WO1998010067A1 - Human homolog of the latheo gene - Google Patents

Abstract

The present invention relates to isolated or recombinant nucleic acids (DNA, RNA) which encode a latheo protein. The nucleic acids of the present invention include nucleic acids encoding human latheo protein (SEQ ID NO. 1), fly latheo protein (SEQ ID NO. 3) and characteristic portions thereof. The invention also relates to isolated or recombinantly produced latheo protein. For example, the latheo protein can be human latheo protein (SEQ ID NO. 4), fly latheo protein (SEQ ID NO. 2) and functional portions thereof. Also encompassed by the present invention is an agent which interacts with latheo directly or indirectly, and inhibits or enhances latheo function. The present invention further relates to a method of detecting latheo protein in a sample (e.g., blood, cerebral spinal fluid) obtained from an individual (e.g., human). The present invention further relates to a method of modulating dopamine levels in a mammal, comprising administering to the mammal an agent which interacts with latheo.

Description

HUMAN HOMOLOG OF THE LATHEO GENE

RELATED APPLICATION

This application is a continuation-in-part of U.S.S.N. 08/876,890, entitled "Human Homolog of the Latheo Gene" filed June 16, 1997 in the U.S. Patent Office, which is a continuation-in-part of U.S.S.N. 08/707,158, entitled "Human Homolog of the Latheo Gene" filed September 3, 1996 in the U.S. Patent Office. The teachings of U.S.STN. 08/876,890 and U.S.S.N. 08/707,158 are incorporated herein by reference.

BACKGROUND OF THE INVENTION

Identification of genes involved in learning and memory is essential for understanding the processes involved in information, acquisition, storage and retrieval. Initial genetic analysis of learning and memory in Drosophila have identified several genes whose products have central roles in the cAMP signal transduction cascade. The dunce mutation affects a gene for cAMP phosphodiesterase (Dudai, Y., et al, Proc . Natl . Acad . Sci, USA, 73:1684-1688 (1976); Byers, D. , et al . , Nature,

289:79-81 (1981)) and the rutabaga mutation affects an adenylate cyclase gene (Livingstone, M.S., et al . , Proc Natl . Acad . Sci . , USA, 82:5992-5996 (1985); Levin, L.R. , et al . , Cell, 68:479-489 (1992)). Several cAMP phosphodiesterase genes have been cloned in humans and found to be associated with behavioral abnormalities.

A greater understanding of genes involved in learning and memory will provide new approaches for the treatment of a variety of conditions affecting learning and memory. SUMMARY OF THE INVENTION

The present invention relates to isolated (e.g., purified, essentially pure) nucleic acids (oligonucleotides, nucleotide sequences) which encode a latheo protein, and include, for example, nucleic acids (RNA, DNA) obtained from natural sources, recombinantly produced or chemically synthesized. The nucleic acids of the present invention include nucleic acids encoding fly latheo protein (SEQ ID NO: 1) , human latheo protein (SEQ ID NO: 3) and characteristic portions thereof. The invention also relates to complementary sequences (i.e., a complement) of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or characteristic portions thereof. The nucleic acids of the present invention also include nucleic acids encoding a fly latheo amino acid sequence (SEQ ID NO: 2), a human latheo amino acid sequence (SEQ ID NO: 4) and characteristic portions thereof.

The present invention further relates to isolated, recombinantly produced or synthetic nucleic acids which hybridize to the nucleotide sequences described herein

(e.g., SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7) and encode latheo protein (a protein having the same amino acid sequence as the amino acid sequences included herein and/or a protein which exhibits the same characteristics as the latheo protein described herein) .

In particular, the invention relates to nucleic acids which hybridize, under moderate or high stringency conditions, to SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or other sequences which encode latheo protein or characteristic portions thereof.

The present invention also relates to a nucleic acid construct comprising nucleic acids which encode a latheo protein (e.g., SEQ ID NO: 1, SEQ ID NO: 3 or characteristic portions thereof) which is expressed when the construct is present in an appropriate host cell. In one embodiment, the nucleic acid construct of the present invention can be operably linked to exogenous regulatory sequences, such as a promoter and/or enhancer, whereby latheo protein is expressed when the host cell is maintained under conditions suitable for expression. The present invention further relates to a host cell comprising nucleic acid encoding a latheo protein.

The present invention also relates to a method for producing a latheo protein (human) . In the method, a nucleic acid construct comprising a nucleotide sequence

(DNA, RNA) which encodes latheo protein is introduced into a host cell, resulting in production of a recombinant host cell which contains the coding sequence operably linked to an (i.e., at least one) expression control sequence. The host cells produced are maintained under conditions appropriate for the nucleotide sequence to be expressed, whereby the encoded latheo is produced.

The invention also relates to isolated (e.g., purified, essentially pure) latheo protein and includes, for example, latheo protein obtained from natural resources, recombinantly produced or chemically synthesized. For example, the latheo protein can be human latheo protein (SEQ ID NO: 4) , fly latheo protein (SEQ ID NO: 2) and functional portions thereof. The present invention further relates to a method of identifying inhibitors or enhancers of latheo. An inhibitor of latheo interferes with the function or bioactivity of latheo, directly or indirectly. An enhancer of latheo enhances the function or bioactivity of latheo, also directly or indirectly.

Also encompassed by the present invention is an agent which interacts with latheo directly or indirectly, and inhibits or enhances latheo function. In one embodiment, the agent is an inhibitor which interferes with latheo directly (e.g., by binding latheo) or indirectly (e.g., by blocking the ability of latheo to interact with tyrosine hydroxylase) . In a particular embodiment, an inhibitor of the latheo protein is an antibody specific for latheo protein or a portion of latheo protein; that is, the antibody binds the latheo protein. For example, the antibody can be specific for the fly latheo protein (SEQ ID NO: 2), the human latheo protein (SEQ ID NO: 4) or functional portions thereof. Alternatively, the inhibitor can be an agent other than an antibody (e.g., small organic molecule, protein, peptide) which binds latheo and blocks its activity. Furthermore, the inhibitor can be an agent which mimics latheo structurally but lacks its function. Alternatively, it can be an agent which binds to or interacts with a molecule which latheo normally binds with or interacts with, thus blocking latheo from doing so and preventing it from exerting the effects it would normally exert. In another embodiment, the agent is an enhancer of latheo which increases the activity of latheo (increases the effect of a given amount or level of latheo) , increases the length of time it is effective (by preventing its degradation or otherwise prolonging the time during which it is active) or both, either directly or indirectly. The present invention also relates to antibodies (monoclonal, polyclonal) or functional portions thereof (e.g., an antigen binding portion such as, Fv, Fab, Fab', or F(ab')₂ fragment) which bind latheo protein.

Isolation of latheo protein makes it possible to detect latheo in a sample (e.g., test sample). The present invention further relates to a method of detecting latheo protein in a sample (e.g., blood, cerebral spinal fluid) obtained from an individual (e.g. , human) . In one embodiment, the sample is treated to render nucleic acids in the sample available for hybridization to a nucleic acid probe (e.g., SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7 or portions thereof which bind to characteristic regions of latheo-encoding nucleic acids) . The treated sample is combined with a nucleic acid probe (labeled or unlabeled) comprising all or a functional portion of the nucleotide sequence of latheo protein, under conditions appropriate for hybridization of complementary nucleic acids. Hybridization of the treated sample with the labeled nucleic acid probe is detected; the occurrence of hybridization indicates the presence of latheo protein in the sample. In another embodiment, the sample is combined with an antibody which binds latheo protein (e.g., SEQ ID NO: 2, SEQ ID NO: 4 and functional portions thereof). Binding of the antibody to a component of the sample is detected; binding of the antibody to a component of the sample indicates the presence of latheo protein in the sample.

The present invention further relates to a method of modulating dopamine levels in a mammal, comprising administering to the mammal an agent which interacts with latheo protein.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 is the nucleotide sequence (SEQ ID NO: 1) of fly latheo.

Figure 2A is an open reading frame map of fly latheo. Figure 2B is the amino acid sequence (SEQ ID NO: 2) of fly latheo.

Figure 3 is the nucleotide sequence (SEQ ID NO: 3) of human latheo.

Figures 4A-4B are an alignment of the EST50150 (human latheo) amino acid sequence (SEQ ID NO: 4) and the dLatheo (fly latheo) amino acid sequence (SEQ ID NO: 2) , showing 50% overall sequence identity and 32% conservation of sequence. Figure 5A is an illustration of the fly latheo genomic region.

Figure 5B is an illustration of the fly latheo cDNA.

Figure 6 is the amino acid sequence of the est (human) latheo protein (SEQ ID NO: 4) .

Figure 7A is the initial reading of the fly latheo nucleotide sequence (SEQ ID NO: 5) .

Figure 7B is the initial reading of the fly latheo amino acid sequence (SEQ ID NO: 6) . Figure 8 is the initial reading of the human latheo nucleotide sequence (SEQ ID NO: 7) .

Figures 9A-9B is a comparison of the initial reading of the fly amino acid sequence (SEQ ID NO: 6 ) and the initial reading of a portion of the human latheo amino acid sequence (SEQ ID NO: 8) .

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to an isolated (e.g. , purified, essentially pure) latheo gene which is involved in associative learning and memory. In particular, the invention relates to nucleic acids (e.g., DNA, RNA, oligonucleotides, polynucleotides) or characteristic portions thereof as described herein, obtained from natural sources, recombinantly produced or chemically synthesized, which encode a latheo protein or a functional portion thereof.

Nucleic acids referred to herein as "isolated" are nucleic acids substantially free of (separated away from) the genomic DNA or cellular RNA of the biological source from which they were obtained (e.g. , as it exists in cells or in a mixture of nucleic acids such as a library) , which may have undergone further processing. "Isolated" nucleic acids include nucleic acids obtained by methods described herein, similar methods or other suitable methods, including essentially pure nucleic acids, nucleic acids produced by chemical synthesis, by combinations of biological and chemical methods, and recombinantly produced nucleic acids which are isolated (see e.g., Daugherty, B.L. et al . , Nucleic Acids Res . , 19 (9) .-2471-2476 (1991); Lewis, A. P. and J.S. Crowe, Gene, 101 : 297-302 (1991)). Nucleic acids referred to herein as "recombinant" are nucleic acids which have been produced by recombinant DNA methodologies (recombinantly produced) . Recombinant DNA methodologies include, for example, expression of latheo in a host cell containing or modified to contain DNA or RNA encoding latheo, or expression of latheo using polymerase chain reaction (PCR) techniques.

This invention includes characteristic portions of the nucleic acids described herein. As used herein, a "characteristic portion" of the nucleic acids described herein refers to portions of a nucleotide sequence which encode a protein or polypeptide having at least one property, function or activity characteristic of latheo protein (e.g., a protein involved in learning and memory and/or interacts with dopamine) . In addition, the term includes a nucleotide sequence which, through the degeneracy of the genetic code, encodes the same peptide as a peptide whose sequence is presented herein (e.g., SEQ ID NO: 2, SEQ ID NO: 4) . The nucleic acids described herein may also contain a modification of the molecule such that the resulting gene product is sufficiently similar to that encoded by the unmodified sequence that it has essentially the same activity. An example of such a modification would be a "silent" codon substitution or an amino acid substitution, for instance, substitution of one codon encoding a hydrophobic amino acid for another codon encoding a hydrophobic amino acid or substitution of one acidic amino acid for another acidic amino acid. See Ausubel, F.M. et al . , Current Protocols in Molecular Biology, Greene Publ. Assoc. and Wiley-Interscience 1989. In one embodiment, the nucleic acid or characteristic portion thereof encodes a protein or polypeptide having at least one property, activity or function characteristic of a latheo protein (as defined herein) , such as activity in learning and memory, and/or interaction with tyrosine hydroxylase.

The present invention also relates more specifically to isolated nucleic acids or a characteristic portion thereof having sequences which encode latheo or variants thereof.

The invention relates to isolated nucleic acids that:

(1) hybridize to (a) a nucleic acid encoding latheo (e.g. human) such as nucleic acid having a nucleotide sequence as set forth or substantially as set forth in Figure 1 (SEQ ID NO: 1), Figure 3 (SEQ ID NO: 3), Figure 7A (SEQ ID NO: 5) and/or Figure 8 (SEQ ID NO: 7) (b) the complement of (a) ; or (c) characteristic portions of either of the foregoing (e.g. , a portion comprising the open reading frame) ;

(2) encode a protein or polypeptide having at least one property, activity or function characteristic of a latheo protein (e.g., activity in learning and memory and/or interaction with tyrosine hydroxylase) ;

(3) encode a polypeptide having the amino acid sequence of a latheo protein as set forth in Figure 2B (e.g., SEQ ID N0:2), or Figure 6 (SEQ ID NO: 4); or

(4) have a combination of these characteristics.

In one embodiment, the nucleic acid sequence shares at least about 50% nucleotide sequence similarity to the nucleotide sequences shown in Figures 1 (SEQ ID NO: 1) or 3 (SEQ ID NO: 3) . More preferably, the nucleic acid sequence shares at least about 75% nucleotide sequence similarity, and still more preferably, at least about 90% nucleotide sequence similarity, to the sequences shown in Figures 1 (SEQ ID N0:l) or 3 (SEQ ID NO: 3). Isolated nucleic acids meeting these criteria include nucleic acids having sequences identical to sequences of naturally occurring latheo or variants of the naturally occurring sequences. Such variants include mutants differing by the addition, deletion or substitution of one or more residues, modified nucleic acids in which one or more residues are modified (e.g., DNA or RNA analogs), and mutants comprising one or more modified residues.

Nucleic acids of the present invention may be RNA or DNA (e.g., cDNA, genomic DNA, and synthetic DNA). The DNA may be double-stranded or single-stranded and, if single stranded, may be the coding strand or non-coding (antisense) strand. The coding sequence which encodes the polypeptide may be identical to the coding sequence shown in Figures 1 (SEQ ID NO:l) or 3 (SEQ ID NO: 3) or may be a different coding sequence which, as a result of the redundancy or degeneracy of the genetic code, encodes the same polypeptide as the DNA of Figures 1 (SEQ ID NO:l) or 3 (SEQ ID NO: 3) . The polynucleotide which encodes a latheo polypeptide may include: only the coding sequence of a polypeptide; the coding sequence for a polypeptide and additional coding sequences such as a leader or secretory sequence; the coding sequence for a polypeptide (and optionally additional coding sequence) and non-coding sequence, such as introns or non-coding sequence 5' and/or 3' of the coding sequence.

Nucleic acids of the present invention, including those which hybridize to a selected nucleic acid as described above, can be detected or isolated under high stringency conditions or moderate stringency conditions, for example. "High stringency conditions" and "moderate stringency conditions" for nucleic acid hybridizations are explained at pages 2.10.1-2.10.16 (see particularly 2.10.8- 11) and pages 6.3.1-6 in Current Protocols in Molecular Biology (Ausubel, F.M. et al . , eds., Vol. 1, Suppl. 26, 1991) , the teachings of which are hereby incorporated by reference. Factors such as probe length, base composition, percent mismatch between the hybridizing sequences, temperature and ionic strength influence the stability of nucleic acid hybrids. Thus, high or moderate stringency conditions can be determined empirically, and depend in part upon the characteristics of the known nucleic acid (e.g. , DNA) and the other nucleic acids to be assessed for hybridization thereto.

Nucleic acids of the present invention that are characterized by their ability to hybridize (e.g., under high or moderate stringency conditions) to (a) a nucleic acid encoding a latheo protein (e.g., the nucleic acid depicted in Figure 1 (SEQ ID NO:l), Figure 3 (SEQ ID NO: 3)) or other sequences described herein (e.g., the nucleic acid depicted in Figure 7 (SEQ ID NO: 5) or Figure 8 (SEQ ID NO: 7)); (b) the complement of such nucleic acids; or (c) a portion of the nucleic acids of (a) , can also encode a protein or polypeptide having at least one property, activity or function characteristic of a latheo as defined herein, (e.g., such as activity in learning and memory, interaction with dopamine) . In a preferred embodiment the nucleic acid encodes a polypeptide which retains substantially the same biological function or activity as the polypeptide encoded by the DNA of Figures 1 (SEQ ID NO:l) or 3 (SEQ ID NO: 3).

Nucleic acids of the present invention can be used in the production of proteins or polypeptides. For example, a nucleic acid (e.g., DNA) encoding a latheo protein can be incorporated into various constructs and vectors created for further manipulation of sequences or for production of the encoded polypeptide in suitable host cells as described above. A further embodiment of the invention is antisense nucleic acid, which is complementary, in whole or in part, to a latheo sense strand, and can hybridize with it. The antisense strand hybridizes to DNA, or its RNA counterpart (i.e., wherein T residues of the DNA are U residues in the RNA counterpart). When introduced into a cell, antisense nucleic acid hybridizes to and inhibits the expression of the sense strand. Antisense nucleic acids can be produced by standard techniques. In another embodiment, the antisense nucleic acid is wholly or partially complementary to and can hybridize with a target nucleic acid which encodes a latheo protein. For example, antisense nucleic acid can be complementary to a target nucleic acid having the sequence shown as the open reading frame in Figures 1 (SEQ ID NO: 1) or 3 ( SEQ ID NO: 3) or to a portion thereof sufficient to allow hybridization.

The nucleic acids can also be used as probes to detect and/or isolate (e.g., by hybridization with RNA or DNA) polymorphic or allelic variants, for example, in a sample (e.g., blood, cerebral spinal fluid) obtained from a host (e.g. mammalian, particularly human) . Moreover, the presence or level of a particular variant in a sample(s) obtained from an individual, as compared with the presence or level in a sample (s) from normal individuals, can be indicative of an association between latheo and a particular condition, which in turn can be used in the diagnosis of the condition.

The present invention also relates to isolated (e.g., purified, including essentially pure) proteins or polypeptides designated latheo and variants of latheo. In a preferred embodiment, the isolated proteins of the present invention have at least one property, activity or function characteristic of a latheo protein as defined herein (e.g., activity in learning and memory; interaction with dopamine) .

Proteins or polypeptides referred to herein as "isolated" are proteins or polypeptides purified to a state beyond that in which they exist in cells. "Isolated" proteins or polypeptides include proteins or polypeptides obtained by methods described herein, similar methods or other suitable methods. They include essentially pure proteins or polypeptides, proteins or polypeptides produced by chemical synthesis (e.g., synthetic peptides), or by combinations of biological and chemical methods, and recombinant proteins or polypeptides which are isolated. The proteins can be obtained in an isolated state of at least about 50 % by weight, preferably at least about 75 % by weight, and more preferably, in essentially pure form. Proteins or polypeptides referred to herein as "recombinant" are proteins or polypeptides produced by the expression of recombinant nucleic acids.

As used herein "latheo protein" refers to naturally occurring or endogenous latheo proteins, proteins having an amino acid sequence which is the same as that of a naturally occurring or endogenous corresponding latheo (e.g. , recombinant proteins) , and functional variants of each of the foregoing (e.g., functional fragments and/or mutants produced via mutagenesis and/or recombinant techniques) . Accordingly, as defined herein, the term includes latheo, glycosylated or unglycosylated latheo proteins, polymorphic or allelic variants, and other isoforms of latheo (e.g., produced by alternative splicing or other cellular processes) , and functional fragments. Naturally occurring or endogenous latheo proteins include wild type proteins such as latheo, polymorphic or allelic variants and other isoforms which occur naturally. Such proteins can be recovered from a source which naturally produces latheo, for example. These proteins have the same amino acid sequence as naturally occurring or endogenous corresponding latheo.

"Functional portions" or "functional variants" of latheo protein include functional fragments, functional mutant proteins, and/or functional fusion proteins.

Generally, fragments or portions of latheo encompassed by the present invention include those having one or more amino acid deletions relative to the naturally occurring latheo protein (such as N-terminal, C-terminal or internal deletions) . Fragments or portions in which only contiguous amino acids have been deleted or in which non-contiguous amino acids have been deleted relative to naturally occurring latheo protein are also envisioned.

Generally, mutants or derivatives of latheo encompassed by the present invention include natural or artificial variants differing by the addition, deletion and/or substitution of one or more contiguous or non-contiguous amino acid residues, or modified polypeptides in which one or more residues is modified, and mutants comprising one or more modified residues. For example, mutants are natural or artificial variants of latheo which differ from wild type latheo by the addition, deletion and/or substitution of one or more contiguous or non-contiguous amino acid residues. A "functional fragment or portion", "functional mutant" and/or "functional fusion protein" of a latheo protein refers to an isolated protein or oligopeptide which has at least one property, activity or function characteristic of a latheo protein (e.g., activity associated with learning and memory, interaction with dopamine) .

Suitable fragments or mutants can be identified by screening. For example, the N-terminal, C-terminal, or internal regions of the protein can be deleted in a step- wise fashion and the resulting protein or polypeptide can be screened using a suitable binding or adhesion assay. Where the resulting protein displays activity in the assay, the resulting protein ("fragment") is functional.

The invention also encompasses fusion proteins, comprising a latheo protein as a first moiety, linked to a second moiety not occurring in the latheo found in nature. Thus, the second moiety can be an amino acid, oligopeptide or polypeptide. The first moiety can be in an N-terminal location, C-terminal location or internal location of the fusion protein. In one embodiment, the fusion protein comprises a latheo protein or portion thereof as the first moiety, and a second moiety comprising an affinity ligand (e.g., an enzyme, an antigen, epitope tag) joined to the first moiety, optionally, the two components can be joined by a linker.

Examples of "latheo" proteins include proteins having an amino acid sequence as set forth or substantially as set forth in Figures 2B (SEQ ID NO: 2) or 6 (SEQ ID NO: 4), and functional portions thereof. In a preferred embodiment, a latheo protein or variant has an amino acid sequence which has at least about 50% identity, more preferably at least about 75% identity, and still more preferably at least about 90% identity, to the protein shown in Figures 2B (SEQ ID N0:2) and 6 (SEQ ID NO: 4). Another aspect of the invention relates to a method of producing a latheo protein or variant (e.g., portion) thereof. Recombinant protein can be obtained, for example, by the expression of a recombinant DNA molecule encoding a latheo protein or variant thereof in a suitable host cell. Constructs suitable for the expression of a latheo protein or variant thereof are also provided. The constructs can be introduced into a suitable host cell, and cells which express a recombinant latheo protein or variant thereof, can be produced and maintained in culture. Such cells are useful for a variety of purposes, and can be used in the production of protein for characterization, isolation and/or purification, (e.g., affinity purification) , and as immunogens, for instance. Suitable host cells can be procaryotic, including bacterial cells such as E. coli, B . subtiliε and or other suitable bacteria (e.g., Streptococci ) or eucaryotic, such as fungal or yeast cells (e.g., Pichia pas tor is, Aspergrillus species, Saccharomyces cerevisiae, Schizosaccharomyces pombe , Neurospora crassa) , or other lower eucaryotic cells, and cells of higher eucaryotes such as those from insects

(e.g., Sf9 insect cells) or mammals (e.g., Chinese hamster ovary cells (CHO) , COS cells, HuT 78 cells, 293 cells) . (See, e.g., Ausubel, F.M. et al . , eds. Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons Inc., (1993)).

Host cells which produce a recombinant latheo protein or variants thereof can be produced as follows. For example, nucleic acid encoding all or part of latheo protein or a functional portion of latheo protein can be inserted into a nucleic acid vector, e.g., a DNA vector, such as a plasmid, virus or other suitable replicon for expression. A variety of vectors is available, including vectors which are maintained in a host cell in single copy or multiple copy, or which become integrated into the host cell chromosome.

The transcriptional and/or translational signals of a latheo gene can be used to direct expression. Alternatively, suitable expression vectors for the expression of a nucleic acid encoding all or part of the desired protein are available. Suitable expression vectors can contain a number of components, including, but not limited to, one or more of the following: an origin of replication; a selectable marker gene; one or more expression control elements, such as a transcriptional control element (e.g. , a promoter, an enhancer, terminator) , and/or one or more translation signals; a signal sequence or leader sequence for membrane targeting or secretion (of mammalian origin or from a heterologous mammal or non-mammalian species) . In a construct, a signal sequence can be provided by the vector, the latheo coding sequence, or other source.

A promoter can be provided for expression in a suitable host cell. Promoters can be constitutive or inducible. The promoter is operably linked to nucleic acid encoding the latheo or variant thereof, and is capable of directing expression of the encoded polypeptide in the host cell. A variety of suitable promoters for procaryotic (e.g., lac, tae, T3, T7 promoters for E. coli) and eucaryotic (e.g., yeast alcohol dehydrogenase (ADHl) , SV40, CMV) hosts is available.

In addition, the expression vectors typically comprise a selectable marker for selection of host cells carrying the vector, and in the case of a replicable expression vector, also comprise an origin of replication. Genes encoding products which confer antibiotic or drug resistance are common selectable markers and may be used in procaryotic (e.g., 3-lactamase gene (ampicillin resistance) , Tet gene for tetracycline resistance) and eucaryotic cells (e.g. , neomycin (G418 or geneticin) , gpt (mycophenolic acid) , ampicillin, or hygromycin resistance genes) . Dihydrofolate reductase marker genes permit selection with methotrexate in a variety of hosts. Genes encoding the gene product of auxotrophic markers of the host (e.g., LEU2, URA3, HIS3 ) are often used as selectable markers in yeast. Use of viral (e.g., baculovirus) or phage vectors, and vectors which are capable of integrating into the genome of the host cell, such as retroviral vectors, are also contemplated. The present invention also relates to cells carrying these expression vectors. For example, a nucleic acid encoding a latheo protein or variant thereof is incorporated into a vector, operably linked to one or more expression control elements, and the construct is introduced into host cells which are maintained under conditions suitable for expression, whereby the encoded polypeptide is produced. The construct is introduced into cells by a method appropriate to the host cell selected (e.g., transformation, transfection, electroporation, infection) . For production of a protein, host cells comprising the construct are maintained under conditions appropriate for expression, (e.g. , in the presence of inducer, suitable media supplemented with appropriate salts, growth factors, antibiotic, nutritional supplements, etc.). The encoded protein (e.g., human latheo) can be isolated from the host cells or medium.

Fusion proteins can also be produced in this manner. For example, some embodiments can be produced by the insertion of a latheo cDNA or portion thereof into a suitable expression vector, such as Bluescript®ll SK +/- (Stratagene) , pGEX-4T-2 (Pharmacia) , pcDNA-3 (Invitrogen) and pET-15b (Novagen) . The resulting construct can then be introduced into a suitable host cell for expression. Upon expression, fusion protein can be isolated or purified from a cell lysate by means of a suitable affinity matrix (see e.g., Current Protocols in Molecular Biology (Ausubel, F.M. et al . , eds., Vol. 2, Suppl. 26, pp. 16.4.1-16.7.8 (1991)). In addition, affinity labels provide a means of detecting a fusion protein. For example, the cell surface expression or presence in a particular cell fraction of a fusion protein comprising an antigen or epitope affinity label can be detected by means of an appropriate antibody.

The latheo nucleic acids and protein can be used in a variety of ways. For example, latheo nucleic acids and proteins can be used to identify agents (e.g., molecules) that modulate (enchance, inhibit) latheo expression and/or function. For example, latheo can be expressed in a host cell and effects of test compounds on the ability of latheo to react with tyrosine hydroxylase could be assessed.

Also encompassed by the present invention is an agent which interacts with latheo directly or indirectly, and inhibits or enhances latheo function. In one embodiment, the agent is an inhibitor which interferes with latheo directly (e.g., by binding latheo) or indirectly (e.g., by blocking the ability of latheo to interact with dopamine) . In a particular embodiment, an inhibitor of the latheo protein is an antibody specific for latheo protein or a portion of latheo protein; that is, the antibody binds the latheo protein. For example, the antibody can be specific for the protein encoded by the amino acid sequence of fly latheo protein (SEQ ID NO:2), the amino acid sequence of human latheo protein (SEQ ID NO: 4) or portions thereof. Alternatively, the inhibitor can be an agent other than an antibody (e.g., small organic molecule, protein, peptide) which binds latheo and blocks its activity. For example, the inhibitor can be an agent which mimics latheo structurally but lacks its function. Alternatively, it can be an agent which binds to or interacts with a molecule which latheo normally binds with or interacts with, thus blocking latheo from doing so and preventing it from exerting the effects it would normally exert. In another embodiment, the agent is an enhancer of latheo which increases the activity of latheo (increases the effect of a given amount or level of latheo) , increases the length of time it is effective (by preventing its degradation or otherwise prolonging the time during which it is active) or both either directly or indirectly.

In another embodiment, the sequences described herein can be used to detect latheo or DNA encoding latheo in a sample. For example, a labeled nucleic acid probe having all or a functional portion of the nucleotide sequence of latheo can be used in a method to detect DNA enoding latheo protein in a sample. In one embodiment, the sample is treated to render nucleic acids in the sample available for hybridization to a nucleic acid probe, which can be DNA or RNA. The resulting treated sample is combined with a labeled nucleic acid probe having all or a portion of the nucleotide sequence of latheo, under conditions appropriate for hybridization of complementary sequences to occur. Detection of hybridization of the sample with the labeled nucleic acid probe indicates the presence of nucleic acids encoding latheo in a sample. The presence of latheo mRNA is indicative of latheo expression. Such a method can be used, for example, as a learning and/or memory screen. Alternatively, a method of detecting latheo in a sample can be accomplished using an antibody directed against latheo or a portion of latheo. Detection of specific binding to the antibody indicates the presence of latheo in the sample (e.g. , ELISA) . This could reflect a pathological state associated with latheo and, thus, can be used diagnostically.

The sample for use in the methods of the present invention includes a suitable sample from, for example, a mammal, particularly a human. For example, the sample can be blood, cerebrospinal fluid (CSF) or tissue from a human. The present invention also relates to a method of modulating (e.g., regulating, altering) a dopamine level(s) in a host (e.g., human) by administering to the host an agent which interacts with latheo, directly or indirectly. As demonstrated herein, latheo physically interacts with tyrosine hydroxylase (TH) , an enzyme involved in the synthesis of catecholamines such as dopamine. In one embodiment, dopamine levels are modulated (e.g., increased, decreaed) in a host (e.g., mammal, particularly human) by administering to the host an agent which interacts with latheo protein, directly or indirectly, such that the interaction between latheo protein and TH is altered, thereby modulating dopamine levels in the mammal. The ability of latheo to modulate dopamine levels provides methods for treating a condition or disease associated with dopamine malfunction (e.g., Parkinson's Disease, schizophrenia, and depression) , such as by gene therapy methods in which an individual is provided with the latheo gene (e.g., by the introduction of the latheo gene into the individual or by transplantation to the individual or by transplantation to the individual of cells modified to contain and express the latheo gene) .

The present invention is illustrated in the following examples, which are not intended to be limiting in any way.

Example 1 Cloning and Behavioral Rescue of the Drosophila Associative Learning Mutant latheo

METHODS/MATERIALS

Stocks

The lat^pl and lat¹^³⁴⁴ flies were maintained using the

CyO 2nd chromosome balancer. Canton-S flies served as the wild type control. The markers (Sb, Sp, Bd^s (Ser) ) and balancers {FM7a, CyO, TM3) are described in Lindsley, D.L. and Zimm, G.G. , The Genome of Drosophila melanogaεter, San

Diego CA:Academic Press, Inc. (1992) . The lat⁺ transgene was either on a lat^pl, lat^{1 344} , or a lat⁺ background. The transgenic stocks used for behavioral rescue were: w hs- lat⁺4b; lat^pl; +, w; lat^P1 ; hs-lat⁺7a, w hs-lat⁺7b; lat^pl: +, and w s-lat⁺8; lat^pl: +. These were abbreviated as 4b; lat^pl, lat^pι; 7a, 7b; lat^pl , and 8; lat^pl respectively.

Flies were raised at 25°C on a 16/8 hr light/dark cycle with lights on at 7 a.m. A standard cornmeal-agar food was prepared from a 94.2 gm/L dextrose, 76.1 gm/L cornmeal, 31.9 gm/L yeast (Nutrex #540), 8.7 gm/L potassium tartrate, 8.4 gm/L agar, 7 gm/L CaCl₂, and 2 gm Tegosept M mold inhibitor.

Locating essential coding region relative to lat P-element Excisions of the lat^w-element were shown to produce lethal and viable alleles of the gene (Boynton, S.C. and Tully, T., Genetics, 131:655-672 (1992)); Boynton, S. C, Behavioral Genetic Studies on Learning and Memory in Drosophila melanogaεter. Ph.D., Brandeis Univ.). Analysis of the molecular changes resulting in lethality indicated where the essential regions of the lat gene were located relative to the P-element. To determine the missing sections of the P-element, left-end and right-end specific P-element probes were made by restriction digests of a 2.9 kb P-element subclone in pBR322 (O'Hare, K. and Rubin, G.M., Cell, 34 : 25-35 (1983)). The iat^-element contains only the w+ gene and two promoters. A PstI digest of this subclone, followed by recircularization using the T4 DNA ligase (New England Biolabs) , eliminated the right end leaving about 1.2 kb of left end specific sequence. An .EcoRI digest removed the left end DNA leaving about 1.7 kb of right-end P-element DNA. P-element probes were ³P- labeled by random priming (Boehringer Mannheim kit; Feinberg A. P., and Vogelstein, B., [Addendum:Anal . Biochem, 137 (1) : 266-267] Anal. Biochem . , 132:6-13 (1983)) and purified on Sephadex-G50 spin columns. Genomic DNA was isolated from lethal and viable P-element excision lines using the proteinase K digestion method (Ashburner, M. , Drosophila : A Laboratory Manual. (Plainview NY: Cold Spring Harbor Laboratory Press) 1989)). DNA was digested with various restriction enzymes, electrophoresed in 0.5% agarose gels, and transferred to Gene Screen membranes (DuPont) with lox ssc. latheo genomic library construction

Genomic DNA was isolated from lat^pl flies and digested to completion with BamHI . Assuming an average fragment size of 4096 bp, genomic DNA was ligated to λGEMll arms at a 2.5:1 molar ratio of insert to arms using a prepared packaging extract (Stratagene) . Following ligation, the library was titered, plated, and 270,000 plaques were screened using an 838 bp Hindlll P-element probe from a 2.9 kb P-element subclone.

genomic and cDNA library screening

A library consisting of Sau3A partial digests of wild- type genomic DNA cloned into arms of λDASHII (Stratgene) was screened at random primed probes made from a 700 bp Hindlll subclone consisting of 660 bp of lat genomic DNA and 40 bp of P-element sequence (Figs. 5A and 5B) . A total of 180,000 plaques were screened. Positive clones were plaque-purified, DNA was prepared by the plate lysate method (Current Protocols) , and the clones were restriction mapped and formed three classes. A Canton-S adult head cDNA library in λgtll was screened using various subcloned genomic fragments (Fig. 5A) determined by restriction mapping to be near the latheo P-element. A total of 360,000 plaques were screened. Positive cDNA phages were processed similarly to genomic phages. All cDNA clones appeared to be truncated reverse transcriptase synthesis products originating from one specific mRNA species.

northern analysis

The general method of Chomczynski, P. and Sacchi, N., Anal. Biochem . , 162:156-159 (1987) was used for all RNA isolation. The RNA used for the developmental Northerns and isolated from the transgenic lines was prepared using the commercially-available Chomczynski reagent TRIzol (GibcoBRL) . All other RNA was prepared following the original reference. For analysis of transgene induction in the transgenic lines following heat-shock, flies were heat- shocked in empty glass vials for 30 min. at 37°C, then placed in 25°C glass vials containing standard cornmeal- agar food for three hr to duplicate the recovery-interval conditions used for classical conditioning. Three hr after heat-shock, flies were frozen in liquid nitrogen and ground to a powder with a liquid nitrogen chilled mortar and pestle. Oligo dT cellulose (Collaborative Research) was used for poly⁺ RNA isolation following the manufacturer's instructions. Poly RNA was electrophoresed in 1.9% formaldehyde/1.0% agarose gels and transferred to GeneScreen membrane (DuPont) using 25mM Na₂HP0₄, pH 7.4. DNA probes for Northerns were random primed and strand- specific RNA probes were prepared using T3 and T7 polymerase reactions on subclones of the latheo* ORF (Figs. 5A and 5B) in BlueScript (Stratgene) subclones.

hybridizations RNA:RNA hybridizations were performed in 50% formamide; 270 mM NaCl; 20 mM Na₂HP0₄; 1.5 mM EDTA; 1% SDS; 0.5% nonfat powdered milk; 0.2 mg/ml yeast tRNA; 0.2 mg/ml salmon sperm DNA. All other hybridizations were carried out in 1% BSA; 200 mM Na₂HP0₄, pH 7.2; 15% formamide; lmM EDTA; 7% SDS. Probes were present at a specific activity of 5 x 10⁶ c. p.m. /ml.

in situ hybridization to polytene chromosomes

Genomic DNA and cDNA fragments were labeled with a digoxigenin labeling and detection kit following the manufacturer's instructions (Boehringer Mannheim) .

Salivary gland polytene chromosomes were removed from 3rd instar larvae raised at 18°C and fixed in a solution of 10% lactic acid: 40% water: 50% glacial acetic acid. Salivary gland tissue was dispersed under a glass coverslip by gentle tapping, and then the slide was placed in a bespoke computer-controlled pneumatic vise ("Tim's Thumb" General Valve, Fairfield, NJ) and compressed. Following compression, slides were snap frozen in liquid nitrogen, and then progressively dehydrated in ethanol. Hybridizations were carried out at 42 °C overnight in a solution of 5X SSC, 0.5% casein, 0.1% sarkosyl, 0.02% SDS, and 50% dextran sulfate using 20 ng of digoxigenin-labeled probe per slide. Excess probe was removed by a IX SSC at 55°C. Anti-digoxigenin antibody and color reactions were carried out following manufacturer's instructions (Boehringer Mannheim, digoxigenin labeling and detection kit) , except that 2% casein was used for blocking instead of the recommended 1%.

strand-specific RNA probes on adult head sections

Wild-type sections, 7μm thick, were prepared as described in Nighorn, A., et al . , Neuron, 6:455-467 (1991) with the following modifications: a) tissue was fixed in PLP containing 8% formaldehyde instead of 4%, and b) prehybridization was for 5 hr at 42°C, instead of 1 hr. Digoxigenin-labeled strand-specific RNA probes were prepared as described in the Boehringer Mannheim Genius labeling kit and alkaline hydrolyzed (Cox, K.H., et al . , Dev . Biol . , 101:485-502 (1984)). The sense and anti-sense probes were not completely overlapping, but each contained most of the open-reading frame. Digoxigenin incorporation into the probes was determined by spotting dilutions of the labeled probes onto nitrocellulose along with known amounts of labeled control DNA included with the kit, and then following manufacturer's instructions for the antibody hybridization and color reactions. Based on the results of this quantification, each slide received 50 ng of labeled probe.

construction of transgenic flies

A 2.4 kb Bell fragment encoding the 1.9 kb putative lat* open-reading frame was cloned into the vector pCaSpeR- hs (Pirrotta, 1988) cut with BglII. pCaSpeR-hs contains the hsp70 promoter, white* gene, and P-element ends for transposition in the Drosophila genome. The resulting vector, pC-hs-Iat⁺, was coinjected with another plasmid, pTurbo (pUChspΔ2-3wc) , which expresses P-element transposase (Tomlinson et al . (1988)). Embryos were collected from a population of lat ^E344/CyO flies, and dechorionated in 40% aqueous bleach for 1 min, rinsed with water for 1 min, and lined up on double sided tape while desiccating (20 min at 18°C) . Prior to injection, embryos were covered with hydrocarbon oil (Alocarbon Products, series 700) and injected using an Eppendorf model 5242 microinjector controlled with a Narishige model HO-202 micromanipulator. Adults were backcrossed to lat^LE344/CyO flies, and the progeny scored for white* eye color.

(w) lat^LE344/CyO flies were crossed to lat^^/CyO and the w* heterozygote progeny crossed inter se to establish lines. Lines were maintained as heterozygotes so that w; lat^LE344/CyO (i . e . , non-transgenics) could be used as a control for induced rescue of lethality.

heat-shock induced rescue of lethality

Embryos were collected from each of the transgenic lines for 1 hr in glass vials containing standard corn eal- agar food. After 24 hr, these vials were placed in an incubator cycling between 25°C for 5 hr and 37 °C for 1 hr; thus, developing larvae/pupae received 4 one hr heat shocks per day. Once wings and eyes were visible through the pupal case, vials were placed at a constant 25°C for eclosion. Adults were scored for the Curly wings phenotype. Flies without Curly wings represented the genotype of lat^LE344/lat^LE344, indicating that induction of the hs-lat* transgene was able to rescue lethality. All adults were counted and rescue of lethality reported as the percentage of eclosed flies that had straight wings, with 33% being maximum (CyO/CyO die in early first instar) .

constructing transgenic latheo lines Transgenic lines that were capable of rescuing lat^{1 344} developmental lethality were selected for the lat^P1 adult behavioral rescue experiment. The corresponding transgenes needed to be crossed into a lat^PI background. The primary complication with this procedure resulted from the W* -eye color marker of the trangene being obscured by the w⁺-eye color produced by the w⁹'³ P-element. Therefore, both the transgene and lat^pl second chromosome needed to be balanced, and the markers on the balancer chromosomes selected against to generate homozygotes. The chromosome carrying the transgene was determined by crossing (hs-lat* ) lat^{1 344}/ CyO females to w;CyO/Sp;TM3 (Ser) /Sb males and (w*) CyO/Sp;TM3 (Ser) /+ or Sb/+ flies were recovered. These flies were backcrossed to w;CyO/Sp;TM3 (Ser) /Sb and any lines yielding (w*) CyO/Sp;TM3 (Ser) /Sb were presumed to have an X-linked hs-lat+ transgene.

For making the X-linked transgene homozygous, it was first balanced over FM7a . By crossing hs- lat* /w;CyO/Sp;TM3 (Ser) /Sb with FM7a/y;+;+, hε- lat*/FM7a;CyO/+;TM3 (Ser) /+ and hs-lat* /Y;Sp/+ ; +/+ flies were recovered. These flies were crossed together and hε- lat* /FM7a;CyO/Sp; +/+ recovered. Separately, FM7a/Y;CyO/lat^PI ; +/+ were made and crossed with hs- lat*/FM7a;CyO/Sp;+/+ . Both hs-lat* /FM7a and hε- lat*/Y;CyO/lat^pl;+/+ were recovered and crossed together to yield hε-lat* /hε-lat* ;lat^pl;+.

To combine the third chromosome transgenes with latheo w;lat^pl/Sp;TM3 (Ser) /+ were crossed with w;Sp/ CyO; hs-lat* /hε- lat* and w;lat^PΪ /CyO; hε-lat* /TM3 (Ser) were recovered. These males and females were crossed together, the balancers selected against, and w ; lat/ lat ;hs-lat* /hε-lat* ho ozygotes procured. The two transgenes capable of rescuing the lat^pl learning phenotype, hε-lat* 4b (X-linked) and hε-lat*7a (3rd chromosome) were combined with lat* to determine the effects of lat* over-expression on a wild-type background. hε-lat* 4b /hε-lat* 4b ;lat^pl /lat^pl was crossed with w/Y;+/CyO and hs-lat* 4b /w ; lat^pl /CyO recovered . These females were crossed with FM7a/Y;+/CyO males and both hε- lat* 4b /FM7a;+ /CyO and hε-lat* 4b/Y;+ /CyO recovered and crossed to yield hε-lat* 4b /hε-lat* 4b ;+/+ and hs- lat*4b/Y;+/+ . The third chromosome linked hs-lat*7a was combined with lat* by crossing lat^pl / ;at^pι ;hε-lat*7a/hε- lat*7a with +/CyO;+/TM3 and recovering females of the genotype +/lat^P1 ;hε-lat*7a/TM3 . These were crossed together and +/+; hε-lat* 7 a hε-lat*7a recovered.

behavioral rescue Four lat transgenic lines were tested for heat-shock induced rescue of the lat^pl learning phenotype. When tested immediately after training, lat flies already show a deficit, and lat^pl was therefore classified as a learning mutation (Boynton, S. and Tully, T., Genetics, 131:655-672 (1992)). Transgenic lines which gave full rescue of the learning deficit were: a) tested for normal sensory responses, b) tested for retention of the conditioned odor avoidance response three hr after training, and c) tested for the effect of induction of the transgene on a wild-type background. The conditioning procedure of Tully, T and Quinn, W.G., ". Comp. Phyεiol . A, 157:263-277 (1985)) was used to train and test the flies with only one modification; the interval between odor presentations has been lengthened from 30 sec to 45 sec to reduce the effect of trace conditioning. The experimenter was blind to the genotypes being examined.

Each evening prior to behavioral tests, approximately 600 1- to 2-day old flies were sequestered in foam-plugged glass bottles containing 125 ml of cornmeal-agar food with a folded paper towel. Bottles were stored overnight at 25°C and 70% relative humidity. In the morning, groups that were destined to receive heat-shock (30 min at 37 °C) were transferred to foam-plugged glass vials with a 10 mm X 70 mm strip of Whatman filter paper for absorbing moisture. After heat-shock, flies were transferred to 25°C vials containing cornmeal-agar food for a 3 hr recovery period at 25°C and 70% relative humidity, and then training began. The protocols for these experiments were identical to those reported in Bolwig, G.B., et al . , Neuron (1995).

bacterial Lat-fusion protein expression

The ORF shown in Figure 5B was directionally cloned into the bacterial protein expression vector pET-30b (Novagen) . A BamHI /EcoRI fragment was removed from the pCaSpeR-hs-lat⁺ transformation vector, with EcoRI site being provided by the pCaSpeR vector. This fragment was ligated into BamHI /EcoR -cut, de-phosphorylated, and gel- purified pET vector. Positive subclones were verified by mini alkaline-lysis plasmid preps followed by dideoxy sequencing to verify the reading frame, and then transformed into the E. coli strain BL21 DE3. A time course following IPTG induction of early log-phase cultures revealed maximal protein production by 60 min after induction. The 75 kDa fusion protein corresponded to the size predicted from the lat⁺ ORF combined with the 5' affinity tags from pET. Using the pET-lat⁺ construct, over 3 mg of the tagged Lat protein using SDS-PAGE followed by electroelution of the protein out of the acrylamide matrix was purified. The protein solution was lyophilized once from the electroelution buffer and twice more from water.

RESULTS molecular analysis of latheo" genomic function

The P-element (w⁺⁹ ) used to create the lat^pl mutation does not contain plasmid rescue sequences, so a lat^pl genomic phage library was constructed and screened with P- element probes to recover genomic DNA adjacent to the insert. First, the region around the P-element was characterized so that an appropriate-sized restriction fragment for cloning into phage could be identified. Various restriction digests of lat^pl genomic DNA were hybridized with left-end and right-end specific P-element probes revealing that a BamHI digest would produce a 17 kb fragment containing 2.7 kb of w*⁹³ P-element DNA and 14.3 kb of lat^pl genomic DNA off the left-end of the P-element. lat^pl genomic DNA was digested to completion, and since fragments smaller than 9 kb cannot be packaged by the phage, this served to enrich for the 17 kb BamHI fragment. Eleven phage were purified from this library, and ten had the same 17 kb insert. The genomic DNA was restriction mapped and a 700 bp Hindlll fragment was found that contained about 40 bp of P-element DNA and 660 bp of genomic DNA immediately flanking the P-element. This fragment was subcloned into pBluescript (Stratagene) , labeled with digoxigenin and hybridized in εitu to salivary gland polytene chromosomes. A positive signal developed in 49F, the site of the latheo P-element, confirming that the DNA was from this region.

Genomic DNA analyzed from lines carrying lethal excisions of the lat^Pl P-element (Boynton, S. and Tully, T, Genetics , 131:655-672 (1992)) was done to identify the essential coding region of the gene relative to the P- element. Lethal excisions of the lat^P1 P-element balanced over CyO revealed that the left-end of the P-element was still present in some of these lines. Other lethal excisions completely removed P-element sequences from the latheo genome. However, the observation that the left-end of the P-element could be intact in some of the lethal excisions suggested that the essential coding region of the gene necessarily was to the right of the P-element. This required additional library screening to obtain DNA from the other side of the insertion site.

Using 700 bp Hindlll fragment as a probe for a Canton- S genomic library, 3 phage clones were isolated that spanned about 25 kb of genomic DNA. Several genomic fragments spanning the insertion site were subcloned and used as probes for a Northern blot analysis and for screening cDNA libraries.

characterization of RNA transcripts near the lat^pι P- element Northern blot analysis of poly⁺ RNA isolated from adult Canton-S and lat^p/ heads revealed a difference between mutant and wild-type. Probing with the 3.7 kb EcoRI genomic fragment detected two transcripts in wild- type flies, 3.9 kb and 2.6 kb, whereas lat^pl flies had three transcripts of sizes 3.9, 3.6, and 2.6 kb. When 1.9 kb EcoRI /Hindlll genomic fragment was used, the same pattern was detected. The genomic DNA fragments indicated in Figures 5A and 5B were subcloned into pBluescript and used as templates for random primed DNA probes. Together, there three probes spanned the site of the P-element insert and revealed that the two lat* transcripts and three lat^P1 transcripts contained sequences from both sides of the P-element.

An examination of transcripts from wild-type and lat^pι heads and bodies revealed that the new transcripts detected in lat^pι appeared to be more abundant in heads. In addition, several changes were obvious when comparing wild- type and lat^w across developmental stages. Primarily, the expression of a 3.6 kb transcript appears to occur in six of eight lat^pl developmental stages, yet only in wild-type 0-4 hr embryos was a transcript of this size detected.

isolation of cDNA's in the latheo region

An adult head cDNA library in λgtll was screened with the 1.9 kb EcoRI /Hindlll fragment. Three positive clones were detected out of 180,000 plaques. These cDNA clones were hybridized to Southern blots containing digests of the genomic phage. Two of the cDNA clones contained sequences that hybridized to restriction fragments from both sides of the P-element insertion position. The phage clones were restriction mapped and appeared to be all of the same transcript. The largest cDNA (3.2 kb) was subcloned into pBluescript, labeled with digoxigenin and used for chromosomal in situ hybridization. A positive signal developed only in region 49F, indicating that the cDNA was from the previously characterized region of the latheo P- element. The genomic region of wild-type, lat^pl and the subcloned cDNA were sequenced and it was shown that the P- element had inserted 134 bp upstream of the only large ORF in this region. Stop codons in all reading frames prior to this ORF and the ATG codon is part of the generally recognized translation-start consensus sequence (GATGG, (SEQ ID NO: 9) Cavener, D.R. , Nucleic Acids Res . , 15:1353- 1361 (1987); Kozak, M. , Nucleic Acids Res . , 15:8125-8148 (1987)). All introns have consensus 5' and 3' splice donor/acceptance sequences (Mount, S., Nucleic Acidε Reε . , 10:459-472 (1982)). An initial reading of the fly latheo nucleotide sequence (SEQ ID NO: 5, see Figure 7) , indicated an ORF encoding a putative 617 amino acid (73 kd) deduced protein (SEQ ID NO: 6, see Figure 7B) , and terminating 433 nucleotides upstream of the identified poly A tail. A consensus polyadenylation site preceded the poly A tail by 21 nucleotides (Proudfoot, N.J. and Brownlee, G.G., Nature, 263:211-214 (1976)). A subsequent reading of the fly latheo nucleotide sequence (SEQ ID NO: 1) shown in Figure 1, resulted in changes in the fly nucleotide and amino acid sequence and indicated a longer amino acid sequence (SEQ ID NO: 2) as shown in Figure 2B. Subsequent DNA and protein (BLAST, Pro-site, Swiss-Protein) database searches have revealed no homologies to known proteins, indicating that lat* encodes a novel protein involved in associative learning.

lat^L£M lethal phenotype rescue by induced expression of an hs-lat* transgene during development Of the 15 transgenic lines tested, seven lines were capable of rescuing the lethality of flies homozygous for a lethal, P-element excision allele of lat^PI, lat^{1 344} (Boynton, S. and Tully, T., Geneticε, 131:655-672 (1992)). Table 1 lists the seven different lines and the corresponding percentage (of total progeny) of lethal excision homozygotes that survived to adulthood due to the heat- shock induction of the hs-lat* transgene. Table 1. line# LE/LE LE/CyO % rescue

3c 8 364 2

4b 15 156 10

6a 1 9 11

7a 1 3 33*

7b 21 197 11

8 10 159 6

13b 2 5 40*

As an internal control, the transgenic lines were not homozygous for the transgene, so it was possible to verify that the heat-shock was not causing non-transgenic lat^LE344/lat^LE344 flies to survive. The only straight-wing flies obtained also had colored-eyes, indicating that the transgene must be present and induced for lethal excision homozygotes to survive. This observation also held true for nonheat-shock conditions: when maintaining the transgenic lines at 25°C, no straight-wing flies were ever present. This indicated that the transgenes were not "leaky" enough to rescue lethality without heat-shock induction (Steller, H. and Pirrotta, V., EMBO J, 4:3765- 3772 (1985); Ewer, J. , et al . , J. Neurogrenet . , 7:31-73, (1990)) .

The heat-shock regimen was quite severe and may have been a limiting factor not only for the degree of rescue but also for overall survivability. Rescue of the lat^{1 344} developmental phenotype was used to assess transgene function. induced expression of the hs-lat⁺ transgene rescues the lat?¹ learning defect

Four transgenic lines (of the 7 that rescued lat¹⁸³⁴⁴ lethality) were tested for heat shock induced rescue of the lat" learning phenotype Pavlovian conditioning procedure of Tully and Quinn, J. Comp. Phyεiol .A, 157:263-277 (1985)). Without heat shock, the four transgenic lines (4b; lat", lat"; 7a, 7b; lat^pl , and 8; lat") were statistically equivalent to lat" (P=0.225, 0.536,0.134, and 0.489, respectively) . Three hr following the 30 min, 37 °C heat- shock, two lines ( 7b; lat" , and 8; lat") partially rescued the initial learning phenotype of the lat" mutation (P<0.001 for 7J ;2at^w(-)hs compared to (+)hs, and P=0.008 for 8;lat" (-)hs vs. (+)hs, yet P=0.004 for wild type (+)hs vs. 7b;lat" (+)hs and P<0.001 for wild type (+)hs vs.

8;lat" (+) hs) . The two remaining transgenic lines, 4b;lat" and lat"; 7a fully rescued the observed lat" learning defect, producing learning scores similar to those of wild- type flies (P=0.271 for 4 ;lat^w(+)hs vs. wild type(+)hs, and P=0.257 for lat"; 7a (+)hs vs. wild type(+)hs).

induced expression of the hs-lat⁺ transgene rescues the lat^P1 memory defect

Three hr retention was then measured in both of the transgenic lines that gave full rescue of learning, 4b; lat" and lat"; 7a . 4b; lat" was capable of full rescue (P=0.450) of the three retention deficit observed in lat^p/ flies, whereas lat"; 7a was capable of only partial rescue (P=0.001 for lat^w;7a(-)hs and (+)hs, yet, P=0.002 for wild-type vs. lat"; 7a (+)hs) . Analysis of the peripheral behaviors in response to the training stimuli at three hr after heat-shock indicated no statistically significant differences between wild-type, lat", 4b; lat"; 7a . induced expression of the hs-lat⁺ transgene does not affect olfactory acuity or shock reactivity

In the two lines giving full behavioral rescue, the peripheral behaviors necessary for proper performance during the classical conditioning experiments were verified. Olfaction to both training odors was measured with and without heat-shock at the concentration used during conditioning (undiluted), and at 1:100 dilution.

The ability to sense and escape from electric shock was measured in both lines with and without heat-shock using the normal training voltage (60V) and at 20 volts.

Responses to the task-relevant stimuli strengths were not statistically different than wild-type.

The weaker stimuli, either odors or shock, were used to determine if heat-shock somehow produced an increased responsiveness to the stimuli which might then have resulted in an enhancement of performance during conditioning. Normal or reduced responses to the weaker stimuli indicated that response thresholds with and without heat-shock were not different and that the enhancement in performance observed following heat-shock was specific to associative learning.

detection of hs-lat⁺ transcript by northern analysis

To determine if the behavioral rescue was supported on a molecular level via heat-shock induced transcription from the hε-lat* transgene, an analysis of mRNA from wild-type, lat", 4b; lat" , and lat" ; 7a was performed. Phosphoimager quantitation of the broad band at 3.5/3.6 kb indicated that the signal increased 2.5-fold in 4b; lat" and 3.8-fold in lat"; 7a three hr after the 30 min, 37 °C heat-shock. induced expression of the hs-lat⁺7a transgene in lat* flies is deleterious

To determine the effects of expression of the hs-Iat⁺ transgene on a wild-type (lat*) genetic background, the hs- lat* 7 a transgenes were crossed into wild-type flies. Learning and three hr memory of the lat* ; s-lat*7a transgenic flies was determined. Both learning and memory (P<0.001) and memory (P=0.006) were reduced by induced expression of the hslat⁺ transgene on a, wild-type background.

expression of lat* RNA in wild-type adult heads

Anti-sense RNA probes generated by T3 and T7 polymerase transcription reactions using a cDNA subclone in pKS were used to probe 7μm frontal sections of wild-type heads. In heads, the sense strand probe failed to detect any signal. The anti-sense probe hybridized with structures in the optic lobes, dorsal deutocerebrum, inferior bridge and fascicles of the inferior bridge. Notably, expression was not preferential to mushroom bodies (Nighorn, A., et al . , Neuron, 6:455-467 (1991): Han, P. et al . , Neuron, 9:619-627 (1992)).

DISCUSSION localization of the P-element insertion

Sequencing of both genomic DNA and cDNA from the latheo region revealed that the P-element has inserted 134 bp 5' from the start of the largest open reading frame (ORF) identified. Both wild-type and lat" adults have transcripts of 3.9 kb and 2.6 kb. Additionally, lat" adults transcribe a 3.6 kb message. Hybridization of the cDNA probes to the genomic sequences flanking the transposon indicated that cDNA sequence was present on both sides of the transposon, verifying that the P-element had inserted in the gene. Strand-specific RNA probes generated from a cDNA subclone revealed that only the anti-sense strand hybridized to the same bands as random primed probes generated from the cDNA. Therefore, with no genes present on the complementary strand, the latheo* gene appeared to be uniquely affected by the P-element.

Northern analysis indicated that a new transcript expressed preferentially in heads in latheo" adults. When comparing the head versus body lanes on the lat" developmental Northern, the high abundance of the 3.6 kb band obscured the signal from the 3.9 kb band directly above it. Perhaps this new transcript resulted from an effect of the P-element on splicing of either the large (3.9 kb) or small (2.6 kb) transcripts normally present, or the aberrant expression of an embryo-specific 3.6 kb transcript in adults.

The developmental Northern analysis pointed towards a mis-expression of the 3.6 kb embryo-specific message as being a potential explanation for the appearance of this size transcript in lat" adults. In wild-type, the 3.6 kb transcript was only detected in the early (0-4 hr) embryo stage. The analysis of lat" revealed that the 3.6 kb transcript was also present in 0-4 hr embryos, but expression continued on into large embryogenesis (>16 hr) embryos) . No signal was detected in 1st or 2nd instar. Beginning in 3rd instar and continuing on into adults, the 3.6 kb transcript was aberrantly expressed. The initiation of the aberrant expression in late 2nd instar corresponds with the detection of a cell proliferation defect in the anterior imaginal disk of larvae carrying the lethal heteroallelic genotype, lat^{1 344} /laV*⁶ (Boynton, S., Behavioral Genetic Studies on Learning and Memory in Drosophila melanogaster . Ph.D. , Brandeis University) . The function of the 3.6 kb transcript in embryos may be to inhibit proliferation or differentiation of certain cell types. Perhaps if this transcript is expressed in the developing adult nervous system, the inhibition of cell proliferation or differentiation (e.g. , inhibition of plasticity) may result in an alteration of the functional status of neurons necessary for associative learning to occur. Alternatively, the lethal phenotype and the behavioral phenotype may be functionally distinct aspects of lat* gene function.

expression in adult head

Strand-specific RNA probes generated from the cDNA subclone were hybridized to 7μ paraffin sections of wild- type heads. Signal was detected only with the anti-sense probe. Staining was seen in the several regions of the brain and was not limited to the mushroom bodies and/or central complex, therefore indicating that preferential expression in these brain regions may not be necessary for genes involved in learning and memory.

rescue of lethality

Rescue of lethality was the first criterion chosen for assessing the specific function of the lat⁺ gene. This decision was primarily based on simplicity: the assay for straight-wing flies following induction of the transgene was easy to score and allowed verification of which transgenic lines could be used for the more difficult test of behavioral rescue. Of the 15 transgenic lines obtained, 7 of these were able to rescue the lethality of lat^{1 344} homozygotes with varying degrees of success. rescue of behavior

The transgenes present in the 7 lines which rescued lethality were crossed off the lat^LE344/CyO background and into a lat" background. Four of these lines were tested for conditional rescue of associative learning phenotype of lat" . Wild-type flies typically score PI=85 immediately after training, compared to a lat" learning score of PI=55 at t=0. This initial difference remained constant throughout a 6 hr retention interval (Boynton, S. and Tully, T. , Genetics, 1331 : 655-672 (1992)), resulting in roughly parallel decay curves for retention of the conditioned odor avoidance response. Two of the transgenic lines (4b; lat" and lat"; 7a ) were statistically equivalent to wild-type following heat-shock, indicating a full rescue of the lat" learning defect, yet were equivalent to lat" without heat-shock. The observation that induction of the transgene in lat" adults was capable of restoring normal learning and memory function indicated that the nervous system of these animals was functional yet lacked sufficient lat* product for normal performance. In addition, learning scores without heat-shock revealed that the slight "leakiness" of the hsp70 promoter provided insufficient lat* product to affect the mutants' behavior. Task-relevant peripheral behaviors were examined in wild- type, lat", 4b; lat" and lat"; 7a and found to be equivalent with or without heat-shock, indicating that the presence of the transgene on a lat" genetic background did not alter the mutants' response to odor or shock. Testing the transgenic lines' olfaction using a hundred-fold dilution of odor and shock-reactivity using a 3-fold reduction in voltage revealed that heat-shock did not produce a hypersensitivity to these stimuli which might then have resulted in increased performance in the conditioning procedure . A three hr delay and training was introduced in order to minimize the non-specific effects of heat-shock on the flies' performance during the training procedure. However, the function of the hε-lat* transgene was sufficient to permit wild-type learning to occur even after this delay. The reduction in the score of wild-type following heat- shock (P=0.019) indicated that some non-specific effects still remain. The conclusion from these experiments was that the rescue of learning must be a specific, central response to normal levels of sensory input.

Extensive homology searches using both the DNA and protein sequences of lat* has yielded no significant similarities with known proteins, meaning that a novel component involved in Droεophila learning has been identified.

Example 2 Developmental effects of mutations in the learning gene latheo METHODS stocks The latheo" P element insertion strain and the excision lethal alleles latheo¹"⁴⁴ and latheo'^E4il (IE344 and IE49) , were isolated from a P-element mutagenesis for mutations affecting learning and memory (Boynton, S. and Tully, T. Geneticε, 131:655-672 (1992)). The vr6.6 and vr6.35 mutations were isolated in an ethylmethane sulfonate mutagenesis for lethals in the veεtigal region (Lasko, P.F. and Pardue, M.L., Genetics, 120:495-502 (1988)). Boynton and Tully (1992) have shown that all of these alleles map within the deficiency chromosome Df (2R) vg56 (vg56 ; Lasko, P.F. and Pardue, M.L., Genetics, 120:495-502 (1988);

Lindsley, D.L. and Zimm, G.G. , The Genome of Drosophila melanogaster. Academic press, San Diego, CA 1992) . All lethal alleles and the deficiency were maintained heterozygous with the balancer chromosome CyO (Lindsley, D.L. and Zimm, G.G., The Genome of Drosophila melanogaster. Academic press, San Diego, CA 1992) .

Mutant larval and pupal samples were distinguished from wild-type siblings using the marker chromosome

In (2LR) Gla which carries the dominant larval marker Black cell (Be) . Wild-type chromosomes were derived from w (cε4) (Boynton, S. and Tully, T. Geneticε, 131:655-672 (1992)).

All Drosophila strains were maintained at 25°C on a 16/8 hr light/dark cycle with lights on at 7:00 a.m., on a food medium consisting of 8.4 g/L agar, 31.9 g/L yeast (Nutrex #540), 94.2 g/L dextrose, 8.7 g/L NaKT, 7 g/L CaCl₂, 76.1 g/L cornmeal and 2 g/L Tegosept M mold inhibitor.

Determination of lethal phase

The lethal latheo allele, vr6 .6, vr6.35, IE49 and le344 were outcrossed to w (cs4) and the lethal /+ males collected. The lethal/+ males were mated to lethal/CyO, vg56/CyO, or +/+ female flies for 2 days. The lethal /CyO females in this cross were carrying either the same allele as the males, or one of the three remaining lethal alleles examined. As a further control for nonspecific lethality, w (cs4) was crossed to itself. Beginning on the third day after mating, eggs were collected on 5% sucrose and 1.8% agar plates sprinkled with yeast for 6 hours at 25°C. Eggs were transferred to a fresh plate with a drop of live yeast and allowed to develop at 25°C for at least 28 hours. Unhatched eggs were dechorionated in bleach and the number of unfertilized eggs which lacked signs of development as evidenced by mouth hooks or segmental bands were counted.

The percentage of eggs hatched then was calculated relative to the total number of fertilized eggs. Hatched larvae were allowed to develop on sucrose- agar-yeast plates until puparium formation. The puparia were collected, counted and transferred to petri plates containing a moistened filter (Whatman #42) . Twenty four hours later, normal development of the pupae as indicated by release of the mouth hooks and eversion of the head, was scored and quantified. On subsequent days, pupal development was monitored and compared to the pupal stages defined by Bainbridge, S.P. and Bownes, M. , J. Embryol . Exp . Morphol . , 66:57-80 (1981). Lastly, the number of flies eclosing was determined.

histology larvae and white prepupae were collected from crosses of lethal /Be males and females. Homozygous mutant animals lacking the Be marker were selected. Prepupae were collected at stage PI (Bainbridge, S.P. and Bownes, M. , J . Embryol . Exp . Morphol . , 66:57-80 (1981)). Larvae were selected at late-second instar or mid-third instar by the shape of the anterior spiracles (Demerec, M. , Biology of Drosophila . John Wiley & Sons, Inc. New York (1950)) and the age from hatching (45 and 72 hours at 25°C, respectively) . The posterior end of the larvae and pupae was cut off and the animals fixed overnight in FAAG (formalin:alcohol:acetic acid;10:85:5 with 1% glutaraldehyde) , dehydrated in an ethanol series, cleared in xylene, and embedded in Paraplast Plus embedding medium (Monojet Scientific; Campos, A. R. , et al . , J . Neurogenetics , 2:197-218 (1985)). To examine genital discs, the anterior end was removed and the animals prepared for sectioning as above. Seven micron thick section were collected and mounted on gelatin coated slides and stained with heraatoxylin (Humason, G.L., Animal Tissue Techniqueε . 3rd ed. , W.H. Freeman, San Francisco (1972)). Neural anatomy of adult brains was examined in latheo" homozygous and latheo" /vg56 heterozygous flies. Since red-eye pigment enhances the autoflourescence used to examine these sections, the behaviorally normal latheo" /+ (Boynton, S. and Tully, T. Geneticε, 131:655-672 (1992)), as opposed to w(cε4) flies, were used as a control in these experiments. Heads were prepared for sectioning according to protocol 112 of Asburner, M. , Droεophila : A Laboratory Manual , Cold Spring Harbor Lab. Press, Cold Spring Harbor, NY (1989)). Use of this mass histology procedure allowed the genotypes to be coded and the sectioned material examined blind. Seven micron thick sections were examined under a florescent microscope attached to a monitor. The sizes of the mushroom bodies, central complex and medulla were recorded. In brief, relevant structures were traced and their area integrated across serial sections.

BUdR staining

Mitotic activity was monitored by incorporation of 5- bromodeoxyuridine (BUdR, Boehringer mannheim) according to Truman, J.W. and Bate, M., £>ev. Biol . , 125:145-157 (1988)). The central nervous system (CNS) of climbing and early- third instar larvae (approximately 96 and 55 hours post- hatching at 25°C respectively) were exposed by removing the posterior end of the animals and opening up the anterior cuticle. The anterior of the larvae was cultured in

Schneider's medium (with L-glutamate, Gibco) containing 15 μg/ml BUdR for 1.5 hours at room temperature. The CNS was fixed in 4% paraformaldehyde for one hour, washed and stained with mouse anti-BUdR (Becton Dickinson) followed by a biotinylated horse anti-mouse secondary antibody.

Antibody was visualized using avidin-Texas red (Vectastain ABC kit, Vector Labs) and examined with a Biorad MRC 600 confocal microscope (Selleck, S.B., et al . , Neuron, 6:253- 255 (1992)).

ELAV immunocytochemistry

Climbing third instar larvae were processed as above for histology and stained for elav immunocytochemistry according to Robinow, S. and White, K. , J. NeuroJiol., 22:443-461 (1991) using a 1:50 dilution of anti-ELAV antibodies in PBT (0.1 M sodium phosphate buffer, pH 7.2, 0.3% Triton X-100) . Immunoreactivity was visualized using an indirect immunoperoxidase procedure (ABD elite kit:

Vector Laboratories) . The slides were then dehydrated and mounted in DPX.

RESULTS

Lethal phase of latheo mutant To determine the lethal period, mutant vr6.6, vr6.35, IE49 and IE344 flies were mated to each other and to a deficiency of the region, vg56. In all of the above FI crosses, 25% of the progeny are expected to bear lethal mutations of latheo on both chromosomes and hence die during the lethal period. As a control for mortality not induced by latheo, lethal/CyO as well as w (cs4) flies were mated to w (cs4) males. The frequency of animals surviving each developmental stage in the experimental crosses were then compared to control crosses to determine any significant difference in survival in the two crosses. The survival frequencies in experimental crosses which were significantly different from controls were then compared to the expected survival frequency which was then defined as the survival frequency in the control lethal /CyO x +/+ cross less 0.25.

Examination of the frequency of animals hatching, pupating or eclosing indicated pupal lethality in all crosses except vr6.35 x vr6.35. The remaining crosses all demonstrate normal frequencies of hatching and pupation while showing a large reduction in eclosion frequency. The cross, vr6.3S x vr6.35 was found not to be significantly different from the 3 :4 ratio anticipated for embryonic lethality induced by homozygous second chromosomes.

Nonetheless, flies heterozygous for vr6.35 and a deficiency of this region, vg56, and flies heterozygous for vr6.35 and any of the other lethal alleles show pupal lethality. Thus it is likely that the embryonic lethality seen in vr6.35 x vr6.35 was induced by a second site lethal mutation on the vr6.35 chromosome. The vr6.35 x vr6.35 cross also indicates the normal viability of the remaining genotypes, vr6.35/+, vr6.35/Cyθ, and +/CyO generated in this cross as the surviving siblings do not show significant reductions in the frequencies of pupation or eclosion. It is concluded, therefore, that lethality occurs during pupation for the four alleles tested when crossed inter se .

During the determination of lethal phase, it was noted that the presumptive homozygous lethal animals appeared to die very early in pupation. Comparison of the uneclosed pupae to the developmental stages described by Bainbridge S.P. and Bownes, M. , J. Embryol . Exp . Morphol . 66:57-80 (1981) suggested that lethality occurred during pupal stage P3. Histological analysis thus was performed on early prepupal larval animals which were compared to age-matched w(cs4) larvae and pupae.

Morphological analysis of lethal alleles

Imaginal disc abnormalities in latheo lethal pupae

In wild-type prepupae, the anterior imaginal discs are seen as well organized folded sheets of cells surrounded by a thin basement membrane. Within the discs lies a lumen known as the peripodial cavity (Madhaven M.M. and Schneiderman, H.A. , W . Roux 'ε Archives, 183:269-305 (1977); Demerec, M. , Biol . of Drosoph . , J. VJiley & Sons, Inc. New York (1950)). Examination of disc morphology in the lethal alleles allowed division of the alleles into two classes. The allelic combinations vr6 , 6/vr6. 6, vr6 . 6/IE344, IE344/IE344, and vr6.35/IE344 completely lacked anterior imaginal discs in late-third instar and early-pupal stages. In the second class, represented by IE49/IE49 and IE49/IE344 animals, the anterior imaginal discs were visible as large masses of cells in the appropriate imaginal disc locations. The "discs", however, appeared quite disorganized with the tissue consisting of loose clumps of cells in which the peripodial cavity was not visible. Instead, the space normally devoid of cells was confluent. The cells were rounded instead of the normal columnar shape (Demerec, M. , Biol . of Droεoph . , J. Wiley & Sons, Inc. New York (1950)). They appeared, however, to be approximately normal in size. Interestingly, homozygous IE344 prepupae did not show any imaginal discs at all, suggesting that the presence of presumptive discs in IE344/IE49 animals was caused by activity of the IE49 allele.

Mutations such as defective dorsal discs (Simcox, A.A. , et al . , Devel . Biol . , 122:559-567 (1987)) and discs large (Murphy, C, Dev . biol . , 39 : 23-36 (1974)) have been found to affect only a subset of the imaginal discs. Thus, the genital discs of IE49 homozygous and vr6 . 6/IE344 heterozygous larvae were examined also. The genital discs showed abnormalities similar to that of the anterior discs with vr6 . 6/IE344 larvae lacking genital discs and IE49/IE49 larvae showing disorganized genital discs. To distinguish between failure to form imaginal structures and degeneration of partially developed discs, earlier larval stages were examined. By late-second instar, the organization and folding of imaginal disc epithelium was apparent in wild-type larvae. During the next instar, the disc tissue continued to enlarge and to form the characteristically infolded structures (Madhaven, M.M. and Schneiderman, H.A., W . Roux 's Archives, 183 : 269- 305 (1977)). Clumps of cells suggestive of rudimentary imaginal discs were present in mutant vrβ .6/IE344 larvae during late-second instar, some of which showed the presence of a peripodial cavity. In older larvae, however, the discs appeared to lose organization and failed to enlarge. In some cases, the mutant discs persisted in third instar, but without significant development. By late-third instar imaginal discs were never found in vr. 6. 6/IE344 .

Maldevelopment of the brain in latheo lethal mutants

In wild-type animals, the brain can be divided into a central brain region and optic lobes. The optic lobes, which began to develop late in larval life, are produced from proliferation centers which are visible as darkly staining clusters of cells (White, K. and Kankel, D.R., Dev. Biol . , 65:296-321 (1978)). As neurons develop, processes are extended leading to the crescent-shaped neuropillar regions of the optic lobe (White, K. and Kankel, D.R., Dev . Biol . , 65:296-321 (1978)).

In all mutant genotypes the central nervous system was abnormally developed. These abnormalities fell into two classes. In IE344/IE344, vr6. 6/vr6. 6, vr6.35/ IE344 -and vr6.6/IE344 prepupae, the nervous system was reduced in size. In particular, the optic lobes were very poorly developed in these genotypes. The optic lobes of TE344/IE344, vr6. 6/vr6. 6, vr6.35/IE344 and vr6. 6/IE344 prepupae, failed to show any optic lobe neurophil. Furthermore, darkly staining proliferation centers were absent or only faintly visible in prepupae.

In prepupae of the remaining two genotypes examined, IE49/IE49 and IE49/IE344 , the nervous system was distended and poorly organized. Neuropil was visible in the optic lobe region, but its size was disproportionately small relative to controls. Darkly staining proliferative centers were present yet disorganized. In contrast to the organized proliferation centers in wild-type prepupae (White, K. and Kankel, D.R. , Dev . Biol . , 65:296-321 (1978)), the darkly stained presumptive proliferation centers of IE49/IE49 and IE49/IE344 consisted of many patches of rounded cells. The less darkly stained cells, however, appeared normal in size and shape. Examination of the CNS in younger vr6.6/lE344 larvae showed the presence of proliferation centers at late-second and mid-third instar. Optic lobe proliferation centers were present in the vr6. 6/IE344 second instar larvae, with both an inner and outer proliferation center developing as bands of columnar cells (White, K. and Kankel, D.R. , Dev . Biol . , 65:296-321 (1978)). In the mutants the proliferation centers were seen to develop until third instar larval stage but failed to produce normal optic lobes in prepupae. These results suggest that growth of the optic lobes is initiated, but subsequently becomes abnormal during the third instar in these genotypes.

Cell division patterns in mutant latheo larvae

The abnormal appearance of proliferating tissue in the mutants suggested that cell division may be altered-in these larvae. Thus, mitotic activity was monitored immunohistochemically via incorporation of 5-bromo-2'- deoxyuridine (BUdR) into the DNA of dividing cells in the CNS's of second or third instar larvae. A single representative of the "discs absent" (vr6.6/IE344 ) and "discs confluent" (IE49/IE49) classes of latheo lethal alleles were used in these experiments.

In wild type early-third instar, a 1.5 hour BUdR pulse led to staining throughout the optic lobes (White, K. and Kankel, D.R., Dev. Biol . , 65:296-321 (1978); Truman, J.W. and Bate, M. , Dev. Biol . , 125:145-157 (1988)). In late third instar larvae, BUdR staining became prominent in the optic lobes with highly organized rings of dividing cells arising from cell division in the two proliferation centers (Selleck, S.B. and Stellar, H. , Neuron, 6:83-99 (1991)).

The nervous systems of early-third instar vr6.6/IE344 and IE49/IE49 larvae showed a staining pattern similar to control larvae of the same age. Both genotypes demonstrated strong superficial staining of the optic lobe. During late-third instar, however, the pattern of mitotic activity changed dramatically in the mutant animals. BUdR immunoreactivity in IE49/IE49 late-third instar nervous systems failed to show the "ring" pattern seen in wild-type optic lobes. In contrast, the staining pattern was reduced to scattered patches of cells. Alterations in BUdR incorporated was even more dramatic in vr6.6/IE344 where no BUdR staining was observed in the late-third instar. Thus in vr6.6/IE344 and IE49/IE49 larvae, mitotic activity showed a decline and loss of organization by late-third instar, respectively.

Neuronal differentiation in mutant latheo larvae

The altered histological appearance and mitotic activity of the latheo mutant larvae suggested that the number of terminally differentiated, non-dividing cells may be altered in the CNS of these animals. To visualize differentiated neurons in the larval CNS, sections were stained with anti-ELAV antibodies. The ELAV protein is expressed ubiquitously in neurons and it is not found in glia, neuroblasts or ganglion mother cells (Robinow, S. and White, K., J. Neurobiol . , 22:443-461 (1991)).

As expected, in wild-type animals nuclear staining was seen throughout the central brain and optic lobes along with the neurons of the developing eye imaginal discs (Robinow, S. and White, K. , J. Neurobiol . , 22:443-461 (1991)). In addition, no staining was seen in the proliferation centers of the optic lobes since those areas are comprised of ganglion mother cells and neuroblasts. The staining patterns in IE49/IE49 and vr6 . 6/IE344 larvae were markedly different. In vr6.6/IE344 animals, ELAV immunoreactive cells comprise a larger proportion of the CNS. Very few cells along the border of the CNS remain unstained in this genotype indicating a reduction of nonneuronal cells. The IE49/IE49 larvae, on the other hand, have large areas of in stained cells. Given the disorganized BUdR staining pattern in the CNS of IE49/IE49 larvae, the unstained cells here are likely to reflect an increased number of dividing cells. These groups of putatively dividing cells are clumped throughout the CNS including the anterior portion where neuroblasts are absent in controls.

Volume of mushroom bodies and central complex in latheo mutant flies

Two regions of the adult brain have been implicated in fly learning and memory; the mushroom bodies and the central complex (Tully, T. , (1994); Davis, R.L. , Neuron, 11:1-14 (1993): Heisenberg, Progreεε in Zoology Vol37, Fundamentalε of memory Formation : Neuronal Plaεticity and Brain Function , edited by G. Rahman, Fischer Verlag,- Stuttgart, pp.1-45, (1989)). The mushroom bodies in flies are clusters of cells located in each brain hemisphere which receive olfactory information (Heisenberg, M.A. , Dev . and Biol . of Droεoph . , O.Siddiqi, P. Babu, L.M. Hall and J.C. hall eds., Plenum, NY, pp. 337-390 (1980)). The central complex is a neuropilar region composed of the ellipsoid body, fan-shaped body, noduli and the protocerbral bridge (Heisenberg, M.A. , J. Neurogenet . , 2 : 1- 30 (1985)). To determine if these regions were normal in latheo mutant flies, heads of latheo" homozygous adult flies were examined in frontal paraffin sections. As a more severe yet viable genotype, latheo" /vg 56 heterozygous flies were examined as well since vg56 , as a deficiency, is an amorphic allele of latheo . To quantify any size differences, the areas of the mushroom body calyces, central complex, and medulla were measured in 7 μ thick serial sections and the total volume of the structure was calculated. There was a significant reduction in the size of the mushroom body calyx neurophile in latheo" /latheo" flies relative to control latheo" /+flieε . The central complex and medulla, however, were not significantly different from controls. In contrast, latheo" /vg 56 flies demonstrate a larger reduction in the mushroom bodies and a significant reduction in the volume of the central complex. The more pronounced mushroom body reduction in latheo" /vg56 flies is consistent with previous indication that the latheo" allele is hypomorphic (Boynton, S and Tully, T., Geneticε, 131:655-672 (1992)). The medulla was of normal size in all of the genotypes examined indicating that the reductions in volume demonstrated do not reflect an overall reduction in brain size.

DISCUSSION Deyelopmental and morphological analysis of lethal latheo mutants suggest a role for this locus in development of adult structures. Mutant latheo flies die during the pupal stage, most likely from abnormal development of the CNS and imaginal discs. BUdR incorporation and ELAV immunoreactivity suggests that the altered development in the CNS reflects improper cell proliferation and differentiation. In viable alleles, the developmental abnormalities were detected in the mushroom bodies and central complex which were reduced in volume.

Different combinations of latheo lethal alleles lead to either reduction or disorganization of the CNS. In vr6 . 6/IE344 , IE344/IE344, vr6.6/vr6 . 6 and IE344 /vr6 .35 nervous systems, the growth associated with adult regions such as the optic lobes appears to be absent, leaving these brains smaller in size than wild-type. The nervous system of IE49/IE49 and IE344/IE49 prepupae, although larger in size, also show little development of the optic lobes along with a generalized disorganization in histological sections.

In contrast to the adult viable genotypes which have a normal medulla volume, the nervous system defect in latheo lethal mutants is most pronounced in the optic lobes.

Reduced development of the optic lobes has been correlated with poor development of the eye (Fischbach, K.F., et al . , Dev Biol . , 95:1-18 (1983); Fischbach, K.F. and Technau, G. , Dev . Biol . , 104 : 219-239 (1984); Selleck, S.B., et al . , Nature, 355:253-255 (1992)). Mosaic analysis of optic lobe formation has indicated that development of the optic lobe is dependent on the genotype of the eye tissue innervating it (Meyerowitz, E.M. and Kankel, D.R., Dev. Biol . , 62:112- 142 (1978)). Nonetheless, eyeless εine oculiε mutants, possess a recognizable medulla and lobula complex -

(Fishbach, K.F., et al . , Dev Biol . , 95:1-18 (1983)). Thus the absence of these regions of neuropile in latheo lethal alleles cannot be accounted for solely by the absence of developing eyes. Instead, the abnormalities apparent in the latheo lethal alleles may be correlated with mitotic activity in the nervous system. In vr6. 6/IE344 larvae, DNA replication appears to occur normally in early-third instar larvae but is absent by the late-third instar. The loss of BUdR incorporation correlates with the loss of proliferation centers in this genotype suggesting that DNA replication and cell division has ceased prematurely.

Altered development also is evidenced by the staining patterns seen using anti-ELAV antibodies. The increase in nonELAV immunoreactive cells in IE49/IE49 animals and its decrease in vr6.6/IE344 animals suggests that neuronal proliferation is improperly controlled in these genotypes leading to differences in the numbers of differentiated neurons. It should be noted, however, that glia also are not ELAV immunoreactive. Thus, it remains possible that the unstained regions indicate a change in proliferation of glia as opposed to a change in the number of dividing neuroblasts. In either case, however, the large number of cells which are not ELAV immunoreactive in IE49/IE49 larvae, suggests that many cells fail to differentiate into neurons, precluding the development of optic lobe neuropilar regions.

The adult-specific epidermal structures (e.g., eyes, wings) also exhibit altered development which was dependent on the alleles examined. In IE344/vr6.6, IE344/IE344, vr6. 6/vr6. 6 and IE344/vr6.35 animals, these structures are absent by the time of transition from larva to pupa whereas in IE49/IE49 and IE344/IE49 pupae they are disorganized. The similarity of the phenotype in both the CNS and the epidermis suggests both are caused by a defect in cell proliferation or differentiation. Histological sections of earlier larval stages indicate that some putative imaginal disc cells are present until early-third instar in vr6.6/IE344 larvae. The apparent lack of these same cells at the time of pupal formation may suggest degeneration of this tissue as well.

In both the epidermis and the CNS of latheo lethal animals, the early larval counterparts appear to have developed normally. Apparently, the putative cell proliferation phenotype is rescued in the embryo by maternal effects or the latheo locus is not necessary for development larval structures. The epidermis of the adult is distinct from that of the larvae and is generated from a cluster of cells detectable during embryogenesis (Bate, M. and Arias, A.M., Development, 112:755-761 (1991)).

Thirteen to fifteen hours after hatching, these imaginal disc precursor cells begin divisions and continue to proliferate during larval life (Madhavan, M.M. and Schneiderman, H.A., W. Roux'ε Archives, 183:269-305 (1977)). AS for the CNS, the precursors of the adult brain are arrested during embryogenesis and then resume cell division during the larval period (Truman, J.W. , J. Neurobiol . , 21:1072-1084 (1990); Truman, J.W. and Bate, M. , Dev. Biol . , 125:145-157 (1988); White, K. and Kankel, D.R., Dev. Biol . , 65:296-321 (1978)). Following pupation, some larval neurons are degenerated (Kimura, K.I. and Truman, J.W. , J . Neurosci . , 10:403-411 (1990)) while others appear to be maintained and join with the newly differentiated "adult" neurons to produce the nervous system of the imago (Valles, A.M. and White, K. , J . Comp . Neurol . , 286:414-428 91988); Budnick, V. and White, K. , J . Comp. Neurol . , 268:400-413 91988); Technau, G.M. and Heisneberg, M. , Nature, 295:405-407 (1982)).

Taken together, the developmental analyses suggest that in latheo lethal alleles, larval development i& initiated normally. At least some precursors of adult- specific cells are generated, and these larvae seem to be normal in appearance. With time, however, the precursors of adult-specific structures fail to develop into the appropriate tissues. If the results of the nervous system mitotic activity can be generalized, it would appear that the growth of imaginal tissue is halted by a failure to continue cell division at all ( IE344/vr6 . 6) or in an organized manner (IE49/IE49) . Finally, without the proper development of adult structure, the animals die in early pupation.

The morphological phenotype of the latheo lethal alleles is similar to a class of mutations affecting pupal development. Selection of mutations which die during the larval-pupal transition have identified several mutant strains which lead to abnormalities in imaginal disc (Stewart, M. , et al . , Dev . Biol . , 27:71-83 (1972);Kiss, I., et al . , Theor. Appl . Genet . , 48:217-226 (1976); Kiss, I., et al., Genetics, 164:77-83 (1978) ;Murphy, C, et al . , Cell Diff . , 6:319-330 (1977)) and nervous system (Gateff, E. , et al . , W . Roux Arch., 176:23-65 (1974); Datta, S., et al . , Genetics, 130:523-537 (1992)) development. The disc and nervous system appearance of the IE344, vr6.6 and vr6.35 alleles is most similar to the "disc degenerate" class of mutations, and the disorganization of IE49 is similar to "discs large" (Stewart, M. , et al . , Dev . Biol . , 27:71-83 (1972)). Several of the late larval lethal mutations which affect disc morphology including the 1(1) discs deg, 1(1) discs large and 1(1) discless mutations are also known to affect cell cycle (Gatti, M. , et al . , Genes dev . , 3:438- 453 (1989); Woods, D.F., et al . , Neurophyεiol . , 27:61-69 (1989)). In fact, Gatti and Baker propose that late lethality and poorly developed imaginal discs is diagnostic of cell cycle mutations.

In ^contrast to the lethal alleles of latheo , only subtle anatomical defects were seen in the adult viable mutants. Preliminary examination had revealed no gross morphological defects in the brains of adult latheo flies (Boynton, S and Tully, T., Genetics, 131 : 655-672 (1992)). Planimetric analysis indicated a reduction in the size of the mushroom body calyx in latheo homozygous flies and a reduction of both the mushroom body calyx and the central complex in latheo /vg56 flies. The volume of the medulla, however, was found to be normal in both genotypes. The normal medulla size is surprising given the alterations in the optic lobes of the lethal mutants. Apart from the longer proliferation period of the mushroom body neuroblasts (Itto, K, et al . , Dev . Biol . , 149:134-148 (1992) which may lead to an enhanced sensitivity to cell cycle disruption in the mushroom bodies, it remains unclear as to why the medulla does not demonstrate a similar reduction in volume.

Previous studies indicated the two structures reduced in the adult viable latheo mutants, the mushroom bodies and central complex, to be relevant to Drosophila behavior, whereas mutations which affect other brain regions such as the optic lobes perform normally in learning tests (Heisenberg, M.A. , J. Neurogenet . , 2:1-30 (1985); Heisenberg, K. A. Progreεε in Zoology Vol37, Fundamentals of memory Formation : Neuronal Plaεticity and Brain Function , edited by G. Rahman, Fischer Verlag, Stuttgart, pp.1-45, (1989)). The mushroom bodies, in particular, appear to be important for normal olfactory learning (Davis, R.L., Neuron, 11:1-14 (1993)). The mushroom body mutations, mushroom bodies deranged and muεhroom bodies miniature have been shown to reduce olfactory learning (Heisenberg, M.A. Progreεs in Zoology Vol37, Fundamentals of memory Formation : Neuronal Plaεticity and Brain Function , edited by G. Rahman, Fischer Verlag, Stuttgart, pp.1-45, (-1989)). Furthermore, two of the existing learning mutants, dnc and rutabaga (rut) have been shown to have enhanced gene expression in this region (Nighorn, A., et al . , Neuron, 6:455-467 (1991); Han, P. et al . , Neuron, 9:619-627 (1992)). The Drosophila evidence corroborates that found in bees. Cooling of the mushroom bodies in bees leads to a reduction in memory of olfactory conditioning (Erber, J., et al . , Physiol . Entemol . , 5:343-358 (1980)) and at least one neuron which mediates olfactory learning is known to arbortize in this region (Hammer, M. , Nature, 366:59-63 (1993) ) .

Subtle neuroanatomical defects of the mushroom bodies have been demonstrated in the learning mutants dnc and rut . Both of these mutations show abnormal development of the mushroom body cells known as Kenyon fibers (Balling, A. , et al . , J . Neurogenet . , 4 : 65-73 (1987)). During the first few days following wild-type emergence, Kenyon cell fiber number increases. In wild-type flies, a reduction in Kenyon cell fibers occurs following isolation and sensory deprivation (Technau, G.M., J. Neurogenet., 1:113-126 (1984)). In contrast, the number of Kenyon cell fibers are abnormally high at emergence and subsequently decline in dnc flies, while the fiber number fails to increase in rut flies. Furthermore, both dnc and rut flies fail to show the experience-dependent modification of fiber number seen in wild-type flies (Balling, A., et al . , J . Neurogenet . ,4:65-73 (1987)). Outside of the CNS, these mutations appear to affect the development of sensory and motor pathways. Motor neurons of dnc and sensory neurons of both dnc and rut show increased branching and varicosities (Zhong, Y., et al . , Neurophysiol . , 27:61-69 (1989); Corfas, G., et al., PNAS, USA, 88:7252-7256 (1991)). Mutations which disrupt the central complex ine-luding the ellipsoid body open, central complex deranged, central complex broad and no-bridge, also have been shown to reduce olfactory learning (Heisenberg, M.A. Progreεε in Zoology Vol37, Fundamentalε of memory Formation : Neuronal Plaεticity and Brain Function , edited by G. Rahman, Fischer

Verlag, Stuttgart, pp.1-45, (1989)). Furthermore, the central complex is implicated in learning via dunce protein expression being found in this region as well as in the mushroom bodies (Nighorn, A., et al . , Neuron, 6:455-467 (1991)). More recently, however, the central complex has been shown to have a strong involvement in locomotor activity (Strauss and Heisneberg, 1993) . The behavioral analysis of latheo flies indicated a learning defect in both latheo/latheo and in latheo/vg65 whereas latheo/vg56 demonstrated a locomotor defect as well (Boynton, S and Tully, T. , Genetics, 131 : 655-672 (1992)). Thus, it is possible that the central complex reduction which is found only in latheo/vg56 is responsible for the locomotor defect in this genotype while the mushroom body defect found in both genotypes leads to the reductions in learning. It must be noted, however, that normal size of the central complex in latheo flies does not preclude defects in the circuitry of the central complex in this genotype. It remains possible that a finer level of analysis might indicate defects in the central complex of latheo" flies which could play a role in the learning defect.

Several single gene mutations have shown defects in both learning behavior and neuroanato y (Tully, 1994) . In fyn mutant mice for example, a knockout mutation of the fyn tyrosine kinase gene leads to mice which are not only defective in behavioral learning assays and LTP but also in the development of the hippocampus (grant, et al . , Science, 258:1903-1910 (1992)). In such cases, the relation between the two phenotypes is unclear. Although the anatomical defect may lead to reductions in learning , it is also possible that the two phenotypes are induced independently through pleiotropic effects of the mutated genes of that the anatomical defects arise as a secondary effect on cellular function. Further molecular and physiological analysis on latheo may allow distinctions to be made between these possibilities. Example 3 Identification of the human homolog of the latheo protein A homology search for the latheo was performed using the Netblast program. An EST cDNA clone 50150(5') accession #H17704 was identified which encodes human latheo.

Whole brain from 73 days post natal female was used. First strand cDNA was primed with a Notl-oligo (dT) primer [ 5 'AACTGGAAGAATTCGCGGCCGCAGGAATTTTTTTTTTTTTTTTTT 3 ' ] (SEQ ID NO: 10); double stranded cDNA was ligated to HIND III adaptors (Pharmacia) , digested with NotI and directionally cloned into the NotI and Hindlll sites of the Lafmid BA vector. Library went through one round of normalization. An initial reading of the human latheo nucleotide sequence (SEQ ID NO: 7) is shown in Figure 8. Figures 9A- 9B are a comparison of the initial reading of the fly latheo amino acid sequence (SEQ ID NO: 5) and a portion of the initial reading of the human latheo amino acid sequence (SEQ ID NO: 8) . However, a subsequent reading of the human latheo nucleotide sequence (SEQ ID NO: 3) resulted in changes in the human latheo amino acid sequence (SEQ ID NO: 4).

Example 4 Latheo physically interacts with Tyrosine hydroxylase, implicating its role in regulation of dopamine synthesis

As a means of identifying unknown components of the latheo pathway, a yeast two-hybrid genetic screen was performed using a LAT bait hybrid to isolate LATHEO- interacting proteins. The latheo open reading frame (894-2881) was cloned into the LexA vector. The L40 reporter expressing the LexA-LAT bait hybrid was transformed with a Drosophila adult cDNA library as well as with a third instar larva library. Transformants were screened for activation of the HISH3 and 0-galactosidase reporter genes. Library plasmids were purified from the Hist β-gal+ colonies. A total of four independent plasmids that were found to interact with LAT were determined by sequence analysis to encode the Drosophila Tyrosine Hydroxylase (TH) .

To test reproducibility and specificity, the library plasmid encoding TH was introduced back into yeast expressing either an original LexA-LAT bait or a negative control LexA-lamin bait. The LexA-Lamin exhibited no detectable histidine or /3-galactosidase activity after transformation. In contrast, the LexA-LAT strain exhibited histidine and 0-galactosidase activity when transformed with the TH clone.

Preliminary analysis of the region of LAT involved in LAT-TH interaction revealed that the carboxyl-half of the LAT protein (1744-3250) is sufficient for the interaction with TH in the two-hybrid assay.

A direct association between LAT and TH was demonstrated in vitro . Glutathione-S-transferase (GST)-TH fusion protein or GST alone were expressed in bacteria and purified on glutathione-agarose beads. The purified proteins were analyzed by SDS polyacrylamide gel electrophoresis (SDS-PAGE) . The purified GST-TH fusion or GST were incubated with an in vitro-translated ³⁵S-labeled LAT. An autoradiograph of SDS-PAGE analysis showed-that ³⁵S-labeled LAt binds to GST-TH but not to GST alone.

The in vitro analysis further corroborates the yeast- two-hybrid results showing the interaction between TH and LAT and demonstrates that the interaction between LAT and TH is a direct interaction. EOUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

Claims

CLAIMS We claim:

1. Isolated or recombinant nucleic acid which encodes a latheo protein.

2. The nucleic acid of Claim 1 wherein the nucleic acid is selected from the group consisting of: nucleic acid encoding human latheo protein and nucleic acid encoding fly latheo protein.

3. A nucleic acid of Claim 2 which comprises a nucleotide sequence selected from the group consisting of: SEQ ID

NO: 1, SEQ ID NO: 3, complements thereof and functional portions thereof.

4. A nucleic acid of Claim 1 which hybridizes under stringent conditions with a second nucleic acid having a nucleotide sequence selected from the group consisting of: SEQ ID NO: 1, a complement of SEQ ID NO: 1, SEQ ID NO: 3, a complement of SEQ ID NO: 3, SEQ ID NO: 5, a complement of SEQ ID NO: 5, SEQ ID NO: 7, a complement of SEQ ID NO: 7 and functional portions thereof.

5. A nucleic acid comprising a nucleotide sequence selected from the group consisting of: SEQ ID NO: 1, complements of SEQ ID NO:l, SEQ ID NO: 3, complements of SEQ ID NO: 3 and functional portions thereof.

6. A nucleic acid encoding a protein, wherein the protein comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4 and functional portions thereof.

7. A nucleic acid which hybridizes under moderate stringency conditions to DNA selected from the group consisting of: SEQ ID NO: 1, complements of SEQ ID NO:l, SEQ ID NO: 3, complements of SEQ ID NO: 3 and functional portions thereof.

8. A nucleic acid construct comprising nucleic acid of Claim 1.

9. An nucleic acid construct comprising nucleic acid selected from the group consisting of: SEQ ID NO: 1, SEQ ID NO: 3 and functional portions thereof.

10. The nucleic acid construct of Claim 8 wherein the nucleic acid is operably linked to an expression control sequence.

11. A host cell comprising the nucleic acid of Claim 1.

12. The host cell of Claim 11 wherein the nucleic acid is operably linked to an expression control sequence, whereby latheo protein is expressed when the host cell is maintained under conditions suitable for expression of latheo protein.

13. Isolated or recombinantly produced latheo protein.

14. The protein of Claim 13 wherein the protein is selected from the group consisting of: human latheo protein and fly latheo protein.

15. The protein of Claim 14 wherein the protein has an amino acid selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4 and functional portions thereof.

16. A protein comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4 and functional portions thereof.

17. An inhibitor of latheo protein.

18. The inhibitor of Claim 17 wherein the inhibitor is an antibody which binds latheo protein or a functional portion of latheo protein.

19. The antibody of Claim 18 which binds a protein wherein the amino acid sequence of the protein is selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4 and functional portions thereof.

20. The antibody of Claim 19 wherein the antibody is selected from the group consisting of: monoclonal antibodies, chimeric antibodies and humanized antibodies.

21. A method of detecting latheo nucleic acid in a sample obtained from an individual, comprising the steps of: a) treating the sample to render nucleic acids in the sample available for hybridization to a- nucleic acid probe, thereby producing a treated sample; b) combining the treated sample with a labeled nucleic acid probe comprising all or a functional portion of the nucleotide sequence of latheo protein, under conditions appropriate for hybridization of complementary nucleic acids; and c) detecting hybridization of the treated sample with the labeled nucleic acid probe, wherein hybridization indicates the presence of latheo nucleic acid in the sample.

22. The method of Claim 21 wherein the nucleic acid probe comprises DNA selected from the group consisting of:

SEQ ID NO: 1, SEQ ID NO: 3 and functional portions thereof.

23. A method of Claim 22 wherein the sample is selected from the group consisting of: blood and cerebral spinal fluid.

24. A method of detecting latheo protein in a sample obtained from an individual, comprising the steps of: a) combining the sample with an antibody which binds latheo protein or a functional portion of latheo protein; and b) detecting binding of the antibody to a component of the sample, wherein binding of the antibody to a component of the sample indicates the presence of latheo protein in the sample.

25. The method of Claim 24 wherein the antibody binds a protein comprising an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, SEQ ID NO: 4 and functional portions thereof.

26. A method of modulating a dopamine level in a mammal, comprising administering to the mammal an agent which interacts with latheo protein wherein the interaction between latheo and tyrosine hyroxylase is altered, thereby modulating the dopamine level in the mammal.

27. Use of an agent which interacts with latheo protein to modulate a dopamine level in a mammal, wherein the interaction between latheo and tyrosine hydroxylase is altered thereby modulating the dopamine level in the mamma1.