US20030013848A1 - Protein-tyrosine kinase genes - Google Patents

Protein-tyrosine kinase genes Download PDF

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US20030013848A1
US20030013848A1 US09/158,722 US15872298A US2003013848A1 US 20030013848 A1 US20030013848 A1 US 20030013848A1 US 15872298 A US15872298 A US 15872298A US 2003013848 A1 US2003013848 A1 US 2003013848A1
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Greg E. Lemke
Cary H. C. Lai
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    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention relates generally to the molecular cloning of genes which encode unique protein-tyrosine kinase receptor subtypes which can be used in an assay to screen various compositions which modulate these receptors.
  • PTKs receptor protein-tyrosine kinases
  • Binding of a polypeptide ligand to its corresponding cell surface receptor results in rapid activation of that receptor's intracellular tyrosine kinase, which in turn results in the tyrosine phosphorylation of the receptor itself and of multiple downstream target proteins (Hunter and Cooper, Annu. Rev. Biochem., 54:897-930, 1985; Hunter, et al, eds. J. B. Hook and G. Poste, Plenum Press, New York and London, pp. 119-139, 1990). For many receptor PTKs, growth factor binding ultimately triggers multiple rounds of cell division.
  • PDGF Platelet-derived growth factor
  • bFGF acidic and basic fibroblast growth factor
  • novel receptor protein tyrosine kinase (PTK) subtype polypeptides have been isolated. These PTKs possess a tyrosine kinase domain and a unique tissue expression pattern different from all previously known receptor PTKs. These novel receptor PTK subtypes have been designated tyro-1 through tyro-8 and tyro-10 through tyro-12. Of particular interest among the new PTK subtypes are tyro-1 through tyro-6 which are found predominantly or exclusively in neural tissue.
  • the invention provides a method for identifying compositions which potentially affect the activity of the receptor PTK subtype. This method comprises (a) contacting cells containing DNA which expresses the PTK polypeptide with the composition under conditions suitable for cell culture; and (b) monitoring the cells for a physiological change resulting from this interaction.
  • the present invention provides unique oligonucleotide which align with the unique flanking regions of the receptor PTK subtypes, thereby allowing amplification of the polynucleotides encoding the receptor PTK subtype by such techniques as polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the present invention also provides a method of gene therapy comprising introducing into a host subject an expression vector comprising a nucleotide sequence encoding a receptor PTK subtype capable of affecting a biological activity of the host subject cell.
  • FIGS. 1A and 1B show the tissue expression profiles of the novel PTK mRNAs.
  • FIG. 2 shows the developmental tissue profiles of the novel PTK mRNAs which were predominantly or exclusively neural in their distribution.
  • the present invention relates to novel protein tyrosine kinase (PTK) gene and polypeptides encoded by these genes.
  • PTK protein tyrosine kinase
  • Various of these PTK subtypes are implicated in neural development where they function primarily as signal transduction molecules.
  • the receptor PTKs of the invention are characterized as having a tyrosine kinase domain and a unique tissue expression pattern which differs from that of all known receptor PTKs.
  • the invention provides polynucleotides, such as DNA, cDNA, and RNA, encoding novel receptor PTK polypeptides. It is understood that all polynucleotides encoding all or a portion of the receptor PTKs of the invention are also included herein, so long as they exhibit at least one protein tyrosine kinase domain and the tissue expression pattern characteristic of a given subtype. Such polynucleotides include both naturally occurring and intentionally manipulated, for example, mutagenized polynucleotides.
  • DNA sequences of the invention can be obtained by several methods.
  • the DNA can be isolated using hybridization procedures which are well known in the art. These include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect shared nucleotide sequences and 2) antibody screening of expression libraries to detect shared structural features.
  • Hybridization procedures are useful for the screening of recombinant clones by using labeled mixed synthetic oligonucleotide probes where each probe is potentially the complete complement of a specific DNA sequence in the hybridization sample which includes a heterogeneous mixture of denatured double-stranded DNA.
  • hybridization is preferably performed on either single-stranded DNA or denatured double-stranded DNA.
  • Hybridization is particularly useful in the detection of cDNA clones derived from sources where an extremely low amount of mRNA sequences relating to the polypeptide of interest are present.
  • a receptor PTK cDNA library can be screened by injecting the various cDNAs into oocytes, allowing sufficient time for expression of the cDNA gene products to occur, and testing for the presence of the desired cDNA expression product, for example, by using antibody specific for the receptor PTK subtype polypeptide or by using functional assays for receptor PTK subtype activity and a tissue expression pattern characteristic of the desired subtype.
  • a cDNA library can be screened indirectly for receptor PTK polypeptides having at least one epitope using antibodies specific for receptor PTK subtypes of the invention.
  • Such antibodies can be either polyclonally or monoclonally derived and used to detect expression product indicative of the presence of protein tyrosine kinase receptor PTK subtype cDNA.
  • DNA sequences encoding receptor PTK subtypes of the invention can also be obtained by: (1) isolation of a double-stranded DNA sequence from the genomic DNA; (2) chemical manufacture of a DNA sequence to provide the necessary codons for the polypeptide of interest; and (3) in vitro synthesis of a double-stranded DNA sequence by reverse transcription of mRNA isolated from a eukaryotic donor cell. In the latter case, a double-stranded DNA complement of mRNA is eventually formed which is generally referred to as cDNA.
  • genomic DNA sequences which encode allelic variant forms.
  • DNA sequences which are degenerate as a result of the genetic code are also be obtained by: (1) isolation of a double-stranded DNA sequence from the genomic DNA; (2) chemical manufacture of a DNA sequence to provide the necessary codons for the polypeptide of interest; and (3) in vitro synthesis of a double-stranded DNA sequence by reverse transcription of mRNA isolated from a eukaryotic donor cell. In the latter case, a double-strande
  • genomic DNA isolates (1) are the least common. This is especially true when it is desirable to obtain the microbial expression of mammalian polypeptides because of the presence of introns.
  • the production of labeled single or double-stranded DNA or RNA probe sequences duplicating a sequence putatively present in the target cDNA may be employed in DNA/DNA hybridization procedures which are carried out on cloned copies of the cDNA which have been denatured into a single-stranded form (Jay, et al., Nucleic Acid Research, 11:2325, 1983).
  • novel DNA sequences of the invention encode essentially all or part of a receptor PTK, it is now a routine matter to prepare, subclone, and express smaller polypeptide fragments of DNA from this or corresponding DNA sequences.
  • DNA fragments disclosed herein which define the unique tyrosine kinase receptor subtype of the invention it is possible, in conjunction with known techniques, to determine the DNA sequences encoding the entire receptor subtypes. Such techniques are described in U.S. Pat. Nos. 4,394,443 and 4,446,235 which are incorporated herein by reference.
  • polypeptide resulting from expression of a DNA sequence of the invention can be further characterized as being free from association with other eukaryotic polypeptides or other contaminants which might otherwise be associated with the protein kinase in its natural cellular environment.
  • Isolation and purification of microbially expressed polypeptides may be by conventional means including, preparative chromatographic separations and immunological separations involving monoclonal and/or polyclonal antibody preparation.
  • receptor PTK subtypes which are homologous to those of the invention can be identified by structural as well as functional similarity.
  • Structural similarity can be determined, for example, by assessing polynucleotide strand hybridization or by screening with antibody, especially a monoclonal antibody, which recognizes a unique epitope present on the subtypes of the invention.
  • hybridization is used as criteria to establish structural similarity, those polynucleotide sequences which hybridize under stringent conditions to the polynucleotides of the invention are considered to be essentially the same as the polynucleotide sequences of the invention.
  • polynucleotide sequences encoding receptor PTK subtype may be introduced into a host cell by means of a recombinant expression vector.
  • recombinant expression vector refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of the polynucleotide sequences of the invention.
  • Such expression vectors typically contain a promotor sequence which facilitates efficient transcription of the inserted sequence in the host.
  • the expression vector also typically contains specific genes which allow phenotypic selection of the transformed cells.
  • nucleotide sequences encoding a receptor PTK subtype can be introduced directly in the form of free nucleotide, for example, by microinjection, or transfection.
  • DNA sequences encoding receptor PTK subtypes of the invention can be expressed in vivo by DNA transfer into a suitable host cell.
  • “Recombinant host cells” or “host cells” are cells in which a vector can be propagated and its DNA expressed. The term includes not only prokaryotes, but also such eukaryotes as yeast, filamentous fungi, as well as animal cells which can replicate and express an intron-free DNA sequence of the invention and any progeny of the subject host cell. It is understood that not all progeny are identical to the parental cell since there may be mutations that occur at replication. However, such progeny are included when the terms above are used.
  • DNA sequences having eukaryotic coding sequences in prokaryotes are well known in the art.
  • Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art. Such vectors are used to incorporate DNA sequences of the invention.
  • Hosts include microbial, yeast and mammalian organisms.
  • Transformation of a host cell with recombinant DNA may be carried out by conventional techniques which are well known to those skilled in the art.
  • the host is prokaryotic, such as E. coli
  • competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl 2 method well known in the art.
  • CaCl 2 method well known in the art.
  • MgCl 2 or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell.
  • Eukaryotic cells can also be cotransformed with foreign cDNA encoding the desired receptor PTK subtype protein, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene.
  • Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein.
  • a eukaryotic viral vector such as simian virus 40 (SV40) or bovine papilloma virus
  • SV40 simian virus 40
  • bovine papilloma virus bovine papilloma virus
  • the cDNA can be expressed by inserting the cDNA into appropriate expression vectors and introducing the product into the host cells.
  • Various shuttle vectors for the expression of foreign genes in yeast have been reported (Heinemann, et al., Nature, 340:205, 1989; Rose, et al., Gene, 60:237, 1987).
  • Isolation and purification of microbially expressed protein, or fragments thereof provided by the invention may be carried out by conventional means including preparative chromatography and immunological separations involving monoclonal or polyclonal antibodies.
  • Antibodies provided in the present invention are immunoreactive with the receptor PTK subtypes of the invention.
  • Antibody which consists essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are provided.
  • Monoclonal antibodies are made from antigen containing fragments of the protein by methods well known in the art (Kohler, et al., Nature, 256:495, 1975; Current Protocols in Molecular Biology, Ausubel, et al., ed., 1989).
  • the invention also discloses a method for identifying a composition which affects the activity of a receptor PTK subtype of the invention.
  • the receptor is, for example, capable of affecting cell division and/or differentiation.
  • the composition is incubated in combination with cells under conditions suitable for cell culture, then subsequently monitoring the cells for a physiologic change.
  • oligonucleotide(s) which are primers for amplification of the genomic polynucleotide encoding PTK receptor. These unique oligonucleotide primers were produced based upon identification of the flanking regions contiguous with the polynucleotide encoding the receptor PTK. These oligonucleotide primers comprise sequences which are capable of hybridizing with the flanking nucleotide sequence encoding a polypeptide having amino acid residues HRDLAAR and/or DVWS(F/Y)G(V/I) and sequences complementary thereto.
  • the primers of the invention embrace oligonucleotides of sufficient length and appropriate sequence so as to provide specific initiation of polymerization on a significant number of nucleic acids in the polynucleotide encoding the receptor PTK subtype.
  • the term “primer” as used herein refers to a sequence comprising two or more deoxyribonucleotides or ribonucleotides, preferably more than three, which sequence is capable of initiating synthesis of a primer extension product, which is substantially complementary to a receptor PTK strand.
  • Environmental conditions conducive to synthesis include the presence of nucleoside triphosphates and an agent for polymerization, such as DNA polymerase, and a suitable temperature and pH.
  • the primer is preferably single stranded for maximum efficiency in amplification, but may be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products.
  • the primer is an oligodeoxyribonucleotide.
  • the primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent for polymerization. The exact length of primer will depend on many factors, including temperature, buffer, and nucleotide composition.
  • the oligonucleotide primer typically contains 15-22 or more nucleotides, although it may contain fewer nucleotides.
  • Primers of the invention are designed to be “substantially” complementary to each strand of polynucleotide encoding the receptor PTK to be amplified. This means that the primers must be sufficiently complementary to hybridize with their respective strands under conditions which allow the agent for polymerization to perform. In other words, the primers should have sufficient complementarity with the flanking sequences to hybridize therewith and permit amplification of the polynucleotide encoding the receptor PTK. Preferably, the primers have exact complementarity with the flanking sequence strand.
  • Oligonucleotide primers of the invention are employed in the amplification process which is an enzymatic chain reaction that produces exponential quantities of polynucleotide encoding the receptor PTK relative to the number of reaction steps involved.
  • one primer is complementary to the negative ( ⁇ ) strand of the polynucleotide encoding the receptor PTK and the other is complementary to the positive (+) strand.
  • the oligonucleotide primers of the invention may be prepared using any suitable method, such as conventional phosphotriester and phosphodiester methods or automated embodiments thereof.
  • diethylphos-phoramidites are used as starting materials and may be synthesized as described by Beaucage, et al. ( Tetrahedron Letters, 22:1859-1862, 1981).
  • Beaucage, et al. Tetrahedron Letters, 22:1859-1862, 1981.
  • One method for synthesizing oligonucleotides on a modified solid support is described in U.S. Pat. No. 4,458,066.
  • any nucleic acid specimen, in purified or nonpurified form, can be utilized as the starting nucleic acid or acids, provided it contains, or is suspected of containing, the specific nucleic acid sequence containing a protein receptor PTK of the invention.
  • the process may employ, for example, DNA or RNA, including messenger RNA, wherein DNA or RNA may be single stranded or double stranded.
  • DNA or RNA may be single stranded or double stranded.
  • enzymes, and/or conditions optimal for reverse transcribing the template to DNA would be utilized.
  • a DNA-RNA hybrid which contains one strand of each may be utilized.
  • a mixture of nucleic acids may also be employed, or the nucleic acids produced in a previous amplification reaction herein, using the same or different primers may be so utilized.
  • the specific nucleic acid sequence to be amplified i.e., the receptor PTK, may be a fraction of a larger molecule or can be present initially as a discrete molecule, so that the specific sequence constitutes the entire nucleic acid. It is not necessary that the sequence to be amplified be present initially in a pure form; it may be a minor fraction of a complex mixture, such as contained in whole human DNA.
  • DNA or RNA utilized herein may be extracted from a body sample, such as brain, or various other tissue, by a variety of techniques such as that described by Maniatis, et al. ( Molecular Cloning, 280:281, 1982). If the extracted sample is impure (such as plasma, serum, or blood), it may be treated before amplification with an amount of a reagent effective to open the cells, fluids, tissues, or animal cell membranes of the sample, and to expose and/or separate the strand(s) of the nucleic acid(s). This lysing and nucleic acid denaturing step to expose and separate the strands will allow amplification to occur much more readily.
  • Strand separation can be effected either as a separate step or simultaneously with the synthesis of the primer extension products. This strand separation can be accomplished using various suitable denaturing conditions, including physical, chemical, or enzymatic means, the word “denaturing” includes all such means.
  • One physical method of separating nucleic acid strands involves heating the nucleic acid until it is denatured. Typical heat denaturation may involve temperatures ranging from about 80° to 105° C. for times ranging from about 1 to 10 minutes.
  • Strand separation may also be induced by an enzyme from the class of enzymes known as helicases or by the enzyme RecA, which has helicase activity, and in the presence of riboATP, is known to denature DNA.
  • an enzyme from the class of enzymes known as helicases or by the enzyme RecA which has helicase activity, and in the presence of riboATP, is known to denature DNA.
  • the reaction conditions suitable for strand separation of nucleic acids. with helicases are described by Kuhn Hoffmann-Berling ( CSH-Quantitative Biology, 43:63, 1978) and techniques for using RecA are reviewed in C. Radding ( Ann. Rev. Genetics, 16:405-437, 1982).
  • nucleic acid containing the sequence to be amplified is single stranded
  • its complement is synthesized by adding one or two oligonucleotide primers. If a single primer is utilized, a primer extension product is synthesized in the presence of primer, an agent for polymerization, and the four nucleoside triphosphates described below. The product will be partially complementary to the single-stranded nucleic acid and will hybridize with a single-stranded nucleic acid to form a duplex of unequal length strands that may then be separated into single strands to produce two single separated complementary strands. Alternatively, two primers may be added to the single-stranded nucleic acid and the reaction carried out as described.
  • the separated strands are ready to be used as a template for the synthesis of additional nucleic acid strands.
  • This synthesis is performed under conditions allowing hybridization of primers to templates to occur. Generally synthesis occurs in a buffered aqueous solution, preferably at a pH of 7-9, most preferably about 8.
  • a molar excess for genomic nucleic acid, usually about 10 8 :1 primer:template
  • a molar excess for genomic nucleic acid, usually about 10 8 :1 primer:template
  • the amount of complementary strand may not be known if the process of the invention is used for diagnostic applications, so that the amount of primer relative to the amount of complementary strand cannot be determined with certainty.
  • the amount of primer added will generally be in molar excess over the amount of complementary strand (template) when the sequence to be amplified is contained in a mixture of complicated long-chain nucleic acid strands. A large molar excess is preferred to improve the efficiency of the process.
  • the deoxyribonucleotide triphosphates dATP, dCTP, dGTP, and dTTP are added to the synthesis mixture, either separately or together with the primers, in adequate amounts and the resulting solution is heated to about 90°-100° C. from about 1 to 10 minutes, preferably from 1 to 4 minutes. After this heating period, the solution is allowed to cool to room temperature, which is preferable for the primer hybridization. To the cooled mixture is added an appropriate agent for effecting the primer extension reaction (called herein “agent for polymerization”), and the reaction is allowed to occur under conditions known in the art. The agent for polymerization may also be added together with the other reagents if it is heat stable. This synthesis (or amplification) reaction may occur at room temperature up to a temperature above which the agent for polymerization no longer functions.
  • the temperature is generally no greater than about 40 ° C. Most conveniently the reaction occurs at room temperature.
  • the agent for polymerization may be any compound or system which will function to accomplish the synthesis of primer extension products, including enzymes.
  • Suitable enzymes for this purpose include, for example, E. coli DNA polymerase I, Klenow fragment of E coli DNA polymerase I, T4 DNA polymerase, other available DNA polymerases, polymerase muteins, reverse transcriptase, and other enzymes, including heat-stable enzymes (i.e., those enzymes which perform primer extension after being subjected to temperatures sufficiently elevated to cause denaturation).
  • Suitable enzymes will facilitate combination of the nucleotides in the proper manner to form the primer extension products which are complementary to each receptor PTK nucleic acid strand.
  • the synthesis will be initiated at the 3′ end of each primer and proceed in the 5′ direction along the template strand, until synthesis terminates, producing molecules of different lengths.
  • the newly synthesized receptor PTK strand and its complementary nucleic acid strand will form a double-stranded molecule under hybridizing conditions described above and this hybrid is used in subsequent steps of the process.
  • the newly synthesized double-stranded molecule is subjected to denaturing conditions using any of the procedures described above to provide single-stranded molecules.
  • the steps of denaturing and extension product synthesis can be repeated as often as needed to amplify the receptor PTK nucleic acid sequence to the extent necessary for detection.
  • the amount of the specific nucleic acid sequence produced will accumulate in an exponential fashion.
  • Sequences amplified by the methods of the invention can be further evaluated, detected, cloned, sequenced, and the like, either in solution or after binding to a solid support, by any method usually applied to the detection of a specific DNA sequence such as PCR, oligomer restriction (Saiki, et al., Bio/Technology, 3:1008-1012, 1985), allele-specific oligonucleotide (ASO) probe analysis (Conner, et al., Proc. Natl. Acad. Sci. USA, 80:278, 1983), oligonucleotide ligation assays (OLAs) (Landegren, et al., Science, 241:1077, 1988), and the like. Molecular techniques for DNA analysis have been reviewed (Landegren, et al., Science, 242:229-237, 1988).
  • the present invention also provides methods for the treatment of disease employing gene therapy that modulates cellular differentiation or maturation.
  • Such therapy can be affected by introduction of polynucleotide sequences of the invention into cells of a subject having a disease. Delivery of polynucleotide can be achieved using techniques well known in the art. For example, a recombinant expression vector, such as a chimeric virus, or a colloidal dispersion system can be employed.
  • RNA virus such as a retrovirus
  • retroviral vector is a derivative of a murine or avian retrovirus.
  • retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV).
  • MoMuLV Moloney murine leukemia virus
  • HaMuSV Harvey murine sarcoma virus
  • MuMTV murine mammary tumor virus
  • RSV Rous Sarcoma Virus
  • a number of additional retroviral vectors can incorporate multiple genes. All of these vectors can incorporate a gene for a selectable marker so that transduced cells can be identified and generated.
  • Retroviral vectors can be made target specific by including in the retroviral vector a polynucleotide encoding a target related binding substance. Preferred targeting is accomplished by using an antibody to target the retroviral vector.
  • helper cell lines that contain plasmids encoding all of the structural genes of the retrovirus under the control of regulatory sequences within the LTR. These plasmids are missing a nucleotide sequence which enables the packaging mechanism to recognize an RNA transcript for encapsidation.
  • Helper cell lines which have deletions of the packaging signal include, but are not limited to, ⁇ 2, PA317 and PA12, for example. These cell lines produce empty virions, since no genome is packaged.
  • the vector can be packaged and vector virion produced.
  • the vector virions produced by this method can then be used to infect a tissue cell line, such as NIH 3T3 cells, to produce large quantities of chimeric retroviral virions.
  • NIH 3T3 or other tissue culture cells can be directly transfected with plasmids encoding the retroviral structural genes gag, pol and env, by conventional calcium phosphate transfection. These cells are then transfected with the vector plasmid containing the genes of interest. The resulting cells release the retroviral vector into the culture medium.
  • colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.
  • the preferred colloidal system of this invention is a liposome.
  • the receptor PTK polypeptide may be indiscriminate in its action with respect to cell type, a targeted delivery system offers a significant improvement over randomly injected non-specific liposomes.
  • a number of procedures can be used to covalently attach either polyclonal or monoclonal antibodies to a liposome bilayer.
  • Antibody-targeted liposomes can include monoclonal or polyclonal antibodies or fragments thereof such as Fab, or F(ab′) 2 , as long as they bind efficiently to an epitope on the target cells. Liposomes may also be targeted to cells expressing receptors for hormones or other serum factors.
  • Liposomes are artificial membrane vesicles which are useful as in vitro and in vivo delivery vehicles. It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 ⁇ m can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules. RNA, DNA, intact virions and peptides can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al., Trends Biochem. Sci., 6:77, 1981).
  • LUV large unilamellar vesicles
  • a liposome In order for a liposome to be an efficient transfer vehicle, the following characteristics should be present: (1) encapsulation of polynucleotides of interest at high efficiency without compromising biological activity; (2) preferential and substantial binding to target cells relative to non-target cells; (3) delivery of aqueous contents of vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino, et al., Biotechniques, 6:682, 1988).
  • the surface of the targeted delivery system may be modified in a variety of ways.
  • lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting receptor in stable association with the liposomal bilayer.
  • Various linking groups can be used for joining the lipid chains to the targeting receptor.
  • the targeted delivery system will be directed to cell surface receptors thereby allowing the delivery system to find and “home in” on the desired cells.
  • the delivery system can be directed to any cell surface molecule preferentially found in the cell population for which treatment is desired and capable of association with the delivery system.
  • Antibodies can be used to target liposomes to specific cell-surface molecules. For example, where a tumor is associated with a receptor PTK of the invention, certain antigens expressed specifically or predominantly on the cells of the tumor may be exploited for the purpose of targeting antibody tyrosine kinase receptor DNA-containing liposomes directly to a malignant tumor, if desired.
  • An alternative use for recombinant retroviral vectors comprises the introduction of polynucleotide sequences into the host by means of skin transplants of cells containing the virus.
  • Long term expression of foreign genes in implants, using cells of fibroblast origin, may be achieved if a strong housekeeping gene promoter is used to drive transcription.
  • a strong housekeeping gene promoter is used to drive transcription.
  • the dihydrofolate reductase (DHFR) gene promoter may be used.
  • Cells such as fibroblasts can be infected with virions containing a retroviral construct containing the receptor PTK gene of interest together with a gene which allows for specific targeting, such as a tumor-associated antigen and a strong promoter.
  • the infected cells can be embedded in a collagen matrix which can be grafted into the connective tissue of the dermis in the recipient subject. As the retrovirus proliferates and escapes the matrix it will specifically infect the target cell population. In this way the transplantation results in increased amounts of receptor PTK being produced in cells manifesting the disease.
  • the present invention identifies nucleotide sequences encoding novel receptor PTKs, it is possible to design therapeutic or diagnostic protocols utilizing these sequences.
  • the polynucleotide sequence encoding the PTK can be utilized to design sequences which interfere with the function of the receptor.
  • This approach utilizes, for example, antisense nucleic acid and ribozymes to block translation of specific receptor mRNA, either by masking the mRNA with antisense nucleic acid or by cleaving it with ribozyme.
  • Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (Weintraub, Scientific American, 262:40, 1990). In the cell, the antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule. The antisense nucleic acids interfere with the translation of the mRNA since the cell will not translate a mRNA that is double-stranded. Antisense oligomers of about 15 nucleotides are preferred, since they are easily synthesized and are less likely to cause problems than larger molecules when introduced into the target receptor-producing cell. The use of antisense methods to inhibit the in vitro translation of genes is well known in the art (Marcus-Sakura, Anal.Biochem., 172:289, 1988).
  • Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA in a manner analogous to DNA restriction endonucleases. Through the modification of nucleotide sequences which encode these RNAs, it is possible to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, J. Amer. Med. Assn., 260:3030, 1988). A major advantage of this approach is that, because they are sequence-specific, only mRNAs with particular sequences are inactivated.
  • ribozymes There are two basic types of ribozymes namely, tetrahymena-type (Hasselhoff, Nature, 334:585, 1988) and “hammerhead”-type. Tetrahymena-type ribozymes recognize sequences which are four bases in length, while “hammerhead”-type ribozymes recognize base sequences 11-18 bases in length. The longer the recognition sequence, the greater the likelihood that the sequence will occur exclusively in the target mRNA species. Consequently, hammerhead-type ribozymes are preferable to tetrahymena-type ribozymes for inactivating a specific mRNA species and 18-based recognition sequences are preferable to shorter recognition sequences.
  • Antisense sequences can be therapeutically administered by techniques as described above for the administration of receptor PTK polynucleotides.
  • Targeted liposomes are especially preferred for therapeutic delivery of antisense sequences.
  • PCR was used to amplify PTK-related sequences located between the degenerate oligonucleotide primer sequences shown in TABLE 1. These primers correspond to the amino acid sequences HRDLAAR (SEQ ID NO:27) (upstream) and DVWS(F/Y)G(I/V) (SEQ ID NO:28) (downstream), which flank a highly conserved region of the kinase domain shared by receptor PTKs (Hanks, et al., Science, 241:42-52, 1988).
  • the upstream primer was chosen to exclude members of the src family of cytoplasmic tyrosine kinases.
  • the downstream primer was chosen such that a second highly conserved amino acid sequence diagnostic or PTKs—P(I/V)(K/R)W(T/M)APE (SEQ ID NO:29)—would be contained within amplified PCR products.
  • the DNA substrates used for amplification were sciatic nerve cDNA populations prepared for use in the construction of subtracted cDNA libraries. Three different subtracted cDNAs were produced. The first two, UN and TWI, were enriched for transcripts expressed predominantly in Schwann cells. The third, BD, was enriched for transcripts shared between Schwann cells and myelinating stage (P17-23) brain. Two initial hybridizations were performed. Both samples contained 500 ng of single-stranded sciatic nerve cDNA mixed with the following poly(A)-selected RNAs: 10 ⁇ g of muscle, 7.5 ⁇ g of liver, and 5 ⁇ g of kidney.
  • Both samples also contained a series of RNAs synthesized in vitro; these encoded portions of the sense strand of the following Schwann cell transcripts: NGF receptor, glial fibrillary acidic protein, proteolipid protein, protein zero, myelin basic protein, and CNPase.
  • the first sample contained, in addition, 10 ⁇ g of poly(A)-selected RNA from rat brain cerebellum (P19) and cortex (P3). Each hybridization was allowed to proceed to approximately R 0 t 2000. Following hybridization, these samples were bound to hydroxylapatite (0.12M phosphate buffer, 65° C.). For the first sample, material not binding to hydroxylapatite (HAP) was collected and converted to a double-stranded form.
  • HAP hydroxylapatite
  • Amplified DNAs were size fractionated on 5% non-denaturing acrylamide gels. The gels were stained with ethidium bromide (1 ⁇ g/ml) and amplified bands of ⁇ 220 bp were excised. These bands were eluted overnight into 0.5 M ammonium acetate. 1 mM EDTA, 0.2% SDS, and eluted DNA was then precipitated with 10 ⁇ g of tRNA carrier. Recovered PCR products were blunt-ended using T4 DNA polymerase, and phosphorylated using T4 polynucleotide kinase. Approximately 40 ng of insert was then ligated with 200 ng of dephosphorylated SmaI/EcoRV-digested pBluescript plasmid. One-tenth of each ligation was used to transform MC1061 bacteria.
  • EGF-R human; Ullrich, et al., Nature 309, 418-425, 1984
  • PDGF-A human; Matsui, et al., Science, 243:800-804, 1989; rat; Lee, et al., Science, 245:57-60, 1989; Reid, et al., Proc. Natl. Acad. Sci.
  • CSF-1 colony-stimulating factor 1
  • IGF-1 insulin-like growth factor 1
  • the kinase domain sequences of tyro-1 through tyro-13 have been grouped by similarity to the equivalent sequences of other PTKs (TABLE 2).
  • the indicated subfamilies were defined with reference to a computer-generated phylogenetic tree, constructed from an analysis of 13 novel partial PTK sequences along with a set of 55 additional PTKs, according to the methods of Fitch and Margoliash ( Science, 15:279-284, 1967) as implemented by the programs of Feng and Doolittle ( J. Mol. Evol., 25:351-360, 1987).
  • the resulting closely related sequence clusters were used to organize the kinase subfamilies presented in TABLE 2.
  • Tyro-1 and tyro-4 are related to the epithelial cell kinase (eck) (Lindberg and Hunter, Mol. Cell. Biol., in press, 1990), tyro-2 to the EGF receptor and the neu proto-oncogene (Bargmann, et et., Nature, 319:226-230, 1986), tyro-5, tyro-6, and tyro-11 to the elk kinase (Letwin, et al., Oncogene, 3:621-627, 1988), tyro-9 to the bFGF receptor, and tyro-10 to trk and trkB (Martin-Zanca, et al., Nature, 319:743-748, 1986; Klein, et al., EMBO J., 8:3701-3709, 1989).
  • eck epithelial cell kinase
  • tyro-2 to the EGF
  • tyro-3, tyro-7, and tyro-12 are listed as a novel subfamily since they are more closely related to each other than to any previously described kinase.
  • the eck- and elk-related sequences are listed in separate subsets, but it is important to note the high degree of similarity between these subfamilies.
  • the sequences of fes, trk, trkB, and Dsrc28 (each marked with an asterisk) are included in TABLE 2 only for comparison, since they were not encountered in these cloning studios.
  • the expression pattern of the 13 novel kinase clones were characterized by first examining the relative levels of mRNA present in a variety of neonatal and adult rat tissues. Radiolabeled cDNA probes for each of these clones, as well as probes prepared from isolates of the bFGF receptor, bek, and elk kinases, were hybridized to a set of eight parallel Northern blots containing RNA isolated from kidney, liver, spleen, heart, skeletal muscle, brain, sciatic nerve, and cultured Schwann cells.
  • RNA from various tissues was prepared by the method of Chomczynski and Sacchi ( Anal. Biochem., 162:156-159, 1987). One additional phenol-chloroform extraction was performed prior to nucleic acid precipitation. Poly(A)-selected RNA samples were purified by either column chromatography or in batch using oligo(dT)-cellulose type III (Collaborative Research). RNA samples were denatured in 50% formamide, 2.2M formaldehyde, and MOPS at 65° C. for 10 min, electrophoresed in 1.0% agarose, 2.2M formaldehyde, and MOPS, transferred to Nytran filters (Schleicher & Schuell) and baked at 80° C.
  • Probes for blot hybridizations were prepared using [ ⁇ - 32 P] dCTP and a random hexamer priming kit, according to instructions provided by the manufacturer (Bethesda Research Laboratories). In all cases, final wash stringency for Northern blots was set at 0.2 ⁇ SSC, 0.2% SDS, 65 ° C.
  • FIG. 1 Poly(A) (left 10 lanes) or total RNA (tot, right 4 lanes) from the indicated rat tissues was analyzed for expression of PTK mRNAs. All tissues were taken from animals 27 days postnatal, except where otherwise indicated. Sciatic nerves (sciatic) were obtained from 7-to-8-day-old rats. Rat Schwann cells were cultured in either the presence (+) or absence ( ⁇ ) of 20 ⁇ M forskolin for 48 hr prior to harvesting. All lanes contain either 2.5 ⁇ g of poly(A) + RNA or 10 ⁇ g of total RNA, except for the cultured Schwann cell poly(A) + lanes, which contain 1.0 ⁇ g each.
  • Filters 1-13 show hybridization with 32 P radiolabeled cDNA probes to tyro-1 through tyro-13. Also shown for comparison is the hybridization observed using isolates of elk, the bFGF receptor (FGFR), and the bek FGF receptor. Exposure times were as follows: 34 hr (1, 5, 6, 7, 11), 41 hr (3, 4, FGFR), 120 hr (2, 9, 10, bek), 158 hr (8, 13, elk), 8 days (12).
  • FIG. 1 The results of this analysis (FIG. 1) demonstrate that 6 of the 11 novel kinase genes (tyro-1 through tyro-6), together with the elk gene, are preferentially expressed by cells of the nervous system.
  • tyro-1 a novel member of the eck kinase subfamily
  • Tyro-4 also a novel member of the eck subfamily, exhibited more modest hybridization to two mRNAs in postnatal day 5(P5) brain, with lower signals evident in older brains as well as kidney and heart.
  • the novel EGF receptor homolog tyro-2 identified a high molecular weight mRNA in brain that could also be detected in kidney and heart. It is possible that the very low tyro-1, tyro-2, and tyro-4 hybridization signals observed in kidney and heart are due to neural contamination from the adrenal gland and cardiac ganglia, respectively. Tyro-3, a member of the novel kinase subfamily with similarity to the insulin receptor, showed intense hybridization to brain mRNA, with very faint signals in perhaps all other tissues.
  • kinase subfamily Members of the same receptor-configured kinase subfamily occasionally exhibited very different patterns of expression.
  • elk subfamily for example, elk itself and the related kinases tyro-5 and tyro-6 were exclusively or predominantly expressed in neural tissues, elk strongly hybridized to two mRNA species in brain and Schwann cells, tyro-5 exhibited strong hybridization to P5 brain mRNA with reduced signals present in later stage brains and in Schwann cells, and tyro-6 gave a strong hybridization signal in cultured Schwann cells, weaker signals in brain, and very faint but detectable signals in other tissues.
  • tyro-9 a member of the FGF receptor subfamily, exhibited a pattern of expression that was distinct from that of either the bFGF receptor or bek. Most strongly expressed in kidney and liver, it exhibited only weak hybridization signals with brain mRNA. At two extremes of expression, tyro-12 yielded weak hybridization signals in all tissues, with expression being somewhat lower in heart and muscle, but tyro-8 (distantly related to Dsrc28) yielded only an extremely faint signal in spleen and heart.
  • cAMP induction of the PDGF receptor appears to account for the synergistic effect on Schwann cell proliferation achieved with combined application of PDGF and forskolin (Weinmaster and Lemke, EMBO J., 9:915-920, 1990), cAMP induction of the FGF receptor may also explain the similar synergistic effect observed for the combination of FGF and forskolin (Davis and Stroobant, J. Cell Biol., 110:1353-1360, 1990).
  • cAMP induction was not observed for most of the receptor PTKs expressed by Schwann cells; the tyro-1, tyro-3, tyro-6, tyro-7, tyro-12, and tyro-13 mRNAs were down-regulated in the presence of forskolin, and expression of the tyro-5 and tyro-11 genes was not affected by the drug.
  • Filters 1-6 show hybridization obtained with 32 P-radiolabled cDNA probes to tyro-1 through tyro-6. Also shown are the hybridization profiles obtained using isolates of elk and the bFGF receptor (FGFR). The relative migration of 18S and 28S ribosomal RNAs, as determined by methylene blue staining, is indicated by the arrowheads. Exposure times are as follows: 15 hr (1, 3, 5, elk, FGFR), 22 hr (4, 6), 50 hr (2).
  • mRNA encoding the elk-related kinase tyro-6 was most abundantly expressed at E12; expression gradually fell until P10 and was relatively constant thereafter.
  • mRNA encoding the closely related kinase tyro-5 was maximally expressed at E14; expression fell sharply after P5 to a much lower steady-state level in the adult brain.
  • the gene encoding the eck-related kinase tyro-4 exhibited a similar, though even more dramatic regulation, with a peak in expression at E14/17, a sharp drop at birth, and a low steady-state level after P10.
  • tyro-2 hybridization signal in the brain was observed in an intercalated nucleus of the amygdala. No signal was evident in the Purkinje cell layer in the cerebellum.
  • the hybridization pattern have observed for tyro-2 is largely consistent with its expression by a subset of 7-amino-n-butyric acid (GABA)-ergic neurons.
  • tyro-4, tyro-5, and tyro-6 exhibited the most restricted patterns of expression in adult brain. Distinct hybridization to tyro-4 was evident in the facial nucleus of the pons, with more modest signals present in the bed nucleus of the anterior commissure and the triangular nucleus of the septum.
  • the tyro-5 gene was expressed weakly in cortex, at a modest level in all fields of the hippocampus, and in a subset of Purkinje cells in the cerebellum.
  • the tyro-6 gene showed a similar pattern of expression, giving a signal in Purkinje cells and weak signals in the hippocampus.
  • the two FGF receptor genes examined those encoding the bFGF receptor and bek, exhibited very different patterns of expression in the brain.
  • mRNA encoding the bFGF receptor was expressed at high levels in hippocampal neurons, but exhibited a field distribution that was nearly the inverse of tyro-3., i.e., expression was reduced in CA1 relative to CA2 and CA3.
  • mRNA levels in the dentate gyrus were lowest of all.

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Abstract

Substantially pure receptor PTK subtypes and methods of using the subtypes are provided.

Description

    CROSS-RELATED APPLICATIONS
  • This application is a divisional of U.S. patent application Ser. No. 08/456,647, filed Jun. 2, 1995, issued on Sep. 22, 1998 as U.S. Pat. No. 5,811,516, which is a divisional of U.S. patent application Ser. No.08/237,401, filed May 2, 1994, which is a continuation of U.S. patent application Ser. No. 07/884,486, filed May 15, 1992.[0001]
  • [0002] This work was supported by Grant Number NS-23896 from the National Institutes of Health. The United States Government may retain certain rights of this invention.
    • BACKGROUND OF THE INVENTION
  • 1. Field of the Invention [0003]
  • This invention relates generally to the molecular cloning of genes which encode unique protein-tyrosine kinase receptor subtypes which can be used in an assay to screen various compositions which modulate these receptors. [0004]
  • 2. Related Art [0005]
  • Among the signal transduction molecules implicated in neural development, the receptor protein-tyrosine kinases (PTKs) are of particular interest: These proteins function as transmembrane receptors for polypeptide growth factors, and contain a tyrosine kinase as an integral part of their cytoplasmic domains (Yarden and Ullrich, [0006] Annu. Rev. Biochem., 57:443-478, 1988; Ullrich and Schlessinger, Cell, 61:203-212, 1990). Binding of a polypeptide ligand to its corresponding cell surface receptor results in rapid activation of that receptor's intracellular tyrosine kinase, which in turn results in the tyrosine phosphorylation of the receptor itself and of multiple downstream target proteins (Hunter and Cooper, Annu. Rev. Biochem., 54:897-930, 1985; Hunter, et al, eds. J. B. Hook and G. Poste, Plenum Press, New York and London, pp. 119-139, 1990). For many receptor PTKs, growth factor binding ultimately triggers multiple rounds of cell division.
  • Molecular studies of mutations that affect cell differentiation have demonstrated that several of these receptor PTKs act as early determinants of cell fate. Loss-of-function mutations in the sevenless gene of Drosophila (Harris, et al., [0007] J. Physiol., 256:415-439, 1976), for example, abolish the tyrosine kinase activity of a transmembrane receptor expressed in the developing ommatidia of the eye and result in the aberrant differentiation of the precursors to the number 7 photoreceptors (Basler and Haten, Cell, 54:299-311, 1988; Rubin, Cell, 57:519-520, 1989). Rather than becoming number 7 photoreceptor cells, these precursors instead differentiate into non-neuronal cone cells, which form the lens. In marked contrast, the remaining complement of photoreceptors (numbers 1-6 and 8) differentiate normally.
  • Mutations in genes encoding other receptor PTKs have also been shown to affect cell differentiation. For example, mutations in the torso gene of Drosophila specifically disrupt the terminal differentiation of extreme anterior and posterior structures in the embryo (Sprenger, et al., [0008] Nature 338:478-483, 1989), and mutations in the Drosophila Ellipse gene, which encodes a homolog of the mammalian epidermal growth factor (EGF) receptor, result in the developmental failure of multiple cell types in the eye (Baker and Rubin, Nature, 340:150-153, 1989). In vertebrates, mutations in the mouse dominant white spotting locus (W), which encodes the c-kit receptor PTK, produce pleiotropic developmental effects that include disruption of the normal proliferation and differentiation of neural crest-derived melanocytes (Chabot, et al., Nature, 335:88-89, 1988; Geissler, et al., Cell, 55:185-192, 1988).
  • Parallel to these studies of the developmental role of receptor PTKs has been the demonstration that many of the ligands for these receptors influence the differentiation of neural cells in culture. Platelet-derived growth factor (PDGF), for example, has been shown to stimulate the proliferation and prevent the premature differentiation of oligodendrocyte/type-2 astrocyte glial progenitor cells in rat optic nerve cultures (Noble, et al., [0009] Nature, 333:560-562, 1988; Raff, et al., Nature, 333:562-565, 1988).
  • Similarly, both acidic and basic fibroblast growth factor (bFGF) have been shown to stimulate the neuronal differentiation of cultured rat pheochromocytoma (PC-12) cells (Togari, et al., [0010] J. Neurosci., 5:307-316, 1985; Wagner and D'Amore, J. Cell Biol., 103:1363-1367, 1986). bFGF has also been reported to prolong survival and stimulate neurite outgrowth in cultures of primary cortical and hippocampal neurons (Morrison, et al., Proc. Natl. Acad. Sci. USA, 83:7537-7541, 1986; Walicke, et al., Proc. Natl. Acad. Sci. USA, 83:3012-3016, 1986), to induce cell division, neuronal differentiation, and nerve growth factor (NGF) dependence in adrenal chromatifin cells (Stemple, et al., Neuron, 1:517-525, 1988), and to function as a survival factor, both in vivo and in vitro, for neural crest-derived non-neuronal cells during the early development of sensory ganglia (Kalcheim, Dev. Biol., 134:1-10, 1989). Recently, the product of the mouse mutant steel gene (Sl), which interacts genetically with W, has been identified as a growth factor ligand for the c-kit receptor (Witte, Cell, 63:5-6, 1990). Genetic and biochemical studies of the expression patterns of the sevenless, torso and c-kit receptors suggest that specification of cell fates can be achieved through the spatially and temporally restricted expression of either the receptors or their ligands (Rubin, Cell, 57:519-520, 1989; Tomlinson and Ready, Biol., 120:366-376, 1987; Reinke and Zipursky, Cell, 55:321-330, 1988; Banerjee and Zipursky, Neuron, 4:177-187, 1990; Stevens, et al., Nature, 346:660-663, 1990; Matsui, et al., Nature, 347:667-669, 1990).
    • SUMMARY OF THE INVENTION
  • In accordance with the present invention, novel receptor protein tyrosine kinase (PTK) subtype polypeptides have been isolated. These PTKs possess a tyrosine kinase domain and a unique tissue expression pattern different from all previously known receptor PTKs. These novel receptor PTK subtypes have been designated tyro-1 through tyro-8 and tyro-10 through tyro-12. Of particular interest among the new PTK subtypes are tyro-1 through tyro-6 which are found predominantly or exclusively in neural tissue. [0011]
  • By providing the polynucleotide sequences and corresponding polypeptide sequences for the new PTK subtypes, it is now possible to obtain polynucleotide sequences encoding the entire receptor PTK for each of the subtypes. [0012]
  • Further, the invention provides a method for identifying compositions which potentially affect the activity of the receptor PTK subtype. This method comprises (a) contacting cells containing DNA which expresses the PTK polypeptide with the composition under conditions suitable for cell culture; and (b) monitoring the cells for a physiological change resulting from this interaction. [0013]
  • In addition, the present invention provides unique oligonucleotide which align with the unique flanking regions of the receptor PTK subtypes, thereby allowing amplification of the polynucleotides encoding the receptor PTK subtype by such techniques as polymerase chain reaction (PCR). [0014]
  • The present invention also provides a method of gene therapy comprising introducing into a host subject an expression vector comprising a nucleotide sequence encoding a receptor PTK subtype capable of affecting a biological activity of the host subject cell. [0015]
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • FIGS. 1A and 1B show the tissue expression profiles of the novel PTK mRNAs. [0016]
  • FIG. 2 shows the developmental tissue profiles of the novel PTK mRNAs which were predominantly or exclusively neural in their distribution.[0017]
    • DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT
  • The present invention relates to novel protein tyrosine kinase (PTK) gene and polypeptides encoded by these genes. Various of these PTK subtypes are implicated in neural development where they function primarily as signal transduction molecules. The receptor PTKs of the invention are characterized as having a tyrosine kinase domain and a unique tissue expression pattern which differs from that of all known receptor PTKs. [0018]
  • The invention provides polynucleotides, such as DNA, cDNA, and RNA, encoding novel receptor PTK polypeptides. It is understood that all polynucleotides encoding all or a portion of the receptor PTKs of the invention are also included herein, so long as they exhibit at least one protein tyrosine kinase domain and the tissue expression pattern characteristic of a given subtype. Such polynucleotides include both naturally occurring and intentionally manipulated, for example, mutagenized polynucleotides. [0019]
  • DNA sequences of the invention can be obtained by several methods. For example, the DNA can be isolated using hybridization procedures which are well known in the art. These include, but are not limited to: 1) hybridization of probes to genomic or cDNA libraries to detect shared nucleotide sequences and 2) antibody screening of expression libraries to detect shared structural features. [0020]
  • Hybridization procedures are useful for the screening of recombinant clones by using labeled mixed synthetic oligonucleotide probes where each probe is potentially the complete complement of a specific DNA sequence in the hybridization sample which includes a heterogeneous mixture of denatured double-stranded DNA. For such screening, hybridization is preferably performed on either single-stranded DNA or denatured double-stranded DNA. Hybridization is particularly useful in the detection of cDNA clones derived from sources where an extremely low amount of mRNA sequences relating to the polypeptide of interest are present. In other words, by using stringent hybridization conditions directed to avoid non-specific binding, it is possible, for example, to allow the autoradiographic visualization of a specific cDNA clone by the hybridization of the target DNA to that single probe in the mixture which is its complete complement (Wallace, et al., [0021] Nucleic Acid Research, 9:879, 1981).
  • A receptor PTK cDNA library can be screened by injecting the various cDNAs into oocytes, allowing sufficient time for expression of the cDNA gene products to occur, and testing for the presence of the desired cDNA expression product, for example, by using antibody specific for the receptor PTK subtype polypeptide or by using functional assays for receptor PTK subtype activity and a tissue expression pattern characteristic of the desired subtype. [0022]
  • Alternatively, a cDNA library can be screened indirectly for receptor PTK polypeptides having at least one epitope using antibodies specific for receptor PTK subtypes of the invention. Such antibodies can be either polyclonally or monoclonally derived and used to detect expression product indicative of the presence of protein tyrosine kinase receptor PTK subtype cDNA. [0023]
  • The development of specific DNA sequences encoding receptor PTK subtypes of the invention can also be obtained by: (1) isolation of a double-stranded DNA sequence from the genomic DNA; (2) chemical manufacture of a DNA sequence to provide the necessary codons for the polypeptide of interest; and (3) in vitro synthesis of a double-stranded DNA sequence by reverse transcription of mRNA isolated from a eukaryotic donor cell. In the latter case, a double-stranded DNA complement of mRNA is eventually formed which is generally referred to as cDNA. Specifically embraced in (1) are genomic DNA sequences which encode allelic variant forms. Also included are DNA sequences which are degenerate as a result of the genetic code. [0024]
  • Of the three above-noted methods for developing specific DNA sequences for use in recombinant procedures, the use of genomic DNA isolates (1), is the least common. This is especially true when it is desirable to obtain the microbial expression of mammalian polypeptides because of the presence of introns. [0025]
  • The synthesis of DNA sequences is frequently the method of choice when the entire sequence of amino acid residues of the desired polypeptide product is known. When the entire sequence of amino acid residues of the desired polypeptide is not known, the direct synthesis of DNA sequences is not possible and the method of choice is the formation of cDNA sequences. Among the standard procedures for isolating cDNA sequences of interest is the formation of plasmid-carrying cDNA libraries which are derived from reverse transcription of mRNA which is abundant in donor cells that have a high level of genetic expression. When used in combination with polymerase chain reaction technology, even rare expression products can be cloned. In those cases where significant portions of the amino acid sequence of the polypeptide are known, the production of labeled single or double-stranded DNA or RNA probe sequences duplicating a sequence putatively present in the target cDNA may be employed in DNA/DNA hybridization procedures which are carried out on cloned copies of the cDNA which have been denatured into a single-stranded form (Jay, et al., [0026] Nucleic Acid Research, 11:2325, 1983).
  • Since the novel DNA sequences of the invention encode essentially all or part of a receptor PTK, it is now a routine matter to prepare, subclone, and express smaller polypeptide fragments of DNA from this or corresponding DNA sequences. Alternatively, by utilizing the DNA fragments disclosed herein which define the unique tyrosine kinase receptor subtype of the invention it is possible, in conjunction with known techniques, to determine the DNA sequences encoding the entire receptor subtypes. Such techniques are described in U.S. Pat. Nos. 4,394,443 and 4,446,235 which are incorporated herein by reference. [0027]
  • The polypeptide resulting from expression of a DNA sequence of the invention can be further characterized as being free from association with other eukaryotic polypeptides or other contaminants which might otherwise be associated with the protein kinase in its natural cellular environment. [0028]
  • Isolation and purification of microbially expressed polypeptides provided by the invention may be by conventional means including, preparative chromatographic separations and immunological separations involving monoclonal and/or polyclonal antibody preparation. [0029]
  • For purposes of the present invention, receptor PTK subtypes which are homologous to those of the invention can be identified by structural as well as functional similarity. Structural similarity can be determined, for example, by assessing polynucleotide strand hybridization or by screening with antibody, especially a monoclonal antibody, which recognizes a unique epitope present on the subtypes of the invention. When hybridization is used as criteria to establish structural similarity, those polynucleotide sequences which hybridize under stringent conditions to the polynucleotides of the invention are considered to be essentially the same as the polynucleotide sequences of the invention. [0030]
  • In the present invention, polynucleotide sequences encoding receptor PTK subtype may be introduced into a host cell by means of a recombinant expression vector. The term “recombinant expression vector” refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of the polynucleotide sequences of the invention. Such expression vectors typically contain a promotor sequence which facilitates efficient transcription of the inserted sequence in the host. The expression vector also typically contains specific genes which allow phenotypic selection of the transformed cells. Alternatively, nucleotide sequences encoding a receptor PTK subtype can be introduced directly in the form of free nucleotide, for example, by microinjection, or transfection. [0031]
  • DNA sequences encoding receptor PTK subtypes of the invention can be expressed in vivo by DNA transfer into a suitable host cell. “Recombinant host cells” or “host cells” are cells in which a vector can be propagated and its DNA expressed. The term includes not only prokaryotes, but also such eukaryotes as yeast, filamentous fungi, as well as animal cells which can replicate and express an intron-free DNA sequence of the invention and any progeny of the subject host cell. It is understood that not all progeny are identical to the parental cell since there may be mutations that occur at replication. However, such progeny are included when the terms above are used. [0032]
  • Methods of expressing DNA sequences having eukaryotic coding sequences in prokaryotes are well known in the art. Biologically functional viral and plasmid DNA vectors capable of expression and replication in a host are known in the art. Such vectors are used to incorporate DNA sequences of the invention. Hosts include microbial, yeast and mammalian organisms. [0033]
  • Transformation of a host cell with recombinant DNA may be carried out by conventional techniques which are well known to those skilled in the art. Where the host is prokaryotic, such as [0034] E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl2 method well known in the art. Alternatively, MgCl2 or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell.
  • When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate co-precipitates, conventional mechanical procedures such as microinjection, insertion of a plasmid encased in liposomes, or virus vectors may be used. Eukaryotic cells can also be cotransformed with foreign cDNA encoding the desired receptor PTK subtype protein, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein. ([0035] Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).
  • Where the eukaryotic host cells are yeast, the cDNA can be expressed by inserting the cDNA into appropriate expression vectors and introducing the product into the host cells. Various shuttle vectors for the expression of foreign genes in yeast have been reported (Heinemann, et al., [0036] Nature, 340:205, 1989; Rose, et al., Gene, 60:237, 1987).
  • Isolation and purification of microbially expressed protein, or fragments thereof provided by the invention, may be carried out by conventional means including preparative chromatography and immunological separations involving monoclonal or polyclonal antibodies. Antibodies provided in the present invention are immunoreactive with the receptor PTK subtypes of the invention. Antibody which consists essentially of pooled monoclonal antibodies with different epitopic specificities, as well as distinct monoclonal antibody preparations are provided. Monoclonal antibodies are made from antigen containing fragments of the protein by methods well known in the art (Kohler, et al., [0037] Nature, 256:495, 1975; Current Protocols in Molecular Biology, Ausubel, et al., ed., 1989).
  • Minor modifications of the receptor PTK primary amino acid sequence may result in proteins which have substantially equivalent activity compared to the receptor PTKs described herein. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. All proteins produced by these modifications are included herein as long as tyrosine kinase activity and the characteristic tissue expression pattern for the subtype is present. [0038]
  • The invention also discloses a method for identifying a composition which affects the activity of a receptor PTK subtype of the invention. The receptor is, for example, capable of affecting cell division and/or differentiation. The composition is incubated in combination with cells under conditions suitable for cell culture, then subsequently monitoring the cells for a physiologic change. [0039]
  • The production of a receptor PTK can be accomplished by oligonucleotide(s) which are primers for amplification of the genomic polynucleotide encoding PTK receptor. These unique oligonucleotide primers were produced based upon identification of the flanking regions contiguous with the polynucleotide encoding the receptor PTK. These oligonucleotide primers comprise sequences which are capable of hybridizing with the flanking nucleotide sequence encoding a polypeptide having amino acid residues HRDLAAR and/or DVWS(F/Y)G(V/I) and sequences complementary thereto. [0040]
  • The primers of the invention embrace oligonucleotides of sufficient length and appropriate sequence so as to provide specific initiation of polymerization on a significant number of nucleic acids in the polynucleotide encoding the receptor PTK subtype. Specifically, the term “primer” as used herein refers to a sequence comprising two or more deoxyribonucleotides or ribonucleotides, preferably more than three, which sequence is capable of initiating synthesis of a primer extension product, which is substantially complementary to a receptor PTK strand. Environmental conditions conducive to synthesis include the presence of nucleoside triphosphates and an agent for polymerization, such as DNA polymerase, and a suitable temperature and pH. The primer is preferably single stranded for maximum efficiency in amplification, but may be double stranded. If double stranded, the primer is first treated to separate its strands before being used to prepare extension products. Preferably, the primer is an oligodeoxyribonucleotide. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent for polymerization. The exact length of primer will depend on many factors, including temperature, buffer, and nucleotide composition. The oligonucleotide primer typically contains 15-22 or more nucleotides, although it may contain fewer nucleotides. [0041]
  • Primers of the invention are designed to be “substantially” complementary to each strand of polynucleotide encoding the receptor PTK to be amplified. This means that the primers must be sufficiently complementary to hybridize with their respective strands under conditions which allow the agent for polymerization to perform. In other words, the primers should have sufficient complementarity with the flanking sequences to hybridize therewith and permit amplification of the polynucleotide encoding the receptor PTK. Preferably, the primers have exact complementarity with the flanking sequence strand. [0042]
  • Oligonucleotide primers of the invention are employed in the amplification process which is an enzymatic chain reaction that produces exponential quantities of polynucleotide encoding the receptor PTK relative to the number of reaction steps involved. Typically, one primer is complementary to the negative (−) strand of the polynucleotide encoding the receptor PTK and the other is complementary to the positive (+) strand. Annealing the primers to denatured nucleic acid followed by extension with an enzyme, such as the large fragment of DNA Polymerase I (Klenow) and nucleotides, results in newly synthesized + and −strands containing the receptor PTK sequence. Because these newly synthesized sequences are also templates, repeated cycles of denaturing, primer annealing, and extension results in exponential production of the region (i.e., the tyrosine kinase receptor polynucleotide sequence) defined by the primer. The product of the chain reaction is a discrete nucleic acid duplex with termini corresponding to the ends of the specific primers employed. [0043]
  • The oligonucleotide primers of the invention may be prepared using any suitable method, such as conventional phosphotriester and phosphodiester methods or automated embodiments thereof. In one such automated embodiment, diethylphos-phoramidites are used as starting materials and may be synthesized as described by Beaucage, et al. ([0044] Tetrahedron Letters, 22:1859-1862, 1981). One method for synthesizing oligonucleotides on a modified solid support is described in U.S. Pat. No. 4,458,066.
  • Any nucleic acid specimen, in purified or nonpurified form, can be utilized as the starting nucleic acid or acids, provided it contains, or is suspected of containing, the specific nucleic acid sequence containing a protein receptor PTK of the invention. Thus, the process may employ, for example, DNA or RNA, including messenger RNA, wherein DNA or RNA may be single stranded or double stranded. In the event that RNA is to be used as a template, enzymes, and/or conditions optimal for reverse transcribing the template to DNA would be utilized. In addition, a DNA-RNA hybrid which contains one strand of each may be utilized. A mixture of nucleic acids may also be employed, or the nucleic acids produced in a previous amplification reaction herein, using the same or different primers may be so utilized. The specific nucleic acid sequence to be amplified, i.e., the receptor PTK, may be a fraction of a larger molecule or can be present initially as a discrete molecule, so that the specific sequence constitutes the entire nucleic acid. It is not necessary that the sequence to be amplified be present initially in a pure form; it may be a minor fraction of a complex mixture, such as contained in whole human DNA. [0045]
  • DNA or RNA utilized herein may be extracted from a body sample, such as brain, or various other tissue, by a variety of techniques such as that described by Maniatis, et al. ([0046] Molecular Cloning, 280:281, 1982). If the extracted sample is impure (such as plasma, serum, or blood), it may be treated before amplification with an amount of a reagent effective to open the cells, fluids, tissues, or animal cell membranes of the sample, and to expose and/or separate the strand(s) of the nucleic acid(s). This lysing and nucleic acid denaturing step to expose and separate the strands will allow amplification to occur much more readily.
  • Where the target nucleic acid sequence of the sample contains two strands, it is necessary to separate the strands of the nucleic acid before it can be used as the template. Strand separation can be effected either as a separate step or simultaneously with the synthesis of the primer extension products. This strand separation can be accomplished using various suitable denaturing conditions, including physical, chemical, or enzymatic means, the word “denaturing” includes all such means. One physical method of separating nucleic acid strands involves heating the nucleic acid until it is denatured. Typical heat denaturation may involve temperatures ranging from about 80° to 105° C. for times ranging from about 1 to 10 minutes. Strand separation may also be induced by an enzyme from the class of enzymes known as helicases or by the enzyme RecA, which has helicase activity, and in the presence of riboATP, is known to denature DNA. The reaction conditions suitable for strand separation of nucleic acids. with helicases are described by Kuhn Hoffmann-Berling ([0047] CSH-Quantitative Biology, 43:63, 1978) and techniques for using RecA are reviewed in C. Radding (Ann. Rev. Genetics, 16:405-437, 1982).
  • If the nucleic acid containing the sequence to be amplified is single stranded, its complement is synthesized by adding one or two oligonucleotide primers. If a single primer is utilized, a primer extension product is synthesized in the presence of primer, an agent for polymerization, and the four nucleoside triphosphates described below. The product will be partially complementary to the single-stranded nucleic acid and will hybridize with a single-stranded nucleic acid to form a duplex of unequal length strands that may then be separated into single strands to produce two single separated complementary strands. Alternatively, two primers may be added to the single-stranded nucleic acid and the reaction carried out as described. [0048]
  • When complementary strands of nucleic acid or acids are separated, regardless of whether the nucleic acid was originally double or single stranded, the separated strands are ready to be used as a template for the synthesis of additional nucleic acid strands. This synthesis is performed under conditions allowing hybridization of primers to templates to occur. Generally synthesis occurs in a buffered aqueous solution, preferably at a pH of 7-9, most preferably about 8. Preferably, a molar excess (for genomic nucleic acid, usually about 10[0049] 8:1 primer:template) of the two oligonucleotide primers is added to the buffer containing the separated template strands. It is understood, however, that the amount of complementary strand may not be known if the process of the invention is used for diagnostic applications, so that the amount of primer relative to the amount of complementary strand cannot be determined with certainty. As a practical matter, however, the amount of primer added will generally be in molar excess over the amount of complementary strand (template) when the sequence to be amplified is contained in a mixture of complicated long-chain nucleic acid strands. A large molar excess is preferred to improve the efficiency of the process.
  • The deoxyribonucleotide triphosphates dATP, dCTP, dGTP, and dTTP are added to the synthesis mixture, either separately or together with the primers, in adequate amounts and the resulting solution is heated to about 90°-100° C. from about 1 to 10 minutes, preferably from 1 to 4 minutes. After this heating period, the solution is allowed to cool to room temperature, which is preferable for the primer hybridization. To the cooled mixture is added an appropriate agent for effecting the primer extension reaction (called herein “agent for polymerization”), and the reaction is allowed to occur under conditions known in the art. The agent for polymerization may also be added together with the other reagents if it is heat stable. This synthesis (or amplification) reaction may occur at room temperature up to a temperature above which the agent for polymerization no longer functions. [0050]
  • Thus, for example, if DNA polymerase is used as the agent, the temperature is generally no greater than about 40 ° C. Most conveniently the reaction occurs at room temperature. [0051]
  • The agent for polymerization may be any compound or system which will function to accomplish the synthesis of primer extension products, including enzymes. Suitable enzymes for this purpose include, for example, [0052] E. coli DNA polymerase I, Klenow fragment of E coli DNA polymerase I, T4 DNA polymerase, other available DNA polymerases, polymerase muteins, reverse transcriptase, and other enzymes, including heat-stable enzymes (i.e., those enzymes which perform primer extension after being subjected to temperatures sufficiently elevated to cause denaturation). Suitable enzymes will facilitate combination of the nucleotides in the proper manner to form the primer extension products which are complementary to each receptor PTK nucleic acid strand. Generally, the synthesis will be initiated at the 3′ end of each primer and proceed in the 5′ direction along the template strand, until synthesis terminates, producing molecules of different lengths. There may be agents for polymerization, however, which initiate synthesis at the 5′ end and proceed in the other direction, using the same process as described above.
  • The newly synthesized receptor PTK strand and its complementary nucleic acid strand will form a double-stranded molecule under hybridizing conditions described above and this hybrid is used in subsequent steps of the process. In the next step, the newly synthesized double-stranded molecule is subjected to denaturing conditions using any of the procedures described above to provide single-stranded molecules. [0053]
  • The above process is repeated on the single-stranded molecules. Additional agent for polymerization, nucleotides, and primers may be added, if necessary, for the reaction to proceed under the conditions prescribed above. Again, the synthesis will be initiated at one end of each of the oligonucleotide primers and will proceed along the single strands of the template to produce additional nucleic acid. After this step, half of the extension product will consist of the specific nucleic acid sequence bounded by the two primers. [0054]
  • The steps of denaturing and extension product synthesis can be repeated as often as needed to amplify the receptor PTK nucleic acid sequence to the extent necessary for detection. The amount of the specific nucleic acid sequence produced will accumulate in an exponential fashion. [0055]
  • Sequences amplified by the methods of the invention can be further evaluated, detected, cloned, sequenced, and the like, either in solution or after binding to a solid support, by any method usually applied to the detection of a specific DNA sequence such as PCR, oligomer restriction (Saiki, et al., [0056] Bio/Technology, 3:1008-1012, 1985), allele-specific oligonucleotide (ASO) probe analysis (Conner, et al., Proc. Natl. Acad. Sci. USA, 80:278, 1983), oligonucleotide ligation assays (OLAs) (Landegren, et al., Science, 241:1077, 1988), and the like. Molecular techniques for DNA analysis have been reviewed (Landegren, et al., Science, 242:229-237, 1988).
  • The present invention also provides methods for the treatment of disease employing gene therapy that modulates cellular differentiation or maturation. Such therapy can be affected by introduction of polynucleotide sequences of the invention into cells of a subject having a disease. Delivery of polynucleotide can be achieved using techniques well known in the art. For example, a recombinant expression vector, such as a chimeric virus, or a colloidal dispersion system can be employed. [0057]
  • Various viral vectors which can be utilized for introduction of polynucleotide according to the present invention, include adenovirus, herpes virus, vaccinia, or, preferably, an RNA virus such as a retrovirus. Preferably, the retroviral vector is a derivative of a murine or avian retrovirus. Examples of retroviral vectors in which a single foreign gene can be inserted include, but are not limited to: Moloney murine leukemia virus (MoMuLV), Harvey murine sarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and Rous Sarcoma Virus (RSV). A number of additional retroviral vectors can incorporate multiple genes. All of these vectors can incorporate a gene for a selectable marker so that transduced cells can be identified and generated. [0058]
  • By inserting a polynucleotide encoding the receptor PTK of interest into a viral vector, along with another gene which encodes ligand for a receptor on a specific target cell, the vector now becomes target specific. Retroviral vectors can be made target specific by including in the retroviral vector a polynucleotide encoding a target related binding substance. Preferred targeting is accomplished by using an antibody to target the retroviral vector. Those of skill in the art will know of, or can readily ascertain without undue experimentation, specific polynucleotide sequences which can be inserted into the retroviral genome to allow target specific delivery of the retroviral vector containing the receptor PTK polynucleotide. [0059]
  • Since recombinant retroviruses are defective, they require assistance in order to produce infectious vector particles. This assistance can be provided, for example, by using helper cell lines that contain plasmids encoding all of the structural genes of the retrovirus under the control of regulatory sequences within the LTR. These plasmids are missing a nucleotide sequence which enables the packaging mechanism to recognize an RNA transcript for encapsidation. Helper cell lines which have deletions of the packaging signal include, but are not limited to, Ψ2, PA317 and PA12, for example. These cell lines produce empty virions, since no genome is packaged. If a retroviral vector is introduced into such cells in which the packaging signal is intact, but the structural genes are replaced by other genes of interest, the vector can be packaged and vector virion produced. The vector virions produced by this method can then be used to infect a tissue cell line, such as NIH 3T3 cells, to produce large quantities of chimeric retroviral virions. [0060]
  • Alternatively, NIH 3T3 or other tissue culture cells can be directly transfected with plasmids encoding the retroviral structural genes gag, pol and env, by conventional calcium phosphate transfection. These cells are then transfected with the vector plasmid containing the genes of interest. The resulting cells release the retroviral vector into the culture medium. [0061]
  • Another targeted delivery system for introduction of polynucleotides encoding the receptor PTKs of the invention is a colloidal dispersion system. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. The preferred colloidal system of this invention is a liposome. [0062]
  • Since the receptor PTK polypeptide may be indiscriminate in its action with respect to cell type, a targeted delivery system offers a significant improvement over randomly injected non-specific liposomes. A number of procedures can be used to covalently attach either polyclonal or monoclonal antibodies to a liposome bilayer. Antibody-targeted liposomes can include monoclonal or polyclonal antibodies or fragments thereof such as Fab, or F(ab′)[0063] 2, as long as they bind efficiently to an epitope on the target cells. Liposomes may also be targeted to cells expressing receptors for hormones or other serum factors.
  • Liposomes are artificial membrane vesicles which are useful as in vitro and in vivo delivery vehicles. It has been shown that large unilamellar vesicles (LUV), which range in size from 0.2-4.0 μm can encapsulate a substantial percentage of an aqueous buffer containing large macromolecules. RNA, DNA, intact virions and peptides can be encapsulated within the aqueous interior and be delivered to cells in a biologically active form (Fraley, et al., [0064] Trends Biochem. Sci., 6:77, 1981). In order for a liposome to be an efficient transfer vehicle, the following characteristics should be present: (1) encapsulation of polynucleotides of interest at high efficiency without compromising biological activity; (2) preferential and substantial binding to target cells relative to non-target cells; (3) delivery of aqueous contents of vesicle to the target cell cytoplasm at high efficiency; and (4) accurate and effective expression of genetic information (Mannino, et al., Biotechniques, 6:682, 1988).
  • The surface of the targeted delivery system may be modified in a variety of ways. In the case of a liposomal targeted delivery system, lipid groups can be incorporated into the lipid bilayer of the liposome in order to maintain the targeting receptor in stable association with the liposomal bilayer. Various linking groups can be used for joining the lipid chains to the targeting receptor. [0065]
  • In general, the targeted delivery system will be directed to cell surface receptors thereby allowing the delivery system to find and “home in” on the desired cells. Alternatively, the delivery system can be directed to any cell surface molecule preferentially found in the cell population for which treatment is desired and capable of association with the delivery system. Antibodies can be used to target liposomes to specific cell-surface molecules. For example, where a tumor is associated with a receptor PTK of the invention, certain antigens expressed specifically or predominantly on the cells of the tumor may be exploited for the purpose of targeting antibody tyrosine kinase receptor DNA-containing liposomes directly to a malignant tumor, if desired. [0066]
  • An alternative use for recombinant retroviral vectors comprises the introduction of polynucleotide sequences into the host by means of skin transplants of cells containing the virus. Long term expression of foreign genes in implants, using cells of fibroblast origin, may be achieved if a strong housekeeping gene promoter is used to drive transcription. For example, the dihydrofolate reductase (DHFR) gene promoter may be used. Cells such as fibroblasts, can be infected with virions containing a retroviral construct containing the receptor PTK gene of interest together with a gene which allows for specific targeting, such as a tumor-associated antigen and a strong promoter. The infected cells can be embedded in a collagen matrix which can be grafted into the connective tissue of the dermis in the recipient subject. As the retrovirus proliferates and escapes the matrix it will specifically infect the target cell population. In this way the transplantation results in increased amounts of receptor PTK being produced in cells manifesting the disease. [0067]
  • Because the present invention identifies nucleotide sequences encoding novel receptor PTKs, it is possible to design therapeutic or diagnostic protocols utilizing these sequences. Thus, where a disease is associated with a receptor PTK of the invention, the polynucleotide sequence encoding the PTK can be utilized to design sequences which interfere with the function of the receptor. This approach utilizes, for example, antisense nucleic acid and ribozymes to block translation of specific receptor mRNA, either by masking the mRNA with antisense nucleic acid or by cleaving it with ribozyme. [0068]
  • Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule (Weintraub, [0069] Scientific American, 262:40, 1990). In the cell, the antisense nucleic acids hybridize to the corresponding mRNA, forming a double-stranded molecule. The antisense nucleic acids interfere with the translation of the mRNA since the cell will not translate a mRNA that is double-stranded. Antisense oligomers of about 15 nucleotides are preferred, since they are easily synthesized and are less likely to cause problems than larger molecules when introduced into the target receptor-producing cell. The use of antisense methods to inhibit the in vitro translation of genes is well known in the art (Marcus-Sakura, Anal.Biochem., 172:289, 1988).
  • Ribozymes are RNA molecules possessing the ability to specifically cleave other single-stranded RNA in a manner analogous to DNA restriction endonucleases. Through the modification of nucleotide sequences which encode these RNAs, it is possible to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it (Cech, [0070] J. Amer. Med. Assn., 260:3030, 1988). A major advantage of this approach is that, because they are sequence-specific, only mRNAs with particular sequences are inactivated.
  • There are two basic types of ribozymes namely, tetrahymena-type (Hasselhoff, [0071] Nature, 334:585, 1988) and “hammerhead”-type. Tetrahymena-type ribozymes recognize sequences which are four bases in length, while “hammerhead”-type ribozymes recognize base sequences 11-18 bases in length. The longer the recognition sequence, the greater the likelihood that the sequence will occur exclusively in the target mRNA species. Consequently, hammerhead-type ribozymes are preferable to tetrahymena-type ribozymes for inactivating a specific mRNA species and 18-based recognition sequences are preferable to shorter recognition sequences.
  • Antisense sequences can be therapeutically administered by techniques as described above for the administration of receptor PTK polynucleotides. Targeted liposomes are especially preferred for therapeutic delivery of antisense sequences. [0072]
  • The following Examples are intended to illustrate, but not to limit the invention. While such Examples are typical of those that might be used, other procedures known to those skilled in the art may alternatively be utilized. [0073]
    • EXAMPLE 1 Isolation of Novel PTK Clones
  • PCR was used to amplify PTK-related sequences located between the degenerate oligonucleotide primer sequences shown in TABLE 1. These primers correspond to the amino acid sequences HRDLAAR (SEQ ID NO:27) (upstream) and DVWS(F/Y)G(I/V) (SEQ ID NO:28) (downstream), which flank a highly conserved region of the kinase domain shared by receptor PTKs (Hanks, et al., [0074] Science, 241:42-52, 1988). The upstream primer was chosen to exclude members of the src family of cytoplasmic tyrosine kinases. The downstream primer was chosen such that a second highly conserved amino acid sequence diagnostic or PTKs—P(I/V)(K/R)W(T/M)APE (SEQ ID NO:29)—would be contained within amplified PCR products.
  • The DNA substrates used for amplification were sciatic nerve cDNA populations prepared for use in the construction of subtracted cDNA libraries. Three different subtracted cDNAs were produced. The first two, UN and TWI, were enriched for transcripts expressed predominantly in Schwann cells. The third, BD, was enriched for transcripts shared between Schwann cells and myelinating stage (P17-23) brain. Two initial hybridizations were performed. Both samples contained 500 ng of single-stranded sciatic nerve cDNA mixed with the following poly(A)-selected RNAs: 10 μg of muscle, 7.5 μg of liver, and 5 μg of kidney. Both samples also contained a series of RNAs synthesized in vitro; these encoded portions of the sense strand of the following Schwann cell transcripts: NGF receptor, glial fibrillary acidic protein, proteolipid protein, protein zero, myelin basic protein, and CNPase. The first sample contained, in addition, 10 μg of poly(A)-selected RNA from rat brain cerebellum (P19) and cortex (P3). Each hybridization was allowed to proceed to approximately R[0075] 0t 2000. Following hybridization, these samples were bound to hydroxylapatite (0.12M phosphate buffer, 65° C.). For the first sample, material not binding to hydroxylapatite (HAP) was collected and converted to a double-stranded form. This material was designated UN (unbound). For the second sample, cDNA not binding to the column was further hybridized with 40 μg of poly(A)-selected RNA from rat cerebellum (equal mix of P17 and P23) until R0t 800. This mixture was re-applied to hydroxylapatite. The unbound material was collected and converted to a double-stranded form and designated TWI (twice unbound). The material that bound to the HAP column was then eluted and also converted to a double-stranded DNA form. This fraction was called BD (bound).
  • Approximately 2-4 ng of the UN and TWI subtracted cDNAs and 1 ng of the BD cDNA were used in each of the amplifications, which were conducted using reagents and instructions provided by U.S. Biochemicals. The final concentration of magnesium ion was increased to 2.1 mM. Thirty-nine cycles of amplification were performed on a water-cooled [0076] vtwb Model 1 cycler (San Diego, Calif.). Amplification parameters included an initial 1 minute denaturation step at 94° C., a 5 minute annealing at 37° C., a 5 minute extension at 65° C., and a 0.3 minute denaturation at 94° C. Approximately 4 μg of each of the degenerate primers (TABLE 1) was included in each amplification. The unusually low annealing temperatures employed in these amplifications may favor polymerase extension from stably-hybridized oligonucleotide primers, resulting in a broader and less-biased amplified population than those obtained with previous protocols (Wilks, Proc. Natl. Acad. Sci. USA, 86:1603-1607, 1989).
    TABLE 1
    (SEQ ID NOS:30-35)
    Figure US20030013848A1-20030116-C00001
  • Amplified DNAs were size fractionated on 5% non-denaturing acrylamide gels. The gels were stained with ethidium bromide (1 μg/ml) and amplified bands of ˜220 bp were excised. These bands were eluted overnight into 0.5 M ammonium acetate. 1 mM EDTA, 0.2% SDS, and eluted DNA was then precipitated with 10 μg of tRNA carrier. Recovered PCR products were blunt-ended using T4 DNA polymerase, and phosphorylated using T4 polynucleotide kinase. Approximately 40 ng of insert was then ligated with 200 ng of dephosphorylated SmaI/EcoRV-digested pBluescript plasmid. One-tenth of each ligation was used to transform MC1061 bacteria. [0077]
  • The DNA sequence of both strands of each PCR product subclone was determined from alkaline lysis miniprep DNA, using the dideoxy chain termination method. In those cases in which clones having apparently identical inserts were isolated multiple times, the sequence of complementary strands was derived from independent clones. [0078]
    • EXAMPLE 2 Sequence Analysis of PCR Subclones
  • Sequence analysis of 168 PCR product subclones yielded 155 with significant similarity to the tyrosine kinase family. TABLE 2 lists the 27 distinct kinase domain sequences contained in this set, which includes those of the abl (human; Shtivelman, et al., [0079] Cell, 47:277-284, 1986) arg (human: Kruh, et al., Science, 234:1545-1548, 1986), and fer cytoplasmic (nonreceptor) kinases (human, Hao, et al., Mol. Cell. Biol., 9:1587-1593, 1989), as well as those of the receptors for EGF-R (human; Ullrich, et al., Nature 309, 418-425, 1984), PDGF-A (human; Matsui, et al., Science, 243:800-804, 1989; rat; Lee, et al., Science, 245:57-60, 1989; Reid, et al., Proc. Natl. Acad. Sci. USA, 87:1596-1600, 1990; Safran, et al., Oncogene, 5:635-643, 1990), colony-stimulating factor 1 (CSF-1; human; Coussens, et al., Nature, 320:277-280, 1986; mouse; Rothwell and Rohrschneider, Oncogene Res., 1:311-324, 1987, and insulin-like growth factor 1 (IGF-1; human; Ullrich, et al., EMBO, J., 5:2503-2512, 1986).
  • Other domain sequences listed include fes (human; Roebroek, et al., [0080] EMBO J., 4:2897-2903, 1985); Dsrc (Drosophila; Gregory, et al., Mol. Cell. Biol., 7:2119-2127, 1987); eph (human; Hirai, et al., Science, 238:1717-1720, 1987; eck (human; Lindberg and Hunter, Mol. Cell. Biol., in press, 1990); elk (rat; Letwin, et al., Oncogene, 3:621-627, 1988); neu (Bargmann, et al., Nature, 319:226-230, 1986); bek (mouse; Kornbluth, et al., Mol. Cell. Biol., 8:5541-5544, 1988); flt (human; Shibuya, et al., Oncogene, 5:519-524,1990); trk, (human; Martin-Zanca, et al., Nature, 319:743-748, 1986), and trk B (mouse; Klein, et al., EMBO J., 8:3701-3709, 1989).
  • Amino acid sequences were deduced from the nucleotide sequence of the 27 different PTK domain cDNAs. Deduced amino acid sequences corresponding to the oligonucleotide primers used for PCR amplification were not included. Kinase domain sequences are segmented according to the subdomains defined by Hanks, et al. ([0081] Science, 241:42-52, 1988). After each sequence is a number indicating the number of times it was identified. Numbers listed parenthetically correspond to clones uniquely obtained from amplification of the BD substrate. The segregation of kinase domain subfamilies is based solely on amino acid sequence conservation; sequences denoted by an asterisk were not encountered in this survey but have been included to facilitate comparisons.
  • The high percentage of isolates encoding tyrosine kinases (92%) and the large number of different kinase clones obtained probably reflect the highly degenerate primers and low temperature annealing and extension parameters used for PCR amplification, as well as the stringent size criteria used in the subcloning and sequencing of PCR products. [0082]
  • Of the 27 different kinases identified in this nervous system survey, 11 (tyro-1 through tyro-8 and tyro-10 through tyro-12) are novel. For the previously identified kinases, several rat isolates differ by 1 or 2 amino acids from the kinase domain sequences reported for other species. Nucleotide sequence comparisons suggest that these differences are accounted for by species variation and do not represent the amplification of novel kinase cDNAs. The novel isolates tyro-1 and tyro-11 were each obtained only a single time. [0083]
  • The kinase domain sequences of tyro-1 through tyro-13 have been grouped by similarity to the equivalent sequences of other PTKs (TABLE 2). The indicated subfamilies were defined with reference to a computer-generated phylogenetic tree, constructed from an analysis of 13 novel partial PTK sequences along with a set of 55 additional PTKs, according to the methods of Fitch and Margoliash ([0084] Science, 15:279-284, 1967) as implemented by the programs of Feng and Doolittle (J. Mol. Evol., 25:351-360, 1987). The resulting closely related sequence clusters were used to organize the kinase subfamilies presented in TABLE 2. Tyro-1 and tyro-4, for example, are related to the epithelial cell kinase (eck) (Lindberg and Hunter, Mol. Cell. Biol., in press, 1990), tyro-2 to the EGF receptor and the neu proto-oncogene (Bargmann, et et., Nature, 319:226-230, 1986), tyro-5, tyro-6, and tyro-11 to the elk kinase (Letwin, et al., Oncogene, 3:621-627, 1988), tyro-9 to the bFGF receptor, and tyro-10 to trk and trkB (Martin-Zanca, et al., Nature, 319:743-748, 1986; Klein, et al., EMBO J., 8:3701-3709, 1989). Although they exhibit similarity to the insulin receptor, tyro-3, tyro-7, and tyro-12 are listed as a novel subfamily since they are more closely related to each other than to any previously described kinase. The eck- and elk-related sequences are listed in separate subsets, but it is important to note the high degree of similarity between these subfamilies. The sequences of fes, trk, trkB, and Dsrc28 (each marked with an asterisk) are included in TABLE 2 only for comparison, since they were not encountered in these cloning studios.
    • EXAMPLE 3 Tissue Expression Profile of Novel PTK mRNAs
  • The expression pattern of the 13 novel kinase clones were characterized by first examining the relative levels of mRNA present in a variety of neonatal and adult rat tissues. Radiolabeled cDNA probes for each of these clones, as well as probes prepared from isolates of the bFGF receptor, bek, and elk kinases, were hybridized to a set of eight parallel Northern blots containing RNA isolated from kidney, liver, spleen, heart, skeletal muscle, brain, sciatic nerve, and cultured Schwann cells. RNA was isolated from Schwann cells cultured in both the presence and absence of the adenylate cyclase activator forskolin, since at least one receptor PTK gene (that encoding the PDGF-B receptor) exhibits ell-specific cAMP induction in these cells (Weinmaster and Lemke, [0085] EMBO J., 9:915-920, 1990) individual blots were in some cases reutilized for as many as four rounds of hybridization.
  • Total RNA from various tissues was prepared by the method of Chomczynski and Sacchi ([0086] Anal. Biochem., 162:156-159, 1987). One additional phenol-chloroform extraction was performed prior to nucleic acid precipitation. Poly(A)-selected RNA samples were purified by either column chromatography or in batch using oligo(dT)-cellulose type III (Collaborative Research). RNA samples were denatured in 50% formamide, 2.2M formaldehyde, and MOPS at 65° C. for 10 min, electrophoresed in 1.0% agarose, 2.2M formaldehyde, and MOPS, transferred to Nytran filters (Schleicher & Schuell) and baked at 80° C. for 2 hr as previously described (Monuki, et al., Neuron, 3:783-793, 1989). Probes for blot hybridizations were prepared using [α-32P] dCTP and a random hexamer priming kit, according to instructions provided by the manufacturer (Bethesda Research Laboratories). In all cases, final wash stringency for Northern blots was set at 0.2×SSC, 0.2% SDS, 65 ° C.
  • In situ hybridization was performed according to Simmons, et al. ([0087] J. Histotechnology, 12:169-181, 1989), with minor modifications. Paraformaldehyde-fixed brain sections (30 μm), from either adult or 33-day-old rats were used. Antisense probes from PCR product Subclones were prepared using 125 μCi or [35S] UTP (1250 Ci/mmol: New England Nuclear) in a 10 μl transcription reaction, with reagents obtained from Stratagene (La Jolla, Calif.). Hybridizations were performed at 55° C. for 22 hr using approximately 75 μl or 5×106 cpm/ml probe per slide. RNAase A digestions were performed in buffer prewarmed to 37° C. The final wash stringency was 0.1×SSC at 60° C. for 35 min. Emulsion-dipped slides were exposed for 2 weeks prior to developing. Slides were counterstained with thionin.
  • The various tissue expression profiles are shown in FIG. 1. Poly(A) (left 10 lanes) or total RNA (tot, right 4 lanes) from the indicated rat tissues was analyzed for expression of PTK mRNAs. All tissues were taken from animals 27 days postnatal, except where otherwise indicated. Sciatic nerves (sciatic) were obtained from 7-to-8-day-old rats. Rat Schwann cells were cultured in either the presence (+) or absence (−) of 20 μM forskolin for 48 hr prior to harvesting. All lanes contain either 2.5 μg of poly(A)[0088] + RNA or 10 μg of total RNA, except for the cultured Schwann cell poly(A)+ lanes, which contain 1.0 μg each. The relative migration of 18S and 28S ribosomal RNAs; as determined by methylene blue staining, is indicated by the arrowheads. Filters 1-13 show hybridization with 32P radiolabeled cDNA probes to tyro-1 through tyro-13. Also shown for comparison is the hybridization observed using isolates of elk, the bFGF receptor (FGFR), and the bek FGF receptor. Exposure times were as follows: 34 hr (1, 5, 6, 7, 11), 41 hr (3, 4, FGFR), 120 hr (2, 9, 10, bek), 158 hr (8, 13, elk), 8 days (12).
  • The results of this analysis (FIG. 1) demonstrate that 6 of the 11 novel kinase genes (tyro-1 through tyro-6), together with the elk gene, are preferentially expressed by cells of the nervous system. For example, tyro-1, a novel member of the eck kinase subfamily, exhibited strong hybridization to brain mRNA, a faint signal in Schwann cells, and very faint signals in kidney and heart. Tyro-4 also a novel member of the eck subfamily, exhibited more modest hybridization to two mRNAs in postnatal day 5(P5) brain, with lower signals evident in older brains as well as kidney and heart. The novel EGF receptor homolog tyro-2 identified a high molecular weight mRNA in brain that could also be detected in kidney and heart. It is possible that the very low tyro-1, tyro-2, and tyro-4 hybridization signals observed in kidney and heart are due to neural contamination from the adrenal gland and cardiac ganglia, respectively. Tyro-3, a member of the novel kinase subfamily with similarity to the insulin receptor, showed intense hybridization to brain mRNA, with very faint signals in perhaps all other tissues. [0089]
  • Members of the same receptor-configured kinase subfamily occasionally exhibited very different patterns of expression. Within the elk subfamily, for example, elk itself and the related kinases tyro-5 and tyro-6 were exclusively or predominantly expressed in neural tissues, elk strongly hybridized to two mRNA species in brain and Schwann cells, tyro-5 exhibited strong hybridization to P5 brain mRNA with reduced signals present in later stage brains and in Schwann cells, and tyro-6 gave a strong hybridization signal in cultured Schwann cells, weaker signals in brain, and very faint but detectable signals in other tissues. In contrast, expression of the elk-related kinase tyro-11 was predominant in heart and kidney, but expressed at lower levels in neural tissue. The distinct hybridization patterns observed between members of this closely related subfamily indicate that despite significant similarity at the nucleotide level, cross-hybridization is not readily detected when hybridizations are carried out at high stringency. Tyro-5 and tyro-6, the most closely related of the PTK domains we analyzed, exhibit 84.2% nucleotide identity over the kinase domain, but their hybridization profiles can be readily distinguished (FIG. 1, compare [0090] profiles 5 and 6).
  • Among those kinases not restricted to neural cells, tyro-9, a member of the FGF receptor subfamily, exhibited a pattern of expression that was distinct from that of either the bFGF receptor or bek. Most strongly expressed in kidney and liver, it exhibited only weak hybridization signals with brain mRNA. At two extremes of expression, tyro-12 yielded weak hybridization signals in all tissues, with expression being somewhat lower in heart and muscle, but tyro-8 (distantly related to Dsrc28) yielded only an extremely faint signal in spleen and heart. [0091]
  • Schwann cell expression of certain kinase genes was strongly regulated by cAMP (FIG. 1). As for the PDGF receptor gene (Weinmaster and Lemke, [0092] EMBO J., 9:915-920, 1990), expression of the elk and FGF receptor genes was significantly up-regulated by 48 hr treatment with forskolin. Since cAMP induction of the PDGF receptor appears to account for the synergistic effect on Schwann cell proliferation achieved with combined application of PDGF and forskolin (Weinmaster and Lemke, EMBO J., 9:915-920, 1990), cAMP induction of the FGF receptor may also explain the similar synergistic effect observed for the combination of FGF and forskolin (Davis and Stroobant, J. Cell Biol., 110:1353-1360, 1990). Importantly, cAMP induction was not observed for most of the receptor PTKs expressed by Schwann cells; the tyro-1, tyro-3, tyro-6, tyro-7, tyro-12, and tyro-13 mRNAs were down-regulated in the presence of forskolin, and expression of the tyro-5 and tyro-11 genes was not affected by the drug.
  • Several receptor PTKs exhibited relatively modest signals in sciatic nerve compared with cultured Schwann cells or other tissues. This is probably a function of both the cellular heterogeneity of the nerve, which contains a substantial number of fibroblasts and endothelial cells, and the great sensitivity of PCR amplification. [0093]
    • EXAMPLE 4 Developmental Expression Profile of Neural PTK mRNAs
  • Since many of the determinative events in mammalian neural development occur near the midpoint of embryogenesis, a study was performed to determine whether any of the novel neural kinase genes were expressed embryonically. To assess their developmental expression, a set of Northern blots containing mRNA isolated from the brains of rats ranging in age from embryonic day 12 (E12) to adult were probed. For comparison, included were the bFGF receptor and elk in this survey, the results of which are presented in FIG. 2. For each of the novel kinase genes, expression was observed in the developing central nervous system at E12, a time at which multiple influences on both neural cell proliferation and differentiation are known to be exercised. [0094]
  • Poly(A)[0095] + RNA (2 μg) from rat brains obtained from animals of the indicated ages (E12 to 7 months postnatal) was analyzed for the expression of PTK mRNAs. Filters 1-6 show hybridization obtained with 32P-radiolabled cDNA probes to tyro-1 through tyro-6. Also shown are the hybridization profiles obtained using isolates of elk and the bFGF receptor (FGFR). The relative migration of 18S and 28S ribosomal RNAs, as determined by methylene blue staining, is indicated by the arrowheads. Exposure times are as follows: 15 hr (1, 3, 5, elk, FGFR), 22 hr (4, 6), 50 hr (2).
  • Although detected in adult brain, three of the novel kinase genes were maximally expressed embryonically. mRNA encoding the elk-related kinase tyro-6, for example, was most abundantly expressed at E12; expression gradually fell until P10 and was relatively constant thereafter. Similarly, mRNA encoding the closely related kinase tyro-5 was maximally expressed at E14; expression fell sharply after P5 to a much lower steady-state level in the adult brain. The gene encoding the eck-related kinase tyro-4 exhibited a similar, though even more dramatic regulation, with a peak in expression at E14/17, a sharp drop at birth, and a low steady-state level after P10. [0096]
  • In contrast to the pronounced drop in expression for tyro-4 and tyro-5, expression of mRNA encoding the eck-like kinase tyro-1, while exhibiting some temporal fluctuation, was relatively constant throughout neural development. A similar, though less variable-developmental profile, was observed for mRNA encoding the bFGF receptor. Although maximal expression was observed at E12, bFGF receptor mRNA levels fell only modestly during the course of brain development and remained high in adult animals. Of the novel kinase genes analyzed in FIG. 2, only tyro-3 exhibited a significant increase in expression during late neural development, with appreciably higher mRNA levels (relative to E12) evident after P20. [0097]
    • EXAMPLE 5 In situ Localization of Novel PTK Transcripts in Brain
  • To determine whether any of the novel neural kinases exhibited cell type-restricted expression in the vertebrate central nervous system, radiolabeled antisense RNA probes for each of the clones were prepared and these probes hybridized in situ to 30 μm brain sections prepared from 33-day-old and adult male rats. For comparison, antisense probes prepared from our isolates of the bFGF receptor and the related FGF receptor bek were included. [0098]
  • Although the profiles of these brain sections represented a selective sampling of the brain, they nonetheless demonstrated that expression of each of the novel neural kinases is highly regionalized. Tyro-1 mRNA was the most widely expressed in adult brain. Tyro-1 probes exhibited exceptionally strong and continuous hybridization in all fields of the hippocampus and the dentate gyrus and throughout the neocortex, with a diffuse band present in [0099] layer 3. Strong hybridization was also seen in the Purkinje cell layer, the inferior olive, and lateral nucleus of the cerebellum, but not in the cerebellar granule cell layers.
  • In contrast, the tryo-2 gene exhibited a much more restricted pattern of expression. Hybridization was again evident throughout all fields of the hippocampus and the dentate gyrus, but signals were restricted to occasional (˜1 in 10) cells. This striking, punctate pattern of hippocampal hybridization was not seen for any other PTK gene. A similarly restricted pattern of tyro-2 hybridization was also observed throughout the neocortex. Stronger and more continuous hybridization was evident in the medial habenula and in the reticular nucleus of the thalamus, but in contrast to tyro-1, no signal above background was observed in the remainder of the thalamus. The strongest tyro-2 hybridization signal in the brain was observed in an intercalated nucleus of the amygdala. No signal was evident in the Purkinje cell layer in the cerebellum. The hybridization pattern have observed for tyro-2 is largely consistent with its expression by a subset of 7-amino-n-butyric acid (GABA)-ergic neurons. [0100]
  • In situ hybridization signals corresponding to tyro-3 mRNA presented an equally striking pattern. In the hippocampus, strong hybridization was observed in the CA1 field. However, upon crossing the border from CA1 to the shorter CA2 field an abrupt drop in the tyro-3 hybridization signal was observed. The tyro-3 signal remained much reduced in CA3 (relative to CA1), and no signal at all in the dentate gyrus was observed. Tyro-3 therefore provides an excellent molecular marker for the CA1/CA2 transition, previously defined on the basis of hippocampal cell size and circuitry. Robust tyro-3 hybridization was also evident in large cells throughout neocortex, with the strongest signals being observed in deeper layers. In the cerebellum, strong hybridization was observed to granule cells, but not to Purkinje cells, a pattern that was the opposite of that observed for tyro-1. [0101]
  • Consistent with their developmental expression profiles, tyro-4, tyro-5, and tyro-6 exhibited the most restricted patterns of expression in adult brain. Distinct hybridization to tyro-4 was evident in the facial nucleus of the pons, with more modest signals present in the bed nucleus of the anterior commissure and the triangular nucleus of the septum. The tyro-5 gene was expressed weakly in cortex, at a modest level in all fields of the hippocampus, and in a subset of Purkinje cells in the cerebellum. The tyro-6 gene showed a similar pattern of expression, giving a signal in Purkinje cells and weak signals in the hippocampus. [0102]
  • The two FGF receptor genes examined, those encoding the bFGF receptor and bek, exhibited very different patterns of expression in the brain. mRNA encoding the bFGF receptor was expressed at high levels in hippocampal neurons, but exhibited a field distribution that was nearly the inverse of tyro-3., i.e., expression was reduced in CA1 relative to CA2 and CA3. mRNA levels in the dentate gyrus were lowest of all. The expression of bFGF receptor mRNA in the choroid plexus and in the central nucleus of the amygdala and in a narrow band of cells in [0103] layer 6 of neocortex, a region not seen in the previous work of Wanaka, et al. (Neuron, 5:267-281, 1990) was also observed. In contrast, expression of bek mRNA was largely confined to non-neuronal cells. High level expression was observed in the choroid plexus, and in the white matter glia of the cerebellum and the pons. Diffusely localized hybridization to a layer of cells that may be Bergmann glia was also apparent in the cerebellum. The cerebellar expression pattern of bek was clearly distinct from the patterns observed for tyro-5 and tyro-6, which marked Purkinje cells, but exhibited no hybridization to white matter glia.
    TABLE 2
    (SEQ ID NOS.:36-54)
    KINASE
    DEDUCED AMINO ACID SEQUENCES FOR PUTATIVE TYROSINE KINASES
    SUB-FAMILY VI VII VIII IX INCIDENCE
    abl abl NCLVGENH LVKVADFGLSRLMTGDTYTAH AGAKFPIKWTAPESL AYNFKSIKS  6
    arg NCLVGENH VVKVADFGLSRLMTGDTYTAH AGAKFPIKWTAPESL AYNFKSIKS  3
    fes/fps* NCLVTEKN VLKISDFGMSREEADGVYAASG GLRQVPVKWTAPEAL NYGRYSSES
    fer NCLVGENN TLKISDFGMSRQEDGGVYSSS GLKQIPIKWTAPEAL NYGRYSSES  2
    src Dsrc28* NCLVGSEN VVKVADFGLARYVLDDQYTSSG GTKFPIKWAPPEVL NYTRFSSKS
    tyro-8 NCLVDSDL SVKVSDFGMTRYVLDDQYVSSV GTKFPVKWSAPEVF HYFKTSSKS  2
    tyro-13 tyro-13 NVLVSEDN VAKVSDFGLTKEASSTQ DTGKLPVKWTAPEAL REKKFSTKS 11
    eph/eck/elk eph* NILVNQNL CCKVSDFGLTRLLDDFDGTYET QGGKIPIRWTAPEAI AHRIFTTAS
    eck NILVNSNL VCKVSDFGLSRVLEDDPEATYTT SGGKIPIRWTAPEAI SYRKFTSAS  5
    tyro-1 NILVNSNL VCKVSDFGMSRVLEDDPEAAYTT RGGKIPIRWTAPEAI AYRKFTSAS  1
    tyro-4 NILINSNL VCKVSDFGLSRVLEDDPEAAYTT RGGKIPIRWTSPEAI AYRKFTSAS  4
    elk NILVNSNL VCKVSDFGLSRYLQDDTSDPTYTSS LGGKIPVRWTAPEAI AYRKFTSAS  1
    tyro-5 NILVNSNL VCKVSDFGLSRFLEDDTSDPTYTSA LGGKIPIRWTAPEAI QYRKFTSAS (3)
    tyro-6 NILVNSNL VCKVSDFGLSRFLEDDPSDPTYTSS LGGKIPIRWTAPEAI AYRKFTSAS  3
    tyro-11 NILVNSNL VCKVSDFGLSRFLEENSSDPTYTSS LGGKIPIRWTAPEAI AFRKFTSAS (1)
    EGF-R EGF-R NVLVKTPQ HVKITDFGLAKLLGAEEKEYHA EGGKVPIKWMALESI LHRIYTHQS  3
    neu NVLVKSPN HVKITDFGLARLLDIDETEYHA DGGKVPIKWMALESI LRRRFTHQS 10
    tyro-2 NVLVKSPN HVKITDFGLARLLEGDEKEYNA DGGKMPIKWMALECI HYRKFTHQS  8
    FGF-R bFGF-R NVLVTEDN VMKIADFGLARDIHHLDYYKKT TNGRLPVKWMPEAL FDRIYTHQS  4
    bek NVLVTENN VMKIADFGLARDINNIDYYKKT TNGRLPVKWMAPEAL FDRVYTHQS  2
    tyro-9 NVLVTEDD VMKIADFGLARGVHHIDYYKKT SNGRLPVKWMAPEAL FDRVYTHQS  1
    PDGF-R PDGF-AR NVLLAQGK IVKICDFGLARDIMHDSNYVSK GSTFLPVKWMAPESI FDNLYTTLS  3
    PDGF-BR NMLICEGK LVKICDFGLARDIMRDSNYISK GSTFLPLKWMAPESI FNSLYTTLS  1
    CSF-1R NVLLTSGH VAKIGDFGLARDIMNDSNYVVK GNARLPVKWMAPESI FDCVYTVQS 20
    flt NLLLSENN VVKICDFGLARDIYKNPDYVRR GDTRLPLKWMAPESI FDKVYSTKS  5
    tyro-3 tyro-3 NCMLAEDM TVCVADFGLSRKIYSGDYYRQG CASKLPVKWLALESL ADNLYTVHS  3
    tyro-7 NCMLNENM SVCVADFGLSKKIYNGDYYRQG PFAKMPVKWIAIESL ADRVYTSKS  4
    tyro-12 NCMLRDDM TVCVADFGLSKKIYSGDYYRQG RIAKMPVKWIAIESL ADRVTYSKS (3)
    Insulin-R trk* NCLVGQGL VVKIGDFGMSRDIYSTDYYRVG GRTMLPIRWMPPESI LYRKETTES
    trkB* NCLVGENL LVKIGDFGMSRDVYSTDYYRVG GHTMLPIRWMPPESI MYRKFTTES
    IGF1R NCMVAEDF TVKIGDFGMTRDIYETDYYRKG GKGLLPVRWMSPESL KDGVFTTHS  2
    tyro-10 NCLVGKNY TIKIADFGMSRNLYSGDYYRIQ GRAVLPIRWMSWESI LLGKFTTAS (6)
  • The foregoing is meant to illustrate, but not to limit, the scope of the invention. Indeed, those of ordinary skill in the art can readily envision and produce further embodiments, based on the teachings herein, without undue experimentation. [0104]
  • 1 54 165 base pairs nucleic acid single linear DNA Tyro-1 CDS 1..165 1 AAC ATT CTG GTA AAC AGC AAC TTG GTC TGC AAG GTG TCT GAT TTC GGC 48 Asn Ile Leu Val Asn Ser Asn Leu Val Cys Lys Val Ser Asp Phe Gly 1 5 10 15 ATG TCC AGG GTG CTT GAG GAT GAC CCG GAA GCA GCC TAT ACT ACC AGG 96 Met Ser Arg Val Leu Glu Asp Asp Pro Glu Ala Ala Tyr Thr Thr Arg 20 25 30 GGC GGC AAG ATT CCC ATC CGG TGG ACT GCA CCA GAA GCA ATT GCG TAT 144 Gly Gly Lys Ile Pro Ile Arg Trp Thr Ala Pro Glu Ala Ile Ala Tyr 35 40 45 CGT AAA TTT ACC TCA GCC AGT 165 Arg Lys Phe Thr Ser Ala Ser 50 55 55 amino acids amino acid linear protein 2 Asn Ile Leu Val Asn Ser Asn Leu Val Cys Lys Val Ser Asp Phe Gly 1 5 10 15 Met Ser Arg Val Leu Glu Asp Asp Pro Glu Ala Ala Tyr Thr Thr Arg 20 25 30 Gly Gly Lys Ile Pro Ile Arg Trp Thr Ala Pro Glu Ala Ile Ala Tyr 35 40 45 Arg Lys Phe Thr Ser Ala Ser 50 55 2437 base pairs nucleic acid single linear DNA Tyro-2 CDS 3..2118 3 CA AAC TGT GTG GAG AAA TGT CCA GAT GGC CTA CAG GGA GCA AAC AGT 47 Asn Cys Val Glu Lys Cys Pro Asp Gly Leu Gln Gly Ala Asn Ser 1 5 10 15 TTC ATT TTT AAG TAT GCA GAT CAG GAT CGG GAG TGC CAC CCT TGC CAT 95 Phe Ile Phe Lys Tyr Ala Asp Gln Asp Arg Glu Cys His Pro Cys His 20 25 30 CCA AAC TGC ACC CAG GGG TGT AAC GGT CCC ACT AGT CAT GAC TGC ATT 143 Pro Asn Cys Thr Gln Gly Cys Asn Gly Pro Thr Ser His Asp Cys Ile 35 40 45 TAC TAC CCA TGG ACG GGC CAT TCC ACT TTA CCA CAA CAC GCT AGA ACT 191 Tyr Tyr Pro Trp Thr Gly His Ser Thr Leu Pro Gln His Ala Arg Thr 50 55 60 CCA CTG ATT GCA GCC GGA GTC ATT GGA GGC CTC TTC ATC CTG GTG ATC 239 Pro Leu Ile Ala Ala Gly Val Ile Gly Gly Leu Phe Ile Leu Val Ile 65 70 75 ATG GCT TTG ACA TTT GCT GTC TAT GTC AGA AGA AAG AGC ATC AAA AAG 287 Met Ala Leu Thr Phe Ala Val Tyr Val Arg Arg Lys Ser Ile Lys Lys 80 85 90 95 AAA CGT GCT TTG AGG AGA TTC CTG GAG ACA GAG CTG GTA GAG CCC TTA 335 Lys Arg Ala Leu Arg Arg Phe Leu Glu Thr Glu Leu Val Glu Pro Leu 100 105 110 ACT CCC AGT GGC ACG GCA CCC AAT CAA GCT CAA CTT CGC ATT TTG AAG 383 Thr Pro Ser Gly Thr Ala Pro Asn Gln Ala Gln Leu Arg Ile Leu Lys 115 120 125 GAA ACC GAA CTA AAG AGG GTA AAG GTC CTT GGC TCG GGA GCT TTT GGA 431 Glu Thr Glu Leu Lys Arg Val Lys Val Leu Gly Ser Gly Ala Phe Gly 130 135 140 ACC GTT TAT AAA GGT ATT TGG GTG CCT GAA GGT GAA ACA GTG AAA ATC 479 Thr Val Tyr Lys Gly Ile Trp Val Pro Glu Gly Glu Thr Val Lys Ile 145 150 155 CCT GTG GCT ATA AAG ATC CTC AAT GAA ACA ACT GGC CCC AAA GCC AAC 527 Pro Val Ala Ile Lys Ile Leu Asn Glu Thr Thr Gly Pro Lys Ala Asn 160 165 170 175 GTG GAG TTC ATG GAT GAG GCT CTG ATC ATG GCA AGT ATG GAT CAC CCA 575 Val Glu Phe Met Asp Glu Ala Leu Ile Met Ala Ser Met Asp His Pro 180 185 190 CAC CTA GTT CGC CTA TTG GGA GTG TGT CTG AGT CCC ACT ATC CAG TTG 623 His Leu Val Arg Leu Leu Gly Val Cys Leu Ser Pro Thr Ile Gln Leu 195 200 205 GTT ACG CAG CTG ATG CCG CAT GCG TGC CTA CTG GAC TAT GTT CAT GAA 671 Val Thr Gln Leu Met Pro His Ala Cys Leu Leu Asp Tyr Val His Glu 210 215 220 CAC AAG GAT AAC ATT GGA TCA CAG CTG CTG TTG AAC TGG TGT GTC CAG 719 His Lys Asp Asn Ile Gly Ser Gln Leu Leu Leu Asn Trp Cys Val Gln 225 230 235 ATT GCT AAG GGA ATG ATG TAC CTA GAA GAA AGA CGG CTT GTT CAT CGG 767 Ile Ala Lys Gly Met Met Tyr Leu Glu Glu Arg Arg Leu Val His Arg 240 245 250 255 GAT CTG GCA GCC CGC AAT GTC TTA GTG AAA TCT CCA AAT CAT GTT AAA 815 Asp Leu Ala Ala Arg Asn Val Leu Val Lys Ser Pro Asn His Val Lys 260 265 270 ATC ACA GAT TTT GGA CTG GCC CGG CTC TTG GAA GGA GAT GAA AAA GAA 863 Ile Thr Asp Phe Gly Leu Ala Arg Leu Leu Glu Gly Asp Glu Lys Glu 275 280 285 TAC AAT GCT GAT GGT GGC AAG ATG CCA ATT AAA TGG ATG GCT CTG GAA 911 Tyr Asn Ala Asp Gly Gly Lys Met Pro Ile Lys Trp Met Ala Leu Glu 290 295 300 TGT ATA CAT TAT AGG AAA TTC ACA CAT CAA AGT GAT GTT TGG AGC TAT 959 Cys Ile His Tyr Arg Lys Phe Thr His Gln Ser Asp Val Trp Ser Tyr 305 310 315 GGC GTC ACT ATA TGG GAA CTG ATG ACC TTT GGA GGA AAG CCC TAT GAT 1007 Gly Val Thr Ile Trp Glu Leu Met Thr Phe Gly Gly Lys Pro Tyr Asp 320 325 330 335 GGA ATT CCA ACC CGA GAA ATC CCC GAT TTA CTG GAG AAA GGA GAG CGT 1055 Gly Ile Pro Thr Arg Glu Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg 340 345 350 CTG CCT CAG CCT CCC ATC TGC ACT ATT GAT GTT TAC ATG GTC ATG GTC 1103 Leu Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr Met Val Met Val 355 360 365 AAA TGT TGG ATG ATC GAT GCT GAC AGC AGA CCT AAA TTC AAA GAA CTG 1151 Lys Cys Trp Met Ile Asp Ala Asp Ser Arg Pro Lys Phe Lys Glu Leu 370 375 380 GCT GCT GAG TTT TCA AGA ATG GCT AGA GAC CCT CAA AGA TAC CTA GTT 1199 Ala Ala Glu Phe Ser Arg Met Ala Arg Asp Pro Gln Arg Tyr Leu Val 385 390 395 ATT CAG GGT GAT GAT CGT ATG AAG CTT CCC AGT CCA AAT GAC AGC AAA 1247 Ile Gln Gly Asp Asp Arg Met Lys Leu Pro Ser Pro Asn Asp Ser Lys 400 405 410 415 TTC TTC CAG AAT CTC TTG GAT GAA GAG GAT TTG GAA GAC ATG ATG GAT 1295 Phe Phe Gln Asn Leu Leu Asp Glu Glu Asp Leu Glu Asp Met Met Asp 420 425 430 GCT GAG GAA TAT TTG GTC CCC CAG GCT TTC AAC ATA CCT CCT CCC ATC 1343 Ala Glu Glu Tyr Leu Val Pro Gln Ala Phe Asn Ile Pro Pro Pro Ile 435 440 445 TAC ACA TCC AGA ACA AGA ATT GAC TCC AAT AGG AAT CAG TTT GTG TAC 1391 Tyr Thr Ser Arg Thr Arg Ile Asp Ser Asn Arg Asn Gln Phe Val Tyr 450 455 460 CAA GAT GGG GGC TTT GCT ACA CAA CAA GGA ATG CCC ATG CCC TAC AGA 1439 Gln Asp Gly Gly Phe Ala Thr Gln Gln Gly Met Pro Met Pro Tyr Arg 465 470 475 GCC ACA ACC AGC ACC ATA CCA GAG GCT CCA GTA GCT CAG GGT GCA ACG 1487 Ala Thr Thr Ser Thr Ile Pro Glu Ala Pro Val Ala Gln Gly Ala Thr 480 485 490 495 GCT GAG ATG TTT GAT GAC TCC TGC TGT AAT GGT ACC CTA CGA AAG CCA 1535 Ala Glu Met Phe Asp Asp Ser Cys Cys Asn Gly Thr Leu Arg Lys Pro 500 505 510 GTG GCA CCC CAT GTC CAA GAG GAC AGT AGC ACT CAG AGG TAT AGT GCT 1583 Val Ala Pro His Val Gln Glu Asp Ser Ser Thr Gln Arg Tyr Ser Ala 515 520 525 GAT CCC ACA GTG TTC GCC CCA GAA CGG AAT CCT CGA GGA GAA CTG GAT 1631 Asp Pro Thr Val Phe Ala Pro Glu Arg Asn Pro Arg Gly Glu Leu Asp 530 535 540 GAA GAA GGC TAC ATG ACT CCA ATG CAT GAC AAG CCC AAA CAA GAA TAT 1679 Glu Glu Gly Tyr Met Thr Pro Met His Asp Lys Pro Lys Gln Glu Tyr 545 550 555 CTG AAT CCT GTG GAA GAG AAC CCT TTT GTG TCC CGA AGG AAG AAT GGA 1727 Leu Asn Pro Val Glu Glu Asn Pro Phe Val Ser Arg Arg Lys Asn Gly 560 565 570 575 GAT CTT CAA GCT TTA GAT AAT CCG GAG TAT CAC AGT GCT TCC AGC GGT 1775 Asp Leu Gln Ala Leu Asp Asn Pro Glu Tyr His Ser Ala Ser Ser Gly 580 585 590 CCA CCC AAG GCG GAG GAT GAA TAC GTG AAT GAG CCT CTA TAC CTC AAC 1823 Pro Pro Lys Ala Glu Asp Glu Tyr Val Asn Glu Pro Leu Tyr Leu Asn 595 600 605 ACC TTC GCC AAT GCC TTG GGG AGT GCA GAG TAC ATG AAA AAC AGT GTA 1871 Thr Phe Ala Asn Ala Leu Gly Ser Ala Glu Tyr Met Lys Asn Ser Val 610 615 620 CTG TCT GTG CCA GAG AAA GCC AAG AAA GCA TTT GAC AAC CCC GAC TAC 1919 Leu Ser Val Pro Glu Lys Ala Lys Lys Ala Phe Asp Asn Pro Asp Tyr 625 630 635 TGG AAC CAC AGC CTG CCA CCC CGG AGC ACC CTT CAG CAC CCA GAC TAC 1967 Trp Asn His Ser Leu Pro Pro Arg Ser Thr Leu Gln His Pro Asp Tyr 640 645 650 655 CTG CAG GAA TAC AGC ACA AAA TAT TTT TAT AAA CAG AAT GGA CGG ATC 2015 Leu Gln Glu Tyr Ser Thr Lys Tyr Phe Tyr Lys Gln Asn Gly Arg Ile 660 665 670 CGC CCC ATT GTG GCA GAG AAT CCT GAG TAC CTC TCG GAG TTC TCG CTG 2063 Arg Pro Ile Val Ala Glu Asn Pro Glu Tyr Leu Ser Glu Phe Ser Leu 675 680 685 AAG CCT GGC ACT ATG CTG CCC CCT CCG CCC TAC AGA CAC CGG AAT ACT 2111 Lys Pro Gly Thr Met Leu Pro Pro Pro Pro Tyr Arg His Arg Asn Thr 690 695 700 GTG GTG T GAGCTTGGCT AGAGTGTTAG GTGGAGAAAC ACACACCCAC TCCATTTCCC 2168 Val Val 705 TTCCCCCTCC TCTTTCTCTG GTGGTCTTCC TTCTTCTCCC AAGGCCAGTA GTTTTGACAC 2228 TTCCAAGTGG AAGCAGTAGA GATGCAATGA TAGTTCTGTG CTTACCTAAC TTGAATATTA 2288 GAAGGAAAGA CTGAAAGAGA AAGACAGGGA TACACACACT GTTTCTTCGT TTCTTCATAT 2348 GGGTTGGTTA ACAGAGTGTC AAAGCTAGAG AAGGTCTAGG AAGTATAAGG CAATACTGCC 2408 TGCTGTCAAA GAGCCCCATC TTTCTTCTC 2437 705 amino acids amino acid linear protein 4 Asn Cys Val Glu Lys Cys Pro Asp Gly Leu Gln Gly Ala Asn Ser Phe 1 5 10 15 Ile Phe Lys Tyr Ala Asp Gln Asp Arg Glu Cys His Pro Cys His Pro 20 25 30 Asn Cys Thr Gln Gly Cys Asn Gly Pro Thr Ser His Asp Cys Ile Tyr 35 40 45 Tyr Pro Trp Thr Gly His Ser Thr Leu Pro Gln His Ala Arg Thr Pro 50 55 60 Leu Ile Ala Ala Gly Val Ile Gly Gly Leu Phe Ile Leu Val Ile Met 65 70 75 80 Ala Leu Thr Phe Ala Val Tyr Val Arg Arg Lys Ser Ile Lys Lys Lys 85 90 95 Arg Ala Leu Arg Arg Phe Leu Glu Thr Glu Leu Val Glu Pro Leu Thr 100 105 110 Pro Ser Gly Thr Ala Pro Asn Gln Ala Gln Leu Arg Ile Leu Lys Glu 115 120 125 Thr Glu Leu Lys Arg Val Lys Val Leu Gly Ser Gly Ala Phe Gly Thr 130 135 140 Val Tyr Lys Gly Ile Trp Val Pro Glu Gly Glu Thr Val Lys Ile Pro 145 150 155 160 Val Ala Ile Lys Ile Leu Asn Glu Thr Thr Gly Pro Lys Ala Asn Val 165 170 175 Glu Phe Met Asp Glu Ala Leu Ile Met Ala Ser Met Asp His Pro His 180 185 190 Leu Val Arg Leu Leu Gly Val Cys Leu Ser Pro Thr Ile Gln Leu Val 195 200 205 Thr Gln Leu Met Pro His Ala Cys Leu Leu Asp Tyr Val His Glu His 210 215 220 Lys Asp Asn Ile Gly Ser Gln Leu Leu Leu Asn Trp Cys Val Gln Ile 225 230 235 240 Ala Lys Gly Met Met Tyr Leu Glu Glu Arg Arg Leu Val His Arg Asp 245 250 255 Leu Ala Ala Arg Asn Val Leu Val Lys Ser Pro Asn His Val Lys Ile 260 265 270 Thr Asp Phe Gly Leu Ala Arg Leu Leu Glu Gly Asp Glu Lys Glu Tyr 275 280 285 Asn Ala Asp Gly Gly Lys Met Pro Ile Lys Trp Met Ala Leu Glu Cys 290 295 300 Ile His Tyr Arg Lys Phe Thr His Gln Ser Asp Val Trp Ser Tyr Gly 305 310 315 320 Val Thr Ile Trp Glu Leu Met Thr Phe Gly Gly Lys Pro Tyr Asp Gly 325 330 335 Ile Pro Thr Arg Glu Ile Pro Asp Leu Leu Glu Lys Gly Glu Arg Leu 340 345 350 Pro Gln Pro Pro Ile Cys Thr Ile Asp Val Tyr Met Val Met Val Lys 355 360 365 Cys Trp Met Ile Asp Ala Asp Ser Arg Pro Lys Phe Lys Glu Leu Ala 370 375 380 Ala Glu Phe Ser Arg Met Ala Arg Asp Pro Gln Arg Tyr Leu Val Ile 385 390 395 400 Gln Gly Asp Asp Arg Met Lys Leu Pro Ser Pro Asn Asp Ser Lys Phe 405 410 415 Phe Gln Asn Leu Leu Asp Glu Glu Asp Leu Glu Asp Met Met Asp Ala 420 425 430 Glu Glu Tyr Leu Val Pro Gln Ala Phe Asn Ile Pro Pro Pro Ile Tyr 435 440 445 Thr Ser Arg Thr Arg Ile Asp Ser Asn Arg Asn Gln Phe Val Tyr Gln 450 455 460 Asp Gly Gly Phe Ala Thr Gln Gln Gly Met Pro Met Pro Tyr Arg Ala 465 470 475 480 Thr Thr Ser Thr Ile Pro Glu Ala Pro Val Ala Gln Gly Ala Thr Ala 485 490 495 Glu Met Phe Asp Asp Ser Cys Cys Asn Gly Thr Leu Arg Lys Pro Val 500 505 510 Ala Pro His Val Gln Glu Asp Ser Ser Thr Gln Arg Tyr Ser Ala Asp 515 520 525 Pro Thr Val Phe Ala Pro Glu Arg Asn Pro Arg Gly Glu Leu Asp Glu 530 535 540 Glu Gly Tyr Met Thr Pro Met His Asp Lys Pro Lys Gln Glu Tyr Leu 545 550 555 560 Asn Pro Val Glu Glu Asn Pro Phe Val Ser Arg Arg Lys Asn Gly Asp 565 570 575 Leu Gln Ala Leu Asp Asn Pro Glu Tyr His Ser Ala Ser Ser Gly Pro 580 585 590 Pro Lys Ala Glu Asp Glu Tyr Val Asn Glu Pro Leu Tyr Leu Asn Thr 595 600 605 Phe Ala Asn Ala Leu Gly Ser Ala Glu Tyr Met Lys Asn Ser Val Leu 610 615 620 Ser Val Pro Glu Lys Ala Lys Lys Ala Phe Asp Asn Pro Asp Tyr Trp 625 630 635 640 Asn His Ser Leu Pro Pro Arg Ser Thr Leu Gln His Pro Asp Tyr Leu 645 650 655 Gln Glu Tyr Ser Thr Lys Tyr Phe Tyr Lys Gln Asn Gly Arg Ile Arg 660 665 670 Pro Ile Val Ala Glu Asn Pro Glu Tyr Leu Ser Glu Phe Ser Leu Lys 675 680 685 Pro Gly Thr Met Leu Pro Pro Pro Pro Tyr Arg His Arg Asn Thr Val 690 695 700 Val 705 3307 base pairs nucleic acid single linear DNA Tyro-3 CDS 237..2859 5 CGGCGGCGGC GGCGGCTGTG GAAGGAGCGC GGTGGCCCAG CCGCAGCCCC GGGGACTCCT 60 CGCTGCTGAC GGCGGTGGCC GCGGCTCTAG GCGGCCGCGG GTCCGGGACG CCCGGGCCGA 120 GCGCCGCCCC CCGCCCCTCC CGCGGGCCTC CCGCCCCTCC TCCGCCACCC TCCTCTCTGC 180 GCTCGCGGGC CGGGCCCGGC ATGGTGCGGC GTCGCCGCCG ATGGCTGAGG CGGAGC 236 ATG GGG TGG CCG GGG CTC CGG CCG CTG CTG CTG GCG GGA CTG GCT TCT 284 Met Gly Trp Pro Gly Leu Arg Pro Leu Leu Leu Ala Gly Leu Ala Ser 1 5 10 15 CTG CTG CTC CCC GGG TCT GCG GCC GCA GGC CTG AAG CTC ATG GGC GCC 332 Leu Leu Leu Pro Gly Ser Ala Ala Ala Gly Leu Lys Leu Met Gly Ala 20 25 30 CCA GTG AAG ATG ACC GTG TCT CAG GGG CAG CCA GTG AAG CTC AAC TGC 380 Pro Val Lys Met Thr Val Ser Gln Gly Gln Pro Val Lys Leu Asn Cys 35 40 45 AGC GTG GAG GGG ATG GAG GAC CCT GAC ATC CAC TGG ATG AAG GAT GGC 428 Ser Val Glu Gly Met Glu Asp Pro Asp Ile His Trp Met Lys Asp Gly 50 55 60 ACC GTG GTC CAG AAT GCA AGC CAG GTG TCC ATC TCC ATC AGC GAG CAC 476 Thr Val Val Gln Asn Ala Ser Gln Val Ser Ile Ser Ile Ser Glu His 65 70 75 80 AGC TGG ATT GGC TTA CTC AGC CTA AAG TCA GTG GAA CGG TCT GAT GCT 524 Ser Trp Ile Gly Leu Leu Ser Leu Lys Ser Val Glu Arg Ser Asp Ala 85 90 95 GGC CTG TAC TGG TGC CAG GTG AAG GAT GGG GAG GAA ACC AAG ATT TCT 572 Gly Leu Tyr Trp Cys Gln Val Lys Asp Gly Glu Glu Thr Lys Ile Ser 100 105 110 CAG TCA GTA TGG CTC ACT GTC GAA GGT GTG CCA TTC TTC ACA GTG GAA 620 Gln Ser Val Trp Leu Thr Val Glu Gly Val Pro Phe Phe Thr Val Glu 115 120 125 CCA AAA GAT CTG GCG GTG CCA CCC AAT GCC CCT TTT CAG CTG TCT TGT 668 Pro Lys Asp Leu Ala Val Pro Pro Asn Ala Pro Phe Gln Leu Ser Cys 130 135 140 GAG GCT GTG GGT CCT CCA GAA CCC GTA ACC ATT TAC TGG TGG AGA GGA 716 Glu Ala Val Gly Pro Pro Glu Pro Val Thr Ile Tyr Trp Trp Arg Gly 145 150 155 160 CTC ACT AAG GTT GGG GGA CCT GCT CCC TCT CCC TCT GTT TTA AAT GTG 764 Leu Thr Lys Val Gly Gly Pro Ala Pro Ser Pro Ser Val Leu Asn Val 165 170 175 ACA GGA GTG ACC CAG CGC ACA GAG TTT TCT TGT GAA GCC CGC AAC ATA 812 Thr Gly Val Thr Gln Arg Thr Glu Phe Ser Cys Glu Ala Arg Asn Ile 180 185 190 AAA GGC CTG GCC ACT TCC CGA CCA GCC ATT GTT CGC CTT CAA GCA CCG 860 Lys Gly Leu Ala Thr Ser Arg Pro Ala Ile Val Arg Leu Gln Ala Pro 195 200 205 CCT GCA GCT CCT TTC AAC ACC ACA GTA ACA ACG ATC TCC AGC TAC AAC 908 Pro Ala Ala Pro Phe Asn Thr Thr Val Thr Thr Ile Ser Ser Tyr Asn 210 215 220 GCT AGC GTG GCC TGG GTG CCA GGT GCT GAC GGC CTA GCT CTG CTG CAT 956 Ala Ser Val Ala Trp Val Pro Gly Ala Asp Gly Leu Ala Leu Leu His 225 230 235 240 TCC TGT ACT GTA CAG GTG GCA CAC GCC CCA GGA GAA TGG GAG GCC CTT 1004 Ser Cys Thr Val Gln Val Ala His Ala Pro Gly Glu Trp Glu Ala Leu 245 250 255 GCT GTT GTG GTT CCT GTG CCA CCT TTT ACC TGC CTG CTT CGG AAC TTG 1052 Ala Val Val Val Pro Val Pro Pro Phe Thr Cys Leu Leu Arg Asn Leu 260 265 270 GCC CCT GCC ACC AAC TAC AGC CTT AGG GTG CGC TGT GCC AAT GCC TTG 1100 Ala Pro Ala Thr Asn Tyr Ser Leu Arg Val Arg Cys Ala Asn Ala Leu 275 280 285 GGC CCT TCT CCC TAC GGC GAC TGG GTG CCC TTT CAG ACA AAG GGC CTA 1148 Gly Pro Ser Pro Tyr Gly Asp Trp Val Pro Phe Gln Thr Lys Gly Leu 290 295 300 GCG CCA CGC AGA GCT CCT CAG AAT TTC CAT GCC ATT CGT ACC GAC TCA 1196 Ala Pro Arg Arg Ala Pro Gln Asn Phe His Ala Ile Arg Thr Asp Ser 305 310 315 320 GGC CTT ATC CTG GAA TGG GAA GAA GTG ATT CCT GAG GAC CCT GGG GAA 1244 Gly Leu Ile Leu Glu Trp Glu Glu Val Ile Pro Glu Asp Pro Gly Glu 325 330 335 GGC CCC CTA GGA CCT TAT AAG CTG TCC TGG GTC CAA GAA AAT GGA ACC 1292 Gly Pro Leu Gly Pro Tyr Lys Leu Ser Trp Val Gln Glu Asn Gly Thr 340 345 350 CAG GAT GAG CTG ATG GTG GAA GGG ACC AGG GCC AAT CTG ACC GAC TGG 1340 Gln Asp Glu Leu Met Val Glu Gly Thr Arg Ala Asn Leu Thr Asp Trp 355 360 365 GTA CCC CAG AAG GAC CTG ATT TTG CGT GTG TGT GCC TCC AAT GCA ATT 1388 Val Pro Gln Lys Asp Leu Ile Leu Arg Val Cys Ala Ser Asn Ala Ile 370 375 380 GGT GAT GGG CCC TGG AGT CAG CCA CTG GTG GTG TCT TCT CAT GAC CAT 1436 Gly Asp Gly Pro Trp Ser Gln Pro Leu Val Val Ser Ser His Asp His 385 390 395 400 GCA GGG AGG CAG GGC CCT CCC CAC AGC CGC ACA TCC TGG GTG CCT GTG 1484 Ala Gly Arg Gln Gly Pro Pro His Ser Arg Thr Ser Trp Val Pro Val 405 410 415 GTC CTG GGC GTG CTC ACC GCC CTG ATC ACA GCT GCT GCC TTG GCC CTC 1532 Val Leu Gly Val Leu Thr Ala Leu Ile Thr Ala Ala Ala Leu Ala Leu 420 425 430 ATC CTG CTT CGG AAG AGA CGC AAG GAG ACG CGT TTC GGG CAA GCC TTT 1580 Ile Leu Leu Arg Lys Arg Arg Lys Glu Thr Arg Phe Gly Gln Ala Phe 435 440 445 GAC AGT GTC ATG GCC CGA GGG GAG CCA GCT GTA CAC TTC CGG GCA GCC 1628 Asp Ser Val Met Ala Arg Gly Glu Pro Ala Val His Phe Arg Ala Ala 450 455 460 CGA TCT TTC AAT CGA GAA AGG CCT GAA CGC ATT GAG GCC ACA TTG GAT 1676 Arg Ser Phe Asn Arg Glu Arg Pro Glu Arg Ile Glu Ala Thr Leu Asp 465 470 475 480 AGC CTG GGC ATC AGC GAT GAA TTG AAG GAA AAG CTG GAG GAT GTC CTC 1724 Ser Leu Gly Ile Ser Asp Glu Leu Lys Glu Lys Leu Glu Asp Val Leu 485 490 495 ATT CCA GAG CAG CAG TTC ACC CTC GGT CGG ATG TTG GGC AAA GGA GAG 1772 Ile Pro Glu Gln Gln Phe Thr Leu Gly Arg Met Leu Gly Lys Gly Glu 500 505 510 TTT GGA TCA GTG CGG GAA GCC CAG CTA AAG CAG GAA GAT GGC TCC TTC 1820 Phe Gly Ser Val Arg Glu Ala Gln Leu Lys Gln Glu Asp Gly Ser Phe 515 520 525 GTG AAA GTG GCA GTG AAG ATG CTG AAA GCT GAC ATC ATT GCC TCA AGC 1868 Val Lys Val Ala Val Lys Met Leu Lys Ala Asp Ile Ile Ala Ser Ser 530 535 540 GAC ATA GAA GAG TTC CTC CGG GAA GCA GCT TGC ATG AAG GAG TTT GAC 1916 Asp Ile Glu Glu Phe Leu Arg Glu Ala Ala Cys Met Lys Glu Phe Asp 545 550 555 560 CAT CCA CAC GTG GCC AAG CTT GTT GGG GTG AGC CTC CGG AGC AGG GCT 1964 His Pro His Val Ala Lys Leu Val Gly Val Ser Leu Arg Ser Arg Ala 565 570 575 AAA GGT CGT CTC CCC ATT CCC ATG GTC ATC CTG CCC TTC ATG AAA CAT 2012 Lys Gly Arg Leu Pro Ile Pro Met Val Ile Leu Pro Phe Met Lys His 580 585 590 GGA GAC TTG CAC GCC TTT CTG CTC GCC TCC CGA ATC GGG GAG AAC CCT 2060 Gly Asp Leu His Ala Phe Leu Leu Ala Ser Arg Ile Gly Glu Asn Pro 595 600 605 TTT AAC CTG CCC CTC CAG ACC CTG GTC CGG TTC ATG GTG GAC ATT CGC 2108 Phe Asn Leu Pro Leu Gln Thr Leu Val Arg Phe Met Val Asp Ile Arg 610 615 620 TGT GGC ATG GAG TAC CTG AGC TCC CGG AAC TTC ATC CAC CGA GAC CTA 2156 Cys Gly Met Glu Tyr Leu Ser Ser Arg Asn Phe Ile His Arg Asp Leu 625 630 635 640 GCA GCT CGG AAT TGC ATG CTG GCC GAG GAC ATG ACA GTG TGT GTG GCT 2204 Ala Ala Arg Asn Cys Met Leu Ala Glu Asp Met Thr Val Cys Val Ala 645 650 655 GAT TTT GGA CTC TCT CGG AAA ATC TAT AGC GGG GAC TAT TAT CGT CAG 2252 Asp Phe Gly Leu Ser Arg Lys Ile Tyr Ser Gly Asp Tyr Tyr Arg Gln 660 665 670 GGC TGT GCC TCC AAA TTG CCC GTC AAG TGG CTG GCC CTG GAG AGC TTG 2300 Gly Cys Ala Ser Lys Leu Pro Val Lys Trp Leu Ala Leu Glu Ser Leu 675 680 685 GCT GAC AAC TTG TAT ACT GTA CAC AGT GAT GTG TGG GCC TTC GGG GTG 2348 Ala Asp Asn Leu Tyr Thr Val His Ser Asp Val Trp Ala Phe Gly Val 690 695 700 ACC ATG TGG GAG ATC ATG ACT CGT GGG CAG ACG CCA TAT GCT GGC ATT 2396 Thr Met Trp Glu Ile Met Thr Arg Gly Gln Thr Pro Tyr Ala Gly Ile 705 710 715 720 GAA AAT GCC GAG ATT TAC AAC TAC CTC ATC GGC GGG AAC CGC CTG AAG 2444 Glu Asn Ala Glu Ile Tyr Asn Tyr Leu Ile Gly Gly Asn Arg Leu Lys 725 730 735 CAG CCT CCG GAG TGC ATG GAG GAA GTG TAT GAT CTC ATG TAC CAG TGC 2492 Gln Pro Pro Glu Cys Met Glu Glu Val Tyr Asp Leu Met Tyr Gln Cys 740 745 750 TGG AGC GCC GAC CCC AAG CAG CGC CCA AGC TTC ACG TGT CTG CGA ATG 2540 Trp Ser Ala Asp Pro Lys Gln Arg Pro Ser Phe Thr Cys Leu Arg Met 755 760 765 GAA CTG GAG AAC ATT CTG GGC CAC CTG TCT GTG CTG TCC ACC AGC CAG 2588 Glu Leu Glu Asn Ile Leu Gly His Leu Ser Val Leu Ser Thr Ser Gln 770 775 780 GAC CCC TTG TAC ATC AAC ATT GAG AGA GCT GAG CAG CCT ACT GAG AGT 2636 Asp Pro Leu Tyr Ile Asn Ile Glu Arg Ala Glu Gln Pro Thr Glu Ser 785 790 795 800 GGC AGC CCT GAG GTC CAC TGT GGA GAG CGA TCC AGC AGC GAG GCA GGG 2684 Gly Ser Pro Glu Val His Cys Gly Glu Arg Ser Ser Ser Glu Ala Gly 805 810 815 GAC GGC AGT GGC GTG GGG GCA GTA GGT GGC ATC CCC AGT GAC TCT CGG 2732 Asp Gly Ser Gly Val Gly Ala Val Gly Gly Ile Pro Ser Asp Ser Arg 820 825 830 TAC ATC TTC AGC CCC GGA GGG CTA TCC GAG TCA CCA GGG CAG CTG GAG 2780 Tyr Ile Phe Ser Pro Gly Gly Leu Ser Glu Ser Pro Gly Gln Leu Glu 835 840 845 CAG CAG CCA GAA AGC CCC CTC AAT GAG AAC CAG AGG CTG TTG TTG CTG 2828 Gln Gln Pro Glu Ser Pro Leu Asn Glu Asn Gln Arg Leu Leu Leu Leu 850 855 860 CAG CAA GGG CTA CTG CCT CAC AGT AGC TGT T AACCCTCAGG CAGAGGAAAG 2879 Gln Gln Gly Leu Leu Pro His Ser Ser Cys 865 870 TTGGGGCCCC TGGCTCTGCT GACCGCTGTG CTGCCTGACT AGGCCCAGTC TGATCACAGC 2939 CCAGGCAGCA AGGTATGGAG GCTCCTGTGG TAGCCCTCCC AAGCTGTGCT GGCGCCTGGA 2999 CGGACCAAAT TGCCCAATCC CAGTTCTTCC TGCAGCCGCT CTGGCCAGCC TGGCATCAGT 3059 TCAGGCCTTG GCTTACAGGA GGTGAGCCAG AGCTGGTTGC CTGAATGCAG GCAGCTGGCA 3119 GGAGGGGAGG GTGGCTATGT TTCCATGGGT ACCATGGTTG TGGATGGCAG TAAGGGAGGG 3179 TAGCAACAGC CCTGTGCGCC CTACCCTCCT GGCTGAGCTG CTCCTACTTT AGTGCATGCT 3239 TGGAGCCGCC TGCAGCCTGG AACTCAGCAC TGCCCACCAC ACTTGGGCCG AAATGCCAGG 3299 TTTGCCCC 3307 874 amino acids amino acid linear protein 6 Met Gly Trp Pro Gly Leu Arg Pro Leu Leu Leu Ala Gly Leu Ala Ser 1 5 10 15 Leu Leu Leu Pro Gly Ser Ala Ala Ala Gly Leu Lys Leu Met Gly Ala 20 25 30 Pro Val Lys Met Thr Val Ser Gln Gly Gln Pro Val Lys Leu Asn Cys 35 40 45 Ser Val Glu Gly Met Glu Asp Pro Asp Ile His Trp Met Lys Asp Gly 50 55 60 Thr Val Val Gln Asn Ala Ser Gln Val Ser Ile Ser Ile Ser Glu His 65 70 75 80 Ser Trp Ile Gly Leu Leu Ser Leu Lys Ser Val Glu Arg Ser Asp Ala 85 90 95 Gly Leu Tyr Trp Cys Gln Val Lys Asp Gly Glu Glu Thr Lys Ile Ser 100 105 110 Gln Ser Val Trp Leu Thr Val Glu Gly Val Pro Phe Phe Thr Val Glu 115 120 125 Pro Lys Asp Leu Ala Val Pro Pro Asn Ala Pro Phe Gln Leu Ser Cys 130 135 140 Glu Ala Val Gly Pro Pro Glu Pro Val Thr Ile Tyr Trp Trp Arg Gly 145 150 155 160 Leu Thr Lys Val Gly Gly Pro Ala Pro Ser Pro Ser Val Leu Asn Val 165 170 175 Thr Gly Val Thr Gln Arg Thr Glu Phe Ser Cys Glu Ala Arg Asn Ile 180 185 190 Lys Gly Leu Ala Thr Ser Arg Pro Ala Ile Val Arg Leu Gln Ala Pro 195 200 205 Pro Ala Ala Pro Phe Asn Thr Thr Val Thr Thr Ile Ser Ser Tyr Asn 210 215 220 Ala Ser Val Ala Trp Val Pro Gly Ala Asp Gly Leu Ala Leu Leu His 225 230 235 240 Ser Cys Thr Val Gln Val Ala His Ala Pro Gly Glu Trp Glu Ala Leu 245 250 255 Ala Val Val Val Pro Val Pro Pro Phe Thr Cys Leu Leu Arg Asn Leu 260 265 270 Ala Pro Ala Thr Asn Tyr Ser Leu Arg Val Arg Cys Ala Asn Ala Leu 275 280 285 Gly Pro Ser Pro Tyr Gly Asp Trp Val Pro Phe Gln Thr Lys Gly Leu 290 295 300 Ala Pro Arg Arg Ala Pro Gln Asn Phe His Ala Ile Arg Thr Asp Ser 305 310 315 320 Gly Leu Ile Leu Glu Trp Glu Glu Val Ile Pro Glu Asp Pro Gly Glu 325 330 335 Gly Pro Leu Gly Pro Tyr Lys Leu Ser Trp Val Gln Glu Asn Gly Thr 340 345 350 Gln Asp Glu Leu Met Val Glu Gly Thr Arg Ala Asn Leu Thr Asp Trp 355 360 365 Val Pro Gln Lys Asp Leu Ile Leu Arg Val Cys Ala Ser Asn Ala Ile 370 375 380 Gly Asp Gly Pro Trp Ser Gln Pro Leu Val Val Ser Ser His Asp His 385 390 395 400 Ala Gly Arg Gln Gly Pro Pro His Ser Arg Thr Ser Trp Val Pro Val 405 410 415 Val Leu Gly Val Leu Thr Ala Leu Ile Thr Ala Ala Ala Leu Ala Leu 420 425 430 Ile Leu Leu Arg Lys Arg Arg Lys Glu Thr Arg Phe Gly Gln Ala Phe 435 440 445 Asp Ser Val Met Ala Arg Gly Glu Pro Ala Val His Phe Arg Ala Ala 450 455 460 Arg Ser Phe Asn Arg Glu Arg Pro Glu Arg Ile Glu Ala Thr Leu Asp 465 470 475 480 Ser Leu Gly Ile Ser Asp Glu Leu Lys Glu Lys Leu Glu Asp Val Leu 485 490 495 Ile Pro Glu Gln Gln Phe Thr Leu Gly Arg Met Leu Gly Lys Gly Glu 500 505 510 Phe Gly Ser Val Arg Glu Ala Gln Leu Lys Gln Glu Asp Gly Ser Phe 515 520 525 Val Lys Val Ala Val Lys Met Leu Lys Ala Asp Ile Ile Ala Ser Ser 530 535 540 Asp Ile Glu Glu Phe Leu Arg Glu Ala Ala Cys Met Lys Glu Phe Asp 545 550 555 560 His Pro His Val Ala Lys Leu Val Gly Val Ser Leu Arg Ser Arg Ala 565 570 575 Lys Gly Arg Leu Pro Ile Pro Met Val Ile Leu Pro Phe Met Lys His 580 585 590 Gly Asp Leu His Ala Phe Leu Leu Ala Ser Arg Ile Gly Glu Asn Pro 595 600 605 Phe Asn Leu Pro Leu Gln Thr Leu Val Arg Phe Met Val Asp Ile Arg 610 615 620 Cys Gly Met Glu Tyr Leu Ser Ser Arg Asn Phe Ile His Arg Asp Leu 625 630 635 640 Ala Ala Arg Asn Cys Met Leu Ala Glu Asp Met Thr Val Cys Val Ala 645 650 655 Asp Phe Gly Leu Ser Arg Lys Ile Tyr Ser Gly Asp Tyr Tyr Arg Gln 660 665 670 Gly Cys Ala Ser Lys Leu Pro Val Lys Trp Leu Ala Leu Glu Ser Leu 675 680 685 Ala Asp Asn Leu Tyr Thr Val His Ser Asp Val Trp Ala Phe Gly Val 690 695 700 Thr Met Trp Glu Ile Met Thr Arg Gly Gln Thr Pro Tyr Ala Gly Ile 705 710 715 720 Glu Asn Ala Glu Ile Tyr Asn Tyr Leu Ile Gly Gly Asn Arg Leu Lys 725 730 735 Gln Pro Pro Glu Cys Met Glu Glu Val Tyr Asp Leu Met Tyr Gln Cys 740 745 750 Trp Ser Ala Asp Pro Lys Gln Arg Pro Ser Phe Thr Cys Leu Arg Met 755 760 765 Glu Leu Glu Asn Ile Leu Gly His Leu Ser Val Leu Ser Thr Ser Gln 770 775 780 Asp Pro Leu Tyr Ile Asn Ile Glu Arg Ala Glu Gln Pro Thr Glu Ser 785 790 795 800 Gly Ser Pro Glu Val His Cys Gly Glu Arg Ser Ser Ser Glu Ala Gly 805 810 815 Asp Gly Ser Gly Val Gly Ala Val Gly Gly Ile Pro Ser Asp Ser Arg 820 825 830 Tyr Ile Phe Ser Pro Gly Gly Leu Ser Glu Ser Pro Gly Gln Leu Glu 835 840 845 Gln Gln Pro Glu Ser Pro Leu Asn Glu Asn Gln Arg Leu Leu Leu Leu 850 855 860 Gln Gln Gly Leu Leu Pro His Ser Ser Cys 865 870 165 base pairs nucleic acid single linear DNA Tyro-4 CDS 1..165 7 AAC ATC TTG ATC AAC AGT AAC TTG GTG TGC AAA GTC TCT GAC TTC GGA 48 Asn Ile Leu Ile Asn Ser Asn Leu Val Cys Lys Val Ser Asp Phe Gly 1 5 10 15 CTT TCT CGA GTG TTG GAA GAT GAC CCT GAA GCT GCT TAC ACC ACC AGA 96 Leu Ser Arg Val Leu Glu Asp Asp Pro Glu Ala Ala Tyr Thr Thr Arg 20 25 30 GGA GGA AAG ATA CCA ATA AGG TGG ACA TCA CCA GAA GCA ATT GCC TAC 144 Gly Gly Lys Ile Pro Ile Arg Trp Thr Ser Pro Glu Ala Ile Ala Tyr 35 40 45 CGC AAG TTC ACA TCA GCC AGC 165 Arg Lys Phe Thr Ser Ala Ser 50 55 55 amino acids amino acid linear protein 8 Asn Ile Leu Ile Asn Ser Asn Leu Val Cys Lys Val Ser Asp Phe Gly 1 5 10 15 Leu Ser Arg Val Leu Glu Asp Asp Pro Glu Ala Ala Tyr Thr Thr Arg 20 25 30 Gly Gly Lys Ile Pro Ile Arg Trp Thr Ser Pro Glu Ala Ile Ala Tyr 35 40 45 Arg Lys Phe Thr Ser Ala Ser 50 55 171 base pairs nucleic acid single linear DNA Tyro-5 CDS 1..171 9 AAC ATC CTT GTC AAT AGC AAC CTG GTG TGC AAG GTG TCT GAC TTC GGG 48 Asn Ile Leu Val Asn Ser Asn Leu Val Cys Lys Val Ser Asp Phe Gly 1 5 10 15 CTC TCA CGC TTC CTG GAG GAC GAC ACA TCT GAC CCC ACC TAC ACC AGC 96 Leu Ser Arg Phe Leu Glu Asp Asp Thr Ser Asp Pro Thr Tyr Thr Ser 20 25 30 GCT CTG GGT GGG AAG ATC CCC ATC CGT TGG ACA GCA CCG GAA GCC ATC 144 Ala Leu Gly Gly Lys Ile Pro Ile Arg Trp Thr Ala Pro Glu Ala Ile 35 40 45 CAG TAC CGG AAA TTC ACC TCA GCC AGT 171 Gln Tyr Arg Lys Phe Thr Ser Ala Ser 50 55 57 amino acids amino acid linear protein 10 Asn Ile Leu Val Asn Ser Asn Leu Val Cys Lys Val Ser Asp Phe Gly 1 5 10 15 Leu Ser Arg Phe Leu Glu Asp Asp Thr Ser Asp Pro Thr Tyr Thr Ser 20 25 30 Ala Leu Gly Gly Lys Ile Pro Ile Arg Trp Thr Ala Pro Glu Ala Ile 35 40 45 Gln Tyr Arg Lys Phe Thr Ser Ala Ser 50 55 171 base pairs nucleic acid single linear DNA Tyro-6 CDS 1..171 11 AAC ATC CTT GTC AAC AGT AAC TTG GTC TGC AAA GTA TCT GAC TTT GGG 48 Asn Ile Leu Val Asn Ser Asn Leu Val Cys Lys Val Ser Asp Phe Gly 1 5 10 15 CTC TCC CGC TTC CTG GAG GAC GAC CCC TCA GAC CCC ACC TAC ACC AGC 96 Leu Ser Arg Phe Leu Glu Asp Asp Pro Ser Asp Pro Thr Tyr Thr Ser 20 25 30 TCC CTG GGT GGG AAG ATC CCT ATC CGT TGG ACC GCC CCA GAG GCC ATA 144 Ser Leu Gly Gly Lys Ile Pro Ile Arg Trp Thr Ala Pro Glu Ala Ile 35 40 45 GCC TAT CGG AAG TTC ACG TCT GCC AGC 171 Ala Tyr Arg Lys Phe Thr Ser Ala Ser 50 55 57 amino acids amino acid linear protein 12 Asn Ile Leu Val Asn Ser Asn Leu Val Cys Lys Val Ser Asp Phe Gly 1 5 10 15 Leu Ser Arg Phe Leu Glu Asp Asp Pro Ser Asp Pro Thr Tyr Thr Ser 20 25 30 Ser Leu Gly Gly Lys Ile Pro Ile Arg Trp Thr Ala Pro Glu Ala Ile 35 40 45 Ala Tyr Arg Lys Phe Thr Ser Ala Ser 50 55 162 base pairs nucleic acid single linear DNA Tyro-7 CDS 1..162 13 AAC TGC ATG CTG AAT GAG AAC ATG TCC GTG TGC GTG GCA GAC TTC GGG 48 Asn Cys Met Leu Asn Glu Asn Met Ser Val Cys Val Ala Asp Phe Gly 1 5 10 15 CTC TCC AAG AAG ATC TAC AAT GGG GAT TAC TAC CGC CAA GGG CGC ATT 96 Leu Ser Lys Lys Ile Tyr Asn Gly Asp Tyr Tyr Arg Gln Gly Arg Ile 20 25 30 GCC AAG ATG CCA GTC AAG TGG ATT GCT ATC GAG AGT CTG GCA GAT CGA 144 Ala Lys Met Pro Val Lys Trp Ile Ala Ile Glu Ser Leu Ala Asp Arg 35 40 45 GTC TAC ACC AGC AAG AGT 162 Val Tyr Thr Ser Lys Ser 50 54 amino acids amino acid linear protein 14 Asn Cys Met Leu Asn Glu Asn Met Ser Val Cys Val Ala Asp Phe Gly 1 5 10 15 Leu Ser Lys Lys Ile Tyr Asn Gly Asp Tyr Tyr Arg Gln Gly Arg Ile 20 25 30 Ala Lys Met Pro Val Lys Trp Ile Ala Ile Glu Ser Leu Ala Asp Arg 35 40 45 Val Tyr Thr Ser Lys Ser 50 159 base pairs nucleic acid single linear DNA Tyro-8 CDS 1..159 15 AAC TGT TTG GTG GAC AGT GAT CTC TCC GTG AAA GTC TCA GAC TTT GGA 48 Asn Cys Leu Val Asp Ser Asp Leu Ser Val Lys Val Ser Asp Phe Gly 1 5 10 15 ATG ACG AGA TAT GTC CTT GAT GAC CAG TAT GTC AGT TCA GTA GGA ACC 96 Met Thr Arg Tyr Val Leu Asp Asp Gln Tyr Val Ser Ser Val Gly Thr 20 25 30 AAG TTT CCA GTC AAG TGG TCG GCC CCA GAG GTG TTT CAC TAT TTC AAA 144 Lys Phe Pro Val Lys Trp Ser Ala Pro Glu Val Phe His Tyr Phe Lys 35 40 45 TAC AGC AGC AAG TCG 159 Tyr Ser Ser Lys Ser 50 53 amino acids amino acid linear protein 16 Asn Cys Leu Val Asp Ser Asp Leu Ser Val Lys Val Ser Asp Phe Gly 1 5 10 15 Met Thr Arg Tyr Val Leu Asp Asp Gln Tyr Val Ser Ser Val Gly Thr 20 25 30 Lys Phe Pro Val Lys Trp Ser Ala Pro Glu Val Phe His Tyr Phe Lys 35 40 45 Tyr Ser Ser Lys Ser 50 162 base pairs nucleic acid single linear DNA Tyro-9 CDS 1..162 17 AAC GTG CTG GTG ACC GAG GAT GAC GTG ATG AAG ATC GCT GAC TTT GGT 48 Asn Val Leu Val Thr Glu Asp Asp Val Met Lys Ile Ala Asp Phe Gly 1 5 10 15 CTG GCC CGT GGT GTC CAC CAC ATC GAC TAC TAT AAG AAA ACC AGC AAT 96 Leu Ala Arg Gly Val His His Ile Asp Tyr Tyr Lys Lys Thr Ser Asn 20 25 30 GGC CGC CTG CCA GTC AAG TGG ATG GCT CCT GAG GCG TTG TTT GAC CGT 144 Gly Arg Leu Pro Val Lys Trp Met Ala Pro Glu Ala Leu Phe Asp Arg 35 40 45 GTA TAC ACA CAC CAG AGT 162 Val Tyr Thr His Gln Ser 50 54 amino acids amino acid linear protein 18 Asn Val Leu Val Thr Glu Asp Asp Val Met Lys Ile Ala Asp Phe Gly 1 5 10 15 Leu Ala Arg Gly Val His His Ile Asp Tyr Tyr Lys Lys Thr Ser Asn 20 25 30 Gly Arg Leu Pro Val Lys Trp Met Ala Pro Glu Ala Leu Phe Asp Arg 35 40 45 Val Tyr Thr His Gln Ser 50 3120 base pairs nucleic acid single linear DNA Tyro-10 CDS 485..3047 19 GGGCCCGGGT CTAAGTGGAC TTCTCTTGGT GTGTCAGGAA AAGTTCGGAA AAGCGGCAGA 60 GGGCAGAGTT TGAATCAGGG CGGAAGGGCA GGGAGCTGGG CTCTTCAAGA CTCAGGACCG 120 AGGCAGATCT CATGTTTTGG GGTCTGGATT TGTGTCAGCG AGGGAAGAAC AGGCGCCAAT 180 AACCAAAGAA GGCTGAAGCG AGGTACAGGA CTCCATAGCA GCTGCAAGTA CAATAAACAG 240 TTTTAGCAGA GCTGGAAATG TTGGCAGGCA AGACAGGCCG ATCGCAGAGT CGGGCTGCTG 300 GAGAGAGGGA AATCTACAAG CGACCTGACA TTTGGTGCTC TAGAGCATTC TAAGGCTTGC 360 TGCTTGACTT CTAAAGAAGC TGAAATAATT GAGGAGGAGC GGGGACCCTC TGTTTCCAAG 420 GACTCTGTTC TGCAGAGAAT GTTCTGCACC CTCTGATACT CCAGATCCAA CTCCGTCTTC 480 TGAA ATG ATC CCG ATT CCC AGA ATG CCC CTG GTG CTG CTC CTG CTC TTG 529 Met Ile Pro Ile Pro Arg Met Pro Leu Val Leu Leu Leu Leu Leu 1 5 10 15 CTC ATC CTG GGT TCT GCA AAA GCT CAG GTT AAT CCA GCC ATA TGC CGC 577 Leu Ile Leu Gly Ser Ala Lys Ala Gln Val Asn Pro Ala Ile Cys Arg 20 25 30 TAT CCT CTG GGC ATG TCA GGA GGC CAC ATT CCA GAT GAG GAC ATC ACA 625 Tyr Pro Leu Gly Met Ser Gly Gly His Ile Pro Asp Glu Asp Ile Thr 35 40 45 GCC TCA AGT CAG TGG TCA GAA TCC ACG GCT GCC AAA TAT GGG AGG CTG 673 Ala Ser Ser Gln Trp Ser Glu Ser Thr Ala Ala Lys Tyr Gly Arg Leu 50 55 60 GAC TCT GAA GAA GGA GAT GGA GCC TGG TGT CCT GAG ATT CCA GTG CAA 721 Asp Ser Glu Glu Gly Asp Gly Ala Trp Cys Pro Glu Ile Pro Val Gln 65 70 75 CCC GAT GAC CTG AAG GAA TTT CTG CAG ATT GAC TTG CGA ACC CTA CAC 769 Pro Asp Asp Leu Lys Glu Phe Leu Gln Ile Asp Leu Arg Thr Leu His 80 85 90 95 TTT ATC ACT CTT GTG GGG ACC CAG GGG CGC CAT GCA GGG GGT CAT GGC 817 Phe Ile Thr Leu Val Gly Thr Gln Gly Arg His Ala Gly Gly His Gly 100 105 110 ATT GAA TTT GCA CCC ATG TAC AAG ATC AAC TAC AGT CGG GAT GGC AGT 865 Ile Glu Phe Ala Pro Met Tyr Lys Ile Asn Tyr Ser Arg Asp Gly Ser 115 120 125 CGC TGG ATC TCC TGG CGT AAC CGG CAT GGG AAG CAG GTG CTT GAT GGA 913 Arg Trp Ile Ser Trp Arg Asn Arg His Gly Lys Gln Val Leu Asp Gly 130 135 140 AAC AGT AAC CCT TAT GAT GTA TTC CTG AAG GAC TTG GAG CCA CCC ATC 961 Asn Ser Asn Pro Tyr Asp Val Phe Leu Lys Asp Leu Glu Pro Pro Ile 145 150 155 GTC GCC AGA TTT GTT CGC CTT ATC CCA GTC ACT GAC CAC TCC ATG AAC 1009 Val Ala Arg Phe Val Arg Leu Ile Pro Val Thr Asp His Ser Met Asn 160 165 170 175 GTG TGC ATG AGG GTT GAG CTT TAT GGT TGT GTC TGG CTA GAT GGC TTG 1057 Val Cys Met Arg Val Glu Leu Tyr Gly Cys Val Trp Leu Asp Gly Leu 180 185 190 GTA TCC TAC AAT GCT CCA GCT GGA CAG CAG TTT GTA CTC CCT GGA GGC 1105 Val Ser Tyr Asn Ala Pro Ala Gly Gln Gln Phe Val Leu Pro Gly Gly 195 200 205 TCC ATC ATT TAT CTG AAT GAT TCT GTC TAT GAT GGA GCT GTT GGG TAC 1153 Ser Ile Ile Tyr Leu Asn Asp Ser Val Tyr Asp Gly Ala Val Gly Tyr 210 215 220 AGC ATG ACT GAA GGG CTA GGC CAG TTG ACT GAT GGA GTA TCC GGC CTG 1201 Ser Met Thr Glu Gly Leu Gly Gln Leu Thr Asp Gly Val Ser Gly Leu 225 230 235 GAT GAT TTT ACC CAG ACC CAT GAA TAC CAC GTG TGG CCT GGC TAT GAC 1249 Asp Asp Phe Thr Gln Thr His Glu Tyr His Val Trp Pro Gly Tyr Asp 240 245 250 255 TAC GTG GGA TGG CGG AAC GAA AGT GCT ACC AAC GGT TTC ATT GAG ATC 1297 Tyr Val Gly Trp Arg Asn Glu Ser Ala Thr Asn Gly Phe Ile Glu Ile 260 265 270 ATG TTT GAA TTT GAC CGA ATC AGG AAT TTT ACT ACC ATG AAG GTC CAC 1345 Met Phe Glu Phe Asp Arg Ile Arg Asn Phe Thr Thr Met Lys Val His 275 280 285 TGC AAC AAC ATG TTT GCT AAA GGT GTG AAG ATT TTT AAG GAG GTC CAG 1393 Cys Asn Asn Met Phe Ala Lys Gly Val Lys Ile Phe Lys Glu Val Gln 290 295 300 TGC TAC TTT CGC TCG GAA GCC AGC GAG TGG GAA CCC ACT GCT GTC TAC 1441 Cys Tyr Phe Arg Ser Glu Ala Ser Glu Trp Glu Pro Thr Ala Val Tyr 305 310 315 TTT CCC CTG GTC CTG GAC GAT GTG AAC CCC AGT GCC CGG TTT GTC ACG 1489 Phe Pro Leu Val Leu Asp Asp Val Asn Pro Ser Ala Arg Phe Val Thr 320 325 330 335 GTG CCC CTC CAC CAC CGA ATG GCC AGT GCC ATC AAG TGC CAA TAC CAT 1537 Val Pro Leu His His Arg Met Ala Ser Ala Ile Lys Cys Gln Tyr His 340 345 350 TTT GCC GAC ACG TGG ATG ATG TTC AGC GAG ATC ACT TTC CAA TCA GAT 1585 Phe Ala Asp Thr Trp Met Met Phe Ser Glu Ile Thr Phe Gln Ser Asp 355 360 365 GCT GCA ATG TAT AAC AAC TCT GGA GCC CTT CCC ACC TCT CCT ATG GCA 1633 Ala Ala Met Tyr Asn Asn Ser Gly Ala Leu Pro Thr Ser Pro Met Ala 370 375 380 CCC ACC ACC TAT GAT CCC ATG CTT AAA GTT GAT GAT AGC AAC ACT CGG 1681 Pro Thr Thr Tyr Asp Pro Met Leu Lys Val Asp Asp Ser Asn Thr Arg 385 390 395 ATC CTG ATT GGT TGC TTG GTG GCC ATC ATC TTC ATC CTG CTG GCT ATC 1729 Ile Leu Ile Gly Cys Leu Val Ala Ile Ile Phe Ile Leu Leu Ala Ile 400 405 410 415 ATC GTC ATC ATC CTG TGG AGG CAG TTC TGG CAG AAG ATG CTA GAA AAG 1777 Ile Val Ile Ile Leu Trp Arg Gln Phe Trp Gln Lys Met Leu Glu Lys 420 425 430 GCT TCA CGG AGG ATG CTG GAT GAT GAA ATG ACA GTC AGC CTT TCC CTG 1825 Ala Ser Arg Arg Met Leu Asp Asp Glu Met Thr Val Ser Leu Ser Leu 435 440 445 CCC AGC GAG TCC AGC ATG TTC AAT AAC AAC CGC TCC TCA TCA CCA AGT 1873 Pro Ser Glu Ser Ser Met Phe Asn Asn Asn Arg Ser Ser Ser Pro Ser 450 455 460 GAA CAG GAG TCC AAC TCT ACT TAT GAT CGA ATC TTC CCC CTT CGC CCT 1921 Glu Gln Glu Ser Asn Ser Thr Tyr Asp Arg Ile Phe Pro Leu Arg Pro 465 470 475 GAC TAC CAG GAG CCA TCC AGA CTG ATC CGA AAG CTT CCA GAG TTT GCT 1969 Asp Tyr Gln Glu Pro Ser Arg Leu Ile Arg Lys Leu Pro Glu Phe Ala 480 485 490 495 CCA GGA GAG GAG GAG TCA GGG TGC AGT GGT GTT GTG AAG CCG GCC CAG 2017 Pro Gly Glu Glu Glu Ser Gly Cys Ser Gly Val Val Lys Pro Ala Gln 500 505 510 CCC AAT GGA CCT GAG GGC GTG CCC CAC TAT GCA GAA GCC GAC ATA GTG 2065 Pro Asn Gly Pro Glu Gly Val Pro His Tyr Ala Glu Ala Asp Ile Val 515 520 525 AAT CTC CAG GGA GTG ACA GGT GGC AAC ACC TAC TGT GTG CCT GCT GTA 2113 Asn Leu Gln Gly Val Thr Gly Gly Asn Thr Tyr Cys Val Pro Ala Val 530 535 540 ACC ATG GAT CTG CTA TCG GGG AAA GAT GTG GCT GTG GAA GAG TTC CCC 2161 Thr Met Asp Leu Leu Ser Gly Lys Asp Val Ala Val Glu Glu Phe Pro 545 550 555 AGG AAA CTG TTG GCC TTC AAG GAG AAG CTG GGA GAA GGC CAG TTT GGG 2209 Arg Lys Leu Leu Ala Phe Lys Glu Lys Leu Gly Glu Gly Gln Phe Gly 560 565 570 575 GAG GTT CAT CTC TGT GAA GTG GAG GGA ATG GAA AAA TTC AAA GAC AAA 2257 Glu Val His Leu Cys Glu Val Glu Gly Met Glu Lys Phe Lys Asp Lys 580 585 590 GAT TTT GCA CTA GAT GTC AGT GCC AAC CAG CCT GTC CTG GTG GCC GTG 2305 Asp Phe Ala Leu Asp Val Ser Ala Asn Gln Pro Val Leu Val Ala Val 595 600 605 AAA ATG CTC CGA GCA GAT GCC AAC AAG AAT GCC AGG AAT GAT TTT CTT 2353 Lys Met Leu Arg Ala Asp Ala Asn Lys Asn Ala Arg Asn Asp Phe Leu 610 615 620 AAG GAG ATC AAG ATC ATG TCT CGG CTC AAG GAC CCA AAC ATC ATC CGT 2401 Lys Glu Ile Lys Ile Met Ser Arg Leu Lys Asp Pro Asn Ile Ile Arg 625 630 635 CTC TTA GCT GTG TGC ATC ACT GAG GAC CCG CTC TGC ATG ATC ACG GAA 2449 Leu Leu Ala Val Cys Ile Thr Glu Asp Pro Leu Cys Met Ile Thr Glu 640 645 650 655 TAC ATG GAG AAT GGA GAT CTT AAT CAG TTT CTT TCT CGC CAC GAG CCT 2497 Tyr Met Glu Asn Gly Asp Leu Asn Gln Phe Leu Ser Arg His Glu Pro 660 665 670 CTG AGT TCC TGT TCT AGT GAT GCC ACA GTC AGT TAC GCC AAC CTG AAG 2545 Leu Ser Ser Cys Ser Ser Asp Ala Thr Val Ser Tyr Ala Asn Leu Lys 675 680 685 TTT ATG GCA ACC CAG ATT GCC TCT GGT ATG AAG TAC CTT TCG TCT CTC 2593 Phe Met Ala Thr Gln Ile Ala Ser Gly Met Lys Tyr Leu Ser Ser Leu 690 695 700 AAC TTT GTC CAC CGA GAT CTG GCC ACA CGA AAC TGT TTA GTG GGC AAG 2641 Asn Phe Val His Arg Asp Leu Ala Thr Arg Asn Cys Leu Val Gly Lys 705 710 715 AAT TAC ACC ATC AAG ATA GCT GAT TTT GGC ATG AGC AGA AAC CTG TAC 2689 Asn Tyr Thr Ile Lys Ile Ala Asp Phe Gly Met Ser Arg Asn Leu Tyr 720 725 730 735 AGT GGT GAT TAC TAC CGG ATC CAG GGC CGG GCG GTG CTC CCC ATT CGC 2737 Ser Gly Asp Tyr Tyr Arg Ile Gln Gly Arg Ala Val Leu Pro Ile Arg 740 745 750 TGG ATG TCC TGG GAA AGC ATC TTG CTG GGC AAA TTC ACC ACG GCA AGT 2785 Trp Met Ser Trp Glu Ser Ile Leu Leu Gly Lys Phe Thr Thr Ala Ser 755 760 765 GAT GTG TGG GCC TTT GGG GTG ACT CTG TGG GAG ACC TTC ACC TTT TGC 2833 Asp Val Trp Ala Phe Gly Val Thr Leu Trp Glu Thr Phe Thr Phe Cys 770 775 780 CAG GAG CAG CCC TAT TCC CAG CTG TCG GAT GAG CAG GTT ATC GAG AAC 2881 Gln Glu Gln Pro Tyr Ser Gln Leu Ser Asp Glu Gln Val Ile Glu Asn 785 790 795 ACT GGA GAG TTC TTC CGA GAC CAA GGG AGG CAG ATC TAT CTC CCT CAA 2929 Thr Gly Glu Phe Phe Arg Asp Gln Gly Arg Gln Ile Tyr Leu Pro Gln 800 805 810 815 CCA GCC CTT TGC CCC GAC TCT GTG TAT AAG CTG ATG CTC AGC TGC TGG 2977 Pro Ala Leu Cys Pro Asp Ser Val Tyr Lys Leu Met Leu Ser Cys Trp 820 825 830 AGA AGA GAA ACC AAG CAC CGG CCA TCC TTC CAG GAA ATA CAC CTC CTG 3025 Arg Arg Glu Thr Lys His Arg Pro Ser Phe Gln Glu Ile His Leu Leu 835 840 845 CTT CTT CAG CAA GGA GCC GAG T GATGATGCAT CAGCACCTGG CAGTGTTCCT 3077 Leu Leu Gln Gln Gly Ala Glu 850 GTGGCCCAGA TCCTTCCCAC AAGACCTACT GCTCACCCAC ATC 3120 854 amino acids amino acid linear protein 20 Met Ile Pro Ile Pro Arg Met Pro Leu Val Leu Leu Leu Leu Leu Leu 1 5 10 15 Ile Leu Gly Ser Ala Lys Ala Gln Val Asn Pro Ala Ile Cys Arg Tyr 20 25 30 Pro Leu Gly Met Ser Gly Gly His Ile Pro Asp Glu Asp Ile Thr Ala 35 40 45 Ser Ser Gln Trp Ser Glu Ser Thr Ala Ala Lys Tyr Gly Arg Leu Asp 50 55 60 Ser Glu Glu Gly Asp Gly Ala Trp Cys Pro Glu Ile Pro Val Gln Pro 65 70 75 80 Asp Asp Leu Lys Glu Phe Leu Gln Ile Asp Leu Arg Thr Leu His Phe 85 90 95 Ile Thr Leu Val Gly Thr Gln Gly Arg His Ala Gly Gly His Gly Ile 100 105 110 Glu Phe Ala Pro Met Tyr Lys Ile Asn Tyr Ser Arg Asp Gly Ser Arg 115 120 125 Trp Ile Ser Trp Arg Asn Arg His Gly Lys Gln Val Leu Asp Gly Asn 130 135 140 Ser Asn Pro Tyr Asp Val Phe Leu Lys Asp Leu Glu Pro Pro Ile Val 145 150 155 160 Ala Arg Phe Val Arg Leu Ile Pro Val Thr Asp His Ser Met Asn Val 165 170 175 Cys Met Arg Val Glu Leu Tyr Gly Cys Val Trp Leu Asp Gly Leu Val 180 185 190 Ser Tyr Asn Ala Pro Ala Gly Gln Gln Phe Val Leu Pro Gly Gly Ser 195 200 205 Ile Ile Tyr Leu Asn Asp Ser Val Tyr Asp Gly Ala Val Gly Tyr Ser 210 215 220 Met Thr Glu Gly Leu Gly Gln Leu Thr Asp Gly Val Ser Gly Leu Asp 225 230 235 240 Asp Phe Thr Gln Thr His Glu Tyr His Val Trp Pro Gly Tyr Asp Tyr 245 250 255 Val Gly Trp Arg Asn Glu Ser Ala Thr Asn Gly Phe Ile Glu Ile Met 260 265 270 Phe Glu Phe Asp Arg Ile Arg Asn Phe Thr Thr Met Lys Val His Cys 275 280 285 Asn Asn Met Phe Ala Lys Gly Val Lys Ile Phe Lys Glu Val Gln Cys 290 295 300 Tyr Phe Arg Ser Glu Ala Ser Glu Trp Glu Pro Thr Ala Val Tyr Phe 305 310 315 320 Pro Leu Val Leu Asp Asp Val Asn Pro Ser Ala Arg Phe Val Thr Val 325 330 335 Pro Leu His His Arg Met Ala Ser Ala Ile Lys Cys Gln Tyr His Phe 340 345 350 Ala Asp Thr Trp Met Met Phe Ser Glu Ile Thr Phe Gln Ser Asp Ala 355 360 365 Ala Met Tyr Asn Asn Ser Gly Ala Leu Pro Thr Ser Pro Met Ala Pro 370 375 380 Thr Thr Tyr Asp Pro Met Leu Lys Val Asp Asp Ser Asn Thr Arg Ile 385 390 395 400 Leu Ile Gly Cys Leu Val Ala Ile Ile Phe Ile Leu Leu Ala Ile Ile 405 410 415 Val Ile Ile Leu Trp Arg Gln Phe Trp Gln Lys Met Leu Glu Lys Ala 420 425 430 Ser Arg Arg Met Leu Asp Asp Glu Met Thr Val Ser Leu Ser Leu Pro 435 440 445 Ser Glu Ser Ser Met Phe Asn Asn Asn Arg Ser Ser Ser Pro Ser Glu 450 455 460 Gln Glu Ser Asn Ser Thr Tyr Asp Arg Ile Phe Pro Leu Arg Pro Asp 465 470 475 480 Tyr Gln Glu Pro Ser Arg Leu Ile Arg Lys Leu Pro Glu Phe Ala Pro 485 490 495 Gly Glu Glu Glu Ser Gly Cys Ser Gly Val Val Lys Pro Ala Gln Pro 500 505 510 Asn Gly Pro Glu Gly Val Pro His Tyr Ala Glu Ala Asp Ile Val Asn 515 520 525 Leu Gln Gly Val Thr Gly Gly Asn Thr Tyr Cys Val Pro Ala Val Thr 530 535 540 Met Asp Leu Leu Ser Gly Lys Asp Val Ala Val Glu Glu Phe Pro Arg 545 550 555 560 Lys Leu Leu Ala Phe Lys Glu Lys Leu Gly Glu Gly Gln Phe Gly Glu 565 570 575 Val His Leu Cys Glu Val Glu Gly Met Glu Lys Phe Lys Asp Lys Asp 580 585 590 Phe Ala Leu Asp Val Ser Ala Asn Gln Pro Val Leu Val Ala Val Lys 595 600 605 Met Leu Arg Ala Asp Ala Asn Lys Asn Ala Arg Asn Asp Phe Leu Lys 610 615 620 Glu Ile Lys Ile Met Ser Arg Leu Lys Asp Pro Asn Ile Ile Arg Leu 625 630 635 640 Leu Ala Val Cys Ile Thr Glu Asp Pro Leu Cys Met Ile Thr Glu Tyr 645 650 655 Met Glu Asn Gly Asp Leu Asn Gln Phe Leu Ser Arg His Glu Pro Leu 660 665 670 Ser Ser Cys Ser Ser Asp Ala Thr Val Ser Tyr Ala Asn Leu Lys Phe 675 680 685 Met Ala Thr Gln Ile Ala Ser Gly Met Lys Tyr Leu Ser Ser Leu Asn 690 695 700 Phe Val His Arg Asp Leu Ala Thr Arg Asn Cys Leu Val Gly Lys Asn 705 710 715 720 Tyr Thr Ile Lys Ile Ala Asp Phe Gly Met Ser Arg Asn Leu Tyr Ser 725 730 735 Gly Asp Tyr Tyr Arg Ile Gln Gly Arg Ala Val Leu Pro Ile Arg Trp 740 745 750 Met Ser Trp Glu Ser Ile Leu Leu Gly Lys Phe Thr Thr Ala Ser Asp 755 760 765 Val Trp Ala Phe Gly Val Thr Leu Trp Glu Thr Phe Thr Phe Cys Gln 770 775 780 Glu Gln Pro Tyr Ser Gln Leu Ser Asp Glu Gln Val Ile Glu Asn Thr 785 790 795 800 Gly Glu Phe Phe Arg Asp Gln Gly Arg Gln Ile Tyr Leu Pro Gln Pro 805 810 815 Ala Leu Cys Pro Asp Ser Val Tyr Lys Leu Met Leu Ser Cys Trp Arg 820 825 830 Arg Glu Thr Lys His Arg Pro Ser Phe Gln Glu Ile His Leu Leu Leu 835 840 845 Leu Gln Gln Gly Ala Glu 850 171 base pairs nucleic acid single linear DNA Tyro-11 CDS 1..171 21 AAC ATC CTG GTC AAC AGT AAC CTG GTC TGC AAG GTG TCC GAC TTT GGC 48 Asn Ile Leu Val Asn Ser Asn Leu Val Cys Lys Val Ser Asp Phe Gly 1 5 10 15 CTC TCC AGA TTC CTG GAG GAG AAC TCC TCT GAT CCC ACC TAC ACA AGT 96 Leu Ser Arg Phe Leu Glu Glu Asn Ser Ser Asp Pro Thr Tyr Thr Ser 20 25 30 TCC CTG GGA GGA AAG ATT CCC ATC CGA TGG ACC GCC CCT GAG GCC ATT 144 Ser Leu Gly Gly Lys Ile Pro Ile Arg Trp Thr Ala Pro Glu Ala Ile 35 40 45 GCC TTC AGG AAA TTC ACG TCT GCC AGT 171 Ala Phe Arg Lys Phe Thr Ser Ala Ser 50 55 57 amino acids amino acid linear protein 22 Asn Ile Leu Val Asn Ser Asn Leu Val Cys Lys Val Ser Asp Phe Gly 1 5 10 15 Leu Ser Arg Phe Leu Glu Glu Asn Ser Ser Asp Pro Thr Tyr Thr Ser 20 25 30 Ser Leu Gly Gly Lys Ile Pro Ile Arg Trp Thr Ala Pro Glu Ala Ile 35 40 45 Ala Phe Arg Lys Phe Thr Ser Ala Ser 50 55 162 base pairs nucleic acid single linear DNA Tyro-12 CDS 1..162 23 AAT TGC ATG TTG CGG GAT GAC ATG ACT GTC TGC GTG GCA GAC TTT GGC 48 Asn Cys Met Leu Arg Asp Asp Met Thr Val Cys Val Ala Asp Phe Gly 1 5 10 15 CTC TCT AAG AAG ATT TAC AGT GGT GAT TAT TAC CGC CAA GGC CGC ATT 96 Leu Ser Lys Lys Ile Tyr Ser Gly Asp Tyr Tyr Arg Gln Gly Arg Ile 20 25 30 GCC AAA ATG CCT GTG AAG TGG ATC GCC ATA GAG AGC CTG GCG GAC CGA 144 Ala Lys Met Pro Val Lys Trp Ile Ala Ile Glu Ser Leu Ala Asp Arg 35 40 45 GTC TAC ACA AGC AAG AGT 162 Val Tyr Thr Ser Lys Ser 50 54 amino acids amino acid linear protein 24 Asn Cys Met Leu Arg Asp Asp Met Thr Val Cys Val Ala Asp Phe Gly 1 5 10 15 Leu Ser Lys Lys Ile Tyr Ser Gly Asp Tyr Tyr Arg Gln Gly Arg Ile 20 25 30 Ala Lys Met Pro Val Lys Trp Ile Ala Ile Glu Ser Leu Ala Asp Arg 35 40 45 Val Tyr Thr Ser Lys Ser 50 147 base pairs nucleic acid single linear DNA Tyro-13 CDS 1..147 25 AAT GTG CTG GTG TCT GAG GAC AAC GTG GCC AAA GTC AGT GAC TTT GGC 48 Asn Val Leu Val Ser Glu Asp Asn Val Ala Lys Val Ser Asp Phe Gly 1 5 10 15 CTC ACT AAG GAA GCT TCC AGC ACT CAG GAC ACA GGC AAA CTG CCA GTC 96 Leu Thr Lys Glu Ala Ser Ser Thr Gln Asp Thr Gly Lys Leu Pro Val 20 25 30 AAG TGG ACA GCT CCT GAA GCC TTG AGA GAG AAG AAA TTT TCC ACC AAG 144 Lys Trp Thr Ala Pro Glu Ala Leu Arg Glu Lys Lys Phe Ser Thr Lys 35 40 45 TCT 147 Ser 49 amino acids amino acid linear protein 26 Asn Val Leu Val Ser Glu Asp Asn Val Ala Lys Val Ser Asp Phe Gly 1 5 10 15 Leu Thr Lys Glu Ala Ser Ser Thr Gln Asp Thr Gly Lys Leu Pro Val 20 25 30 Lys Trp Thr Ala Pro Glu Ala Leu Arg Glu Lys Lys Phe Ser Thr Lys 35 40 45 Ser 7 amino acids amino acid Not Relevant linear protein 27 His Arg Asp Leu Ala Ala Arg 1 5 7 amino acids amino acid Not Relevant linear protein 28 Asp Val Trp Ser Xaa Gly Xaa 1 5 8 amino acids amino acid Not Relevant linear protein 29 Pro Xaa Xaa Trp Xaa Ala Pro Glu 1 5 68 amino acids amino acid Not Relevant linear protein 30 His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Lys Thr Pro Gln His 1 5 10 15 Val Lys Ile Thr Asp Phe Gly Leu Ala Asp Leu Leu Gly Ala Glu Glu 20 25 30 Lys Glu Tyr His Ala Glu Gly Gly Lys Val Pro Ile Lys Trp Met Ala 35 40 45 Leu Glu Ser Ile Leu His Arg Ile Tyr Thr His Gln Ser Asp Val Trp 50 55 60 Ser Tyr Gly Val 65 68 amino acids amino acid Not Relevant linear protein 31 His Arg Asp Leu Ala Ala Arg Asn Cys Met Val Ala His Asp Phe Thr 1 5 10 15 Val Lys Ile Gly Asp Phe Gly Met Thr Arg Asp Ile Tyr Glu Thr Asp 20 25 30 Tyr Tyr Arg Lys Gly Gly Lys Gly Leu Leu Pro Val Arg Trp Met Ala 35 40 45 Pro Glu Ser Leu Lys Asp Gly Val Phe Thr Thr Ser Ser Asp Met Trp 50 55 60 Ser Phe Gly Val 65 68 amino acids amino acid Not Relevant linear protein 32 His Arg Asp Leu Ala Ala Arg Asn Val Leu Ile Cys Glu Gly Lys Leu 1 5 10 15 Val Lys Ile Cys Asp Phe His Leu Ala Arg Asp Ile Met Arg Asp Ser 20 25 30 Asn Tyr Ile Ser Lys Gly Ser Thr Tyr Leu Pro Leu Lys Trp Met Ala 35 40 45 Pro Glu Ser Ile Phe Asn Ser Leu Tyr Thr Thr Leu Ser Asp Val Trp 50 55 60 Ser Phe Gly Ile 65 68 amino acids amino acid Not Relevant linear protein 33 His Arg Asp Leu Ala Ala Arg Asn Val Leu Ile Cys Glu Gly Lys Leu 1 5 10 15 Val Lys Ile Cys Asp Phe Gly Leu Ala Arg Asp Ile Met Arg Asp Ser 20 25 30 Asn Tyr Ile Ile Asp Gly Ser Thr Tyr Leu Pro Leu Lys Trp Met Ala 35 40 45 Pro Glu Ser Ile Phe Asn Ser Leu Tyr Thr Thr Leu Ser Asp Val Trp 50 55 60 Ser Phe Gly Ile 65 43 amino acids amino acid Not Relevant linear protein 34 His Arg Asp Leu Ala Ala Arg Asn Val Leu Val Val Lys Ile Asp Phe 1 5 10 15 Gly Leu Ala Arg Asp Ile Tyr Gly Leu Pro Lys Trp Met Ala Pro Glu 20 25 30 Ser Tyr Thr Ser Asp Val Trp Ser Phe Gly Val 35 40 27 base pairs nucleic acid Not Relevant linear protein 35 GGAATTCCAT CGNGATTTNG CNGCNCG 27 55 amino acids amino acid Not Relevant linear protein 36 Asn Cys Leu Val Gly Glu Asn Ile Ile Leu Val Lys Val Ala Asp Phe 1 5 10 15 Gly Leu Ser Arg Leu Met Thr Gly Asp Thr Tyr Thr Ala Ile Ile Ala 20 25 30 Gly Ala Lys Phe Pro Ile Lys Trp Thr Ala Pro Glu Ser Leu Ala Tyr 35 40 45 Asn Lys Phe Ser Ile Lys Ser 50 55 55 amino acids amino acid Not Relevant linear protein 37 Asn Cys Leu Val Gly Glu Asn Ile Ile Val Val Lys Val Ala Asp Phe 1 5 10 15 Gly Leu Ser Arg Leu Met Thr Gly Asp Thr Tyr Thr Ala Ile Ile Ala 20 25 30 Gly Ala Lys Phe Pro Ile Lys Trp Thr Ala Pro Glu Ser Leu Ala Tyr 35 40 45 Asn Thr Pro Ser Ile Lys Ser 50 55 54 amino acids amino acid Not Relevant linear protein 38 Asn Cys Leu Val Thr Glu Lys Asn Val Leu Lys Ile Ser Asp Phe Gly 1 5 10 15 His Ser Arg Glu Glu Ala Asp Gly Val Tyr Ala Ala Ser Gly Gly Leu 20 25 30 Arg Gln Val Pro Val Lys Trp Thr Ala Pro Glu Ala Leu Asn Tyr Gly 35 40 45 Arg Tyr Ser Ser Glu Ser 50 53 amino acids amino acid Not Relevant linear protein 39 Asn Cys Leu Val Gly Glu Asn Asn Thr Leu Lys Ile Ser Asp Phe Gly 1 5 10 15 Met Ser Arg Gln Glu Asp Gly Gly Val Tyr Ser Ser Ser Gly Leu Lys 20 25 30 Gln Ile Pro Ile Lys Trp Thr Ala Pro Glu Ala Leu His Tyr Gly Arg 35 40 45 Tyr Ser Ser Glu Ser 50 53 amino acids amino acid Not Relevant linear protein 40 Asn Cys Leu Val Gly Ser Glu Asn Val Val Lys Val Ala Asp Phe Gly 1 5 10 15 Leu Ala Arg Tyr Val Leu Asp Asp Gln Tyr Thr Ser Ser Gly Gly Thr 20 25 30 Lys Phe Pro Ile Lys Trp Ala Pro Pro Glu Val Leu Asn Tyr Thr Arg 35 40 45 Phe Ser Ser Lys Ser 50 54 amino acids amino acid Not Relevant linear protein 41 Asn Ile Leu Val Asn Gln Asn Leu Cys Cys Lys Val Ser Asp Phe Gly 1 5 10 15 Leu Thr Arg Leu Leu Asp Asp Phe Asp Gly Thr Tyr Glu Thr Gln Gly 20 25 30 Gly Lys Ile Pro Ile Arg Trp Thr Ala Pro Glu Ala Leu Ala His Arg 35 40 45 Ile Phe Thr Thr Ala Ser 50 55 amino acids amino acid Not Relevant linear protein 42 Asn Ile Leu Val Asn Ser Asn Leu Val Cys Lys Val Ser Asp Phe Gly 1 5 10 15 Leu Ser Arg Val Leu Glu Asp Asp Pro Glu Ala Thr Tyr Thr Thr Ser 20 25 30 Gly Gly Lys Ile Pro Ile Arg Trp Thr Ala Pro Glu Ala Ile Ser Tyr 35 40 45 Arg Lys Phe Thr Ser Ala Ser 50 55 57 amino acids amino acid Not Relevant linear protein 43 Asn Ile Leu Val Asn Ser Asn Leu Val Cys Lys Val Ser Asp Phe Gly 1 5 10 15 Leu Ser Arg Tyr Leu Gln Asp Asp Thr Ser Asp Pro Thr Tyr Thr Ser 20 25 30 Ser Leu Gly Gly Lys Ile Pro Val Arg Trp Thr Ala Pro Glu Ala Ile 35 40 45 Ala Tyr Arg Lys Phe Thr Ser Ala Ser 50 55 54 amino acids amino acid Not Relevant linear protein 44 Asn Val Leu Val Lys Thr Pro Gln His Val Lys Ile Thr Asp Phe Gly 1 5 10 15 Leu Ala Lys Leu Leu Gly Ala Glu Glu Lys Glu Tyr His Ala Glu Gly 20 25 30 Gly Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu His Arg 35 40 45 Ile Tyr Thr His Gln Ser 50 54 amino acids amino acid Not Relevant linear protein 45 Asn Val Leu Val Lys Ser Pro Asn His Val Lys Ile Thr Asp Phe Gly 1 5 10 15 Leu Ala Arg Leu Leu Asp Ile Asp Glu Thr Glu Tyr His Ala Asp Gly 20 25 30 Gly Lys Val Pro Ile Lys Trp Met Ala Leu Glu Ser Ile Leu Arg Arg 35 40 45 Arg Phe Thr His Gln Ser 50 54 amino acids amino acid Not Relevant linear protein 46 Asn Val Leu Val Thr Glu Asp Asn Val Met Lys Ile Ala Asp Phe Gly 1 5 10 15 Leu Ala Arg Asp Ile His His Ile Asp Tyr Tyr Lys Lys Thr Thr Asn 20 25 30 Gly Arg Leu Pro Val Lys Trp Met Ala Pro Glu Ala Leu Phe Asp Arg 35 40 45 Ile Tyr Thr His Gln Ser 50 54 amino acids amino acid Not Relevant linear protein 47 Asn Val Leu Val Thr Glu Asn Asn Val Met Lys Ile Ala Asp Phe Gly 1 5 10 15 Leu Ala Arg Asp Ile Asn Asn Ile Asp Tyr Tyr Lys Lys Thr Thr Asn 20 25 30 Gly Arg Leu Pro Val Lys Trp Met Ala Pro Glu Ala Leu Phe Asp Arg 35 40 45 Val Tyr Thr His Gln Ser 50 54 amino acids amino acid Not Relevant linear protein 48 Asn Val Leu Leu Ala Gln Gly Lys Ile Val Lys Ile Cys Asp Phe Gly 1 5 10 15 Leu Ala Arg Asp Ile Met His Asp Ser Asn Thr Val Ser Lys Gly Ser 20 25 30 Thr Phe Leu Pro Val Lys Trp Met Ala Pro Glu Ser Ile Phe Asp Asn 35 40 45 Leu Thr Tyr Tyr Leu Ser 50 54 amino acids amino acid Not Relevant linear protein 49 Asn Met Leu Ile Cys Glu Gly Lys Leu Val Lys Ile Cys Asp Phe Gly 1 5 10 15 Leu Ala Arg Asp Ile Met Arg Asp Ser Asn Tyr Ile Ser Lys Gly Ser 20 25 30 Thr Phe Leu Pro Leu Lys Trp Met Ala Pro Glu Ser Ile Phe Asn Ser 35 40 45 Leu Tyr Thr Thr Leu Ser 50 54 amino acids amino acid Not Relevant linear protein 50 Asn Val Leu Leu Thr Ser Gly His Val Ala Lys Ile Gly Asp Phe Gly 1 5 10 15 Leu Ala Arg Asp Ile Met Asn Asp Ser Asn Tyr Val Val Lys Gly Asn 20 25 30 Ala Arg Leu Pro Val Lys Trp Met Ala Pro Glu Ser Ile Phe Asp Cys 35 40 45 Val Tyr Thr Tyr Gln Ser 50 54 amino acids amino acid Not Relevant linear protein 51 Asn Ile Leu Leu Ser Glu Asn Asn Val Val Lys Ile Cys Asp Phe Gly 1 5 10 15 Leu Ala Arg Asp Ile Tyr Lys Asn Pro Asp Tyr Val Arg Arg Gly Asp 20 25 30 Thr Arg Leu Pro Leu Lys Trp Met Ala Pro Glu Ser Ile Phe Asp Lys 35 40 45 Val Tyr Ser Thr Lys Ser 50 54 amino acids amino acid Not Relevant linear protein 52 Asn Cys Leu Val Gly Gln Gly Leu Val Val Lys Ile Gly Asp Phe Gly 1 5 10 15 Met Ser Arg Asp Ile Tyr Ser Thr Asp Tyr Tyr Arg Val Gly Gly Arg 20 25 30 Thr Met Leu Pro Ile Arg Trp Met Pro Pro Glu Ser Ile Leu Tyr Arg 35 40 45 Lys Phe Thr Thr Glu Ser 50 54 amino acids amino acid Not Relevant linear protein 53 Asn Cys Leu Val Gly Glu Asn Leu Leu Val Lys Ile Gly Asp Phe Gly 1 5 10 15 Met Ser Arg Asp Val Tyr Ser Thr Asp Tyr Tyr Arg Val Gly Gly Arg 20 25 30 Thr Met Leu Pro Ile Arg Trp Met Pro Pro Glu Ser Ile Met Tyr Arg 35 40 45 Lys Phe Thr Thr Glu Ser 50 54 amino acids amino acid Not Relevant linear protein 54 Asn Cys Met Val Ala Glu Asp Phe Thr Val Lys Ile Gly Asp Phe Gly 1 5 10 15 Met Thr Arg Asp Ile Tyr Glu Thr Asp Tyr Tyr Arg Lys Gly Gly Lys 20 25 30 Gly Leu Leu Pro Val Arg Trp Met Ser Pro Glu Ser Leu Lys Asp Gly 35 40 45 Val Phe Thr Thr His Ser 50

Claims (1)

1. Substantially pure protein(s), or functional fragments thereof, characterized as having a tyrosine kinase domain and a tissue expression pattern characteristic of at least one receptor protein-tyrosine kinase subtype selected from the group consisting of tyro-1, tyro-2, tyro-4, tyro-5, tyro-6, tyro-7, tyro-8, tyro-10, tyro-11, and tyro-12.
US09/158,722 1992-05-15 1998-09-22 Protein-tyrosine kinase genes Abandoned US20030013848A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US09/158,722 US20030013848A1 (en) 1992-05-15 1998-09-22 Protein-tyrosine kinase genes

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US88448692A 1992-05-15 1992-05-15
US08/237,401 US5837448A (en) 1992-05-15 1994-05-02 Protein-tyrosine kinase genes
US08/456,647 US5811516A (en) 1992-05-15 1995-06-02 Tyro-3 protein tyrosine kinase
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