PROTEIN-TYROSINE KINASE GENES

Abstract

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

Description

CROSS-RELATED APPLICATIONS

[0001] 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.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] 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.

[0005] 2. Related Art

[0006] 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, 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.

[0007] 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., 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.

[0008] 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., 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).

[0009] 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., Nature, 333:560-562, 1988; Raff, et al., Nature, 333:562-565, 1988).

[0010] 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., 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

[0011] 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.

[0012] 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.

[0013] 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.

[0014] 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).

[0015] 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.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIGS. 1A and 1B show the tissue expression profiles of the novel PTK mRNAs.

[0017] FIG. 2 shows the developmental tissue profiles of the novel PTK mRNAs which were predominantly or exclusively neural in their distribution.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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., Nucleic Acid Research, 9:879, 1981).

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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., Nucleic Acid Research, 11:2325, 1983).

[0027] 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.

[0028] 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.

[0029] 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.

[0030] 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.

[0031] 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.

[0032] 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.

[0033] 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.

[0034] 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 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.

[0035] 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. (Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982).

[0036] 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., Nature, 340:205, 1989; Rose, et al., Gene, 60:237, 1987).

[0037] 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).

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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. (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.

[0045] 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.

[0046] 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.

[0047] 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 (CSH-Quantitative Biology, 43:63, 1978) and techniques for using RecA are reviewed in C. Radding (Ann. Rev. Genetics, 16:405-437, 1982).

[0048] 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.

[0049] 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 108:1 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.

[0050] 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.

[0051] 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.

[0052] 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. 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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).

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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, &PSgr;2, 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.

[0061] 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.

[0062] 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.

[0063] 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′)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.

[0064] 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 &mgr;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). 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).

[0065] 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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).

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

EXAMPLE 1

Isolation of Novel PTK Clones

[0074] 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.

[0075] 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 &mgr;g of muscle, 7.5 &mgr;g of liver, and 5 &mgr;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 &mgr;g of poly(A)-selected RNA from rat brain cerebellum (P19) and cortex (P3). Each hybridization was allowed to proceed to approximately R0t 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 &mgr;g 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).

[0076] 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 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 &mgr;g 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). 1 TABLE 1 (SEQ ID NOS:30-35) 1

[0077] Amplified DNAs were size fractionated on 5% non-denaturing acrylamide gels. The gels were stained with ethidium bromide (1 &mgr;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 &mgr;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.

[0078] 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.

EXAMPLE 2

Sequence Analysis of PCR Subclones

[0079] 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., 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).

[0080] Other domain sequences listed include fes (human; Roebroek, et al., 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).

[0081] 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. (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.

[0082] 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.

[0083] 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.

[0084] 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, 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

[0085] 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, EMBO J., 9:915-920, 1990) individual blots were in some cases reutilized for as many as four rounds of hybridization.

[0086] Total 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. for 2 hr as previously described (Monuki, et al., Neuron, 3:783-793, 1989). Probes for blot hybridizations were prepared using [&agr;-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.

[0087] In situ hybridization was performed according to Simmons, et al. (J. Histotechnology, 12:169-181, 1989), with minor modifications. Paraformaldehyde-fixed brain sections (30 &mgr;m), from either adult or 33-day-old rats were used. Antisense probes from PCR product Subclones were prepared using 125 &mgr;Ci or [35S] UTP (1250 Ci/mmol: New England Nuclear) in a 10 &mgr;l transcription reaction, with reagents obtained from Stratagene (La Jolla, Calif.). Hybridizations were performed at 55° C. for 22 hr using approximately 75 &mgr;l 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.

[0088] 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 &mgr;M forskolin for 48 hr prior to harvesting. All lanes contain either 2.5 &mgr;g of poly(A)+ RNA or 10 &mgr;g of total RNA, except for the cultured Schwann cell poly(A)+ lanes, which contain 1.0 &mgr;g 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).

[0089] 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.

[0090] 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 profiles 5 and 6).

[0091] 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.

[0092] Schwann cell expression of certain kinase genes was strongly regulated by cAMP (FIG. 1). As for the PDGF receptor gene (Weinmaster and Lemke, 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.

[0093] 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.

EXAMPLE 4

Developmental Expression Profile of Neural PTK mRNAs

[0094] 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.

[0095] Poly(A)+ RNA (2 &mgr;g) 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).

[0096] 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.

[0097] 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.

EXAMPLE 5

In situ Localization of Novel PTK Transcripts in Brain

[0098] 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 &mgr;m 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.

[0099] 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 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.

[0100] 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.

[0101] 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.

[0102] 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.

[0103] 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 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. 2 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)

[0104] 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.

Claims

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.