WO1996003043A1 - Human brain specific kinase - Google Patents

Abstract

The present invention relates, in general, to human brain specific kinase, hBsk. In particular, the present invention relates to nucleic acid molecules coding for hBsk; hBsk polypeptides; recombinant nucleic acid molecules; cells containing the recombinant nucleic acid molecules; antibodies having binding affinity specifically to hBsk; hybridomas containing the antibodies; nucleic acid probes for the detection of hBsk nucleic acid; a method of detecting hBsk nucleic acid or polypeptide in a sample; and kits containing nucleic acid probes or antibodies. This invention further relates to bioassays using the nucleic acid sequence, receptor protein or antibodies of this invention to diagnose, assess, or prognose a mammal afflicted with neurodegenerative disease. Therapeutic uses for the hBsk receptor-like tyrosine kinase are also provided. This invention also relates to the ligand for the hBsk receptor, and diagnostic and therapeutic uses for the hBsk ligand.

Description

HUMAN BRAIN SPECIFIC KINASE

Background of the Invention

Field of the Invention

The present invention relates, in general, to human brain specific kinase, hBsk. In particular, the present invention relates to nucleie acid molecules coding for hBsk; hBsk polypeptides; recombinant nucleic acid molecules; cells containing the recombinant nucleic acid molecules; antibodies having binding affinity specifically to hBsk; hybridomas containing the antibodies; nucleic acid probes for the detection of hBsk nucleic acid; a method of detecting hBsk nucleic acid or polypeptide in a sample; and kits containing nucleic acid probes or antibodies.

Background Information

Neuronal degeneration has been shown to be involved in many neurological disorders (Price et al., in Selective Neuronal Death, Ciba Foundation Symposium 126, Wiley & Chichester, eds. (1987), pp. 30-48).

For example, in Alzheimer's Disease (AD), neuronal loss has been reported in a variety of brain structures including the locus coemleus and raphe complex of the brainstem, the basal forebrain cholinergic system, amygdala, hippocampus and neocortex (Coleman & Flood, Neurobiol. Aging 5:521-545 (1987)). Although the pattern of cell loss in AD has similarities to that in the aging brain, the speed and amount of loss is far greater. The most striking loss of neurons compared with the age-matched controls occurs in the hippocampal region, with a loss of up to 57% of the pyramidal cells (Coleman and Flood, Neurobiol. Aging 5:521-545 (1987)). These observation indicate that the hippocampus is a key structure in the neurobiology of AD. The extent of cell loss is most evident in the CA1 and subiculum, while areas CA3 and CA4 and granular cells of the dentate gy s are largely spared (Van Hoesen and Hyman, Progress in Brain Research 55:445-457 (1990)).

Neuronal degeneration in the hippocampus has long been known to be a site of pathological change in epileptic patients (Nadler, in The

Hippocampus-New Vistas, Chan-Palay V., Koehler C, eds.„ Alan R. Liss, Inc., New York (1989), pp. 463-481). CA3-CA4 damage is nearly always observed in pharmacologically intractable complex partial (limbic, temporal lobe, psychomotor) epileptic patients, while CA1 damage is also frequently observed (Nadler, in The Hippocampus-New Vistas, Chan-Palay V.,

Koehler C, eds.„ Alan R. Liss, Inc., New York (1989), pp. 463-481). Since frequently prominent cell loss in the CA3-CA4 area may be present without obvious damage to area CA1 , it is believed that the CA3 pyramidal cells and the morphologically diverse CA4 neurons are most vulnerable. Although the relationship between hippocampal sclerosis and seizure has been a controversy for over a century, recent studies with animal models indicate that although other etiologies may exist, hippocampal lesions can arise from prolonged febrile convulsions/status epilepticus (Sutula, Epilepsia 57:345-554 (1990)). Furthermore, whatever the original cause of the sclerotic lesion, the damage serves as a focus for hyperexcitability and eventually causes spontaneous seizures. Formation of aberrant excitatory circuitry through axon sprouting and permanently depressed synaptic inhibition were thought to be two major factors linking hippocampal damage to the subsequent development of an epileptic focus (Nadler, in The Hippocampus-New Vistas, Chan-Palay V., Koehler C, eds.,, Alan R. Liss, Inc., New York (1989), pp. 463-481; Sutula, Epilepsia 31 (Suppl. 3):545-554 (1990)).

Hippocampal defects are also suggested to be involved in schizophrenia (Bogerts et al., Arch. Gen. Psychiatry 42:184-191 (1985)). Significant reductions in hippocampal volume were found in chronic schizophrenic patients, possibly due to degenerative shrinkages of unknown etiology (Bogerts et al., Arch. Gen. Psychiatry 42:1 '84-791 (1985); Bogerts et al , Biol. Psychiatry 55:236-246 (1993). The reduced volume in hippocampus and other limbic system structures such as amygdala and parahippocampal gyms was associated with increased severity of psychopathology (Bogerts et al, Biol. Psychiatry 55:236-246 (1993)). These changes in the limbic system in schizophrenia are rather specific since the volumes of the putamen, caudate nucleus, nucleus accumbens, and the red nucleus of the stria teπninalis did not differ between patients and controls (Bogerts et al, Biol Psychiatry 55:236-246 (1993)).

The hippocampus and its adjacent, anatomically related entorhinal, perύhinal, and parahippocampal cortices play an essential, although temporal, role for establishing long-term memory for facts and events (Squire and Zola-Morgan, Science 255:1380-1386 (1991)). The widespread and reciprocal connections between hippocampal structures and neocortex may explain their degeneration in a variety of neurological diseases. Understanding the mechanism of neuronal survival in the hippocampus may help to develop effective treatments of neural degenerative diseases or disorders as well as neoplasms involving neuronal tissue. It is known that growth/trophic factors promote the differentiation and survival of neurons during development and regeneration of the nervous system, with specific types of neurons requiring specific growth factors (Barde, Neuron 2:1525-1534 (1989)). Nerve growth factor (NGF) has been a model trophic factor (Levi-Montalcini, Science 257: 1154-1162 (1987); Black et al , in Current Topics in Developmental Biology,

Volume 24, Academic Press Inc., (1990), pp. 161-182; Gage et al, Current Topics in Microbiology and Immunology, Volume 165, Both well, M., ed., Springer Verlag, (1991), pp. 71-92). In vivo depletion by inducing an auto-immune reaction to NGF in rats and guinea pigs during embryonic development results in the destruction of up to 85 % of the dorsal root ganglion neurons and the destmction of peripheral sympathetic neurons (Colkart-Gorin an Johnson, Proc. Natl. Acad. Sci. USA 76:5382- 5386 (1979); Johnson et al, Science 270:916-918 (1980)). Other polypeptide growth factors also have trophic effects on neurons (Nieto- Sampedro and Vovolenta, in Progress in Brain Research, Vol. 83,

Mathison, Zimmer, Otterson, eds., Elsevier, New York (1990), pp. 341- 355). The fibroblast growth factors are well known- mitogens (Gospodarowicz, in Current Topics in Developmental Biology, Vol. 24, Hilsen-Hamilton, M. ed., Academic Press Inc., San Diego (1990), pp. 57-93) that exhibit potent neurotrophic activity both in vivo (Anderson et al, Nature 552:360-361 (1988)) and on cultured neurons from many brain regions (Morrison et al, Proc. Natl. Acad. Sci. USA 55:7537-7541 (1986); Morrison, Neuroscience Res. 77:99-101 (1987); Walicke J. Neursci. 6:1114-1121 (1988); Wagner, in Current Topics in Microbiology and Immunology, Vol. 165, Bothwell, M. ed., Springer Verlag, New York

(1991), pp. 95-112) including the hippocampus (Walicke et al. , Proc. Natl. Acad. Sci. USA 55:3012-3016 (1986). Epidermal growth factor (EFG) was shown to enhance the survival and process outgrowth of primary cultures of subneocortical telencephalic neurons of neonatal rat brain (Morrison et al, Science 255:72-75 (1987)). Brain derived neurotrophic factor

(BDNF) (Leibrock et al, Nature 341:149-152 (1989), and neurotrophin-3 (NT-3) (Masisonpierre et al, Science 247:1446-1451 (1990); Hohn et al, Nature 344:339-341 (1990)) are two neurotrophic factors cloned recently. BDNF, related to NGF, has neurotrophic activity for sensory and retinal ganghon neurons and rescues spinal motor neurons in vivo from axotomy- induced cell death (Sendtner et al, Nature 360 (1992); Yan et al, Nature 360:153-155 (1992)). NT-3 was shown to support the growth of neurons from dorsal root ganghon, the neural placode-derived nodose ganghon, and the paravertebral chain sympathetic ganghon (Maisonpierre et al. , Science 247:1446-1451 (1990)). The potential involvement of growth factors in neuronal regeneration aft^- injuries or in disease is demonstrated by the fact that brain injury causes a time dependent increase in neurotrophic activity at the lesion site (Nieto-Sampedro et al, Science 277:860-861 (1982). Furthermore, intraventricular administration of NGF prevents retrograde degeneration of axotomized septal cholinergic neurons (Hefti J. Neurosci. 6:2155-2162 (1986); Kromer, Science 255:214-216 (1987); Williams et al , Proc. Natl. Acad. Sci. USA 55:9231-9235 (1986)) and local appUcation of BDNF prevents spinal motor neuron degeneration after nerve section (Sendtner et al. , Nature 360:153-155 (1992)). The therapeutic value of neurotrophic factors for nerve injury or neurodegenerative disease is shown by the observation that the symptoms of he mice with progressive motor neuropathy is relieved by the use of ciliary neurotrophic factor (Sendtner et al, Nature 255:502-504 (1992)). Growth/trophic factors function through their receptors which often possess intrinsic protein tyrosine kinase activity (Schlessinger & Ullrich, Neuron 9:383-931 (1992)). In general, the receptor protein-tyrosine kinases are composed of an extracellular domain, a membrane spanning domain and a catalytic domain (Schlessinger and Ullrich, Neuron 9:383-391 (1992). Binding of the growth/trophic factor to the extracellular domain activates the catalytic domain inside the cell and results in phosphorylation of substrates within the cell. Activation of the receptor is believed to mediate a variety of cellular processes including cell growth and differentiation. In addition, many receptor tyrosine kinases are expressed during embryogenesis and are therefore believed to be important in the mechanisms underlying oncogenesis and cellular growth (Wilks, Advances in Cancer Research 60:43-13 (1993)). Increased or aberrant expressions of tyrosine kinase receptors has been associated with several human neoplasms, including glioblastomas, squamous carcinomas, breast and gastric cancers (Carpenter, Ann. Rev. Biochem. 56: 881-914 (1987); Muller et al., Cell 54:105-109 (1989); Kraus et al, Proc. Natl. Acad. Sci. USA 56^9193-9197 (1989)). Recently, it has been demonstrated that expression of trk, the tyrosine kinase receptor for NGF, in neuroblastomas is indicative of a better prognosis for the patient than those patients having neuroblastomas without trk expression (Kogner, et al. , Cancer Research

55:2044-2050 (1993); Nakagawara et al, New Eng. J. Med. 525:847-854 (1993); Suzuki et al, J. Natl. Can. Inst. 55:377-384 (1993)), suggesting a therapeutic value for the neurotrophic receptors as well as their ligands. Recently, a new family of tyrosine kinase receptors have been discovered and designated the eph/elk family (Zhou et al , J. Neuroscience

Re. 37(1): 129-143 (1994); Sajjadi et al, Oncogene 5:1807-1813 (1993)). Members of the eph/elk family have also been demonstrated to have aberrant expression in certain neoplasm as well as transfoirming ability. For example, elevated expression of EPH bus been detected in carcinomas of the liver, lung, breast and colon (Hirai et al, Science 255:1717-1720

(1987); Mam et al., Mol. Cell Biol. 5:3770-3776 (1988)). Overexpression of eph has been shown to result in transformation of cells as well as development of tumors in nude mice (Mam et al , Oncogene 5:445-447 (1990)). The distinct tissue distributions of the eph/elk family members suggest that each member may serve specific functions.

These findings demonstrate the extensive involvement of growth factors and their corresponding receptors in the survival of neurons and their potential therapeutic value in neurodegenerative diseases, neuronal disorders and neoplasms. Alzheimer's epilepsy and schizophrenia are but a few of the diseases associated with degeneration of neurons in the hippocampus. However, the factors needed for the regeneration and survival of neurons in the hippocampus and its associated limbic system are poorly characterized. The identification of factors which promote the regeneration and survival of these neurons will be potentially useful in the treatment of the neoplasms, neurodegenerative diseases or disorders and brain injuries involving the limbic system.

Zhou et al. , J. ofNeuroscience Res. 57:129-143 (1994) describes the isolation and characterization of a mouse Bsk.

Summary of the Invention

The invention provides an isolated nucleic acid molecule coding for a polypeptide comprising an amino acid sequence corresponding to human brain specific kinase, hBsk.

The invention further provides a substantially pure polypeptide comprising an amino acid sequence corresponding to hBsk.

The invention also provides a nucleic acid probe for the specific detection of the presence of hBsk in a sample.

The invention further provides a method of detecting hBsk nucleic acid in a sample. The invention also provides a kit for detecting the presence of hBsk nucleic acid in a sample.

The invention further provides a recombinant nucleic acid molecule comprising, 5' to 3', a promoter effective to initiate transcription in a host cell and the above-described isolated nucleic acid molecule. The invention also provides a recombinant nucleic acid molecule comprising a vector and the above-described isolated nucleic acid molecule.

The invention further provides a recombinant nucleic acid molecule comprising a sequence complimentary to an RNA sequence encoding an amino acid sequence corresponding to the above-described polypeptide. The invention also provides a cell that contains the above-described recombinant nucleic acid molecule.

The invention further provides a non-human organism that contains the above-described recombinant nucleic acid molecule. The invention also provides an antibody having binding affinity specifically to a hBsk polypeptide.

The invention further provides a method of detecting a hBsk polypeptide in a sample. The invention also provides a method of measuring the amount of hBsk in a sample.

The invention further provides a diagnostic kit comprising a first container means containing the above-described antibody, and a second container means containing a conjugate comprising a binding partner of said monoclonal antibody and a label.

The invention also provides a hybridoma which produces the above- described monoclonal antibody.

The invention further provides diagnostic methods for human disease, in particular neurodegenerative diseases, disorders, and injuries. The invention also provides methods for therapeutic uses involving all or part of the nucleic acid sequence encoding hBsk and its corresponding protein.

The invention provides assays for the isolation of the ligand or ligands capable of activating the hBsk receptor and therapeutic uses for said ligand.

The invention also provides assays for the assessment and development of dmgs capable of activating the hBsk receptor and therapeutic uses for said dmgs.

Further objects and advantages of the present invention will be clear from the description that follows.

Brief Description of the Drawings

FIGURE 1. Schematic of overlapping human Bsk cDNA Isolates. Human Bsk cDNA clones are shown with their extent of overlap under a schematic of the mouse Bsk gene comprising: an extracellular domain (E), transmembrane domain (TM) and a kinase domain (K). The nucleotide numbers at the 3' and/or 5' ends of the isolates represent the corresponding nucleotide number within the mouse Bsk sequence (See, Zhou et al. (1994) J. Neuroscience Res. 37: 129- 143.

FIGURE 2A-2J. Partial Nucleotide Sequence of a hBsk gene.

Figure 2A, 2C, 2E, 2G, and 21 describe a partial nucleotide sequence of

Clones 6-1, 7-2, 8-1, 16-1, and 19-1, respectively, using an sk primer.

Figure 2B, 2D, 2F, 2H, and 2J describe a partial nucleotide sequence of Clones 6-1, 7-2, 8-1, 16-1, and 19-1, respectively, using a T7 primer.

Definitions

In the description that follows, a number of terms used in recombinant DNA (rDNA) technology are extensively utilized. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided.

Isolated Nucleie Acid Molecule. An "isolated nucleic acid molecule", as is generally understood and used herein, refers to a polymer of nucleotides, and includes but should not be limited to DNA and RNA. DNA Segment. A DNA segment, as is generally understood and used herein, refers to a molecule comprising a linear stretch of nucleotides wherein the nucleotides are present in a sequence that may encode, through the genetic code, a molecule comprising a linear sequence of amino acid residues that is referred to as a protein, a protein fragment or a polypeptide.

Gene. A DNA sequence related to a single polypeptide chain or protein, and as used herein includes the 5' and 3' untranslated ends. The polypeptide can be encoded by a full-length sequence or any portion of the coding sequence, so long as the functional activity of the protein is retained.

Complementary DNA (cDNA). Recombinant nucleic acid molecules synthesized by reverse transcription of messenger RNA ("mRNA").

Structural Gene. A DNA sequence that is transcribed into mRNA that is then translated into a sequence of amino acids characteristic of a specific polypeptide.

Restriction Endonuclease. A restriction endonuclease (also restriction enzyme) is an enzyme that has the capacity to recognize a specific base sequence (usually 4, 5, or 6 base pairs in length) in a DNA molecule, and to cleave the DNA molecule at every place where this sequence appears. For example, EcόSl recognizes the base sequence GAATTC/CTTAAG. Restriction Fragment. The DNA molecules produced by digestion with a restriction endonuclease are referred to as restriction fragments. Any given genome may be digested by a particular restriction endonuclease into a discrete set of restriction fragments.

Agarose Gel Electrophoresis. To detect a polymorphism in the length of restriction fragments, an analytical method for fractionating double-stranded DNA molecules on the basis of size is required. The most commonly used technique (though not the only one) for achieving such a fractionation is agarose gel electrophoresis. The principle of this method is that DNA molecules migrate through the gel as though it were a sieve that retards the movement of the largest molecules to the greatest extent and the movement of the smallest molecules to the least extent. Note that the smaller the DNA fragment, the greater the mobility under electrophoresis in the agarose gel.

The DNA fragments fractionated by agarose gel electrophoresis can be visualized directly by a staining procedure if the number of fragments included in the pattern is small. The DNA fragments of genomes can be visualized successfully. However, most genomes, including the human genome, contain far too many DNA sequences to produce a simple pattem of restriction fragments. For example, the human genome is digested into approximately 1,000,000 different DNA fragments by -EcøRI. In order to visualize a small subset of these fragments, a methodology referred to as the Southern hybridization procedure can be applied.

Southern Transfer Procedure. The purpose of the Southern transfer procedure (also referred to as blotting) is to physically transfer DNA fractionated by agarose gel electrophoresis onto a nitrocellulose filter paper or another appropriate surface or method, while retaining the relative positions of DNA fragments resulting from the fractionation procedure. The methodology used to accomplish the transfer from agarose gel to nitrocellulose involves drawing the DNA from the gel into the nitrocellulose paper by capillary action.

Nucleic Acid Hybridization. Nucleic acid hybridization depends on the principle that two single-stranded nucleic acid molecules that have complementary base sequences will reform the meπnodynamically favored double-stranded structure if they are mixed under the proper conditions. The double-stranded structure will be formed between two complementary single-stranded nucleic acids even if one is immobilized on a nitrocellulose filter. In the Southern hybridization procedure, the latter situation occurs. As noted previously, the DNA of the individual to be tested is digested with a restriction endonuclease, fractionated by agarose gel electrophoresis, converted to the single-stranded form, and transferred to nitrocellulose paper, making it available for reannealing to the hybridization probe.

Hybridization Probe. To visualize a particular DNA sequence in the Southern hybridization procedure, a labeled DNA molecule or hybridization probe is reacted to the fractionated DNA bound to the nitrocellulose filter. The areas on the filter that carry DNA sequences complementary to the labeled DNA probe become labeled themselves as a consequence of the rea πealing reaction. The areas of the filter that exhibit such labeling are visualized. The hybridization probe is generally produced by molecular cloning of a specific DNA sequence. Oligonucleotide or Otigomer. A molecule comprised of two or more deoxyribonucleotides or ribonucleotides, preferably more than three. Its exact size will depend on many factors, which in turn depend on the ultimate function or use of the oligonucleotide. An oligonucleotide may be derived synthetically or by cloning. Sequence Amplification. A method for generating large amounts of a target sequence. In general, one or more amplification primers are annealed to a nucleic acid sequence. Using appropriate enzymes, sequences found adjacent to, or in between the primers are amplified.

Amplification Primer. An oligonucleotide which is capable of annealing adjacent to a target sequence and serving as an initiation point for

DNA synthesis when placed under conditions in which synthesis of a primer extension product which is complementary to a nucleic acid strand is initiated.

Vector. A plasmid or phage DNA or other DNA sequence into which DNA may be inserted to be cloned. The vector may replicate autonomously in a host cell, and may be further characterized by one or a small number of endonuclease recognition sites at which such DNA sequences may be cut in a deteπninable fashion and into which DNA may be inserted. The vector may further contain a marker suitable for use in die identification of cells transformed with the vector. Markers, for example, are tetracycline resistance or ampicillin resistance. The words "cloning vehicle" are sometimes used for "vector."

Expression. Expression is the process by which a structural gene produces a polypeptide. It involves transcription of the gene into mRNA, and the translation of such mRNA into polypeptide(s). Expression Vector. A vector or vehicle similar to a cloning vector but? which is capable of expressing a gene which has been cloned into it, after transformation into a host. The cloned gene is usually placed under the control of (i.e., operably linked to) certain control sequences such as promoter sequences.

Expression control sequences will vary depending on wheύier the vector is designed to express the operably linked gene in a prokaryotic or eukaryotic host and may additionally contain transcriptional elements such as enhancer elements, teπnination sequences, tissue-specificity elements, and/or translational initiation and termination sites.

Functional Derivative. A "functional derivative" of a sequence, either protein or nucleic acid, is a molecule that possesses a biological activity (either functional or stmctural) that is substantially similar to a biological activity of the protein or nucleic acid sequence. A functional derivative of a protein may or may not contain post-translational modifications such as covalently linked carbohydrate, depending on the necessity of such modifications for the performance of a specific function. The term "functional derivative" is intended to include e "fragments," "segments," "variants," "analogs," or "chemical derivatives" of a molecule. As used herein, a molecule is said to be a "chemical derivative" of anouier molecule when it contains additional chemical moieties not normally a part of the molecule. Such moieties may improve the molecule's solubility, absorption, biological half life, and me like. The moieties may alternatively decrease the toxicity of die molecule, etøiiinate or attenuate any undesirable side effect of the molecule, and the like.

Moieties capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences (1980). Procedures for coupling such moieties to a molecule are well known in the art.

Variant. A "variant" of a protein or nucleic acid is meant to refer to a molecule substantially similar in structure and biological activity to either the protein or nucleic acid. Thus, provided that two molecules possess a common activity and may substitute for each other, they are considered variants as that term is used herein even if the composition or secondary, tertiary, or quaternary stmcture of one of the molecules is not identical to that found in the other, or if the amino acid or nucleotide sequence is not identical.

Substantially Pure. A "substantially pure" protein or nucleic acid is a protein or nucleic acid preparation that is generally lacking in other cellular components. Ligand. Ligand refers to any protein or proteins that may interact with the hBsk receptor binding domain. Said ligand or ligands may be soluble or membrane bound. The ligand or ligands may be a naturally occurring protein, or synthetically or recombinantly produced. The ligand may also be a nonprotein molecule that acts as ligand when it interacts with the Bsk receptor binding domain. Interactions between the ligand and receptor binding domain include, but are not limited to, any covalent or non-covalent interactions. The receptor binding domain is any region of the hBsk receptor molecule that interacts directly or indirectly with the hBsk ligand. Neurodegenerative disease. The term neurodegenerative disease includes, but is not limited to, states in a mammal which can include chromosomal abnormalities, degenerative growth and developmental disorders, viral infections, bacterial infections, brain injuries, or neoplastic conditions. Examples of neurodegenerative diseases that can be diagnosed, assessed or treated by methods described in the present appUcation include, but are not limited to, Alzheimer's disease, epilepsy, schizophrenia. In a preferred embodiment diseases characterized by neurodegeneration in the limbic system are diagnosed, assessed or treated by methods disclosed in the present appUcation. Examples of injuries to the nervous system include, but are not limited to, stroke and cerebral ischemia due to stroke or cardiac arrest. Also considered within this definition is the treatment of injury to the nervous system. Further, neoplasms involving neuronal tissue may be diagnosed, assessed or therapeutically treated by methods suggested herein. Drug. Dmgs include, but are not limited to proteins, peptides, degenerate peptides, agents purified form conditioned cell medium, organic molecules, inorganic molecules, antibodies or oUgonucleotides. Other • candidate dmgs include analogs of the hBsk ligand or Ugands. The drug may be naturally occurring or synthetically or recombinantiy produced. One skilled in the art will understand that such dmgs may be developed by the assays described below.

Detailed Description of the Invention

For purposes of clarity of disclosure, and not by way of limitation, the detailed description of the invention is divided into the foUowing subsections:

I. Isolated Nucleic Acid Molecules Coding for hBsk

Polypeptides. π. Substantially Pure hBsk Polypeptides. in. A Nucleic Acid Probe for the Specific Detection of hBsk. IV. A Method of Detecting The Presence of hBsk in a Sample.

V. A Kit for Detecting the Presence of hBsk in a Sample.

VI. DNA Constructs Comprising a hBsk Nucleic Acid Molecule and Cells Containing These Constructs.

VII . An Antibody Having Binding Affinity to an hBsk Polypeptide and a Hybridoma Containing the Antibody.

Viπ. A Mediod of Detecting an hBsk Polypeptide in a Sample. IX. A Diagnostic Kit Comprising Antibodies to hBsk. - X. Diagnostic Screening and Treatment

/. Isolated Nucleic Acid Molecules Coding for hBsk Polypeptides

In one embodiment, the present invention relates to an isolated nucleic acid molecule coding for a polypeptide having an amino acid sequence corresponding to human brain specific kinase, hBsk. In one preferred embodiment, e isolated nucleic acid molecule comprises the hBsk nucleotide sequence present in Clones 6-1 and 8-1 (See Figure 1) as deposited with the ATCC. In another preferred embodiment, the isolated nucleic acid molecule encodes the hBsk amino acid sequence present in

Clones 6-1 and 8-1 (See Figure 1) as deposited witii the ATCC. The hBsk sequence within Clones 6-1 and 8-1 is identified by its homology to the mouse bsk sequence (See Figure 1).

Due to the degeneracy of nucleotide coding sequences, other DNA sequences which encode the same amino acid sequence as found in Clones

6-1 and 8-1 may be used in the practice of the present invention.

A. Isolation of Nucleic Acid

In one aspect of the present invention, isolated nucleic acid molecules coding for polypeptides having amino acid sequences corresponding to hBsk are provided. In particular, the nucleic acid molecule may be isolated from a biological sample containing human RNA or DNA.

The nucleic acid molecule may be isolated from a biological sample containing human RNA using the techniques of cDNA cloning and subtractive hybridization. The nucleic acid molecule may also be isolated from a cDNA Ubrary using a homologous probe. The nucleic acid molecule may be isolated from a biological sample containing human genomic DNA or from a genomic Ubrary. Suitable biological samples include, but are not limited to, blood, semen and tissue.

The method of obtaining the biological sample will vary depending upon the nature of me sample.

One skilled in the art wiU realize that the human genome may be subject to sUght alleUc variations between individuals. Therefore, the isolated nucleic acid molecule is also intended to include alleUc variations, so long as the sequence is a functional derivative of the hBsk gene. When a hBsk allele does not encode the identical sequence to that found in Clones

6-1 and 8-1, it can be isolated and identified as hBsk using the same techniques used herein, and especially PCR techniques to amplify the appropriate gene with primers based on the sequences disclosed herein.

B. Synthesis of Nucleic Acid

Isolated nucleic acid molecules of the present invention are also meant to include mose chemicaUy synthesized. For example, a nucleic acid molecule with me nucleotide sequence which codes for the expression product of a hBsk gene may be designed and, if necessary, divided into appropriate smaUer fragment. Then an oUgomer which corresponds to the nucleic acid molecule, or to each of me divided fragments, may be synthesized. Such synύietic oUgonucleotides may be prepared, for example, by me triester method of Matteucci et al , J. Am. Chem. Soc. 705:3185-3191 (1981) or by using an automated DNA synthesizer.

An oUgonucleotide may be derived synthetically or by cloning. If necessary, the 5 '-ends of the oUgomers may be phosphorylated using T4 polynucleotide kinase. Kinasing of single strands prior to annealing or for labeling may be achieved using an excess of the enzyme. If kinasing is for the labeling of probe, the ATP may contain high specific activity radioisotopes. Then, the DNA oUgomer may be subjected to annealing and Ugation with T4 Ugase or the like.

//. Substantially Pure hBsk Polypeptides

In another embodiment, title present invention relates to a substantially pure polypeptide having an amino acid sequence corresponding to hBsk. In a preferred embodiment, the polypeptide has the amino acid sequence set forth in Clones 6-1 and 8-1 (See Figure 1).

A variety of methodologies known in the art can be utilized to obtain the peptide of the present invention. In one embodiment, the peptide is purified from human tissues or cells which naturally produce the peptide.

Alternatively, the above-described isolated nucleic acid fragments could be used to expressed the hBsk protein in any organism. The samples of the present invention include cells, protein extracts or membrane extracts of cells, or biological fluids. The sample will vary based on the assay format, the detection method and title nature of the tissues, ceUs or extracts used as the sample.

One skilled in the art can readily follow known methods for isolating proteins in order to obtain the peptide free of natural contaminants. These include, but are not limited to: immunochromotography, size-exclusion chromatography, HPLC, ion-exchange chromatography, and immunoaffinity chromatography.

777. A Nucleic Acid Probe for the Specific Detection of hBsk

In another embodiment, the present invention relates to a nucleic acid probe for the specific detection of the presence of hBsk in a sample comprising the above-described nucleic acid molecules or at least 18 contiguous nucleotides thereof (preferably at least 20, 25, 30, 35, 40, or 50 thereof). The probe is designed such that it does not have 100% homology with a similarly located mouse Bsk probe.

The nucleic acid probe may be used to probe an appropriate chromosomal or cDNA Ubrary by usual hybridization methods to obtain another nucleic acid molecule of the present invention. A chromosomal

DNA or cDNA Ubrary may be prepared from appropriate ceUs according to recognized methods in the art (cf. Molecular Cloning: A Laboratory

Manual, second edition, edited by Sambrook, Fritsch, & Maniatis, Cold

Spring Harbor Laboratory, 1989). In the alternative, chemical synthesis is carried out in order to obtain nucleic acid probes having nucleotide sequences which correspond to

N-terminal and C-teπninal portions of the amino acid sequence of the hBsk.

Thus, the synuiesized nucleic acid probes may be used as primers in a polymerase chain reaction (PCR) carried out in accordance with recognized PCR techniques, essentially according to PCR Protocols, A Guide to

Methods and Applications, edited by Michael et al., Academic Press, 1990, utilizing the appropriate chromosomal or cDNA Ubrary to obtain the fragment of the present invention.

One skilled in the art can readily design such probes based on the sequence disclosed herein using methods of computer aUgnment and sequence analysis known in title art (cf . Molecular Cloning: A Laboratory

Manual second edition, edited by Sambrook, Fritsch, & Maniatis, Cold

Spring Harbor Laboratory, 1989).

The hybridization probes of the present invention can be labeled by standard labeling techniques such as with a radiolabel, enzyme label, fluorescent label, biotin-avidin label, chemuuminescence, and the like.

After hybridization, the probes may be visualized using known methods. The nucleic acid probes of the present invention include RNA, as weU as DNA probes, such probes being generated using techniques known in the art. In one embodiment of the above described method, a nucleic acid probe is immobilized on a soUd support. Examples of such soUd supports include, but are not limited to, plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, and acryhc resins, such as polyacrylamide and latex beads. Techniques for coupling nucleic acid probes to such soUd supports are well known in the art.

The test samples suitable for nucleic acid probing methods of the . present invention include, for example, cells or nucleic acid extracts of cells, or biological fluids. The sample used in the above-described methods will vary based on the assay format, the detection method and the nature of the tissues, ceUs or extracts to be assayed. Methods for preparing nucleic acid extracts of cells are weU known in the art and can be readily adapted in order to obtain a sample which is compatible with the method utilized.

IV. A Method of Detecting The Presence of hBsk in a Sample

In another embodiment, the present invention relates to a method of detecting the presence of hBsk in a sample comprising a) contacting said sample with d e above-described nucleic acid probe, under conditions such that hybridization occurs, and b) detecting the presence of said probe bound to said nucleic acid molecule. One skilled in the art would select the nucleic acid probe according to techniques known in the art as described above. Samples to be tested include but should not be limited to RNA samples of human tissue. hBsk has been found to be expressed in brain ceUs. Accordingly, hBsk probes may be used detect the presence of RNA from brain ceUs in a sample. Further, altered expression levels of hBsk RNA in an individual, as compared to normal levels, may indicate the presence of disease. The hBsk probes may further be used to assay ceUular activity in general and specifically in brain tissue.

V. A Kit for Detecting the Presence of hBsk in a Sample

In another embodiment, the present invention relates to a kit for detecting the presence of hBsk in a sample comprising at least one container means having disposed therein me above-described nucleic acid probe. In a preferred embodiment, the kit further comprises other containers comprising one or more of the following: wash reagents and reagents capable of detecting the presence of bound nucleic acid probe. Examples of detection reagents include, but are not limited to radiolabeUed probes, enzymatic labeled probes (horse radish peroxidase, alkaline phosphatase), and affinity labeled probes (biotin, avidin, or steptavidin).

In detail, a compartmentalized kit includes any kit in which reagents are contained in separate containers. Such containers include small glass containers, plastic containers or strips of plastic or paper. Such containers allow the efficient transfer of reagents from one compartment to another compartment such that the samples and reagents are not cross-contaminated and me agents or solutions of each container can be added in a quantitative fashion from one compartment to another. Such containers will include a container which will accept the test sample, a container which contains the probe or primers used in the assay, containers which contain wash reagents (such as phosphate buffered saline, Tris-buffers, and the like), and containers which contain the reagents used to detect the hybridized probe, bound antibody, ampUfied product, or the like. One skiUed in the art will readily recognize that the nucleic acid probes described in the present invention can readily be incorporated into one of the estabUshed kit formats which are well known in the art. VI. DNA Constructs Comprising a hBsk Nucleic Acid Molecule and Cells Containing These Constructs

In another embc liment, the present invention relates to a recombinant DNA molecule comprising, 5' to 3', a promoter effective to initiate transcription in a host cell and the above-described nucleic acid molecules. In another embodiment, the present invention relates to a recombinant DNA molecule comprising a vector and an above-described nucleic acid molecule.

In another embodiment, the present invention relates to a nucleic acid molecule comprising a transcriptional control region functional in a ceU, a sequence complimentary to an RNA sequence encoding an amino acid sequence corresponding to the above-described polypeptide, and a transcriptional termination region functional in said ceU.

Preferably, the above-described molecules are isolated and/or purified DNA molecules.

In another embodiment, the present invention relates to a ceU or non-human organism that contains an above-described nucleic acid molecule.

In another embodiment, me peptide is purified from ceUs which have been altered to express the peptide.

As used herein, a cell is said to be "altered to express a desired peptide" when the cell, through genetic manipulation, is made to produce a protein which it normally does not produce or which the ceU normaUy produces at low levels. One skilled in the art can readily adapt procedures for introducing and expressing either genomic, cDNA, or synthetic sequences into either eukaryotic or prokaryotic ceUs.

A nucleic acid molecule, such as DNA, is said to be "capable of expressing" a polypeptide if it contains nucleotide sequences which contain transcriptional and translational regulatory information and such sequences are "operably linked" to nucleotide sequences which encode the polypeptide. An operable linkage is a linkage in which the regulatory DNA sequences and the DNA sequence sought to be expressed are connected in such a way as to permit gene sequence expression. The precise nature of the regulatory regions needed for gene sequence expression may vary from organism to organism, but shaU in general include a promoter region which, in prokaryotes, contains both the promoter (which directs the initiation of RNA transcription) as weU as the DNA sequences which, when transcribed into RNA, wiU signal synthesis initiation. Such regions will normally include those 5 '-non-coding sequences involved with initiation of transcription and translation, such as the TATA box, capping sequence, CAAT sequence, and title like.

If desired, the non-coding region 3' to the sequence encoding an hBsk gene may be obtained by the above-described methods. This region may be retained for its transcriptional termination regulatory sequences, such as termination and polyadenylation. Thus, by retaining the 3 '-region naturally contiguous to the DNA sequence encoding a hBsk gene, the transcriptional termination signals may be provided. Where the transcriptional termination signals are not satisfactorily functional in the expression host ceU, then a 3' region functional in the host ceh may be substituted.

Two DNA sequences (such as a promoter region sequence and an hBsk sequence) are said to be operably linked if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region sequence to direct the transcription of a hBsk gene sequence, or (3) interfere with title ability of the hBsk gene sequence to be transcribed by the promoter region sequence. Thus, a promoter region would be operably linked to a DNA sequence if die promoter were capable of effecting transcription of that DNA sequence. The present invention encompasses the expression of the hBsk gene (or a functional derivative thereof) in either prokaryotic or eukaryotic cells. Prokaryotic hosts are, generally, the most efficient and convenient for the production of recombinant proteins and, therefore, are preferred for title expression of the hBsk gene.

Prokaryotes most frequendy are represented by various strains of E. coli. However, other microbial strains may also be used, including other bacterial strains. In prokaryotic systems, plasmid vectors that contain repUcation sites and control sequences derived from a species compatible witih the host may be used. Examples of suitable plasmid vectors may include pBR322, pUC118, ρUC119 and me Uke; suitable phage or bacteriophage vectors may include λgtlO, λgtl 1 and the like; and suitable virus vectors may include pMAM-neo, pKRC and die Uke. Preferably, die selected vector of me present invention has the capacity to repUcate in title selected host ceU.

Recognized prokaryotic hosts include bacteria such as E. coli, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, and the like. However, under such conditions, the peptide will not be glycosylated. The prokaryotic host must be compatible with the rephcon and control sequences in the expression plasmid.

To express hBsk in a prokaryotic ceU, it is necessary to operably link the hBsk sequence to a functional prokaryotic promoter. Such promoters may be either constitutive or, more preferably, regulatable (i.e., inducible or derepressible). Examples of constitutive promoters include the int promoter of bacteriophage λ, the bla promoter of the j8-lactamase gene sequence of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene sequence of pBR325, and the like. Examples of inducible prokaryotic promoters include the major right and left promoters of bacteriophage λ (P_L and P_R), the trp, recA, lacZ, lad, and gal promoters of E. coli, the α-amylase (Ulmanen et al, J. Bacteriol. 762: 176-182 (1985)) and the ς-28-specific promoters of B. subtilis (Gilman et al, Gene sequence 52:11-20 (1984)), the promoters of the bacteriophages of Bacillus (Gryczan, In: The Molecular Biology of the Bacilli, Academic Press, Inc., NY (1982)), and Streptomyces promoters (Ward et al, Mol. Gen. Genet. 205:468-478 (1986)). Prokaryotic promoters are reviewed by GUck

(J. Ind. Microbiol. 7:277-282 (1987)); Cenatiempo (Biochimie 65:505-516 (1986)); and Gottesman (Ann. Rev. Genet. 75:415-442 (1984)).

Proper expression in a prokaryotic ceU also requires the presence of a ribosome binding site upstream of the gene sequence-encoding sequence. Such ribosome binding sites are disclosed, for example, by Gold et al

(Ann. Rev. Microbiol 55:365-404 (1981)).

The selection of control sequences, expression vectors, transformation methods, and the like, are dependent on die type of host cell used to express d e gene. As used herein, "ceU", "ceU line", and "cell culture" may be used interchangeably and all such designations include progeny. Thus, the words "tramformants" or "transformed ceUs" include the primary subject ceU and cultures derived therefrom, without regard to d e number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deUberate or inadvertent mutations. However, as defined, mutant progeny have the same functionaUty as tiiat of me originaUy transformed ceU.

Host ceUs which may be used in the expression systems of the present invention are not strictiy limited, provided tiiat tiiey are suitable for use in the expression of the hBsk peptide of interest. Suitable hosts may often include eukaryotic ceUs.

Preferred eukaryotic hosts include, for example, yeast, fungi, insect cells, mammalian ceUs either in vivo, or in tissue culture. Mammalian cells which may be useful as hosts include HeLa ceUs, cells of fibroblast origin such as VERO or CHO-K1 , or cells of lymphoid origin and their derivatives. In addition, plant cells are also available as hosts, and control sequences compatible widi plant cells are available, such as the cauUfiower mosaic vims 35S and 19S, and nopaline synthase promoter and polyadenylation signal sequences. Another preferred host is an insect cell, for example Drosophila larvae. Using insect cells as hosts, the Drosophila alcohol dehydrogenase promoter can be used. Rubin, Science 240:1453-1459 (1988).. Alternatively, baculovirus vectors can be engineered to express large amounts of hBsk in insects ceUs (Jasny, Science 255:1653 (1987); Miller et al, In: Genetic Engineering (1986), Setlow, J.K., et al , eds., Plenum,

Vol. 8, pp. 277-297).

Different host ceUs have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation, cleavage) of proteins. Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed. For example, expression in a bacterial system can be used to produce an unglycosylated core protein product. Expression in yeast will produce a glycosylated product. Expression in mammalian ceUs can be used to ensure "native" glycosylation of the heterologous hBsk protein. Furthermore, different vector/host expression systems may effect processing reactions such as proteolytic cleavages to different extents.

Any of a series of yeast gene sequence expression systems can be utilized which incorporate promoter and teπnination elements from the actively expressed gene sequences coding for glycolytic enzymes are produced in large quantities when yeast are grown in mediums rich in glucose. Known glycolytic gene sequences can also provide very efficient transcriptional control signals.

Yeast provides substantial advantages in that it can also carry out post-translational peptide modifications. A number of recombinant DNA strategies exist which utilize strong promoter sequences and high copy number of plasmids which can be utilized for production of die desired proteins in yeast. Yeast recognizes leader sequences on cloned mammalian gene sequence products and secretes peptides bearing leader sequences (i.e., pre-peptides). For a mammalian host, several possible vector systems are available for the expression of hBsk.

A wide variety of transcriptional and translational regulatory sequences may be employed, depending upon die nature of the host. The transcriptional and translational regulatory signals may be derived from viral sources, such as adenovims, bovine papilloma vims, simian virus, or the Uke, where the regulatory signals are associated with a particular gene sequence which has a high level of expression. Alternatively, promoters from mammalian expression products, such as actin, collagen, myosin, and die like, may be employed. Transcriptional initiation regulatory signals may be selected which aUow for repression or activation, so that expression of the gene sequences can be modulated. Of interest are regulatory signals which are temperature-sensitive so that by varying the temperature, expression can be repressed or initiated, or are subject to chemical (such as metabohte) regulation.

As discussed above, expression of hBsk in eukaryotic hosts requires the use of eukaryotic regulatory regions. Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis. Preferred eukaryotic promoters include, for example, the promoter of the mouse metaUothionein I gene sequence (Hamer et al. , J. Mol Appl. Gen. 7:273-288 (1982)); the TK promoter of Herpes vims (McKnight, Cell 57:355-365 (1982)); the SV40 early promoter (Benoist et al, Nature (London) 290:304-310 (1981)); the yeast gal4 gene sequence promoter (Johnston et al., Proc. Natl Acad. Sci. (USA) 79:6971-6975

(1982); Silver et al., Proc. Natl. Acad. Sci. (USA) 57:5951-5955 (1984)).

As is widely known, translation of eukaryotic mRNA is initiated at the codon which encodes the first methionine. For this reason, it is preferable to ensure that the linkage between a eukaryotic, promoter and a DNA sequence which encodes hBsk does not contain any intervening codons which are capable of encoding a methionine (i.e., AUG). The presence of such codons results either in a formation of a fusion protein (if the AUG codon is in die same reading frame as the hBsk coding sequence) or a frame-shift mutation (if the AUG codon is not in the same reading frame as the hBsk coding sequence).

An hBsk nucleic acid molecule and an operably linked promoter may be introduced into a recipient prokaryotic or eukaryotic ceU either as a non- repUcating DNA (or RNA) molecule, which may either be a linear molecule or, more preferably, a closed covalent circular molecule. Since such molecules are incapable of autonomous rephcation, the expression of the gene may occur through the transient expression of the introduced sequence. Alternatively, permanent expression may occur through the integration of the introduced DNA sequence into the host chromosome.

In one embodiment, a vector is employed which is capable of integrating the desired gene sequences into the host ceU chromosome. CeUs which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which aUow for selection of host ceUs which contain the expression vector.

The marker may provide for prototrophy to an auxotrophic host, biocide resistance, e.g., antibiotics, or heavy metals, such as copper, or the like. The selectable marker gene sequence can either be directly linked to the DNA gene sequences to be expressed, or introduced into die same ceU by co-transfection. Additional elements may also be needed for optimal synthesis of single chain binding protein mRNA. These elements may include sphce signals, as weU as transcription promoters, enhancers, and termination signals. cDNA expression vectors incorporating such elements include tiiose described by Okayama, Molec. Cell. Biol. 5:280 (1983). In a preferred embodiment, d e introduced nucleic acid molecule will be incorporated into a plasmid or viral vector capable of autonomous repUcation in the recipient host. Any of a wide variety of vectors may be employed for tiαis purpose. Factors of importance in selecting a particular plasmid or viral vector include: die ease with which recipient ceUs tiiat contain the vector may be recognized and selected from those recipient ceUs which do not contain the vector; die number of copies of the vector which are desired in a particular host; and whetitier it is desirable to be able to "shuttle" die vector between host ceUs of different species. Preferred prokaryotic vectors include plasmids such as those capable of repUcation in

E. coli (such as, for example, pBR322, ColEl, pSClOl, pACYC 184, TΓVX. Such plasmids are, for example, disclosed by Sambrook (cf. Molecular Cloning: A Laboratory Manual, second edition, edited by Sambrook, Fritsch, & Maniatis, Cold Spring Harbor Laboratory, 1989)). Bacillus plasrnids include pC194, pC221, pT127, and die Uke. Such plasmids are disclosed by Gryczan (In: The Molecular Biology of the Bacilli, Academic Press, NY (1982), pp. 307-329). Suitable Streptomyces plasmids include pJJlOl (KendaU et al, J. Bacteriol. 769:4177-4183 (1987)), and streptomyces bacteriophages such as ΦC31 (Chater et al, In: Sixth International Symposium on Actinomycetales Biology, Akademiai

Kaido, Budapest, Hungary (1986), pp. 45-54). Pseudomonas plasmids are reviewed by John et al. (Rev. Infect. Dis. 5:693-704 (1986)), and Izaki (Jpn. J. Bacteriol. 55:729-742 (1978)).

Preferred eukaryotic plasmids include, for example, BPV, vaccinia, SV40, 2-micron circle, and die like, or their derivatives. Such plasmids are weU known in the art (Botstein et al, Miami Wntr. Symp. 79:265-274 (1982); Broach, In: The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 445-470 (1981); Broach, Cell 25:203-204 (1982); BoUon et al, J. Clin. Hematol. Oncol. 70:39-48 (1980); Maniatis, In: Cell Biology: A Comprehensive Treatise, Vol. 3, Gene Sequence Expression, Academic Press, NY, pp. 563-608 (1980)).

Once die vector or nucleic acid molecule containing d e constructs) has been prepared for expression, die DNA constmct(s) may be introduced into an appropriate host ceU by any of a variety of suitable means, i.e., transformation, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate-precipitation, direct microinjection, and die like. After the introduction of the vector, recipient ceUs are grown in a selective medium, which selects for the growtii of vector-containing ceUs. Expression of the cloned gene molecule(s) results in the production of hBsk. This can take place in the transformed ceUs as such, or foUowing die induction of tiiese ceUs to differentiate (for example, by administration of bromodeoxyuracil to neuroblastoma ceUs or the like).

VII. An Antibody Having Binding Affinity to an hBsk Polypeptide and a Hybridoma Containing the Antibody

In another embodiment, die present invention relates to an antibody having binding affinity specificaUy to a hBsk polypeptide as described above. Those which bind selectively to hBsk would be chosen for use in mediods which could include, but should not be limited to, die analysis of altered hBsk expression in tissue containing hBsk.

The hBsk proteins of the present invention can be used in a variety of procedures and methods, such as for die generation of antibodies, for use in identifying pharmaceutical compositions, and for studying DNA/protein interaction. The hBsk peptide of the present invention can be used to produce antibodies or hybridomas. One skilled in the art will recognize that if an antibody is desired, such a peptide would be generated as described herein and used as an immunogen. The antibodies of title present invention include monoclonal and polyclonal antibodies, as weU as fragments of these antibodies. The invention further includes single chain antibodies. Antibody fragments which contain the idiotype of me molecule can be generated by known techniques. For example, such fragments include but are not limited to: die F(ab')₂ fragment; the Fab' fragments, Fab fragments, and Fv fragments. Of special interest to the present invention are antibodies to hBsk which are produced in humans, or are "humanized" (i.e. non-immunogenic in a human) by recombinant or other technology. Humanized antibodies may be produced, for example by replacing an immunogenic portion of an antibody with a corresponding, but non-immunogenic portion (i.e. chimeric antibodies) (Robinson, R.R. et al, International Patent PubUcation PCT/US86/02269; Akira, K. et al, European Patent AppUcation 184,187; Taniguchi, M., European Patent AppUcation 171,496; Morrison, S.L. et al. , European Patent AppUcation 173,494; Neuberger, M.S. et al., PCT

AppUcation WO 86/01533; Cabilly, S. et al , European Patent AppUcation 125,023; Better, M. et al, Science 240:1041-1043 (1988); Liu, A.Y. et al, Proc. Natl. Acad. Sci. USA 54:3439-3443 (1987); Liu, A.Y. et al., J. Immunol. 759:3521-3526 (1987); Sun, L.K. et al, Proc. Natl. Acad. Sci. USA 54:214-218 (1987); Nishimura, Y. et al, Cane. Res. 47:999-1005

(1987); Wood, CR. et al, Nature 574:446-449 (1985)); Shaw et al, J. Natl. Cancer Inst. 50:1553-1559 (1988). General reviews of "humanized" chimeric antibodies are provided by Morrison, S.L. (Science, 229:1202- 1207 (1985)) and by Oi, V.T. et al, BioTechniques 4:214 (1986)). Suitable "humanized" antibodies can be alternatively produced by CDR or

CEA substitution (Jones, P.T. et al, Nature 527:552-525 (1986); Verhoeyan et al, Science 259: 1534 (1988); Beidler, C.B. et al, J. Immunol. 747:4053-4060 (1988)).

In anodier embodiment, die present invention relates to a hybridoma which produces die above-described monoclonal antibody. A hybridoma is an immortalized ceU line which is capable of secreting a specific monoclonal antibody.

In general, techniques for preparing monoclonal antibodies and hybridomas are weU known in die art (Campbell, "Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular

Biology," Elsevier Science PubUshers, Amsterdam, The Netherlands (1984); St. Groth et al, J. Immunol. Methods 55:1-21 (1980)).

Any animal (mouse, rabbit, and the like) which is known to produce antibodies can be immunized widi die selected polypeptide. Mediods for immunization are weU known in the art. Such methods include subcutaneous or interperitoneal injection of the polypeptide. One skilled in the art will recognize that die amount of polypeptide used for immunization will vary based on die animal which is immunized, the antigenicity of the polypeptide and the site of injection. The polypeptide may be modified or administered in an adjuvant in order to increase the peptide antigenicity. Mediods of increasing the antigenicity of a polypeptide are weU known in the art. Such procedures include coupling the antigen widi a heterologous protein (such as globulin or /S-galactosidase) or through the inclusion of an adjuvant during immunization.

For monoclonal antibodies, spleen ceUs from d e immunized animals are removed, fused widi myeloma ceUs, and aUowed to become monoclonal antibody producing hybridoma ceUs.

Any one of a number of methods well known in the art can be used to identify the hybridoma ceU which produces an antibody widi die desired characteristics. These include screening the hybridomas with an ELISA assay, western blot analysis, or radioimmunoassay (Lutz et al, Exp. Cell Res. 775:109-124 (1988)).

Hybridomas secreting the desired antibodies are cloned and die class and subclass is determined using procedures known in the art (CampbeU, Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology, supra (1984)).

For polyclonal antibodies, antibody containing antisera is isolated from the immunized animal and is screened for the presence of antibodies widi the desired specificity using one of die above-described procedures.

In another embodiment of the present invention, the above-described antibodies are detectably labeled. Antibodies can be detectably labeled through the use of radioisotopes, affinity labels (such as biotin, avidin, and die like), enzymatic labels (such as horse radish peroxidase, alkaline phosphatase, and the like) fluorescent labels (such as FITC or rhodamine, and die like), paramagnetic atoms, and die like. Procedures for accompUshing such labeling are weU-known in the art, for example, see (Sternberger et al., J. Histochem. Cytochem. 75:315 (1970); Bayer et al., Meth. Enzym. 62:308 (1979); Engval et al., Immunol. 709:129 (1972); Goding, J. Immunol. Meth. 75:215 (1976)). The labeled antibodies of the present invention can be used for in vitro, in vivo, and in situ assays to identify ceUs or tissues which express a specific peptide.

In another embodiment of the present invention the above-described antibodies are immobilized on a soUd support. Examples of such soUd supports include plastics such as polycarbonate, complex carbohydrates such as agarose and sepharose, acryhc resins and such as polyacrylamide and latex beads. Techniques for coupling antibodies to such soUd supports are well known in the art (Weir et al. , "Handbook of Experimental Immunology" 4dι Ed., Blackwell Scientific PubUcations, Oxford, England, Chapter 10 (1986); Jacoby et al., Meth. Enzym. 34 Academic Press, N.Y.

(1974)). The immobilized antibodies of the present invention can be used for in vitro, in vivo, and in situ assays as weU as in immunochromotography .

Furthermore, one skilled in the art can readily adapt currently available procedures, as weU as die techniques, methods and kits disclosed above with regard to antibodies, to generate peptides capable of binding to a specific peptide sequence in order to generate rationally designed antipeptide peptides, for example see Hurby et al. , "AppUcation of Synthetic Peptides: Antisense Peptides", In Synthetic Peptides, A User's Guide, W.H. Freeman, NY, pp. 289-307 (1992), and Kaspczak et al,

Biochemistry 25:9230-8 (1989).

Anti-peptide peptides can be generated in one of two fashions. First, the anti-peptide peptides can be generated by replacing the basic amino acid residues found in the hBsk peptide sequence widi acidic residues, while maintaining hydrophobic and uncharged polar groups. For example, lysine, arginine, and/or histidine residues are replaced widi aspartic acid or glutamic acid and glutamic acid residues are replaced by lysine, arginine or histidine.

VIII. A Method of Detecting an hBsk Polypeptide in a Sample

In another embodiment, the present invention relates to a method of detecting a hBsk polypeptide in a sample, comprising: a) contacting the sample wid an above-described antibody, under conditions such that immunocomplexes form, and b) detecting the presence of said antibody bound to the polypeptide. In detail, die methods comprise incubating a test sample widi one or more of die antibodies of die present invention and assaying whether die antibody binds to the test sample. Altered levels of hBsk in a sample as compared to normal levels may indicate a specific disease.

Conditions for incubating an antibody widi a test sample vary. Incubation conditions depend on the format employed in the assay, die detection methods employed, and the type and nature of the antibody used in die assay. One skilled in the art wiU recognize that any one of the commonly available immunological assay formats (such as radioimmunoassays, enzyme-linked immunosorbent assays, diffusion based Ouchterlony, or rocket immunofluorescent assays) can readily be adapted to employ the antibodies of die present invention. Examples of such assays can be found in Chard, An Introduction to Radioimmunoassay and Related Techniques, Elsevier Science PubUshers, Amsterdam, The Netherlands

(1986); Bullock et al, Techniques in Immunocytochemistry, Academic Press, Orlando, FL Vol. 1 (1982), Vol. 2 (1983), Vol. 3 (1985); Tijεsen, Practice and Theory of Enzyme Immunoassays: Laboratory Techniques in Biochemistry and Molecular Biology, Elsevier Science PubUshers, Amsterdam, The Netherlands (1985).

The immunological assay test samples of the present invention include ceUs, protein or membrane extracts of ceUs, or biological fluids such as blood, serum, plasma, or urine. The test sample used in the above- described method will vary based on the assay format, nature of the detection method and the tissues, ceUs or extracts used as the sample to be assayed. Mediods for preparing protein extracts or membrane extracts of ceUs are weU known in the art and can be readily be adapted in order to obtain a sample which is capable widi d e system utilized.

LX. A Diagnostic Kit Comprising Antibodies to hBsk

In another embodiment of the present invention, a kit is provided which contains aU the necessary reagents to carry out the previously described mediods of detection. The kit may comprise: i) a first container means containing an above-described antibody, and u) second container means containing a conjugate comprising a binding partner of the antibody and a label. In anotiier preferred embodiment, die kit further comprises one or more other containers comprising one or more of die foUowing: wash reagents and reagents capable of detecting die presence of bound antibodies. Examples of detection reagents include, but are not limited to, labeled secondary antibodies, or in the alternative, if the primary antibody is labeled, the chromophoric, enzymatic, or antibody binding reagents which are capable of reacting with die labeled antibody. The compartmentalized kit may be as described above for nucleic acid probe kits.

One skilled in the art will readily recognize that the antibodies described in die present invention- can readily be incorporated into one of die established kit formats which are weU known in the art.

X. Diagnostic Screening and Treatment

The diagnostic and screening methods of the invention are especially useful for a patient suspected of being at risk for developing a disease associated with an altered expression level of hBsk based on family history, or a patient in which it is desired to diagnose an hBsk-related disease.

According to die invention, presymptomatic screening of an individual in need of such screening is now possible using DNA encoding die hBsk protein of the invention. The screening method of the invention allows a presymptomatic diagnosis, including prenatal diagnosis, of die presence of a missing or aberrant hBsk gene in individuals, and thus an opinion concerning die likelihood tiiat such individual would develop or has developed an hBsk-associated disease. This is especiaUy valuable for the identification of carriers of altered or missing hBsk genes, for example, from individuals widi a family history of a hBsk-associated disease. Early diagnosis is also desired to maximize appropriate timely intervention.

In one preferred embodiment of die method of screening, a tissue sample would be taken from such individual, and screened for (1) the presence of die "normal" hBsk gene; (2) the presence of hBsk mRNA and/or (3) die presence of hBsk protein. The normal human gene can be characterized based upon, for example, detection of restriction digestion patterns in "normal" versus the patient's DNA, including, RFLP analysis, using DNA probes prepared against the hBsk sequence (or a functional fragment thereof) taught in the invention. Similarly, hBsk mRNA can be characterized and compared to normal hBsk mRNA (a) levels and/or (b) size as found in a human population not at risk of developing hBsk- associated disease using similar probes. Lastly, hBsk protein can be (a) detected and/or (b) quantitated using a biological assay for hBsk activity or using an immunological assay and hBsk antibodies. When assaying hBsk protein, the omunological assay is preferred for its speed. An (1) aberrant hBsk DNA size pattem, and/or (2) aberrant hBsk mRNA sizes or levels and/or (3) aberrant hBsk protein levels would indicate tiiat die patient is at risk for developing an hBsk-associated disease.

The screening and diagnostic methods of the invention do not require that die entire hBsk DNA coding sequence be used for the probe. Ratiher, it is only necessary to use a fragment or length of nucleic acid that is sufficient to detect die presence of the hBsk gene in a DNA preparation from a normal or affected individual, the absence of such gene, or an altered physical property of such gene (such as a change in electrophoretic migration pattem). Prenatal diagnosis can be performed when desired, using any known method to obtain fetal cells, including amniocentesis, chorionic villous sampling (CVS), and fetoscopy. Prenatal chromosome analysis can be used to determine if the portion of the chromosome possessing the normal hBsk gene is present in a heterozygous state. In the metiiod of treating an hBsk-associated disease in a patient in need of such treatment, functional hBsk DNA can be provided to the ceUs of such patient in a manner and amount tiiat permits die expression of the hBsk protein provided by such gene, for a time and in a quantity sufficient to treat such patient. Many vector systems are known in the art to provide such deUvery to human patients in need of a gene or protein missing from the ceU. For example, retro virus systems can be used, especially modified retjpovims systems and especiaUy herpes simplex vims systems. Such methods are provided for, in, for example, the teachings of Breakefield, X.A. et al, The New Biologist 5:203-218 (1991); Huang, Q. et al, Experimental Neurology 775:303-316 (1992), WO93/03743 and

WO90/09441. DeUvery of a DNA sequence encoding a functional hBsk protein will effectively replace: the missing or mutated hBsk gene of die invention.

In an alternative embodiment stem ceU populations for either neuronal or glial ceUs can be genetically engineered to express a functional hBsk receptor. Such ceUs recombinantly expressing the hBsk receptor, can be transplanted to die diseased or injured region of the mammal's limbic system (Neural Transplantation. A Practical Approach, Donnet & Djorklund, eds., Oxford University Press, New York, NY (1992)). In yet another alternative embodiment, embryonic tissue or fetal neurons can be geneticaUy engineered to express functional hBsk receptor and transplanted to the diseased or injured region of the mammal's limibic system. The feasibility of transplanting fetal dopamine neurons into Parkinsonian patients has recendy been demonstrated. (LindvaU et al , Archives of Neurology 46:615-631 (1989)).

Studies of die molecular interactions between Ugands and their receptors showed tiiat only the extraceUular domain of the receptor is involved in die special physical interaction between the molecules (Riedel et al , Nature 524:68-70 (1986); Riedel et al . EMBP J. 5:2943-2945 (1989)). Thus, the extracellular domain of a receptor can be used as a probe to screen an expression cDNA Ubrary for die hBsk Ugand or Ugands. In one approach for detection of the receptor probe, placenta! alkaline phosphatase will be fused to die extraceUular domain of a receptor, and positive clones will be detected by the presence of alkaline phosphatase activity. An alternative approach is to isolate the putative hBsk Ugand is to utilize die findings tiiat co-expression of a receptor and its Ugand in the same ceUs results in uncontroUed proliferation and maUgnant transformation (Klein et al , Cell 66:395-403 (1991); Gazit et al, Cell 59:89-97 (1984)). A eukaryotic cDNA expression Ubrary can be transfected into ceUs expressing a receptor, and die presence of a Ugand will create an autocrine loop, resulting in a transformed phenotype. This approach has been successfuUy used by Miki et al, Science 257:72-75 (1991), to isolate die receptor of die keratinocyte growth factor (KGF) using ceUs expressing KGF. In yet another alternative approach the hBsk receptor protein can be expressed in a ceU line or in Xenopus oocytes by the recombinant techniques described above and its Ugand stimulated activation of tyrosine kinase activity, as detected by an anti-phosphotryosine antibody (UBI, Happauge, New York) can be used to assay and purify the Ugand. For example, cells expressing the recombinant hBsk receptor can be exposed to mammalian brain extract. The brain extracts can be fractionated by chromatography and used to assay for die presence of the Ugand activity. Once an activity is identified in a particular fraction, it can be further purified by conventional biochemical techniques. In another alternative approach, die hBsk extraceUular domain can be used to screen a random peptide Ubrary (Cull et al , Proc. Natl. Acad. Sci. USA 59:1865-1869 (1982); Lam et al , Nature 554:82-84 (1991)). Peptides isolated can be assayed for dieir Ugand activity in the above- described assays for drug screening. In another embcrdiment of tins invention, the hBsk Ugand is expressed as a recombinant gene in a ceU, so tiiat die ceUs may be transplanted into a mammal, preferably a human in need of gene dierapy. To provide gene dierapy to an individual, a genetic sequence which encodes for aU or part of the hBsk Ugand is inserted into a vector and introduced into a host ceU. Examples of diseases tiiat may be suitable for gene dierapy include, but are not limited to, neurodegenerative diseases or disorders, Alzheimer's, schizophrenia, epilepsy, neoplasms and cancer. Examples of vectors that may be used in gene dierapy include, but are not limited to, defective retroviral, adenoviral, or other viral vectors (Mulligan, R.C., Science 260:926-932 (1993)). The means by which the vector carrying the gene may be introduced into the ceU include but is not limited to, microinjection, electroporation, transduction, or transfection using DEAE-Dextran, Upofection, calcium phosphate or other procedures known to one skilled in the art (Molecular Cloning, A Laboratory Manual, Sambrook et al, eds., Cold Spring Harbor Press, Plainview, New York

(1989)).

In another embodiment, the present invention relates to a method of administering hBsk to an animal (preferably, a mammal (specifically, a human)) in an amount sufficient to effect an altered level of hBsk in said animal. The administered hBsk could specificaUy effect hBsk associated functions. Further, since hBsk is expressed in brain tissue, administration of hBsk could be used to alter hBsk levels in the brain.

One skilled in the art will appreciate that the amounts to be administered for any particular treatment protocol can readily be determined. The dosage should not be so large as to cause adverse side effects, such as unwanted cross-reactions, anaphylactic reactions, and the like. GeneraUy, die dosage wiU vary with die age, condition, sex and extent of disease in die patient, counter indications, if any, and other such variables, to be adjusted by the individual physician. Dosage can vary from .001 mg/kg to 50 mg/kg of hBsk, in one or more aάjninistrations daily, for one or several days. hBsk can be administered parenteraUy by injection or by gradual perfusion over time. It can be administered intravenously, intraperitoneaUy, intramuscularly, or subcutaneously.

Preparations for parenteral administration include sterile or aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non- aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such_ as oUve oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohoUc/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose and sodium chloride, lactated Ringer- s, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the Uke. Preservatives and otiier additives may also be present, such as, for example, antimicrobials, antioxidants, chelating agents, inert gases and die like. See, generaUy, Remington's

Pharmaceutical Science, 16th Ed., Mack Eds. (1980).

In anotiier embodiment, the present invention relates to a pharmaceutical composition comprising hBsk in an amount sufficient to alter hBsk associated activity, and a pharmaceuticaUy acceptable diluent, carrier, or excipient. Appropriate concentrations and dosage unit sizes can be readily determined by one skilled in die art as described above (See, for example, Remington 's Pharmaceutical Sciences (16th ed., Osol, A., Ed., Mack, Easton PA (1980) and WO 91/19008).

The present invention is described in further detail in the foUowing non-Umiting examples.

Example 1 Isolation of hBsk

A λ ZAP Ubrary made from mRNA isolated from the brain of a

18-week-old female human fetus was used to isolate hBsk. hBsk was isolated using a 4.3 kb mouse Bsk probe labeled with [³²P] by random priming. 10⁶ phage plaques were lifted onto nitroceUulose filters and prehybridized and hybridized under non-stringent conditions (5X SSPE,

10X Denhardt's solution, 100 μg/ml freshly denatured salmon sperm DNA,

37 % freshly deionized formamide, 2% SDS). Hybridization was performed at 42 °C for 16 hrs after prehybridization in the same buffer without the labeled probe for 2 hrs. After hybridization, the filters were washed under increasingly stringent conditions: 2X SSC, 0.5% SDS, 30 min, 50°C, 2 times; 0.1X SSC, 0.1 % SDS, 50°C, 30 min, 2 times; 0.1X SSC, 0.1 % SDS, 65 °C. Filters were exposed to X-ray films to assess the extent of signal loss after each wash. Positive plaques were picked and purified to homogeneity. cDNA inserts in the purified positive clones were tiien excised from the phage vector into plasmid DNA using M13 helper phage. Partial sequence analysis (for general techniques See, Molecular Cloning: A Laboratory Manual, second edition, edited by Sambrook, Fritsch, &

Maniatis, Cold Spring Harbor Laboratory, 1989) was then performed to identify hBsk clones (See Figure 2).

Thirty-four independent potential hBsk cDNA clones were isolated from mRNA purified from a human fetal brain using a mouse Bsk probe under stringent washing conditions. Partial sequence of these demonstrate tiiat five of these clones represent different parts of the hBs as shown schematicaUy in Figure 1.

Clones 6-1 and 8-1 contain an entire hBsk gene and were deposited

A clone containing die entire hBsk gene is constructed by 1) identifying a restriction site shared by Clones 6-1 and 8-1 which is only present in one location within these clones, 2) digesting die clones with die corresponding restriction enzyme and a restriction enzyme(s) which cuts at die 5' end of the 8-1 clone and die 3' end of the 6-1 clone, and 3) linking the fragments together widi, for example, Ugase. If convenient restriction enzyme sites are not present witiiin the clones, they may be inserted into the clones using site specific mutagenesis (cf. Molecular Cloning: A Laboratory Manual, second edition, edited by Sambrook, Fritsch, & Maniatis, Cold Spring Harbor Laboratory, 1989). Example 2 The Biological function of hBsk

Construction of a Chimeric Receptor

To construct a M-CSFR hBsk chimeric receptor, a combination of restriction enzyme digestion and PCR approaches will, be used to Ugate die

M-CSFR extraceUular domain and title hBsk transmembrane and intraceUular domains (M-CSFR/Bsk). An alternative construct, M- CSFR/hBsk 2, would contain M-CSFR extraceUular and transmembrane domains and hBsk intraceUular domain. The chimeric receptor will be expressed under the LTR promoter in the pMEX expression vector (Oskam et al, Proc. Natl Acad. Sci. USA 55:2964-2968 (1988)) in NIH/3T3 cells to study whether it has a mitogenic effect and in PC 12 ceUs to study whether it induces differentiation. The expression cassette pMEX-CR (CR for chimeric receptor) will be cotransfected widi pSV2Neo. Neo-resistant colonies will be grown up and tested for the expression of the chimeric receptor using immunoprecipitation or Western blot analysis (Ausubel et al. , in Current Protocols in Molecular Biology, John Wiley & Sons, New York, NY (1987)). To smdy if the chimeric receptor is properly localized in the cytoplasmic membrane, ceU surface labeling will be performed. The labeled ceUs wiU be lysed and immunoprecipitated widi either Bsk- or M-CSFR-specific antibodies. If the receptor is properly localized on d e ceU surface, a positive labeling of the chimeric product should result.

To examiner die binding property of the chimeric receptor to M-CSF, [^I25I]-MCSF will be used to bind intact transfected or control ceUs.

To measure die receptor binding affinity and specificity, the concentration of M-CSF needed to cause 50% inhibition of maximal [¹²⁵I]-MCSF binding to die ceUs wiU be determined (IC50). Scratchard analysis will be performed on die binding competition data (Scatchard, Ann. NY Acad. Sci. 57:660-672 (1949)) and the dissociation constant (Kd) will be calculated. CeUs transfected widi vector alone will be used as controls.

To smdy if the chimeric receptor functions properly, the chimeric receptor will be immunoprecipitated widi eitiier M-CSFR or hBsk-specific antibodies from M-CSF-stimulated ceUs for various times, using unstimulated ceUs as a control. The precipitated receptor will be analyzed by die Western blot technique with anti-phosphotyrosine antibody to examine the activation of the receptor tyrosine kinase. The biological effects of the stimulation of the chimeric receptor with M-CSF wiU be studied in NTH/3T3 cells by stimulation of DNA synthesis under low serum (calf serum at 0.3 %) conditions. To determine die functions of hBsk in neuronal differentiation, the effects of the chimeric receptor activation on neurite outgrowth in PC12 cells will be studied.

f⁵SJ Methionine/Cysteine Labeling of Proteins

CeUs including, but not limited to, PC 12 cells, and NIH/3T3 ceUs, are grown to 80% confluence in 100-mm tissue culture dishes, washed twice in metitdonine/cysteine-free DMEM, (Dulbecco's Modified Eagle Media) starved in die same medium supplemented widi 5% dialyzed fetal calf serum for 30 min, and then incubated for 2, 4, or 8 hours in the same medium widi Translabel (Amersham, 70% ["S] cysteine. The labeled ceUs will be lysed and immunoprecipitated as described below.

Cell Surface Labeling

CeUs expressing the M-CSFR/hBsk chimeric receptor will be grown to 80% confluence on 175-cm² flasks. CeUs are washed twice widi PBS, removed gently widi a ceU scraper, and resuspended in PBS containing 6 U of lactoperoxidase, 20 U of glucose oxidase and 2mCi of [^,25rj. After 0, 10,; and 20 min, 140 μl of 1M glucose will be added. At 30 min, the reaction is stopped by three washes in PBS. The ceUs are then lysed and immunoprecipitated using appropriate antibodies.

Preparation of Cell Extracts and lmmunoprecipitation

CeUs will be washed in Wash Buffer (HEPES 50 mM, pH 7.4, NaCl 150 mM, glycerol 10%, EDTA 10 mM, NaF 100 mM, vanadate 2 mM, Na₄P₂0₇ 10 mM, trypsin inhibitor 1000 U/ml, PMSF ImM, aprotinin ImM, leupeptin 10 μM); the ceUs are lysed for 30 min at 4°C in 200 μL of lysis buffer (Wash Buffer containing 1 % Triton X-100) and centrifuged for 30 min at 150,000 g in a Beckman TL-100 ultracentrifuge. The extracts are then cleared twice by 15 min incubation with protein A Sepharose (40 μl of 10% gel for 200 μl of ceU extracts). After a 5 min centrifugation, supernatants are mixed widi appropriate antibodies adsorbed on protein-A-Sepharose and incubated for 2 h at 4 °C widi agitation. The samples are then centrifuged for 30 sec and die peUets are washed 6 times (3 times widi wash buffer, 3 times with wash buffer supplemented with 500 mM NaCl, 0.1 % Triton X-100, 0.1 % SDS). The washed peUets are dien resuspended in SDS-PAGE buffer and subjected to SDS-PAGE analysis. Labeled proteins are visualized by autoradiography.

Ligand Binding Study

CeUs are grown in 100 mm culture dishes in DMEM to 80% confluence and tiien washed widi PBS and incubated widi 5 ml of 25 mM

EDTA in PBS for 2 min. CeUs are then removed from die plate, washed once with binding Buffer (100 mM HEPES, pH 7.6, 120 mM NaCl, 5 mM

KC1, 1.2 mM MgS0₄, 1 mM EDTA, 10 mM glucose, 15 mM sodium acetate, 1 % dialyzed BSA), and resuspended in 5 ml of Binding buffer to deteπnine die ceU number. 400 μl of this ceU suspension is tiien incubated widi [¹²⁵rj-M-CSF (5 pM) and increasing concentrations of unlabeled M-CFS (from 0 to 10^*6 M) in a total volume of 500 μl for 90 min at 15 °C. After incubation, ceUs are washed with Binding Buffer. Free [¹²⁵I]-MCSF is removed by six washes in Binding Buffer. FinaUy, the [¹²⁵I] radioactivity bound to d e ceUs is determined in a λ-counter. Data obtained will be analyzed by die method of Scatchard (Scatchard, Ann. N.Y. Acad. Sci. 57:660-672 (1949).

Thymidine Incorporation Assay

Confluent ceU monolayers in 12-weU culture dishes will be grown to quiescence in medium containing 0.5 % fetal bovine serum for 24 hours (h). DNA synthesis wiU be stimulated by adding various amount of M-CSF. Eighteen hours later, cells will be labeled for 4 h with 0.5 μCi [methyl-³H] thymidine at 3 TBq/mmol, then washed tiiree times with ice cold PBS, incubated widi 1 ml of 10% trichloroacetic acid for 30 min, and washed twice widi die same solution at 4°C. CeUs will be then solubilized in 0.5 ml of 0.2 N NaOH, 1 % SDS for 1 h at 37°C and die lysate will be. brought to neutral pH with Tris buffer. The incorporated radioactivity wiU be determined in a Uquid scintillation counter.

Construction of Recombinant Adenovirus (Ad-CR)

The recombinant adenovirus is constructed by in vivo homologous recombination between an adenoviral vector containing the chimeric receptor and an adenovirus deletion mutant Ad 1327 genomic DNA (Stratgord-Perricaudt et al. , J. Clin. Invest. 90:626-630 ( 1992)) in 293 ceUs which express adenoviral early genes (Graham et al , J. Gen. Virol. 56:59-72 (1977)). Briefly, 293 ceUs are cotransfected widi 5 μg of linearized plasmid pAd-CR and 5 μg of die Large Cla I fragment (2.6-100 mu) of Ad5 DNA. After overlaying with agar and incubating for 10 days at 37 °C, plaques containing recombinant adenovimses are isolated and amplified in 293 ceUs, viral DNA is purified, and recombinant adenovims plaques containing die Bsk chimeric receptor are identified by restriction cleavage and Soutiiern analysis.

Hippocampal Neuron Cultures

Hippocampi are dissected from El 8 rate embryos and coUected in F10 medium (Gibco). The tissues are minced, rinsed twice widi F10 medium, and the ceUs are dissociated by gentle trituration and coUected by low speed centrifugation (500 rpm) for 30 sec. The peUet is washed again in the same medium by resuspension and centrifugation. The ceU peUets are resuspended in MEM supplemented widi 10% fetal calf serum, 2 mM glutamine, 25 U/ml PenicilUn and 25 μg/ml) and laminin (10 μg/ml) coated

6 mm microtiter weUs at a density of 70,000 ceUs/cm². Six hours foUowing the plating of cells, the medium is changed to a serum-free medium containing 25 μg/ml insulin, 100 μg/ml transferrin, 60 μM putrescine, 20 nM progesterone, 30 nM selenium, 6 mg/ml glucose (Lu et al, Proc. Natl Acad. Sci. USA 55:6289-6292 (1991) and pemcillin- streptomycin (25 U/ml and 25 μg/ml, respectively), and infected widi die viruses at a moi of 10, 5, 2, and 1 , respectively. M-CSF is added at die same time. Medium is changed every 3-4 days widi die re-addition of fresh factors. Measurement of Neurofilament Protein

CeUs are fixed widi 4% (v/v) paraformaldehyde for 4 hr at 4°C, permeabilized with 0.1 % (v/v) Triton X-100 in PBS for 15 min, and blocked with 10% FCS in PBS for 1 h. The ceUs are then incubated widi anti-neurofilament 20 antibody for 1 h at room temperature, washed twice widi PBS containing 10% FCS, and incubated widi die secondary antibody (horseradish peroxidase-conjugated) for 1 h. FoUowing sequential washing with PBS and water, the ceUs are incubated widi 0.2% (w/v) 0-phenylenediamine and 0.02% (v/v) H₂0₂ in citrate buffer (50 mM) for 30 min. The reaction is topped by adding an equal volume of 4.5 M

H₂S0₄. Product formation will be quantitated by reading die optical density of the reaction product at 492 nm.

Immunocytochemistry

CeUs are rinsed twice widi PBS, fixed widi 4% paraformaldehyde for 30 min at room temperature, and blocked widi 10% FCS in PBS containing

0.1 % Triton X-100. The ceUs are then incubated widi the primary antibodies overnight at 4°C, washed widi 0.1 % Triton X-100 in PBS three times and incubated widi Texas Red conjugated secondary antibodies for 90 min at room temperature. The ceUs are washed again and positive ceUs are visualized under a fluorescent microscope.

Measurement of High-Affinity Uptake of GAB A

ffigh-affinity GABA uptake will be measured as described (Ip et al. ,

J. Neuro. Sci. 77:3124-3234 (1991)). CeUs are washed in die GABA uptake buffer containing 140 mM NaCl, 2.5 mM KC1, ImM KH₂P0₄, ImM N-i_jHPO^ 6mg/ml glucose, lMgCl₂, 1 mM CaCl₂, and 0.1 % BSA. FoUowing washing, ceUs are incubated in the GABA uptake buffer for 5 min at 37 °C. pHj-GABA is then added to a final concentration of 12 nM, and incubated at 37°C for 10 min. CeUs are kept on ice and washed tihree times with the uptake buffer. CeUs are then solubilized with 0.14 N NaOH for 2 h at room temperature, and pHJ-GABA in the extract is counted.

Uptake of GABA into non-neuronal ceUs is inhibited by die addition of 2 mM jS-alanine, whereas uptake specific for neurons is verified by inhibition with nipecotic acid at 1 mM. Specific neuronal GABA uptake is determined as GABA uptake that is blocked in die presence of 1 mM mpecotic acid.

Placenta! Alkaline Phosphatase Activity Assay

The assay is performed by heating a portion of the supernatant at 65 °C for 10 min to inactivate background phosphatase activity and tiien measuring the optical density at 405 nm after incubation with 1 M diethanolamine (pH 9.8), 0.5 mM MgCl, 10 μM L-homoarginine (a phosphatase inhibitor), 0.5 mg/ml BSA, and 12 mM p-nitrophenyl phosphate. The highest alkaline phosphatase-expressing clone will be selected for the purification of AP-tag-hBsk fusion protein. To concentrate and purify AP-tag-hBsk protein, the supernatant will be incubated widi a monoclonal antibody to placental alkaline phosphatase coupled to CNBr- activated Sepharose. SpecificaUy bound protein wiU be eluted with 144 mM NaCl, 1 mM MgCl₂, 1 mM CaCl₂, 50 mM sodium citrate (pH 2.5), and will be tiien immediately neutralized with 1 M HEPES (pH 8.0). The purified protein wiU be used as a probe to screen a brain expression Ubrary. Library Screening Using AP-tag hBsk

A brain expression cDNA Ubrary will be plated at a density of 50,000 pfu per 150 mm plate. DupUcate filters will be lifted from me plates and rinsed in TBST. The filters are then blocked with TBST widi 10% goat serum, rinsed once in TBST, and incubated in TBST widi AP- tag-Bsk probe for 3 hours. The FUters are then washed in tiiree changes of TBST, 3 min each. The positive clones will be detected by color formation when the filters are incubated wid alkaline phosphatase substrates 5-bromo-4-chloro-3-inodyl-phosphate (BCIP, 0.017 mg/ml) and nitrobluetetrazoUum (NBT, 0.33 mg/ml) in 100 mM Tris-HCl (pH 9.5),

100 mM NaCl, 5 mM MgCl₂. A non-specific alkaline phosphatase inhibitor, L-homoarginine (10 mM), wiU be added if required.

Effect of the Chimeric Receptor in Hippocampal Neurons

A vector system which is based on an adenovims (Stratford- Perricaudet et al, J. Clin, Invest. 90:626-630 (1992)) wiU be used to deUver CSFR/Bsk into hippocampal neurons from El 8 rat embryos. This vector has been used successfuUy in the nervous system and no cytotoxicity was observed (LeGal LeSaUe et al , Science 259:988-990 (1993)). In addition, long-term expression of genes was achieved widi this vector (LeGal LeSaUe et al. , Science 259:988-990 (1993)).

To clone M-CSF/Bsk chimeric receptor into an adenoviral promoter, a vector plasmid, pAd-Cr, containing a chimeric receptor expressing cassette driven by M-MLV LTR promoter will be constructed. The cassette will be bordered at the 5' end by the left end (map unit 0-1.3) of adenovims type 5 (Ad5) and at die 3' end by sequences from mu 9.4-17

(Bgl II-Hind II fragment of Ad5) to aUow homologous recombination with the adenoviral genome to generate recombinant vims. The resulting recombinant virus will lack the early gene El and therefore will be repUcation competent except when provided widi El function in 293 ceUs (Graham et al, J. Gen Virol. 56:59-72 (1977)). To smdy die effect of hBsk chimeric receptor on the survival of hippocampal neurons, hippocampal neuron culture will be estabUshed in polyornithine- and laminin-coated plastic dishes in MEM supplemented widi 10% fetal calf serum (FCS) and glutamine and infected widi Ad-CR or control virus. Later, the medium will be changed to a serum-free medium containing hormone supplements. M-CSF will be added at this time. After various times of treatment, ceUs will be stained widi antibody against neuron-specific enolase to identify neurons in the culture. The number of neurons in cultures infected witih vims containing M-CSFR/hBsk or with viral vector only will be compared to determine the effect of M-CSF stimulation on the survival of specific neurons. ParaUel infected cultures will be studied for hBsk protein expression at various time points using Western blot or immunoprecipitation and for Ugand-dependent receptor (Kaplan et al, Nature 550:158-160 (1991); Klein et al. , Cell 65:189-197 (1991)). To examine if hBsk has any effect on the neurite outgrowth of the hippocampal neurons, changes in the level of neurofilament protein upon M-CSF treatment will be examined. Hippocampal neurons wiU be infected with virus carrying M-CSF/hBsk and treated widi various concentrations of M-CSF (0.001-10 ng/ml) of M-CSF for 8 days and neurofilament protein levels will be measued by ELISA. Neurons infected widi vector alone wiU be used as controls. To delineate which neuronal population which respond to hBsk kinase activation, die effect of M-CSF treatment of neurons expressing the chimeric receptor on the number of GABAergic and calbindin-positive neurons wiU be studied. Infected neurons will be treated witih various concentrations of M-CSF (0.001-100 ng/ml). After 8 days of treatment, ceUs will be stained widi anti-GABA receptor or anti-calbindin antibodies to study die effect of hBsk activation on the survival of various neuronal populations. In addition to d e immunostaining wid different antibodies, die changes of the Wgh-affinity uptake for GABA will be studied after various times of M-CSF treatment. pHJ-GABA binding by die neurons in cultures infected widi the vims expressing d e chimeric receptor or with control vims will be compared. _/S-alanine wiU be used to inhibit die uptake of GABA into non-neuronal ceUs (Ip et al. , Neurosci. 77:3124-3134 (1991)). An alternative vector system which is based on a herpes vims may also be used to dehver CSFR/hBsk into hippocampal neurons (Anderson et al, Human Gene Therapy 5:487-499 (1992); Fink et al , Human Gene Therapy 5:11-19 (1992)).

Example 3 Isolation of an hBsk Ligand

Screening of a cDNA Expression Library For hBsk Ligand Using an ExtraceUular Domain-Alkaline Phosphatase Fusion Protein As a Probe

To construct a fusion protein between the extraceUular domain of hBsk and die secreted placental alkaline phosphatase (SEAP), a vector named APtag-1, constructed by Flannagan & Leder, Cell 65:185-194

(1990), will be used. APtag-1 contains a set of restriction sites for the insertion of the region of die hBsk cDNA encoding die extraceUular domain. Downstream of the insertion sites is the fuU length sequence of SEAP, which will be fused to die upstream sequence. To generate a hBsk receptor fusion protein, the 5' end of die hBsk cDNA sequence wiU be inserted into APtag-1 , including sequences encoding die hBsk secretion signal peptide and die entire extraceUular domain, ending immediately before the first hydrophobic amino acid of die transmembrane region. The resulting plasmid will merefore encode a fusion protein with die hBsk extraceUular domain joined to a SEAP. A fusion protein will be expressed from a Moloney Murine Leukemia virus LTR promoter. The fusion construct will be transfected into NIH/3T3 ceUs which have been shown to express high levels of an APtag-Kit fusion protein (Flannagan & Leder, Cell 65:185-194 (1990)). The fusion construct will be contransfected with a selectable marker plasmid pSV2neo, and selected with G418 (400-800 μg/ml). Neo-resistant colonies will be grown in 96-weU plates and screened for secretion of SEAP activity into the media (See above). The fusion protein will be concentrated, purified and used as a probe to screen a cDNA expression Ubrary from mammalian brain, preferably mouse.

Three types of positive clones are expected: (1) clones having background alkaline phosphatase activity; (2) clones which bind non- specificaUy to die fusion protein; and (3) clones encoding die putative hBsk Ugand. Background phosphatase clones will be positive without the added probe in die presence of alkaline phosphatase substrates. To distinguish the specific from the non-specific interacting clones, extracts from bacteria expressing these clones will be used to stimulate die tyrosine kinase activity of hBsk in a hBsk expressing NIH/3T3 ceUs. Only the Ugand will be able to stimulate activation of hBsk tyrosine activity.

It is preferable to produce die receptor probe in NTH/3 T3 ceUs rather than bacteria to receive proper glycosylation of the hBsk extraceUular domain. It has however been demonstrated tiiat glycosylation of growth factors is often not necessary for their activity. For example, M-CSF (Metcalf, Blood 67:257-267 (1986) and NGF (available from Boehringer Mannheim) produced in bacteria are biologically active. Therefore, die glycosylated receptor probe should interact properly with its Ugand syntiiesized by E. coli in a phase plaque during die screening. In addition to using the Ap-tagged hBsk probe to screen for putative Ugand in vitro, die probe can also be used in histological staining on mammalian brain section to localize expression of the Ugand. Determination of the loci of expression of the Bsk Ugand will aUow for biochemical purification of the Ugand from that tissue ceU source further for analysis.

Functional Screening of the hBsk Ligand

An alternative approach to isolate the hBsk Ugand is to utilize a functional screening approach. FuU lengtii cDNA of hBsk will be cloned into an expression vector pMEX under a MMLV LTR promoter. The hBsk expression vector will be co-transfected into NTH/3T3 ceUs together with pSV2Hygro containing a hygromycin 3-phosphotransferase gene which confers hygromycin resistance (Gritz & Davies, Gene 25:179-188 (1983)). The transfected ceUs will be selected widi hygromycin B at a concentration of 350 μg/ml. The resistant clones will be grown in 12-weU plates and screened for hBsk expression widi anti-hBsk antibody by Western blot analysis.

The vector system developed by Miki et al , Gene 55:137-146 (1989), will be used to construct a directional eukaryotic cDNA Ubrary from mouse brain mRNA. The vector has a MMLV LTR promoter for the expression of cDNA inserts and a SV40 early promoter-driven Neo gene as a selectable marker. In addition, tiiis vector contains a pBR322 repUcation origin, and die cDNA inserts of interest can be obtained easily by Not I digestion of crude Lambda DNA preparations and Ugation foUowed by transfection of bacterial ceUs. The cDNA Ubrary will be constructed as described in detail by Miki et al. Gene 83:131-146 (1989).

The cDNA Ubrary will be transfected into hBsk-expressing NIH/3T3 mouse embryo fibroblasts. Foci from transfected ceUs wUl be isolated and tested for Neo resistance to eliminate the background transformation in NIH/3T3 ceUs. Genomic DNA from each Neo-resistant transformant will be cleaved by Not I which will release the plasmid. Digested DNA will be Ugated under diluted conditions and used to transform competent bacteria. Plasmid DNA from each focus will be purified and transfected in NIH/3T3 ceUs with or witiiout hBsk expression. The transformation by the putative hBsk Ugand but not otiier oncogenes is expected to be dependent on die presence of hBsk expression. Putative clones will then be further analyzed by sequencing, the encoded protein purified and assayed for hBsk binding.

AU pubUcations mentioned hereinabove are hereby incorporated in their entirety by reference.

WhUe the foregoing invention has been described in some detail for purposes of clarity and understanding, it will be appreciated by one skilled in the art from a reading of tins disclosure tiiat various changes in form and detail can be made witiiout departing from the true scope of the invention and appended claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Rutgers, The State University of New, Jersey

(ii) TITLE OF INVENTION: Human Brain Specific Kinase (iii) NUMBER OF SEQUENCES: 10

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: STERNE, KESSLER, GOLDSTEIN & FOX

(B) STREET: 1100 New York Ave . , N.W.

(C) CITY: Washington

(D) STATE: D.C.

(E) COUNTRY: U.S.A.

(F) ZIP: 20005

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentln Release #1.0, Version #1.25

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: To Be Assigned

(B) FILING DATE: 26-JUL-1995

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: US 08/279,855

(B) FILING DATE: 26-JUL-1994

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Goldstein, Jorge A.

(B) REGISTRATION NUMBER: 29,021

(C) REFERENCE/DOCKET NUMBER: 1459.020PC0O

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 202-371-2600

(B) TELEFAX: 202-371-2540

(2) INFORMATION FOR SEQ ID NO: 1 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 174 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1 : GCACCCCGGG CCCCGAGGCG CTGCTACCTG CACCTCGACG GGCTCCCCTC TGGACGTTCC 60 TTCTCCTGTG CGCCGCTACC GGACCCTCCT GGCCAGCCCC AGTAACGAAT GAATTATGAT 120 CACGCACTGT CATGGACTGA TGATCGTTCA AATGTGAGAT GATGATGATG CCTA 174

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 216 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2 :

TGCATAATTT GTATCACAAA TATAAGTCTT CATCATGTGT GGTATATAGA GCATACATAG 60

AAGATATGTT ATATTCCTTA ATAAGCCTAA TTACATAATA CCTTGGATTG GTTCAATAAA 120

ATAAACTCAA AGCCATGTAA CTGAATACAA ACTGAAATTA ACAATGAATA AACATCCATT 180

AAAATAAACT TATCAATTAT TGAATAATTC TGGAGG 216 (2) INFORMATION FOR SEQ ID NO: 3 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 262 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3 :

AGGCTTGGCT TGGCAAACGA AAACAAGATC AGAAGAGGAA AAGATGCATT TTCATAATGG 60

GCACATTAAA CTGCCAGGAG TAAGAACTAC ATTGATCCAC ATACCTATGA GGATCCCAAT 120

CAAGCTGTCC ACGAATTTGC TAAGGAGATA GAAGCATCAT GTATCACCAT TGAGAGAGTT 180

ATTGGAGCAG GTGAATTTGG TGAGTTTGTA GTGGACATTT GAACTACCAG GAAAGAGATT 240 ACCTGTGCTA TCAACCTAAG TG 262

(2) INFORMATION FOR SEQ ID NO:4 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 223 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4 :

ATTTTAGGAA GGCATTCTTT CCTCTTTTTT AGGGAGAGTA CTTGTTGTTT GACAGTCTCT 60

TTATTCATTC TAAAGTCATA TTAACCTTCC TTAGAAATTC TCAACCAAAT TGAGATCGAA 120

AGAAACTATG TGGCTATTTA ACTTTCCTTC TTCAATTTAC TTCAAATGAC ACCAAGATCG 180

AAGAGGCAAT AATTAGCATA TAATCTCTTG TAAGGAACAA CGC 223 (2) INFORMATION FOR SEQ ID NO : 5 :

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 245 base pairs WO 96/03043 _ -._g PO7US95/09334

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(li) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

GAGGTTGAAG CTTCCTCATG GGTATAACTA TGAGGTGGAC ATTTGCCGCA GCTCTGGATG 60

TGAGGTGAGG CTTTGAAGAA CCCAGGTCTG CACACTTGAC AGGTGCCATT TTTCTCTTCA 120

TATCCTGCCT TGCACATGCA TTTCCCGATG GCACCAGCAC TCCCCTTCGC TGCAGTACAT 180

TTTGGGAGGT TCATCGTCAC AGATGGTTGA CACAGAGCTG ACACTCGAGC ATGGAGATCA 240

GCTCA 245 (2) INFORMATION FOR SEQ ID NO:6 :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 176 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6 :

ATAGACAGAC CACCATCATT CACGAGAGCA CTCAGCCCGC AGCTATTTCC TTCTGCCAGT 60

CTCTTTGAAC TCTGGATCTT TGCAAAAGCT CGCTGCTCTC CTGTTTTTCA TTCTCCACAT 120

TTTCTCAAGG TCCTCTTTCT TATCCTTAAG CACCTGCTTT TCTCTTTTAA AGAGTG 176 (2) INFORMATION FOR SEQ ID NO:7 :

(l) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 95 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: Single

(D) TOPOLOGY: linear

(n) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION. SEQ ID NO:7. AACACGAAGG CTGCAAAGAA CCGCACCTCC CCTAGCGGAT TTAAAAACTC TAACCGAAAA 60 AGCTGAAAGG CAAGGACAGG ACCCAGGACC TCTGA 95

(2) INFORMATION FOR SEQ ID NO:8 :

(l) SEQUENCE CHARACTERISTICS-

(A) LENGTH: 163 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS. single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) -by-

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8 : TTTGATGTAT TGGTTTTCCT TGATGTTTCT CCCATTCTGA TCACTGACTC AAAGTAATAC 60 ATATTAAAGG TCCTTACAGG TCCCAGTCCT CCAGGAAGGC TGTTGCAGTC CGCAGTAAAT 120 TGAGTCTATG AGATCTGAGC ACTCATGAGA CCACGCAAGC AGA ^' 163

(2) INFORMATION FOR SEQ ID NO:9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 68 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9 : TAAACTACCA GGAAAAAGAA TTAATGTGGC TATAAAACCC TTAAAGTGGC TATACTGAAA 60 AGCGCCGG 68

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 133 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

CACAACTTAC ATTACTATTC CATAACTCCA GACATCACTG GCAGAAGTAA ACTTTCGGAA 60

AGCTATGCTT CTGGGCGTCC ATCTGATTGG AATTTTCCTT GTGTGTAGCT GCTCGGACAC 120

TCAGACCGAG CAG 133

INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule 13bts)

For international Bureau use only

I I This sheet was received by the International Bureau on:

Aulhorizcd officer

Form PCT/RO/134 (July 1992) INDICATIONS RELATING TO A DEPOSITED MICROORGANISM

(PCT Rule bis)

A. The indications made below relate to the microorganism referred to in the description on page 16 , line 8.

B. IDENTIFICATION OF DEPOSIT Further deposits are identified on an additional sheet | |

Name of depositary institution

American Type Culture Collection

Address of depositary institution (including postal code and country)

12301 Parklawn Drive Rockville, Maryland 20852 United States of America

Date of deposit Accession Number

22 July 1994 ATCC 75839

C. ADD I IONAL INDICATIONS (leave blank if not applicable) This information is continued on an additional sheet | \

Plasmid, pBSK-Human clone 8-1

. DESIGNATED STATES FOR WHICH INDICATIONS ARE MADE (if the indications are not for all designated States)

. SEPARATE FURNISHING OF INDICATIONS (leave blank if not applicable) he indications listed below will be submitted to the International Bureau later (specify the general nature of the indications eg., 'Accession umber of Deposit")

For International Bureau use only

I j This sheet was received by the International Bureau on:

Authorized officer

rm PCT RO/134 (July 1992)

Claims

What Is Claimed Is:

1. An isolated nucleic acid molecule coding for a polypeptide comprising an amino acid sequence corresponding to human brain specific kinase, hBsk.

2. The isolated nucleic acid molecule according to claim 1 , wherein the molecule has the nucleic acid sequence of the hBsk present in Clones 6-1 and 8-1 which are deposited with the ATCC.

3. The isolated nucleic acid molecule according to claim 1, wherein the molecule encodes the amino acid sequence of the hBsk present in Clones 6-1 and 8-1 which are deposited with the ATCC.

4. A substantially pure polypeptide comprising an amino acid sequence corresponding to hBsk.

5. The polypeptide according to claim 4, wherein the polypeptide comprises the amino acid sequence of the hBsk present in Clones 6-1 and 8-1 which are deposited with the ATCC.

6. A nucleic acid probe for the detection of the presence of hBsk in a DNA sample from an individual comprising a nucleic acid molecule sufficient to specifically detect under stringent hybridization conditions the presence of the molecule according to claim 1 in said sample.

7. A method of detecting hBsk nucleic acid in a sample comprising: a) contacting said sample with the nucleic acid probe according to claim 6, under conditions such that hybridization occurs, and b) detecting the presence of said probe bound to hBsk nucleic acid.

8. A kit for detecting the presence of hBsk nucleic acid in a sample comprising at least one container means having disposed therein the nucleic acid probe according to claim 6.

9. A recombinant nucleic acid molecule comprising, 5' to 3', a promoter effective to initiate transcription in a host cell and the isolated nucleic acid molecule according to claim 1.

10. A recombinant nucleic acid molecule comprising a vector and the isolated nucleic acid molecule according to claim 1.

11. A cell that contains the recombinant nucleic acid molecule according to any one of claims 9 or 10.

12. A non-human organism that contains the recombinant nucleic acid molecule according to any one of claims 9 or 10.

13. An antibody having binding affinity to the polypeptide of claim 4.

14. A method of detecting a hBsk polypeptide in a sample, comprising: a) contacting said sample with an antibody according to claim 13, under conditions such that immunocomplexes form, and -o4-

b) detecting the presence of said antibody bound to said polypeptide.

15. A diagnostic kit comprising: a) a first container means containing the antibody according to claim 13 and b) second container means containing a conjugate comprising a binding partner of said monoclonal antibody and a label.

16. A hybridoma which produces the monoclonal antibody according to claim 13.

17. A bioassay for assessing candidate drugs or ligands of the hBsk receptor comprising: a) contacting a candidate drug or ligand with a cell producing functional hBsk receptors; and b) evaluating the biological activity mediated by said contact.

18. The bioassay of claim 17 wherein said cell is selected from the group consisting of PC 12 cells, primary culture of hippocampal neurons and NIH-3T3 cells.

19. The bioassay of claim 17 wherein a source of said candidate drug or said ligand is selected from the group consisting of medium from primary cultures of hippocampal neurons, conditioned medium from PC 12 cells, conditioned medium from NIH/3T3 cells and mammalian brain homogenate.

20. A method of treatment of limbic system disease in a mammal, comprising administering a therapeutically effective amount of an hBsk receptor gene in a gene delivery system to said mammal.

21. The method of claim 20, wherein said method of administering comprises microinjecting said hBsk receptor gene in said gene delivery system into said limbic system of said mammal.

22. The method of claim 20, wherein said disease is selected from the group consisting of neurodegenerative diseases, neurdegenerative disorders and neurodegenerative injuries.

23. A ligand or ligands that mediate hBsk biologic activity.

24. A method of treatment of limbic system disease in a mammal, comprising administering a therapeutically effective amount of a hBsk ligand or ligands to a mammal afflicted with limbic system disease.