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WO1995006116A1 - Mammalian stromal cell proteins and dna sequences coding therefor - Google Patents

Mammalian stromal cell proteins and dna sequences coding therefor Download PDF

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Publication number
WO1995006116A1
WO1995006116A1 PCT/US1994/009854 US9409854W WO9506116A1 WO 1995006116 A1 WO1995006116 A1 WO 1995006116A1 US 9409854 W US9409854 W US 9409854W WO 9506116 A1 WO9506116 A1 WO 9506116A1
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Prior art keywords
protein
stromalin
cells
sequence
polypeptide
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PCT/US1994/009854
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French (fr)
Inventor
Dov Zipori
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Yeda Research And Development Company Limited
Rycus, Avigail
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Priority to AU77182/94A priority Critical patent/AU7718294A/en
Publication of WO1995006116A1 publication Critical patent/WO1995006116A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues

Definitions

  • the present invention concerns new mammalian proteins found in stromal cells.
  • a protein of the kind discovered in accordance with the invention is designated herein as "stromalin " .
  • the present invention also concerns DNA sequences encoding stromalin.
  • Blood cells have a short life span, and the continuity of vital hemopoietic functions is strictly dependent on the constant production of new cells. All types of blood cells are derived from a common hemopoietic stem cell. These stem cells are pluripotent and form various types of colonies (erythroid, granulocytic, etc.). The first step in the differentiation of stem cells involves the generation of progenitors, committed to either the erythroid, granulocytic and megakaryocytic lineages or to lymphoid stem cells.
  • CSF colony-stimulating factors
  • Stromal cells support long-term hemopoiesis with prolonged stem cell renewal, as was shown in a number of studies.
  • a clonal population of endothelial-adipocytes from mouse bone marrow represented by the cell line 14F1.1
  • the very stromal cells responsible for the stem cell maintaining activity of the heterogeneous stromal cell population (Zipori and Lee, 1987).
  • These cells did not produce substantial amounts of any CSF and their conditioned media did not have biological activities that could account for their effects on the stem cells.
  • stromal cell factor IL-7 was found to be a proliferation factor for pro-B, pre-B and pro-T cells (Henney, 1989). This factor is partially responsible for lymphopoiesis supported by stromal cells but does not account for long-term hemopoiesis and stem cell renewal.
  • SCF Stem Cell Factor
  • the stromal CSF has a relatively poor colony-forming ability, but is a potent synergistic factor. So far, however, it has not been shown to be a stem cell renewal factor, and it is secreted in ample amounts by stromal cells incapable of supporting hemopoiesis.
  • SUBSTITUTE SHEET (RULE 28) cultures was Thy-1.2 + , CD4 " , CDS ' , Pgp-1 + (Tamir et ai, 1990) and had the T cell receptor gene in the germline configuration (Tamir et ai, 1989).
  • the incidence of cells with such a phenotype is very low in the fresh thymocyte population initially seeded with the stroma.
  • the proliferation of the precursor T cells depended strictly on the support of cloned stromal cell lines. Addition of IL-2 to the culture system augmented T cell yield but was not mandatory. When tested after removal from the stromal layer, the cells had no cytotoxic activity. They became highly cytotoxic when removed from the stroma and propagated with IL-2 for a week or longer, prior to testing.
  • stromal cells exhibit a capacity to support the renewal and accumulation of double-negative thymocytes (precursor T cells) but do not allow their further differentiation even in the presence of IL-2.
  • the 14F1.1 stromal cell line supports myelopoiesis and lymphopoiesis of cells of both the B and the T lineage.
  • fibroblasts In contrast to hemopoietic cells that can be classified precisely into a variety of lineages, cells of the stromal family are usually referred to mostly with respect to their morphological appearance (i.e., "fibroblasts" or
  • epidermal cells etc.
  • This is mainly due to the fact that cell surface markers for this heterogeneous cell population are unknown. Further, it has been found that stromal cells differ markedly with respect to the composi ⁇ tion of the extracellular matrix that they deposit. This is clearly an important feature, since many cell functions are directly influenced by the mode of the cells' attachment to surfaces and to each other. To date, very little is known about the molecular basis for the differences between the various types of stromal cells, e.g. differences in gene expression. Further, very little is known about the molecular basis for the above-mentioned biological activity of stromal cells, i.e. their ability to support long-term hemopoiesis with prolonged stem cell renewal.
  • Elucidation of this interaction may provide the means for modulation of stem cell growth in individuals suffering from various diseases associated with abnormal stem cell modulation, e.g. various hemopoietic diseases such as aplastic anemia, myelofibrosis as well as various leukemias. Moreover, many of the problems associated with bone marrow transplantation may be resolved when stem cell growth can be augmented. Further, elucidation of the above interaction may also provide the means for diagnosing, at an early stage, diseases caused by an alteration in the normal mechanism of stem cell modulation.
  • B92 a hybridoma, designated B92, obtained following immunization of mice with mice stromal cells of the line 14F1.1, produced a monoclonal antibody, designated herein as B92 monoclonal antibody (B92 MAb), that recognizes, i.e. binds to, a novel antigen found in 14F1.1 cells.
  • This antigen was designated as "stromalin- 1 ".
  • the cDNA encoding the stromalin-1 was isolated and cloned. By homology to the cDNA encoding murine stromalin-1, a human gene encoding a human "stromalin- 1 " was identified and cloned.
  • stromalin-2 On the basis of homology with stromalin-1, a human stromal protein "stromalin-2", was also identified and cloned.
  • stromal cells such as the mouse endothelial-adipocyte stromal cell line, 14F1.1, support long- term hemopoiesis and stem cell renewal. Accordingly, the novel stromalin protein may be related to the ability of 14F1.1 to interact with stem cells and thus these proteins, as well as their analogs or fragments (as defined
  • stromalin-1 and stromalin-2 proteins from other mammals, and other stromalins from both human and non-human mammals, may be isolated.
  • the present invention thus provides novel proteins or polypeptides expressed in stromal cells. These proteins or polypeptides may be used for various therapeutic or diagnostic purposes. These proteins or polypeptides may be purified from biological samples or may be obtained by genetic engineering methods. Proteins obtained by means of genetic engineering are very often produced as fusion proteins and fusion proteins of the invention may be used as such for various purposes.
  • the present invention provides a protein or a polypeptide being a member of the group consisting of:
  • the present invention further provides a DNA molecule comprising a sequence coding for the proteins or polypeptides of the invention.
  • the DNA molecules according to the invention are, for example, those selected from the group consisting of:
  • nucleic acid sequences encoding the same protein or polypep ⁇ tide as the nucleic acid sequence of (a);
  • SUBSTITUTE SHEET (RULE 2 ⁇ ) (e) a nucleic acid sequence which hybridizes to any of sequences under (a), (b), (c) or (d), under high stringency conditions; and
  • DNA molecules defined under (f) are various DNA vectors comprising also a sequence required for the propagation and replication of the vector in a host cell and at times also a sequence controlling expression of said nucleic acid sequence.
  • the invention will now be illustrated by the following non- limiting example and by the figures.
  • Fig. 1 shows the map of plasmid pGEMSA-1, described in Example 3;
  • Fig. 2 shows the results of the Northern blotting of extracts from 14F1.1 cells probed with a -actin probe as described in Example 3;
  • Fig. 3 shows the results of the Northern blotting analysis of mRNA extracted from various tissues and probed with a pGEMSA-I or -actin probe, also described in Example 3;
  • Fig. 4 shows the results of the PCR analysis of cDNA from various tissues using a stromalin-1 3' probe also described in Example 3;
  • Fig. 5 shows the immunoprecipitation products of in vitro translated RNA transcripts from the expression of a pGEMSA-1 clone, described in Example 4;
  • Fig. 6 shows the nucleotide sequence of the murine stromalin-1 gene as described in Example 5;
  • Fig. 7 shows the amino acid sequence of the murine stromalin- 1 protein as described in Example 5;
  • Fig. 8 shows the nucleotide sequence of the human stromalin-1 gene as described in Example 5;
  • Fig. 9 shows the amino acid sequence of the human stromalin-1 protein as described in Example 5;
  • Fig. 10 shows the nucleotide sequence of the human stromalin-2 gene sequence, described in Example 6;
  • Fig. 11 shows the amino acid sequence of the human stromalin-2 protein amino acid sequence, also described in Example 6;
  • Fig. 12 depicts MBA-1.1.1 stromal cells grown on a Labtek slides fixed in methanol and stained with a B92 MAb as described in Example 7:
  • Fig. 12A depicts the cells after normal growth
  • Fig. 12B depicts these cells first treated with antisense oligonucleotide of the stromalin-1 gene prior to staining;
  • Fig. 13 shows immuno precipitation results of MBA 15 stromal cell lysates with antisera obtained following immunization of rabbits with 796 (lane 1) and 795 (lane 2) peptides. Immuno precipitative proteins were subjected to Western blot analysis using B92 monoclonal antibody;
  • Fig. 14 shows results of Western blotting of nuclear proteins with B92 antibody (left lane) and of cytoplasmic proteins (right lane);
  • Fig. 15 depicts Ml myeloid cells fixed and then stained with 740 polyclonal antiserum, as described in Example 9:
  • Fig. 15A shows cells fixed with formaldehyde (FA) followed by Triton X100 and then reacted with the 740 antiserum;
  • Fig. 15B shows the same cells after fixation with methanol followed by reaction with the 740 antiserum
  • Fig. 15C is a conrol - cells fixed with FA followed by Triton and then reacted with a control non-immune serum.
  • Fig. 16 depicts results of an immuno staining of MBA-1.1.1 stromal cells with 740 polyclonal antiserum as described in Example 9:
  • Fig. 16A shows cells fixed with FA and Triton
  • Fig. 16B shows cells fixed with FA without Triton
  • Fig. 16C shows the same cells fixed with FA and Triton and with a control non-immune serum
  • Fig. 17 shows COS-7 monkey cells shown in a similar manner as in
  • Fig. 18 is a schematic representation of the manner of preparation of a construct containing an antisense stromalin- 1 fragment, described in Example 10;
  • Fig. 19 shows the morphological changes occurring in a clone of
  • MBA-15 cells permanently transfected with a plasmid containing an antisenses form of stromalin-1:
  • Fig. 19A shows a lower magnification depicting dead cells in the center of the colony (upper right portion of the picture) with viable cells at the periphery;
  • Fig. 19B shows a higher magnification view of the cells
  • Fig. 20 shows a transformed foci after myc and ras transformation (upper plate), following cotransfection of the cells with p53 (middle plate) and following cotransfection with the stromalin-1 (SA1; lower plate).
  • B92 stands for epitope recognized by B92 antibody
  • Cho stands for a carbohydrate group
  • Glc stands for a glycoaminoglycan
  • Myr stands for a myristyl group
  • NH2 stands for an amide group
  • SO4 stands for a sulfate group
  • SCBS stands for stem cell binding site
  • SPCS stands for signal peptide cleavage site.
  • DMEM Dulbecco's Modified Eagle's Medium, supplemented with 2mM glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, and
  • DMEM-FCS DMEM supplemented with fetal calf serum (FCS) inactivated at (56°C, 30 min).
  • HAT-medium DMEM-FCS supplemented with 1 ml of each of following components for each 100 ml HAT medium:
  • Hypoxanthine 10 4 M, prepared as a 100 x stock solution containing 136 mg hypoxanthine dissolved in 100 ml distilled water, dissolution at 60-70°C;
  • HT-medium DMEM-FCS supplemented with hypoxanthine and thymidine as above, but without aminopterin.
  • PEG Polyethylene glycol
  • SUBSTITUTE SHEET (RULE 28) solution still warm.
  • 20 g of PEG in a 100 ml flask is sterilized in an autoclave for 15-20 min, cooled to 50°C to which is added 28 ml of DMEM, mixed and stored at room temperature (R.T.).
  • Myeloma cells - Myeloma cells used for hybridization were the NSO cells. This cell line does not produce any immunoglobulin, it grows in the presence of 20 g/ml 8-azoguanine (8-AZG) and therefore dies in HAT-medium.
  • NSO cell line is of H-2 d and grows in BALB/c mice. NSO cells are grown in petri dishes or tissue culture bottles to a concentration of 1.0 - 2.5 x 10 6 cells/ml in DMEM-HS then transferred to fresh flasks at cell concentration of about 5 x 10 4 /ml. To avoid revertants it is recommended to grow the NSO cells in the presence of 8-AZG for at least one week before the fusion.
  • Immuno-spleen cells BALB/c female mice were immunized at the age of 6-8 weeks followed by a booster immunization (prefusion boost) 3-4 days before fusion, by either an I.V. injection, or if not possible by interperitoneal injection. Hyperimmune animals were allowed to rest at least 3 weeks before the prefusion boost.
  • Spleen cells were prepared by removing spleens from mice in a sterile hood (metal-hood) and placing them into DMEM in a petri dish. The spleens were then teased apart with sterile forceps, the cells were resuspended and transferred to a 10 ml conical tube, which was left in a vertical position for about 10 min on ice, during which large pieces of tissue (spleen debris) precipitated. The single cell suspension was then removed
  • Myeloma cells were prepared by removing NSO cells from an incubation flask, at a logarithmic growth phase of the cells, and placed into a 50 ml conical tube from which a sample was taken for counting (diluted 1:1 with 0.1% Trypan-blue), and the remainder of cells in the conical tube were centrifuged at 1000 rpm (200 x g) for 10 min. The precipitated cells were then resuspended in DMEM to a cell concentration of 10 x 10 6 cells/ml.
  • DMEM-FCS-HAT prewarmed DMEM-FCS-HAT
  • 5 ml of prewarmed DMEM-FCS-HAT was added and the cells resuspended very carefully to prevent breaking the fused cells apart.
  • About 10 ml DMEM- FCS-HAT was added and a sample was taken for counting the viable cells, in particular the number of viable NSO cells which is larger than the number of the viable spleen cells.
  • the remaining fused cells were then suspended in DMEM-FCS-HAT to a concentration of 2-5 x 10 4 viable NSO cells/ml and 0.1 ml aliquots were distributed into each well of 96 well microplate, which was incubated at 37°C in an 10% CO 2 humid incubator.
  • the cells obtained according to above procedure were diluted to 0.25 cell/well. After 10 days the clones were tested by radioimmuno- assay (RIA). The positive clones were grown, and the cloning efficiency determined.
  • RIA radioimmuno- assay
  • hybridomas selected from cloned hybridomas according to (d) above, were grown in DMEM supplemented with 10% FCS until they reached confluence. The cells were removed by centrifugation and the supernatant was filtered. (ii) Ammonium Sulfate precipitation of Immunoglobulins:
  • Immunoglobulins (Ig) were precipitated at 33-50% saturated ammonium sulfate at 4°C.
  • SUBSTITUTE SHEET (RULE 28) completely dispersed in the ammonium sulfate solution each time, each wash being followed by centrifugation as before.
  • the pellet after the second wash was dissolved in water in 1/2 - 1/10 of the original serum volume and then transferred into a prewashed dialysis tube and dialyzed against more than 10 6 volumes of saline or PBS at 4°C and centrifuged to remove precipitate.
  • the so-obtained monoclonal antibodies were frozen at -70°C, or with 0.1% NaN 3 at 4°C, for storage purposes, (iv) Determination of the antibody class by the Ouchterlony test:
  • the double immunodiffusion test where antigen and antibody form a precipitating line in agar which is the easiest assay to determine the Ig class and subclass was used.
  • the sensitivity of this assay (-10 mg/ml) allows for the analysis of the monoclonal antibody (mAb) class already in a high density hybridoma culture supernatant without the need to first prepare large amounts of mAb by the ascite fluid method (see (e)(iii) above).
  • the immunoglobulin (Ig) from the hybridoma culture supernatant could be precipitated in 50% saturated ammonium sulfate and the pellet dissolved in 1/10 of the original volume with water (as in (e)(ii) and (e)(iii) above).
  • standard SerotecTM mouse Ig RID plates were used for determination of the mAb class.
  • the uv-2237 IP3 ⁇ gtl l library (8 x 10 9 PFU/ml) was plated on 12 plates (150 mm) 30,000 PFU/plate. Each plate contained LB, pH 7.5, with Mg (lOmM), Amp (lOOmg/ml). Cells Y1090 (Promega), were grown at 37°C overnight, while agitating with LB Mg, pH 7.5, Maltose 0.2%. The overnight culture was centrifuged and resuspended in 1/2 volume lOmM MgS0 4 , and kept at 4°C. The phage from the above library was absorbed to the above cells for 20 min, 37°C, 9 ml Top Agar (0.7%) was then added and the mixture plated. The plates were incubated at 42°C, 3
  • the filter was then treated as follows: 10 min with 10% LFM, PBS, followed by 10 min with 10% LFM, PBS, 0.1% Tween 20 and then 10 min with 10% LFM, PBS. Incubation was then carried out with the 2nd antibody (1 -1.5 hrs at R.T.), 2nd antibody being I 125 labelled rabbit-anti-mouse (Jackson) 10 6 cpm/ml, 200 ml in 10% LFM in PBS. The filter was subsequently washed as before and exposed to X-ray.
  • Tissue Expression of stromalin-1 at the mRNA Level mRNA was extracted by standard procedures from 14F1.1 stromal cells and subjected to Northern blotting using the plasmid pGEMSA-I (see Fig. 1 showing the plasmid's map) or a probe for ⁇ -actin oligolabelled with 32 P as hybridization probe.
  • the Northern blotting analysis results indicate the presence of a single 6 kbp transcript in the mRNA from 14F1.1 cells shows hybridization of the ⁇ -actin probe by the mRNA from 14F1.1 and IP-3 cells.
  • stromalin-1 The expression of the stromalin-1 gene was then examined in various tissues and again Northern blots showed a 6 kbp transcript. Smaller signals were also seen. These results are shown in Fig. 3, wherein mRNA was extracted from the various indicated tissues and subjected to Northern blotting using hybridization probes for the stromalin-1 mRNA (this being a 1.4 kbp cDNA insert) of pGEMSA-1 and a probe for ⁇ -actin mRNA. Ribosomal mRNA (18S and 28S) served as markers.
  • PCR was carried out by standard techniques using cDNA from the various tissues.
  • the probes chosen were derived from a 307 base segment from the 3' end of the stromalin- 1 coding sequence. It was observed that various tissues gave a signal.
  • the bone marrow that expresses a 50 kDa protein showed a PCR product indistinguishable from that found in other tissues.
  • 5'-GTdTCTAACTGGCTCCGTG-3' both from the 3' end of the B92 coding sequence, reveals stromalin-1 cDNA amplification products of 307 bp in different hemopoietic and non-hemopoietic tissues.
  • the plasmid SA-I containing the 1.4Kb stromalin-1 insert was linearized for transcription from either the T7 or the SP6 sites.
  • In vitro transcription of RNA from T7 or SP6 promotors was performed using the PromegaTM kit (Madison, Wl, USA).
  • In vitro translation of the transcripts was done using the Strategene TM kit (La Jolla, CA., USA).
  • the products were immunoprecipitated with B92 antibody and run on a 13% acrylamide SDS gel. The results of the immunoprecipitation are depicted in Fig. 5 and as can be seen there is a band of about 32 kDa from the T7 transcript, shown by an arrow. No transcription product was obtained from the SP6 direction.
  • Example 2 By the procedure set forth in Example 1, about 500,000 individual clones from a ⁇ gtl l cDNA library were screened for positive plaques reactive with B92 MAb, 29 candidates were selected and 4 of them were eventually shown to be specifically reactive with the antibody. Protein extracts from these were reacted with antibodies to ⁇ -galactosidase and separately with B92 antibody. The results indicated that the 4 clones contained fusion proteins of about 160 kDa. This corresponds to a cDNA insert of about 1.4 kbp. An insert of this size was indeed observed when the phage DNA from all 4 clones was cut with EcoRI restriction endonuclease.
  • SUBSTITUTE SHEET Proteins translated in vitro in a reticulocyte lysate from transcription products (mRNA) from the expression of SA-I insert cloned into EcoRI site of pGEM (pGEM-SA-I clone), (see Example 4 and Fig. 4), were examined by immunoprecipitation. One protein product, detectable by the B92 MAb, was observed at 32 kDa. The sequence of cDNA insert in the pGEM-SA-I showed an open reading frame corresponding to a protein of only 18 kDa.
  • the pGEM-SA-I clone was cut with StuI restriction endonuclease, resulting in maintenance of all 540 bases of the open reading frame with additional 56 bases as template for in vitro transcription, and removal of the remainder of the putative non-coding region.
  • This clone was in vitro transcribed and translated in a reticulocyte system and the product was observed to migrate in a gel (polyacrylamide) as a 32 kDa doublet.
  • stromalin-1 murine and human full length genes that were designated as stromalin-1: (i) cloning by anchored polymerase chain reaction (PCR) of the unknown 5' region of the gene using mRNA from 14F1.1 cells and (ii) screening a human thymus cDNA library with labelled oligonucleotide probes.
  • PCR polymerase chain reaction
  • a mean of about 500 new nucleotides corresponding to the 5' coding region were obtained, and at least 2-3 clones were sequenced and compared for the sake of accuracy.
  • Figs. 6 and 7 show the nucleotide and amino acid sequences, respectively, corresponding to the murine full length open reading frame.
  • the complete open reading frame of the murine stromalin-1 gene spans about 3774 bases and encodes a protein of about 144.7 kDa.
  • 4 different screenings of the human thymus cDNA library have been carried out; in each one, 500,000 plaque forming units were screened.
  • the nucleotide and amino acid sequence of the human stromalin-1 protein is shown in Figs. 8 and 9, respectively.
  • the sequence of the stromalin-1 gene does not show homology with any sequence found in updated gene data banks, i.e. it represents a new gene sequence. It is important to mention the presence of a REDV sequence (positions 974-977 of the murine sequence and positions 1038-1041 of the human sequence), which has been described as an adhesion sequence found in the IIICS alternatively spliced region of fibronectin and interacts with NLA-4 integrin, namely, this is a putative binding site for the adhesion of early stem cells (day 12 CFU-S) to fibronectin, through the cell surface protein, integrin VLA-4.
  • the protein contains a potential glycosaminoglycan binding site at the C- terminal end (position 1187-1190 of the murine sequence and 1251-1254 of the human sequence).
  • An alignment of the murine and human sequences shows a very high homology (about 92.9%) between the murine and human genes and an extremely high homology (about 98.9%) between the murine proteins suggesting a very important role for this molecule.
  • a plaque forming unit (clone) with reduced but significant hybridization was obtained; the cD ⁇ A insert was sequenced and a fragment of about 1200 nucleotides (Fig. 10) was obtained. It codes for a gene showing a 68% nucleotide homology and a 71 % amino acid homology (Fig. 11) with the human stromalin-1, and was designated stromalin-2 gene. The presence of the stromalin-2 gene has also been observed by PCR amplification in the human K562 leukemic cell line and in the murine 14F1.1 stromal cell line.
  • Example 8 Derivatives of polyclonal antibodies that detect stromalin-l gene product by a combination of immunoprecipitation and immunoblotting and further evidence of nuclear localization of stromalin-l protein
  • the peptides were purified by chromatography on Sephadex G-25 and fractions were pooled and lyophilized. The purified peptides were dissolved in distilled water and were conjugated to Keyhole lymphet hemocyanine (KLH).
  • KLH Keyhole lymphet hemocyanine
  • New Zealand white rabbits were immunized by multisite intradermal injections of 1 mg of peptide conjugates in 0.5 ml of PBS emulsified in 0.75 ml of complete freund's adjuvant (CFA). Booster injections were administered 3 weeks later. Each peptide was injected to two rabbits. Antisera were purified either by a protein A column or by
  • stromalin recognized the recombinant stromalin as a 100 kDa and 130 kDa band.
  • the signals obtained by western blotting with the 796 antibodies were very weak compared with a strong background of numerous other nonspecific bands.
  • immunoprecipitation was combined with immunoblotting and results, shown in Fig. 13, indicate that a specific band at 100 kDa, as well as some higher molecular weight proteins, react with the antibody.
  • the B92 MAB showed that stromalin is indeed found in the nucleous in titer or that its antigenic epitope is more accessible there than in the cytoplasm, as can be seen in Fig. 14.
  • Example 9 Further properties of stromalin derivatives by immune staining using stromalin specific polyclonal antibodies
  • a series of polyclonal antibodies against synthetic peptides from straomalin-1 were prepared.
  • the synthetic peptides were chosen according to the Chou-Fasman prediction. Regions with the highest
  • SUBSTITUTE SHEET (RULE 28) antigenicity were chosen for the preparation of synthetic peptides. One of them was an antiserum against a peptide, designated herein as "740" (which corresponds to residues 451-465 of the murine stromalin-l), which had the following sequence: LAKRRGRNSPNGNLI
  • the antiserum to the 740 peptides was purified either by protein A or by peptide affinity column.
  • Ml myeloid cells were fixed with either 3% formaldehyde
  • the second antibody was a Texas Red conjugated doneky anti-rabbit
  • Figs. 15A and 15B Results from cells fixed with FA and with methanol are shown in Figs. 15A and 15B respectively. Some cultures were incubated with a control non-immune rabbit serum and the results are shown in Fig. 15C.
  • cytoplasmic staining was observed in COS-7 monkey cells, as shown in Fig. 17. These cells were later verified by Hoechst staining to be dividing cells.
  • An antisense construct of the stromalin-l gene containing the antisense nucleotides between positions 371 to 3358 of the stromalin gene was introduced into a PCEP4 plasmid, as shown schematically in Fig. 18. This construct was then transferred into a variety of cell types using a standard transfection. A series of clones were obtained from the MBA-15 cell lines wich showed an increase growth rate, crowding at confluence and loss of their contact inhibition.
  • Transformed foci assay is a standard assay used to demonstrate the function of tumor suppressor genes such as p53.
  • this assay the procedure of which is depicted schematically in . Fig. 20, formation of transformed foci by rat embryo fibroblasts upon transfection with a combination of myc and ras oncogens is tested following the addition of a suppressor gene, such as p53.
  • SA-1 stromalin-l gene

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Abstract

New mammalian proteins are disclosed, found in stromal cells and designated herein as stromalin. One such protein has been isolated from murine cells and two such proteins from human cells.

Description

MAMMALIAN STROMAL CELL PROTEINS AND DNA SEQUENCES CODING THEREFOR
FIELD OF THE INVENTION
The present invention concerns new mammalian proteins found in stromal cells. A protein of the kind discovered in accordance with the invention is designated herein as "stromalin " . The present invention also concerns DNA sequences encoding stromalin.
PRIOR ART
The following listed publications will be referred to in the present application:
1. Dexter, T.M. and Spooncer, E. (1987) Growth and differentiation in the hemopoietic system. Ann. Rev. Cell Biol. 3, 423-441. 2. Zipori, D. and Lee, F. (1988) Introduction of interleukin-3 gene into stromal cells from the bone marrow alters hemopoietic differentiation but does not modify stem cell renewal. Blood 71, 586-596.
3. Kalai, M. and Zipori, D. (1988) A quantitative assay for stroma- dependent hemopoiesis. In: Baum SJ, Dicke KA, Lotzova E, Pluznik
DH, eds. Experimental Hematology Today, Springer- Verlag, New
York, p. 25-30.
4. Peled, A., Kalai, M., Toledo, J. and Zipori, D. (1991) Stroma cell dependent hemopoiesis. Seminar in Hematology 28, 132. 5. Henney C. (1989) Interleukin-7 effects on early events in lympho¬ poiesis. Immunology Today 10, 170-173. 6. Tamir M, Eren R, and Globerson A. (1990) Selective accumulation of lymphocyte precursor cells mediated by stromal cells of hemopoi¬ etic origin. Exp. Hematol. 18, 332-340. 7. Tamir M, Harris N, Trainin N, Toledo J. and Zipori D. (1989) Multilineage hemopoiesis induced by cloned stromal cells. Int. J. Cell Cloning 7, 373-384.
BACKGROUND OF THE INVENTION AND PRIOR ART Blood cells have a short life span, and the continuity of vital hemopoietic functions is strictly dependent on the constant production of new cells. All types of blood cells are derived from a common hemopoietic stem cell. These stem cells are pluripotent and form various types of colonies (erythroid, granulocytic, etc.). The first step in the differentiation of stem cells involves the generation of progenitors, committed to either the erythroid, granulocytic and megakaryocytic lineages or to lymphoid stem cells. Certain cytokines, designated colony-stimulating factors (CSF) are known and are character¬ ized by an ability to induce differentiation coupled with extensive prolifera- tion and while these CSFs not only affect progenitors but also stimulate the in vitro differentiation of stem cells, their action is not coupled with a process of stem cell renewal. The molecular nature of the mechanism that protects the stem cell pool from exhaustion and enables the maintenance of potent stem cells throughout the entire life span of mammals is yet unknown, but, however, it seems to be connected with functions of stromal cells (Dexter & Spooncer, 1987).
Stromal cells support long-term hemopoiesis with prolonged stem cell renewal, as was shown in a number of studies. In one study a clonal population of endothelial-adipocytes from mouse bone marrow (represented by the cell line 14F1.1) was identified as the very stromal cells responsible for the stem cell maintaining activity of the heterogeneous stromal cell population (Zipori and Lee, 1987). These cells did not produce substantial amounts of any CSF and their conditioned media did not have biological activities that could account for their effects on the stem cells. A study of anti-CSF antibodies in a stem cell/stroma assay (Kalai & Zipori, 1988) indicated that CSFs do not contribute to the interactions between the hemopoietic cells and the stroma (Peled et al , 1991). The stromal cell factor IL-7 was found to be a proliferation factor for pro-B, pre-B and pro-T cells (Henney, 1989). This factor is partially responsible for lymphopoiesis supported by stromal cells but does not account for long-term hemopoiesis and stem cell renewal.
Stem Cell Factor (SCF), the stromal CSF, has a relatively poor colony-forming ability, but is a potent synergistic factor. So far, however, it has not been shown to be a stem cell renewal factor, and it is secreted in ample amounts by stromal cells incapable of supporting hemopoiesis.
In the mouse, precursor T cells capable of repopulating an irradiated thymus, proliferated for more than 90 days with the above-noted 14F1.1 mouse stromal cells. The predominant cell population in these
SUBSTITUTE SHEET (RULE 28) cultures was Thy-1.2+, CD4", CDS', Pgp-1+ (Tamir et ai, 1990) and had the T cell receptor gene in the germline configuration (Tamir et ai, 1989). The incidence of cells with such a phenotype is very low in the fresh thymocyte population initially seeded with the stroma. The proliferation of the precursor T cells depended strictly on the support of cloned stromal cell lines. Addition of IL-2 to the culture system augmented T cell yield but was not mandatory. When tested after removal from the stromal layer, the cells had no cytotoxic activity. They became highly cytotoxic when removed from the stroma and propagated with IL-2 for a week or longer, prior to testing.
Thus, stromal cells exhibit a capacity to support the renewal and accumulation of double-negative thymocytes (precursor T cells) but do not allow their further differentiation even in the presence of IL-2. Thus, in addition to supporting stem cells, the 14F1.1 stromal cell line supports myelopoiesis and lymphopoiesis of cells of both the B and the T lineage.
In contrast to hemopoietic cells that can be classified precisely into a variety of lineages, cells of the stromal family are usually referred to mostly with respect to their morphological appearance (i.e., "fibroblasts" or
"epithelioid cells", etc.). This is mainly due to the fact that cell surface markers for this heterogeneous cell population are unknown. Further, it has been found that stromal cells differ markedly with respect to the composi¬ tion of the extracellular matrix that they deposit. This is clearly an important feature, since many cell functions are directly influenced by the mode of the cells' attachment to surfaces and to each other. To date, very little is known about the molecular basis for the differences between the various types of stromal cells, e.g. differences in gene expression. Further, very little is known about the molecular basis for the above-mentioned biological activity of stromal cells, i.e. their ability to support long-term hemopoiesis with prolonged stem cell renewal. It has therefore been a long-felt want to elucidate the molecular mechanism for the interaction of stroma with hemopoietic stem cells, which leads to prolonged stem cell renewal and hemopoiesis. Elucidation of this interaction may provide the means for modulation of stem cell growth in individuals suffering from various diseases associated with abnormal stem cell modulation, e.g. various hemopoietic diseases such as aplastic anemia, myelofibrosis as well as various leukemias. Moreover, many of the problems associated with bone marrow transplantation may be resolved when stem cell growth can be augmented. Further, elucidation of the above interaction may also provide the means for diagnosing, at an early stage, diseases caused by an alteration in the normal mechanism of stem cell modulation.
GENERAL DESCRIPTION OF THE INVENTION In accordance with the invention it was found that a hybridoma, designated B92, obtained following immunization of mice with mice stromal cells of the line 14F1.1, produced a monoclonal antibody, designated herein as B92 monoclonal antibody (B92 MAb), that recognizes, i.e. binds to, a novel antigen found in 14F1.1 cells. This antigen was designated as "stromalin- 1 ". The cDNA encoding the stromalin-1 was isolated and cloned. By homology to the cDNA encoding murine stromalin-1, a human gene encoding a human "stromalin- 1 " was identified and cloned. On the basis of homology with stromalin-1, a human stromal protein "stromalin-2", was also identified and cloned. As noted above in the Background Section, stromal cells such as the mouse endothelial-adipocyte stromal cell line, 14F1.1, support long- term hemopoiesis and stem cell renewal. Accordingly, the novel stromalin protein may be related to the ability of 14F1.1 to interact with stem cells and thus these proteins, as well as their analogs or fragments (as defined
SUBSTITUTE SHEET (RULE 20) below), or antibodies directed against these proteins, and their analogs or fragments may have potential use as agents for the treatment of various diseases or disorders associated with abnormalities in hemopoiesis and stem cell renewal. Human stromalin-1 cDNA sequence shows a very high (above
90%) homology with the corresponding sequence of the mouse stromalin-1. The homology between the mouse and human stromalin-1 is much higher (above 98%) when comparing the amino acid sequence of the two proteins. Furthermore, there is also a relatively high nucleotide homology between the cDNA's coding for stromalin-1 and stromalin-2 (about 68% homology), and a 71% amino acid homology between these two proteins. On the basis of homology, stromalin-1 and stromalin-2 proteins from other mammals, and other stromalins from both human and non-human mammals, may be isolated. The present invention thus provides novel proteins or polypeptides expressed in stromal cells. These proteins or polypeptides may be used for various therapeutic or diagnostic purposes. These proteins or polypeptides may be purified from biological samples or may be obtained by genetic engineering methods. Proteins obtained by means of genetic engineering are very often produced as fusion proteins and fusion proteins of the invention may be used as such for various purposes.
By a first of its embodiments, the present invention provides a protein or a polypeptide being a member of the group consisting of:
(a) stromalin having an amino acid sequence as depicted in Figs. 7, 9 and 13;
(b) proteins or polypeptides which are homologous, i.e. at least about 60%, preferably at least 70%, to those of (a) and having a similar biological activity; (c) analogs of (a) or (b) obtained by deletion, addition, replace¬ ment or chemical modification of one or more amino acid residues without substantially affecting the biological activity thereof; (d) fragments of the protein, polypeptides or analogs of (a), (b) or
(c) which essentially retain the biological activity of the non- fragmented polypeptide or protein; and (e) a fusion protein of a first protein or polypeptide being that of (a), (b), (c) or (d), and a second protein or peptide, the fusion protein essentially retaining the biological properties of said first protein or polypeptide.
The present invention further provides a DNA molecule comprising a sequence coding for the proteins or polypeptides of the invention. The DNA molecules according to the invention are, for example, those selected from the group consisting of:
(a) nucleotide sequence depicted in Figs. 6, 8 and 12;
(b) nucleic acid sequences, encoding the same protein or polypep¬ tide as the nucleic acid sequence of (a); (c) nucleic acid sequences homologous to the sequences under (a) or (b), e.g. above about 60% homology, preferably above about 70% homology, and encode a protein or polypeptide having a similar biological activity to the protein or polypep¬ tide encoded by the sequences under (a) or (b); (d) fragments of any of the nucleic acid sequences of (a), (b) or
(c), which encode a polypeptide which retain some of the biological activity of the non-fragmented protein or polypep¬ tide;
SUBSTITUTE SHEET (RULE 2β) (e) a nucleic acid sequence which hybridizes to any of sequences under (a), (b), (c) or (d), under high stringency conditions; and
(f) a DNA molecule comprising any of the sequences under (a), (b), (c), (d) or (e).
A particular example of the DNA molecules defined under (f), are various DNA vectors comprising also a sequence required for the propagation and replication of the vector in a host cell and at times also a sequence controlling expression of said nucleic acid sequence. The invention will now be illustrated by the following non- limiting example and by the figures.
BRIEF DESCRIPTION OF THE FIGURES
Fig. 1 shows the map of plasmid pGEMSA-1, described in Example 3;
Fig. 2 shows the results of the Northern blotting of extracts from 14F1.1 cells probed with a -actin probe as described in Example 3;
Fig. 3 shows the results of the Northern blotting analysis of mRNA extracted from various tissues and probed with a pGEMSA-I or -actin probe, also described in Example 3;
Fig. 4 shows the results of the PCR analysis of cDNA from various tissues using a stromalin-1 3' probe also described in Example 3;
Fig. 5 shows the immunoprecipitation products of in vitro translated RNA transcripts from the expression of a pGEMSA-1 clone, described in Example 4;
Fig. 6 shows the nucleotide sequence of the murine stromalin-1 gene as described in Example 5;
Fig. 7 shows the amino acid sequence of the murine stromalin- 1 protein as described in Example 5; Fig. 8 shows the nucleotide sequence of the human stromalin-1 gene as described in Example 5;
Fig. 9 shows the amino acid sequence of the human stromalin-1 protein as described in Example 5; Fig. 10 shows the nucleotide sequence of the human stromalin-2 gene sequence, described in Example 6;
Fig. 11 shows the amino acid sequence of the human stromalin-2 protein amino acid sequence, also described in Example 6;
Fig. 12 depicts MBA-1.1.1 stromal cells grown on a Labtek slides fixed in methanol and stained with a B92 MAb as described in Example 7:
Fig. 12A depicts the cells after normal growth;
Fig. 12B depicts these cells first treated with antisense oligonucleotide of the stromalin-1 gene prior to staining;
Fig. 13 shows immuno precipitation results of MBA 15 stromal cell lysates with antisera obtained following immunization of rabbits with 796 (lane 1) and 795 (lane 2) peptides. Immuno precipitative proteins were subjected to Western blot analysis using B92 monoclonal antibody;
Fig. 14 shows results of Western blotting of nuclear proteins with B92 antibody (left lane) and of cytoplasmic proteins (right lane); Fig. 15 depicts Ml myeloid cells fixed and then stained with 740 polyclonal antiserum, as described in Example 9:
Fig. 15A shows cells fixed with formaldehyde (FA) followed by Triton X100 and then reacted with the 740 antiserum;
Fig. 15B shows the same cells after fixation with methanol followed by reaction with the 740 antiserum;
Fig. 15C is a conrol - cells fixed with FA followed by Triton and then reacted with a control non-immune serum.
Fig. 16 depicts results of an immuno staining of MBA-1.1.1 stromal cells with 740 polyclonal antiserum as described in Example 9:
SUBSTITUTE SHEET (RULE 28) Fig. 16A shows cells fixed with FA and Triton; Fig. 16B shows cells fixed with FA without Triton; Fig. 16C shows the same cells fixed with FA and Triton and with a control non-immune serum; Fig. 17 shows COS-7 monkey cells shown in a similar manner as in
Fig. 12;
Fig. 18 is a schematic representation of the manner of preparation of a construct containing an antisense stromalin- 1 fragment, described in Example 10; Fig. 19 shows the morphological changes occurring in a clone of
MBA-15 cells permanently transfected with a plasmid containing an antisenses form of stromalin-1:
Fig. 19A shows a lower magnification depicting dead cells in the center of the colony (upper right portion of the picture) with viable cells at the periphery;
Fig. 19B shows a higher magnification view of the cells; and Fig. 20 shows a transformed foci after myc and ras transformation (upper plate), following cotransfection of the cells with p53 (middle plate) and following cotransfection with the stromalin-1 (SA1; lower plate).
In Figs. 7, 9 and 11 several sites are annotated on the amino acid sequence which are abbreviated as follows:
B92: stands for epitope recognized by B92 antibody;
Cho: stands for a carbohydrate group; Glc: stands for a glycoaminoglycan;
Myr: stands for a myristyl group;
NH2: stands for an amide group;
(P): stands for a phosphate group;
SO4: stands for a sulfate group; SCBS: stands for stem cell binding site;
SPCS: stands for signal peptide cleavage site. EXAMPLES
Example 1
(a) Fusion procedure for mouse cells (based on the standard Kohler- Milstein method)
Materials:
(i) DMEM: Dulbecco's Modified Eagle's Medium, supplemented with 2mM glutamine, 100 U/ml penicillin, 100 U/ml streptomycin, and
ImM Na-pyruvate. (ii) DMEM-FCS: DMEM supplemented with fetal calf serum (FCS) inactivated at (56°C, 30 min). (iii) HAT-medium: DMEM-FCS supplemented with 1 ml of each of following components for each 100 ml HAT medium:
Hypoxanthine 10"4M, prepared as a 100 x stock solution containing 136 mg hypoxanthine dissolved in 100 ml distilled water, dissolution at 60-70°C;
Aminopterin 4 x 10"7M, in 100 ml distilled water, dissolution of aminopterin by first adding thereto 25 ml H20 and 0.5 ml of 5N
NaOH, then neutralizing with 5N HC1, followed by adjustment to final volume of 180 ml with H2O, this solution then filter sterilized through a 0.2 μm millipore filter and stored at 20°C; and Thymidine 1.6 x 10"5M, prepared as a 100 x stock solution contain- ing 38.7 mg in 100 ml distilled H2O.
(iv) HT-medium: DMEM-FCS supplemented with hypoxanthine and thymidine as above, but without aminopterin. (v) Polyethylene glycol (PEG).
To make a 41% (w/v) solution, 4.2 ml of DMEM, is added to 3 g of PEG 1500 (MW 1300-1600), dissolved in a well-capped vial at 56-
60°C and then sterilized by filtration through 0.45 μm. filter while
SUBSTITUTE SHEET (RULE 28) solution still warm. Alternatively, 20 g of PEG in a 100 ml flask is sterilized in an autoclave for 15-20 min, cooled to 50°C to which is added 28 ml of DMEM, mixed and stored at room temperature (R.T.).
Cells:
Myeloma cells - Myeloma cells used for hybridization were the NSO cells. This cell line does not produce any immunoglobulin, it grows in the presence of 20 g/ml 8-azoguanine (8-AZG) and therefore dies in HAT-medium. NSO cell line is of H-2d and grows in BALB/c mice. NSO cells are grown in petri dishes or tissue culture bottles to a concentration of 1.0 - 2.5 x 106 cells/ml in DMEM-HS then transferred to fresh flasks at cell concentration of about 5 x 104/ml. To avoid revertants it is recommended to grow the NSO cells in the presence of 8-AZG for at least one week before the fusion.
Immuno-spleen cells: BALB/c female mice were immunized at the age of 6-8 weeks followed by a booster immunization (prefusion boost) 3-4 days before fusion, by either an I.V. injection, or if not possible by interperitoneal injection. Hyperimmune animals were allowed to rest at least 3 weeks before the prefusion boost.
Procedure:
(i) Preparation of cells:
Spleen cells were prepared by removing spleens from mice in a sterile hood (metal-hood) and placing them into DMEM in a petri dish. The spleens were then teased apart with sterile forceps, the cells were resuspended and transferred to a 10 ml conical tube, which was left in a vertical position for about 10 min on ice, during which large pieces of tissue (spleen debris) precipitated. The single cell suspension was then removed
SUBSTITUTE -SHEET (RULE 26) and placed into a new tube (the above spleen debris was kept on ice), and aliquots of single cells were taken for cell counting, the cells were then centrifuged and resuspended in DMEM (5-10 ml volume).
Myeloma cells were prepared by removing NSO cells from an incubation flask, at a logarithmic growth phase of the cells, and placed into a 50 ml conical tube from which a sample was taken for counting (diluted 1:1 with 0.1% Trypan-blue), and the remainder of cells in the conical tube were centrifuged at 1000 rpm (200 x g) for 10 min. The precipitated cells were then resuspended in DMEM to a cell concentration of 10 x 106 cells/ml.
(ii) Cell Fusion:
Before starting the fusion, separate containers having about 3 ml PEG, 50 ml DMEM and 100 ml DMEM-FCS-HAT were prewarmed in a 37°C water bath. Splenic lymphocytes and NSO cells were then mixed at a ratio of 5:1 in a 50 ml tube (not more than 10s spleen cells), to which DMEM to a final volume of 40 ml was added. The cell mixture was centrifuged and all supernatant was removed by careful aspiration, and the cell pellet was loosened by gentle flicking. 2.0 ml of prewarmed (37°C) PEG was dropwise added to the loosened cell pellet with a pipette having a wide opening and the cell pellet was gently resuspended for 1 min, so as not to destroy small cell aggregates. The resuspended cells were incubated for an additional 1 min at 37°C and prewarmed DMEM is dropwise added to dilute the PEG. The addition was done slowly with gentle mixing of cells, by adding 5 ml in the first 5 min, then 10 ml in the next 5 min and an additional 15-20 ml of DMEM in the last 5 min. The resulting cell suspension was then centrifuged, the supernatant was removed and 5 ml of prewarmed DMEM-FCS-HAT was added and the cells resuspended very carefully to prevent breaking the fused cells apart. About 10 ml DMEM- FCS-HAT was added and a sample was taken for counting the viable cells, in particular the number of viable NSO cells which is larger than the number of the viable spleen cells. The remaining fused cells were then suspended in DMEM-FCS-HAT to a concentration of 2-5 x 104 viable NSO cells/ml and 0.1 ml aliquots were distributed into each well of 96 well microplate, which was incubated at 37°C in an 10% CO2 humid incubator. After 24-48 hr. 0.1 ml of prewarmed DMEM-FCS-HAT was added to each well and 5-7 days following fusion, the cultures were fed by aspirating half of the culture fluid and replacing it with prewarmed fresh DMEM- FCS-HAT medium. This feeding was repeated every 3-4 days. The cultures were inspected for hybrid cell growth regularly and usually about 10-14 days following fusion supernatants could be removed to screen for antibody activity. Two-three weeks following fusion, the HAT-medium was replaced by HT-medium and one week later by regular DMEM-FCS medium. Positive wells from the 96-well plate were transferred into wells of a 24 well plate and then to flasks. The positive hybridomas were frozen in liquid N2 and cloned on agar or by limiting dilution (see (b) below).
(b) Cloning of hybrid cells (i) Cloning under conditions of limiting dilution:
The cells (obtained according to above procedure) were diluted to 0.25 cell/well. After 10 days the clones were tested by radioimmuno- assay (RIA). The positive clones were grown, and the cloning efficiency determined.
(ii) Cloning in Methylcellulose (MC):
The cloning was done in DMEM supplemented with 0.8% MC, 10% FCS. Cells (10 -2xl03), obtained according to the procedure in (c) above, were embedded in MC and seeded into 35 mm plates. Colonies were picked after 10 days, grown as before and analyzed for cloning efficiency.
(c) Production of Monoclonal Antibodies (i) Growth under Tissue culture conditions:
The hybridomas, selected from cloned hybridomas according to (d) above, were grown in DMEM supplemented with 10% FCS until they reached confluence. The cells were removed by centrifugation and the supernatant was filtered. (ii) Ammonium Sulfate precipitation of Immunoglobulins:
Immunoglobulins (Ig) were precipitated at 33-50% saturated ammonium sulfate at 4°C.
(iii) Large-scale monoclonal activity production procedure using ascite method: The ascites were prepared by injecting (BALB/c x C57BL/6)
Fl mice interperitoneally (IP) with 0.5 ml pristine (2,6,10,1,4-tetramethyl- pentadecane, Aldrich Chemical Co.). Two weeks later the mice were injected with 107 hybridoma cells (see (e)(i) above and ten days after the injection ascites fluid was collected. The ascite fluid (or serum) was then cleared of ascites by centrifugation (17000 x g for 15 min, at 4°C), the floating lipids removed by suction and the clear supernatant collected, its volume measured and then kept on ice. An equal volume of saturated ammonium sulfate (see (e) (ii) above) was then added with gradual stirring (750 g solid (NH4)2S04 in 1 liter of water and neutralized with NH4OH). This mixture is then kept at 0-4°C for at least 2 hr. Alternatively, 50% saturation could be obtained by adding 31.3 g of (NH4)2SO4 to 100 ml of serum. The mixture was transferred into Sorval centrifuge tubes and spun in rotor SS34 at 10000 rpm for 10 min at 4°C. The pellet was collected and washed twice with 50% of cold ammonium sulfate, the pellet being
SUBSTITUTE SHEET (RULE 28) completely dispersed in the ammonium sulfate solution each time, each wash being followed by centrifugation as before. The pellet after the second wash was dissolved in water in 1/2 - 1/10 of the original serum volume and then transferred into a prewashed dialysis tube and dialyzed against more than 106 volumes of saline or PBS at 4°C and centrifuged to remove precipitate. The so-obtained monoclonal antibodies were frozen at -70°C, or with 0.1% NaN3 at 4°C, for storage purposes, (iv) Determination of the antibody class by the Ouchterlony test:
The double immunodiffusion test where antigen and antibody form a precipitating line in agar (Ouchterlony) which is the easiest assay to determine the Ig class and subclass was used. The sensitivity of this assay (-10 mg/ml) allows for the analysis of the monoclonal antibody (mAb) class already in a high density hybridoma culture supernatant without the need to first prepare large amounts of mAb by the ascite fluid method (see (e)(iii) above). For lower concentrations, the immunoglobulin (Ig) from the hybridoma culture supernatant could be precipitated in 50% saturated ammonium sulfate and the pellet dissolved in 1/10 of the original volume with water (as in (e)(ii) and (e)(iii) above). For determination of the mAb class, standard Serotec™ mouse Ig RID plates were used.
(d) Screening of λgtll library
The uv-2237 IP3 λgtl l library (8 x 109 PFU/ml) was plated on 12 plates (150 mm) 30,000 PFU/plate. Each plate contained LB, pH 7.5, with Mg (lOmM), Amp (lOOmg/ml). Cells Y1090 (Promega), were grown at 37°C overnight, while agitating with LB Mg, pH 7.5, Maltose 0.2%. The overnight culture was centrifuged and resuspended in 1/2 volume lOmM MgS04, and kept at 4°C. The phage from the above library was absorbed to the above cells for 20 min, 37°C, 9 ml Top Agar (0.7%) was then added and the mixture plated. The plates were incubated at 42°C, 3
SUBSTITUTE SHEET (RULE 28) hrs, blotted with nitrocellulose filters (Schleicher and Schull) and embedded in lOmM IPTG overnight, 37°C. The filters were then treated for 20 min in 10% LFM (Low fat milk) in PBS, and incubated with a 1st antibody, B92 (1:100 dilution), 2-3 hrs at R.T. (B92 obtained according to (d) and (e) above, was pre-treated with E.Coli extract to reduce background, and kept in 2.5% BSA, 0.02% Azide in PBS at 4°C). The filter was then treated as follows: 10 min with 10% LFM, PBS, followed by 10 min with 10% LFM, PBS, 0.1% Tween 20 and then 10 min with 10% LFM, PBS. Incubation was then carried out with the 2nd antibody (1 -1.5 hrs at R.T.), 2nd antibody being I125 labelled rabbit-anti-mouse (Jackson) 106 cpm/ml, 200 ml in 10% LFM in PBS. The filter was subsequently washed as before and exposed to X-ray. On the basis of the X-ray results, positive clones were identified on the above plates and were picked and kept in TMG buffer (lOmM Tris pH 7.5, 10 mM MgCl2 and 0.01% Gelatin and Chloroform, 4°C.
Second, third and fourth cycles of the above procedure were carried out using the positive clones to ensure the isolation of only the true- positive (as opposed to any false-positive) clones, these times using 90mm plates.
Example 2
Derivation of the B92 Hybridoma
A series of monoclonal antibodies were prepared (see Example 1(a) and (b) above) following immunization with the 14FIL3.CB6 mouse (BALB/c) bone marrow derived stromal cell subclone obtained from the mouse endothelial-adipocyte stromal cell line, 14F1.1. Out of 10 so- immunized allogeneic mice (C57B1/6J) the sera of which were tested by RIA, one mouse was selected and its spleen cells were used for fusion. Out of 1500 hybrids obtained in the fusion, 11 secreted monoclonal antibodies (MAbs) that recognized the immunizing cells. These were then cloned by limiting dilution and the type of immunoglobulin secreted by each one was determined (see Example 1(b) and (c)). As is set forth hereinbelow, upon analysis of the various cloned hybridomas, the hybridoma designated B92 was observed to secrete a monoclonal antibody, designated herein as B92 MAb that recognized an antigen, designated herein as "stromalin-1".
Example 3
Tissue Expression of stromalin-1 at the mRNA Level mRNA was extracted by standard procedures from 14F1.1 stromal cells and subjected to Northern blotting using the plasmid pGEMSA-I (see Fig. 1 showing the plasmid's map) or a probe for β-actin oligolabelled with 32P as hybridization probe. The Northern blotting analysis results, indicate the presence of a single 6 kbp transcript in the mRNA from 14F1.1 cells shows hybridization of the β-actin probe by the mRNA from 14F1.1 and IP-3 cells.
The expression of the stromalin-1 gene was then examined in various tissues and again Northern blots showed a 6 kbp transcript. Smaller signals were also seen. These results are shown in Fig. 3, wherein mRNA was extracted from the various indicated tissues and subjected to Northern blotting using hybridization probes for the stromalin-1 mRNA (this being a 1.4 kbp cDNA insert) of pGEMSA-1 and a probe for β-actin mRNA. Ribosomal mRNA (18S and 28S) served as markers.
PCR was carried out by standard techniques using cDNA from the various tissues. The probes chosen were derived from a 307 base segment from the 3' end of the stromalin- 1 coding sequence. It was observed that various tissues gave a signal. The bone marrow that expresses a 50 kDa protein showed a PCR product indistinguishable from that found in other tissues.
SUBSTITUTE SHEET (RULE 28) The above results are depicted in Fig. 4, wherein the PCR analysis procedure using primers 5'-TGGACCCTTGCCAACCC-3' and
5'-GTdTCTAACTGGCTCCGTG-3', both from the 3' end of the B92 coding sequence, reveals stromalin-1 cDNA amplification products of 307 bp in different hemopoietic and non-hemopoietic tissues.
Example 4
The plasmid SA-I containing the 1.4Kb stromalin-1 insert was linearized for transcription from either the T7 or the SP6 sites. In vitro transcription of RNA from T7 or SP6 promotors was performed using the Promega™ kit (Madison, Wl, USA).. In vitro translation of the transcripts was done using the Strategene™ kit (La Jolla, CA., USA). The products were immunoprecipitated with B92 antibody and run on a 13% acrylamide SDS gel. The results of the immunoprecipitation are depicted in Fig. 5 and as can be seen there is a band of about 32 kDa from the T7 transcript, shown by an arrow. No transcription product was obtained from the SP6 direction.
Example 5 Cloning of a cDNA encoding a protein recognized by the B92 antibody
By the procedure set forth in Example 1, about 500,000 individual clones from a λgtl l cDNA library were screened for positive plaques reactive with B92 MAb, 29 candidates were selected and 4 of them were eventually shown to be specifically reactive with the antibody. Protein extracts from these were reacted with antibodies to β-galactosidase and separately with B92 antibody. The results indicated that the 4 clones contained fusion proteins of about 160 kDa. This corresponds to a cDNA insert of about 1.4 kbp. An insert of this size was indeed observed when the phage DNA from all 4 clones was cut with EcoRI restriction endonuclease.
SUBSTITUTE SHEET (RULE 28) Proteins translated in vitro in a reticulocyte lysate from transcription products (mRNA) from the expression of SA-I insert cloned into EcoRI site of pGEM (pGEM-SA-I clone), (see Example 4 and Fig. 4), were examined by immunoprecipitation. One protein product, detectable by the B92 MAb, was observed at 32 kDa. The sequence of cDNA insert in the pGEM-SA-I showed an open reading frame corresponding to a protein of only 18 kDa. The pGEM-SA-I clone was cut with StuI restriction endonuclease, resulting in maintenance of all 540 bases of the open reading frame with additional 56 bases as template for in vitro transcription, and removal of the remainder of the putative non-coding region. This clone was in vitro transcribed and translated in a reticulocyte system and the product was observed to migrate in a gel (polyacrylamide) as a 32 kDa doublet.
Using the 3 '-end region of the above original clone two approaches were followed to obtain murine and human full length genes that were designated as stromalin-1: (i) cloning by anchored polymerase chain reaction (PCR) of the unknown 5' region of the gene using mRNA from 14F1.1 cells and (ii) screening a human thymus cDNA library with labelled oligonucleotide probes. In each anchored PCR experiment, a mean of about 500 new nucleotides corresponding to the 5' coding region were obtained, and at least 2-3 clones were sequenced and compared for the sake of accuracy. Figs. 6 and 7 show the nucleotide and amino acid sequences, respectively, corresponding to the murine full length open reading frame.
From the sequencing results it was observed that the complete open reading frame of the murine stromalin-1 gene spans about 3774 bases and encodes a protein of about 144.7 kDa. Taking advantage of this presently determined sequence of the murine stromalin-1 gene, 4 different screenings of the human thymus cDNA library have been carried out; in each one, 500,000 plaque forming units were screened. The nucleotide and amino acid sequence of the human stromalin-1 protein is shown in Figs. 8 and 9, respectively.
The sequence of the stromalin-1 gene does not show homology with any sequence found in updated gene data banks, i.e. it represents a new gene sequence. It is important to mention the presence of a REDV sequence (positions 974-977 of the murine sequence and positions 1038-1041 of the human sequence), which has been described as an adhesion sequence found in the IIICS alternatively spliced region of fibronectin and interacts with NLA-4 integrin, namely, this is a putative binding site for the adhesion of early stem cells (day 12 CFU-S) to fibronectin, through the cell surface protein, integrin VLA-4. In addition the protein contains a potential glycosaminoglycan binding site at the C- terminal end (position 1187-1190 of the murine sequence and 1251-1254 of the human sequence). An alignment of the murine and human sequences shows a very high homology (about 92.9%) between the murine and human genes and an extremely high homology (about 98.9%) between the murine proteins suggesting a very important role for this molecule.
Example 6
Human Stromalin-1
During the screening of the human thymus cDΝA library a plaque forming unit (clone) with reduced but significant hybridization was obtained; the cDΝA insert was sequenced and a fragment of about 1200 nucleotides (Fig. 10) was obtained. It codes for a gene showing a 68% nucleotide homology and a 71 % amino acid homology (Fig. 11) with the human stromalin-1, and was designated stromalin-2 gene. The presence of the stromalin-2 gene has also been observed by PCR amplification in the human K562 leukemic cell line and in the murine 14F1.1 stromal cell line.
SUBSTITUTE SHEET (RULE 28) Example 7
Nuclear and membrane expression of stromalin-1
Cells from the stromal cell line MBA-1.1.1 were immune stained with B92 monoclonal antibody by using a donkey anti-mouse IgG antibody carrying a Texas Red marker. The antibody stained the cells extensively in the peripheral zone of the outer membrane, as well as in the cytoplasm and nucleus, as can be seen in Fig. 12A. The MBA-1.1.1 cells were incubated with an antisense oligonucleotide which had the following sequence: TGGACTGTTCCTTCCTGTCCTC
The above sequence is antisense to the sequence in mouse stromalin-1 from positions 1655 (5') to 1675 (3'). Following incubation with this antisense nucleotide, the nuclear and membrane stainings were eliminated, whereas the cytoplasmic staining remaining intense, as can be seen in Fig. 12B. The above results tentatively suggest that stromalin is expressed both in the nucleus and in the peripheral zone of the membrane of the MBA-1.1.1 cells.
Example 8 Derivatives of polyclonal antibodies that detect stromalin-l gene product by a combination of immunoprecipitation and immunoblotting and further evidence of nuclear localization of stromalin-l protein
Three synthetic peptides derived from the predicted amino acid sequence of murine stromalin-l gene were synthesized by the solid-phase method known per se. These peptides designated herein as "794" (corre¬ sponding to amino acid residues 41-55), "795" (corresponding to amino acid residues 161-175), and "796" (corresponding to amino acid residues 1095- 1110), had the following amino acid sequences: 794: GRPPSTNKKPRKSPG
795: DYPLTMPGPQWKKFR
SUBSTITUTE SHEET (RULE 28) 796: NTWLNRTDTMIQTPG
The peptides were purified by chromatography on Sephadex G-25 and fractions were pooled and lyophilized. The purified peptides were dissolved in distilled water and were conjugated to Keyhole lymphet hemocyanine (KLH).
New Zealand white rabbits were immunized by multisite intradermal injections of 1 mg of peptide conjugates in 0.5 ml of PBS emulsified in 0.75 ml of complete freund's adjuvant (CFA). Booster injections were administered 3 weeks later. Each peptide was injected to two rabbits. Antisera were purified either by a protein A column or by
(NH4)2SO4 precipitation. These purified antibodies were characterized by western blot analysis either with tissues or stromalin-l recombinant protein.
Only the antisera obtained following immunization with the
796 peptide recognized the recombinant stromalin as a 100 kDa and 130 kDa band. The signals obtained by western blotting with the 796 antibodies were very weak compared with a strong background of numerous other nonspecific bands. To circumvent this problem, immunoprecipitation was combined with immunoblotting and results, shown in Fig. 13, indicate that a specific band at 100 kDa, as well as some higher molecular weight proteins, react with the antibody. In addition, the B92 MAB showed that stromalin is indeed found in the nucleous in titer or that its antigenic epitope is more accessible there than in the cytoplasm, as can be seen in Fig. 14.
Example 9 Further properties of stromalin derivatives by immune staining using stromalin specific polyclonal antibodies
A series of polyclonal antibodies against synthetic peptides from straomalin-1 were prepared. The synthetic peptides were chosen according to the Chou-Fasman prediction. Regions with the highest
SUBSTITUTE SHEET (RULE 28) antigenicity were chosen for the preparation of synthetic peptides. One of them was an antiserum against a peptide, designated herein as "740" (which corresponds to residues 451-465 of the murine stromalin-l), which had the following sequence: LAKRRGRNSPNGNLI
The antiserum to the 740 peptides was purified either by protein A or by peptide affinity column.
Ml myeloid cells were fixed with either 3% formaldehyde
(FA) followed by 5% Triton XI 00 (30 mins. in room temperature) or with 100% methanol for ten minutes in 20°C. The fixed cells were stained for immunofluorescence with polyclonal antibodies from the 740 antiserum.
The second antibody was a Texas Red conjugated doneky anti-rabbit
Ig-Fab. Slides were incubated for one hour with the 740 antibody, worked in an incubator for a further 45 minutes with the second antibody. Slides were then examined in a fluorescence sized microscope.
Results from cells fixed with FA and with methanol are shown in Figs. 15A and 15B respectively. Some cultures were incubated with a control non-immune rabbit serum and the results are shown in Fig. 15C.
As can be seen, there is extensive staining following FA fixation while the staining is almost absent following the methanol fixation. Thus, it is conceivable that the antigen to which the 740 antiserum reacts is sensitive to methyl fixation.
MBA-1.1.1 stromal cells were reacted with 740 polyclonal antiserum in the same manner as that described above and the results can be seen in Fig. 16. As can be seen, nuclear staining is evidenced following fixation with FA and Triton (Fig. 16A) while FA without Triton gave full results (Fig. 16B) as compared to the negative control (Fig. 16C) of the second antibody only.
SUBSTITUTE SHEET (RULE 28) Nuclear staining was also seen in human skin fibroblast cells.
Against this, cytoplasmic staining was observed in COS-7 monkey cells, as shown in Fig. 17. These cells were later verified by Hoechst staining to be dividing cells.
Example 10
Preparation and properties of cell lines transfected with a plasmid containing an antisense form of the stromalinOl cDNA.
An antisense construct of the stromalin-l gene containing the antisense nucleotides between positions 371 to 3358 of the stromalin gene was introduced into a PCEP4 plasmid, as shown schematically in Fig. 18. This construct was then transferred into a variety of cell types using a standard transfection. A series of clones were obtained from the MBA-15 cell lines wich showed an increase growth rate, crowding at confluence and loss of their contact inhibition.
When grown in colonies, in clones which contained stromalin- 1 antisense, dells in the center of the colonies died while the cells in the periphery, which are dividing, were viable as can be seen in Fig. 19. In fact, it became difficult to maintain these cell clones. An even clearer pattern was observed when attempting to obtain clones of the 14F1.1 stromal cell lines expressing stromalin-l antisense. Control clones of 14F1.1 stromal cells transfected with a pCEP4 plasmid which do not contain any gene insert, were normally growing; against this, in most transfections where the pCEP4 contained stromalin-l antisense, no clones could be obtained and only two clones that were eventually derived out of the transformed cells grew albeit very slowly. It thus appears that prolonged expression of stromalin-l antisense is detrimental to those cell's prolifera- tion.
SUBSTITUTE SHEET (RULE 28) Example 11
Putative role of stromalin-l as a tumor suppressor gene
Transformed foci assay is a standard assay used to demonstrate the function of tumor suppressor genes such as p53. In this assay, the procedure of which is depicted schematically in . Fig. 20, formation of transformed foci by rat embryo fibroblasts upon transfection with a combination of myc and ras oncogens is tested following the addition of a suppressor gene, such as p53.
The assay was utilized in order to test the stromalin-l gene (SA-1) and as can be seen from the results depicted in Fig. 11, SA-1 seems to suppress the formation of foci induced by myc and ras and points to a possibility that stromalin-l is a candidate tumor suppressor gene.
SUBSTITUTE SHEET (RULE 28)

Claims

CLAIMS:
1. A protein or a polypeptide being a member of the group consisting of:
(a) stromalin being one of the following (i), (ii) or (iii) -
(i) murine stromalin-l having the folliwng amino acid sequence
1 MITSELPVLQ DSTNETTAHS DHGRQLEETE VKGKRKRGRP GRPPSTNKKP
RKSPGEKSRI EAGIRGAGRG RANGHPQQNG DGDPVTLFEV VKLGKSRMQS
101 VVDDWIELYK QDRDIALLDL INFFIQCSGC RGTVRIEMFR NMQNAEIIRK
MTEEFDEDSG DYPLTMPGPQ WKKFRSNFCE FIGVLIRQCQ YSIIYDEYMM
201 DTVISLLTGL SDSQVRAFRH TSTLAAMKLM TALVNVALNL SIHQDNTQRQ
YEAERNKMIG KRANERLELL LQKRKELQEN QDEIENMMNS IFKGIFVHRY
301 RDAIAEIRAI CIEEIGVWMK MYSDAFLNDS YLKYVGWTLH DRQGEVRLKC
LKALQSLYTN RELFPKLELF TNRFKDRIVS MTLDKEYDVA VEAIRLVTLI
401 LHGSEEALSN EDCENVYHLV YSAHRPVAVA AGEFLHKKLF SRHDPQAEEA
LAKRRGRNSP NGNLIRMLVL FFLESELHEH AAYLVDSLWE SSQELLKDWE
501 CMTELLLEEP VQGEEAMSDR QESALIELMV CTIRQAAEAH PPVGRGTGKR
VLTAKERKTQ IDDRNKLTEH FIITLPMLLS KYSADAEKVA NLLQIPQYFD
601 LEIYSTGRME KHLDALLKQI KFVVEKHVES DVLEACSKTY SILCSEEYTI
QNRVDIARSQ LIDEFVDRFN HSVEDLLQEG EEADDDDIYN VLSTLKRLTS
701 FHNAHDLTKW DLFGNCYRLL KTGIEHGAMP EQIVVQALQC SHYSILWQLV
KITDGSPSKE DLLVLRKTVK SFLAVCQQCL SNVNTPVKEQ AFMLLCDLLM
801 IFSHQLMTGG REGLQPLVFN PDTGLQSELL SFVMDHVFID QDEENQSMEG
DEEDEANKIE ALHKRRNLLA AFSKLIIYDI VDMHAAADIF KHYMKYYNDY
901 GDIIKETLSK TRQIDKIQCA KTLILSLQQL FNELVQEQGP NLDRTSAHVS
GIKELARRFA LTFGLDQIKT REAVATLHKD GIEFAFKYQN QKGQEYPPPN
1001 LAFLEVLSEF SSKLLRQDKK TVHSYLEKFL TEQMMERRED VWLPLISYRN
SLVTGGEDDR MSVNSGSSSS KTSSVRSKKG RPPLHRKRVE DESLDNTWLN
1101 RTDTMIQTPG PLPTPQLTST VLRENSRPMG EQIQEPESEH GSEPDFLHNP
QMQISWLGQP KLEDLNRKDR TGMNYMKVRA GVRHAVRGLM EEDAEPIFED
1201 VMMSSRSQLE DMNEEFEDTM VIDLPPSRNR RERAELRPDF FDSAAIIEDD SGFGMPMF,
(ii) human stromalin-l having the following amino acid sequence
1 MITSELPVLQ DSTNETTAHS DAGSELEETE VKGKRKRGRP GRPPSTNKKP
RKSPGEKSRI EAGIRGAGRG RANGHPQQNG EGEPVTLFEV VKLGKSAMQS
101 VVDDWIESYK QDRDIALLDL INFFIQCSGC RGTVRIEMFR NMQNAEIIRK
MTEEFDEDSG DYPLTMPGPQ WKKFRSNFCE FIGVLIRQCQ YSIIYDEYMM
201 DTVISLLTGL SDSQVRAFRH TSTLAAMKLM TALVNVALNL SIHQDNTQRQ
SUBSTITUTE SHEET (RULE 2β; YEAERNKMIG KRANERLELL LQKRKELQEN QDEIENMMNS IFKGIFVHRY
301 RDAIAEIRAI CIEEIGVWMK MYSDAFLNDS YLKYVGWTLH DRQGEVRLKC
LKALQSLYTN RELFPKLELF TNRFKDRIVS MTLDKEYDVA VEAIRLVTLI
401 LHGSEEALSN EDCENVYHLV YSAHRPVAVA AGEFLHKKLF SRHDPQAEEA
LAKRRGRNSP NGNLIRMLVL FFLESELHEH AAYLVDSLWE SSQELLKDWE
501 CMTELLLEEP VQGEEAMSDR QESALIELMV CTIRQAAEAH PPVGRGTGKR
VLTAKERKTQ IDDRNKLTEH FIITLPMLLS KYSADAEKVA NLLQIPQYFD
601 LEIYSTGRME KHLDALLKQI KFVVEKHVES DVLEACSKTY SILCNEEYTI
QNRVDIARSQ LIDEFVDRFN HSVEDLLQEG EEADDDDIYN VLSTLKRLTS
701 FQNAHDLTKW DLFGNCYRLL KTGIEHGAMP EQIVVQALQC SHYSILWQLV
KITDGSPSKE DLLVLRKTVK SFLAVCQQCL SNVNTPVKEQ AFMLLCDLLM
801 IFSHQLMTGG REGLQPLVFN PDTGLQSELL SFVMDHVFID QDEENQSMEG
DEEDEANKIE ALHKRRNLLA AFSKLIIYDI VDMHAAADIF KHYMKYYNDY
901 GDIIKETLSK TRQIDKIQCA KTLILSLQQL FNELVQEQGP NLDRTSAHVS
GIKELARRFA LTFGLDQIKT REAVATLHKD GIEFAFKYQN QKGQEYPPPN
1001 LAFLEVLSEF SSKLLRQDKK TVHSYLEKFL TEQMMERRED VWLPLISYRN
SLVTGGEDDR MSVNSGSSSS KTSSVRNKKG RPPLHKKRVE DESLDNTWLN
1101 RTDTMIQTPG PLPAPQLTST VLRENSRPMG DQIQEPESEH GSEPDFLHNP
QMQISWLGQP KLEDLNRKDR TGMNYMKVRT GVRHAVRGLM EEDAEPIFED
1201 VMMSSRSQLE DMNEEFEDTM VIDLPPSRNR RERAELRPDF FDSAAIIEDD SGFGMPMF,
(iii) human stromalin-2 having the following amino acid sequence
1 MNGHHQQNGV ENMMLFEVVK MGKSAMQSVV DDWIESYKHD RDIALLDLIN
FFIQCSGCKG VVTAEMFRHM QNSEIIRKMT EEFDEDSGDY PLTMAGPQWK
101 KFKSSFCEFI GVLVRQCQYS IIYDEYMMDT VISLLTGLSD SQVRAFRHTS
TLAAMKLMTA LVNVALNLSI NMDNTQRQYE AERNKMIGKR ANERLELLLQ
201 KRKELQENQD EIENMMNAIF KGVFVHRYRD AIREIRAICI EEIGIWMKMY
SDAFLNDSYL KYVGWTMHDK QGEVRLKCLT ALQGLYYNKE LNSKLELFTS
301 RFKDRIVSMT LDKEYDVAVQ AIKLLTLVLQ SSEEVLTAED CENVYHLVYS
AHRPVAVAAG EFLYKKLFSR RDPEEDGMMK RRGRQGPNAN LVKTLVFFFL
401 ESELHEHAAY LVDSMWDCAT ELLKDWECMN SLLLEEPLSG EEALTDRQES
ALIEIMLCTI RQAAECHPPV GRGTGKRVLT AKEKKTQLDD RTKITELFAV
501 ALPQLLAKYS VDAEKVTNLL QLPQYFDLEI YTTGRLENDL DALLRQIRNI
VEKHTDTDVL EACSKTYHAL CNEEFTIFNR VDISRSQLID ELADKFNRLL
601 EDFLQEGEEP DEDDAYQVLS TLKRITAFHN AHDLSKRDLF ACNYKLLKTG
IENGDMPEQI VIHALQCTHY VILWQLAKIT ESSSTKEDLL RLKKQMRVFC
701 QICQHYLTNV NTTVKEQAFT ILCDILMIFS HQIMSGGRDM LEPLVYTPDS
SLQSELLSFI LDHVFIEQDD DNNSADGQQE DEASKIEALH KRRNLLAAFC
801 KLIVYTWEM NTAADIFKQY MKYYNDYGDI IKETMSKTRQ IDKIQCAKTL
ILSLQQLFNE MIQENGYNFD RSSSTFSGIK ELARRFALTF GLDQLKTREA
901 IAMLHKDGIE FAFKEPNPQG ESHPPLNLAF LDILSEFSSK LLRQDKRTVY
VYLEKFMTFQ MSLRREDVWL PLMSYRNSLL AGGDDDTMSV ISGISSRGST
1001 VRSKKSKPST GKRKVVEGMQ LSLTEESSSS DSMWLTREQT LHTPVMMQTP
QLTSTIMREP KRLRPEDSFM SVYPKQTEHH QTPLDYNRRG TSLMEDDEEP
IVEDVMMSSE GRIEDLNEGM DFDTMDIDLP PSKNRRERTE LKPDFFDPAS
IMDESVLGVS MF;
SUBSTITUTE SHEET (RULE 2φ (b) proteins or polypeptides which are homologs of those of (a) and having a similar biological activity;
(c) analogs of (a) or (b) obtained by deletion, addition, replace¬ ment or chemical modification of one or more amino acid residues without substantially affecting the biological activity thereof;
(d) fragments of the protein, polypeptides or analogs of (a), (b) or (c) which essentially retain the biological activity of the non- fragmented polypeptide or protein; and
(e) a fusion protein of a first protein or polypeptide being that of (a), (b), (c) or (d), and a second protein or peptide, the fusion protein essentially retaining the biological properties of said first protein or polypeptide.
2. A protein or polypeptide according to Claim 1, being homologs as defined under (b) in Claim 1, the degree of homology being at least about 60%.
3. A protein or polypeptide according to Claim 1, wherein the degree of homologs is at least about 70%.
4. A DNA molecule comprising a sequence coding for the protein or polypeptide of any one of Claim 1.
5. A DNA molecule according to Claim 4, being a member of the group consisting of:
(a) a nucleotide sequence coding for stromalin, being one of the following sequences under (i), (ii) or (iii) - (i) a DNA sequence coding for murine stromalin-l and having the following sequence
GAGCAGAGGG CGGTCGGGAC CCGAGTCTGC AGCGGCGCCA TTGGCGTGTG
GAAAATGCCA CCAGATGGCG GGTTAGGATT GCAGCTCCGT TGAAGGCGCG GCCCCCGCTC CCGAACCCCC GGCGACCACC CCGTAACCAC CCCCCCACCT
CGGGAATAAC ACACCGGAGA CTTTTGGGGG GAAACTAGGT CGACGGCCGA CAGCGCCCGG ATGGGCAGCT GAGGTGTGAC TTTGAGGTTG AATAACCAGT
SUBSTITUTESHEET(RULE28) TTGAATTATA CAGAAATTTC TGTACTGTGG AATAGCTTCT CCAGCAATGA
301 TTACTTCAGA GTTACCAGTG TTACAGGATT CAACTAATGA AACTACTGCC
CACTCTGATC ATGGCAGGCA ACTTGAAGAA ACAGAGGTCA AAGGAAAAAG
401 AAAAAGGGGT CGTCCTGGCC GGCCTCCATC AACAAATAAG AAACCTCGAA
AATCTCCAGG AGAAAAGAGC AGAATTGAAG CTGGAATTCG AGGAGCAGGT
501 CGTGGAAGAG CCAATGGGCA CCCCCAACAG AACGGCGACG GGGATCCTGT
CACCTTATTT GAGGTGGTGA AGCTGGGGAA GAGTCGAATG CAGTCCGTGG
601 TGGATGACTG GATTGAATTA TATAAACAAG ACAGGGACAT CGCACTTCTG
GATTTAATCA ACTTTTTTAT CCAGTGTTCA GGATGTCGAG GTACGGTCAG
701 AATAGAGATG TTTCGAAATA TGCAGAACGC AGAAATAATC AGAAAAATGA
CTGAAGAATT TGATGAGGAC AGTGGTGACT ACCCCCTTAC AATGCCTGGT
801 CCTCAGTGGA AAAAATTTCG TTCCAATTTT TGTGAATTTA TTGGAGTCCT
GATTCGACAG TGTCAATATA GCATAATTTA TGATGAATAT ATGATGGACA
901 CCGTAATTTC CCTTTTGACT GGTTTGTCAG ACTCCCAAGT CAGAGCTTTT
AGGCATACAA GTACCCTTGC TGCAATGAAG CTGATGACTG CTCTGGTGAA
1001 TGTTGCTTTA AACCTCAGTA TTCATCAAGA TAATACACAG AGGCAATATG
AAGCTGAGAG AAATAAAATG ATTGGGAAGA GAGCCAATGA AAGACTGGAG
1101 TTACTACTTC AGAAACGTAA AGAGCTACAA GAAAATCAAG ATGAAATTGA
AAATATGATG AACTCTATCT TTAAGGGTAT ATTTGTTCAT CGATACCGTG
1201 ATGCAATTGC TGAAATTCGA GCCATCTGTA TTGAAGAAAT TGGAGTGTGG
ATGAAAATGT ATAGCGATGC CTTCCTAAAT GACAGTTACT TGAAATATGT
1301 AGGATGGACT CTTCACGATA GGCAAGGGGA AGTCAGACTG AAGTGTTTGA
AAGCTCTACA AAGCCTGTAT ACCAATAGAG AATTATTCCC CAAATTGGAG
1401 CTGTTTACAA ATCGATTCAA GGATCGCATT GTATCAATGA CTCTTGACAA
AGAATATGAT GTTGCTGTGG AAGCAATCCG ATTGGTTACT CTGATCCTTC
1501 ATGGCAGTGA AGAAGCTCTT TCCAACGAAG ACTGTGAAAA TGTTTACCAT
TTGGTGTATT CAGCACATCG CCCTGTTGCT GTGGCAGCTG GAGAGTTCCT
1601 ACACAAAAAG CTGTTCAGCA GACATGACCC ACAAGCAGAG GAAGCGTTAG
CGAAGAGGAG AGGAAGGAAC AGTCCAAACG GGAACCTCAT TAGGATGCTT
1701 GTTCTTTTCT TTCTGGAAAG TGAGTTACAT GAACATGCAG CCTACTTGGT
GGACAGCTTG TGGGAGAGCT CTCAAGAACT GTTGAAAGAC TGGGAATGTA
1801 TGACAGAGTT ACTATTAGAA GAACCTGTTC AAGGAGAAGA AGCAATGTCT
GACCGTCAAG AGAGTGCTCT TATAGAGCTA ATGGTCTGTA CAATTCGTCA
1901 GGCAGCTGAG GCACATCCTC CAGTGGGAAG GGGTACTGGC AAGAGAGTGC
TAACAGCCAA AGAAAGGAAA ACTCAAATTG ATGATAGGAA CAAATTAACT
2001 GAACATTTTA TCATTACACT TCCTATGTTA CTATCAAAGT ATTCTGCAGA
TGCTGAGAAG GTAGCAAACT TGCTGCAAAT TCCACAGTAT TTTGATCTAG
2101 AAATCTACAG CACAGGTCGG ATGGAAAAGC ACCTGGATGC TTTATTAAAA
CAAATTAAGT TTGTTGTAGA GAAACATGTA GAATCAGATG TCCTAGAAGC
2201 CTGCAGTAAA ACCTACAGCA TTTTATGCAG TGAAGAGTAT ACCATTCAGA
ATCGAGTTGA TATTGCTCGG AGCCAACTGA TTGATGAATT TGTAGATCGA
2301 TTCAATCATT CTGTAGAAGA CCTGTTGCAA GAGGGTGAAG AAGCTGACGA
TGATGATATC TACAATGTTC TTTCCACCTT AAAGCGTTTA ACTTCTTTTC
2401 ACAATGCTCA TGATCTCACT AAATGGGATC TATTTGGTAA TTGCTACAGA
CTATTGAAGA CTGGAATTGA ACATGGAGCT ATGCCAGAAC AGATAGTCGT
2501 ACAAGCACTG CAGTGTTCCC ATTACTCAAT TCTGTGGCAG TTGGTGAAAA
TTACTGACGG ATCTCCTTCC AAAGAGGATT TGTTGGTATT GAGGAAAACA
2601 GTGAAGTCTT TTCTGGCTGT TTGCCAACAG TGCCTATCTA ATGTTAATAC
TCCAGTTAAG GAACAGGCTT TCATGCTGCT CTGTGATCTT TTAATGATTT
2701 TTAGCCACCA ATTAATGACA GGTGGCAGAG AGGGCCTTCA GCCTTTGGTG
TTTAATCCAG ATACTGGACT CCAGTCTGAA CTCCTCAGTT TTGTGATGGA
2801 TCATGTTTTC ATTGACCAAG ATGAGGAAAA CCAAAGCATG GAGGGTGATG
AGGAAGATGA AGCTAATAAA ATTGAAGCCT TACATAAAAG GAGGAACCTG
2901 CTTGCTGCCT TCAGCAAACT TATCATTTAT GATATTGTTG ACATGCACGC
AGCTGCAGAC ATCTTTAAAC ACTACATGAA GTATTACAAT GACTATGGTG
SUBSTITUTE SHEET (RULE 28) 3001 ATATTATTAA GGAAACACTG AGTAAGACCA GGCAGATTGA TAAAATTCAG
TGTGCTAAGA CTCTCATTCT CAGTCTGCAA CAGTTGTTTA ATGAACTTGT
3101 TCAAGAACAA GGTCCTAATC TGGATAGGAC ATCTGCCCAC GTCAGTGGGA
TTAAAGAACT GGCACGTCGT TTTGCCCTTA CATTTGGATT GGACCAGATC
3201 AAGACCCGAG AAGCTGTTGC CACACTTCAC AAGGATGGAA TAGAGTTTGC
CTTTAAATAC CAGAATCAGA AAGGACAAGA ATACCCACCC CCTAATCTGG
3301 CTTTTCTAGA AGTACTAAGT GAATTTTCTT CTAAACTCCT TCGACAGGAC
AAGAAAACTG TTCATTCGTA TCTTGAAAAA TTCCTCACCG AGCAGATGAT
3401 GGAAAGGAGG GAGGATGTGT GGCTTCCACT CATCTCGTAT AGAAACTCAT
TAGTCACTGG AGGTGAAGAT GACAGGATGT CTGTGAACAG TGGAAGTAGC
3501 AGCAGTAAAA CGTCCTCAGT AAGGAGTAAG AAAGGAAGAC CCCCACTGCA
CAGAAAACGA GTAGAAGATG AAAGTCTGGA TAACACATGG CTAAATAGGA
3601 CTGACACGAT GATTCAGACT CCTGGACCCT TGCCAACCCC ACAGCTCACC
TCCACGGTAC TTAGAGAAAA CAGCCGTCCC ATGGGAGAGC AGATTCAGGA
3701 ACCTGAGTCT GAGCATGGCT CTGAACCAGA CTTTTTACAT AATCCCCAGA
TGCAGATCTC TTGGTTAGGC CAGCCGAAGT TAGAAGACTT GAATCGGAAG
3801 GACAGAACAG GGATGAACTA CATGAAAGTA AGAGCTGGAG TCCGGCACGC
CGTTCGGGGT CTAATGGAGG AAGATGCTGA GCCCATCTTT GAAGATGTGA
3901 TGATGTCATC ACGGAGCCAG TTAGAAGACA TGAATGAAGA ATTTGAAGAC
ACCATGGTTA TTGATTTGCC CCCATCAAGA AATCGCCGAG AGAGAGCTGA
4001 GCTAAGGCCA GACTTCTTTG ACTCTGCAGC TATCATAGAA GATGATTCAG
GATTTGGAAT GCCTATGTTC TGAAGTCTGA AGAAAATTTA CAAATCTGGA
4101 ACTCTATTAT TTAGAGCTAG AGGCCTATAT ACTGTGATAG CTTGTATGGG,
(ii) a DNA sequence coding for human stromalin-l and having the following sequence
1 GGCTGTGACA CTAATACTTA ACATGGTGGT TGTGTCTCTT TATGCCTGAC
TCAATCAGTT GAAATCCAAA AGTAAGTTCT TCCTTGATTT ACCTGCCAAG
101 ACCTGAGTTC AGGCCCTCAG GGTGCTGAGG TTTTCCTTTG TGGGAGAAAA
TGCCACCAGA TGGCGGGTTA GGATTGCAGC TCCGTTGAAG GCGCGGCCCC
201 CGCTCCCGAA CCCCCGGCGA CCACCCCGTA ACAACCCCCC CACATCGGGA
ATAACACACC GGAGACTTTT GGGGGGAAAC TAGGTCGATG GTCGGCGGCG
301 CCGGATGGGC AGCTGAGGAT TGCCTTTGAG GTTATTTTAA AAGTTTTGAG
TTGTACAGCA CTTGATTATT TTGCTGCATT GTGAAAGGAC CTCTCCAGCA
401 ATGATTACTT CAGAATTACC AGTGTTACAG GATTCAACTA ATGAAACTAC
TGCCCATTCC GATGCTGGCA GCGAGCTTGA AGAAACAGAG GTCAAAGGAA
501 AAAGAAAAAG GGGTCGTCCT GGCCGGCCTC CATCTACAAA TAAGAAACCT
CGAAAATCTC CAGGTGAGAA GAGCAGAATT GAAGCTGGAA TTAGAGGAGC
601 AGGCCGTGGA AGAGCTAATG GACACCCTCA ACAGAATGGG GAAGGGGAGC
CTGTCACATT ATTTGAGGTG GTGAAACTGG GGAAAAGTGC AATGCAGTCC
701 GTGGTGGATG ACTGGATTGA ATCATATAAA CAAGACAGGG ACATCGCACT
TCTGGATTTA ATCAACTTTT TTATCCAGTG TTCAGGATGT CGAGGTACTG
801 TGAGAATAGA GATGTTTCGA AATATGCAGA ATGCAGAAAT CATCAGAAAA
ATGACTGAAG AATTTGATGA GGACAGTGGT GATTATCCTC TTACCATGCC
901 TGGACCTCAG TGGAAAAAAT TTCGTTCAAA CTTTTGTGAA TTTATTGGAG
TCCTGATTCG ACAGTGTCAG TATAGCATAA TTTATGATGA GTATATGATG
1001 GACACAGTAA TCTCCCTTTT GACGGGTTTG TCAGACTCCC AGGTCAGAGC
TTTTAGGCAT ACAAGTACCC TGGCTGCCAT GAAGCTCATG ACTGCTCTGG
1101 TGAATGTTGC CTTAAACCTC AGTATTCATC AGGATAATAC CCAGAGACAA
TATGAAGCCG AGAGAAATAA AATGATTGGG AAGAGAGCCA ATGAAAGGTT
SUBSTITUTE SHEET (RULE 28) 1201 GGAGTTACTA CTTCAGAAAC GCAAAGAGCT GCAAGAAAAT CAGGATGAAA
TCGAAAATAT GATGAACTCT ATTTTTAAGG GTATATTTGT TCATAGATAC
1301 CGTGATGCTA TTGCTGAGAT TAGAGCCATT TGTATTGAAG AAATTGGAGT
ATGGATGAAA ATGTATAGTG ATGCCTTCCT AAATGACAGT TACCTAAAAT
1401 ATGTTGGCTG GACTCTTCAT GACAGGCAAG GGGAAGTCAG GCTGAAGTGT
TTGAAAGCTC TGCAGAGTCT ATATACCAAT AGAGAATTAT TCCCCAAATT
1501 GGAACTATTC ACTAACCGAT TCAAGGATCG CATTGTATCA ATGACACTTG
ATAAAGAATA TGATGTTGCT GTGGAAGCTA TTCGATTGGT TACTCTGATA
1601 CTTCATGGAA GTGAAGAAGC TCTTTCCAAT GAAGACTGTG AAAATGTTTA
CCACTTGGTG TACTCGGCAC ATCGCCCTGT TGCTGTGGCA GCTGGAGAGT
1701 TCCTTCACAA AAAGCTATTT AGCAGACATG ACCCACAAGC AGAAGAAGCA
TTAGCAAAGA GGAGGGGAAG AAACAGCCCG AATGGAAACC TCATTAGGAT
1801 GCTGGTTCTT TTCTTTCTTG AAAGTGAGTT ACATGAACAT GCAGCCTACT
TGGTGGACAG TTTATGGGAG AGCTCTCAAG AACTGTTGAA AGACTGGGAA
1901 TGTATGACAG AGTTGCTATT AGAAGAACCT GTTCAAGGAG AGGAAGCAAT
GTCTGATCGT CAAGAGAGTG CTCTTATAGA GCTAATGGTT TGTACAATTC
2001 GTCAAGCTGC TGAGGCACAT CCTCCAGTGG GAAGGGGTAC CGGCAAGAGA
GTGCTAACTG CCAAAGAAAG GAAAACTCAA ATTGATGATA GAAACAAATT
2101 GACTGAACAT TTTATTATTA CACTTCCTAT GTTACTGTCA AAGTATTCTG
CAGATGCAGA GAAGGTAGCA AACTTGCTAC AAATCCCACA GTATTTTGAT
2201 TTAGAAATCT ACAGCACAGG TAGAATGGAA AAGCATCTGG ATGCTTTATT
AAAACAGATT AAGTTTGTTG TGGAGAAACA CGTAGAATCA GATGTTCTAG
2301 AAGCCTGCAG TAAAACCTAT AGTATCTTAT GCAATGAAGA ATATACCATC
CAGAACAGAG TTGACATAGC TCGAAGCCAG CTGATTGATG AGTTTGTAGA
2401 TCGATTCAAT CATTCTGTGG AAGACCTATT GCAAGAGGGA GAAGAAGCTG
ATGATGATGA CATTTACAAT GTTCTTTCTA CATTAAAGCG GTTAACTTCT
2501 TTTCAGAATG CACATGATCT CACAAAATGG GATCTCTTTG GTAATTGCTA
CAGATTATTG AAGACTGGAA TTGAACATGG AGCCATGCCA GAACAGATAG
2601 TCGTGCAAGC ACTGCAGTGT TCCCATTATT CGATTCTTTG GCAGTTGGTG
AAAATTACTG ATGGCTCTCC TTCCAAAGAG GATTTGTTGG TATTGAGGAA
2701 AACGGTGAAA TCCTTTTTGG CTGTTTGCCA GCAGTGCCTG TCTAATGTTA
ATACTCCAGT GAAAGAACAG GCTTTCATGT TACTCTGTGA TCTTCTGATG
2801 ATTTTCAGCC ACCAATTAAT GACAGGTGGC AGAGAGGGCC TTCAGCCTTT
GGTGTTCAAT CCAGATACTG GACTCCAATC TGAACTCCTC AGTTTTGTGA
2901 TGGATCACGT TTTTATTGAC CAAGACGAGG AGAACCAGAG CATGGAGGGT
GATGAAGAAG ATGAAGCTAA TAAAATTGAG GCCTTACATA AAAGAAGGAA
3001 TCTACTTGCT GCTTTCAGCA AACTTATCAT TTATGACATT GTTGACATGC
ATGCAGCTGC AGACATCTTC AAACACTACA TGAAGTATTA CAATGACTAT
3101 GGTGATATTA TTAAGGAAAC ACTGAGTAAA ACCAGGCAGA TTGATAAAAT
TCAGTGTGCC AAGACTCTCA TTCTCAGTTT GCAACAGTTA TTTAATGAAC
3201 TTGTTCAAGA GCAAGGTCCC AACCTAGATA GGACATCTGC CCATGTCAGT
GGCATTAAAG AACTGGCACG TCGCTTTGCC CTTACATTTG GATTGGACCA
3301 GATTAAGACA CGAGAAGCAG TTGCCACACT TCACAAGGAT GGCATAGAGT
TTGCATTTAA ATACCAAAAT CAGAAAGGAC AAGAGTATCC ACCTCCTAAT
3401 CTGGCTTTTC TTGAAGTACT AAGTGAATTT TCTTCTAAAC TTCTTCGACA
GGACAAAAAG ACAGTTCATT CATACCTAGA GAAATTCCTT ACCGAGCAGA
3501 TGATGGAAAG GAGGGAGGAT GTATGGCTTC CACTCATCTC CTATAGAAAT
TCATTAGTCA CTGGGGGTGA AGATGATAGA ATGTCTGTGA ACAGTGGAAG
3601 TAGCAGCAGC AAAACCTCAT CAGTAAGGAA TAAGAAAGGA CGACCTCCAC
TTCATAAAAA ACGAGTAGAA GATGAGAGTC TGGATAACAC ATGGCTAAAC
3701 AGGACTGACA CCATGATTCA GACTCCTGGC CCCCTGCCAG CACCACAACT
CACATCCACT GTACTGCGGG AGAACAGTCG GCCCATGGGA GACCAGATTC
3801 AAGAACCTGA GTCTGAACAT GGTTCTGAAC CAGACTTTTT ACACAATCCT
CAGATGCAGA TCTCTTGGTT AGGCCAGCCG AAGTTAGAAG ACTTAAATCG
SUBSTITUTE SHEET (RULE 28) 3901 GAAGGACAGA ACAGGAATGA ACTACATGAA AGTGAGAACT GGAGTGAGGC
ATGCTGTTCG GGGTCTAATG GAGGAAGATG CTGAGCCCAT CTTTGAAGAT
4001 GTGATGATGT CATCCCGAAG CCAGTTAGAA GATATGAATG AAGAATTTGA
GGACACCATG GTTATTGATC TGCCTCCATC AAGAAATCGG CGAGAGAGAG
4101 CTGAGCTAAG GCCAGACTTC TTTGACTCTG CAGCTATCAT AGAAGATGAT
TCAGGATTTG GAATGCCTAT GTTCTGAAGT CTGAAGAAAA TTTACAAATC
4201 TGGAACTCTA TTATTTAGAG CTAGAGGCCT ATATACTGTG ATAGCTTGTA
TGGGGAAAAA CAACTTTTGA TGTGATCTGA TTTGTTTTTT AATCAAATGA
4301 TTAAGGTCAA TCCCTTTTTG CAGTGACAGA AGAGGAG,
(iii) a DNA sequence coding for human stromalin-2 and having the following sequence
1 TCAACATATG AAAAATCAAT GTAATATTTC ATATTAACAG TTGGAAGACT
CACAATTCAT AGTTTCAGAA TTTACAACAC AAACCTACAG GAATAAAGAC
101 AATGTAGTAT TAGAATAAGG TCAGATATAT AGATCAATGG ACAGAATTGA
CAGTCCAGAA ATACACCCAT ACGATTACGG TCACCTGATT TTTGACTAAG
201 GTTCCAAAAC AATTCACTAG GGGAAAGAAT AGTCTTGTCA AAAAATGGTG
CTCAGACAAG TGGATAGCCA TAAGCACAGA ATGAGTTTGG ACCCCTATCT
301 CAAATCTCAA ATACAAACAT TAACTCCGAA TTAATCAAAT GCCTAAATTT
AAGAGCTAAA ATTTTAAAGC TCTTAGAAGA AAACATAGGT ACACATCTTT
401 GTGACTTTGA ATTAGGTCAC GGTTTCCTTG ACATGACACT TAAAAGTACA
AGCAACAAAA GAAAAAAATA GATAAAATGA ACTTCAGCAA AATTAGAATG
501 TTTATGCTTC AGAAAACACT GTGAAGAAAG TGATCAGACA ACTCACAGAA
TGGGAAAAAT ATTTGTGAAT CATATCTCTT AATAAGGGTC CAGCAGAAAA
601 GGGCAAAGGT GGAAATGGAG GAGGAAAACC TCCTTCTGGT CCAAACCGAA
TGAATGGTCA TCACCAACAG AATGGAGTGG AAAACATGAT GTTGTTTGAA
701 GTTGTTAAAA TGGGCAAGAG TGCTATGCAG TCGGTGGTAG ATGATTGGAT
AGAATCATAC AAGCATGACC GAGATATAGC ACTTCTTGAC CTTATCAACT
801 TTTTTATTCA GTGTTCAGGC TGTAAAGGAG TTGTCACAGC AGAAATGTTT
AGACATATGC AGAACTCTGA GATAATTCGA AAAATGACTG AAGAATTCGA
901 TGAGGATAGT GGAGATTATC CACTTACCAT GGCTGGTCCT CAGTGGAAGA
AGTTCAAATC CAGTTTTTGT GAATTCATTG GCGTGTTAGT ACGGCAATGT
1001 CAATATAGTA TCATATATGA TGAGTATATG ATGGATACAG TCATTTCACT
TCTTACAGGA TTGTCTGACT CACAAGTCAG AGCATTTCGA CATACAAGCA
1101 CCCTGGCAGC TATGAAGTTG ATGACAGCTT TGGTGAATGT GGCACTAAAT
CTTAGCATTA ATATGGATAA TACACAAAGA CAATATGAAG CAGAACGAAA
1201 TAAAATGATT GGGAAACGAG CCAATGAGAG GCTAGAACTC CTGCTACAAA
AGCGGAAAGA GCTTCAGGAA AATCAAGATG AAATAGAAAA TATGATGAAT
1301 GCAATATTTA AAGGAGTGTT TGTACATAGA TACCGTGATG CGATACGTGA
AATTCGAGCT ATTTGCATTG AAGAGATTGG CATTTGGATG AAGATGTATA
1401 GTGATGCCTT TCTTAATGAC AGTTATTTAA AATATGTTGG TTGGACTATG
CATGATAAGC AAGGTGAAGT AAGACTCAAA TGTCTTACTG CTCTACAAGG
1501 GCTTTATTAT AACAAAGAGC TTAATTCCAA ACTGGAACTT TTTACCAGTC
GGTTCAAGGA TAGAATTGTG TCTATGACCC TTGACAAAGA ATATGATGTT
1601 GCAGTACAAG CAATAAAATT ACTCACTCTT GTTTTACAGA GTAGTGAAGA
AGTTCTCACT GCAGAAGATT GTGAAAATGT CTATCATCTG GTTTATTCAG
SUBSTITUTE SHEET (RULE 28) 1701 CTCACCGGCC AGTAGCAGTA GCAGCTGGAG AATTTCTCTA CAAAAAGCTC
TTCAGTCGTA GAGATCCAGA GGAGGATGGA ATGATGAAAA GAAGAGGAAG
1801 ACAAGGTCCA AATGCCAACC TTGTTAAGAC ATTGGTTTTT TTCTTTCTAG
AAAGTGAGTT ACATGAGCAT GCAGCATACC TTGTGGATAG CATGTGGGAC
1901 TGTGCTACTG AGCTGCTGAA AGACTGGGAA TGTATGAATA GCTTGTTACT
GGAAGAGCCA CTTAGTGGAG AGGAAGCACT AACAGATAGG CAAGAGAGTG
2001 CTCTGATTGA AATAATGCTT TGTACCATTA GACAAGCGGC TGAATGTCAT
CCTCCCGTGG GAAGAGGGAC AGGAAAAAGG GTGCTTACAG CAAAGGAGAA
2101 GAAGACACAG TTGGATGATA GGACAAAAAT CACTGAGCTT TTTGCCGTGG
CCCTTCCTCA GTTATTAGCA AAATACTCTG TAGATGCAGA AAAGGTGACT
2201 AACTTGTTGC AGTTGCCTCA GTACTTTGAT TTGGAAATAT ATACCACTGG
ACGATTAGAA AACGATTTGG ATGCCTTATT GCGACAGATC CGGAATATTG
2301 TAGAGAAGCA CACAGATACA GATGTTTTGG AAGCATGTTC TAAAACTTAC
CATGCACTCT GTAATGAAGA GTTCACAATC TTCAACAGAG TAGATATTTC
2401 AAGAAGTCAA CTGATAGATG AATTGGCAGA TAAATTTAAC CGGCTTCTTG
AAGATTTTCT GCAAGAGGGT GAAGAACCTG ATGAAGATGA TGCATATCAG
2501 GTATTGTCAA CATTGAAGAG GATCACTGCT TTTCATAATG CCCATGACCT
TTCAAAGAGG GATTTATTTG CTTGTAATTA CAAACTCTTG AAAACTGGAA
2601 TCGAAAATGG AGACATGCCT GAGCAGATTG TTATTCACGC ACTGCAGTGT
ACTCACTATG TAATCCTTTG GCAACTTGCT AAGATAACTG AAAGCAGCTC
2701 TACAAAGGAG GACTTGCTGC GTTTAAAGAA ACAAATGAGA GTATTTTGTC
AGATATGTCA ACATTACCTG ACCAACGTGA ATACTACTGT TAAGGAACAG
2801 GCCTTCACTA TTCTGTGTGA TATTTTGATG ATCTTCAGCC ATCAGATTAT
GTCAGGAGGG CGTGACATGT TAGAGCCATT AGTGTATACC CCTGATTCTT
2901 CATTGCAGTC TGAGTTGCTC AGCTTTATTT TGGATCATGT CTTCATTGAA
CAGGATGATG ATAATAATAG TGCAGATGGT CAGCAAGAGG ATGAAGCCAG
3001 TAAAATTGAA GCTCTGCACA AGAGAAGAAA TTTACTTGCA GCATTTTGTA
AGCTAATTGT ATATACTGTG GTGGAGATGA ATACAGCTGC AGATATCTTC
3101 AAACAGTATA TGAAGTATTA TAATGACTAT GGAGATATCA TCAAAGAAAC
AATGAGTAAA ACAAGGCAGA TAGACAAAAT TCAGTGTGCT AAGACCCTTA
3201 TTCTCAGTCT GCAACAGCTT TTTAATGAAA TGATACAAGA AAATGGCTAT
AATTTTGATA GATCATCCTC TACATTTAGT GGCATAAAAG AACTTGCTCG
3301 ACGTTTTGCT TTAACTTTTG GACTTGATCA GTTGAAAACA AGAGAAGCCA
TTGCCATGCT ACACAAAGAT GGCATAGAAT TTGCTTTTAA AGAGCCTAAT
3401 CCGCAAGGGG AGAGCCATCC ACCTTTAAAT TTGGCATTTC TTGATATTCT
GAGTGAATTT TCTTCTAAAC TACTTCGACA AGACAAAAGA ACAGTGTATG
3501 TTTACTTGGA AAAGTTCATG ACCTTTCAGA TGTCACTCCG AAGAGAGGAT
GTGTGGCTTC CACTGATGTC TTACCGAAAT TCTTTGCTAG CTGGTGGTGA
3601 TGATGACACC ATGTCAGTCA TTAGTGGAAT CAGCAGCCGG GGGTCAACAG
TACGGAGTAA AAAATCAAAA CCATCTACAG GAAAACGGAA AGTGGTTGAG
3701 GGCATGCAGC TTTCACTCAC TGAAGAAAGT AGTAGTAGTG ACAGTATGTG
GTTAACGAGA GAACAAACAC TGCACACCCC TGTTATGATG CAGACACCAC
3801 AACTCACCTC CACTATTATG AGAGAGCCCA AAAGATTACG GCCTGAGGAT
AGCTTCATGA GTGTTTATCC TAAGCAGACT GAACATCATC AAACACCTCT
3901 TGATTATAAT CGGCGTGGCA CAAGCCTAAT GGAAGATGAT GAAGAGCCAA
TTGTGGAAGA TGTTATGATG TCCTCAGAAG GGAGGATTGA GGATCTTAAT
4001 GAGGGAATGG ATTTTGACAC CATGGATATA GATTTGCCAC CATCAAAGAA
CAGACGAGAG AGAACAGAAC TGAAGCCTGA TTTCTTTGAT CCAGCTTCAA
4101 TTATGGATGA ATCAGTTCTT GGAGTGTCAA TGTTTTAATA CCAGTACACA
ATTAAATCTG TGGTGAAGTC AAAAAAAAAA AAAAAAAAAA AAA;
(b) nucleic acid sequences, encoding the same protein or polypep¬ tide as the nucleic acid sequence of (a); (c) nucleic acid sequences homologous to the sequences under (a) or (b) and encode a protein or polypeptide having a similar biological activity to the protein or polypeptide encoded by the sequences under (a) or (b);
(d) fragments of any of the nucleic acid sequences of (a), (b) or (c), which encode a polypeptide which retain some of the biological activity of the non-fragmented protein or polypep¬ tide;
(e) a nucleic acid sequence which hybridizes to any of sequences under (a), (b), (c) or (d), under high stringency conditions; and
(f) a DNA molecule comprising any of the sequences under (a), (b), (c), (d) or (e).
6. A DNA molecule according to Claim 1, being a vector comprising a nucleic acid sequence as defined in Claim 5 and a sequence required for the propagation and replication of said vector in a host cell.
7. A vector according to Claim 6 comprising a sequence required for control of expression of said nucleotide sequence.
SUBSTITUTE SHEET (RULE 28)
PCT/US1994/009854 1993-08-27 1994-08-26 Mammalian stromal cell proteins and dna sequences coding therefor WO1995006116A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
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Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IL106820A IL106820A0 (en) 1993-08-27 1993-08-27 Mammalian stromal cell proteins and dna sequences coding therefor
IL106820 1993-08-27

Publications (1)

Publication Number Publication Date
WO1995006116A1 true WO1995006116A1 (en) 1995-03-02

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Country Status (4)

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AU (1) AU7718294A (en)
IL (1) IL106820A0 (en)
WO (1) WO1995006116A1 (en)
ZA (1) ZA946448B (en)

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS, Volume 197, No. 3, issued 30 December 1993, D. BENAYAHU et al., "Marrow Adipocytes Regulate Growth and Differentiation of Osteoblasts", pages 1245-1252. *
SEMINARS IN HEMATOLOGY, Volume 28, No. 2, issued April 1991, A. PELED et al., "Stroma-Cell Dependent Hematopoiesis", pages 132-137. *
STEM CELLS, Volume 12, issued 1994, T. OTSUKA et al., "Expression of the c-kit Ligand and Interleukin 6 Genes in Mouse Bone Marrow Stromal Cell Lines", pages 409-415. *

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IL106820A0 (en) 1993-12-08
ZA946448B (en) 1995-05-02

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