WO2000017359A1 - Nouveaux polynucleotides associes au cartilage, polypeptides codes par ces derniers et utilisations associees - Google Patents

Nouveaux polynucleotides associes au cartilage, polypeptides codes par ces derniers et utilisations associees Download PDF

Info

Publication number
WO2000017359A1
WO2000017359A1 PCT/US1999/022108 US9922108W WO0017359A1 WO 2000017359 A1 WO2000017359 A1 WO 2000017359A1 US 9922108 W US9922108 W US 9922108W WO 0017359 A1 WO0017359 A1 WO 0017359A1
Authority
WO
WIPO (PCT)
Prior art keywords
sequence
polypeptide
cdna
polynucleotides
sequences
Prior art date
Application number
PCT/US1999/022108
Other languages
English (en)
Inventor
Dominik R. Haudenschild
Francios Binette
Original Assignee
Genzyme Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Genzyme Corporation filed Critical Genzyme Corporation
Priority to AU60591/99A priority Critical patent/AU6059199A/en
Publication of WO2000017359A1 publication Critical patent/WO2000017359A1/fr

Links

Classifications

    • 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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the present invention is directed to novel polynucleotides the polypeptides they encode and their use in preparing antibodies and in assays.
  • the ability to modulate cartilage and bone growth either positively or negatively is extremely important. There are a number of conditions where it would be desirable to be able to enhance or "up-regulate” bone growth as well as a number of conditions where it would be desirable to inhibit or "down-regulate” such bone growth.
  • Extensive bone growth along with associated cellular changes occurs during embryonic development.
  • the formation of long bones during embryonic limb development is a highly controlled process that requires precise coordination and interaction of many cellular pathways and the regulated expression of many gene products.
  • the process begins with a condensation of mesenchymal cells, followed by controlled proliferation, and subsequent differentiation initially into mature chondrocytes and then into hypertrophic cells.
  • the hypertrophic chondrocytes synthesize a specialized matrix which is thought to signal the remodeling process as well as the vascularization necessary for ossification to occur.
  • the process begins with a condensation of mesenchymal cells, followed by controlled proliferation, and subsequent differentiation initially into mature chondrocytes and then into hypertrophic cells (Fell and Canti, 1934) (Pechak et al., 1986) (Searls et al., 1972) (Reddi, 1995) (Linsenmayer et al., 1973) (Goetinck, 1991).
  • the hypertrophic chondrocytes synthesize a specialized matrix, which is thought to signal the remodeling process as well as the vascularization necessary for ossification to occur (Vu et al., 1998).
  • This chondrocyte differentiation in the developing long bone is under strict positional and temporal regulation. While the chondrocytes in the center of the cartilaginous bone terminally differentiate into hypertrophic cells, the chondrocytes at the ends maintain their cartilaginous phenotype and form the articular cartilage. That process is extremely intricate involving several different pathways and many different types of genes, both structural and enzymatic. It has been possible to simulate some of these conditions in vitro.
  • chondrocyte transplantation it would also be useful to have a simple and effective means of identifying and distinguishing chondrocytes from other cells.
  • these polynucleotides correspond to portions of genes which are differentially regulated in human articular chondrocytes 48 hours after transfer to an alginate suspension culture as compared to those expressed in a dedifferentiated monolayer culture.
  • One embodiment of the present invention is directed to isolated polynucleotides encoding human chrondrocyte differentiation-related polypeptides or immunogenic portions thereof, including mRNAs, DNAs, cDNAs, genomic DNAs, as well as antisense, analogs and biologically active and diagnostically or therapeutically useful fragments thereof, which include polynucleotides as set forth in the sequence listings.
  • the polypeptides which include fragments of the full length protein, are those expressed in the proper reading frame by the full length gene that the present polynucleotide sequences are part of.
  • Another embodiment is directed to polynucleotides and/or proteins that can be used as markers to identify chondrocytes.
  • the clone 2B9 (a partial sequence for which is provided in SEQ ID NO: 25) and unique fragments thereof of at least about 17 nucleotides can be used to distinguish chondrocytes from other cells.
  • Another marker can be an antibody to a polypeptide encoded by a polynucleotide, e.g., an antibody to a polynucleotide encoded by 2B9.
  • Another embodiment of the present invention is directed to nucleic acid probes comprising the novel polynucleotides of the invention or portions thereof of sufficient length to specifically hybridize to RNA transcribed from the human genes to which they correspond or to
  • Yet another embodiment is directed to human chondrocyte differentiation-related polypeptides or immunogenic portions thereof encoded by a DNA sequence comprising a polynucleotide of this invention.
  • a still further embodiment of the present invention is directed to a process for producing the polypeptides as well as immunogenic, biologically active or diagnostically useful fragments, analogs and derivatives thereof.
  • a still further embodiment of the present invention is directed to a process for producing such polypeptides by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells containing a polynucleotide of the present invention under conditions promoting expression of said proteins and subsequent recovery of said proteins.
  • Yet a further embodiment of the present invention is directed to antibodies specific to such polypeptides.
  • Another embodiment of the present invention is directed to processes for using one or more polypeptides encoded by the novel polynucleotides of the present invention to screen for compounds which interact with the polypeptides, for example, compounds which inhibit or activate the polypeptides of the present invention.
  • Still another embodiment of the present invention uses the antibodies directed against the polypeptides encoded by the novel polynucleotides of the present invention to create "knockouts" of the specific genes to which they correspond, allowing one to more fully analyze their function. For example, by intracellularly expressing a single-chain antibody that specifically binds to any of the polypeptides, that antibody by binding to the polypeptide, prevents the polypeptide from functioning. In this manner one can determine the precise effect a particular polypeptide has on a cell. Another method is to use the antibodies to bind extra-cellular or transmembrane proteins to similarly prevent function. In this manner one can readily determine the particular function of the protein expressed by a particular gene.
  • Figure 1 shows a northern blot analysis of cDNAs enriched during chondrocyte monolayer culture.
  • Total RNA was isolated from articular chondrocytes growing in monolayer: (M) or in alginate suspension: (S), for the indicated period of time in days: (d). Five ⁇ g of RNA was separated on formaldehyde 1% agarose gel and blotted onto Hybond-N+ membranes.
  • Blot A was hybridized with a CTGF cDNA probe and blot B with cDNA probe from clone 2G5. After washes in 0.1X SSC at 55°C, the blots were exposed and analyzed with a fugi BAS-1500 phosphorimager. The blots were then stripped and reprobed with a radiolabelled GAPDH probe. The patient age is indicated in parenthesis.
  • Figure 2 shows a northern blot analysis of cDNA enriched during suspension culture.
  • Total RNA was isolated from articular chondrocytes growing in monolayer: (M) or in alginate suspension: (S), for the indicated period of time in days: (d). Five ⁇ g of RNA was separated on formaldehyde 1% agarose gel and blotted onto Hybond-N+ membranes. Blot A was by hybridized with a clusterin cDNA probe and blot B with cDNA probe from clone 2G7. After washes in 0.1X SSC at 55°C, the blots were exposed and analyzed with a fugi BAS-1500 phosphorimager.
  • FIG 4 shows tissue distribution of 2G7 expression.
  • Multiple Tissue Blots tm were hybridized with a labelled 2G7 cDNA probe.
  • the blots were washed in 0.1X SSC at 55°C and analyzed with a fugi BAS-1500 phosphorimager.
  • the blots were then stripped and reprobed with a radiolabelled GAPDH probe.
  • Figure 5 shows 2G7 gene conservation amongst animal species. Genomic DNA from different species on a Zooblot tm were probed with labelled 2G7 cDNA and washed in 0.2X SSC at 68°C and analyzed with a fugi BAS-1500 phosphorimager.
  • the present invention is based on the discovery of novel polynucleotides involved in modulation of cartilage growth and differentiation.
  • the novel polynucleotides disclosed herein correspond to genes that are differentially expressed during chondrocyte growth and differentation. Chondrocyte differentiation is a complex process involving numerous pathways with intricate regulation of the involved genes. For example, the same progenitor cell may develop differently depending upon neighboring cells. Thus, chondrocytes in the center of the cartilaginous bone terminally differentiate into hypertrophic cells, whereas those at the ends maintain the cartilaginous phenotype, forming the articular cartilage.
  • the 2B9 clone is uniquely expressed in chondrocytes.
  • a probe based on a unique portion of the 2B9 sequence e.g. SEQ ID NO: 25 or a unique fragment thereof, one can readily distinguish chondrocytes from other cells.
  • a unique fragment is typically at least 15 nucleotides, preferably at least 17 nucleotides, more preferably at least 20 nucleotides, and still more preferably at least 30 nucleotides.
  • a unique fragment can be determined by a computer comparison against public data bases as of the filing date of this application.
  • the polynucleotides according to this invention were isolated by one method of identifying differentially expressed genes, subtractive hybridization of cDNA libraries, generated from dedifferentiated articular chondrocytes grown in monolayer, and from the same cell strains at 48 hours after transfer to an alginate suspension.
  • This in vitro system for the induction of chondrocyte differentiation involves the tryptic release of articular chondrocytes from the culture vessel, suspension of the cells in alginate, and gelling of the alginate by calcium chloride (Benya and Shaffer 1982; Bon Rush, et al. 1994).
  • Differential display techniques such as substractive hybridization are known in the art [Diatchenko et al, Proc. Natl. Acad Sci USA 93: 6025-6030 (1996); Gurskaya, N.G. et al, Anal. Biochem. 240: 90-97 (1996)]. They enable one to compare two populations of, for example mRNA, to identify those genes or rnRNA expressed in one population, but not the other. In this manner one can identify those genes that are specifically associated with a particular condition.
  • subtractive hybridization confirms that the novel polynucleotides disclosed herein are involved during the early events of articular chondrocyte dedifferentiation and redifferentiation.
  • cytokine IL-6 can bind to a receptor complex which includes gp-130 and IL-6R (Taga and Kishimoto, 1997; Hibi, et al. 1996). Activation of gp-130 will affect the transcription factor Acute Phase Response Factor (Stat3) (Akina, et al., 1994), whose activity results in transcriptional gene activation (Darnell, 1996).
  • Stat3 Acute Phase Response Factor
  • Stat3 has been implicated in the regulation of collagenase in gingival cells (Reddy, et al., 1998), as well as the differentiation of embryonic stem cells (Niwa, et al., 1998) and osteoblasts (Bellido, et al., 1997).
  • IL-6 has also been demonstrated to be involved in the regulation of manganese superoxide dismutase (Dougall and Nick, 1991).
  • Clusterin apolipoprotein J
  • novel polynucleotides of the present invention are of great interest. By their further characterization in cartilage and other tissues, a better ability to understand and control cartilage development and maintenance will be obtained.
  • the novel polynucleotides will be able to be used per se, to develop agonists and antagonists, preferably as small molecules, that can be used to more finely regulate the process.
  • agonists and antagonists preferably as small molecules, that can be used to more finely regulate the process.
  • differentially expressed sequences disclosed herein will provide important clues about the early induction of articular chondrocyte differentiation during embryonic development, which will be useful in a range of activities such as modulating cartilage repair after chondrocyte transplantation.
  • novel polynucleotides disclosed herein may, as discussed above, also be used as markers for specific cell types or states of differentiation.
  • clone 2B9 deposited as deposit C24 and comprising the partial sequence set forth in SEQ ID NO: 25, was found to be expressed in chondrocytes and not in synoviocytes or other adult human tissues, indicating it will be useful as a chondrocyte specific marker.
  • the isolated and purified polynucleotides of the present invention may be utilized in the form of RNA or in the form of DNA, which DNA includes cDNA, genomic DNA, and synthetic DNA.
  • the DNA may be double-stranded or single-stranded.
  • single stranded it may be either the coding (sense) strand or non-coding (anti-sense) strand.
  • the sequence may include DNA identical to one or more of the Seq. ID Nos. 1-99 or identical to that of the deposited clones to which the SEQ ID NO. corresponds or may be a different sequence which, as a result of the redundancy or degeneracy of the genetic code, would encode the same polypeptide as the DNA sequences of one of the above-described clones.
  • polynucleotides according to this invention when used to express a polyeptide, they may include additional coding sequences such as a leader or secretory sequence or a proprotein sequence; and non-coding sequences, such as introns or non-coding sequence 5' and/or 3' of the coding sequence.
  • polynucleotide encoding a polypeptide can encompass a polynucleotide which includes only coding sequence for the polypeptide as well as a polynucleotide which includes additional coding and/or non-coding sequence.
  • polynucleotides of the present invention can also be used as probes to identify other nucleic acid sequences, for example, by their ability to hybridize to other sequences under stringent conditions.
  • stringent conditions means hybridization is carried out under conditions typically referred to as “high stringency”. Such conditions are equivalent to hybridization overnight at 65 - 68°C without formamide in .2X SSC.
  • high stringency Such conditions are equivalent to hybridization overnight at 65 - 68°C without formamide in .2X SSC.
  • the skilled artisan knows that by varying the reagents and temperature equivalent conditions can be created.
  • nucleic acid sequences which hybridize to the above described polynucleotides in a preferred embodiment encode polypeptides which retain substantially the same biological function or activity as the gene product of a gene which corresponds to a polynucleotide according to this invention or to the deposited cDNA(s).
  • the nucleic acid sequences may have at least 17 or 20 bases, preferably at least 20 bases, more preferably at least 30 bases, and still more preferably at least 50 bases which hybridize to a polynucleotide of the present invention and which has an identity thereto, as hereinabove described, and which may or may not encoded a polypeptide which retains activity.
  • Such nucleic acid sequences may be employed as probes, for example, for recovery of a corresponding gene or as a diagnostic probe or as a PCR primer.
  • the present invention is also directed to polynucleotides having at least 75% identity, preferably at least 85%, more preferably at least 90% and more preferably at least 95% identity to a polynucleotide that includes the DNA of one of SEQ ID Nos: 1-99 or to a corresponding cDNA deposit as well as fragments thereof, which fragments have at least 30 bases and preferably at least 50 bases, and to polypeptides encoded by such polynucleotides.
  • a polypeptide encoded by these polynucleotides should be from the same reading frame as that of the differentially expressed gene. As explained herein in cases where the deposited cDNA is not full length one can use these clones to obtain the full length gene to readily determine the correct reading frame. In most instances one can use standard techniques to determine the appropriate reading frame.
  • the polynucleotides of this invention can serve to uniquely identify messenger RNA molecules.
  • the complete sequence of that messenger RNA, in the form of cDNA can be determined using the partial sequence as a probe to identify a cDNA clone corresponding to a full-length transcript, followed by sequencing of that clone.
  • the partial cDNA clone can also be used as a probe to identify a genomic clone or clones that contain the complete gene including regulatory and promoter regions, exons, and introns.
  • polynucleotides disclosed herein that are not full length may be used to identify the corresponding full length gene to which they correspond by standard means.
  • a partial sequence can be nick-translated or end-labelled with 2 P using polynucleotide kinase and labelling methods known to those with skill in the art (see, e.g., Basic Methods in Molecular Biology, L. G. Davis, M. D. Dibner, and J. F. Battey, ed., Elsevier Press, N.Y., 1986).
  • a library such as a lambda cDNA library prepared from cartilage tissue can be directly screened with the labelled sequences of interest or the library can be converted en masse to, for example, pBluescript (Stratagene Cloning Systems, La Jolla, Calif. 92037) to facilitate bacterial colony screening (see Sambrook et al., Molecular Cloning-A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), pg. 1.20. These methods are well known in the art. Briefly, filters with bacterial colonies containing the library in pBluescript or bacterial lawns containing lambda plaques are denatured and the DNA is fixed to the filters.
  • the filters are hybridized with the labelled probe using standard hybridization conditions such as those described by Davis et al., supra.
  • the partial sequences, cloned into lambda or pBluescript, can be used as positive controls to assess background binding and to adjust the hybridization and washing stringencies necessary for accurate clone identification.
  • the resulting autoradiograms are compared to duplicate plates of colonies or plaques; each exposed spot corresponds to a positive colony or plaque.
  • the colonies or plaques are selected, expanded and the DNA is isolated from the colonies for further analysis and sequencing.
  • Longer cDNA clones which hybridize to the polynucleotides according to this invention may be analyzed to determine the amount of additional sequence they contain using PCR with one primer from the known sequence and the other primer from the vector.
  • Clones with a larger vector-insert PCR product than the original partial sequence are analyzed by restriction digestion and DNA sequencing to determine whether they contain an insert of the same size or similar as the mRNA size determined from Northern blot analysis
  • longer cDNAs corresponding to the polynucleotides according to this invention may be purified by endonuclease digestion to release it from vector DNA, gel electrophoresis, and isolation of the cDNA by removal from low melting agarose gel.
  • the isolated insert DNA may be radiolabeled e.g., with 32 P labels, preferably by nick translation or random primer labeling. If necessary, the labeled insert can be used as a probe to screen e.g., a lambda phage cDNA library or a plasmid cDNA library to obtain even longer clones or clones with overlapping sequence. Colonies containing clones related to the probe cDNA may be identified and purified by known purification methods.
  • the newly purified clones may be nucleotide sequenced to identify full length sequences. Complete sequencing of full length clones may be then performed by known means. Northern blots of the mRNA from various tissues using at least part of the cDNA clones deposited herein can optionally be performed to check the size of the mRNA against that of the pu ⁇ orted full length cDNA.
  • the following procedures can be used to obtain full length genes or full length coding portions of genes corresponding to the cDNA clones disclosed herein that are not full length.
  • the partial cDNA clones disclosed herein can be made full-length by a variety of methods. For example, utilizing the rapid amplification of cDNA ends (RACE) procedure described in Frohman, M. A., Dush, M. K. and Martin, G. R. (1988) Proc. Nat'l. Acad. Sci. U.S.A., 85:8998-9002.
  • RACE rapid amplification of cDNA ends
  • poly A + or total RNA is reverse transcribed with Superscript II (Gibco/BRL) and an antisense or complementary primer specific to the cDNA sequence.
  • the primer is removed from the reaction with a Microcon Concentrator (Amicon).
  • the first-strand cDNA is then tailed with dATP and terminal deoxynucleotide transferase (Gibco/BRL).
  • an anchor sequence is produced which is needed for PCR amplification.
  • the second strand is synthesized from the dA-tail in PCR buffer, Taq DNA polymerase (Perkin-Elmer Cetus), an oligo-dT primer containing three adjacent restriction sites (Xhol, Sail and Clal) at the 5 1 end and a primer containing just these restriction sites.
  • This double-stranded cDNA is PCR amplified for 40 cycles with the same primers as well as a nested cDNA-specific antisense primer.
  • the PCR products are size-separated on an ethidium bromide-agarose gel and the region of gel containing cDNA products the predicted size of missing protein-coding DNA is removed.
  • cDNA is purified from the agarose with the Magic PCR Prep kit (Promega), restriction digested with Xhol or Sail, and ligated to a plasmid such as pBluescript SKII (Stratagene) at Shol and EcoRV sites.
  • This DNA is transformed into bacteria and the plasmid clones sequenced to identify the correct protein-coding inserts. Correct 5' ends are confirmed by comparing this sequence with the putatively identified homologue and overlap with the partial cDNA clone.
  • Several quality-controlled kits are available for purchase. Similar reagents and methods to those above are supplied in kit form from Gibco/BRL.
  • RNA alkaline hydrolyzed after reverse transcription and RNA ligase is used to join a restriction site-containing anchor primer to the first-strand cDNA. This obviates the necessity for the dA-tailing reaction which results in a polyT stretch that is difficult to sequence past.
  • a known alternative to generating 5' cDNA from RNA is to use cDNA library double- stranded DNA.
  • An asymmetric PCR-amplified antisense cDNA strand is synthesized with an antisense cDNA-specific primer and a plasmid-anchored primer. These primers are removed and a symmetric PCR reaction is performed with a nested cDNA-specific antisense primer and the plasmid-anchored primer.
  • a method similar to 5' RACE can also be used for generating the missing 5' end of a desired full-length gene.
  • a specific RNA oligonucleotide is ligated to the 5' ends of a population of RNA presumably containing full-length gene RNA transcript and a primer set containing a primer specific to the ligated RNA oligonucleotide.
  • a primer specific to a known sequence of the gene of interest is used to PCR amplify the 5' portion of the desired full length gene which may then be sequenced and used to generate the full length gene.
  • RNA isolation may then be treated with phosphatase if necessary to eliminate 5' phosphate groups on degraded or damaged RNA which may interfere with the later RNA ligase step.
  • the phosphatase if used is then inactivated and the RNA is treated with e.g. tobacco acid pyrophosphatase in order to remove the cap structure present at the 5' ends of messenger RNAs. This reaction leaves a 5' phosphate group at the 5' end of the cap-cleaved RNA which can then be ligated to an RNA oligonucleotide using T4RNA ligase.
  • This modified RNA preparation can then be used as a template for first strand cDNA synthesis using a gene-specific oligonucleotide.
  • the first strand synthesis reaction can then be used as a template for PCR amplification of the desired 5' end using a primer specific to the ligated RNA oligonucleotide and a primer specific to a specific partial sequence obtained from one of the SEQ ID NOs or one of the deposited clones.
  • the resultant product is then sequenced and analyzed to confirm that the 5' end sequence belongs to the partial sequence.
  • the complete sequence of the clones can be determined by a variety of methods. For example, using exonuclease III digestion (McCombie, W. R, Kirkness, E., Fleming, J. T., Kerlavage, A. R., Iovannisci, D. M., and Martin- Gallardo, R., Methods, 3:33-40, 1991).
  • a series of deletion clones are generated, each of which is sequenced.
  • the resulting overlapping sequences are assembled into a single contiguous sequence of high redundancy (usually three to five overlapping sequences at each nucleotide position), resulting in a highly accurate final sequence.
  • the present invention further relates to variants of the hereinabove described polynucleotides which encode fragments, analogs and derivatives of a polypeptide product of a gene corresponding to the polynucleotides of the present invention.
  • the variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non- naturally occurring variant of the polynucleotide.
  • Such nucleotide variants include deletion variants, substitution variants and addition or insertion variants.
  • the variant has at least about 85% homology to the present polypeptides. More preferably, at least about 90% o homology, still more preferably at least about 95%> homology.
  • % homology can be determined by any standard algorithm used to compare homologies. These include, but are not limited to BLAST 2.0 such as BLAST 2.0.4 and i. 2.0.5 available from the NIH (See www.ncbi.nlm.nkh.gov/BLAST/newblast.html)
  • the polynucleotides of the invention include those with coding sequences which are naturally occurring allelic variants of the human genes whose partial sequences are shown in SEQ ID NOs. 1-99 or of the sequences of the cDNA's in the deposited clones.
  • an allelic variant is an alternate form of a polynucleotide sequence which may have a substitution, deletion or addition of one or more nucleotides, which does not substantially alter the function of the encoded polypeptide.
  • the polynucleotides of the present invention may also be fused in frame to a marker sequence which allows for purification of the encoded polypeptide of the present invention.
  • the marker sequence may be a hexahistidine tag such as supplied by a pQE-9 vector to provide for purification of the mature polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used.
  • the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al, Cell, 37:767 (1984)).
  • this invention provides deposits of the cDNA clones described herein, either alone on in combination and containing the SEQ ID NOs. as set forth below:
  • Microorganisms transformed with each of the clones disclosed herein have been deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110-2209 as deposits and given ATCC DEPOSIT NOS. 98889-98894 (Identification by Depositor: ATCC Designation 98889 - Mixture of 22 Escherichia coli DH10B clones. C1-C18, C20-C23 ; Identification by Depositor: ATCC Designation 98890 - Mixture of 24 Escherichia coli DH10B clones, C25-C48;
  • the deposits as indicated above in four instances represent a combination deposit.
  • a known technique such as hybridization one can select a particular clone based upon the present disclosure that one is interested in and readily isolate it from the remainder of the deposit.
  • One can readily obtain a particular clone by using a probe based upon all or part of the sequence set forth for that clone, e.g. in the specific SEQ ID NO., by carrying out hybridization to obtain the clones from an appropriate sample or from one of the deposits.
  • PCR based upon the partial sequences set forth in the SEQ ID NOs., for example to prepare PCR primers.
  • a typical primer ranges from 17 - 23 bases.
  • the primers can then be prepared by standard olignonucleotide synthesis. PCR can be carried out using a sample from the deposit. Licenses may be needed to use the resultant sequence and no such license is hereby granted.
  • the deposit(s) referred to herein will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for pu ⁇ oses of Patent Procedure. These deposits are provided merely as convenience to those of skill in the art and are not an admission that a deposit is required under 35 U.S.C. ⁇ 112.
  • the sequence of the polynucleotides contained in the deposited materials, as well as the amino acid sequence of the polypeptides encoded thereby, are inco ⁇ orated herein by reference and are controlling in the event of any conflict with any description of sequences herein. A license may be required to make, use or sell the deposited materials, and no such license is hereby granted.
  • the polynucleotide sequences which hybridize as described above may be used to hybridize to and detect the expression of the human genes to which they correspond for use in diagnostic assays as hereinafter described.
  • cDNA polynucleotide complementary to the mRNA sequence
  • PCR polymerase chain reaction
  • reverse transcriptase Saiki et al., Nature, 234:163-166 (1986)
  • PCR polymerase chain reaction
  • reverse transcriptase Saiki et al., Nature, 234:163-166 (1986)
  • One application of this technology in nucleic acid probe technology is to bring up nucleic acid sequences present in low copy numbers to a detectable level. Numerous diagnostic and scientific applications of this method have been described. See e.g., H. A. Erlich (ed.) in PCR Technology-Principles and Applications for DNA Amplification, Stockton Press, U.S.A., 1989, and by M. A. Inis (ed.) in PCR Protocols, Academic Press, San Diego, U.S.A., 1990.
  • the present invention further relates to polypeptides encoded by the polynucleotides disclosed or deposited herein as well as fragments, analogs and derivatives of such polypeptides.
  • polypeptides encoded by the polynucleotides disclosed or deposited herein as well as fragments, analogs and derivatives of such polypeptides.
  • polypeptides may be recombinant polypeptides, natural polypeptides or synthetic polypeptides, preferably recombinant polypeptides.
  • the fragment, derivative or analog of the polypeptides encoded by the polynucleotides of the invention may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the polypeptide, such as a leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide or a proprotein sequence.
  • a conserved or non-conserved amino acid residue preferably a conserved amino acid residue
  • substituted amino acid residue may or may not be one encoded
  • polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
  • isolated means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring).
  • a naturally-occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated.
  • Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment.
  • similarity between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one polypeptide to the sequence of a second polypeptide.
  • Fragments or portions of the polypeptides of the present invention may be employed for producing the corresponding full-length polypeptide by peptide synthesis; therefore, the fragments may be employed as intermediates for producing the full-length polypeptides. Fragments or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.
  • the present invention also relates to vectors which include polynucleotides of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention by recombinant techniques.
  • Host cells may be genetically engineered (transduced or transformed or transfected) with the vectors of this invention which may be, for example, a cloning vector or an expression vector.
  • the vector may be, for example, in the form of a plasmid, a viral particle, a phage, etc.
  • the engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes.
  • the culture conditions such as temperature, pH and the like, are those previously used with the host cell selected for expression, and will be apparent to those of ordinarily skill in the art.
  • Vectors include chemical conjugates such as described in WO 93/04701, which has targeting moiety (e.g. a ligand to a cellular surface receptor), and a nucleic acid binding moiety (e.g. polylysine); viral vectors (e.g. a DNA or RNA viral vector); fusion proteins such as described in PCT/US 95/02140 (WO 95/22618) which is a fusion protein containing a target moiety (e.g. an antibody specific for a target cell) and a nucleic acid binding moiety (e.g. a protamine); plasmids; phage, etc.
  • the vectors can be chromosomal, non-chromosomal or synthetic.
  • Preferred vectors include plasmid vectors, viral vectors, fusion proteins and chemical conjugates.
  • Retroviral vectors are known to the skilled artisan and include those based on moloney murine leukemia viruses and HIV-based viruses.
  • One preferred HIV-based viral vector comprises at least two vectors wherein the gag and pol genes are from an HIV genome and the env gene is from another virus.
  • DNA viral vectors are preferred. These vectors include he ⁇ es virus vectors such as a he ⁇ es simplex I virus (HSV) vector [Geller, A.I. et al. J. Neurochem 64: 487 (1995); Lim, F. et al, in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ.
  • HSV simplex I virus
  • Useful vectors include chromosomal, nonchromosomal and synthetic DNA sequences, e.g., derivatives of SV40; bacterial plasmids; phage DNA; baculovirus; yeast plasmids; vectors derived from combinations of plasmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies.
  • any other vector may be used as long as it is replicable and viable in the host.
  • the appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures known in the art. Such procedures and others are deemed to be within the scope of those skilled in the art.
  • the DNA sequence in the expression vector is operatively linked to an appropriate expression control sequence(s) (promoter) to direct mRNA synthesis. Representative examples of such promoters, include a retioviral LTR or SV40 promoter, the E. coli. lac or t ⁇ , the phage lambda P[L ]promoter and other promoters known to control expression of genes in prokaryotic or eukaryotic cells or their viruses.
  • the expression vector also contains a ribosome binding site for translation initiation and a transcription terminator.
  • the vector may also include appropriate sequences for amplifying expression.
  • the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
  • the vector containing the appropriate DNA sequence as hereinabove described, as well as an appropriate promoter or control sequence, may be employed to transform an appropriate host to permit the host to express the protein.
  • bacterial cells such as E. coli, Streptomyces, Salmonella typhimurium
  • fungal cells such as yeast
  • insect cells such as
  • Drosophila S2 and Spodoptera Sf9 Drosophila S2 and Spodoptera Sf9; animal cells such as CHO, COS or Bowes melanoma; plant cells, etc.
  • animal cells such as CHO, COS or Bowes melanoma
  • plant cells etc.
  • the selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.
  • the present invention also includes recombinant constructs comprising one or more of the sequences as broadly described above.
  • the constructs comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inserted, in a forward or reverse orientation.
  • the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence.
  • a promoter operably linked to the sequence.
  • Bacterial pQ ⁇ 70, pQE60, pQE-9 (Qiagen), pBS, pDIO, phagescript, ⁇ siX174, pbluescript SK, pBSKS, pNH8A, pkrHl ⁇ a, pNH18A, pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 (Pharmacia).
  • Eukaryotic pWLNEO, pSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia).
  • any other plasmid or vector may be used as long as they are replicable and viable in the host.
  • Promoter regions can be selected from any desired gene using CAT (chloramphenicol transferase) vectors or other vectors with selectable markers.
  • Two appropriate vectors are pKK232-8 and pCM7.
  • Particular named bacterial promoters include lad, lacZ, T3, T7, gpt, lambda P[R], P[L ]and t ⁇ .
  • Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retro virus, and mouse metallothionein-I.
  • the present invention relates to host cells containing the above- described constructs.
  • the host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
  • Introduction of the construct into the host cell can be effected by a variety of methods including calcium phosphate transfection, DEAE-Dextran mediated transfection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)).
  • constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
  • the polypeptides of the invention can be synthetically produced by conventional peptide synthesizers.
  • Proteins can be expressed in mammalian cells, yeast, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteins using RNAs derived from the DNA constructs of the present invention. Appropriate cloning and expression vectors for use with prokaryotic and eukaryotic hosts are described by Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, N.Y., (1989).
  • recombinant expression vectors will include origins of replication and selectable markers permitting transformation of the host cell, e.g., the ampicillin resistance gene of E. coli and S. cerevisiae TRP1 gene, and a promoter derived from a highly-expressed gene to direct transcription of a downstream structural sequence.
  • promoters can be derived from operons encoding glycolytic enzymes such as 3-phosphoglycerate kinase (PGK), alpha-factor, acid phosphatase, or heat shock proteins, among others.
  • PGK 3-phosphoglycerate kinase
  • the heterologous structural sequence is assembled in appropriate phase with translation initiation and termination sequences.
  • the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant product.
  • Useful expression vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signals in operable reading frame with a functional promoter.
  • the vector will comprise one or more phenotypic selectable markers and an origin of replication to ensure maintenance of the vector and to, if desirable, provide amplification within the host.
  • Suitable prokaryotic hosts for transformation include E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.
  • Various mammalian cell culture systems can also be employed to express recombinant protein.
  • mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts, described by Gluzman, Cell, 23:175 (1981), and other cell lines capable of expressing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lines.
  • Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and also any necessary ribosome binding sites, polyadenylation site, splice donor and acceptor sites, transcriptional termination sequences, and 5' flanking nontranscribed sequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
  • polypeptides or fragments thereof produced by from the present genes can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the mature protein. Finally, high performance liquid chromatography (HPLC) can be employed for final purification steps.
  • the polynucleotides of the present invention may have the coding sequence fused in frame to a marker sequence which allows for purification of the polypeptide of the present invention.
  • a marker sequence is a hexahistidine tag which may be supplied by a vector, preferably a pQE-9 vector, which provides for purification of the polypeptide fused to the marker in the case of a bacterial host, or, for example, the marker sequence may be a hemagglutinin (HA) tag when a mammalian host, e.g. COS-7 cells, is used.
  • the HAtag corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson, I., et al., Cell, 31:161 (1984)).
  • polypeptides of the present invention may be a naturally purified product, or a product of chemical synthetic procedures, or produced by recombinant techniques from a prokaryotic or eukaryotic host (for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture). Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non- glycosylated. Polypeptides of the invention may also include an initial methionine amino acid residue.
  • antibodies is meant to include monoclonal antibodies, polyclonal antibodies and antibodies prepared by recombinant nucleic acid techniques that are selectively reactive with polypeptides encoded by the nucleotide sequences of the present invention.
  • selectively reactive refers to those antibodies that react with one or more antigenic determinants on one of the polypeptides and do not react with other polypeptides of differing antigenicity.
  • Antigenic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains and have specific three dimensional structural characteristics as well as specific charge characteristics.
  • Antibodies can be used for diagnostic applications or for research pu ⁇ oses, as well as to block binding interactions.
  • a cDNA clone of the present invention such as 2B9, (having the partial sequence set forth in SEQ ID NO: 25) may be expressed in a host using standard techniques (see above; see Sambrook et al., Molecular Cloning; A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, New York: 1989). Recovered proteins can be electrophoresed using PAGE and the appropriate protein band can be cut out of the gel. The desired protein sample can then be eluted from the gel slice and prepared for immunization. Preferably, one would produce a stably transformed cell to express high levels of the proteins which be selected and used to generate antibodies
  • mice can be immunized twice intraperitoneally with approximately 50 micrograms of protein immunogen per mouse. Sera from such immunized mice can be tested for antibody activity by immunohistology or immunocytology on any host system expressing such polypeptide and by ELISA with the expressed polypeptide.
  • active antibodies of the present invention can be identified using a biotin-conjugated anti-mouse immunoglobulin followed by avidin-peroxidase and a chromogenic peroxidase substrate.
  • Preparations of such reagents are commercially available; for example, from Zymad Co ⁇ ., San Francisco, California. Mice whose sera contain detectable active antibodies according to the invention can be sacrificed three days later and their spleens removed for fusion and hybridoma production. Positive supernatants of such hybridomas can be identified using the assays described above and by, for example, Western blot analysis.
  • any technique that provides for the production of antibody molecules by continuous cell lines may be used.
  • SCA single-chain antibody
  • the monoclonal antibodies may be human monoclonal antibodies or chimeric human- mouse (or other species) monoclonal antibodies.
  • the present invention provides for antibody molecules as well as fragments of such antibody molecules.
  • polypeptides, their fragments or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies thereto.
  • These antibodies can be, for example, polyclonal or monoclonal antibodies.
  • the present invention also includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expression library. Various procedures known in the art may be used for the production of such antibodies and fragments.
  • Antibodies may be used to do in vivo imaging, for example, by labeling the antibodies to facilitate scanning of an individual.
  • Another aspect of the present invention relates to assays which detect the expression of the gene products corresponding to the polynucleotides of the present invention.
  • Assays used to detect levels of a particular polypeptide in a sample are well-known to those skilled in the art and include radioimmunoassays, competitive-binding assays, Western blot analysis, ELISA assays and "sandwich" assays.
  • the sample may be a biological sample which may include, but is not limited to, tissue extracts, cell samples or biological fluids.
  • cartilage was minced, digested 18 hours in 0.1% collagenase (SOURCE), then any remaining cartilage pieces were digested again with 0.25%) collagenase/0.5% trypsin for 3 hours.
  • SOURCE 0.1% collagenase
  • the released cells were seeded into tissue culture flasks for expansion in monolayer, and cultured in Dulbecco's Modified Eagle's Medium (DMEM, GIBCO, Grand Island NY) supplemented with 10% fetal bovine serum (FBS, Hyclone, Logan UT).
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS Hyclone, Logan UT
  • chondrocytes were transferred to alginate suspension culture at a density of 10 6 cells/ml for re-differentiation, as described previously (Binette et al. 1998, Yaeger et al. 1997). Cells from three individual strains were used.
  • RNA was prepared from articular chondrocytes growing in monolayer and two days after their transfer to alginate suspension using Oligotex Direct mRNA kits (QIAGEN, Valencia CA). Two micrograms of these mRNAs were used with a PCR-Select cDNA Subtraction kit (Clontech, Palo Alto, CA) to generate two libraries of partial cDNAs, one enriched for sequences that are up-regulated in monolayer culture (uninduced), the other for sequences that are up-regulated when the cells are induced to differentiate for two days in alginate suspension.
  • Oligotex Direct mRNA kits QIAGEN, Valencia CA
  • Two micrograms of these mRNAs were used with a PCR-Select cDNA Subtraction kit (Clontech, Palo Alto, CA) to generate two libraries of partial cDNAs, one enriched for sequences that are up-regulated in monolayer culture (uninduced), the other for sequences that are up-regulated when the cells are induced to differentiate for two days
  • the libraries were ligated into the Srfl site of the pCR-Script vector (Stratagene, La Jolla, CA) and transformed into ElextroMax DH10B bacteria (GIBCO, Grand Island NY) using an Electroporator II apparatus (Invitrogen, Carlsbad CA).
  • the transformed bacteria were plated on selective LB agar plates containing 100 g/ml ampicillin. 500 clones that grew on the plates were picked and grown in 96 well plates in 1.0 ml of LB with lOO ⁇ g/ml ampicillin. After overnight growth, sterile glycerol was added to 20% and the clones were kept frozen at -80°C.
  • RNA was isolated using RNA/DNA kits (Qiagen) from three independent strains of adult human articular chondrocytes grown as monolayer or induced to differentiate in alginate suspension for various times. Two ⁇ g of RNA were separated on formaldehyde denaturing 1% agarose gels, blotted onto Hybond-N+ (Amersham, Arlington Heights IL) using a posiblotter (Stratagene, La Jolla, CA), and UV-crosslinked to the membrane. Purified cDNA inserts were radioactively labeled with the PrimelT II random priming kit (Stratagene) and hybridized to the northern blots in QuickHyb solution (Stratagene) for two hours.
  • RNA/DNA kits Qiagen
  • tissue northern blots (Clonetech) were used to evaluate the tissue distribution of some of the differentially expressed cDNAs using the same conditions described above.
  • Southern hybridization was performed using a blot which contains genomic DNA from different animal species (Zooblot, Clonetech). The cDNA was labeled and hybridized in the same conditions as for the northern blot analysis. The hybridized nylon membrane was washed twice at room temperature for 10 minutes in 2XSSC, 0.1 % SDS, followed by two washes at 68°C in 0.2 X SSC, 0.1% SDS for 30 minutes each.
  • Plasmid DNA was prepared from 200 clones of each library, and partial sequence was obtained. The sequences were compared to the non-redundant GenBank+EMBL+DDBJ+PDB databases using the BLAST 2.04 algorithm available from the NIH. Some clones contained sequences corresponding to ribosomal RNA, and these were not included in further analysis. Table I represents a list of the known genes which were found in the libraries. Some sequences were identified many times, and others only once. The sequences that were identified in the libraries were classified into groups according to the reported location and function of their gene products. The sequences identified were diverse, and included genes that code for nuclear, cytoplasmic, transmembrane, soluble secreted and extracellular matrix proteins.
  • proteins ranges from structural to enzymatic, and include chromosomal organization, transcription factors, metabolism, cell shape and attachment, signal transduction, extracellular matrix assembly and remodeling, and growth factors.
  • chromosomal organization includes chromosomal organization, transcription factors, metabolism, cell shape and attachment, signal transduction, extracellular matrix assembly and remodeling, and growth factors.
  • sequences from both libraries had little homology to known sequences in the databases at the time the searches were done, and code for novel proteins.
  • the remaining clone did not show a detectable signal in the RNA from either growth condition.
  • a more extensive time course study was performed on select genes, in order to closely follow their expression during the redifferentiation process. These experiments showed that the genes tested from the uninduced library were most highly represented in the monolayer RNA, and that their expression was reduced as the cells were transferred to alginate culture, and continued to decline with extended time in suspension culture. Similarly, the genes tested from the induced library showed reduced expression in monolayer culture, and sustained enhanced expression levels in suspension culture. Fifteen randomly chosen clones that had no homology to existing sequences in Genbank were also tested by northern blot hybridization.
  • the expression of 2B9 was also tested on multiple tissue blots and no signal was detected in any other human tissue (not shown).
  • the clone 2G7 the induced library (suspension cells) was also tested on multiple tissue blots. Unlike 2B9, it detected a band in RNA isolated from trachea and bone marrow ( Figure 4).
  • Clone 2G7 codes for a sequence that was differentially expressed in chondrocytes, and also in trachea and bone marrow. To test whether this sequence was conserved between species, a genomic Southern blot was performed. Upon hybridization of labeled 2G7 to a genomic southern blot containing DNA from several vertebrate species and yeast, we found a detectable pattern of band in all of the vertebrate species tested ( Figure 5).
  • Partial nucleotide sequence was obtained from purified plasmid containing either cDNA enriched during monolayer culture arrow pointing downward, or cDNA enriched during redifferentiation in alginate suspension culture: arrow pointing upward.
  • Chromosomal organization Transcription factor 4 S l/SNF complex 60 kOa subunit (U66618) 4 protein similar to mouse cornichon 1 non-histone chromosomal protein (U90549) i ertdomefial PAS domain protein- 1 (1181904) t acute phase response factor (L29277)
  • Matrix Formation Growth Factors & Cytokines 4 bigiyca ⁇ (J04599) 4 connective tissue growth factor (X78947) i decorin (M14219) 4 extracellular protein SI -5 (U03877) 4 alpha-1 type XV collagen (L25286) T interleukin 6 (X04403) 4 lysyl oxidase-like protein (L21186)
  • T MMP-3/stromelysi ⁇ (X05232) T alpha- 1-a ⁇ rtitry ⁇ stn-(r« « 1465) t MMP-1/collagenase (X54925) T MHC protein homologous to chicken T cartilage gp-39 (Y08374) B-complex protein (M24194) T chitinase precursor (U58514) T complement factor B (X72875)
  • SWI/SNF complex 60KDa subunit 1 Not Detected unknown m1 E2 1 1 unknown m2B1 1 Not Detected unknown m2B9 1 3 unknown m2D6 1 Not Detected unknown m2G5 1 3
  • APRF DNA binding protein 3 cartilage gp-39 protein 15 1 clusterin (apoiipoprotei ⁇ J, SP-40) 2 3 fibronectin 8 1 peanut agglutinin core protein (MGC-24) 3 2 protein-tyrosine phosphatase D1 2 Not Detected skin collagenase 42 1 stromelysin 13 1 unknown s1A4 1 unknown s1C6 Not Detected unknown s2A1 1 Not Detected unknown s2D8 Not Detected unknown s2D10 2 unknown s2E12 Not Detected unknown s1F3 2 unknown s2F10 2 unknown s1G3 Not detected unknown s2G7 3

Landscapes

  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne de nouveaux polynucléotides impliqués dans la modulation de la croissance et de la différenciation du cartilage. De manière plus spécifique, les nouveaux polynucléotides de l'invention correspondent à des gènes qui sont exprimés de manière différentielle pendant la croissance et la différenciation des chondrocytes. On décrit également des polypeptides codés par les polynucléotides de l'invention, leur utilisation dans divers dosages et procédés, ainsi que des anticorps dirigés contre ces polypeptides.
PCT/US1999/022108 1998-09-24 1999-09-23 Nouveaux polynucleotides associes au cartilage, polypeptides codes par ces derniers et utilisations associees WO2000017359A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU60591/99A AU6059199A (en) 1998-09-24 1999-09-23 Novel cartilage-associated polynucleotides, polypeptides encoded thereby, and uses thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US10159898P 1998-09-24 1998-09-24
US60/101,598 1998-09-24

Publications (1)

Publication Number Publication Date
WO2000017359A1 true WO2000017359A1 (fr) 2000-03-30

Family

ID=22285487

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1999/022108 WO2000017359A1 (fr) 1998-09-24 1999-09-23 Nouveaux polynucleotides associes au cartilage, polypeptides codes par ces derniers et utilisations associees

Country Status (2)

Country Link
AU (1) AU6059199A (fr)
WO (1) WO2000017359A1 (fr)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989000164A1 (fr) * 1987-07-01 1989-01-12 Max-Planck-Gesellschaft Zur Förderung Der Wissensc Proteines membranaires specifiques des chondroblastes et procede de diagnostic et de controle de reactions auto-immunitaires contre des chondroblastes
WO1998035043A1 (fr) * 1997-02-06 1998-08-13 Genetics Institute, Inc. Proteines sdf-5 humaines et compositions associees

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1989000164A1 (fr) * 1987-07-01 1989-01-12 Max-Planck-Gesellschaft Zur Förderung Der Wissensc Proteines membranaires specifiques des chondroblastes et procede de diagnostic et de controle de reactions auto-immunitaires contre des chondroblastes
WO1998035043A1 (fr) * 1997-02-06 1998-08-13 Genetics Institute, Inc. Proteines sdf-5 humaines et compositions associees

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK 21 April 1999 (1999-04-21), DUBB K.L.: "H. sapiens clone UWGC:djs124 from 7p14-15, complete sequence", XP002128000 *
DHARMAVARAM R.M. ET AL.: "Detection and characterization of Sp1 binding activity in human chondrocytes and its alterations during chondrocyte dedifferentiation", J. BIOL. CHEM., vol. 272, no. 43, 24 October 1997 (1997-10-24), pages 26918 - 26925, XP002127999 *
SHEN M ET AL: "MOLECULAR CHARACTERIZATION OF THE NOVEL BASIC HELIX-LOOP-HELIX PROTEIN DEC1 EXPRESSED IN DIFFERENTIATED HUMAN EMBRYO CHONDROCYTES", BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS,US,ACADEMIC PRESS INC. ORLANDO, FL, vol. 236, 1997, pages 294-298, XP002914516, ISSN: 0006-291X *

Also Published As

Publication number Publication date
AU6059199A (en) 2000-04-10

Similar Documents

Publication Publication Date Title
EP0750628B1 (fr) Facteur-10 de croissance du fibroblaste
Südhof et al. A synaptic vesicle protein with a novel cytoplasmic domain and four transmembrane regions
US5773252A (en) Fibroblast growth factor 15
US6342370B1 (en) Human slit polypeptide and polynucleotides encoding same
JP2001514024A (ja) ５０個のヒト分泌タンパク質
JP2003525566A (ja) １２５個のヒト分泌タンパク質
US5763214A (en) Fibroblast growth factor 11
US6368822B1 (en) Fibroblast growth factor 13
JP2001519179A (ja) ５３個のヒト分泌タンパク質
US5654172A (en) Gabaa receptor epsilon subunit
US20020090694A1 (en) Human Hox C10 and polynucleotides encoding
WO2000017359A1 (fr) Nouveaux polynucleotides associes au cartilage, polypeptides codes par ces derniers et utilisations associees
US7160994B2 (en) Osteoclast-specific genes and proteins and uses thereof
WO1996039508A1 (fr) Facteur de croissance des fibroblastes 13
US6337195B1 (en) Colon specific genes and proteins
WO2000063382A1 (fr) Genes et produits d'expression provenant de cellules hematopoietiques
EP0905239A2 (fr) Gène codant Afadin-1
US6787640B2 (en) Fibroblast growth factor 14
EP1033403A1 (fr) Gene provenant de chondrocytes foetaux humain
AU748429B2 (en) Fibroblast growth factor 11
EP1038960A1 (fr) BSMAP, une protéine de membrane spécifiquement exprimée dans le cerveau
US20030100485A1 (en) Human neurotrimin homolog
JPH10262680A (ja) Ｒｈｏ標的タンパク質ヒトｍＤｉａおよびその遺伝子
CA2340307A1 (fr) Epimorphine destinee a des artiodactyles
Li et al. Gaba a receptor epsilon subunit

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AU CA JP US

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase