EP0862620A1 - Tyrosine kinase gene and gene product - Google Patents

Tyrosine kinase gene and gene product

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Publication number
EP0862620A1
EP0862620A1 EP96936414A EP96936414A EP0862620A1 EP 0862620 A1 EP0862620 A1 EP 0862620A1 EP 96936414 A EP96936414 A EP 96936414A EP 96936414 A EP96936414 A EP 96936414A EP 0862620 A1 EP0862620 A1 EP 0862620A1
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EP
European Patent Office
Prior art keywords
polynucleotide
polypeptide
seq
leu
tnkl
Prior art date
Legal status (The legal status 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 status listed.)
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EP96936414A
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German (de)
French (fr)
Inventor
Curt I. Civin
Donald Small
Gerard T. Hoehn
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Johns Hopkins University
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Johns Hopkins University
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Publication of EP0862620A1 publication Critical patent/EP0862620A1/en
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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10Transferases (2.)
    • C12N9/12Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61KPREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00Medicinal preparations containing peptides

Definitions

  • This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention has been identified as a tyrosine kinase gene, sometimes hereafter referred to as "Tnkl” and splice variant gene "Tnkl ⁇ .”. The invention also relates to inhibiting the action of such polypeptides.
  • HSC lymphohematopoietic stem cells
  • CD38 antigen is expressed on most CD34+ cells, but the 1-10% of CD34+ cells lacking CD38 antigen expression (CD34+/CD38 cells) , also lack expression antigens characterizing mature blood cell ("lineage antigens") (Terstappen, et al., Blood, 77:1218-1226 (1991)).
  • CD34+/Lin/CD38- cells can be replated in vi tro, and give rise to myeloid and lymphoid colonies in long-term culture assays (Huang, S. and Terstappen, W. M. M. , Nature, 360:867-870 (1992); Huang, S. and Terstappen, W. M. M., Blood 83:1515-1526 (1994)).
  • this CD34+/Lin7CD38- cell population is an enriched population of early progenitors and likely HSC.
  • CD34+ cells obtained from umbilical cord blood contain a larger fraction of CD34+/Lin" /CD38 " cells as compared to adult bone marrow (Cardoso A. A.
  • cord blood contains higher numbers of cells with high proliferative potency in in vivo hematopoietic assays (Broxmeyer, H. E., et al. , PNAS, USA, 89:4109-4113 (1992)); Lu, L., et al., Blood, 81:41-48 (1993); Cardoso, A. A., et al., PNAS, USA, 90:8707-8711 (1993)).
  • Tyrosine kinases are a large, and rapidly growing family of protein ⁇ important in regulating cell growth and differentiation (Fantl, . J., et al., Annu. Rev. Biochem., 62: 453-481 (1993)).
  • Receptor tyrosine kinases contain an extracellular ligand-binding domain, a transmembrane domain, and a highly conserved catalytic kinase domain. Binding of ligand to receptor initiates a cascade of signals, culminating in a biological response. Early events include receptor dimerization, autophosphorylation on tyrosine residue( ⁇ ), and subsequent activation of catalytic activity (Ullrich, A. and Schlessinger, J.
  • non-receptor tyrosine kinases including members of the src and Jak families are involved further downstream, in intracellular signalling pathways. These non-receptor tyrosine kinases also contain the conserved catalytic kinase domain, and often contain domains that mediate protein: rotein interactions, such as SH2 and SH3 domains (Bolen, (1993); Musacchio, A., et al., FEBS Letters, 307:55- 61 (1992); Schlessinger, J. , Curr. Opin. Genet. Devel., 4:25- 30 (1994)).
  • a variety of receptor and non-receptor tyrosine kinases are important in the survival, proliferation and differentiation of hematopoietic cells.
  • the c- fms and c-Jit receptor tyrosine kinases have been shown to be required for the survival, proliferation and differentiation of monocytic cells and early hematopoietic progenitor cells, respectively (Sherr, C. J., Blood, 75:1-12 (1990); Chabot, B., et al., Nature, 335:88-89 (1988); Geissler, E. N. , et al., Cell, 55: 185-192 (1988)).
  • the non-receptor tyrosine kinase pp56 lck a src family member, is specifically expressed in T lymphocytes and is critical for their maturation (Amrein, K. E., et al., Proc. Nat. Aca. Sci. USA, 89:3343- 3346 (1992)), activation (Glaichenhaus, N. , et al., Cell, 64:511-520 (1991)), and IL-2 secretion (Karnitz, L., et al. , Mol. Cell. Biol., 12:4521-4530 (1992)).
  • pp56T' rt mediates signals derived by CD4 and CD8 receptors (Veilette, A., et al., Nature, 338:257-259 (1989)) .
  • the gene (SEQ ID N0:1, as shown in Figure 1) and gene product (SEQ ID N0:2, as shown in Figure 1) (and the splice variant gene (SEQ ID NO:3) and its gene product (SEQ ID NO:4) of the present invention have been putatively identified as the Tnkl gene and gene product (and the Tnkl ⁇ and gene product) as a result of homology to known tyrosine kinases.
  • novel mature polypeptides as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof.
  • the polypeptides of the present invention are of human origin.
  • nucleic acid molecules encoding a polypeptide of the present invention including mRNAs, DNAs, cDNAs, genomic DNAs a ⁇ well as analogs and biologically active and diagnostically or therapeutically useful fragments thereof.
  • a proces ⁇ for producing such polypeptides by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing a nucleic acid sequence encoding a polypeptide of the present invention, under conditions promoting expression of said protein and sub ⁇ equent recovery of said protein.
  • a process for utilizing such polypeptides, or polynucleotide encoding such polypeptides for therapeutic purposes for example, to suppress the transforming activity of certain genes by acting in a regulatory role, to regulate changes in the survival, proliferation and differentiation status of cells.
  • Preferred cells are hematopoietic stem and progenitor cells for bone marrow transplantation and support for cancer therapy.
  • antibodies against such gene and gene product (or to the splice variant gene) which may be employed, for example in labeled form, as markers.
  • Tnkl and Tnkl ⁇ agonists which elicit biological responses similar to native Tnkl and Tnkl ⁇ .
  • nucleic acid probe ⁇ comprising nucleic acid molecules of sufficient length to specifically hybridize to a nucleic acid sequence of the present invention.
  • diagnostic assays for detecting diseases or susceptibility to disease ⁇ related to mutation ⁇ in the nucleic acid sequences encoding a polypeptide of the present invention are provided.
  • FIG. 1 Polynucleotide (SEQ ID N0:1) and predicted amino acid sequence (SEQ ID NO:2) of Tnkl cDNA.
  • the conserved ATP binding motif present in all tyrosine kinases (GxGxxG) is underlined.
  • the conserved kinase region initially amplified from CD34+/Lin-/CD38- umbilical cord blood cDNA is shown in bold face type.
  • the double-underline indicates the putative SH3 domain.
  • Asterisks (*) denote potential SH3 binding motifs of the consensu ⁇ (P-x-x-P) .
  • the triangle ( ⁇ ) indicates a potential phosphotyrosine binding site for a recently described domain which recognizes the motif (N-x-x-Y) (Kavanaugh, W. M., et al., Science, 268:1177- 1180 (1995)).
  • the numbers on the left denote the DNA sequence; the numbers on the right refer to the amino acid sequence.
  • the Tnkl ⁇ splice variant cDNA (SEQ ID NO:3) and predicted amino acid sequence (SEQ ID NO:4) are the same as for Tnkl which are shown in Figure 1, except that nucleotide ⁇ 1359-1374 of SEQ ID NO:l are ab ⁇ ent, and corre ⁇ ponding amino acids 412-416 of SEQ ID NO:2 are absent.
  • FIG. 1 Alignment of the predicted Tnkl amino acid sequence with Ack. Homology begins at the NH2-terminus of both Tnkl (AA 27) and Ack (AA 83) and continues uninterrupted for 413 amino acids. This region includes the kinase domain (AA 123-372) and the putative SH3 domain (AA 396-441) . Homologous regions in the proline-rich domain (AA 530-556) and near the COOH-terminus (AA 638-664) are also shown. (+) indicate similar amino acids. The dashes in the sequence represent gaps to maximize alignment.
  • FIG. 3 Diagram of the structure of the Tnkl protein. The numbers refer to the amino acid sequence.
  • PTK350 refers to the original 210 bp PCR product amplified from CD34+/Lin- /CD38- umbilical cord blood cDNA. 1273 and 1623 refer to the region amplified in RT-PCR experiments.
  • the asteri ⁇ k ⁇ (*) denote the position of potential binding sites for SH3 domain ⁇ ; the triangle ( ⁇ ) refers to the po ⁇ ition of a potential binding site for a newly described pho ⁇ photyrosine binding domain.
  • the ⁇ olid line underneath denotes regions of homology to Ack.
  • p21 BD indicates a putative p21 binding domain.
  • nucleic acid (polynucleotide) sequence which encodes for the mature polypeptide having the deduced amino acid sequence of Figure l or for the mature polypeptide encoded by the cDNA of the clone deposited as ATCC Deposit No. 69924 on October 5, 1995 with the ATCC 12301 Parklawn Drive, Rockville, MD 20852.
  • the gene of the present invention was isolated using degenerate PCR to clone tyrosine kinase genes from an enriched population of human umbilical cord blood hematopoietic stem/progenitor cell ⁇ .
  • the ⁇ equence of the complete Tnkl coding region predicts a 72 kD protein. Compari ⁇ on of Tnkl to ⁇ equence ⁇ in protein databa ⁇ e ⁇ reveal ⁇ that it i ⁇ mo ⁇ t homologous to Ack, an intracellular tyrosine kinase.
  • Tnkl consists of an N-terminal kinase domain, a putative SH3 domain immediately C-terminal to the kinase domain, and a proline-rich C-terminal region. Homology between Tnkl and Ack diverges immediately following the SH3 domain. In Ack, this region is required for binding to the GTP-bound form of p21cdc42Hs (Manser et al., Nature. 363:364-367 (1993)). Analysis of Tnkl mRNA expression demonstrate ⁇ that Tnkl i ⁇ expressed in all cord blood, bone marrow and adult blood subpopulation ⁇ , as well as in most of the leukemia cell lines examined (16 of 20) .
  • Hybridization to fetal multi-tissue Northern blots detected several different Tnkl transcripts in all 4 fetal tissues examined. In contrast, a single 3.0 Kb Tnk transcript was detected in only 5 of 16 adult tissues examined (colon, prostate, and ovary, ⁇ mall intestine, and testis) .
  • Tnkl Fluorescence in si tu hybridization (FISH) analysis of metaphase chromosomes localized the Tnkl gene to the short arm of chromosome 17 (17pl3.1), near the p53 locus.
  • FISH Fluorescence in si tu hybridization
  • Tnkl i ⁇ just N-terminal to, and in frame with, a region of high homology Tnkl share ⁇ with another tyrosine kinase gene, Ack (Manser, E. et al., Nature, 363, 364-367 (1993)) .
  • Ack tyrosine kinase gene
  • this homologous region is al ⁇ o near the N-terminu ⁇ .
  • the Tnkl open reading frame terminate ⁇ at nt 2115, encoding a 666 amino acid protein with a molecular weight of about 72kD.
  • the Tnkl kinase domain contains all 12 conserved structural domains present in the tyrosine kinase family, including the ATP binding site (GxGxxG, aa 123-128) and the highly conserved tyrosine kinase domains (VHRDLAA and SDVWSFG) (Hanks, S.K. et al. , Science, 241, 42-52 (1988)). Tnkl has a methionine residue in this latter conserved motif, instead of the second conserved serine residue.
  • the tyrosine kinases Ack and Fak (Manser, E. et al., Nature, 363, 364-367 (1993); Whitney, G.S., et al.
  • kinase domain of Tnkl is near the N-terminu ⁇ (AA 123-372) .
  • N-x-x-Y AA 74-77
  • This region may serve as a possible binding site for a recently described phosphotyro ⁇ ine binding domain (Kavanaugh, W.M. et al., Science, 268, 1177-1180 (1995)).
  • C-terminal to the kinase domain of Tnkl are several proline-rich areas, including P-x- x-P motifs (around AA' ⁇ 345 and 535), which may serve as binding site ⁇ for SH3 domain ⁇ of other ⁇ ignal tran ⁇ duction proteins (Cohen, G.B. et al., Cell, 80, 237-248 (1995); Alexandropoulo ⁇ , K. et al., Proc. Nat. Acad. Sci. USA, 92, 3110-3114 (1995)).
  • there is a region (AA 396- 431) which is highly homologous to the SH3 domain of Ack (Manser, E. et al., Nature, 363, 364-367 (1993)).
  • Tnkl is 60% homologous to Ack (45% identical, 60% conserved) , over a 413 amino acid span (AA 28-441 of Tnkl) including the entire kinase domain (AA123-372, 43% identical, 57% conserved) and the putative SH3 domain (AA 396-441, 44% identical, 56% conserved) (Figure 2) .
  • the structure of the predicted Tnkl polypeptide i ⁇ similar to Ack in that it contains an N-terminal kinase domain, followed by an SH3 domain and a proline-rich C- terminal region ( Figure 3) .
  • Tnkl mRNA is present in total mononuclear cells, CD34+/Lin- cells and CD34- cells from umbilical cord blood and bone marrow. In addition, adult blood mononuclear cell and granulocytes both express Tnkl mRNA.
  • Tnkl is a rare transcript
  • RT-PCR was used to investigate Tnkl expression in these cells lines.
  • a cDNA was synthe ⁇ ized from Poly(A) RNA (approximately -1 ⁇ g) from 20 different human leukemia cell line ⁇ , amplified with Tnkl specific primers, electrophoresed and then probed with an internal Tnkl specific oligonucleotide. Of the 20 lines examined, 16 expres ⁇ ed detectable Tnkl mRNA. Thu ⁇ , Tnkl may be expre ⁇ sed ubiquitously during hematopoiesis.
  • Tnkl In contrast, only 5 of the 16 adult tis ⁇ ues expres ⁇ ed detectable Tnkl.
  • a single 3.0 Kb transcript is found only in adult prostate, ovary, small intestine, testis and colon. Tnkl transcript ⁇ are not found in adult lung, liver, kidney or brain. Thu ⁇ , Tnkl mRNA expre ⁇ ion appears to be higher in fetal tissues than in adult tissues.
  • the Tnkl gene was localized on human chromosome 17 via fluorescence in si tu hybridization (FISH) analysis on metaphase chromosomes from human lymphocytes. All 30 metaphase cells analyzed had at least 1 paired signal (involving both chromatids of a ⁇ ingle chromosome) . Of 46 paired signals, all were located on the p-arm of an E-group chromosome.
  • FPG Fluorescence plus Giemsa, Bhatt, B. et al., Nucl. Acids Res., 16, 3951-3961 (1988)
  • the polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA include ⁇ cDNA, genomic DNA, and ⁇ ynthetic DNA.
  • the DNA may be double- stranded or single-stranded, and if single ⁇ tranded may be the coding ⁇ trand or non-coding (anti-sense) strand.
  • the coding ⁇ equence which encode ⁇ the mature polypeptide may be identical to the coding sequence shown in Figure 1 or that of the deposited clone or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same mature polypeptide as the DNA of Figure 1 or the deposited cDNA.
  • the polynucleotide which encodes for the mature polypeptide of Figure l and/or for the mature polypeptide encoded by the deposited cDNA may include, but is not limited to: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequence,- the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5' and/or 3' of the coding sequence for the mature polypeptide.
  • polynucleotide encoding a polypeptide encompas ⁇ e ⁇ a polynucleotide which include ⁇ only coding sequence for the polypeptide a ⁇ well as a polynucleotide which includes additional coding and/or non-coding sequence.
  • the present invention further relates to variant ⁇ of the hereinabove described polynucleotides, such as ⁇ plice variant
  • Tnklc- which encode for fragments, analog ⁇ and derivative ⁇ of the polypeptide having the deduced amino acid ⁇ equence of
  • the variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.
  • the present invention includes polynucleotides encoding the same mature polypeptide as shown in Figure 1 and/or the ⁇ ame mature polypeptide encoded by the cDNA of the deposited clone as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptide of Figure 1 and/or the polypeptide encoded by the cDNA of the deposited clone.
  • Such nucleotide variant ⁇ include deletion variants, substitution variants and addition or insertion variants, such as the polypeptide of SEQ ID NO:4.
  • the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in Figure 1 and/or of the coding sequence of the deposited clone.
  • an allelic variant is an alternate form of a polynucleotide sequence which may have a sub ⁇ titution, deletion or addition of one or more nucleotide ⁇ , which does not sub ⁇ tantially alter the function of the encoded polypeptide.
  • the Tnkl ⁇ polypeptide ha ⁇ been shown by testing to have the ⁇ ame or ⁇ ub ⁇ tantially the ⁇ ame function as the Tnkl polypeptide.
  • gene means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons) .
  • Fragments of the full length gene of the present invention may be used as a hybridization probe for a cDNA library to isolate the full length cDNA and to isolate other cDNAs which have a high ⁇ equence ⁇ imilarity to the gene or ⁇ imilar biological activity.
  • Probes of this type preferably have at least 15 ba ⁇ e ⁇ and may contain, for example, at least 30 base ⁇ or 50 or more bases.
  • the probe may also be u ⁇ ed to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete gene including regulatory and promotor regions, exons, and introns.
  • An example of a screen comprises isolating the coding region of the gene by using the known DNA sequence to synthesize an oligonucleotide probe.
  • Labeled oligonucleotide ⁇ having a ⁇ equence complementary to that of the gene of the present invention are used to ⁇ creen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
  • the present invention further relate ⁇ to polynucleotide ⁇ which hybridize to the hereinabove-de ⁇ cribed sequences if there is at least 70%, preferably at least 90%, and more preferably at least 95% identity between the sequence ⁇ .
  • the present invention particularly relates to polynucleotide ⁇ which hybridize under ⁇ tringent condition ⁇ to the hereinabove-described polynucleotides.
  • stringent conditions means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences.
  • polypeptide ⁇ which either retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNAs of Figure l and/or the depo ⁇ ited cDNA(s) .
  • the polynucleotide may have at least 20 bases, preferably at least 30 bases, and more preferably at least 50 ba ⁇ es which hybridize to a polynucleotide of the pre ⁇ ent invention and which has an identity thereto, as hereinabove described, and which may or may not retain activity.
  • such polynucleotides may be employed as probes for the polynucleotide of, for example, for recovery of the polynucleotide or as a diagnostic probe or as a PCR primer.
  • the present invention is directed to polynucleotides having at least a 70% identity, preferably at least 90% and more preferably at lea ⁇ t a 95% identity to a polynucleotide which encode ⁇ the polypeptide of Figure l a ⁇ well a ⁇ fragment ⁇ thereof, which fragment ⁇ have at lea ⁇ t 15 ba ⁇ es and preferably at least 30 base ⁇ or at lea ⁇ t 50 base ⁇ and to polypeptides encoded by such polynucleotides .
  • the deposit (s) referred to herein will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for purposes of Patent Procedure.
  • the deposit is an E. coli bacterial strain DH5 ⁇ harboring a plasmid (pTnkl) that contains the full- length 2,790 bp Tnkl cDNA.
  • the Tnkl cDNA has been cloned into the EcoRI ⁇ ite of pBlue ⁇ cript KS(-) (Stratagene, La Jolla, Ca) .
  • the present invention further relates to a polypeptide which has the deduced amino acid sequence of Figure 1 and/or which has the amino acid sequence encoded by the deposited cDNA, as well as fragments, analogs and derivatives of ⁇ uch polypeptide.
  • fragment when referring to the polypeptide of Figure 1 and/or that encoded by the deposited cDNA, mean ⁇ a polypeptide which retain ⁇ e ⁇ entially the same biological function or activity as ⁇ uch polypeptide. Thu ⁇ , an analog include ⁇ a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
  • the polypeptide of the pre ⁇ ent invention may be a recombinant polypeptide, a natural polypeptide or a ⁇ ynthetic polypeptide, preferably a recombinant polypeptide.
  • the fragment, derivative or analog of the polypeptide of Figure 1 and/or that encoded by the depo ⁇ ited cDNA may be (i) one in which one or more of the amino acid re ⁇ idue ⁇ 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 re ⁇ idue ⁇ include ⁇ a ⁇ ubstituent group, or (iii) one in which the mature polypeptide is fused with another compound, ⁇ uch as a compound to increa ⁇ e the half-life of the polypeptide (for example, polyethylene glycol) , or (iv) one in which the additional amino acid ⁇ are fu ⁇ ed to the mature polypeptide for purification of the mature polypeptide.
  • Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings
  • polypeptide ⁇ and polynucleotide ⁇ 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 polynucleotide ⁇ 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.
  • polypeptides of the present invention include the polypeptide of Figure 1 (in particular the mature polypeptide) as well as polypeptides which have at lea ⁇ t 70% similarity (preferably at least 70% identity) to the polypeptide of Figure 1 and more preferably at least 90% similarity (more preferably at least 90% identity) to the polypeptide of Figure 1 and still more preferably at least 95% similarity (still more preferably at least 95% identity) to the polypeptide of Figure 1 and also include portions of ⁇ uch polypeptide ⁇ with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids.
  • similarity between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid sub ⁇ titute ⁇ 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. Fragment ⁇ or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.
  • the pre ⁇ ent invention al ⁇ o 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 technique ⁇ .
  • Ho ⁇ t cells are genetically engineered (transduced or tran ⁇ formed or tran ⁇ fected) 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 ho ⁇ t cell ⁇ can be cultured in conventional nutrient media modified a ⁇ appropriate for activating promoters, selecting transformant ⁇ or amplifying the genes of the present invention.
  • the culture conditions, ⁇ uch a ⁇ temperature, pH and the like, are tho ⁇ e previously used with the host cell ⁇ elected for expre ⁇ ion, and will be apparent to the ordinarily skilled artisan.
  • the polynucleotide ⁇ of the present invention may be employed for producing polypeptide ⁇ by recombinant technique ⁇ . Thu ⁇ , for example, the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide.
  • Such vectors include chromosomal, nonchromosomal and synthetic DNA sequence ⁇ , e.g. , derivatives of SV40; bacterial plasmids,- phage DNA,- baculovirus; yeast plasmids; vectors derived from combination ⁇ of pla ⁇ mids 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.
  • 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 ⁇ equence in the expression vector is operatively linked to an appropriate expres ⁇ ion control sequence(s) (promoter) to direct mRNA synthe ⁇ i ⁇ .
  • promoter an appropriate expres ⁇ ion control sequence(s) (promoter) to direct mRNA synthe ⁇ i ⁇ .
  • a ⁇ repre ⁇ entative example ⁇ of ⁇ uch promoter ⁇ there may be mentioned: LTR or SV40 promoter, the E. coli . lac or Trp, the phage lambda P L promoter and other promoter ⁇ 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 resi ⁇ tance in E. coli .
  • the vector containing the appropriate DNA ⁇ equence a ⁇ 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 cell ⁇ such as yeast
  • insect cells such as Drosophila S2 and Spodoptera Sf9
  • animal cells such as CHO, COS or Bowes melanoma,- adenoviru ⁇ e ⁇ ,- plant cell ⁇ , etc.
  • the present invention al ⁇ o include ⁇ recombinant con ⁇ tructs compri ⁇ ing one or more of the sequences as broadly described above.
  • the construct ⁇ comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been in ⁇ erted, in a forward or rever ⁇ e orientation.
  • the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence.
  • suitable vectors and promoters are known to those of skill in the art, and are commercially available.
  • Bacterial pQE70, pQE60, pQE-9 (Qiagen), pBS, pDIO, phagescript, p ⁇ iX174, pBlue ⁇ cript SK, pBSKS, pNH8A, pNH16a, 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 marker ⁇ .
  • Two appropriate vector ⁇ are pKK232-8 and pCM7.
  • Particular named bacterial promoter ⁇ include lad, lacZ, T3, T7, gpt, lambda P R , P L and Trp.
  • Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art.
  • the pre ⁇ ent invention relate ⁇ to ho ⁇ t cell ⁇ 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 ho ⁇ t cell can be a prokaryotic cell, such as a bacterial cell.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE- Dextran mediated transfection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)) .
  • the construct ⁇ in host cells can be u ⁇ ed in a conventional manner to produce the gene product encoded by the recombinant ⁇ equence.
  • the polypeptides of the invention can be synthetically produced by conventional peptide synthe ⁇ izer ⁇ .
  • Mature protein ⁇ can be expre ⁇ ed in mammalian cell ⁇ , yea ⁇ t, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such protein ⁇ 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) , the disclosure of which is hereby incorporated by reference.
  • Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increa ⁇ e it ⁇ transcription. Examples include the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • recombinant expre ⁇ ion vector ⁇ will include origin ⁇ of replication and selectable markers permitting tran ⁇ formation of the host cell, e.g., the ampicillin resi ⁇ tance gene of E. coli and S. cerevisiae TRPl gene, and a promoter derived from a highly-expre ⁇ ed gene to direct transcription of a downstream structural sequence.
  • promoters can be derived from operon ⁇ encoding glycolytic enzyme ⁇ ⁇ uch as 3-phosphoglycerate kinase (PGK) , ⁇ -factor, acid phosphata ⁇ e, or heat ⁇ hock proteins, among others.
  • the heterologous structural sequence is as ⁇ embled in appropriate pha ⁇ e with translation initiation and termination ⁇ equences.
  • the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristic ⁇ , e.g., ⁇ tabilization or simplified purification of expressed recombinant product.
  • Useful expres ⁇ ion vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signal ⁇ in operable reading pha ⁇ e 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 ho ⁇ t ⁇ for tran ⁇ formation include E. coli , Bacillus subtilis, Salmonella typhimurium and variou ⁇ species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.
  • useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic element ⁇ of the well known cloning vector pBR322 (ATCC 37017) .
  • cloning vector pBR322 ATCC 37017
  • Such commercial vector ⁇ include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA) .
  • pBR322 "backbone" ⁇ ections are combined with an appropriate promoter and the structural ⁇ equence to be expre ⁇ ed.
  • the selected promoter is induced by appropriate mean ⁇ (e.g., temperature ⁇ hift or chemical induction) and cells are cultured for an additional period.
  • appropriate mean ⁇ e.g., temperature ⁇ hift or chemical induction
  • Cell ⁇ are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
  • Microbial cell ⁇ employed in expre ⁇ ion of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well known to those ⁇ killed in the art.
  • mammalian cell culture ⁇ ystems can also be employed to expres ⁇ recombinant protein.
  • Example ⁇ of mammalian expre ⁇ ion ⁇ y ⁇ tem ⁇ include the COS-7 lines of monkey kidney fibrobla ⁇ t ⁇ , de ⁇ cribed by Gluzman, Cell, 23:175 (1981) , and other cell lines capable of expres ⁇ ing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell line ⁇ .
  • Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and al ⁇ o any nece ⁇ ary ribo ⁇ ome binding ⁇ ite ⁇ , polyadenylation ⁇ ite, ⁇ plice donor and acceptor sites, transcriptional termination sequence ⁇ , and 5' flanking nontran ⁇ cribed ⁇ equences.
  • DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
  • the polypeptide 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.
  • HPLC high performance liquid chromatography
  • 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) .
  • a prokaryotic or eukaryotic host for example, by bacterial, yeast, higher plant, insect and mammalian cells in culture
  • the polypeptides of the present invention may be glycosylated or may be non-glycosylated.
  • Polypeptides of the invention may al ⁇ o include an initial methionine amino acid re ⁇ idue.
  • Tnkl may be ⁇ ignalled, directly or via intermediate protein ⁇ , by the intracellular portion of a cell membrane receptor (after ligand bound to the receptor) directly or indirectly,- the receptor or intermediate protein may bind Tnkl and perhap ⁇ enzymatically activate Tnkl, for example, by phosphorylation of Tnkl. Activated Tnkl may then bind to some other protein in the cascade until eventually a transcription factor is involved and caused to effect expres ⁇ ion of ⁇ ome gene. Expre ⁇ ion of these genes may effect the cells' survival, proliferation and/or differentiation.
  • Tnkl and Tnkl ⁇ are each thought to function ⁇ imilarly to Ack, by binding to a ras- like protein in a pathway controlling cell proliferation.
  • Such pathway ⁇ are important in the biology of both cancer cells and lymphohematopoietic stem and progenitor cells, among other cell types.
  • either of the Tnkl or Tnkl ⁇ gene ⁇ may be employed to control the growth of cancer cells by cau ⁇ ing cancer cells to cea ⁇ e proliferating and die, and ⁇ timulating lymphohematopoietic ⁇ tem and progenitor ⁇ urvival and proliferation.
  • Tnkl or Tnkl ⁇ may also be employed to transiently cause stem and progenitor cells to pause in self-cycling, for example, while the patient with cancer receives chemotherapy or radiotherapy which may then more selectively kill cancer cells but not the normal stem and progenitor cells.
  • Tnkl or Tnkl ⁇ may also be employed to affect differentiation, ⁇ timulation or inhibition of certain cells.
  • Tnkl or Tnkl ⁇ may be employed to regulate the GTPase activity of a p21 ras-like protein by binding thereto, and therefore, suppress its transforming activity.
  • the gene and gene products of the present invention may also be employed to regulate signaling pathways during fetal development.
  • Tnkl or Tnkl ⁇ may also be employed to stimulate proliferation and differentiation of hematopoietic stem and/or progenitor cells for bone marrow transplantation or peripheral blood stem/progenitor cell tran ⁇ plantation.
  • polynucleotides and polypeptide ⁇ of the pre ⁇ ent invention may also be employed as research reagent ⁇ and materials for di ⁇ covery of treatment ⁇ and diagno ⁇ tic ⁇ to human disease.
  • Thi ⁇ invention provides a method for identification of the cell membrane receptor which functions to regulate Tnkl or Tnkl ⁇ activity, and a ligand which interact ⁇ with this receptor and to identify potential intermediate proteins.
  • Tnkl potentially functions in a signal transduction pathway downstream of events occurring on the cell surface.
  • immunoprecipitation followed by Western blotting, could be performed. This may show the interaction of two known proteins, usually shown using antibodies to both.
  • immunoprecipitation with Jak3 antibodies resulted in the co-immunoprecipitation of the IL-4 receptor, demonstrating that Jak3 associates with the IL-4 receptor (Rolling et al. , Oncogene, 10:1757-17611).
  • the protein(s) binding to Tnkl or Tnkl ⁇ can then be purified, partially ⁇ equenced, then cloned.
  • Tnkl can be cloned in-frame with a vector containing only the GAL4 binding domain, resulting in a GAL4-Tnkl fusion protein.
  • a cDNA library is then constructed in a vector containing only the GAL4 activation domain. These are then co-transformed into the appropriate yea ⁇ t strain ⁇ .
  • Po ⁇ itive clones i.e., clones that code for proteins that interact with Tnkl, are identified by growth in selective medium and blue/white screening.
  • the ligand may be identified by expres ⁇ ion cloning, as was done with the cloning of the FH3/FLK2 ligand (Lyman et al., Cell, 75:1157-1167). In this method, the extracellular domain of the receptor was fused to the Fc portion of the IgG and expressed as a fusion protein in 293 cells. Cell lines were a ⁇ sayed for their ability to bind to the Flt3/Fc fusion protein.
  • the method for determining whether a ligand can bind to the receptor which regulates Tnkl or Tnkl ⁇ function comprises transfecting a cell population (one pre ⁇ umed not to contain the receptor) with the appropriate vector expre ⁇ sing this receptor, ⁇ uch that the cell will now expres ⁇ thi ⁇ receptor.
  • a ⁇ uitable response sy ⁇ tem i ⁇ obtained by tran ⁇ fection of the DNA into a suitable host containing the desired second me ⁇ enger pathway ⁇ including cAMP, ion channel ⁇ , pho ⁇ phoinositide kinase, or calcium response.
  • Such a transfection sy ⁇ tem provide ⁇ a re ⁇ pon ⁇ e ⁇ ystem to analyze the activity of various ligands exposed to the cell.
  • Ligands chosen could be identified through a rational approach by taking known ligands that interact with similar types of receptors or u ⁇ ing ⁇ mall molecules, cell supernatants or extracts or natural products.
  • This invention provides a method of screening compounds to identify those which enhance (agonist ⁇ ) interaction of Tnkl or Tnkl ⁇ to it ⁇ receptor.
  • a mammalian cell or membrane preparation expressing a Tnkl or Tnkl ⁇ receptor is incubated with labeled Tnkl or Tnkl ⁇ in the presence of the compound.
  • the ability of the compound to enhance thi ⁇ interaction could then be measured.
  • the response of Tnkl after interaction of the compound to the receptor can be compared and measured in the presence and absence of the compound.
  • the present invention also relates to an a ⁇ ay for identifying potential antagonists against Tnkl or Tnkl ⁇ .
  • An example of such an assay combines Tnkl or Tnkl ⁇ and a potential antagoni ⁇ t with membrane-bound Tnkl or Tnkl ⁇ receptor ⁇ or recombinant Tnkl or Tnkl ⁇ receptor ⁇ under appropriate condition ⁇ for a competitive inhibition assay.
  • Tnkl or Tnkl ⁇ may be labeled, such as by radio activity, such that the number of Tnkl or Tnkl ⁇ molecule ⁇ bound to the receptor can determine the effectivene ⁇ of the potential antagonist.
  • Tnkl gene and gene product (or Tnkl ⁇ and its gene product) of the present invention act intracellularly. Accordingly, the only practical mode of administration of the Tnkl gene (or Tnkl ⁇ gene) is via gene therapy, hereinafter described, where it then acts to produce the Tnkl protein (or Tnkl ⁇ ) intracellularly.
  • Tnkl (or Tnkl ⁇ ) may be administered to a patient in an amount ⁇ ufficient to produce the Tnkl protein (or the Tnkl ⁇ protein) at level ⁇ that prevent, retard or reduce the severity of a disorder or its clinical manifestation ⁇ which i ⁇ related to an underexpre ⁇ ion of Tnkl protein (or Tnkl ⁇ protein) .
  • Tnkl is admini ⁇ tered in an amount and over a period of time with a frequency and duration sufficient to yield a "therapeutically effective" amount, i.e., an amount sufficient to produce Tnkl protein at levels that reduce the ⁇ everity of the disorder ⁇ or their manifestation ⁇ which are related to an underexpre ⁇ sion of the Tnkl protein (or Tnkl ⁇ protein) .
  • the pharmaceutical administrations should provide a quantity of the Tnkl (or the Tnkl ⁇ ) sufficient to effectively treat the patient.
  • Tnkl polypeptide or Tnkl ⁇
  • agonists which are polypeptides may be employed in accordance with the present invention by expression of such polypeptide ⁇ in vivo, which i ⁇ often referred to a ⁇ "gene therapy.”
  • cell ⁇ from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cell ⁇ then being provided to a patient to be treated with the polypeptide.
  • a polynucleotide DNA or RNA
  • cell ⁇ may be engineered by the u ⁇ e of a retroviral pla ⁇ mid vector containing RNA encoding a polypeptide of the present invention.
  • cells may be engineered in vivo for expres ⁇ ion of a polypeptide in vivo by, for example, procedure ⁇ known in the art.
  • a packaging cell i ⁇ tran ⁇ duced with a retroviral pla ⁇ mid vector containing RNA encoding a polypeptide of the pre ⁇ ent invention such that the packaging cell now produces infectious viral particle ⁇ containing the gene of intere ⁇ t.
  • These producer cells may be admini ⁇ tered to a patient for engineering cells in vivo and expre ⁇ ion of the polypeptide in vivo.
  • Retroviruses from which the retroviral plasmid vectors hereinabove mentioned may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruse ⁇ such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.
  • the retroviral plasmid vector is derived from Moloney Murine Leukemia Virus.
  • the vector include ⁇ one or more promoters.
  • Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques. Vol. 7, No. 9, 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and 3-actin promoter ⁇ ) .
  • CMV human cytomegalovirus
  • viral promoter ⁇ which may be employed include, but are not limited to, adenoviru ⁇ promoters, thymidine kinase (TK) promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to tho ⁇ e ⁇ killed in the art from the teachings contained herein.
  • Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter,- or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoter ⁇ , ⁇ uch a ⁇ the MMT promoter, the metallothionein promoter,- heat shock promoters; the albumin promoter,- the ApoAI promoter,- human globin promoters,- viral thymidine kina ⁇ e promoter ⁇ , ⁇ uch a ⁇ the Herpes Simplex thymidine kina ⁇ e promoter; retroviral LTRs (including the modified retroviral LTR ⁇ hereinabove described) ,- the ⁇ -actin promoter,- and human growth hormone promoters.
  • the promoter also may
  • the retroviral pla ⁇ mid vector i ⁇ employed to tran ⁇ duce packaging cell line ⁇ to form producer cell lines.
  • Example ⁇ of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, -2 , ⁇ -AM, PA12, T19-14X, VT-19-17-H2, CHE , GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy. Vol. l, pgs. 5-14 (1990), which is incorporated herein by reference in its entirety.
  • the vector may transduce the packaging cells through any means known in the art. Such mean ⁇ include, but are not limited to, electroporation, the u ⁇ e of lipo ⁇ ome ⁇ , and CaP0 4 precipitation.
  • the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.
  • the producer cell line generates infectious retroviral vector particle ⁇ which include the nucleic acid ⁇ equence( ⁇ ) encoding the polypeptide ⁇ .
  • retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vi tro or in vivo.
  • the transduced eukaryotic cells will expres ⁇ the nucleic acid sequence (s) encoding the polypeptide.
  • Eukaryotic cells which may be transduced include, but are not limited to, embryonic ⁇ tem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibrobla ⁇ ts, myoblast ⁇ , keratinocyte ⁇ , endothelial cells, and bronchial epithelial cell ⁇ .
  • Thi ⁇ invention is also related to the use of the Tnkl gene (or the Tnkl ⁇ gene) of the present invention as a diagnostic. Detection of a mutated form of the gene will allow a diagnosis of a disease or a susceptibility to a disea ⁇ e, for example cancer, which re ⁇ ult ⁇ from overexpre ⁇ sion or underexpression of Tnkl (or Tnkl ⁇ ) , or abnormal function of mutated Tnkl.
  • Nucleic acids for diagnosis may be obtained from a patient's cells, including but not limited to blood, urine, saliva, tis ⁇ ue biopsy and autopsy material.
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by u ⁇ ing PCR (Saiki et al., Nature, 324:163-166 (1986)) prior to analy ⁇ i ⁇ .
  • RNA or cDNA may also be u ⁇ ed for the ⁇ ame purpose.
  • PCR primers complementary to the nucleic acid encoding Tnkl can be used to identify and analyze mutations.
  • deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype.
  • Point mutations can be identified by hybridizing amplified DNA to radiolabeled RNA or alternatively, radiolabeled anti ⁇ en ⁇ e DNA ⁇ equence ⁇ . Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.
  • Sequence differences between the reference gene and genes having mutations may be revealed by the direct DNA sequencing method.
  • cloned DNA segments may be employed as probe ⁇ to detect ⁇ pecific DNA ⁇ egments.
  • the sensitivity of this method is greatly enhanced when combined with PCR.
  • a sequencing primer is used with double-stranded PCR product or a ⁇ ingle- ⁇ tranded template molecule generated by a modified PCR.
  • the ⁇ equence determination i ⁇ performed by conventional procedures with radiolabeled nucleotide or by automatic sequencing procedures with fluorescent-tags.
  • DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragment ⁇ in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized by high re ⁇ olution gel electrophore ⁇ i ⁇ .
  • DNA fragments of different ⁇ equence ⁇ may be di ⁇ tingui ⁇ hed on denaturing formamide gradient gel ⁇ in which the mobilitie ⁇ of different DNA fragment ⁇ are retarded in the gel at different po ⁇ ition ⁇ according to their specific melting or partial melting temperatures (see, e.g., Myers et al., Science, 230:1242 (1985) ) .
  • Sequence changes at specific locations may also be revealed by nuclease protection a ⁇ say ⁇ , ⁇ uch a ⁇ RNa ⁇ e and SI protection or the chemical cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (1985)).
  • the detection of a specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the u ⁇ e of re ⁇ triction enzyme ⁇ , (e.g., Restriction Fragment Length Polymorphisms (RFLP) ) and Southern blotting of genomic DNA.
  • methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the u ⁇ e of re ⁇ triction enzyme ⁇ , (e.g., Restriction Fragment Length Polymorphisms (RFLP) ) and Southern blotting of genomic DNA.
  • RFLP Restriction Fragment Length Polymorphisms
  • mutations can also be detected by in si tu analysis.
  • the polypeptides, their fragments or other derivatives, or analog ⁇ thereof, or cells expres ⁇ ing them can be u ⁇ ed a ⁇ an immunogen to produce antibodie ⁇ thereto.
  • the ⁇ e antibodie ⁇ can be, for example, polyclonal or monoclonal antibodie ⁇ .
  • the present invention al ⁇ o includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expres ⁇ ion library. Various procedures known in the art may be used for the production of such antibodies and fragments.
  • Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodie ⁇ can then be u ⁇ ed to isolate the polypeptide from tissue expressing that polypeptide.
  • any technique which provide ⁇ antibodie ⁇ produced by continuou ⁇ cell line culture ⁇ can be u ⁇ ed.
  • Example ⁇ include the hybridoma technique (Kohler and Mil ⁇ tein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV- hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
  • Antibodies specific to the Tnkl gene and protein may be employed as markers for cells which express the protein.
  • Human Bone Marrow or Cord Blood Cell ⁇ Normal human bone marrow wa ⁇ collected from the po ⁇ terior iliac cre ⁇ t of con ⁇ enting healthy adults under an Institutional Review Board approved protocol. Umbilical cord blood was obtained from post-delivery placenta in the Johns Hopkin ⁇ Ho ⁇ pital. CD34+/Lin- and other hematopoietic cell ⁇ ub ⁇ et ⁇ were prepared by immunomagnetic micro ⁇ phere ⁇ eparation as previou ⁇ ly de ⁇ cribed (Gore, S.D., et al. , Exp. Hetnatol., 23:413-421 (1995)) .
  • CD38- cell ⁇ For preparation of CD38- cell ⁇ , CD34+/Lin- cells, isolated as above, were incubated for an additional 30 minutes at 4°C in the presence of CD38 coated immunomagnetic microspheres, at 20 microspheres per cell. CD38- and CD38+ cell fractions were then separated on the magnetic particle concentrator (Dynal, Lake Succes ⁇ , NY) . Blood was drawn from 2 consenting, healthy adult donors, and mononuclear cell and granulocyte subpopulations were separated by density gradient centrifugation (Mono-Poly Resolving Medium, ICN, Aurora, OH) .
  • RNA Isolation Total RNA wa ⁇ prepared from purified marrow, cord blood, and peripheral blood cell fraction ⁇ , u ⁇ ing the method of Chomczyn ⁇ ki, P. & and Sacchi, N. , Anal. Biochem., 162:156-159 (1987)) .
  • total RNA wa ⁇ treated with DNase (Gibco BRL, Gaithersburg, MD) , phenol:chloroform extracted, ethanol precipitated and resu ⁇ pended in sterile DEPC-treated dH,0.
  • PolyA RNA was purified from leukemic cell lines by the Ribosep mRNA kit (Becton Dickinson, Bedford, MA) .
  • RNA from CD34+/Lin-/CD38- umbilical cord cells (approximately 1.2 x 10 J cell ⁇ ) was treated with DNase, phenol:chloroform extracted, ethanol precipitated and resuspended in DEPC-treated H 2 0.
  • DNase- treated RNA was reverse transcribed using random primers and MMLV reverse transcriptase (Gibco BRL) at 37°C.
  • sequences of the primers were a ⁇ follows.- PTK1: 5' CCCGCGGCCGCTNCAT(T/C) (A/C)GNGA(T/C) (T/OTNGCNGC 3' (SEQ ID NO:5); PTK2: 5'CCCGTCGACCC(A/G)TAN(C/G) (T/A)CCANAC(A/G)TC 3' (SEQ ID N0:6). Degenerate PCR wa ⁇ carried out at 94°C for 1 min. 37°C for 1.5 min., 72°C for 2 min for 35 cycle ⁇ with 10 min. extension at 72°C following the last cycle.
  • PCR products were separated by agaro ⁇ e gel electrophoresis, and appropriate sized products (-234 bp) were eluted using GeneClean (BIO 101, Vista, CA) . Products were digested with NotI and Sail, ligated into pBluescript KS (Stratagene, La Jolla, CA) and transformed into competent E. coli DH5 ⁇ .
  • DNA Sequencing Dideoxy sequencing was performed on plasmid DNA using Sequenase (USB, Cleveland, OH) and resolved on 8% polyacrylamide gels. To quickly eliminate repetitive clones, sequencing gels containing only T-lane ⁇ , were run. Alternatively, double- ⁇ tranded pla ⁇ mid DNA wa ⁇ ⁇ equenced on an automated fluorescent DNA ⁇ equencer in the John ⁇ Hopkin ⁇ Univer ⁇ ity School of Medicine Genetics Core facility. DNA sequences were compared to known sequence ⁇ in Genbank, using the BlastN program from National Center for Biotechnology Information (NIH, Bethe ⁇ da, MD) .
  • NIH National Center for Biotechnology Information
  • 5' and 3' RACE. were performed es ⁇ entially a ⁇ de ⁇ cribed in (Frohman, M.A. , Meth. Enzym. , 218, 340-356 (1993)). Briefly, for 5' RACE, K562 mRNA was reverse- transcribed with Tnkl specific primer B 5' ATCCGAGGC AGACGAGAAGG 3' (SEQ ID NO:7) at 37°C for 2 h with MMLV, diluted to 2 ml in Tri ⁇ -EDTA (TE) buffer, and primers were removed u ⁇ ing a Centricon-100 spin filter (Amicon, Beverly, MA) and washed once in 0.2x TE.
  • TdT terminal nucleotidyl transferase
  • fir ⁇ t round PCR product ⁇ were diluted 1:20 in TE, and 5 ⁇ l was used as templated with Tnkl specific primer D 5' ACCAGGTGTAGGGGATAG 3' (SEQ ID NO:11) and RACE primer #2 for PCR, as above except the initial 2 min. annealing step and 45 min. extension step were eliminated.
  • Products were cloned into the TA vector (Invitrogen, San Diego, CA) , and colonie ⁇ containing 5' Tnkl ⁇ equence ⁇ were identified by hybridizing with internal Tnkl specific primer A 5' CCATCAAGGTGGCTGACTTC 3' (SEQ ID NO:12).
  • K562 mRNA was reverse transcribed with oligo- (dT) using Super ⁇ cript II (Gibco BRL) at 42°C for 2 h and then diluted to 1 ml in TE.
  • Products were diluted 1:20 and 5 ⁇ l of this used as template for second round of PCR using 25 pmol of RACE primer #2 and Tnk specific prier Forward 1 5' CTGGTGTGCCCCAGAGAG 3' (SEQ ID NO:13) , as de ⁇ cribed for 5'-RACE.
  • 3'-RACE products were cloned into the TA vector and transformants containing 3 ' Tnkl sequence ⁇ were identified using an internal Tnkl specific oligonucleotide (Tnk Primer C) .
  • Reverse-Tran ⁇ criptase PCR For expres ⁇ ion ⁇ tudie ⁇ using RT-PCR, total RNA (u ⁇ ually 1-5 ⁇ g) or mRNA (1 ⁇ g) wa ⁇ rever ⁇ e-tran ⁇ cribed to cDNA using random hexamer primers and MMLV-RT at 37°C for 2 hours. An aliquot of this reaction (3 ⁇ l) was amplified with Tnkl specific primers, Tnkl 1623 5' GAGCGAATTCACAGAACTGACTCCAGACTCCTGG 3' (SEQ ID NO:14) at 94°C for 1 min, 55°C for 1 min., 72°C for 1 min., for 35 cycles.
  • Example 2 Chromosome-mapping with DNA hybrid panel. From the cDNA sequence of Tnkl, 2 different primer pairs were made that amplified over an intron on genomic DNA. From the 5'-part of the cDNA, the primer ⁇ were Tnkl 224, 5' GCACTT CGACTTTGTAAAGCCTGAG 3' (SEQ ID NO:16) and Tnkl, 5' TTTTCAG AGCTTCGGACAGTCTG 3' (SEQ ID NO:17) .
  • the primer ⁇ were Tnkl 1270, 5' CATGTTGTGTGAGGGATGCCAC 3' (SEQ ID NO:18) and Tnkl 1392, 5' TGAAGGTGCGACCATTCTGG 3' (SEQ ID NO:19) .
  • the ⁇ e primers were used to amplify DNA from the NIGMS Hybrid DNA PANEL #2 (Drwinga et al. , Genomics, 16:311-314 (1993); Duboi ⁇ and Naylor, Genomics, 16:315-319 (1993)) , which has nearly all the human chromosomes completely separated in hybrids with mou ⁇ e or hamster cell ⁇ .
  • PCR was run separately with the 2 primer-pairs at 94°C for l min, 63°C for 1 min, and 72°C for 1 min for 35 cycles. PCR products were electrophoresed on a 1.0% agarose gel, transferred to nylon filters (Nytran, Schleicher & Schuell) , and hybridized with an 32 P end-labeled internal oligonucleotide.
  • a genomic Tnkl gene isolated from a human Pl genomic DNA library (Human Fore ⁇ kin Fibrobla ⁇ t Pl LIBRARY #1, Du Pont Merck Pharmaceutical Co., St. Loui ⁇ , MO) by PCR using Tnkl specific primers.
  • a method utilized is generating a fusion protein to glutathione-S-transfera ⁇ e(GST) (Smith, D.B. and John ⁇ on, K.S., Gene, 67:31-40 (1988); ⁇ ee also Au ⁇ ubel, F.M. , et al., Current Protocol ⁇ in Molecular Biology, Vol. 2, Chapter 16.7.1-16.7.8 (1993)) .
  • the cloning vectors contain the neces ⁇ ary control elements required for efficient tran ⁇ cription and tran ⁇ lation, including an inducible promoter and multiple termination codon ⁇ .
  • Production of GST-fusion proteins is under the control of the E. coli lacZ promoter, and thus is inducible with IPTG.
  • GST fusion proteins can be purified from bacterial lysates by affinity chromatography using glutathione-agarose beads.
  • Two peptides were ⁇ ynthesized from amino acids u ⁇ ing a peptide ⁇ ynthe ⁇ izer in ⁇ trument ( ⁇ tandard procedure ⁇ ) .
  • Two different anti ⁇ era were rai ⁇ ed by immunizing 2 rabbit ⁇ with 2 different Tnkl ⁇ ynthetic peptide ⁇ (LPATCPVHRGTPARGDQHPG for the fir ⁇ t rabbit and IDGDRKKANLWDAP for the ⁇ econd rabbit) , each conjugated to Keyhole Limpet Hemocyanin (KLH) .
  • Both antibodie ⁇ recognize the appropriate peptide and the fu ⁇ ion protein.
  • ADDRESSEE CARELLA, BYRNE, BAIN, GILFILLAN,
  • CTGCCTCGGA CGTGTGGATG TTTGGGGTGA CGCTGTGGGA GATGTTCTCC GGGGGCGAGG 1080
  • CTGGAGGCCT CTTGTCCGAT CCTGAGTTGC AGAGGAAGAT TATGGAAATG GAGCTGAGTG 1920
  • Val Tyr Ly ⁇ lie Leu Gly Gly Phe Ala Pro Glu Hi ⁇ Ly ⁇ Glu Pro
  • Arg lie Leu Glu His Tyr Gin Trp Asp Leu Ser Ala Ala Ser Arg
  • MOLECULE TYPE CDNA
  • Xi SEQUENCE DESCRIPTION: SEQ ID NO:3:
  • CTGCCTCGGA CGTGTGGATG TTTGGGGTGA CGCTGTGGGA GATGTTCTCC GGGGGCGAGG 1080
  • MOLECULE TYPE Oligonucleotide
  • MOLECULE TYPE Oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:8: AGACGAGAAG GCTCCGTG 18
  • MOLECULE TYPE Oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:9: GATGGATCCT GCAGAAGCT 19
  • MOLECULE TYPE Oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 10: GATGGATCCT GCAGAAGC 18
  • MOLECULE TYPE Oligonucleotide
  • Xi SEQUENCE DESCRIPTION: SEQ ID NO:12: CCATCAAGGT GGCTGACTTC 20
  • MOLECULE TYPE Oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:13: CTGGTGTGCC CCAGAGAG 18
  • MOLECULE TYPE Oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 14: GAGCGAATTC ACAGAACTGA CTCCAGACTC CTGG 34
  • MOLECULE TYPE Oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:16: GCACTTCGAC TTTGTAAAGC CTGAG 25 (2) INFORMATION FOR SEQ ID NO:17:
  • MOLECULE TYPE Oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:17: TTTTCAGAGC TTCGGACAGT CTG 23
  • MOLECULE TYPE Oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:18: CATGTTGTGT GAGGGATGCC AC 22
  • MOLECULE TYPE Oligonucleotide
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:19: TGAAGGTGCG ACCATTCTGG 20

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Abstract

A human intracellular tyrosine kinase polypeptide and DNA (RNA) encoding such polypeptide and a procedure for producing such polypeptide by recombinant techniques is disclosed. Also disclosed are methods for utilizing such polypeptide to suppress the transforming activity of certain genes by acting in a regulatory role, to regulate changes in the proliferation and differentiation status of cells, namely, hematopoietic stem cells for bone marrow transplantation and support for cancer therapy. Diagnostic assays for identifying mutations in nucleic acid sequences encoding a polypeptide of the present invention for detecting diseases, for example, cancer, are also disclosed.

Description

TYROSINE KINASE GENE AND GENE PRODUCT
This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention has been identified as a tyrosine kinase gene, sometimes hereafter referred to as "Tnkl" and splice variant gene "Tnklα.". The invention also relates to inhibiting the action of such polypeptides.
In the complex developmental process of hematopoiesis, mature blood cells senesce and are continuously being replaced from a small pool of undifferentiated pluripotent lymphohematopoietic stem cells (HSC) (Ogawa, M., Blood, 81:2844-2853 (1993)). Immunologic purification of cells expressing the CD34 membrane phosphoglycoprotein from bone marrow, adult blood, umbilical cord blood, or fetal hematopoietic tissue yields an early, yet heterogeneous population. This CD34+ cell population contains a small frequency of HSC among the more abundant and more differentiated committed progenitor cells of all the blood cell lineages (Civin, C.I., et al., J. Immun., 133:157-165 (1984); Civin, C.I., et al., Exp. Hematol., 15:10-17 (1987); Loken, M. R., et al., Blood, 70:1316-1324 (1987)) . The CD38 antigen is expressed on most CD34+ cells, but the 1-10% of CD34+ cells lacking CD38 antigen expression (CD34+/CD38 cells) , also lack expression antigens characterizing mature blood cell ("lineage antigens") (Terstappen, et al., Blood, 77:1218-1226 (1991)). CD34+/Lin/CD38- cells can be replated in vi tro, and give rise to myeloid and lymphoid colonies in long-term culture assays (Huang, S. and Terstappen, W. M. M. , Nature, 360:867-870 (1992); Huang, S. and Terstappen, W. M. M., Blood 83:1515-1526 (1994)). Thus, this CD34+/Lin7CD38- cell population is an enriched population of early progenitors and likely HSC. CD34+ cells obtained from umbilical cord blood contain a larger fraction of CD34+/Lin" /CD38" cells as compared to adult bone marrow (Cardoso A. A. , et al., PNAS, USA, 90:8707-8711 (1993)). In addition, cord blood contains higher numbers of cells with high proliferative potency in in vivo hematopoietic assays (Broxmeyer, H. E., et al. , PNAS, USA, 89:4109-4113 (1992)); Lu, L., et al., Blood, 81:41-48 (1993); Cardoso, A. A., et al., PNAS, USA, 90:8707-8711 (1993)). This suggests the possibility that cord blood contains a higher frequency of HSC per CD34+ cell, as compared to adult bone marrow.
Tyrosine kinases are a large, and rapidly growing family of proteinε important in regulating cell growth and differentiation (Fantl, . J., et al., Annu. Rev. Biochem., 62: 453-481 (1993)). Receptor tyrosine kinases contain an extracellular ligand-binding domain, a transmembrane domain, and a highly conserved catalytic kinase domain. Binding of ligand to receptor initiates a cascade of signals, culminating in a biological response. Early events include receptor dimerization, autophosphorylation on tyrosine residue(ε), and subsequent activation of catalytic activity (Ullrich, A. and Schlessinger, J. , Cell, 61:203-212 (1990)). Many non-receptor tyrosine kinases, including members of the src and Jak families are involved further downstream, in intracellular signalling pathways. These non-receptor tyrosine kinases also contain the conserved catalytic kinase domain, and often contain domains that mediate protein: rotein interactions, such as SH2 and SH3 domains (Bolen, (1993); Musacchio, A., et al., FEBS Letters, 307:55- 61 (1992); Schlessinger, J. , Curr. Opin. Genet. Devel., 4:25- 30 (1994)).
A variety of receptor and non-receptor tyrosine kinases are important in the survival, proliferation and differentiation of hematopoietic cells. For example, the c- fms and c-Jit receptor tyrosine kinases have been shown to be required for the survival, proliferation and differentiation of monocytic cells and early hematopoietic progenitor cells, respectively (Sherr, C. J., Blood, 75:1-12 (1990); Chabot, B., et al., Nature, 335:88-89 (1988); Geissler, E. N. , et al., Cell, 55: 185-192 (1988)). The non-receptor tyrosine kinase pp56lck, a src family member, is specifically expressed in T lymphocytes and is critical for their maturation (Amrein, K. E., et al., Proc. Nat. Aca. Sci. USA, 89:3343- 3346 (1992)), activation (Glaichenhaus, N. , et al., Cell, 64:511-520 (1991)), and IL-2 secretion (Karnitz, L., et al. , Mol. Cell. Biol., 12:4521-4530 (1992)). In addition, pp56T'rt mediates signals derived by CD4 and CD8 receptors (Veilette, A., et al., Nature, 338:257-259 (1989)) .
Recently, several groups (Wilks, A.F., PNAS, USA, 86:1603-1607 (1989); Matthews, W. , et al. , Cell, 65:1143-1152 (1991); Small, D. et al., PNAS, USA, 91:459-463 (1994)) have used degenerate oligonucleotide primers to conserved motifs in tyrosine kinases to identify and clone novel tyrosine kinases expressed in hematopoietic stem/progenitor cells. However, in a recent search for novel tyrosine kinases in CD34+ bone marrow cells, no new tyrosine kinase genes from over 250 recombinants were found. It was hypothesized that in a more highly enriched population of HSC, i.e., CD34+/Lin" /CD38' cells from cord blood, there would be found expression of previously unidentified tyrosine kinases that might be involved in HSC εurvival, proliferation and/or differentiation.
The gene (SEQ ID N0:1, as shown in Figure 1) and gene product (SEQ ID N0:2, as shown in Figure 1) (and the splice variant gene (SEQ ID NO:3) and its gene product (SEQ ID NO:4) of the present invention have been putatively identified as the Tnkl gene and gene product (and the Tnklα and gene product) as a result of homology to known tyrosine kinases.
In accordance with one aspect of the present invention, there is provided novel mature polypeptides, as well as biologically active and diagnostically or therapeutically useful fragments, analogs and derivatives thereof. The polypeptides of the present invention are of human origin.
In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding a polypeptide of the present invention including mRNAs, DNAs, cDNAs, genomic DNAs aε well as analogs and biologically active and diagnostically or therapeutically useful fragments thereof.
In accordance with yet a further aspect of the present invention, there is provided a procesε for producing such polypeptides by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing a nucleic acid sequence encoding a polypeptide of the present invention, under conditions promoting expression of said protein and subεequent recovery of said protein.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotide encoding such polypeptides for therapeutic purposes, for example, to suppress the transforming activity of certain genes by acting in a regulatory role, to regulate changes in the survival, proliferation and differentiation status of cells. Preferred cells are hematopoietic stem and progenitor cells for bone marrow transplantation and support for cancer therapy. In accordance with yet a further aspect of the present invention, there are provided antibodies against such gene and gene product (or to the splice variant gene) which may be employed, for example in labeled form, as markers.
In accordance with another aspect of the present invention, there are provided Tnkl and Tnklα agonists which elicit biological responses similar to native Tnkl and Tnklα. In accordance with yet a further aspect of the present invention, there is also provided nucleic acid probeε comprising nucleic acid molecules of sufficient length to specifically hybridize to a nucleic acid sequence of the present invention.
In accordance with still another aspect of the present invention, there are provided diagnostic assays for detecting diseases or susceptibility to diseaseε related to mutationε in the nucleic acid sequences encoding a polypeptide of the present invention.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotides encoding such polypeptides, for in vi tro purposes related to scientific research, for example, to identify a putative receptor of the Tnkl or Tnklα molecule for the identification of its ligand, synthesis of DNA and manufacture of DNA vectors.
These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.
BRIEF DESCRIPTION OF DRAWINGS The following drawings are illustrative of embodiments of the invention and are not meant to limit the scope of the invention as encompassed by the claims.
Figure 1. Polynucleotide (SEQ ID N0:1) and predicted amino acid sequence (SEQ ID NO:2) of Tnkl cDNA. The conserved ATP binding motif present in all tyrosine kinases (GxGxxG) is underlined. The conserved kinase region initially amplified from CD34+/Lin-/CD38- umbilical cord blood cDNA is shown in bold face type. The double-underline indicates the putative SH3 domain. Asterisks (*) denote potential SH3 binding motifs of the consensuε (P-x-x-P) . The triangle (Δ) indicates a potential phosphotyrosine binding site for a recently described domain which recognizes the motif (N-x-x-Y) (Kavanaugh, W. M., et al., Science, 268:1177- 1180 (1995)). The numbers on the left denote the DNA sequence; the numbers on the right refer to the amino acid sequence. The Tnklα splice variant cDNA (SEQ ID NO:3) and predicted amino acid sequence (SEQ ID NO:4) are the same as for Tnkl which are shown in Figure 1, except that nucleotideε 1359-1374 of SEQ ID NO:l are abεent, and correεponding amino acids 412-416 of SEQ ID NO:2 are absent.
Figure 2. Alignment of the predicted Tnkl amino acid sequence with Ack. Homology begins at the NH2-terminus of both Tnkl (AA 27) and Ack (AA 83) and continues uninterrupted for 413 amino acids. This region includes the kinase domain (AA 123-372) and the putative SH3 domain (AA 396-441) . Homologous regions in the proline-rich domain (AA 530-556) and near the COOH-terminus (AA 638-664) are also shown. (+) indicate similar amino acids. The dashes in the sequence represent gaps to maximize alignment.
Figure 3. Diagram of the structure of the Tnkl protein. The numbers refer to the amino acid sequence. PTK350 refers to the original 210 bp PCR product amplified from CD34+/Lin- /CD38- umbilical cord blood cDNA. 1273 and 1623 refer to the region amplified in RT-PCR experiments. The asteriεkε (*) denote the position of potential binding sites for SH3 domainε; the triangle (Δ) refers to the poεition of a potential binding site for a newly described phoεphotyrosine binding domain. The εolid line underneath denotes regions of homology to Ack. p21 BD indicates a putative p21 binding domain. In accordance with an aspect of the present invention, there is provided an isolated nucleic acid (polynucleotide) sequence which encodes for the mature polypeptide having the deduced amino acid sequence of Figure l or for the mature polypeptide encoded by the cDNA of the clone deposited as ATCC Deposit No. 69924 on October 5, 1995 with the ATCC 12301 Parklawn Drive, Rockville, MD 20852.
The gene of the present invention was isolated using degenerate PCR to clone tyrosine kinase genes from an enriched population of human umbilical cord blood hematopoietic stem/progenitor cellε. The εequence of the complete Tnkl coding region predicts a 72 kD protein. Compariεon of Tnkl to εequenceε in protein databaεeε revealε that it iε moεt homologous to Ack, an intracellular tyrosine kinase. Like Ack, Tnkl consists of an N-terminal kinase domain, a putative SH3 domain immediately C-terminal to the kinase domain, and a proline-rich C-terminal region. Homology between Tnkl and Ack diverges immediately following the SH3 domain. In Ack, this region is required for binding to the GTP-bound form of p21cdc42Hs (Manser et al., Nature. 363:364-367 (1993)). Analysis of Tnkl mRNA expression demonstrateε that Tnkl iε expressed in all cord blood, bone marrow and adult blood subpopulationε, as well as in most of the leukemia cell lines examined (16 of 20) . Hybridization to fetal multi-tissue Northern blots detected several different Tnkl transcripts in all 4 fetal tissues examined. In contrast, a single 3.0 Kb Tnk transcript was detected in only 5 of 16 adult tissues examined (colon, prostate, and ovary, εmall intestine, and testis) .
Fluorescence in si tu hybridization (FISH) analysis of metaphase chromosomes localized the Tnkl gene to the short arm of chromosome 17 (17pl3.1), near the p53 locus. Thus, Tnkl is a tyrosine kinase that may be involved in signaling pathways utilized broadly during fetal development, more selectively in adult tissues, and in cells of the lymphohematopoietic system.
The ATG site of Tnkl iε just N-terminal to, and in frame with, a region of high homology Tnkl shareε with another tyrosine kinase gene, Ack (Manser, E. et al., Nature, 363, 364-367 (1993)) . In the Ack protein, this homologous region is alεo near the N-terminuε. The Tnkl open reading frame terminateε at nt 2115, encoding a 666 amino acid protein with a molecular weight of about 72kD.
The Tnkl kinase domain contains all 12 conserved structural domains present in the tyrosine kinase family, including the ATP binding site (GxGxxG, aa 123-128) and the highly conserved tyrosine kinase domains (VHRDLAA and SDVWSFG) (Hanks, S.K. et al. , Science, 241, 42-52 (1988)). Tnkl has a methionine residue in this latter conserved motif, instead of the second conserved serine residue. The tyrosine kinases Ack and Fak (Manser, E. et al., Nature, 363, 364-367 (1993); Whitney, G.S., et al. , DNA Cell Biol., 12:823-830 (1992)) also have a methionine residue at this position. Similar to Ack but unlike most tyrosine kinases, the kinase domain of Tnkl is near the N-terminuε (AA 123-372) . Just N- terminal to the kinase domain is a short stretch of amino acids, N-x-x-Y (AA 74-77) . This region may serve as a possible binding site for a recently described phosphotyroεine binding domain (Kavanaugh, W.M. et al., Science, 268, 1177-1180 (1995)). C-terminal to the kinase domain of Tnkl are several proline-rich areas, including P-x- x-P motifs (around AA'ε 345 and 535), which may serve as binding siteε for SH3 domainε of other εignal tranεduction proteins (Cohen, G.B. et al., Cell, 80, 237-248 (1995); Alexandropouloε, K. et al., Proc. Nat. Acad. Sci. USA, 92, 3110-3114 (1995)). In addition, there is a region (AA 396- 431) which is highly homologous to the SH3 domain of Ack (Manser, E. et al., Nature, 363, 364-367 (1993)). Tnkl is 60% homologous to Ack (45% identical, 60% conserved) , over a 413 amino acid span (AA 28-441 of Tnkl) including the entire kinase domain (AA123-372, 43% identical, 57% conserved) and the putative SH3 domain (AA 396-441, 44% identical, 56% conserved) (Figure 2) . In addition, there is homology between Tnkl and Ack at the NH2-terminus of both proteinε. The structure of the predicted Tnkl polypeptide iε similar to Ack in that it contains an N-terminal kinase domain, followed by an SH3 domain and a proline-rich C- terminal region (Figure 3) . Homology between Tnkl and Ack diverges immediately following the SH3 domain. In Ack, this region is required for binding to the GTP-bound form of p21cdc42Hs (Manεer, E. et al., Nature, 363, 364-367 (1993)). Following the putative p21 binding domain, there are 2 other εmall regionε of homology between Tnkl and Ack (Figure 3) , 1 in the proline-rich region (AA 530-556, 46%, 57% conserved) and the other close to the C-terminus (AA 638-664, 30% identical, 65% conserved) .
Tnkl mRNA is present in total mononuclear cells, CD34+/Lin- cells and CD34- cells from umbilical cord blood and bone marrow. In addition, adult blood mononuclear cell and granulocytes both express Tnkl mRNA.
Since preliminary analysis of Tnkl expression in human leukemia cells lines indicate that Tnkl is a rare transcript, RT-PCR was used to investigate Tnkl expression in these cells lines. A cDNA was syntheεized from Poly(A) RNA (approximately -1 μg) from 20 different human leukemia cell lineε, amplified with Tnkl specific primers, electrophoresed and then probed with an internal Tnkl specific oligonucleotide. Of the 20 lines examined, 16 expresεed detectable Tnkl mRNA. Thuε, Tnkl may be expreεsed ubiquitously during hematopoiesis.
Commercially obtained filters (Clontech) containing RNA from fetal and adult tissues were probed with a random-primed Tnkl specific probe. In 4 fetal tissues examined, several different Tnkl transcripts were easily detected. All 4 fetal tissue sampleε expreεεed a εmall 1.0 Kb tranεcript . in addition, fetal lung and kidney expressed a major 3.0 Kb transcript and a weakly hybridizing 9.5 Kb tranεcript. A 7.5 Kb Tnkl transcript was found only in fetal liver. The expression of different sized Tnkl transcriptε in the fetal tissue sampleε εuggests the poεεibility of developmental and tissue-specific alternative splicing. In contrast, only 5 of the 16 adult tisεues expresεed detectable Tnkl. A single 3.0 Kb transcript is found only in adult prostate, ovary, small intestine, testis and colon. Tnkl transcriptε are not found in adult lung, liver, kidney or brain. Thuε, Tnkl mRNA expreεεion appears to be higher in fetal tissues than in adult tissues.
The Tnkl gene was localized on human chromosome 17 via fluorescence in si tu hybridization (FISH) analysis on metaphase chromosomes from human lymphocytes. All 30 metaphase cells analyzed had at least 1 paired signal (involving both chromatids of a εingle chromosome) . Of 46 paired signals, all were located on the p-arm of an E-group chromosome. The hybridized slide waε G-banded by FPG (Fluorescence plus Giemsa, Bhatt, B. et al., Nucl. Acids Res., 16, 3951-3961 (1988)) , photographed, and aligned with the color slideε to determine εub-band location. After banding, all analyzable metaphaεeε had signals on chromosome 17p. The predominant signalε (8 of 12) hybridize to band 17pl3.1.
The polynucleotide of the present invention may be in the form of RNA or in the form of DNA, which DNA includeε cDNA, genomic DNA, and εynthetic DNA. The DNA may be double- stranded or single-stranded, and if single εtranded may be the coding εtrand or non-coding (anti-sense) strand. The coding εequence which encodeε the mature polypeptide may be identical to the coding sequence shown in Figure 1 or that of the deposited clone or may be a different coding sequence which coding sequence, as a result of the redundancy or degeneracy of the genetic code, encodes the same mature polypeptide as the DNA of Figure 1 or the deposited cDNA.
The polynucleotide which encodes for the mature polypeptide of Figure l and/or for the mature polypeptide encoded by the deposited cDNA may include, but is not limited to: only the coding sequence for the mature polypeptide; the coding sequence for the mature polypeptide and additional coding sequence,- the coding sequence for the mature polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5' and/or 3' of the coding sequence for the mature polypeptide.
Thus, the term "polynucleotide encoding a polypeptide" encompasεeε a polynucleotide which includeε only coding sequence for the polypeptide aε well as a polynucleotide which includes additional coding and/or non-coding sequence.
The present invention further relates to variantε of the hereinabove described polynucleotides, such as εplice variant
Tnklc-, which encode for fragments, analogε and derivativeε of the polypeptide having the deduced amino acid εequence of
Figure l and/or the polypeptide encoded by the cDNA of the deposited clone. The variant of the polynucleotide may be a naturally occurring allelic variant of the polynucleotide or a non-naturally occurring variant of the polynucleotide.
Thus, the present invention includes polynucleotides encoding the same mature polypeptide as shown in Figure 1 and/or the εame mature polypeptide encoded by the cDNA of the deposited clone as well as variants of such polynucleotides which variants encode for a fragment, derivative or analog of the polypeptide of Figure 1 and/or the polypeptide encoded by the cDNA of the deposited clone. Such nucleotide variantε include deletion variants, substitution variants and addition or insertion variants, such as the polypeptide of SEQ ID NO:4. Aε hereinabove indicated, the polynucleotide may have a coding sequence which is a naturally occurring allelic variant of the coding sequence shown in Figure 1 and/or of the coding sequence of the deposited clone. As known in the art, an allelic variant is an alternate form of a polynucleotide sequence which may have a subεtitution, deletion or addition of one or more nucleotideε, which does not subεtantially alter the function of the encoded polypeptide. For example, the Tnklα polypeptide haε been shown by testing to have the εame or εubεtantially the εame function as the Tnkl polypeptide.
The term "gene" means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening sequences (introns) between individual coding segments (exons) .
Fragments of the full length gene of the present invention may be used as a hybridization probe for a cDNA library to isolate the full length cDNA and to isolate other cDNAs which have a high εequence εimilarity to the gene or εimilar biological activity. Probes of this type preferably have at least 15 baεeε and may contain, for example, at least 30 baseε or 50 or more bases. The probe may also be uεed to identify a cDNA clone corresponding to a full length transcript and a genomic clone or clones that contain the complete gene including regulatory and promotor regions, exons, and introns. An example of a screen comprises isolating the coding region of the gene by using the known DNA sequence to synthesize an oligonucleotide probe. Labeled oligonucleotideε having a εequence complementary to that of the gene of the present invention are used to εcreen a library of human cDNA, genomic DNA or mRNA to determine which members of the library the probe hybridizes to.
The present invention further relateε to polynucleotideε which hybridize to the hereinabove-deεcribed sequences if there is at least 70%, preferably at least 90%, and more preferably at least 95% identity between the sequenceε. The present invention particularly relates to polynucleotideε which hybridize under εtringent conditionε to the hereinabove-described polynucleotides. Aε herein uεed, the term "stringent conditions" means hybridization will occur only if there is at least 95% and preferably at least 97% identity between the sequences. The polynucleotides which hybridize to the hereinabove deεcribed polynucleotideε in a preferred embodiment encode polypeptideε which either retain substantially the same biological function or activity as the mature polypeptide encoded by the cDNAs of Figure l and/or the depoεited cDNA(s) .
Alternatively, the polynucleotide may have at least 20 bases, preferably at least 30 bases, and more preferably at least 50 baεes which hybridize to a polynucleotide of the preεent invention and which has an identity thereto, as hereinabove described, and which may or may not retain activity. For example, such polynucleotides may be employed as probes for the polynucleotide of, for example, for recovery of the polynucleotide or as a diagnostic probe or as a PCR primer.
Thus, the present invention is directed to polynucleotides having at least a 70% identity, preferably at least 90% and more preferably at leaεt a 95% identity to a polynucleotide which encodeε the polypeptide of Figure l aε well aε fragmentε thereof, which fragmentε have at leaεt 15 baεes and preferably at least 30 baseε or at leaεt 50 baseε and to polypeptides encoded by such polynucleotides .
The deposit (s) referred to herein will be maintained under the terms of the Budapest Treaty on the International Recognition of the Deposit of Micro-organisms for purposes of Patent Procedure. The deposit is an E. coli bacterial strain DH5α harboring a plasmid (pTnkl) that contains the full- length 2,790 bp Tnkl cDNA. The Tnkl cDNA has been cloned into the EcoRI εite of pBlueεcript KS(-) (Stratagene, La Jolla, Ca) .
These depositε are provided merely aε 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 depoεited materialε, aε well aε the amino acid εequence of the polypeptideε encoded thereby, are incorporated herein by reference and are controlling in the event of any conflict with any deεcription of sequences herein. A license may be required to make, use or sell the deposited materialε, and no such license is hereby granted.
The present invention further relates to a polypeptide which has the deduced amino acid sequence of Figure 1 and/or which has the amino acid sequence encoded by the deposited cDNA, as well as fragments, analogs and derivatives of εuch polypeptide.
The termε "fragment," "derivative" and "analog" when referring to the polypeptide of Figure 1 and/or that encoded by the deposited cDNA, meanε a polypeptide which retainε eεεentially the same biological function or activity as εuch polypeptide. Thuε, an analog includeε a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide.
The polypeptide of the preεent invention may be a recombinant polypeptide, a natural polypeptide or a εynthetic polypeptide, preferably a recombinant polypeptide.
The fragment, derivative or analog of the polypeptide of Figure 1 and/or that encoded by the depoεited cDNA may be (i) one in which one or more of the amino acid reεidueε 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 reεidueε includeε a εubstituent group, or (iii) one in which the mature polypeptide is fused with another compound, εuch as a compound to increaεe the half-life of the polypeptide (for example, polyethylene glycol) , or (iv) one in which the additional amino acidε are fuεed to the mature polypeptide for purification of the mature polypeptide. Such fragments, derivatives and analogs are deemed to be within the scope of those skilled in the art from the teachings herein.
The polypeptideε and polynucleotideε of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity.
The term "isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring) . For example, 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 polynucleotideε 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.
The polypeptides of the present invention include the polypeptide of Figure 1 (in particular the mature polypeptide) as well as polypeptides which have at leaεt 70% similarity (preferably at least 70% identity) to the polypeptide of Figure 1 and more preferably at least 90% similarity (more preferably at least 90% identity) to the polypeptide of Figure 1 and still more preferably at least 95% similarity (still more preferably at least 95% identity) to the polypeptide of Figure 1 and also include portions of εuch polypeptideε with such portion of the polypeptide generally containing at least 30 amino acids and more preferably at least 50 amino acids. As known in the art "similarity" between two polypeptides is determined by comparing the amino acid sequence and its conserved amino acid subεtituteε 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. Fragmentε or portions of the polynucleotides of the present invention may be used to synthesize full-length polynucleotides of the present invention.
The preεent invention alεo 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 techniqueε.
Hoεt cells are genetically engineered (transduced or tranεformed or tranεfected) 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 hoεt cellε can be cultured in conventional nutrient media modified aε appropriate for activating promoters, selecting transformantε or amplifying the genes of the present invention. The culture conditions, εuch aε temperature, pH and the like, are thoεe previously used with the host cell εelected for expreεεion, and will be apparent to the ordinarily skilled artisan.
The polynucleotideε of the present invention may be employed for producing polypeptideε by recombinant techniqueε. Thuε, for example, the polynucleotide may be included in any one of a variety of expression vectors for expressing a polypeptide. Such vectors include chromosomal, nonchromosomal and synthetic DNA sequenceε, e.g. , derivatives of SV40; bacterial plasmids,- phage DNA,- baculovirus; yeast plasmids; vectors derived from combinationε of plaεmids and phage DNA, viral DNA such as vaccinia, adenovirus, fowl pox virus, and pseudorabies. However, 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 εequence in the expression vector is operatively linked to an appropriate expresεion control sequence(s) (promoter) to direct mRNA syntheεiε. Aε repreεentative exampleε of εuch promoterε, there may be mentioned: LTR or SV40 promoter, the E. coli . lac or Trp, the phage lambda PL promoter and other promoterε 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.
In addition, 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 resiεtance in E. coli .
The vector containing the appropriate DNA εequence aε 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.
As representative examples of appropriate hosts, there may be mentioned: bacterial cells, such as E. coli , Streptomyces, Salmonella typhimurium; fungal cellε, such as yeast; insect cells such as Drosophila S2 and Spodoptera Sf9 ; animal cells such as CHO, COS or Bowes melanoma,- adenoviruεeε,- plant cellε, etc. The εelection of an appropriate hoεt iε deemed to be within the scope of those skilled in the art from the teachings herein.
More particularly, the present invention alεo includeε recombinant conεtructs compriεing one or more of the sequences as broadly described above. The constructε comprise a vector, such as a plasmid or viral vector, into which a sequence of the invention has been inεerted, in a forward or reverεe orientation. In a preferred aεpect of thiε embodiment, the construct further comprises regulatory sequences, including, for example, a promoter, operably linked to the sequence. Large numbers of suitable vectors and promoters are known to those of skill in the art, and are commercially available. The following vectors are provided by way of example; Bacterial: pQE70, pQE60, pQE-9 (Qiagen), pBS, pDIO, phagescript, pεiX174, pBlueεcript SK, pBSKS, pNH8A, pNH16a, pNH18A, pNH46A (Stratagene); pTRC99a, pKK223- 3, pKK233-3, pDR540, pRIT5 (Pharmacia); Eukaryotic: pWLNEO, PSV2CAT, pOG44, pXTl, pSG (Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia) . However, 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 markerε. Two appropriate vectorε are pKK232-8 and pCM7. Particular named bacterial promoterε include lad, lacZ, T3, T7, gpt, lambda PR, PL and Trp. Eukaryotic promoters include CMV immediate early, HSV thymidine kinase, early and late SV40, LTRs from retrovirus, and mouse metallothionein-I. Selection of the appropriate vector and promoter is well within the level of ordinary skill in the art. In a further embodiment, the preεent invention relateε to hoεt cellε 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 hoεt cell can be a prokaryotic cell, such as a bacterial cell. Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE- Dextran mediated transfection, or electroporation (Davis, L., Dibner, M., Battey, I., Basic Methods in Molecular Biology, (1986)) .
The constructε in host cells can be uεed in a conventional manner to produce the gene product encoded by the recombinant εequence. Alternatively, the polypeptides of the invention can be synthetically produced by conventional peptide syntheεizerε.
Mature proteinε can be expreεεed in mammalian cellε, yeaεt, bacteria, or other cells under the control of appropriate promoters. Cell-free translation systems can also be employed to produce such proteinε 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) , the disclosure of which is hereby incorporated by reference.
Transcription of the DNA encoding the polypeptides of the present invention by higher eukaryoteε is increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp that act on a promoter to increaεe itε transcription. Examples include the SV40 enhancer on the late side of the replication origin bp 100 to 270, a cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. Generally, recombinant expreεεion vectorε will include originε of replication and selectable markers permitting tranεformation of the host cell, e.g., the ampicillin resiεtance gene of E. coli and S. cerevisiae TRPl gene, and a promoter derived from a highly-expreεεed gene to direct transcription of a downstream structural sequence. Such promoters can be derived from operonε encoding glycolytic enzymeε εuch as 3-phosphoglycerate kinase (PGK) , α-factor, acid phosphataεe, or heat εhock proteins, among others. The heterologous structural sequence is asεembled in appropriate phaεe with translation initiation and termination εequences. Optionally, the heterologous sequence can encode a fusion protein including an N-terminal identification peptide imparting desired characteristicε, e.g., εtabilization or simplified purification of expressed recombinant product.
Useful expresεion vectors for bacterial use are constructed by inserting a structural DNA sequence encoding a desired protein together with suitable translation initiation and termination signalε in operable reading phaεe 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 hoεtε for tranεformation include E. coli , Bacillus subtilis, Salmonella typhimurium and variouε species within the genera Pseudomonas, Streptomyces, and Staphylococcus, although others may also be employed as a matter of choice.
As a representative but nonlimiting example, useful expression vectors for bacterial use can comprise a selectable marker and bacterial origin of replication derived from commercially available plasmids comprising genetic elementε of the well known cloning vector pBR322 (ATCC 37017) . Such commercial vectorε include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and GEM1 (Promega Biotec, Madison, WI, USA) . These pBR322 "backbone" εections are combined with an appropriate promoter and the structural εequence to be expreεεed.
Following tranεformation of a suitable hoεt εtrain and growth of the hoεt εtrain to an appropriate cell density, the selected promoter is induced by appropriate meanε (e.g., temperature εhift or chemical induction) and cells are cultured for an additional period.
Cellε are typically harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract retained for further purification.
Microbial cellε employed in expreεεion of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents, such methods are well known to those εkilled in the art.
Various mammalian cell culture εystems can also be employed to expresε recombinant protein. Exampleε of mammalian expreεεion εyεtemε include the COS-7 lines of monkey kidney fibroblaεtε, deεcribed by Gluzman, Cell, 23:175 (1981) , and other cell lines capable of expresεing a compatible vector, for example, the C127, 3T3, CHO, HeLa and BHK cell lineε. Mammalian expression vectors will comprise an origin of replication, a suitable promoter and enhancer, and alεo any neceεεary riboεome binding εiteε, polyadenylation εite, εplice donor and acceptor sites, transcriptional termination sequenceε, and 5' flanking nontranεcribed εequences. DNA sequences derived from the SV40 splice, and polyadenylation sites may be used to provide the required nontranscribed genetic elements.
The polypeptide 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 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 alεo include an initial methionine amino acid reεidue.
Many intracellular tyroεine kinaεes have been shown to play a role in εignalling pathwayε which govern survival, proliferation and/or differentiation of certain cell types. Tnkl may be εignalled, directly or via intermediate proteinε, by the intracellular portion of a cell membrane receptor (after ligand bound to the receptor) directly or indirectly,- the receptor or intermediate protein may bind Tnkl and perhapε enzymatically activate Tnkl, for example, by phosphorylation of Tnkl. Activated Tnkl may then bind to some other protein in the cascade until eventually a transcription factor is involved and caused to effect expresεion of εome gene. Expreεεion of these genes may effect the cells' survival, proliferation and/or differentiation.
Based on itε homology to Ack, Tnkl and Tnklα are each thought to function εimilarly to Ack, by binding to a ras- like protein in a pathway controlling cell proliferation. Such pathwayε are important in the biology of both cancer cells and lymphohematopoietic stem and progenitor cells, among other cell types. Accordingly either of the Tnkl or Tnklα geneε may be employed to control the growth of cancer cells by cauεing cancer cells to ceaεe proliferating and die, and εtimulating lymphohematopoietic εtem and progenitor εurvival and proliferation.
Tnkl or Tnklα may also be employed to transiently cause stem and progenitor cells to pause in self-cycling, for example, while the patient with cancer receives chemotherapy or radiotherapy which may then more selectively kill cancer cells but not the normal stem and progenitor cells.
Tnkl or Tnklα may also be employed to affect differentiation, εtimulation or inhibition of certain cells.
Tnkl or Tnklα may be employed to regulate the GTPase activity of a p21 ras-like protein by binding thereto, and therefore, suppress its transforming activity.
The gene and gene products of the present invention may also be employed to regulate signaling pathways during fetal development.
Tnkl or Tnklα may also be employed to stimulate proliferation and differentiation of hematopoietic stem and/or progenitor cells for bone marrow transplantation or peripheral blood stem/progenitor cell tranεplantation.
The polynucleotides and polypeptideε of the preεent invention may also be employed as research reagentε and materials for diεcovery of treatmentε and diagnoεticε to human disease.
Thiε invention provides a method for identification of the cell membrane receptor which functions to regulate Tnkl or Tnklα activity, and a ligand which interactε with this receptor and to identify potential intermediate proteins.
As described above, Tnkl potentially functions in a signal transduction pathway downstream of events occurring on the cell surface. To look for proteins that interact with Tnkl, immunoprecipitation followed by Western blotting, could be performed. This may show the interaction of two known proteins, usually shown using antibodies to both. For example, immunoprecipitation with Jak3 antibodies resulted in the co-immunoprecipitation of the IL-4 receptor, demonstrating that Jak3 associates with the IL-4 receptor (Rolling et al. , Oncogene, 10:1757-17611). However, it is also possible to use immunoprecipitation to identify novel proteins (if Tnkl or Tnklα do indeed bind to novel proteins) . The protein(s) binding to Tnkl or Tnklα can then be purified, partially εequenced, then cloned.
Another method that might be utilized to identify novel proteins that interact with Tnkl or Tnklα is the yeast two- hybrid εyεtem (Fieldε and Strong, (Nature, 340:245-247)). Thiε εyεtem takes advantage of the yeaεt GAL4 tranεcription factor, which haε two domains needed for function, one which is essential for DNA binding, and the other required for activation of transcription. Tnkl can be cloned in-frame with a vector containing only the GAL4 binding domain, resulting in a GAL4-Tnkl fusion protein. A cDNA library is then constructed in a vector containing only the GAL4 activation domain. These are then co-transformed into the appropriate yeaεt strainε. Poεitive clones, i.e., clones that code for proteins that interact with Tnkl, are identified by growth in selective medium and blue/white screening.
If a novel receptor is identified as a protein binding ("upstream") to Tnkl or Tnklα using one of these approacheε (re-iterationε are poεsible, if no receptor is identified as binding directly to Tnkl) , the ligand may be identified by expresεion cloning, as was done with the cloning of the FH3/FLK2 ligand (Lyman et al., Cell, 75:1157-1167). In this method, the extracellular domain of the receptor was fused to the Fc portion of the IgG and expressed as a fusion protein in 293 cells. Cell lines were aεsayed for their ability to bind to the Flt3/Fc fusion protein. A cDNA library waε generated from a positive cell line. Poolε of the cDNA library were transfected into CV-1/EBNA cells, and positives identified by meaεuring binding of the Flt3-Fc to the tranεfected pools. This process was repeated until a single cDNA clone waε purified.
The method for determining whether a ligand can bind to the receptor which regulates Tnkl or Tnklα function comprises transfecting a cell population (one preεumed not to contain the receptor) with the appropriate vector expreεsing this receptor, εuch that the cell will now expresε thiε receptor. A εuitable response syεtem iε obtained by tranεfection of the DNA into a suitable host containing the desired second meεεenger pathwayε including cAMP, ion channelε, phoεphoinositide kinase, or calcium response. Such a transfection syεtem provideε a reεponεe εystem to analyze the activity of various ligands exposed to the cell. Ligands chosen could be identified through a rational approach by taking known ligands that interact with similar types of receptors or uεing εmall molecules, cell supernatants or extracts or natural products.
This invention provides a method of screening compounds to identify those which enhance (agonistε) interaction of Tnkl or Tnklα to itε receptor. As an example, a mammalian cell or membrane preparation expressing a Tnkl or Tnklα receptor is incubated with labeled Tnkl or Tnklα in the presence of the compound. The ability of the compound to enhance thiε interaction could then be measured. The response of Tnkl after interaction of the compound to the receptor can be compared and measured in the presence and absence of the compound.
The present invention also relates to an aεεay for identifying potential antagonists against Tnkl or Tnklα. An example of such an assay combines Tnkl or Tnklα and a potential antagoniεt with membrane-bound Tnkl or Tnklα receptorε or recombinant Tnkl or Tnklα receptorε under appropriate conditionε for a competitive inhibition assay. Tnkl or Tnklα may be labeled, such as by radio activity, such that the number of Tnkl or Tnklα moleculeε bound to the receptor can determine the effectiveneεε of the potential antagonist.
The Tnkl gene and gene product (or Tnklα and its gene product) of the present invention act intracellularly. Accordingly, the only practical mode of administration of the Tnkl gene (or Tnklα gene) is via gene therapy, hereinafter described, where it then acts to produce the Tnkl protein (or Tnklα) intracellularly. Tnkl (or Tnklα) may be administered to a patient in an amount εufficient to produce the Tnkl protein (or the Tnklα protein) at levelε that prevent, retard or reduce the severity of a disorder or its clinical manifestationε which iε related to an underexpreεεion of Tnkl protein (or Tnklα protein) . An amount adequate to accomplish any of theεe effects iε referred to as a "therapeutically effective" amount. Dosages effective for thiε use will depend upon the severity of the disorder and the general state of the patient's health. Tnkl is adminiεtered in an amount and over a period of time with a frequency and duration sufficient to yield a "therapeutically effective" amount, i.e., an amount sufficient to produce Tnkl protein at levels that reduce the εeverity of the disorderε or their manifestationε which are related to an underexpreεsion of the Tnkl protein (or Tnklα protein) . In any event, the pharmaceutical administrations should provide a quantity of the Tnkl (or the Tnklα) sufficient to effectively treat the patient.
The Tnkl polypeptide (or Tnklα) and agonists which are polypeptides may be employed in accordance with the present invention by expression of such polypeptideε in vivo, which iε often referred to aε "gene therapy."
Thuε, for example, cellε from a patient may be engineered with a polynucleotide (DNA or RNA) encoding a polypeptide ex vivo, with the engineered cellε then being provided to a patient to be treated with the polypeptide. Such methodε are well-known in the art and are apparent from the teachingε herein. For example, cellε may be engineered by the uεe of a retroviral plaεmid vector containing RNA encoding a polypeptide of the present invention.
Similarly, cells may be engineered in vivo for expresεion of a polypeptide in vivo by, for example, procedureε known in the art. For example, a packaging cell iε tranεduced with a retroviral plaεmid vector containing RNA encoding a polypeptide of the preεent invention such that the packaging cell now produces infectious viral particleε containing the gene of intereεt. These producer cells may be adminiεtered to a patient for engineering cells in vivo and expreεεion of the polypeptide in vivo. These and other methods for administering a polypeptide of the preεent invention by εuch method εhould be apparent to those skilled in the art from the teachings of the present invention.
Retroviruses from which the retroviral plasmid vectors hereinabove mentioned may be derived include, but are not limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, retroviruseε such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human immunodeficiency virus, adenovirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus. In one embodiment, the retroviral plasmid vector is derived from Moloney Murine Leukemia Virus.
The vector includeε one or more promoters. Suitable promoters which may be employed include, but are not limited to, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus (CMV) promoter described in Miller, et al., Biotechniques. Vol. 7, No. 9, 980-990 (1989), or any other promoter (e.g., cellular promoters such as eukaryotic cellular promoters including, but not limited to, the histone, pol III, and 3-actin promoterε) . Other viral promoterε which may be employed include, but are not limited to, adenoviruε promoters, thymidine kinase (TK) promoters, and B19 parvovirus promoters. The selection of a suitable promoter will be apparent to thoεe εkilled in the art from the teachings contained herein.
The nucleic acid sequence encoding the polypeptide of the present invention is under the control of a suitable promoter. Suitable promoters which may be employed include, but are not limited to, adenoviral promoters, such as the adenoviral major late promoter,- or heterologous promoters, such as the cytomegalovirus (CMV) promoter; the respiratory syncytial virus (RSV) promoter; inducible promoterε, εuch aε the MMT promoter, the metallothionein promoter,- heat shock promoters; the albumin promoter,- the ApoAI promoter,- human globin promoters,- viral thymidine kinaεe promoterε, εuch aε the Herpes Simplex thymidine kinaεe promoter; retroviral LTRs (including the modified retroviral LTRε hereinabove described) ,- the β-actin promoter,- and human growth hormone promoters. The promoter also may be the native promoter which controlε the gene encoding the polypeptide.
The retroviral plaεmid vector iε employed to tranεduce packaging cell lineε to form producer cell lines. Exampleε of packaging cells which may be transfected include, but are not limited to, the PE501, PA317, -2 , ψ-AM, PA12, T19-14X, VT-19-17-H2, CHE , GP+E-86, GP+envAml2, and DAN cell lines as described in Miller, Human Gene Therapy. Vol. l, pgs. 5-14 (1990), which is incorporated herein by reference in its entirety. The vector may transduce the packaging cells through any means known in the art. Such meanε include, but are not limited to, electroporation, the uεe of lipoεomeε, and CaP04 precipitation. In one alternative, the retroviral plasmid vector may be encapsulated into a liposome, or coupled to a lipid, and then administered to a host.
The producer cell line generates infectious retroviral vector particleε which include the nucleic acid εequence(ε) encoding the polypeptideε. Such retroviral vector particles then may be employed, to transduce eukaryotic cells, either in vi tro or in vivo. The transduced eukaryotic cells will expresε the nucleic acid sequence (s) encoding the polypeptide. Eukaryotic cells which may be transduced include, but are not limited to, embryonic εtem cells, embryonic carcinoma cells, as well as hematopoietic stem cells, hepatocytes, fibroblaεts, myoblastε, keratinocyteε, endothelial cells, and bronchial epithelial cellε.
Thiε invention is also related to the use of the Tnkl gene (or the Tnklα gene) of the present invention as a diagnostic. Detection of a mutated form of the gene will allow a diagnosis of a disease or a susceptibility to a diseaεe, for example cancer, which reεultε from overexpreεsion or underexpression of Tnkl (or Tnklα) , or abnormal function of mutated Tnkl.
Individuals carrying mutations in the gene of the present invention may be detected at the DNA level by a variety of techniques. Nucleic acids for diagnosis may be obtained from a patient's cells, including but not limited to blood, urine, saliva, tisεue biopsy and autopsy material. The genomic DNA may be used directly for detection or may be amplified enzymatically by uεing PCR (Saiki et al., Nature, 324:163-166 (1986)) prior to analyεiε. RNA or cDNA may also be uεed for the εame purpose. As an example, PCR primers complementary to the nucleic acid encoding Tnkl can be used to identify and analyze mutations. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to radiolabeled RNA or alternatively, radiolabeled antiεenεe DNA εequenceε. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.
Sequence differences between the reference gene and genes having mutations may be revealed by the direct DNA sequencing method. In addition, cloned DNA segments may be employed as probeε to detect εpecific DNA εegments. The sensitivity of this method is greatly enhanced when combined with PCR. For example, a sequencing primer is used with double-stranded PCR product or a εingle-εtranded template molecule generated by a modified PCR. The εequence determination iε performed by conventional procedures with radiolabeled nucleotide or by automatic sequencing procedures with fluorescent-tags.
Genetic testing based on DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragmentε in gels with or without denaturing agents. Small sequence deletions and insertions can be visualized by high reεolution gel electrophoreεiε. DNA fragments of different εequenceε may be diεtinguiεhed on denaturing formamide gradient gelε in which the mobilitieε of different DNA fragmentε are retarded in the gel at different poεitionε according to their specific melting or partial melting temperatures (see, e.g., Myers et al., Science, 230:1242 (1985) ) .
Sequence changes at specific locations may also be revealed by nuclease protection aεsayε, εuch aε RNaεe and SI protection or the chemical cleavage method (e.g., Cotton et al., PNAS, USA, 85:4397-4401 (1985)).
Thuε, the detection of a specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the uεe of reεtriction enzymeε, (e.g., Restriction Fragment Length Polymorphisms (RFLP) ) and Southern blotting of genomic DNA.
In addition to more conventional gel-electrophoresis and DNA sequencing, mutations can also be detected by in si tu analysis.
The polypeptides, their fragments or other derivatives, or analogε thereof, or cells expresεing them can be uεed aε an immunogen to produce antibodieε thereto. Theεe antibodieε can be, for example, polyclonal or monoclonal antibodieε. The present invention alεo includes chimeric, single chain, and humanized antibodies, as well as Fab fragments, or the product of an Fab expresεion library. Various procedures known in the art may be used for the production of such antibodies and fragments.
Antibodies generated against the polypeptides corresponding to a sequence of the present invention can be obtained by direct injection of the polypeptides into an animal, preferably a nonhuman. The antibody so obtained will then bind the polypeptides itself. In this manner, even a sequence encoding only a fragment of the polypeptides can be used to generate antibodies binding the whole native polypeptides. Such antibodieε can then be uεed to isolate the polypeptide from tissue expressing that polypeptide.
For preparation of monoclonal antibodies, any technique which provideε antibodieε produced by continuouε cell line cultureε can be uεed. Exampleε include the hybridoma technique (Kohler and Milεtein, 1975, Nature, 256:495-497), the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV- hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
Techniqueε deεcribed for the production of εingle chain antibodies (U.S. Patent 4,946,778) can be adapted to produce single chain antibodieε to immunogenic polypeptide products of this invention. Also, transgenic mice may be used to express humanized antibodies to immunogenic polypeptide products of this invention.
Antibodies specific to the Tnkl gene and protein may be employed as markers for cells which express the protein.
The present invention will be further described with reference to the following examples; however, it is to be understood that the present invention is not limited to such exampleε. All parts or amountε, unless otherwise specified, are by weight.
Example 1 Isolation and Expresεion of Tnkl Gene
Human Bone Marrow or Cord Blood Cellε. Normal human bone marrow waε collected from the poεterior iliac creεt of conεenting healthy adults under an Institutional Review Board approved protocol. Umbilical cord blood was obtained from post-delivery placenta in the Johns Hopkinε Hoεpital. CD34+/Lin- and other hematopoietic cell εubεetε were prepared by immunomagnetic microεphere εeparation as previouεly deεcribed (Gore, S.D., et al. , Exp. Hetnatol., 23:413-421 (1995)) . For preparation of CD38- cellε, CD34+/Lin- cells, isolated as above, were incubated for an additional 30 minutes at 4°C in the presence of CD38 coated immunomagnetic microspheres, at 20 microspheres per cell. CD38- and CD38+ cell fractions were then separated on the magnetic particle concentrator (Dynal, Lake Succesε, NY) . Blood was drawn from 2 consenting, healthy adult donors, and mononuclear cell and granulocyte subpopulations were separated by density gradient centrifugation (Mono-Poly Resolving Medium, ICN, Aurora, OH) .
RNA Isolation. Total RNA waε prepared from purified marrow, cord blood, and peripheral blood cell fractionε, uεing the method of Chomczynεki, P. & and Sacchi, N. , Anal. Biochem., 162:156-159 (1987)) . For reverεe-tranεcriptaεe PCR, total RNA waε treated with DNase (Gibco BRL, Gaithersburg, MD) , phenol:chloroform extracted, ethanol precipitated and resuεpended in sterile DEPC-treated dH,0. PolyA RNA was purified from leukemic cell lines by the Ribosep mRNA kit (Becton Dickinson, Bedford, MA) .
Degenerate PCR. Total RNA from CD34+/Lin-/CD38- umbilical cord cells (approximately 1.2 x 10J cellε) was treated with DNase, phenol:chloroform extracted, ethanol precipitated and resuspended in DEPC-treated H20. DNase- treated RNA was reverse transcribed using random primers and MMLV reverse transcriptase (Gibco BRL) at 37°C. An aliquot (5 μl) of this reaction was PCR amplified using degenerate oligonucleotides that targeted the conserved amino acid sequences VHRDLA (PTK1) and SDVWSYG (PTK2) from protein tyrosine kinaseε (Hankε, S.K. et al., Science, 241, 42-52 (1988); Small, D. et al., Proc. Nat. Acad. Sci. USA, 91, 459- 463 (1994)). The sequences of the primers were aε follows.- PTK1: 5' CCCGCGGCCGCTNCAT(T/C) (A/C)GNGA(T/C) (T/OTNGCNGC 3' (SEQ ID NO:5); PTK2: 5'CCCGTCGACCC(A/G)TAN(C/G) (T/A)CCANAC(A/G)TC 3' (SEQ ID N0:6). Degenerate PCR waε carried out at 94°C for 1 min. 37°C for 1.5 min., 72°C for 2 min for 35 cycleε with 10 min. extension at 72°C following the last cycle. PCR products were separated by agaroεe gel electrophoresis, and appropriate sized products (-234 bp) were eluted using GeneClean (BIO 101, Vista, CA) . Products were digested with NotI and Sail, ligated into pBluescript KS (Stratagene, La Jolla, CA) and transformed into competent E. coli DH5α.
DNA Sequencing. Dideoxy sequencing was performed on plasmid DNA using Sequenase (USB, Cleveland, OH) and resolved on 8% polyacrylamide gels. To quickly eliminate repetitive clones, sequencing gels containing only T-laneε, were run. Alternatively, double-εtranded plaεmid DNA waε εequenced on an automated fluorescent DNA εequencer in the Johnε Hopkinε Univerεity School of Medicine Genetics Core facility. DNA sequences were compared to known sequenceε in Genbank, using the BlastN program from National Center for Biotechnology Information (NIH, Betheεda, MD) .
5' and 3' RACE. were performed esεentially aε deεcribed in (Frohman, M.A. , Meth. Enzym. , 218, 340-356 (1993)). Briefly, for 5' RACE, K562 mRNA was reverse- transcribed with Tnkl specific primer B 5' ATCCGAGGC AGACGAGAAGG 3' (SEQ ID NO:7) at 37°C for 2 h with MMLV, diluted to 2 ml in Triε-EDTA (TE) buffer, and primers were removed uεing a Centricon-100 spin filter (Amicon, Beverly, MA) and washed once in 0.2x TE. Samples were concentrated to about 10 μl in a vacuum centrifuge (Savant, Holbrook, NY) , and cDNA products were tailed with poly(A) using terminal nucleotidyl transferase (TdT) according to manufacturer's instructionε (Boehringer Mannheim, Indianapoliε, IN) . Productε were then diluted to 500 μl in TE. For the firεt round of PCR, an aliquot of the diluted cDNA pool (5 μl) was amplified with 25 pmol of Tnkl specific primer C 5' AGACGAGAAGGCTCCGTG 3' (SEQ ID NO:8) , 2 pmol Of RACE PRIMER #1 5' GATGGATCCTGCAGAAGC(T)173' (SEQ ID NO:9) and 25 Pmol of race primer #2 5' GATGGATCCTGCAGAAGC 3' (SEQ ID NO:10) in 10% DMSO, 1.5 mM deoxynucleotidetriphophates (dNTPS) and IX PCR buffer (67 mM Triε-HCl, pH 9.0, 6.7 mM MgCl2, 170 μg/ml BSA, 16.6 mM (NH jSO . Samples were heated to 97°C for 5 min., cooled to 75°C, and then 2.5U of Taq polymerase (Gibco BRL) were added. Products were annealed at 50°C for 2 min. , and extended at 72°C for 45 min. , followed with 35 cycles of 94°C for 1 min., 50°C for 1 min., 72°C for 3 min., with a final 15 min. extension at 72°C. For the second round of amplification, firεt round PCR productε were diluted 1:20 in TE, and 5 μl was used as templated with Tnkl specific primer D 5' ACCAGGTGTAGGGGATAG 3' (SEQ ID NO:11) and RACE primer #2 for PCR, as above except the initial 2 min. annealing step and 45 min. extension step were eliminated. Products were cloned into the TA vector (Invitrogen, San Diego, CA) , and colonieε containing 5' Tnkl εequenceε were identified by hybridizing with internal Tnkl specific primer A 5' CCATCAAGGTGGCTGACTTC 3' (SEQ ID NO:12). For 3' RACE, K562 mRNA was reverse transcribed with oligo- (dT) using Superεcript II (Gibco BRL) at 42°C for 2 h and then diluted to 1 ml in TE. For the firεt round of amplification, 3 μl of diluted cDNA waε amplified with 25 pmol of RACE primer #1 and Tnk εpecific primer A aε in the first round of 5'-RACE. Products were diluted 1:20 and 5 μl of this used as template for second round of PCR using 25 pmol of RACE primer #2 and Tnk specific prier Forward 1 5' CTGGTGTGCCCCAGAGAG 3' (SEQ ID NO:13) , as deεcribed for 5'-RACE. 3'-RACE products were cloned into the TA vector and transformants containing 3 ' Tnkl sequenceε were identified using an internal Tnkl specific oligonucleotide (Tnk Primer C) .
Reverse-Tranεcriptase PCR. For expresεion εtudieε using RT-PCR, total RNA (uεually 1-5 μg) or mRNA (1 μg) waε reverεe-tranεcribed to cDNA using random hexamer primers and MMLV-RT at 37°C for 2 hours. An aliquot of this reaction (3 μl) was amplified with Tnkl specific primers, Tnkl 1623 5' GAGCGAATTCACAGAACTGACTCCAGACTCCTGG 3' (SEQ ID NO:14) at 94°C for 1 min, 55°C for 1 min., 72°C for 1 min., for 35 cycles. Products were separated on an agarose gel, transferred to Nylon membranes (Schleicher and Schuell, Keene, NH) , and hybridized with an internal 32-P end-labeled, Tnkl specific oligonucleotide, Tnkl 1473, 5' TGCCTCTGTGGACTGGACGG 3' (SEQ ID N0:15) according to established protocols (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Preεε, Cold Spring Harbor, NY (1989) ) .
Example 2 Chromosome-mapping with DNA hybrid panel. From the cDNA sequence of Tnkl, 2 different primer pairs were made that amplified over an intron on genomic DNA. From the 5'-part of the cDNA, the primerε were Tnkl 224, 5' GCACTT CGACTTTGTAAAGCCTGAG 3' (SEQ ID NO:16) and Tnkl, 5' TTTTCAG AGCTTCGGACAGTCTG 3' (SEQ ID NO:17) . For the amplification of a segment towardε the 3' part of the Tnkl cDNA, the primerε were Tnkl 1270, 5' CATGTTGTGTGAGGGATGCCAC 3' (SEQ ID NO:18) and Tnkl 1392, 5' TGAAGGTGCGACCATTCTGG 3' (SEQ ID NO:19) . Theεe primers were used to amplify DNA from the NIGMS Hybrid DNA PANEL #2 (Drwinga et al. , Genomics, 16:311-314 (1993); Duboiε and Naylor, Genomics, 16:315-319 (1993)) , which has nearly all the human chromosomes completely separated in hybrids with mouεe or hamster cellε. About 0.5μg of DNA prepared from these cellε waε used for each PCR-reaction. PCR was run separately with the 2 primer-pairs at 94°C for l min, 63°C for 1 min, and 72°C for 1 min for 35 cycles. PCR products were electrophoresed on a 1.0% agarose gel, transferred to nylon filters (Nytran, Schleicher & Schuell) , and hybridized with an 32P end-labeled internal oligonucleotide.
Fluorescence In Situ Hybridization. A genomic Tnkl gene isolated from a human Pl genomic DNA library (Human Foreεkin Fibroblaεt Pl LIBRARY #1, Du Pont Merck Pharmaceutical Co., St. Louiε, MO) by PCR using Tnkl specific primers. DNA from a 70 Kb Pl plasmid containing the human Tnkl gene (Clone #5494 and #5495) waε purified according to manufacturer'ε instructions, labeled with biotin by nick translation (BioNick Kit, Gibco, BRL) , and hybridized on human cytogenetic preparations with metaphase chromosomes. Slides with chromosome spreads were made from normal male lymphocyteε cultured with BrdU (Bhatt, B., et al., Nucl. Acids Res., 16:3951-3961 (1988)). Fluorescence in si tu hybridization was performed as described (Lichter, P., et al., Science, 247:64-69 (1990)) with modifications. Probe mix (2X SSCP, 60% formamide, 10% dextran sulfate, 2 ng/μl biotinylated probe, 200 ng/μl Cot-1 DNA (BRL)) was denatured at 70°C for 5 min, preannealed at 37°C for 60 min, placed on slideε and hybridized at 37°C overnight. Slideε were waεhed in 60% formamide/2x SSC at 45°C for 20 min., then waεhed 2x in 2x SSC at 37°C for 5 min each. Biotinylated probe waε detected with FITC-avidin and amplified with biotinylated anti-avidin, uεing reagents from in si tu hybridization kit (Oncor Inc., Gaithersburg, MD) following manufacturer's instructions.
Example 3 Bacterial Expresεion of Tnkl Tnkl polypeptide in itε entirety, or in fragments, can be expressed in a bacterial syεtem. A method utilized is generating a fusion protein to glutathione-S-transferaεe(GST) (Smith, D.B. and Johnεon, K.S., Gene, 67:31-40 (1988); εee also Auεubel, F.M. , et al., Current Protocolε in Molecular Biology, Vol. 2, Chapter 16.7.1-16.7.8 (1993)) . The Tnkl coding region iε cloned into the appropriate GST vector εuch that Tnkl coding region iε fuεed in-frame with the GST reading frame. The cloning vectors contain the necesεary control elements required for efficient tranεcription and tranεlation, including an inducible promoter and multiple termination codonε. Production of GST-fusion proteins is under the control of the E. coli lacZ promoter, and thus is inducible with IPTG. GST fusion proteins can be purified from bacterial lysates by affinity chromatography using glutathione-agarose beads.
The protocol used is as follows:
1) Grow bacteria containing the fusion plasmid ON at 37°C.
2) The following morning, dilute the ON culture 1:10, and incubate for an additional 3 hours at 37°C.
3) Induce fusion protein by adding IPTG (0.1 mM final cone.) and incubate an additional 3 hours at 37°C.
4) Harvest cells by centrifugation, resuspend in buffer containing detergent. The detergent is MTBS which is 150 mM NaCl, 16 mM NaHP04 and 4 mM NaH2P04 which contains 1% Triton X-100. The cells are then lysed by sonication.
5) Do 2 freeze/thaw cycles on sonicated extracts.
6) Remove cellular debris by centrifugation.
7) Bind fusion-protein to glutathione-agarose beads (prepared as a 50% slurry in MTBS) and incubate at
4°C for 1 hour.
8) Collect beads by centrifugation; Wash 3 times in MTBS + 0.1% Triton. 9) Elute fuεion protein using excess glutathione (5mM in 50 mM Tris pH 8.0) ; elution step can be repeated several times, and elutions pooled and concentrated.
Example 4 Generation of rabbit antibodies against Tnkl peptides
Two peptides were εynthesized from amino acids uεing a peptide εyntheεizer inεtrument (εtandard procedureε) . Two different antiεera were raiεed by immunizing 2 rabbitε with 2 different Tnkl εynthetic peptideε (LPATCPVHRGTPARGDQHPG for the firεt rabbit and IDGDRKKANLWDAP for the εecond rabbit) , each conjugated to Keyhole Limpet Hemocyanin (KLH) . Each rabbit waε booεted (re-immunized) multiple timeε with the εame preparationε (done by Hazleton Reεearch Productε Denver, PA) by their εtandard immunization and bleed protocol) . The antiεera (collected by Hazleton) waε aεsayed by ELISA and immunoblot against Tnkl peptide and bacterially expressed GST-Tnkl fusion protein (the portion of Tnkl expressed in the fusion protein contained both peptide sequenceε) . Both antibodieε recognize the appropriate peptide and the fuεion protein.
Numerouε modifications and variations of the present invention are poεsible in light of the above teachings and, therefore, within the scope of the appended claims, the invention may be practiced otherwise than as particularly described.
SEQUENCE LISTING
(1) GENERAL INFORMATION: (i) APPLICANT: Curt Civin, Donald Small and Gerard Hoehn
(ii) TITLE OF INVENTION: Tyrosine Kinaεe Gene, Splice
Variant and Gene Products
(iii) NUMBER OF SEQUENCES: 19
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: CARELLA, BYRNE, BAIN, GILFILLAN,
CECCHI, STEWART & OLSTEIN
(B) STREET: 6 BECKER FARM ROAD
(C) CITY: ROSELAND
(D) STATE: NEW JERSEY
(E) COUNTRY: USA
(F) ZIP: 07068
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: 3.5 INCH DISKETTE
(B) COMPUTER: IBM PS/2
(C) OPERATING SYSTEM: MS-DOS
(D) SOFTWARE: WORD PERFECT 5.1
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER: TBA
(B) FILING DATE: Filed Herewith
(C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA
(A) APPLICATION NUMBER: 60/005,286
(B) FILING DATE: U.S Provisional Application (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: MULLINS, J.G.
(B) REGISTRATION NUMBER: 33,073
(C) REFERENCE/DOCKET NUMBER: 290770-05
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 201-994-1700
(B) TELEFAX: 201-994-1744
(2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
( ii ) MOLECULE TYPE : cDNA
(xi ) SEQUENCE DESCRIPTION : SEQ ID NO : l :
GAGTCGCCGC TTCCGCCTTG ACCAGGTGGA GCTGGAGACC TGGTCTCTCT AGGGCCTGCC 60
CTGAGCTCAC CATCTGAAGG AGAGTGCCAT CATCCTTAGG AACTCCTTCT CCAGACATGC 120
TTCCCGAGGC TGGCTCCCTG TGGCTACTGA AGCTGCTCCG GGACATCCAG TTGGCCCAGT 180
TTTACTGGCC CATCCTAGAG GAGCTTAATG TCACCCGGCC AGGGCACTTC GACTTTGTAA 240
AGCCTGAGGA CCTGGACGGC ATTGGCATGG GCCGGCCTGC CCAACGCAGA CTGTCCGAAG 300
CTCTGAAAAG CCTACGTTC GGGCCTAAGT CTAAGAACTG GGTCTACAAG ATCCTTGGAG 360
GTTTTGCCCC TGAGCACAAG GAGCCCACCC TGCCCACGGA CAGCCCACGG CACCTCCCTG 420
AGCCAGAGGG GGGCCTCAAG TGTCTGATCC CAGAGGGTGC TGTTTGCAGA GGGGAGCTGC 480
TGGGTTCAGG CTGCTTCGGT GTGGTGCACC GAGGGCTGTG GACGCTGCCC AGTGGCAAGA 540
GTGTCCCAGT GGCTGTCAAG TCCCTCCGGG TAGGTCCCGA AGGCCCGATG GGCACAGAAC 600
TGGGGGACTT CCTGCGAGAG GTATCGGTCA TGATGAACTT GGAGCACCCA CACGTGCTGC 660
GTCTGCACGG CCTTGTACTG GGCCAGCCTC TGCAGATGGT GATGGAGCTG GCGCCACTGG 720
GCTCCCTGCA CGCGCGCTTA ACGGCCCCGG CCCCGACACC CCCGCTGCTC GTGGCCCTGC 780
TCTGCCTC T CCTGCGGCAG CTGGCGGGAG CCATGGCGTA CCTGGGGGCC CGCGGGCTGG 840
TGCACCGAGA CCTCGCTACG CGCAACCTAC AGCTGGCGTC GCCGCGCACC ATCAAGGTGG 900
CTGACTTCGG GCTGGTGCGG CCTCTGGGCG GTGCCCGGGG CCGCTACGTC ATGGGCGGGC 960
CCCGCCCTAT CCCCTACACC TGGTGTGCCC CAGAGAGCCT GCGCCACGGA GCCTTCTCGT 1020
CTGCCTCGGA CGTGTGGATG TTTGGGGTGA CGCTGTGGGA GATGTTCTCC GGGGGCGAGG 1080
AACCCTGGCC CGGGGTCCCA CCGTACCTCA TCCTGCAGCG GCTGGAGGAC AGAGCCCGGC 1140 TGCCTAGGCC TCCCCCCTCC TCCAGGGCCC TCTACTCCCT CGCCTTGCGC TGCTGGGCCC 1200
CCCACCCTGC CGACCGGCCT AGCTTTTCCC ACCTGGAGGG GCTGCTGCAA GAGGCCGGGC 1260
CTTCGGAAGC ATGTTGTGTG AGGGATGCCA CAGAACCAGG CGCCCTGAGG ATGGAGACTG 1320
GTGACCCCAT CACAGTCATC GAGGGCAGCT CCTCTTTCCA CAGCCCCGAC TCCACAATCT 1380
GGAAGGACCA GAATGGTCGC ACCTTCAAAG TGGGCAGCTT CCCAGCCTCG GCAGTGACGC 1440
TGACAGATGC GGGGGGCTTG CCAGCCACCC GTCCAGTCCA CAGAGGCACC CCTGCCCGGG 1500
GAGATCAACA CCCAGGAACC ATAGATGGAG ACAGAAAGAA GGCAAATCTT TGGGATGCGC 1560
CCCCAGCACG GGGCCAGAGG AGGAACATGC CCCTGGAGAG GATGAAAGGC ATTTCCAGGA 1620
GTCTGGAGTC AGTTCTGTCC CTCGGTCCTC GTCCCACAGG GGGTGGTTCA AGCCCCCCTG 1680
AAATTCGACA AGCCAGAGCT GTGCCCCAGG GACCTCCAGG CCTGCCTCCA CGCCCACCTT 1740
TATCCTCTAG CTCTCCTCAG CCCAGCCAGC CCTCTAGGGA GAGGCTTCCC TGGCCCGAAA 1800
GAAAACCCCC ACACAATCAC CCCATGGGAA TGCCTGGAGC CCGTAAAGCC GCTGCCCTCT 1860
CTGGAGGCCT CTTGTCCGAT CCTGAGTTGC AGAGGAAGAT TATGGAAATG GAGCTGAGTG 1920
TGCATTGGGT CACCCACCAG GAGTGCCAGA CAGCACTAGG AGCCACTGGG GGAGATGTGG 1980
CTTCTGCCAT CCGGAACCTC AAGGTAGATC AGCTCTTCCT CCTGAGTAGC CGGTCCAGAG 2040
CTGACTGCTG GCGCATCCTG GAGCATTACC AGTGGGACCT CTCAGCTGCC AGCCGTTATG 2100
TCCTGGCCAG GCCCTGAGCC CAGCTTCTGC GGGCACAGAC ACCAGCATGA AAAGCCTAGG 2160
TCCCTGAGGG CCTGGCCACA TGGGACCAAG TGGAACCAGA ACAAGGCCCC GACAGGGGTA 2220
GACGTTCCAC TTGTGGAGAT CCCACCTGCC GCTAGGCACG TGGAGGAGGA GCCCAGAGTT 2280
GGGCACTGGC AAATGTCTCC TCCCTCCCAT GCTCCTTGGC TTCTGAAGGC TGAAGCCCCT 2340
TTGGCTGGGC CAAGAAGGAT CTAGTCTGCC CACTACATTC TCAAACAAGA GGACTTGGAG 2400
GAAAAGAGCT ACTATACATC ATATGCAGAG GAAGCTTCTA CGCGCTAGAG AGGATCAAGG 2460
GGCCACACTG GACCATGTGA ACAGCCATCC GGAACTGCCA TCAGCTACCA CACTGGACTC 2520
TGCAGGGCAG CCATCCTGGA TGATGGAAGC CACCATATTG ACCTGGGGTA TAGGCCCAAA 2580
CTGCCTTCGT TTGGTCCAGG GCCATCGTGG GTGATGACGA TTGCTCTCTT GCACTCATGG 2640
ACATTTGATG CTGGTAGTAT GGATTATGAG ATGGACTAGC CCCTGCTCCA GCCCAGTTCT 2700
CACATTCCCC TTTGTTTTTT CCCATACCAA CTGCTTCTAC CCTCCCCTAT TACATACATC 2760
TTTCAATGTC CAAAAAAAAA AAAAAAAAA 2789
(3) INFORMATION FOR SEQ ID NO:2: (i) SEQUENCE CHARACTERISTICS
(A) LENGTH : 666 AMIΝO ACIDS
(B) TYPE: AMIΝO ACID
(C) STRANDEDNESS:
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: PROTEIN
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
Met Leu Pro Glu Ala Gly Ser Leu Trp Leu Leu Lys Leu Leu Arg
5 10 15 Aεp lie Gin Leu Ala Gin Phe Tyr Trp Pro lie Leu Glu Glu Leu
20 25 30
Aεn Val Thr Arg Pro Gly Hiε Phe Asp Phe Val Lyε Pro Glu Aεp
35 40 45
Leu Aεp Gly lie Gly Met Gly Arg Pro Ala Gin Arg Arg Leu Ser
50 55 60
Glu Ala Leu Lyε Ser Leu Arg Ser Gly Pro Lys Ser Lys Asn Trp
65 70 75
Val Tyr Lyε lie Leu Gly Gly Phe Ala Pro Glu Hiε Lyε Glu Pro
80 85 90
Thr Leu Pro Thr Aεp Ser Pro Arg Hiε Leu Pro Glu Pro Glu Gly
95 100 105
Gly Leu Lyε Cyε Leu lie Pro Glu Gly Ala Val Cyε Arg Gly Glu
110 115 120
Leu Leu Gly Ser Gly Cyε Phe Gly Val Val His Arg Gly Leu Trp
125 130 135
Thr Leu Pro Ser Gly Lys Ser Val Pro Val Ala Val Lys Ser Lys
140 145 150
Arg Val Gly Pro Glu Gly Pro Met Gly Thr Glu Lys Gly Asp Phe
155 160 165
Leu Arg Glu Val Ser Val Met Met Aεn Leu Glu Hiε Pro Hiε Val
170 175 180
Leu Arg Leu Hiε Gly Leu Val Leu Gly Gin Pro Leu Gin Met Val
185 190 195 Met Glu Leu Ala Pro Leu Gly Ser Leu His Ala Arg Leu Thr Ala
200 205 210
Pro Ala Pro Thr Pro Pro Leu Leu Val Ala Leu Leu Cys Leu Phe
215 220 225
Leu Arg Gin Leu Ala Gly Ala Met Ala Tyr Leu Gly Ala Arg Gly
230 235 240
Leu Val His Arg Asp Leu Ala Thr Arg Asn Leu Gin Leu Ala Ser
245 250 255
Pro Arg Thr lie Lys Val Ala Asp Phe Gly Leu Val Arg Pro Leu
260 265 270
Gly Gly Ala Arg Gly Arg Tyr Val Met Gly Gly Pro Arg Pro lie
275 280 285
Pro Tyr Thr Trp Cyε Ala Pro Glu Ser Leu Arg Hiε Gly Ala Phe
290 295 300
Ser Ser Ala Ser Aεp Val Trp Met Phe Glu Val Thr Leu Trp Glu
305 310 315
Met Phe Ser Gly Gly Glu Glu Pro Trp Pro Gly Val Pro Pro Tyr
320 325 330
Leu lie Leu Gin Arg Leu Glu Asp Arg Ala Arg Leu Pro Arg Pro
335 340 345
Pro Pro Ser Ser Arg Ala Leu Tyr Ser Leu Ala Leu Arg Cys Trp
350 355 360
Ala Pro His Pro Ala Aεp Arg Pro Ser Phe Ser His Leu Glu Gly
365 370 375 Leu Leu Gin Glu Ala Gly Pro Ser Glu Ala Cys Cys Val Arg Asp
380 385 390
Ala Thr Glu Pro Gly Ala Leu Arg Met Glu Thr Gly Asp Pro lie
395 400 405
Thr Val lie Glu Gly Ser Ser Ser Phe Hiε Ser Pro Asp Ser Thr
410 415 420
lie Trp Lys Aεp Gin Aεn Gly Arg Thr Phe Lyε Val Gly Ser Phe
425 430 435
Pro Ala Ser Ala Val Thr Leu Thr Aεp Ala Gly Gly Leu Pro Ala
440 445 450
Thr Arg Pro Val His Arg Gly Thr Pro Ala Arg Gly Aεp Gin Hiε
455 460 465
Pro Gly Ser lie Asp Gly Aεp Arg Lyε Lyε Ala Aεn Leu Trp Aεp
470 475 480
Ala Pro Pro Ala Arg Gly Gin Arg Arg Aεn Met Pro Leu Glu Arg
485 490 495
Met Lyε Gly lie Ser Arg Ser Leu Glu Ser Val Lyε Ser Leu Gly
500 505 510
Pro Arg Pro Thr Gly Gly Gly Ser Ser Pro Pro Glu lie Arg Gin
515 520 525
Ala Arg Ala Val Pro Gin Gly Pro Pro Gly Leu Pro Pro Arg Pro
530 535 540
Pro Leu Ser Ser Ser Ser Pro Gin Pro Ser Gin Pro Ser Arg Glu
545 550 555 Arg Leu Pro Trp Pro Glu Arg Lys Pro Pro His Asn His Pro Met
560 565 570
Gly Met Pro Gly Ala Arg Lys Ala Ala Ala Leu Ser Gly Gly Leu
575 580 585
Leu Ser Asp Pro Glu Leu Gin Arg Lys lie Met Glu Met Glu Leu
590 595 600
Ser Val His Trp Val Thr His Gin Glu Cyε Gin Thr Ala Leu Gly
605 610 615
Ala Thr Gly Gly Aεp Val Ala Ser Ala lie Arg Aεn Leu Lys Val
620 625 630
Asp Gin Leu Phe Leu Leu Ser Ser Arg Ser Arg Ala Aεp Cys Trp
635 640 645
Arg lie Leu Glu His Tyr Gin Trp Asp Leu Ser Ala Ala Ser Arg
650 655 660
Tyr Val Leu Ala Arg Pro
665
(4) INFORMATION FOR SEQ ID NO:3:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: CDNA (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
GAGTCGCCGC TTCCGCCTTG ACCAGGTGGA GCTGGAGACC TGGTCTCTCT AGGGCCTGCC 60
CTGAGCTCAC CATCTGAAGG AGAGTGCCAT CATCCTTAGG AACTCCTTCT CCAGACATGC 120
TTCCCGAGGC TGGCTCCCTG TGGCTACTGA AGCTGCTCCG GGACATCCAG TTGGCCCAGT 180
TTTACTGGCC CATCCTAGAG GAGCTTAATG TCACCCGGCC AGGGCACTTC GACTTTGTAA 240
AGCCTGAGGA CCTGGACGGC ATTGGCATGG GCCGGCCTGC CCAACGCAGA CTGTCCGAAG 300
CTCTGAAAAG CCTACGTTCT GGGCCTAAGT CTAAGAACTG GGTCTACAAG ATCCTTGGAG 360
GTTTTGCCCC TGAGCACAAG GAGCCCACCC TGCCCACGGA CAGCCCACGG CACCTCCCTG 420
AGCCAGAGGG GGGCCTCAAG TGTCTGATCC CAGAGGGTGC TGTTTGCAGA GGGGAGCTGC 480
TGGGTTCAGG CTGCTTCGGT GTGGTGCACC GAGGGCTGTG GACGCTGCCC AGTGGCAAGA 540
GTGTCCCAGT GGCTGTCAAG TCCCTCCGGG TAGGTCCCGA AGGCCCGATG GGCACAGAAC 600
TGGGGGACTT CCTGCGAGAG GTATCGGTCA TGATGAACTT GGAGCACCCA CACGTGCTGC 660
GTCTGCACGG CCTTGTACTG GGCCAGCCTC TGCAGATGGT GATGGAGCTG GCGCCACTGG 720
GCTCCCTGCA CGCGCGCTTA ACGGCCCCGG CCCCGACACC CCCGCTGCTC GTGGCCCTGC 780
TCTGCCTCTT CCTGCGGCAG CTGGCGGGAG CCATGGCGTA CCTGGGGGCC CGCGGGCTGG 840
TGCACCGAGA CCTCGCTACG CGCAACCTAC AGCTGGCGTC GCCGCGCACC ATCAAGGTGG 900
CTGACTTCGG GCTGGTGCGG CCTCTGGGCG GTGCCCGGGG CCGCTACGTC ATGGGCGGGC 960
CCCGCCCTAT CCCCTACACC TGGTGTGCCC CAGAGAGCCT GCGCCACGGA GCCTTCTCGT 1020
CTGCCTCGGA CGTGTGGATG TTTGGGGTGA CGCTGTGGGA GATGTTCTCC GGGGGCGAGG 1080
AACCCTGGCC CGGGGTCCCA CCGTACCTCA TCCTGCAGCG GCTGGAGGAC AGAGCCCGGC 1140
TGCCTAGGCC TCCCCCCTCC TCCAGGGCCC TCTACTCCCT CGCCTTGCGC TGCTGGGCCC 1200
CCCACCCTGC CGACCGGCCT AGCTTTTCCC ACCTGGAGGG GCTGCTGCAA GAGGCCGGGC 1260
CTTCGGAAGC ATGTTGTGTG AGGGATGCCA CAGAACCAGG CGCCCTGAGG ATGGAGACTG 1320
GTGACCCCAT CACAGTCATC GAGGGCAGCC CCGACTCCAC AATCTGGAAG GACCAGAATG 1380
GTCGCACCTT CAAAGTGGGC AGCTTCCCAG CCTCGGCAGT GACGCTGACA GATGCGGGGG 1440
GCTTGCCAGC CACCCGTCCA GTCCACAGAG GCACCCCTGC CCGGGGAGAT CAACACCCAG 1500
GAAGCATAGA TGGAGACAGA AAGAAGGCAA ATCTTTGGGA TGCGCCCCCA GCACGGGGCC 1560
AGAGGAGGAA CATGCCCCTG GAGAGGATGA AAGGCATTTC CAGGAGTCTG GAGTCAGTTC 1620
TGTCCCTCGG TCCTCGTCCC ACAGGGGGTG GTTCAAGCCC CCCTGAAATT CGACAAGCCA 1680
GAGCTGTGCC CCAGGGACCT CCAGGCCTGC CTCCACGCCC ACCTTTATCC TCTAGCTCTC 1740
CTCAGCCCAG CCAGCCCTCT AGGGAGAGGC TTCCCTGGCC CGAAAGAAAA CCCCCACACA 1800
ATCACCCCAT GGGAATGCCT GGAGCCCGTA AAGCCGCTGC CCTCTCTGGA GGCCTCTTGT 1860
CCGATCCTGA GTTGCAGAGG AAGATTATGG AAATGGAGCT GAGTGTGCAT TGGGTCACCC 1920
ACCAGGAGTG CCAGACAGCA CTAGGAGCCA CTGGGGGAGA TGTGGCTTCT GCCATCCGGA 1980
ACCTCAAGGT AGATCAGCTC TTCCTCCTGA GTAGCCGGTC CAGAGCTGAC TGCTGGCGCA 2040
TCCTGGAGCA TTACCAGTGG GACCTCTCAG CTGCCAGCCG TTATGTCCTG GCCAGGCCCT 2100
GAGCCCAGCT TCTGCGGGCA CAGACACCAG CATGAAAAGC CTAGGTCCCT GAGGGCCTGG 2160
CCACATGGGA CCAAGTGGAA CCAGAACAAG GCCCCGACAG GGGTAGACGT TCCACTTGTG 2220
GAGATCCCAC CTGCCGCTAG GCACGTGGAG GAGGAGCCCA GAGTTGGGCA CTGGCAAATG 2280 TCTCCTCCCT CCCATGCTCC TTGGCTTCTG AAGGCTGAAG CCCCTTTGGC TGGGCCAAGA 2340
AGGATCTAGT CTGCCCACTA CATTCTCAAA CAAGAGGACT TGGAGGAAAA GAGCTACTAT 2400
ACATCATATG CAGAGGAAGC TTCTACGCGC TAGAGAGGAT CAAGGGGCCA CACTGGACCA 2460
TGTGAACAGC CATCCGGAAC TGCCATCAGC TACCACACTG GACTCTGCAG GGCAGCCATC 2520
CTGGATGATG GAAGCCACCA TATTGACCTG GGGTATAGGC CCAAACTGCC TTCGTTTGGT 2580
CCAGGGCCAT CGTGGGTGAT GACGATTGCT CTCTTGCACT CATGGACATT TGATGCTGGT 2640
AGTATGGATT ATGAGATGGA CTAGCCCCTG CTCCAGCCCA GTTCTCACAT TCCCCTTTGT 2700
TTTTTCCCAT ACCAACTGCT TCTACCCTCC CCTATTACAT ACATCTTTCAATGTC CAAAA 2760
AAAAAAAAAA AAAA 2774
( 5 ) INFORMATION FOR SEQ ID NO : 4 :
( i ) SEQUENCE CHARACTERISTICS
(A) LENGTH: AMINO ACIDS
(B) TYPE: AMINO ACID
(C) STRANDEDNESS:
(D) TOPOLOGY: LINEAR 2
(ii) MOLECULE TYPE: PROTEIN
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Met Leu Pro Glu Ala Gly Ser Leu Trp Leu Leu Lys Leu Leu Arg
5 10 15
Asp lie Gin Leu Ala Gin Phe Tyr Trp Pro lie Leu Glu Glu Leu
20 25 30
Asn Val Thr Arg Pro Gly His Phe Asp Phe Val Lys Pro Glu Asp
35 40 45
Leu Asp Gly lie Gly Met Gly Arg Pro Ala Gin Arg Arg Leu Ser
50 55 60
Glu Ala Leu Lys Ser Leu Arg Ser Gly Pro Lys Ser Lys Asn Trp
65 70 75 Val Tyr Lys lie Leu Gly Gly Phe Ala Pro Glu His Lys Glu Pro
80 85 90
Thr Leu Pro Thr Asp Ser Pro Arg His Leu Pro Glu Pro Glu Gly
95 100 105
Gly Leu Lys Cyε Leu lie Pro Glu Gly Ala Val Cyε Arg Gly Glu
110 115 120
Leu Leu Gly Ser Gly Cys Phe Gly Val Val His Arg Gly Leu Trp
125 130 135
Thr Leu Pro Ser Gly Lys Ser Val Pro Val Ala Val Lys Ser Lys
140 145 150
Arg Val Gly Pro Glu Gly Pro Met Gly Thr Glu Lys Gly Asp Phe
155 160 165
Leu Arg Glu Val Ser Val Met Met Asn Leu Glu His Pro His Val
170 175 180
Leu Arg Leu His Gly Leu Val Leu Gly Gin Pro Leu Gin Met Val
185 190 195
Met Glu Leu Ala Pro Leu Gly Ser Leu His Ala Arg Leu Thr Ala
200 205 210
Pro Ala Pro Thr Pro Pro Leu Leu Val Ala Leu Leu Cyε Leu Phe
215 220 225
Leu Arg Gin Leu Ala Gly Ala Met Ala Tyr Leu Gly Ala Arg Gly
230 235 240
Leu Val Hiε Arg Aεp Leu Ala Thr Arg Aεn Leu Gin Leu Ala Ser
245 250 255 Pro Arg Thr lie Lys Val Ala Asp Phe Gly Leu Val Arg Pro Leu
260 265 270
Gly Gly Ala Arg Gly Arg Tyr Val Met Gly Gly Pro Arg Pro lie
275 280 285
Pro Tyr Thr Trp Cys Ala Pro Glu Ser Leu Arg His Gly Ala Phe
290 295 300
Ser Ser Ala Ser Asp Val Trp Met Phe Glu Val Thr Leu Trp Glu
305 310 315
Met Phe Ser Gly Gly Glu Glu Pro Trp Pro Gly Val Pro Pro Tyr
320 325 330
Leu lie Leu Gin Arg Leu Glu Asp Arg Ala Arg Leu Pro Arg Pro
335 340 345
Pro Pro Ser Ser Arg Ala Leu Tyr Ser Leu Ala Leu Arg Cys Trp
350 355 360
Ala Pro His Pro Ala Asp Arg Pro Ser Phe Ser His Leu Glu Gly
365 370 375
Leu Leu Gin Glu Ala Gly Pro Ser Glu Ala Cys Cys Val Arg Asp
380 385 390
Ala Thr Glu Pro Gly Ala Leu Arg Met Glu Thr Gly Aεp Pro lie
395 400 405
Thr Val lie Glu Gly Ser Pro Aεp Ser Thr lie Trp Lyε Aεp Gin
410 415 420
Asn Gly Arg Thr Phe Lys Val Gly Ser Phe Pro Ala Ser Ala Val
425 430 435 Thr Leu Thr Asp Ala Gly Gly Leu Pro Ala Thr Arg Pro Val His
440 445 450
Arg Gly Thr Pro Ala Arg Gly Asp Gin Hiε Pro Gly Ser lie Aεp
455 460 465
Gly Aεp Arg Lyε Lyε Ala Aεn Leu Trp Asp Ala Pro Pro Ala Arg
470 475 480
Gly Gin Arg Arg Asn Met Pro Leu Glu Arg Met Lys Gly lie Ser
485 490 495
Arg Ser Leu Glu Ser Val Lys Ser Leu Gly Pro Arg Pro Thr Gly
500 505 510
Gly Gly Ser Ser Pro Pro Glu lie Arg Gin Ala Arg Ala Val Pro
515 520 525
Gin Gly Pro Pro Gly Leu Pro Pro Arg Pro Pro Leu Ser Ser Ser
530 535 540
Ser Pro Gin Pro Ser Gin Pro Ser Arg Glu Arg Leu Pro Trp Pro
545 550 555
Glu Arg Lys Pro Pro His Asn His Pro Met Gly Met Pro Gly Ala
560 565 570
Arg Lys Ala Ala Ala Leu Ser Gly Gly Leu Leu Ser Asp Pro Glu
575 580 585
Leu Gin Arg Lys lie Met Glu Met Glu Leu Ser Val His Trp Val
590 595 600
Thr His Gin Glu Cys Gin Thr Ala Leu Gly Ala Thr Gly Gly Asp
605 610 615 Val Ala Ser Ala lie Arg Asn Leu Lys Val Asp Gin Leu Phe Leu
620 625 630
Leu Ser Ser Arg Ser Arg Ala Asp Cys Trp Arg lie Leu Glu His
635 640 645
Tyr Gin Trp Asp Leu Ser Ala Ala Ser Arg Tyr Val Leu Ala Arg
650 655 660
Pro 661
(2) INFORMATION FOR SEQ ID NO:5:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 27 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
CCCGCGGCCG CTCATTAGGA TTTGCGC 31
(2) INFORMATION FOR SEQ ID NO:6:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 24 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
CCCGTCGACC CATACTCCAA CATC 24
(2) INFORMATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 20 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
ATCCGAGGCA GACGAGAAGG 20
(2) INFORMATION FOR SEQ ID NO:8:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 18 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: AGACGAGAAG GCTCCGTG 18
(2) INFORMATION FOR SEQ ID NO:9:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 19 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: GATGGATCCT GCAGAAGCT 19
(2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 18 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: GATGGATCCT GCAGAAGC 18
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 18 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: ACCAGGTGTA GGGGATAG 18
(2) INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 20 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: CCATCAAGGT GGCTGACTTC 20
(2) INFORMATION FOR SEQ ID NO:13:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 18 BASE PAIRS (B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: CTGGTGTGCC CCAGAGAG 18
(2) INFORMATION FOR SEQ ID NO:14:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 34 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: GAGCGAATTC ACAGAACTGA CTCCAGACTC CTGG 34
(2) INFORMATION FOR SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 20 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: TGCCTCTGTG GACTGGACGG 20
(2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 25 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: GCACTTCGAC TTTGTAAAGC CTGAG 25 (2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 23 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: TTTTCAGAGC TTCGGACAGT CTG 23
(2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 22 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: CATGTTGTGT GAGGGATGCC AC 22
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS
(A) LENGTH: 20 BASE PAIRS
(B) TYPE: NUCLEIC ACID
(C) STRANDEDNESS: SINGLE
(D) TOPOLOGY: LINEAR
(ii) MOLECULE TYPE: Oligonucleotide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: TGAAGGTGCG ACCATTCTGG 20

Claims

WHAT IS CLAIMED IS:
1. An iεolated polynucleotide compriεing a polynucleotide having at leaεt a 95% identity to a member εelected from the group conεiεting of :
(a) a polynucleotide encoding a polypeptide compriεing amino acidε 2 to 666 of SEQ ID NO:2;
(b) a polynucleotide encoding a polypeptide compriεing amino acids 2 to 661 of SEQ ID N0:4; and
(c) the complement of (a) or (b) .
2. The iεolated polynucleotide of claim 1 wherein said member iε (a) .
3. The iεolated polynucleotide of claim 1 wherein εaid member is (b) .
4. The isolated polynucleotide of claim l wherein said member (a) and said polypeptide comprises amino acids l to 666 of SEQ ID NO:2.
5. The isolated polynucleotide of claim 1 wherein εaid member (b) and εaid polypeptide compriεeε amino acidε 1 to 661 Of SEQ ID NO:4.
6. The iεolated polynucleotide of claim 1, wherein the polynucleotide is DNA.
7. The iεolated polynucleotide of claim l, compriεing a
polynucleotide εelected from the group conεiεting of :
(a) a polynucleotide encoding a polypeptide compriεing an amino acid εequence identical to amino acidε 1 to 666 of SEQ ID NO:2; and (b) a polynucleotide encoding a polypeptide comprising an amino acid sequence identical to amino acids l to 661 of SEQ ID NO:4.
8. The isolated polynucleotide of claim 1, wherein said polynucleotide is RNA.
9. A method of making a recombinant vector comprising inserting the isolated polynucleotide of claim l into a vector, wherein said polynucleotide is DNA.
10. A recombinant vector comprising the polynucleotide of claim 1, wherein said polynucleotide iε DNA.
11. A recombinant host cell comprising the polynucleotide of claim 1, wherein said polynucleotide is DNA.
12. A method for producing a polypeptide comprising expressing from the recombinant cell of claim ll the polypeptide encoded by said polynucleotide.
13. The iεolated polynucleotide of claim 1 comprising a polynucleotide member selected from the group consisting
Of:
(a) a polynucleotide, which includes nucleotides 115 to 2115 Of SEQ ID N0:1; and
(b) a polynucleotide, which includes nucleotides 115 to 2100 Of SEQ ID NO:3.
14. The isolated polynucleotide of claim l comprising a polynucleotide member selected from the group consisting of:
(a) the polynucleotide of SEQ ID N0:l; and
(b) the polynucleotide of SEQ ID NO:3.
15. An isolated polynucleotide comprising a polynucleotide having at least a 95% identity to a polynucleotide encoding the same polypeptide encoded by the human cDNA in ATCC
Depoεit No. 69924; and
(b) the complement of (a) .
16. An iεolated polynucleotide comprising a polynucleotide identical to a polynucleotide encoding the same mature polypeptide encoded by the human cDNA in ATCC Deposit No. 69924.
17. The isolated polynucleotide of claim 15 wherein said polynucleotide compriseε DNA identical to the coding portion of the human cDNA in ATCC Depoεit No. 69924.
18. A method of making a recombinant vector compriεing inserting the isolated polynucleotide of claim 15 into a vector, wherein said polynucleotide is DNA.
19. A recombinant vector comprising the polynucleotide of claim 15, wherein said polynucleotide is DNA.
20. A recombinant host cell comprising the polynucleotide of claim 15, wherein εaid polynucleotide iε DNA.
21. A method for producing a polypeptide compriεing expreεsing from the recombinant cell of claim 20 the polypeptide encoded by said polynucleotide.
22. An isolated polypeptide comprising: a polypeptide having an amino acid sequence encoded by a polynucleotide which is at least 95% identical to a polynucleotide selected from the group conεisting of: (a) a polynucleotide encoding a polypeptide having an amino acid εequence comprising amino acidε 2 to 666 of SEQ ID No. 2; and
(b) a polynucleotide encoding a polypeptide having an amino acid sequence comprising amino acids 2 to 661 of SEQ ID No. 4.
23. An isolated polypeptide comprising: a polypeptide sequence encoded by a polynucleotide which is at least 95% identical to a polynucleotide encoding a polypeptide having the amino acid sequence encoded by the human cDNA contained in ATCC Deposit No. 69924.
24. The isolated polypeptide of claim 22, comprising the polypeptide sequence of SEQ ID NO:2.
25. The isolated polypeptide of claim 22, comprising the polypeptide sequence of SEQ ID NO:4.
26. The isolated polypeptide of claim 23 compriεing the mature polypeptide encoded by the human cDNA in ATCC Deposit No. 69924.
27. An isolated polypeptide produced from a host cell transformed with a polynucleotide, comprising a polynucleotide sequence which is at least 95% identical to a member selected from the group consiεting of:
(i) a polynucleotide encoding an amino acid sequence comprising amino acidε 2 to 666 of SEQ ID NO:2; and
(ii) a polynucleotide encoding an amino acid εequence comprising amino acids 2 to 661 of SEQ ID N0:4.
28. An isolated polypeptide according to claim 27, wherein the host cell is transformed with a polynucleotide having a polynucleotide sequence according to SEQ ID NO: 1.
29. An isolated polypeptide according to claim 27, wherein the host cell is transformed with a polynucleotide having a polynucleotide sequence according to SEQ ID NO: 3.
30. An isolated polypeptide according to claim 27, wherein the host cell is transformed with a polynucleotide which is identical to a polynucleotide encoding the polypeptide of SEQ ID NO:2.
31. An isolated polypeptide according to claim 27, wherein the host cell is transformed with a polynucleotide which iε identical to a polynucleotide encoding the polypeptide of SEQ ID NO:4.
32. An isolated polypeptide produced from a host cell transformed with a polynucleotide, comprising a polynucleotide sequence which is at least 95% identical to a polynucleotide encoding the same mature polypeptide encoded by the human cDNA of ATCC Depoεit No. 69924.
33. An iεolated polypeptide according to claim 32, wherein the hoεt cell iε tranεformed with a polynucleotide encoding a polypeptide identical to the polypeptide encoded by the human cDNA of ATCC Deposit No. 69924.
34. A method for the treatment of a patient having need of Tnkl comprising: administering to the patient a therapeutically effective amount of the polypeptide of claim 22.
35. The method of claim 34 wherein said therapeutically effective amount of the polypeptide iε adminiεtered by providing to the patient DNA encoding εaid polypeptide and expreεsing said polypeptide in vivo.
36. An antagoniεt against the polypeptide of claim 22.
37. A method for the treatment of a patient having a need to inhibit Tnkl comprising: administering to the patient a therapeutically effective amount of the antagonist of claim 36.
38. A process for diagnosing a disease or a susceptibility to a disease related to an under-expresεion of the polypeptide of claim 22 compriεing: determining a mutation in a nucleic acid εequence encoding said polypeptide.
39. A proceεs for inhibiting transformation of cells comprising adminiεtering the polynucleotide of claim l in a therapeutically effective amount to regulate the GTPaεe activity of a p21 raε-like protein.
40. A proceεs for regulating fetal or post-natal development comprising administering a therapeutically effective amount of the polynucleotide of claim 1 to a fetus or child or adult.
41. A process for stimulating survival, proliferation and/or differentiation of hematopoietic stem cells for transplantation compriεing adminiεtering a therapeutically effective amount of the polynucleotide of claim 1.
EP96936414A 1995-10-12 1996-10-11 Tyrosine kinase gene and gene product Withdrawn EP0862620A1 (en)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US528695P 1995-10-12 1995-10-12
US5286P 1995-10-12
PCT/US1996/016359 WO1997013846A1 (en) 1995-10-12 1996-10-11 Tyrosine kinase gene and gene product

Publications (1)

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WO (1) WO1997013846A1 (en)

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DE102004026045A1 (en) * 2004-05-25 2005-12-22 Universität Ulm Inhibitor of the nuclear transcription factor kappa B (NFxb)

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US5444149A (en) * 1992-05-11 1995-08-22 Duke University Methods and compositions useful in the recognition, binding and expression of ribonucleic acids involved in cell growth, neoplasia and immunoregulation

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Title
See references of WO9713846A1 *

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