture was 50 vol. formamide, 4 x SSC (1 x SSC = 0.15 M NaCl and 0.015 M sodium citrate), 1 x Denhardt's solution (0.02% polyvinylpyrrolidone, BSA and Ficoll each), 1 vol. sarcosyl (N-lauryl sarcosine, Sigma), 0.02 M phosphate buffer (pH 7.0), 10% w / v. dextrin sulfate (Pharmacia, Sweden), 250 pg / ml yeast tRNA (Sigma), 500 pg / ml DNA of grated and heat denatured salmon semen and 200 mM dithiothreitol (DTT, LKB, Sweden), in the control sections the specificity of both probes was checked by adding 20 - fold excess of unlabelled probe to the hybridization mixture. In addition, adjacent sections were hybridized with a probe unrelated to the sections used in this study, resulting in a different expression pattern. After hybridization, the sections were washed several times in 1x SSC at 55 ° C, dehydrated with ethanol and immersed in an emulsion for detection of NTB2 radioactivity (Kodak, USA). After 3-5 weeks, sections were developed in a D-19 developer (Kodak, USA) and covered with a coverslip, in some cases sections were streaked onto an autoradiographic film (Beta-max autoradiography film from Amersham Ltd., UK) prior to immersion in the emulsion .

different probes gave identical hybridization images in all examined tissues. The expression of mouse VRF was already detected in embryo E8, in which a positive signal was recorded over the structure most likely corresponding to the neural tube, in the sagittal sections of the mouse embryo E14 the strongest hybridization signal was found over the heart and in the nervous system, especially in the cerebral cortex (Fig. 14A). Low expression levels were found in all other tissues, in later gestational age, E17, high mVRF mRNA signal was attributed to the heart and brown adipose tissue in the back and around the neck (Fig. 14B). Clearly positive hybridization signals were present in the gray matter of the spinal cord and in the tongue (Fig. 14B). Expression in the cerebral cortex was clearly weaker than on day 14. The weak background expression observed in embryo E14, eg in muscle, decreased at this gestational age. The strong mRNA hybridization signal of mVRF in young adult mice was present only over the heart and in brown adipose tissue (Fig. 14C). The signal above the heart was evenly distributed throughout the wall of the atrium, including the papillary muscles (Fig. 14D). No specific labeling above background level was detected in sections of cardiac tissue hybridized with an excess of cold probe (Fig. 14E).

Outside the heart, mVRF mRNA signal was present in certain tissues outside the chest which morphologically resemble brown fat. This was confirmed by counterstaining with Sudan Black, which showed strong staining in the same areas (Figures 15A and 15B). in the transverse sections of the spinal cord of an adult mouse, mVRF probes show a neural pattern of staining above the gray matter (Fig. 15C). Toluidine counterstaining (Fig. 15D) showed that motor neurons in the ventral horn (Figs. 15C and 15D), insertion neurons (Fig. 15C) in the deep dorsal horn and around the central canal (not shown) were strongly positive for mRNA. mVRF.

Example 7. Effect of VEGF and SOM175 proteins on chicken sensory neurons

The effect of VEGF and SOM175 proteins on the sensory neurons of an 8-day-old chicken embryo was determined using the method of Nurcombre et al. (1992). The neuronal test was read after 48 hours using 2000 cells / well. Results were obtained by counting ³ H-thymidine. Percentage of neuron survival, neurite outgrowth, and average length

185,293 neurites in pm were determined using NGF as a positive control and VEGF at various concentrations, VEGF in the presence of heparin and VEGF in the absence of heparin and 5 pM 5'-fluoroureeyl (5FU). 5FU kills the glial cells.

The results are shown in Figure 16. These results indicate that VEGF is effective in maintaining neuronal survival, however, it requires the presence of glial cells. Fig. 17 shows the results of the effects of VEGF and SOM175 on 3 types of chicken glial. CNS glias, peripheral glazes and CNS oligodendrocytes were examined. Heparin was used at a concentration of 10 pg / ml in all cultures, and the test was read after 24 hours. Results were determined as counts ³ H-thymidine incorporation using a 2000 cells / well.

The results indicated that for central and peripheral chicken neurons, astroglia proliferation was significantly stimulated by SOM175 in the presence of heparin, while for chicken oligodacdronites a minimal increase in the rate of division was observed.

Example 8. Effect of SOM175 proteins on major and central neurons

The results obtained in Example 7 indicate that the VEGF isoform exerts an effect on major and central chicken neurons by acting on astroglial cells. Similar experiments were repeated on mouse cells.

Breeding conditions

The neurocal and glial cells for all in vitro experiments were prepared and cultured according to the techniques described in "Methods in Neurosniannes (Vol. 2): Cell Culture", edited by P.M. Conn, Academin Press, San Diego, 1990, pp. 33-46 for astroglial cells, pp. 56-74 for oligodendroglial cells and 87-102 for central neurons.

Cells were seeded in 24-well culture plates (Nunc) coated with poly-L-ammonium (0.1 mg / ml, 1 hour) at 2000 cells / well. After culturing for 48 hours, neurons were counted in the wells using reverse phase light using well known techniques (Maruta et al., 1993), and glial cells were assessed by [3 H] thymidine uptake to monitor the rate of cell division as described. below. Heparin (10 pg / ml, low molecular weight fraction. Sigma Chemical Corp.) was present in the culture in all cases with the exceptions noted. Keurocal cultures were supplemented with 5 mM 5-fluoro-2-deoxyuridine (Sigma) to inhibit background glial growth.

Study of the influence of 3H-thymidine introduction on the proliferation of glial cells

Cells were pulsed for 14 hours with 3H-thymidine (specific activity 103 pCi / µg) as a stock solution at 0.1 mCi / ml in standard medium, resulting in a final incubation volume of 20 µl / well. The contents of the wells were collected and absorbed onto nitrocellulose paper (Titertek, Flow). Remaining stuck cells were removed by 5-minute incubation with trypsin / verse (CLS Limited, Victoria, Australia). This procedure was repeated twice. The nitrocellulose discs were washed in a standard Titertek cell harvester (Flow) using first distilled water and then methanol. The nitrocellulose discs were dried, scintillation fluid (containing 5% v / v of Triton-N agent) was added and the discs were counted in a scintillation counter.

The greatest activity was observed for the SOM175 preparations without exon 6 of SOMaX6 in cultures of murine astroglial cells, where there was a significant stimulation of their proliferation when using the preparations in combination with heparin (Fig. 16). There was little stimulation of proliferation with oligodendroglial cells (Fig. 17), and very little enhancement of survival of isolated forebrain neurons (Fig. 18). Standard deviation in all three graphs for each point was less than 8%.

The viability of neurons can be maintained by promoting the proliferation of glial cells. Furthermore, SOMAX6 is a good inducer of astroglial proliferation and can be expressed in conjunction with the formation of astroglial end plates on the epithelial cells of the central carousal system.

185 293

It will be understood by those skilled in the art that changes and modifications other than those specifically described may be made to the described invention. It should be understood that all such changes and modifications are included in the invention. The invention also includes within its scope all of the steps, features, compositions and compounds referred to or indicated in the specification, individually or in combination, and each and all combinations of two or more of such steps or features.

Table 3

Splice junctions of the mouse VRF gene

5 'UTR ... Exon 1 > 223bp CCCAGgtacgtgcgt Intron I 495bp ttccccacagGCCC Exon 2 43bp GAAAGgtaataaatag Intron II 288bp ctgcccacagTGGTG Exon 3 197bp TGCAGgtaccagggc Intron III 196bp ctgagcacagATCCT Exon 4 74bp TGCAGgtgccagccc Intron IV 182bp ctcttttcagACCTA Exon 5 36bp GACAGattcttggtg Intron V 191bp ctcctcctagGGTTG Exon 6 101bp (no intron) CCCACTCCAGCCCCA Exon 7 135bp TGTAGgtaaggagtc Intron VI ~ 2200bp cactccccagGTGGC Exon 8 394bp AGAGATGGAGACACT

Uppercase and lowercase letters represent exon and intron sequences respectively. * Indicates that the 5 'end of exon 1 has not yet been determined.

185 293

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185 293

Sequence overview (1) General information:

(1) Applicants:

(countries other than the US): AMRAD OPERATIONS PTY. LTD.

(USA only): N. Hayward, G. Weber (ii) Title of invention: New growth factor and its genetic sequence (iii) Number of sequences: 14 (iv) Correspondence address:

(A) Addressee: Davies Collison Cave (b) Street: 1 Little Collins Street (C) City: Melbourne (D) State: Victoria (E) Country: Australia (F) Code: 3000 (v) Computer-readable form:

(A) Media: Floppy disk (b) Computer: IBM PC compatible (c) Operating system: PC-DOS / MS-DOS (D) Software: Patent Release # 1.0, Version # 1.25 (vi) Details of this application:

(A) Filing Number: PCT International (b) Filing Date: February 22, 1996 (vii) Previous Filing Details:

(A) Filing number: AU PN 1457 (B) Filing date: March 2, 1995 (A) Filing number: AU PN6647 (B) Filing date: November 20, 1995 (A) Filing number: AU PN7274 (B) Filing date: 22 December 1995 (viii) Information about the attorney / advocate:

(A) Name: Dr. E. John L. Hughes (C) Registration number: EJH / EK (ix) Ιηί'οπηΗφι (A) Telephone: +61 3 9254 2777 (B) Fax: +61 3 9254 2770 (2) Information on SEQ ID NO: 1:

(i) Characteristics of the sequence:

(A) Length: 649 base pairs (B) Type: nucleic acid (C) Stranded: single (D) Topology: linear (ii) Molecular type: DNA (ix) Feature:

(A) Name / key: CDS (b) Location: 17 ... 589 (xi) Sequence description: SEQ ID NO: l:

185 293

TCGGGCCTCC GAAACC ATG AAC TTT CTG CTG TCT TGG GTG CAT TGG AGC Met Asn Phe Leu Leu Ser Trp Val His Trp Ser

10

CTT GCC TTG CTG CTC TAC CTC CAC CAT GCC AAG TGG TCC CAG GCT GCA Leu Ala Leu Leu Leu Tyr Leu His His Ala Lys Trp Gin Ala Ala cheese 15 twenty 25 CCC ATG GCA GAA GGA GGA GGG CAG AAT CAT CAC GAA GTG GTG AAG TTC Pro Underworld Ala Glu Gly Gly Gly Gin Asn His His Glu Val Val Lys Phe thirty 35 40 ATG GAT GTC TAT CAG CGC AGC TAC TGC CAT CCA ATC GAG ACC CTG GTG Underworld Asp Val Tyr Gin Arg Cheese Tyr Cys His Pro Ile Glu Thr Leu Val 45 50 55 GAC ATC TTC CAG GAG TAC CCT GAT GAG ATC GAG TAC ATC TTC AAG CCA Asp How much Phe Gin Glu Tyr Pro Asp Glu Ile Glu Tyr How Much Phe Lys Pro 60 65 70 75 TCC TGT GTG CCC CTG ATG CGA TGC GGG GGC TGC TGC, AAT GAC GAG GGC Cheese Cys Val Pro Leu Underworld Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly 80 85 90 CTG GAG TGT GTG CCC ACT GAG GAG TCC AAC ATC ACC ATG CAG ATT ATG Leu Glu Cys Val Pro Thr Glu Glu Asn Ile Thr Met Gin Ile Met 95 100 105 CGG ATC AAA CCT CAC CAA GGC CAG i CAC ATA GGA GAG ATG AGC TTC CTA Arg How much Lys Pro His Gin Gly Gin His Ile Gly Glu Met Cheese Phe Leu 110 115 120

5

193

241

289

33?

3Θ5

CAG CAC AAC AAA TGT GAA TGC AGA CCA AAG AAA GAT AGA GCA AGA CAA Gin His Asn Lys Cys Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gin

125 130 135

GAA AAT CCC TGT GGG CCT TGC TCA GAG CGG AGA AAG CAT TTG TTT GTA Glu Asn Pro Cys Gly Pro Cys Ser Glu Arg Arg Lys His Leu Phe Val 140 145 ISO 155

CAA GAT CCG CAG ACG TGT AAA TGT TCC TGC AAA AAC ACA GAC TCG CGT Gin Asp Pro Gin Thr Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser Arg

160 165 170

TGC AAG GCG AGG CAG CTT GAG TTA AAC GAA CGT ACT TGC AGA TGT GAC Cys Lys Ala Arg Gin Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys Asp

175 180 185

AAG CCG AGG CGG TGAGCCGGGC AGGAGGAAGG AGCCTCCCTC AGCGTTTCGG Lys Pro Arg Arg

190

433

481

529

577

629

GAACCAGATC T.CTCACCAGG

649

185 293 (2) Information on SEQ ID NO: 2:

(i) Characteristics of the sequence:

(A) Length: 191 amino acids (B) Type: amino acid (D) Topology: linear (ii) Molecular type: protein (xi) Sequence description: SEQ ID NO: 2:

Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu 15 10 15

Tyr Leu His His Ala Lys Trp Ser Gin Ala Ala Pro Met Ala Glu Gly 20 25 30

Gly Gly Gin Asn His His Glu Val Val Lys Phe Met Asp Val Tyr Gin 35 40 45

Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe Gin Glu 50 55 60

Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Cheese Cys Val Pro Leu

70 75 80

Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro

90 95

Thr Glu Glu : Ser 100 Asn , How much Thr Underworld Gin 105 How much ¹ Met Arg How much Lys 110 Pro His Gin Gly Gin 115 His How much Gly Glu Underworld 120 Cheese Phe Leu Gin His 125 Asn Lys Cys Glu Cys 130 Arg Pro Lys Lys Asp 135 Arg Ala Arg Gin Glu 140 Asn Pro Cys Gly Pro 145 Cys Cheese Glu Arg Arg 150 Lys His Leu Phe Val 155 Gin Asp Pro Gin Thr 160 Cys Lys Cys Cheese Cys 165 Lys Asn Tnr Asp Cheese 170 Arg Cys Lys Ala Arg 175 Gin Leu Glu Leu Asn 180 Glu Arg Thr Cys Arg 185 Cys Asp Lys Pro Arg 190 Arg

185 293 (2) Information about SEQ ID NO: 3:

(i) Characteristics of the sequence:

(A) Length: 1094 base pairs (B) Type: nucleic acid (C) Linearity: single (D) Topology: linear (ii) Molecule type: DNA (ix) Feature:

(A) Name / key: CDS (b) Location: 3 ... 624 (xi) Sequence description: SEQ ID NO: 3:

CC ATG AGC CCT CTG CTC CGC CGC CTG CTG CTC GCC GCA CTC CTG CAG 47

Met Ser Pro Leu Leu Arg Arg Leu Leu Leu Ala Ala Leu Leu Gln

10 15

CTG GCC CCC GCC CAG GCC CCT GTC TCC CAG CCT GAT GCC CCT GGC CAC 95 Leu Ala Pro Ala Main Ala Pro Val Cheese Main Pro Asp Ala Pro Gly His twenty 25 thirty CAG AGG AAA GTG GTG TCA TGG ATA GAT GTG ' TAT ACT CGC GCT ACC TGC 143 Main Arg Lys Val Val Cheese Trp How much Asp Val Tyr Thr Arg Ala Thr Cys 35 40 45

CAG CCC CGG GAG GTG GTG GTG CCC TTG ACT GTG GAG CTC ATG GGC ACC 191.

Gln Pro Arg Glu Val Val Val Pro Leu Thr Val Glu Leu Met Gly Thr

55 60

GTG GCC AAA CAG CTG GTG CCC AGC TGC GTG ACT GTG CAG CGC TGT GGT 239

Val Ala Lys Gln Leu Val Pro Ser Cys Val Thr Val Gln Arg Cys Gly

70 75

GGC TGC TGC CCT GAC GAT GGC CTG GAG TGT GTG CCC ACT GGG CAG CAC 237

Gly Cys Cys Pro Asp Asp Gly Leu Glu Cys Val Pro Thr Gly Gln His

75 90 95

CAA GTC CCG7 ATG CIAG ATC CTC ATG ATC CGG TAC CCG AGC AGT CAG CTG 3 3 3

Gln Val Arg Met Gln Ile Leu Met Ile .Arg Tyr Pro Cheese Ser Gln Leu

900 10t 110

GGG GAG ATT CCC CTG CGA CGA CCAC AGC CAG TGT GAA TGC AGA CCT AAA 3 9 7

Gly Glu Mmi tse Leu Glu Glu His Ser Gln Cys Glu Cys Arg Pro Lys

995 920 935

AAA AAG GAC AGT GCT GTG AAAG CCA CGAC ACG GCT GCC ACT CCC CCAC CAC 443. Lys Lys Asp tse Ala Val Lys Pro Asp Arg AHa AHa Thr Pro His His 930 935 940 CGT CCC CAC GCC CGT TTCT GGC CCG CGC TTG CAC CCT CCC CCC CGA GCA 479 Arg Pro Main P ro Arrg Cheese Val Pro Gly Trp Asp Ser AHa Pro Gly AHa 945 950 9S5 CCC TCC CCA GCT GAC ATC ACC CAT, CCC ACT CCA GCC CCA GGC CCC TCT 557 Pro Cheese Pro AHa Assp I1c Thr His PrC Thr Pro AH. a rro Gly Pro Ssi

960 16t 970 197

GCC CAC GCT GCA CCC AGC ACC ACC AGC GCC CTG ACC CCC GGA CCT GCC 5 * 7 5

Ala His AALa Ala Pro Ser Thr Thr Ser ALa Leu Thr Pro Gly Pro Ala

170 18C 190

185 293

GCT GCC GCT GCC GAC GCC GCA GCT TCC TCC GTT GCC AAG GGC GGG GCT T 624 Ala Ala Ala Ala Asp Ala Ala Ala Ser Ser Val Ala Lys Gly Gly Ala

195 200 205

AGAGCTCAAC CCAGACACCT GCAGGTGCCG GAAGCTGCGA AGGTGACACA TGGCTTTTCA 684

GACTCAGCAG GGTGACTTGC CTCAGAGGCT ATATCCCAGT GGGGGAACAA AGGGGAGCCT 744

GGTAAAAAAC AGCCAAGCCC CCAAGACCTC AGCCCAGGCA GAAGCTGCTC TAGGACCTGG B04

GCCTCTCAGA GGGCTCTTCT GCCATCCCTT GTCTCCCTGA GGCCATCATC AAACAGGACA 864

GAGTTGGAAG AGGAGACTGG GAGGCAGCAA GAGGGGTCAC ATACCAGCTC AGGGGAGAAT 924

GGAGTACTGT CTCAGTTTCT AACCACTCTG TGCAAGTAAG CATCTTACAA CTGGCTCTTC 984

CTCCCCTCAC TAAGAAGACC CAAACCTCTG CATAATGGGA TTTGGGCTTT GGTACAAGAA 1044

CTGTGACCCC CAACCCTGAT AAAAGAGATG GAAGGAAAAA AAAAAAAAAA 1094 (2) Information on SEQ ID N0: 4:

(i) Characteristics of the sequence:

(A) Length: 207 amino acids (B) Type: amino acid (D) Topology: linear (ii) Molecular type: protein (xi) Sequence description: SEQ ID NO: 4:

Met Ser Pro Leu Leu Arg Arg Leu Leu Leu [Ala l Ala Leu Leu and Gln Leu 1 5 10 15 Ala Pro Ala Gin All And after Vs1 Cheese Main Pro Asp Alu Pro Gly His Gin twenty 25 thirty Arg Lys Val val Cheese STp How much Asp Val Tyr Thr Arg Ala TTr Vcs on S5 40 45 Pro Arg Glu val Val Val Pro Leu Thr Val du Leu Underworld Gly Tho Val 50 55 60 Ala Lys Main Leu Val Pro S er CCs Val Thr Val Main Arg Cyy Ul for 65 70 77 80 Cys Cys Pro Asp Asp G ly Lsu for Syy name Po Tho Gly Gin Hii Gin 85 90 95 Val JAg Underworld Min How much Irnu Underworld How much Arg Tyv Pao Cheese Cheese Glo Seu d v 100 105 110 Glu Underworld Cheese Igo Glu Glu His Cheese Gin Cys GSg Sys all sso Lys lys 115 120 125

Lys Αυρ Ser Ala Val Lys Prr Asp Arg W. a Ala Thr Pro His His Aro 130 11S 140

185 293

Pro 145 Main Pro Arg Cheese Val 150 Pro Gly Trp Asp Cheese 155 AVa Pro Gly A. a Pro 160 Cheese Pro Ala Asp How much 16S Thr His Pro Thr Pro 170 ALa Pro Gly Pro Cheese 17 5 Ala His Ala Ala Pro 180 Cheese Tir Tlhr Cheese JA.a 115 Leu Tlhr Pro Gly Pro 190 ALs ALa Ala Ala AL a Asp Ala ALa ALa Cheese Cheese Val ALa Lys Gly Gly Ala

195 200 205 (2) Information regarding SEQ ID NO: 5:

(i) Characteristics of the sequence:

(A) Length: 993 base pairs (B) Type: nucleic acid (C) Strandedness: single (D) Topology: linear (ii) Type of molecule: DNA (ix) Feature:

(A) Name / key: CDS (b) Location: 3 ... 566 (xi) Sequence description: SEQ ID NO: 5:

CC ATG AGC CCT CTG CTC CGC CGC CTG CTG CTC GCC GCA CTC CTG CAG n Underworld Cheese Pro Leu Leu Arg Arg Leu Leu Leu Ala Ala Leu Leu Gln 1 5 10 15 CTG GCC CCC GCC CAG GCC CCT GTC TCC CAG CCT GAT GCC CCT GGC CAC 95 Leu Ala Pro Ala Main . Ala , Pro Val Ser Gln Pro Asp Ala Pro Gly His twenty 25 30 CAG AGG AAA GTG GTG TCA TGG ATA GAT GTG TAT ACT CGC GCT ACC TGC 143 Main Arg Lys Val Val Ser Tjp> Ile Asp Val Tyr ΤΓ Arg Ala Thr Cys 35 40 45 CAG CCC CGG GAG GTG GTG GTG CCC TTG ACT GTG GAG CTC ATG GGC ACC 111 Main Pro Arg Glu Val Val Val Pro Leu Thr Val Glu Leu Met Gly Thr 50 55 60 GTG GCC AAA CAG CTG GTG CCC AGC TGC GTG ACT GTG CAG CGC TGT GGT 23 9 Val Ala Lys Main Leu Val Pro Ser Cys Val Thr Val Gln Taoj SCys Gly 65 77 77 GGC TGC TGC CCT GAC GAT GGC CTG GAG TGT GTG CCC ACT GGG CAG CAC 280 Gly Cys Cys Pro Asp Asp Gly Leu Glu Cys Val Poo TIot Gly Gln His 80 85 90 95 CAA GTC CGG ATG CAG ATC CTC ATG ATC CGG TAC CCG AGC AGT CAG CTG 333 Main Val JArg Mes Main Nov Leu Met ISe leg fig Pro res Sos O1v Leu 100 1105 HO GGG GAG ATG TCC CTG GAA GAA CAC AGC CAG TGT GAA TGC AGA CCT AAA 333 Gly Glu Underworld Cheese Ie * u Glu Glu His Ser Gln Cys Glu Cys ŁArg Spo Lys 115 120 112 AAA AAG GAC AGT GCT GTG AAG CCA GAT AGC CCC AGG CCC CTC TGC CCA 431 Lys Lys JAp See Ala VaS Lys Sro LU »p Arg ProLeu Cys Pro cheese 130 13S 140

185 293

CGC TGC ACC CAG CAC CAC CAG CGC CCT GAC CCC CGG ACC TGC CGC TGC 4 7 9

Arg Cys Thr Gln His His Gln Arg Pro Asp Pro Arg Thr Cys Arg Cys

145 150 155

CGC TGC CGA CGC CGC AGC TTC CTC CGT TGC CAA GGG CGG GGC TTA GAG 52 7

Arg Cys Arg Arg Arg Ser Phe Leu Arg Cys Gln Gly Arg Gly Leu Glu 160 165 170 175

CTC AAC CCA GAC ACC TGC AGG TGC CGG AAG CTG CGA AGG TGACACATGG 557

Leu Asn Pro Asp Thr Cys Arg Cys Arg Lys Leu Arg Arg

180 1S5

CTTTTCAGAC TCAGCAGGGT GACTTGCCTC AGAGGCTATA TCCCAGTGGG GGAACAAAGG 636

GGAGCCTGGT AAAAAACAGC CAAGCCCCCA AGACCTCAGC CCAGGCAGAA GCTGCTCTAG 639

GACCTGGGCC TCTCAGAGGG CTCTTCTGCC ATCCCTTGTC TCCCTGAGGC CATCATCAAA 776

CAGGACAGAG TTGGAAGAGG AGACTGGGAG GCAGCAAGAG GGGTCACATA CCAGCTCAGG 811

GGAGAATGGA GTACTGTCTC AGTTTCTAAC CACTCTGTGC AAGTAAGCAT CTTACAACTG 0 0 7

GCTCTTCCTC CCCTCACTAA GAAGACCCAA ACCTCTGCAT AATGGGATTT GGGCTTTGGT 996

ACAAGAACTG TGACCCCCAA CCCTGATAAA AGAGATGGAA GGAAAAAAAA AAAAAAA 999 (2) Information on SEQ ID NO: 6:

(i) Characteristics of the sequence:

(A) Length: 188 amino acids (B) Type: amino acid (D) Topology: linear (ii) Molecular type: protein (xi) Sequence description: SEQ ID NO: 6:

Underworld See Pro Leu How many Arr Arg Seu Se »u Lsu Gal Gla Leu Leu Main Llu 1 5 11 15 Ala Prp Ala Main Ala Pro Val Cheese Main Pro ASp KLa Drank Gly His Main twenty 25 thirty Arg Lys Val Gal Cheese Tir? How much Val Tyr Tir JArg GAL a Thr Main 35 40 40 Pro Arg Gla Val Val Val Pro Leu Thr Val Glu Leu Underworld Gly Thr Val 50 55 60 Ala Lys Main lan Val Vaa corner ce ^ s Val Thr Val hla lrg AIb Gly Gly 65 70 75 80

Cys Cys Pro Asp Asp Gly Leu Glu Cys Val Pro Thr Gly Gln His Gln 85 99 95

Val Arg Met Gln Ile Leu Met liis Arg T / r Pro Ser Ser Gln Leu dy 100 105 110

185 293

Glu Underworld Cheese Leu Glu . Gl s HZ p Se r Gin Cys Glu Cys ZArg Pos Lss Lys 115 120 115 Lys JAp Cheese FLOWS Val Lys Pro Asp Cheese Pro phage Pro Leu Cys Pro ZLrp 130 115 170 Cys Thr Gin His His Gin Arg Pos Asp Pro tAg Thr Cys Arg Cys ZArg 145 150 155 110 Cys Arg ZAg Arg Cheese Phe Leu frrg Cys Gin dy Ztrg Gly Leu Glu LLe 165 170 175 Asn Pro Asp Thr Cys Arg Cys Arg Lys Leu Arg Arg

180 185 (2) Information on SEQ ID NO: 7:

(i) Characteristics of the sequence:

(A) Length: 858 base pairs (B) Type: nucleic acid (C) Thread: single (D) Topology: linear (ii) Molecule type: DNA (ix) Feature:

(A) Name / key: CDS (b) Location: 3 ... 431 (xi) Sequence description: SEQ ID NO: 7:

CC ATG AGC CCT CTG CTC CGC CGC CTG CTG CTC GCC GCA CTC CTG CAG 4 7

Met Ser Pro Leu Leu Arg Arg Leu Leu Leu Ala Ala Leu Leu Gln

1 5 10 15 CTG GCC CCC GCC CAG GCC CCT GTC TCC CAG CCT GAT GCC CCT GGC CAC 95 Leu Ala Pro Ala Main Ala Pro Val Ser Gln Pro Asp Ala Pro Gly Hia twenty 25 thirty CAG AGG AAA GTG GTG TCA TGG ATA GAT GTG TAT ACT CGC GCT ACC TGC 143 Main Arg Lys Val Val Cheese Trp How many Asp Val Type Thr Arg Ala Thr Cys 35 40 45 CAG CCC CGG GAG GTG GTG GTG CCC TTG ACT GTG GAG CTC ATG GGC ACC 191 Main Pro Arg Glu Val Val Val Pro Leu Thr Val Glu Leu Met Gly Thr 50 55 00 GTG GCC AAA CAG CTG GTG CCC AGC TGC GTG ACT GTG CAG CGC TGT GGT 23Z Val Ala Lys Main Leu Val Pro Spz Cy'z VaZ TPz VlZ Gln As. Cyz Gly 05 70 75 GGC TGC TGC CCT GAC GAT CGC CTG GAG TGT GTG CCC ACT GGG CAG CAC 28Z Gly Cys Cyy Pro Asp Asp Gly Leu Glu Cys Val Pro Thr Gly Gln His 80 85 90 95 CAA GTC CGG. ATG - CAG ATC CTC ATG ATC CGG TAC CCG AGC AGT CAG CTG 3Z 3 Main Val Arg Underworld Main lie Leu Met lie Arg Typ Pro Ser Gln Leu cheese 100 105 110

185 293

GGG GAG ATG TCC CTG GAA GAA CAC AGC CAG TGT GAA TGC AGA CCT AAA 383

Gly Glu Met Ser Leu Glu Glu His Ser Gln Cys Glu Cys Arg Pro Lys

115 120 125

AAA AAG GlA AGT GCT GTG AAG CCA GAT AGG TGC CGG AAG CTG CGA AGG 511

Lys Lys Aap Ser Ala Val Lys Pro Asp Arg Cys Arg Lys Leu Arg Arg

130 135 140

TGACACATGG CTTTTCAGAC TCAGCAGGGT GACTTGCCTC AGAGGCTATA TCCCAGTdlG 591

GGACCCACGG GGAGCCTGGT CACCAACCGC CAAGCCCCCA AGACCTCAGC CCAlAlCAGAA 5 51

GCTGCTCTAG GACCTGGGCC TCTCAGAGGG CTCTTCTGCC ATCCCTTGTC TCC <CTGA (AlC 611

CCTCATCCCA CAGGACAGAG TTGGCCGCGG AGACTGGGAG GCAGCAAGAG GGCTCAGCTA (5 71

CCAGCTCAGG GGAGAATGGA GTACTGTCTC AGTTTCTAAC CACTCTGTGC GAGT ^^ O ^ 731

CTTCCACCTG GCTCTTCCTC CCCTCACTAA GCCGΑCCCCΑ ACCTCTGCAT AATtUlATir 791

GGGCTTTGGT ΑCCCGΑCCTG TGACCCCCAA CCCTGATAAA CGCGCTGGCC GGA ^ CACCACC 8 51

CACAACC 8 58 (2) Information on SEQ ID NO: 8:

(i) Characteristics of the sequence:

(A) Length: 143 amino acids (B) Type: amino acid (D) Topology: linear (ii) Molecular type: protein (xi) Sequence description: SEQ ID NO: 8:

Underworld 1 Cheese Pro Leu Leu 5 Arg Arg Leu Leu Leu 10 Ala Ala Leu Luu Main 11 Leu Ala Pro Ala Main twenty Ala Pro Val Cheese Main 25 Pro Asp Ala Pro Gly thirty Hii Main Arg Lys Val 35 Val Cheese Trp How much Asp 40 Val iyr Thr Alg Ala 45 Thr Cys Main Pro Arg Glu VvU Vu5 Val Pro leu Thr VvU Glu Leu Underworld Gly Thr Val

55 60

Ala Lys 65 Cys Cys for Ueu Ppo Usp Val Asp 85 Pro aeu Cpo eel 70 TPu Vu1 Gln Hg lys gly Gly 80 GUn euU 90 75 Main Hu 95 Gly Leu Glu uys Pro Thr Gly Val Arg MMt Main He Iueu MeU How much Utrg Tyr Pro Cheese Cheese Main Lee 05 y 100 105 110 Glu Underworld See ULe Glu Glu His Cheese Main Cys du Cys Hg Pro Lys Lys 115 120 H2 Lys Asp See Ala Val Lys Puu ALy roo Aps Hg Lys jg ^ u Arg Arg 130 115 140

185 293 (2) Information about SEQ ID NO: 9:

(i) Characteristics of the sequence:

(A) Length: 910 base pairs (B) Type: nucleic acid (C) Strand: single (D) Topology: linear (ii) Type of molecule: DNA (ix) Feature:

(A) Name / key: CDS (b) Location: 3 ... 305 (xi) Sequence description: SEQ ID NO: 9:

CC ATG AGC CCT CTG CTC CGC CGC CTG CTG CTC GCC GCA CTC CTG CAG 45 Underworld 1 Cheese Pro Leu Leu 5 Arg Arg Leu Leu Leu 00 Ala Ala Leu Leu Gln 15 CTG GCC CCC GCC CAG GCC CCT GTC TCC CAG CCT GAT GCC CCT GGC CAC 95 Leu Ala Pro Ala Gln Ala Pro Val Ser Main , Pro Asp Ala Pro Gly His twenty 25 thirty CAG AGG AAA GTG GTG TCA TGG ATA GAT GTG TAT ACT CGC GCT ACC TGC 113 Main Arg Lys Val Val Ser Trp He Asp Val Tyr Thr Arg Ala Thr Cys 35 50 55 CAG CCC CGG GAG GTG GTG GTG CCC TTG. ACT and GTG GAG CTC ATG GGC ACC 195 Main Pro Arg Glu Val Val Val Pro Leu Thr Val Glu Leu Met Gly Thr 50 55 thirty GTG GCC AAA CAG CTG GTG CCC AGC TGC. GTG. ACT GTG CAG CGC TGT GGT 239 Val Ala Lys Main Leu Val Pro Cheese Cys Val Thr Val Gln Arg Cys Gly 35 70 75 GGC TGC TGC CCT GAC GAT GGC CTG GAG ' TGT. GTG CCC ACT GGG CAG CAC 228 Gly Cys Cys Pro Asp Asp Gly .Leu Glu Cys Val Pro Thr Gly Gln Hii 80 85 90 95 CAA GTC CGG ATG CAG ACC TΑGΑΑGAGAG GGCAGCGCCG TGGGGCCGGG 333 Main Val Arg Underworld Gln Thr

100

CAGGGCTGCC ACTCCCCACC ACCGTCCCCA GCCCCGTTCT GTTCCGGGCT GGGACTCTGC 395

CCCCGGAGCA CCCTCCCCAG CTGACATCAC CCATCCCACT CCAGCCCCAG GCCCCTCTGC 455

CCACGCTGCA CCCAGCACCA CCAGCGCCCC GACCCCCtG »CCTGCCGCTG CCCGCTGCCG 551

CGCCGCAGCT TCCTCCGTTG CCAAKKGGCG GGCTTAGAtGZ TCAACCCAGA OA ^ (7TGCIAG> 575

TGCCGGAAGC TGCGAAGGTG ACACATOGCT TTTCAGACTC AGC ^ GGGTGA CrTCCCCnCA; 639

AGGCTATATC CCAGTGGGGA ACAAAlG ^ tGGA C ^ C ^ CTGGTAAA AiACCAGCCAA. (GCC ^ t ^ CCCAAGA 635

CCTCAGCCCA GGCAGAAGCT GCTCTACG3AC CGGGGCCTCT CAGAGGCCCC TTCTGCCATC 75.

CCTTGTCTCC CTGGGGCCGC CA’ΓCGGGCGG ACAAG, A (3 ^ (3 GGAGAGGAAA CTGGGAGGCA 815

GCG.GGGGGGG CCGCGCGCCG GCTO ^ GGGGA AAATGGAGTA CGTTCTAAGT TTCTAiCCAC 9 '5

CCCGTGCAGG CG.GGCGTCCC ACAACTGGCT CCTTC

950

185 293 (2) Information about SEQ ID NO: 10:

(i) Characteristics of the sequence:

(A) Length: 101 amino acids (B) Type: amino acid (D) Topology: linear (ii) Molecular type: protein (xi) Sequence description: SEQ ID NO: 10:

Underworld 1 Cheese Pro Leu Leu 5 Arg Arg Leu Leu Leu 10 Ala Ala Leu Leu Main 15 Leu Ala Pro Ala Main twenty Ala Pro Val Cheese Main 25 Pro Asp Ala Pro Gly thirty His Main Arg Lys Val 35 Val Cheese Trp How much Asp 40 Val Tyr Thr Arg Ala 45 Thr Cys Main Pro Arg 50 Glu Val Val Val Pro 55 Leu Thr Val Glu Leu 60 Underworld Gly Thr Val Ala 65 Lys Main Leu Val Pro 70 Cheese Cys Val Thr Val 75 Main Arg Cys Gly Gly 80 Cys Cys Pro Asp Asp 85 Gly Leu Glu Cys Val 90 Pro Thr Gly Main His 95 Main

Val Arg Met Gln Thr 100 (2) Information on SEQ ID NO: 11:

(i) Characteristics of the sequence:

(A) Length: 42 base pairs (B) Type: Nucleic acid (C) Strand: Single (D) Topology: Linear (ii) Molecular Type: Oligonucleotide (xi) Sequence Description: SEQ ID NO: 11:

ACCACCACCT CCCTGGGCTG GCATGTGGCA CGTGCATAAA CG 42 (2) Information on SEQ ID NO: 12:

(i) Characteristics of the sequence:

(A) Length: 42 base pairs (B) Type: nucleic acid (C) Stranded: single (D) Topology: linear (ii) Molecule type: OLigonucleotide (xi) Sequence description: SEQ ID NO: 12:

AGTTGTTTGA CCACATTGCC CATGAGTTCC ATGCTCAGAG GC

185 293 (2) Information about SEQ ID NO: 13:

(i) Characteristics of the sequence:

(A) Length: 38 base pairs (B) Type: nucleic acid (C) Stranded: single (D) Topology: linear (ii) Molecular type: Oligonucleotide (xi) Sequence description: SEQ ID NO: 13:

GATCCTGGGG CTGGAGTGGG ATGGATGATG TCAGCTGG 313 (2) Information on SEQ ID NO: 14:

(i) Characteristics of the sequence:

(A) Length: 40 base pairs (B) Type: nucleic acid (C) Strandedness: single (D) Topology: linear (ii) Molecular type: Oligonucleotide (xi) Sequence description: SEQ ID NO: 14:

GCGGGCAGAG GATCCTGGGG CTGTCTGGCC. TCACAGCACT 40

185 293

2/52 3/52 Ftg.1 (i) Fig. Kii} 4/52 5/52 Fig.Kiii) Fig. 1 (i v)

185 293

TCGGCCTCC GAAACC ATG AAC TTT CTG

Met Asn Phe Leu

50 CTT Leu GCC Ala TTG Leu CTG Leu 15 CTC Leu TAC CTC CAC Tyr Leu His 98 CCC ATG GCA GAA GGA GGA GGG CAG Pro Underworld Ala thirty Glu Gly Gly Gly Gin 35 146 ATG GAT GTC TAT CAG CGC AGC TAC Underworld Asp 45 Val Tyr Gin Arg Cheese 50 Tyr 194 GAC ATC TTC CAG GAG TAC CCT GAT Asp 60 How much Phe Gin Glu Tyr 65 Pro Asp 242 TCC TGT GTG CCC CTG ATG CGA TGC Cheese Cys Val Pro Leu 80 Underworld Arg Cys 290 CTC GAG TGT GTG CCC ACT GAG GAG Leu Glu Cys Val 95 Pro Thr Glu Glu 338 CGG ATC AAA CCT CAC CAA GGC CAG Arg Ily Lys 110 Pro His Gin Gly Gin 115

Fig. Ki)

185 293

CTG TCT TGG GTG CAT TGG AGC 49

Leu Ser Trp Val His Trp Ser

10

CAT GCC AAG TGG TCC CAG GCT GCA 97

His twenty Ala Lys Trp Cheese Main 25 Ala Ala AAT CAT CAC GAA GTG GTG AAG TTC 145 Asn His His Glu Val 40 Val Lys Phe TGC CAT CCA ATC GAG ACC CTG GTG 193 Cys His Pro How much 55 Glu Thr Leu Val GAG ATC GAG TAC ATC TTC AAG CCA 241 Glu How much Glu 70 Tyr How much Phe Lys Pro 75 GGG GGC TGC TGC AAT GAC GAG GGC 289 Gly Gly 85 Cys Cys Asn Asp Glu 90 Gly TCC AAC ATC ACC ATG CAG ATT ATG 337 Cheese 100 Asn How much Thr Underworld Main 105 How much Underworld CAC ATA GGA GAG ATG AGC TTC CTA 385 His How much Gly Glu Underworld 120 Cheese Phe Leu

Fig. Kii)

185 293

386 CAG CAC AAC AAA TGT GAA TGC AGA Gln His Asn Lys Cys Glu Cys Arg 125 130 434 GAA AAT CCC TGT GGG CCT TGC TCA Glu Asn Pro Cys Gly Pro Cys Ser 140 145 482 CAA GAT CCG CAG ACG TGT AAA TGT Gln Asp Pro Gln Thr Cys Lys Cys 160 530 TGC AAG GCG AGG CAG CTT GAG TTA Cys Lys Ala Arg Gln Leu Glu Leu 175 578 AAG CCG AGG CGG TGAGCCGGGC AGGAG Lys Pro Arg Arg. 190 630 GAACCAGATC TCTCACCAGG

Fig. 1 (iii)

185 293

CCA Pro AAG Lys AAA Lys GAT Asp 135 AGA Arg GCA Ala AGA Arg CAA Main 433 GAG CGG AGA AAG CAT TTG TTT GTA 481 Glu Arg Arg 150 Lys His Leu Phe Val 155 TCC TGC AAA AAC ACA GAC TCG CGT 529 Cheese Cys 165 Lys Asn Thr Asp Cheese 170 Arg AAC GAA CGT ACT TGC AGA TGT GAC 577 Asn 180 Glu Arg Thr Cys Arg 185 Cys Asp

GAAGG AGCCTCCCTC AGCGTTTCGG 629

Fig. 1 (i v)

649

185 293

7/52 8/52 Fig. 2 (i) Fig. 2 (ii) 9/52 10/52 Fig 2 (iii) Fig 2 (iv) 11/52 12/52 Fig 2 (v) Fig 2 (vi)

185 293

CC ATG AGC CCT CTG CTC CGC CGC

Underworld 1 Cheese Pro Leu Leu 5 Arg Arg 48 CTG • GCC CCC GCC CAG GCC CCT GTC Leu Ala , Pro Ala . Main twenty . Ala . Pro Val 96 CAG AGG AAA GTG GTG TCA TGG ATA Main Arg Lys Val 35 Val Cheese Trp How much 144 CAG CCC CGG GAG GTG GTG GTG CCC Main Pro Arg 50 Glu Val Val Val Pro 55 192 GTG GCC AAA CAG CTG GTG CCC AGC Val Ala 65 Lys Main Leu Val Pro 70 Cheese 240 GGC TGC TGC CCT GAC GAT GGC CTG Gly 80 Cys Cys Pro Asp Asp 85 Gly Leu 288 CAA GTC CGG ATG CAG ATC CTC ATG Main Val Arg Underworld Main 100 How much Leu Underworld 336 GGG GAG ATG TCC CTG GAA GAA CAC Gly Glu Underworld Cheese 115 Leu Glu Glu His

Fig. 2 (i)

185 293

CTG • CTG • CTC GCC GCA CTC CTG CAG 47 Leu Leu Leu 10 Ala Ala Leu Leu Main 15 TCC CAG CCT GAT GCC CCT GGC CAC 95 Cheese Main 25 Pro Asp Ala Pro Gly thirty His GAT GTG TAT AGT CGC GCT ACC TGC 143 Asp 40 Val Tyr Thr Arg Ala 45 Thr Cys TTG ACT GTG GAG CTC ATG GGC ACC 191 Leu Thr Val Glu Leu 60 Underworld Gly Thr TGC GTG ACT GTG CAG CGC TGT GGT 239 Cys Val Thr Val 75 Main Arg Cys Gly GAG TGT GTG CCC ACT GGG CAG CAC 287 Glu Cys Val 90 Pro Thr Gly Main His 95 ATC CGG TAC CCG AGC AGT CAG CTG 335 How much Arg 105 Tyr Pro Cheese Cheese Main 110 Leu AGC CAG TGT GAA TGC AGA CCT AAA 383 Cheese 120 Main Cys Glu Cys Arg 125 Pro Lys

Fig. 2 Fu)

185 293

384 AAA Lys AAG Lys GAC Asp 130 AGT Cheese GCT Ala GTG Val AAG Lys CCA Pro 135 432 CGT CCC CAG CCC CGT TCT GTT CCG Arg Pro Main Pro Arg Cheese Val Pro 145 150 480 CCC TCC CCA GCT GAC ATC ACC CAT Pro Cheese Pro Ala Asp How much Thr His 160 165 528 GCC CAC GCT GCA CCC AGC ACC ACC Ala His Ala Ala Pro Cheese Thr Thr

180

576 GCT GCC GCT GCC GAC GCC GCA GCT Ala Ala Ala Ala Asp Ala Ala Ala

195

Fig. 2 (iii)

185 293

GAC Asp AGG Arg GCT Ala GCC Ala ACT Thr 140 CCC Pro CAC His CAC His 431 GGC TGG GAC TCT GCC CCC GGA GCA 479 Gly Trp Asp Cheese 155 Ala Pro Gly Ala CCC ACT CCA GCC CCA GGC CCC TCT 527 Pro Thr Pro 170 Ala Pro Gly Pro Cheese 175 AGC GCC CTG ACC CCC GGA CCT GCC 575 Cheese Ala 185 Leu Thr Pro Gly Pro 190 Ala TCC TCC GTT GCC AAG GGC GGG GCT T 624 Cheese 200 Cheese Val Ala Lys Gly 205 Gly Ala

Fig. 2 (iv)

185 293

AGAGCTCAAC CCAGACACCT GCAGGTGCCG GACTCAGCAG GGTGACTTGC CTCAGAGGCT GGTAAAAAAC AGCCAAGCCC CCAAGACCTC GCCTCTCAGA GGGCTCTTCT GCCATCCCTT GAGTTGGAAG AGGAGACTGG GAGGCAGCAA GGAGTACTGT CTCAGTTTCT AACCACTCTG CTCCCCTCAC TAAGAAGACC CAAACCTCTG CTGTGACCCC CAACCCTGAT AAAAGAGATG

Fig. 2 (v)

185 293

GAAGCTGCGA AGGTGACACA TGGCTTTTCA 684 ATATCCCAGT GGGGGAACAA AGGGGAGCCT 744 AGCCCAGGCA GAAGCTGCTC TAGGACCTGG 804 GTCTCCCTGA GGCCATCATC AAACAGGACA 864 GAGGGGTCAC ATACCAGCTC AGGGGAGAAT 924 TGCAAGTAAG CATCTTACAA CTGGCTCTTC 984 CATAATGGGA TTTGGGCTTT GGTACAAGAA 1044 GAAGGAAAAA AAAAAAAAAA 1094

Fig. 2 (vi)

185 293

14/52

Fig. 3 (ι)

15/52

Fig 3 (ii)

185 293> VEGF_HUMAN VEGF_ PRECURSOR OF HUMAN VASCULAR (VASCULAR 215 AA.

LENGTH = 215

RATING = 181 (92.4 BITS), STANDBY = 6.4e-20, IDENTICAL = 33/75 (44%), POSITIVE = 48/75

INQUIRY SUBJECT: 31 HQRKWSWIDVYTRATCQPREVWPLTVEL 36 +++ W + DVY R + C + P E + V - E NHHEWKFMDVYQRSYCHPIETLVDIFQEY INQUIRY; 91 PTGQHQVRMQILMIR 105 PT + + MQI + 1+ SUBJECT _: 96 PTEESNITMQIMRIK 110 RATING = 76 (38.8 BITS), WAIT = 0.0011, IDENTICAL = 12/19 (63%), POSITIVE = 16/19 INQUIRY: 110 QLGEMSLEEHSQCECRPKK 128 ++ GEMS + H + CECRPKK SUBJECT _: 116 HIGEMSFLQHNKCECRPKK 134 RATING - 72 (36.8 BITS), WAIT = 0.0046, IDENTICAL = 14/21 (66%), POSITIVE = 15/21 INQUIRY. 202 RCQGRGLELNPDTCRCRKLRR 222 RC + R LELN TCRC K RR SUBJECT _: 195 RCKARQLELNERTCRCDKPRR 215 RATING - 46 (23.5 BITS), WAIT = 47. , IDENTICAL = = 6/10 (60%), POSITIVE = 9/10 INQUIRY; 187 DPRTCRCRCR 196 DP + TC + C C + SUBJECT _: 1-81, DPOTCKCSCK 190 F'gJ (')

SUBJECT

Fig. 3 (i)

185 293

CUBIC GROWTH FACTOR (VEGF)

P = 6.4e-20 (64%)

MGTVAKQLVPSCVTVQRCGGCCPDDGLECV 90 + PSCV + RCGGCC D + GLECV

PDEIEYIFKPSCVPLMRCGGCCNDEGLECV 9 5

POISSON P (2) = 9.1e-12 (84%)

POISSON P (3) = 3.6e-18 (71%)

POISSON P (4) = 7.3e-10 (90%)

Fig. 3 (i)

185 293

17/52 18/52 Fig 4 (i) Fig. 4 (u) 19/52 20/52 Fig. 4 (iii) Fig. 4 (iv]

185 293

Weighted range _: 3.00 Weighted length.-0.100

Quality: 100.9 Ratio.-0.175

Percent Similarity. -69.70:

Medium fit _; 1.o 0 0 Average mismatch: - 0.9 00

Length _; 7 3 9 Rifts 3 0

Percent identity: 69,703

8 ATGAGCCCTCTGCTCCGCCGCCTGC

ATGAACTTTCTGCT ..... GTCT. .

TGCAGCTGGCCCCCGCCCAGGCCCC

III III II I III III

TGCTGCTCTACCTCCACCATGCCAA

118 CACCAGAGGA ...............

him

106 AGAAGGAGGAGGGCAGAATCATCAC

140 GTGTATACTCGC.GCTACCTGCCAG

II III III INI III

152 GTCTATCAGCGCAGCTA. CTGCCAT

194 T .... GA ..... CTGTGGAGCTCAT

I II III III II

201 TCCAGGAGTACCCTGATGAGATCGA

235 CCCAGCTGCGTGACTGTGCAGCGCT

II III III I II III I

239 CCATCCTGTGTGCCCCTGATGCGAT

285 CCTGGAGTGTGTGCCCACTGGGCAG

CCTGGAGTGTGTGCCCACTGAGGAG

Fig. 4 (i)

289

185 293

TGCTCGCCGCACT .......... CC 67

I I III I I

... TGGGTGCATTGGAGCCTTGCCT 56

TGTCTCCCAGCCTGATGCCCCTGGC 117

GTGGTCCCAGGCTGCA.CCCATGGC 105 .AAGTGGTG .... TCATGGATAGAT 147 llllllll lily III

GAAGTGGTGAAGTTCATG .... GAT 151

CCCCGGGAG ... GTGGTGGTGCCCT 193

CCAATCGAGACCCTGGTGGACATCT 200

GGGCACCGTGGCCAAACAGCTGGTG 234

GTACATCTT. . . CAA ......... G 238

GTGGTGGCTGCTGCCCTGACGATGG 284

GCGGGGGCTGCTGCAATGACGAGGG 288

CACCAAGTCCGGATGCAGAT ..... 329

I I II IIIlll

TCCAACATCACCATGCAGATTATGC 338

Fig4 (ii)

185 293

3 0 CCTCATGATCCGGTACC

OTHERS

3 9 GGATCAAACCTCA ........... C

369 GTCCCTGGAAGAACACAGCCAGTGT

III II INI I I III

376 GAGCTTCCTACAGCACAACAAATGT

419 GTGCTGTGAAGCCAGACAGGGCTGC

I III lily I

423 G ........ AGCAAGAC AAG .....

469 CGTTCTGTTCCGGGCTGGGACTCTG

443 ... TGTGGGCCTTGCTCAGA .....

519 CATCACCCATCCCACTCCAGCCCCA

468 .........................

569 GC .......... ACCACCAGCGCCC

469 GCATTTGTTTGTACAA .........

609 TGCCGACGCCGCAGCTTCCTCCGTT

II I I I III II INI

509 TG.CAAAAACACAGACTC..GCGTT

657 AACCCAGACACCTGCAGGTGCCGGA

554 AACGAACGTACTTGCAGATGTGACA

Fg

185 293

CGAGCAGTCAGC ... TGGGGGAGAT

CAAG. .GCCAGCACATAGGAGAGAT

GAATGCAGACCTAAAAAAAAGGACA iiiiiiimi m ii ιι i

GAATGCAGACC. . . AAAGAAAGATA

CACTCCCCACCACCGTCCCCAGCCC

........... AAAATCCC ......

CCCCCGGAGCACCCTCCCCAGCTGA. . .GCGGAGAA ..............

GGCCCCTCTGCCCACGCTGCACCCA

........................AND

TGACCCCCGGACCTGCCGCTGCCGC .GATCCGCAGACGTGTAAATGTTCC

GCCAAGGGCGGGGC..TTAGAGCTC

GC..AAGGCGAGGCAGCTTGAGTTA

AGCTGCGAAGGTGA

AGCCGAGGCGGTGA

Fig. 4 (iv)

368

375

418

422

468

442

518

467

568

468

608

508

656

553

695

592

185 293

22/52 23/52 24/52 Fig.5li) Fig. 5 (i i Fig 51 iii) 25/52 26/52 27/52 Fig. 5 (iv) Fig. 5 (v) Fig. 5 (vi)

185 293

165SOMSQ.MSF.msf MSF.-687 Type: D Tuesday, June 20, 1995

Attempt: 3140 1 VEGF165 SOM175 SOM175-e6 SOMl75-e6 & 7 SOM175-e4 X ATGAACTTTCTGCTGTCTTGGGTG ATGAGCCCTCTGCTCCGCCGCCTG ATGAGCCCTCTGCTCCGCCGCCTG ATGAGCCCTCTGCTCCGCCGCCTG ATGAGCCCTCTGCTCCGCCGCCTG VEGF165 SOM175 SOM175-e6 SOM175-e6 & 7 SOM175-e4 81 CACCCATGGCAGAAGGAGGAGGGC TGCCCCTGGCCACCAGAGGAAAGT TGCCCCTGGCCACCAGAGGAAAGT TGCCCCTGGCCACCAGAGGAAAGT TGCCCCTGGCCACCAGAGGAAAGT VEGF165 SOM175 SOM175-e6 SOMl75-e6 & 7 SOM175-e4 161 CCAATCGAGACCCTGGTGGACATC GTGGTGGTGCCCTTGACTG.TGGA GTGGTGGTGCCCTTGACTG.TGGA GTGGTGGTGCCCTTGACTG.TGGA GTGGTGGTGCCCTTGACTG.TGGA VEGF165 SOM175 SOM175-e6 SOM175-e6 & 7 SOM175-e4 241 GATGCGATGCGGGGGCTGCTGCAA GCAGCGCTGTGGTGGCTGCTGCCC GCAGCGCTGTGGTGGCTGCTGCCC GCAGCGCTGTGGTGGCTGCTGCCC GCAGCGCTGTGGTGGCTGCTGCCC

Fig. 5 (i)

185 293

CATTGGAGCCTTGCCTTGCTGCTCTACC

CTGCTCGCCGCACTCCTGCAGCTGGCCC

CTGCTCGCCGCACTCCTGCAGCTGGCCC

CTGCTCGCCGCACTCCTGCAGCTGGCCC

CTGCTCGCCGCACTCCTGCAGCTGGCCC

AGAATCATCACGAAGTGGTGAAGTTCAT

GGTGTCATGGATAGATGTGTATACTCGC

GGTGTCATGGATAGATGTGTATACTCGC

GGTGTCATGGATAGATGTGTATACTCGC

GGTGTCATGGATAGATGTGTATACTCGC

TTCCAGGAGTACCCTGATGAGATCGAGT GCTCATGGGCACCGTGGCCAAAC..AGC GCTCATGGGCACCGTGGCCAAAC..AGC GCTCATGGGCACCGTGGCCAAAC..AGC GCTCATGGGCACCAACGTAGGC

TGACGAGGGCCTGGAGTGTGTGCCCACT

TGACGATGGCCTGGAGTGTGTGCCCACT

TGACGATGGCCTGGAGTGTGTGCCCACT

TGACGATGGCCTGGAGTGTGTGCCCACT

TGACGATGGCCTGGAGTGTGTGCCCACT

Fig. 5 (ii)

185 293

TCCACCATGCCAAGTGGTCCCAGGCTG.

CCGCCCAGGCCCCTGTCTCCCAGCCTGA

CCGCCCAGGCCCCTGTCTCCCAGCCTGA

CCGCCCAGGCCCCTGTCTCCCAGCCTGA

CCGCCCAGGCCCCTGTCTCCCAGCCTGA

160

GGATGTCTATCAGCGCAGCTACTGCCAT

G ..... CTACCTGC. CAGCC. CCGGGAG

G ..... CTACCTGC. CAGCC. CCGGGAG

G ..... CTACCTGC. CAGCC. CCGGGAG

G ..... CTACCTGC. CAGCC. CCGGGAG

240

ACATCTTCAAGCCATCCTGTGTGCCCCT

TGGTGCCCAG ...... CTGCGTGACTGT

TGGTGCCCAG ...... CTGCGTGACTGT

TGGTGCCCAG ...... CTGCGTGACTGT

TGGTGCCCAG ...... CTGCGTGACTGT

320

GAGGAGTCCAACATCACCATGCAGATTA GGGCAGCACCAAGTCCGGATGCAGATCC GGGCAGCACCAAGTCCGGATGCAGATCC GGGCAGCACCAAGTCCGGATGCAGATCC GGGCAGCACCAAGTCCGGATGCAGA ...

Fig. 5 (iii)

185 293

VEGF165

SOM175

SOM175-e6

SOMl75-e6 & 7

SOMl75-e4

VEGF165

SOM175

SOMl75-e6

SOM175-e6 & 7

SOMl75-e4

VEGF165

SOM175

SOMl75-e6

SOMl75-e6 & 7

SOMl75-e4

VEGF165

SOM175

SOM175-E6

SOMl75-e6 & 7

SOMl75-e4

VEGF165

SOM175

SOMl75-e6

SOMl75-e6 & 7

SOM175-e4

321

TGCGGATCAAACCTCACCAAGGCC TCATGATCCGG ... TACCCGAGCA TCATGATCCGG ... TACCCGAGCA TCATGATCCGG ... TACCCGAGCA

401

AAGAAAGATAG ........ AGCAA

AAAAAGGACAGTGCTGTGAAGCCA

AAAAAGGACAGTGCTGTGAAGCCA

AAAAAGGACAGTGCTGTGAAGCCA

AAAAAGGACAGTGCTGTGAAGCCA

481

........... AAGCA ........

CTCTGCCCCCGGAGCACCCTCCCC

CTCTGCCCCCGGAGCACCCTCCCC

561

A ............... GATCCGCA

GCACCACCAGCGCCCTGACCCCCG

GCACCACCAGCGCCCTGACCCCCG

GCACCACCAGCGCCCTGACCCCCG

641

TTGAGTTAAACGAACGTACTTGCA

TAGAGCTCAACCCAGACACCTGCA

TAGAGCTCAACCCAGACACCTGCA

TAGAGCTCAACCCAGACACCTGCA

Fig5 (iv)

185 293

AGCACATAGGAGAGATGAGCTTCCTACA

GTCAGCTGGGGGAGATGTCCCTGGAAGA

GTCAGCTGGGGGAGATGTCCCTGGAAGA

GTCAGCTGGGGGAGATGTCCCTGGAAGA

GACAAGAA .... AATCCCTGTGG .....

GACAGGGCTGCCACTCCCCACCACCGTC

GATAG .......................

GATAG .......................

GACAGGGCTGCCACTCCCCACCACCGTC

AGCTGACATCACCCATCCCACTCCAGCC .......................... CC

AGCTGACATCACCCATCCCACTCCAGCC

GACGTGTAAATGTTCCTGCAAAAAC.AC GACCTGCCGCTGCCGCTGCCGACGCCGC GACCTGCCGCTGCCGCTGCCGACGCCGC

GACCTGCCGCTGCCGCTGCCGACGCCGC

687

GATGTGACAAGCCGAGGCGGTGA

GGTGCCGGAAGCTGCGAAGGTGA

GGTGCCGGAAGCTGCGAAGGTGA

GTGCCGGAAGCTGCGAAGGTGA

GGTGCCGGAAGCTGCGAAGGTGA

Fig. 5 (v)

185 293

400

GCACAACAAATGTGAATGCAGACC ... A ACACAGCCAGTGTGAATGCAGACCTAAA ACACAGCCAGTGTGAATGCAGACCTAAA ACACAGCCAGTGTGAATGCAGACCTAAA ...................... CCTAAA

480

......... GCCTTGCTCAGAGCGGAGA

CCCAGCCCCGTTCTGTTCCGGGCTGGGA

CCCAGCCCCGTTCTGTTCCGGGCTGGGA

560

......... TTTGTT ..... TGTAC. . AND

CCAGGCCCCTCTGCCCACGCTGCACCCA

CCAGGCCCCTCTGCCCACGCTGCACCCA

CCAGGCCCCTCTGCCCACGCTGCACCCA

640

AGACTCG..CGTTGCAAGGCGAGGCAGC AGCTTCCTCCGTTGCCAAGGGCGGGGCT AGCTTCCTCCGTTGCCAAGGGCGGGGCT

AGCTTCCTCCGTTGCCAAGGGCGGGGCT

Fig. 5 (vi)

185 293

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185 293

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185 293

Regions with 100% homology are boxed, and the residues retained despite participating in homodimerization are underlined.

The depicted VEGF sequence contains a leader sequence of 26 amino acids (deletion of which leads to mature VEGF ₁₆₅ ), and has a total length of 191 amino acids.

SOM175 homology with VEGF is 27% (33%) at the 165th protein level, although there are blocks with 100% homology.

In particular, many structural residues have been retained, including those that nevertheless play a role in homodimerization

VEGF (by comparison to PDGF) e.g. Cysteine-47

Proline-70, Cysteine-72, Waliba-74, Arginine-77, Cysteine-78, Glycine-80, Cysteine 81 and 82, Cystein-89, Proline-91

Cysteine 122 and 124

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185 293

GENOMIC STRUCTURE OF HUMAN SOM175

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36/52 37/52 Fig. 9 (i) Fig. 9 (i) 38/52 39/52 Fig. 9 (iii) Fig. 9 (iv)

185 293

-163 gcacgagctcaggccgtcgctgcggcgc tg -103 gggggccgcggaggagccgccccctgcgcc

-43 ggcggctctggctgacccccccccacaccg

CGTCGCCTGCTGCTTGTTGCACTGCTGCAG RRLLLVALLQ

AND

6 TTTGATGGCCCCAGTCACCAGAAGAAAGTG FDGPSHQKKV

136 ACATGCCAGCCCAGGGAGGTGGTGGTGCCT TCQPREVVVP

196 AAACAACTAGTGCCCAGCTGTGTGACTGTG KQLVPSCVTV

256 GGCCTGGAATGTGTGCCCACTGGGCAACAC GLECVPTGQH

316 TACCCGAGCAGTCAGCTGGGGGAGATGTCC YPSSQLGEMS

6 CCTAAAAAAAAGGAGAGTGCTGTGAGGCCA PKKKESAVRP

436 CAGCCCCGCTCTGTTCCGGGCTGGGACTCT QPRSVPGWDS

Fig. 9 (i)

185 293 cgttgcgctgcctgcgcccagggctcggga ccgccccgggtccccgggtccgcgccatgg ccgggctagggcccgATGAGCCCCCTGCTG

M S P L L -17 (

CTGGCTCGCACCCAGGCCCCTGTGTCCCAG LARTQAPVSQ 4

GTGCCATGGATAGACGTTTATGCACGTGCC VPWIDVYARA 24

CTGAGCATGGAACTCATGGGCAATGTGGTC LSMELMGNVV 44

CAGCGCTGTGGTGGCTGCTGCCCTGACGAT QRCGGCCPDD 64

CAAGTCCGAATGCAGATCCTCATGATCCAG QVRMQILMIQ 84

AND

CTGGGAGAACACAGCCAATGTGAATGCAGA LGEHSQCECR 104

AND

GACAGGGTTGCCATACCCCACCACCGTCCC D R V A I PHHRP 124

ACCCCGGGAGCACCCTCCCCAGCTGACATC TPGAPSPADI 144

Fig. 9 (ii)

185 293

496 ATCCATCCCACTCCAGCCCCAGGATCCTCT IHPTPAPGSS

S P R I L

556 CTGACCCCCGGACCTGCCGTTGCCGCTGTA LTPGPAVAAV

PDPRTCRCRC

616 GGGGCTTAGAGCTCAACCCAGACACCTGTA GA *

RGLELNPDTC

676

736

796

856

916

976

1036 ctttccagactccacgggcccggctgcttt agcacaggcgtaacctcctcagtctgggag gagctctctcgccatcttttatctcccaga atgtctcacctcaggggccagggtactctc ttctggctaacctactattaacacgcactatgaacta gggctactattaacacgcactatgaacta

Fig. 9 (iii)

185 293

GCCCGCCTTGCACCCAGCGCCGCCAACGCC ARLAPSAANA 164

CPPCTQRRQR 130

GACGCCGCCGCTTCCTCCATTGCCAAGGGC DAAASSIAKG 184

RRRRFLHCQG 150 i

GGTGCCGGAAGCCGCGAAAGTGĄcaagctg

186

RCRKPRK * 167 acacac tatggccctgcttcacagggagaagagtgg gtcactgccccaggacctggaccttttaga gctgccatctaacaattgtcaaggaacctc tcacttaaccaccctggtcaagtgagcatc aaccccaaacttaagaagaaaacttacaagaagataacgcgaagaactaa

Fi g. 9 (iv)

185 293

47/52

Fig 10 (i)

42/52

Fig (0 (H)

185 293

AND

AND

hVRF167 -21 MSPLLRRLLLAALLQLAPAQAP 1 1 1 I II 1 II II 1 II I 1 1 III mVRF167 1 II I 1 i 1 1 1 1 1 I 1 1 I I 1 III -21 MSPLLRRLLLVALLQLARTQAP hVRF167 3 0 EVWPLTVELMGTVAKQLVPSC 1 1 1 1 1 1 -llll 1 1 1 1 1 1 1 1 mVRFl67 1 1 1 1 1 1 · 1 1 1 1 1 1 1 1 1 1 1 1 3 0 EVWPLSMELMGNWKQLVPSC hVRF167 80 ILMIRYPSSQLGEMSLEEHSQC mVRFl67 80 ILMIQYPSSQLGEMSLGEHSQC hVRF167 130 RPDPRTCRCRCRRRSFLRCQGR mVRFl67 130 RPDPRTCRCRCRRRRFLHCQGR

Β

hVRF186 116 RAATPHHRPQPRSVPGWDSAPG mVRFl86 116 RVAIPHHRPQPRSVPGWDSTPG hVRF186 166 TPGPAAAAADAAASSVAKGGA * mVRF186 166 TPGPAVAAVDAAASSIAKGGA * Fig. 10 (i)

185 293

VSQPDAPGHQRKWSWIDVYTRATCQPR 2 9

VSQFDGPSHQKKWPWIDVYARATCQPR 2 9

VTVQRCGGCCPDDGLECVPTGQHQVRMQ 7 9

111II11IIIIII11IIIII11II11II

VTVQRCGGCCPDDGLECVPTGQHQVRMQ 7 9

ECRPKKKDSAVKPDSPRPLCPRCTQHHQ 129 lilies Ml MIII II III MM

ECRPKKKESAVRPDSPRILCPPCTQRRQ 129

GLELNPDTCRCRKLRR * 167

111111111111111:

GLELNPDTCRCRKPRK * 167

APSPADITHPTPAPGPSAHAAPSTTSAL 165

APSPADIIHPTPAPGSSARLAPSAANAL 165

186

186

Fig. Odi)

185 293

44/52

Fig (((ι)

45/52

Fig ((ii)

185 293

mVRF167 -21 MSPLLRRL..LLVALLQL .. mVEGFl88 -26 MNFLLSWVHWTLALLLYLHH mVRF167 25 TCQPREWVPLSMELMGNW mVEGF188 24 YCRPIETLVDIFQEYPDEIE mVRFl67 75 QVRMQILMIQYPSSQ.LGEM mVEGFl88 74 NITMQIMRIKPHQSQHIGEM mVRFl67 119 .............. ILCPPC • 1 1 1 mVEGF188 124 1 and 1 QKRKRKKSRFKSWSVHCEPC mVRFl67 152 GLELNPDTCRCRKPRK mVEGFl88 173 QLELNERTCRCDKPRR

Figure 11 (i)

185 293

AR. TQAPVSQFDGPSHQKKWPWIDVYARA 24 AKWSQAAPTT.EGEQKSHEVIKFMDVYQRS 23 KQLVPSCVTVQRCGGCCPDDGLECVPTGQH 74 YIFKPSCVPLMRCAGCCNDEALECVPTSES 73 SLGEHSQCECRPKKKESAVRPDSPR ..... And. . II II I I (I 1 II 118 1 * - 1 1 1 1 1 1 1 1 1 1 I. SFLQHSRCECRPKKDRTKPEKKSVRGKGKG 123 TQRRQR ... PDPRTCRCRCRRRRFLHCQGR 151 SERRKHLFVQDPQTCKCSCKNTDS.RCKAR 172 167

188

Fig. (((Ii)

185 293

Fig. 12 <Ν W & a co 2Ζ223 σ} ππνΛ <? Μ νππη <ν

185 293

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185 293

185 293

Fig. 15

185 293

AVERAGE NEURITE LENGTH (um)% NEURITE EXTENSION

185 293

6000

5000 "4000 ε

3000

2000

1000 cerebral glia cns nl

OLIGODENDROCITES

and 00 ng

185 293

MOUSE ASTROGLE CELLS

MOUSE OLIGODENDROGLE CELLS

MOUSE NEURONS IN THE PRE-BRAIN

Publishing Department of the UP RP. Circulation of 70 copies

Price PLN 6.00.

Claims (36)

Patent claims 1. An isolated polypeptide having at least one of the following characteristics: (i) the ability to induce proliferation of vascular epithelial cells; (ii) the ability to interact with the flt-1 / flk-1 receptor family, and (iii) the ability to induce cell migration, cell survival, and / or an increase in intracellular levels of alkaline phosphatase; wherein the polypeptide comprises: a. Amino acid sequence encoded by the nucleotide sequence shown in SEQ ID No. 3 or a nucleotide sequence capable of hybridizing to SEQ ID No. 3 or complementary thereto under stringent hybridization conditions in the presence of 0.1-1 x SSC / 0.1% w / w / wt. SDS at 60 ° C for 1-3 hours, or b. the amino acid sequence shown in SEQ ID No. 4, or an amino acid sequence having at least 70% similarity thereto; or a derivative of such a polypeptide, showing at least one of the features (i) - (iii). 2. An isolated polypeptide according to claim 1 The method according to claim 1, characterized in that it comprises the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID No. 3. 3. An isolated polypeptide according to claim 1 The method of claim 1, comprising the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID No. 5. 4. An isolated polypeptide according to claim 1 7. The method of claim 1, comprising the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID No. 7. 5. An isolated polypeptide according to claim 1 The method according to claim 1, characterized in that it comprises the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID No. 9. 6. An isolated polypeptide according to claim 1 The method of claim 1, comprising the amino acid sequence shown in SEQ ID No. 4. 7. The isolated polypeptide of claim 1 The method of claim 1, comprising the amino acid sequence shown in SEQ ID No. 6. 8. An isolated polypeptide according to claim 1 The method of claim 1, comprising the amino acid sequence shown in SEQ ID No. 8. 9. An isolated polypeptide according to claim 1 The method of claim 1, comprising the amino acid sequence shown in SEQ ID No. 10. 10. An isolated polypeptide according to claim 1 4. The process of claim 1 which is of human origin. 11. An isolated polypeptide according to claim 1 A method according to claim 1, characterized in that it has at least one of the following characteristics: ability to induce astroglial proliferation, ability to promote neuronal viability and / or proliferation in mammals. 12. An isolated polypeptide according to claim 1 The polypeptide of claim 1, which is the mature form of the polypeptide with a cleaved leader sequence. 13. An isolated nucleic acid molecule having the nucleotide sequence shown in SEQ ID No. 3 or a nucleotide sequence capable of hybridizing to or complementary to SEQ ID No. 3 under stringent hybridization conditions in the presence of 0.1 -1 x SSC / 0.1 wt%. / wt. SDS at 60 ° C for 1-3 hours. 14. An isolated nucleic acid molecule according to claim 1 13. The method according to claim 13, characterized in that it comprises the nucleotide sequence shown in SEQ ID No. 3. 185 293 15. An isolated nucleic acid molecule according to claim 1 13. The method according to claim 13, characterized in that it comprises the nucleotide sequence shown in SEQ ID No. 5. 16. An isolated nucleic acid molecule according to claim 1. 13. The method according to claim 13, characterized in that it comprises the nucleotide sequence shown in SEQ ID No. 7. 17. An isolated nucleic acid molecule according to claim 1 13. The method according to claim 13, characterized in that it comprises the nucleotide sequence shown in SEQ ID No. 9. 18. An isolated nucleic acid molecule according to claim 1 13. The method of claim 13, which encodes a polypeptide having the amino acid sequence shown in SEQ ID No. 4 or an amino acid sequence having at least 70% similarity thereto. 19. An isolated nucleic acid molecule according to claim 1 The method of claim 13, which encodes a polypeptide which comprises the amino acid sequence shown in SEQ ID No. 4. 20. An isolated nucleic acid molecule according to claim 1 13. The method of claim 13, which encodes a polypeptide which comprises the amino acid sequence shown in SEQ ID No. 6. 21. An isolated nucleic acid molecule according to claim 1 The method of claim 13, which encodes a polypeptide which comprises the amino acid sequence shown in SEQ ID No. 8. 22. An isolated nucleic acid molecule according to claim 1 13. The method of claim 13, which encodes a polypeptide having the amino acid sequence shown in SEQ ID No. 10. 23. An isolated nucleic acid molecule according to claim 23. The method of claim 13, which encodes a polypeptide having at least one of the following characteristics: (i) the ability to induce proliferation of vascular epithelial cells; (ii) the ability to interact with a family of receptors; and (iii) the ability to induce cell migration, cell survival, and / or an increase in intracellular levels of alkaline phosphatase; or a derivative of such a polypeptide, showing at least one of the features (i) - (iii). 24. An isolated nucleic acid molecule according to claim 24. The method of claim 23, wherein the polypeptide further has the ability to induce astroglial proliferation. 25. An isolated nucleic acid molecule according to claim 1 23. The method of claim 23, further encoding a polypeptide having the ability to promote neural viability and / or proliferation in a mammal. 26. An isolated nucleic acid molecule according to claim 1 13. The method of claim 13, wherein it or the polypeptide encoded by it is of human origin. 27. A method of obtaining a polypeptide having at least one of the following characteristics: (i) the ability to induce proliferation of vascular epithelial cells; (ii) the ability to interact with a family of receptors, flt-1 / flk-1 _9, and (iii) the ability to induce cell migration, cell survival and / or an increase in intracellular levels of alkaline phosphatase, characterized in that it comprises expressing the nucleic acid molecule encoding a polypeptide comprising : a. Amino acid sequence encoded by the nucleotide sequence shown in SEQ ID No. 3 or a nucleotide sequence capable of hybridizing to SEQ ID No. 3 or complementary thereto under stringent hybridization conditions in the presence of 0.1 - 1 x SSC / 0.1% w / w / wt. SDS at 60 ° C for 1-3 hours, or b. the amino acid sequence shown in SEQ ID No. 4, or an amino acid sequence having at least 70% similarity thereto; or a derivative of such a polypeptide in a suitable host grown under conditions suitable for polypeptide synthesis. 28. The method according to p. 27, characterized in that the polypeptide comprises an amino acid sequence encoded by a nucleotide sequence selected from SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7 and SEQ ID No. 9, or an amino acid sequence selected from SEQ ID No. 4, SEQ ID No. 6 , SEQ ID No. 8, SEQ ID No. 10, wherein preferably the isolated polypeptide is of human origin. 185 293 29. An antisense molecule that hybridizes to a nucleic acid molecule having or complementary to the nucleotide sequence shown in SEQ ID No. 3, or to a nucleic acid molecule having a nucleotide sequence capable of hybridizing to SEQ ID No. 3 or complementary to it under stringent hybridization conditions in the presence of 0, 1 - 1 x SSC / 0.1% w / w SDS at 60 ° C for 1-3 hours. 30. An antisense molecule according to claim 1 29, characterized in that it hybridizes with a nucleic acid molecule containing a nucleotide sequence selected from SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, or a nucleic acid molecule that encodes a polypeptide containing an amino acid sequence selected from SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, or an amino acid sequence having at least 70% similarity to SEQ ID No. 4. 31. A pharmaceutical for inducing the proliferation of astroglia in a mammal and / or increasing the survival and / or proliferation of mammalian neurons comprising an active agent and one or more pharmaceutically acceptable carriers and / or diluents, characterized in that the active agent comprises an isolated polypeptide having at least one among the following features: (i) the ability to induce proliferation of vascular epithelial cells; (ii) the ability to interact with a family of receptors, and (iii) the ability to induce cell migration, cell survival, and / or an increase in intracellular levels of alkaline phosphatase; wherein the polypeptide comprises: a. Amino acid sequence encoded by the nucleotide sequence shown in SEQ ID No. 3 or a nucleotide sequence capable of hybridizing to SEQ ID No. 3 or complementary thereto under stringent hybridization conditions in the presence of 0.1 - 1 x SSC / 0.1 wt%. / wt. SDS at 60 ° C for 1-3 hours, or b. the amino acid sequence shown in SEQ ID No. 4, or an amino acid sequence having at least 70% similarity thereto; or a derivative of such a polypeptide, showing at least one of the features (i) - (iii). 32. The pharmaceutical composition according to claim 32 31, characterized in that the isolated polypeptide comprises an amino acid sequence encoded by a nucleotide sequence selected from SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7 and SEQ ID No. 9, or an amino acid sequence selected from SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, wherein the isolated polypeptide is preferably of human origin or the mature form of the polypeptide with a cleaved leader sequence. 33. A host cell characterized in that it comprises a nucleic acid molecule having the nucleotide sequence shown in SEQ ID No. 3 or a nucleotide sequence capable of hybridizing to or complementary to SEQ ID No. 3 under stringent hybridization conditions in the presence of 0.1 -1 x SSC / 0.1% w / w SDS at 60 ° C for 1-3 hours. 34. The host cell of claim 34 33, characterized in that the nucleic acid molecule comprises a nucleotide sequence selected from SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9, or encodes a polypeptide comprising an amino acid sequence selected from SEQ ID No. 4, SEQ ID No. 6 , SEQ ID No. 8, SEQ ID No. 10, or an amino acid sequence having at least 70% similarity to SEQ ID No. 4. 35. A vector, characterized in that it comprises a nucleic acid molecule, a nucleic acid molecule having the nucleotide sequence shown in SEQ ID No. 3 or a nucleotide sequence capable of hybridizing to SEQ ID No. 3 or complementary to it under stringent hybridization conditions in the presence of 0.1-1. x SSC / 0.1% w / w SDS at 60 ° C for 1-3 hours. 36. The vector of claim 36 35, characterized in that the nucleic acid molecule comprises a nucleotide sequence selected from SEQ ID No. 3, SEQ ID No. 5, SEQ ID No. 7, SEQ ID No. 9 185,293 or encodes a polypeptide having an amino acid sequence selected from SEQ ID No. 4, SEQ ID No. 6, SEQ ID No. 8, SEQ ID No. 10, or an amino acid sequence having at least 70% similarity to SEQ ID No. 4. * * * The disclosure generally relates to a secretion molecule having properties similar to those of the vascular epithelial growth factor and the genetic sequence encoding it. The molecule will be useful in the development of a variety of therapeutic and diagnostic agents useful in the treatment, prevention and / or diagnosis of conditions requiring increased or decreased vascular and / or vascular permeability. The molecule of the disclosure also serves as a useful effector of major or central neurons and is capable of inducing astroglial proliferation. The molecule is also a useful effector of major and central neurons and can induce astroglial proliferation. The bibliographic details of the publications referred to in the description by the author's name are gathered at the end of the description. The sequence identification numbers (SEQ ID NO.) Relating to the nucleotide and amino acid sequences mentioned in the specification are indicated after the bibliography. Throughout the specification, unless otherwise indicated, the word "include" or variations thereof such as "includes" or "including" refers to the inclusion of a specified item or unit, or group of elements or units, but does not exclude any other element or entity or group of elements. or units. Vascular epithelial growth factor (hereinafter referred to as "VEGF"), also known as vascular permeability factor, is a secreted, covalently linked homodimeric glycoprotein that specifically activates epithelial tissue (Senger et al., 1993). A number of functions attributed to VEGF, such as its role in normal angiogenesis, include yellow body formation (Yan et al., 1993) and placenta development (Sharkey et al., 1993), regulation of vascular permeability (Senger et al., 1993), inflammation in angiogenesis (Sunderkotter et al., 1994) and autotransplantation (Dissen et al., 1994), as well as a role in human disease such as tumor-inducing angiogenesis (Folkman and Shing, 1992), rheumatoid arthritis (Koch et al., 1994), and retinopathy diabetic (Folkman and Shing, 1992). Therefore, VEGF is an important molecule that represents a potentially valuable target for research into therapeutic, prophylactic and diagnostic agents based on VEGF or its activity. There is also a need to identify homologues or other related molecules for use as substitutes for VEGF or in combination with VEGF. In the works leading to the invention, the gene susceptibility to numerous tumors of the endocrine gland type I system (MEN1) was searched for. It was surprisingly found that the genetic sequence that was excluded as a candidate for the MEN1 gene was a new growth factor showing some similarity to VEGF. Moreover, the disclosed growth factor is an effector molecule for major and central neurons. In one aspect, the present specification discloses a biologically isolated protein molecule comprising an amino acid sequence which: (i) it is at least 15% similar to the amino acid sequence given in SEQ ID No. 2 and (ii) it is at least 5% dissimilar to the sequence given in SEQ ID No. 2. In another aspect, the disclosure relates to a biologically isolated proteinaceous molecule which: (i) comprises an amino acid sequence showing at least about 15% similarity, but also at least about 5% dissimilarity with all or part of the amino acid sequence set forth in SEQ ID No. 2; and 185 293 (ii) has at least one property in common with VEGE In a related aspect, the disclosure relates to a biologically isolated protein molecule which: (i) it comprises an amino acid sequence which is at least about 15% similar, but also at least about 5% similar, with all or part of the amino acid sequence set forth in SEQ ID No. 2; and (ii) shows at least one of the following properties: (a) the ability to induce the proliferation of vascular epithelial cells; (b) the ability to interact with a family of receptors; and (c) the ability to induce cell migration, cell survival, and / or increase intracellular levels of alkaline phosphatase. The term "biologically separated" means that the molecule has undergone at least one purification step from a biological source. Preferably, the molecule is also biologically pure, meaning that the composition comprises at least about 20%, more preferably at least about 40%, particularly preferably at least about 65%, and even more preferably at least about 80-90% or more by weight of the molecule. to other compounds in the composition when determined by weight, on an activity basis, or in any other convenient manner. Most preferably the molecule is sequentially pure. In another preferred aspect, the disclosure relates to the molecule in recombinant form. In accordance with this aspect, the disclosure relates to a recombinant molecule comprising an amino acid sequence which: (i) it is at least 15% similar to the amino acid sequence given in SEQ ID No. 2 and (ii) it is at least 5% dissimilar to the sequence given in SEQ ID No. 2. In a related aspect, the disclosure relates to a recombinant molecule which: (i) it comprises an amino acid sequence which is at least about 15% similar, but also at least about 5% similar, with all or part of the amino acid sequence set forth in SEQ ID No. 2; and (ii) has at least one property in common with VEGR In another related aspect, the disclosure relates to a recombinant molecule which: (i) it comprises an amino acid sequence which is at least about 15% similar, but also at least about 5% similar, with all or part of the amino acid sequence set forth in SEQ ID No. 2; and (ii) shows at least one of the following properties: (a) the ability to induce the proliferation of vascular epithelial cells; (b) the ability to interact with the flt-1 / flk-1 receptor family, and (c) the ability to induce cell migration, cell survival and / or increase intracellular levels of alkaline phosphatase. The disclosure also relates to genomic or partial genomic clones encoding a protein molecule that exhibits at least about 15% similarity, but also at least about 5% similarity with SEQ ID NO: 1. The amino acid sequence set forth in SEQ ID NO: 2 corresponds to human VEGF (referred to herein as "VEGF ₁₆₅ "). thus, the disclosed molecule is VEGF-like or a homologue of VEGF except that it contains an amino acid sequence similar, but not identical to, a VEGF amino acid sequence. While the invention exemplifies a human VEGF-like molecule, it is realized that the disclosure also relates to a homologous molecule and coding sequence from other mammals such as farm animals (e.g., sheep, pigs, horses and cows), domestic animals ( e.g. dogs and cats) and laboratory experimental animals (e.g. mice, rats, rabbits and guinea pigs), and also from non-mammals such as birds (e.g. poultry), fish and reptiles. In a most preferred embodiment, the VEGF-like molecule is 185,293 of human origin and is encoded by the gene located on chromosome 11q13. thus, the disclosure extends to the genomic sequence, or portion thereof, encoding a VEGF-like molecule of disclosure. Preferably, the percent similarity is at least about 30%, even more preferably at least about 40%, particularly preferably at least about 50%, even more preferably at least about 60-70% and particularly preferably at least about 80-95%, based on all or part of the amino acid sequence given in SEQ ID No. 2. In a particularly preferred embodiment, the VEGF-like molecule comprises the amino acid sequence given in SEQ ID No. 4, or a part, fragment, derivative or analog thereof. The particularly favorable similarities are around 19-20% and 29-30%. References to derivatives also include spliced variants. thus, the disclosure extends to the folded variants of the SOM175. Exemplary folding variants encompassed by the scope of the disclosure include, but are not limited to, variants with an amino acid sequence substantially equal to that given in at least one of SEQ ID No. 6, q6, SEQ ID No. 8, and / or SEQ ID No. 10, or mutants thereof or derivatives and subsequent folding variants. In another embodiment, the disclosure relates to a recombinant molecule which: (i) comprises an amino acid sequence essentially identical to that given in SEQ ID No. 4, or at least about 15% similar to all or part thereof, provided that the amino acid sequence is at least 5% dissimilar to all or part of it. the amino acid sequence given in sEQ ID NO. 2; and (ii) it has at least one biological property in common with VEGF. In a further embodiment, the disclosure relates to a recombinant molecule which: (i) has an amino acid sequence essentially identical to that given in SEQ ID No. 6, or at least about 15% similar to all or part thereof, provided that the amino acid sequence is at least 5% dissimilar to all or part of it. the amino acid sequence given in sEq ID NO. 2; and (ii) it has at least one biological property in common with VEGF. In yet another embodiment, the disclosure relates to a recombinant molecule which: (i) has an amino acid sequence essentially identical to that given in SEQ ID No. 8, or at least about 15% similar to all or part thereof, provided that the amino acid sequence is at least 5% dissimilar to all or part of it. the amino acid sequence given in SEQ ID NO: 2; and (ii) it has at least one biological property in common with VEGF. In a further embodiment, the disclosure relates to a recombinant molecule which: (i) comprises an amino acid sequence essentially identical to that given in SEQ ID No. 10, or at least about 15% similar to all or part thereof, provided that the amino acid sequence is at least 5% dissimilar to all or part of it. the amino acid sequence given in sEq ID NO. 2; and (ii) it has at least one biological property in common with VEGF. Such properties of VEGF include at least one of the following: (a) the ability to induce the proliferation of vascular epithelial cells; (b) the ability to interact with the flt-1 / flk-1 receptor family; and, (c) the ability to induce cell migration, cell survival, and / or increase intracellular levels of alkaline phosphatase. Preferably, the similarity is at least about 40%, even more preferably at least about 50% and particularly preferably at least about 65%. In yet another aspect, the disclosure relates to a peptide fragment corresponding to a portion of the amino acid sequence set forth in SEQ ID No. 4, a splice variant thereof, such as the variant set out in SEQ ID No. 6, SEQ ID No. 8 or SEQ ID No. 10, or a chemical equivalent thereof. The biologically secreted or recombinant molecule of the disclosure may be naturally glycosylated, or may contain an altered glycosylation pattern depending on the cells from which it is isolated or in which it has been isolated. 185 293 synthesized. For example, if a molecule has been produced recombinantly in prokaryotes, it will not be glycosylated. The molecule may be full-length naturally, or it may be truncated or otherwise modified. In yet another aspect, the disclosure relates to a nucleic acid molecule encoding a VEGF-like molecule described herein. in particular, the disclosure relates to a nucleic acid molecule comprising a nucleotide sequence substantially as shown in SEQ ID No. 3, or having at least 15% similarity to all or part thereof, or capable of hybridizing under low stringency conditions to the inverse complement of the nucleotide sequence shown in SEQ ID No. 3. SEQ ID No. 3, provided that the nucleic acid sequence is at least 15% similar, but also at least 30% dissimilar to the nucleotide sequence shown in SEQ ID No. 3. The nucleotide sequence shown in SEQ ID No. 3 is also referred to herein as "SOM175". Preferably, the percent dissimilarity is about 35%, even more preferably about 39% and particularly preferably about 40-50% or greater. For the determination of the level of stringency, reference is conveniently made to Sambrook et al. (1989), pp. 9.47-9.51, which is incorporated by reference, wherein the stated conditions of the washing steps are considered to be of high stringency. Low stringency conditions are defined as washing in 4-6 x SSC / 0.1 -0.5% w / v SDS at 37-45 ° C for 2-3 hours, depending on the source and concentration of the nucleic acid involved in the hybridization, other stringency conditions may be used, e.g., medium stringency conditions, which are considered herein to be washed in 1-4x SSC / 0.25-0.5% w / v SDS at> 45 ° C for 2-3 hours, or high stringency conditions, which are considered washing in 0.1-1 x SSC / 0.1% w / v SDS at 60 ° C for 1-3 hours. The disclosure also relates to a nucleic acid molecule that encodes a VEGF-like molecule as defined above having at least 15% homology with SEQ ID NO: 3. Preferably, the homology level is at least about 40%, and even more preferably about 60-70%. The disclosure further relates to a mouse homologue of human VEGF (referred to herein as "mVEGF"). mVEGF shows approximately 85% identity and 92% retention of amino acid residues over the entire coding region to human VEGF. mVEGF is encoded by a nucleic acid molecule containing a nucleotide sequence essentially as shown in Figure 9. The VEGF-like molecule will be useful, alone or in combination with other molecules such as VEGF, in the development of a variety of therapeutic and / or diagnostic applications, therefore the disclosure also relates to pharmaceutical compositions comprising a VEGF-like molecule or parts, fragments, derivatives thereof, homologs or analogs, together with one or more pharmaceutically acceptable carriers and / or diluents. The disclosure further encompasses vectors comprising, or similar to, the nucleic acid sequence set forth in SEQ ID No. 3 at least about 15%, even more preferably about 40%, and particularly preferably about 60-70%, but simultaneously at least 30%, even more preferably about 39% dissimilarity, as well as host molecules containing them. In addition, the disclosure includes ribozymes and antisense molecules based on SEQ ID No. 3, as well as neutralizing antibodies to a VEGF-like molecule. Such molecules may be useful to ameliorate effects such as, for example, overexpression of VEGF-like genes leading to angiogenesis and vascularization of tumors. In another aspect, the disclosure relates to a method of inducing astroglial proliferation in a mammal comprising administering to the mammal an effective amount of a recombinant proteinaceous molecule which: (i) it comprises an amino acid sequence showing at least about 15% similarity, but also at least about 5% dissimilarity with the sequence given in SEQ ID NO: 2; and (Ii) exhibits at least one property in common with vascular epithelial growth factor (VEGF). Preferably, the recombinant protein molecule comprises the amino acid sequence set out in SEQ ID No. 3 or SEQ ID No. 6. In a further embodiment, the disclosure provides a method for increasing the survival and / or proliferation of neurons in a mammal by administering to the mammal an effective amount of the recombinant protein molecule. which: (i) it comprises an amino acid sequence which is at least about 15% similar, but also at least about 5% similar, to the sequence set out in SEQ ID No. 2; and (ii) has at least one property in common with vascular epithelial growth factor (VEGF), such administration being for a period of time and under conditions sufficient to cause astroglial proliferation. Preferably, the recombinant protein molecule comprises the amino acid sequence given in SEQ ID No. 3 or SEQ ID No. 6. The disclosure also relates to antibodies to a VEGF-like molecule or nucleic acid probes for a gene encoding a VEGF-like molecule useful as diagnostic agents.