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WO1998010067A1 - Homologue d'origine humaine du gene latheo - Google Patents

Homologue d'origine humaine du gene latheo Download PDF

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Publication number
WO1998010067A1
WO1998010067A1 PCT/US1997/015134 US9715134W WO9810067A1 WO 1998010067 A1 WO1998010067 A1 WO 1998010067A1 US 9715134 W US9715134 W US 9715134W WO 9810067 A1 WO9810067 A1 WO 9810067A1
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WO
WIPO (PCT)
Prior art keywords
latheo
protein
seq
nucleic acid
lat
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PCT/US1997/015134
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English (en)
Inventor
Timothy Tully
Robert M. Mihalek, Jr.
Christopher Jones
Shirley Pinto
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Cold Spring Harbor Laboratory
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Publication date
Application filed by Cold Spring Harbor Laboratory filed Critical Cold Spring Harbor Laboratory
Priority to CA002264493A priority Critical patent/CA2264493A1/fr
Priority to EP97940656A priority patent/EP0941325A1/fr
Publication of WO1998010067A1 publication Critical patent/WO1998010067A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43563—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects
    • C07K14/43577—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies
    • C07K14/43581—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from insects from flies from Drosophila
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • 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
  • 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.
  • 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.
  • a latheo protein e.g., SEQ ID NO: 1, SEQ ID NO: 3 or characteristic portions thereof
  • 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) .
  • 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.
  • 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.
  • an agent which interacts with latheo directly or indirectly, and inhibits or enhances latheo function is an agent which interacts with latheo directly (e.g., by binding latheo) or indirectly (e.g., by blocking the ability of latheo to interact with tyrosine hydroxylase) .
  • 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.
  • 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.
  • the inhibitor can be an agent other than an antibody (e.g., small organic molecule, protein, peptide) which binds latheo and blocks its activity.
  • the inhibitor can be an agent which mimics latheo structurally but lacks its function.
  • 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.
  • 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.
  • 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) .
  • a sample e.g., blood, cerebral spinal fluid
  • 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) .
  • 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.
  • 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.
  • 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) .
  • the present invention relates to an isolated (e.g. , purified, essentially pure) latheo gene which is involved in associative learning and memory.
  • 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 .
  • 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.
  • 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) .
  • 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) .
  • 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.
  • 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:
  • latheo e.g. human
  • 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 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.
  • 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) .
  • the nucleic acid encodes a polypeptide which retains substantially the same biological function or activity as the polypeptide encoded by the DNA of Figures
  • Nucleic acids of the present invention can be used in the production of proteins or polypeptides.
  • a nucleic acid e.g., DNA
  • a nucleic acid 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).
  • antisense nucleic acid 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.
  • the antisense nucleic acid is wholly or partially complementary to and can hybridize with a target nucleic acid which encodes a latheo protein.
  • 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) .
  • a sample e.g., blood, cerebral spinal fluid
  • a host e.g. mammalian, particularly human
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • an affinity ligand e.g., an enzyme, an antigen, epitope tag
  • 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.
  • 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 .
  • subtili ⁇ and or other suitable bacteria e.g., Streptococci
  • 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
  • Host cells which produce a recombinant latheo protein or variants thereof can be produced as follows.
  • 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 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.
  • 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) .
  • 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.
  • suitable promoters for procaryotic e.g., lac, tae, T3, T7 promoters for E. coli
  • eucaryotic e.g., yeast alcohol dehydrogenase (ADHl) , SV40, CMV
  • 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 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) .
  • 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
  • Fusion proteins can also be produced in this manner.
  • 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.
  • 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.
  • affinity labels provide a means of detecting a fusion protein.
  • 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.
  • latheo nucleic acids and protein can be used in a variety of ways.
  • latheo nucleic acids and proteins can be used to identify agents (e.g., molecules) that modulate (enchance, inhibit) latheo expression and/or function.
  • agents e.g., molecules
  • 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.
  • an agent which interacts with latheo directly or indirectly, and inhibits or enhances latheo function is an agent which interacts with latheo directly (e.g., by binding latheo) or indirectly (e.g., by blocking the ability of latheo to interact with dopamine) .
  • 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.
  • 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.
  • the inhibitor can be an agent other than an antibody (e.g., small organic molecule, protein, peptide) which binds latheo and blocks its activity.
  • the inhibitor can be an agent which mimics latheo structurally but lacks its function.
  • 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.
  • 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.
  • sequences described herein can be used to detect latheo or DNA encoding latheo in a sample.
  • 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.
  • 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.
  • 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.
  • the sample can be blood, cerebrospinal fluid (CSF) or tissue from a human.
  • CSF cerebrospinal fluid
  • 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.
  • latheo physically interacts with tyrosine hydroxylase (TH) , an enzyme involved in the synthesis of catecholamines such as dopamine.
  • TH tyrosine hydroxylase
  • 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.
  • a host e.g., mammal, particularly human
  • 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) .
  • a condition or disease associated with dopamine malfunction e.g., Parkinson's Disease, schizophrenia, and depression
  • 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 lat pl and lat 1 ⁇ 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 melanoga ⁇ ter, San
  • 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 p ⁇ ; 7a, 7b; lat pl , and 8; lat pl respectively.
  • 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 CaCl 2 , 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 melanoga ⁇ ter. 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.
  • 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 3 P- 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 ⁇ GEMll 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.
  • 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 ⁇ gtll was screened using various subcloned genomic fragments (Fig.
  • RNA isolation 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.
  • Chomczynski reagent TRIzol GibcoBRL
  • All other RNA was prepared following the original reference.
  • 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.
  • 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.
  • RNA:RNA hybridizations were performed in 50% formamide; 270 mM NaCl; 20 mM Na 2 HP0 4 ; 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 Na 2 HP0 4 , pH 7.2; 15% formamide; lmM EDTA; 7% SDS. Probes were present at a specific activity of 5 x 10 6 c. p.m. /ml.
  • 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%.
  • Wild-type sections 7 ⁇ m 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.
  • 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 (pUChsp ⁇ 2-3wc) , 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.
  • hydrocarbon oil Alocarbon Products, series 700
  • 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.
  • 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) .
  • 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 w 9 ' 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.
  • the X-linked transgene homozygous, it was first balanced over FM7a .
  • FM7a By crossing hs- lat* /w;CyO/Sp;TM3 (Ser) /Sb with FM7a/y;+;+, h ⁇ - lat*/FM7a;CyO/+;TM3 (Ser) /+ and hs-lat* /Y;Sp/+ ; +/+ flies were recovered. These flies were crossed together and h ⁇ - lat* /FM7a;CyO/Sp; +/+ recovered.
  • FM7a/Y;CyO/lat PI ; +/+ were made and crossed with hs- lat*/FM7a;CyO/Sp;+/+ .
  • Both hs-lat* /FM7a and h ⁇ - lat*/Y;CyO/lat pl ;+/+ were recovered and crossed together to yield h ⁇ -lat* /h ⁇ -lat* ;lat pl ;+.
  • h ⁇ -lat* 4b X-linked
  • h ⁇ -lat*7a 3rd chromosome
  • the third chromosome linked hs-lat*7a was combined with lat* by crossing lat pl / ;at p ⁇ ;h ⁇ -lat*7a/h ⁇ - lat*7a with +/CyO;+/TM3 and recovering females of the genotype +/lat P1 ;h ⁇ -lat*7a/TM3 . These were crossed together and +/+; h ⁇ -lat* 7 a h ⁇ -lat*7a recovered.
  • 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.
  • 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.
  • 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.
  • 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 ⁇ itu 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 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.
  • 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.
  • RNA transcripts near the lat p ⁇ 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.
  • 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)).
  • 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
  • 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.
  • 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.
  • Anti-sense RNA probes generated by T3 and T7 polymerase transcription reactions using a cDNA subclone in pKS were used to probe 7 ⁇ m 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)).
  • 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.
  • 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.
  • 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.
  • the lethal phenotype and the behavioral phenotype may be functionally distinct aspects of lat* gene function.
  • RNA probes generated from the cDNA subclone were hybridized to 7 ⁇ 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.
  • 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" .
  • Example 2 Developmental effects of mutations in the learning gene latheo METHODS stocks
  • the latheo" P element insertion strain and the excision lethal alleles latheo 1 " 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. Genetic ⁇ , 131:655-672 (1992)).
  • the vr6.6 and vr6.35 mutations were isolated in an ethylmethane sulfonate mutagenesis for lethals in the ve ⁇ tigal region (Lasko, P.F. and Pardue, M.L., Genetics, 120:495-502 (1988)).
  • (2LR) Gla which carries the dominant larval marker Black cell (Be) .
  • Wild-type chromosomes were derived from w (c ⁇ 4) (Boynton, S. and Tully, T. Genetic ⁇ , 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 CaCl 2 , 76.1 g/L cornmeal and 2 g/L Tegosept M mold inhibitor.
  • 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.
  • 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.
  • 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) .
  • 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
  • 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)).
  • mutant vr6.6, vr6.35, IE49 and IE344 flies were mated to each other and to a deficiency of the region, vg56.
  • 25% of the progeny are expected to bear lethal mutations of latheo on both chromosomes and hence die during the lethal period.
  • 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.
  • 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/Cy ⁇ , 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 .
  • 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 ' ⁇ 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.
  • the anterior imaginal discs were visible as large masses of cells in the appropriate imaginal disc locations.
  • 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)).
  • 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)).
  • IE49/IE49 and IE49/IE344 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.
  • 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.
  • 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)).
  • 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)).
  • heads of latheo" homozygous adult flies were examined in frontal paraffin sections.
  • latheo" /vg 56 heterozygous flies were examined as well since vg56 , as a deficiency, is an amorphic allele of latheo .
  • the areas of the mushroom body calyces, central complex, and medulla were measured in 7 ⁇ 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" /+flie ⁇ .
  • the central complex and medulla were not significantly different from controls.
  • latheo" /vg 56 flies demonstrate a larger reduction in the mushroom bodies and a significant reduction in the volume of the central complex.
  • 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 defect in latheo lethal mutants is most pronounced in the optic lobes.
  • the adult-specific epidermal structures (e.g., eyes, wings) also exhibit altered development which was dependent on the alleles examined.
  • 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.
  • 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)).
  • 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 .
  • 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.
  • mushroom body mutations mushroom bodies deranged and mu ⁇ hroom bodies miniature have been shown to reduce olfactory learning (Heisenberg, M.A. Progre ⁇ s in Zoology Vol37, Fundamentals of memory Formation : Neuronal Pla ⁇ ticity and Brain Function , edited by G. Rahman, Fischer Verlag, Stuttgart, pp.1-45, (-1989)).
  • 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) ) .
  • Kenyon cell fibers are abnormally high at emergence and subsequently decline in dnc flies, while the fiber number fails to increase in rut flies.
  • 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)).
  • 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 .
  • 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.
  • 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.
  • 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.
  • 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) .
  • 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
  • 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 ⁇ -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) .
  • TH Drosophila Tyrosine Hydroxylase
  • 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.
  • the LexA-LAT strain exhibited histidine and 0-galactosidase activity when transformed with the TH clone.
  • 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) .
  • SDS-PAGE SDS polyacrylamide gel electrophoresis
  • the purified GST-TH fusion or GST were incubated with an in vitro-translated 35 S-labeled LAT.
  • An autoradiograph of SDS-PAGE analysis showed-that 35 S-labeled LAt binds to GST-TH but not to GST alone.

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Abstract

La présente invention se rapporte à des acides nucléiques (ADN, ARN) isolés ou recombinés qui codent une protéine latheo. Les acides nucléiques de la présente invention incluent les acides nucléiques codant la protéine latheo humaine (SEQ ID NO:1), la protéine latheo de la mouche (SEQ ID NO:3) et des parties caractéristiques de ces protéines. Cette invention se rapporte également à une protéine latheo isolée ou produite par recombinaison. Ladite protéine latheo peut être par exemple une protéine latheo humaine (SEQ ID NO:4), une protéine latheo de mouche (SEQ ID NO:2) ou des parties fonctionnelles de ces protéines. L'invention se rapporte également à un agent qui agit directement ou indirectement avec la protéine latheo et inhibe ou renforce la fonction latheo. La présente invention se rapporte en outre à un procédé permettant de déceler la protéine latheo dans un échantillon (de sang ou de liquide céphalo-rachidien, par exemple) prélevé chez un individu (par exemple un sujet humain). La présente invention se rapporte enfin à un procédé de modulation des taux de dopamine chez un mammifère, qui consiste à administrer audit mammifère un agent qui agit avec la protéine latheo.
PCT/US1997/015134 1996-09-03 1997-08-28 Homologue d'origine humaine du gene latheo WO1998010067A1 (fr)

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Cited By (2)

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Publication number Priority date Publication date Assignee Title
WO2000060078A3 (fr) * 1999-04-01 2001-01-18 Cold Spring Harbor Lab Latheo codant une sous-unite de proteines complexes de reconnaissance d'origine
EP2333112A2 (fr) 2004-02-20 2011-06-15 Veridex, LLC Pronostics de cancer du sein

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000060078A3 (fr) * 1999-04-01 2001-01-18 Cold Spring Harbor Lab Latheo codant une sous-unite de proteines complexes de reconnaissance d'origine
EP2333112A2 (fr) 2004-02-20 2011-06-15 Veridex, LLC Pronostics de cancer du sein

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