WO2000020585A1 - Proteine mammifere timeless et ses procedes d'utilisation - Google Patents

Proteine mammifere timeless et ses procedes d'utilisation Download PDF

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WO2000020585A1
WO2000020585A1 PCT/US1999/022777 US9922777W WO0020585A1 WO 2000020585 A1 WO2000020585 A1 WO 2000020585A1 US 9922777 W US9922777 W US 9922777W WO 0020585 A1 WO0020585 A1 WO 0020585A1
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protein
seq
tim
mammalian
per
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PCT/US1999/022777
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WO2000020585A9 (fr
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Ashvin M. Sangoram
Joseph Takahashi
Michael W. Young
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The Rockefeller University
Northwestern University
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Publication of WO2000020585A1 publication Critical patent/WO2000020585A1/fr
Publication of WO2000020585A9 publication Critical patent/WO2000020585A9/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
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; CARE OF BIRDS, FISHES, INSECTS; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/07—Animals genetically altered by homologous recombination
    • A01K2217/075—Animals genetically altered by homologous recombination inducing loss of function, i.e. knock out
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • This invention relates to mammalian proteins involved in maintaining circadian rhythms.
  • the invention also relates to variants and mutants of the protein, nucleic acid and amino acid sequences encoding the purified proteins, as well as methods of using the proteins.
  • Patterns of activity with periodicities of approximately 24 hours are termed circadian rhythms, and are governed by an internal clock that functions autonomously, but can be entrained by environmental cycles of light or temperature. These behaviors can be entrained to a "zeitgeiber" (most commonly light), but are sustained under conditions of constant darkness and temperature, revealing activity of an endogenous biological clock. Circadian rhythms produced in constant darkness, for example, can also be reset by pulses of light. Such light pulses will shift the phase of the clock in different directions (advance or delay) and to varying degrees in a fashion that depends on the time of light exposure [Pittendrigh, in Handbook of Behavioral Neurobiology, 4, J. Aschoff, Ed., New York: Plenum, 1981, pp. 95- 124].
  • Circadian rhythms are a fundamental property of living systems and impose a 24-hour temporal organization regulating the physiology and biochemistry of most organisms [Pittendrigh, Annu. Rev. Physiol., 55:17-54 (1993)]. In mammals circadian rhythms are controlled by the suprachiasmatic nucleus (SCN) [Ralph et al, Science 247:975-978 (1990)]. These rhythms have been shown to be under the control of cellular pacemakers [Welsh et al, Neuron 14:697-706 (1995)] which, in turn, are under genetic control [Vitaterna et al, Science 264:719-725 (1994)].
  • SCN suprachiasmatic nucleus
  • PER protein fluctuates with a circadian rhythm [Edery et al Proc. Natl. Acad. Sci. U.S.A 91:2260 (1994)], the protein is phosphorylated with a circadian rhythm [Edery et al., Proc. Natl. Acad. Sci. U.S.A 91:2260 (1994)], and PER is observed in nuclei at night but not late in the day [Siwicki et al Neuron 1:141 (1988); Saez and Young Moi. Cell. Biol. 8:5378 (1988); Zerr et al J. Neurosci 10:2749 (1990)].
  • the expression of per RNA is also cyclic. However, peak mRNA amounts are present late in the day, and the smallest amounts are present late at night [Hardin et al,
  • PERIOD protein The per gene is expressed in many cell types at various stages of development. In most cell types, the PERIOD protein (PER) is found in nuclei [James et al EMBO J. 5:2313 (1986); Liu et al Genes Dev. 2:228 (1988); Saez and Young Moi. Cell. Biol. 8, 5378 (1988); Liu et al J. Neurosci. 12:2735 (1992) Siwicki et al Neuron 1:141 (1988); Zerr et al J. Neurosci. 10:2749 (1990); Edery et al Proc. Natl. Acad. Sci. U.S.A. 91:2260 (1994)].
  • a domain within PER is also found in the Drosophila single-minded protein (SIM) and in subunits of the mammalian aryl hydrocarbon receptor [Crews et al Cell 52:143 (1988); Hoffman et al Science 252:954 (1991); Burbach et al Proc. Natl. Acad. Sci. U.S.A. 89:8185 (1992); Reyes et al Science 256:1193 (1992)], and this domain (PAS, for PER, ARNT, and SIM) mediates dimerization of PER [Huang et al Nature 364:259 (1993)].
  • SIM Drosophila single-minded protein
  • Timeless is a second gene which has been associated with circadian rhythms in Drosophila [U.S. Patent Application No. 08/619,198 filed March 21, 1996, hereby incorporated by reference in its entirety].
  • PER is unstable in the cytoplasm of Drosophila, in the absence of the TIMELESS protein, TIM. Upon binding to TIM in the cytoplasm, PER is stabilized and translocated into the nucleus. Once in the nucleus, PER acts to inhibit the production of its own RNA. Both the tim and per genes are transcribed cyclically in Drosophila, and this transcription drives behavior.
  • the TIM protein not only acts as a nuclear translocation factor for the PER protein, but the PER protein also serves as a nuclear translocation factor for the TIM protein, thus indicating that PER and TIM act as mutual and reciprocal nuclear translocation factors.
  • the nuclear translocation of the PER-TIM heterodimer is a crucial step in the regulation of both tim DNA and per DNA transcription in Drosophila.
  • the Drosophila TIM protein also plays an important role in entraining the circadian rhythm of Drosophila to environmental cycles of light. This property of the TIM protein is due to its requirement for stabilizing the Drosophila PER protein; its role in regulating per DNA transcription; and the TIM protein's extreme sensitivity to light. Unlike the PER protein which requires TIM for stability, the stability of the Drosophila TIM protein is independent of the PER protein.
  • the TIM and PER proteins affect expression of their own mRNAs and this activity requires nuclear entry of the two proteins [Hunter-Ensor et al, Cell 84:677-685 (1996); Lee et al., Science 271:1740-1744 (1996); Myers, et al, Science 271:1736-1740 (1996); Sehgal et al. Science 270:808-10 (1995)].
  • RNA and protein products of the genes oscillate with a circadian rhythm in wild-type flies.
  • a physical interaction of the PER protein and the TIM protein is required for nuclear localization of either protein, and nuclear activity of these proteins coordinately regulates per and tim transcription through a negative feedback loop [Sehgal et al, Science, 263:1603-1606 (1994); Vosshall et al, Science, 263: 1606-1609 (1994); Seghal et al, Science, 270:808-810 (1995); Gekakis ef al. Science, 270:811-815 (1995); Hunter-Ensor et al.
  • circadian rhythms in Drosophila require periodic interaction of the PERIOD (PER) and TIMELESS (TIM) proteins. Physical associations of PER and TIM allow their nuclear translocation, and autoregulation of per and tim transcription through a negative feedback loop.
  • PER PERIOD
  • TIMELESS TIMELESS
  • the CLOCK-BMAL1 complex transactivates the mPerl promoter specifically via E-box elements contained within the first 1.2 kb upstream of the gene [Gekakis, et al, Science 280:1564-1569 (1998)].
  • the dCLOCK-dBMAL complex therefore became a candidate for being the hypothetical transcription factor in the PER/TIM system, that had appeared to be necessary to mediate repression in response to nuclear PER/TIM complexes, since neither PER nor TIM has a recognizable DNA-binding motif [(reviewed by Rosbash ef al, Harb. Symp. Quant. Biol, 76:265-278 (1996); Young ef al, Harb. Symp. Quant. Biol, 61:279-284 (1996)].
  • the dCLOCK-dBMAL complex was shown to activate the transcription of both per and tim through E-box elements found in their respective promoters [Darlington ef al, Science 280:1599-1603 (1998)].
  • dCLOCK-dBMAL defined a critical site for both positive and negative regulation of the circadian cycle in Drosophila and in addition appears to have completed the identification of the four major factors involved in circadian rhythm in Drosophila.
  • the identification of all of the corresponding factors involved in circadian rhythm in mammals has remained elusive.
  • the present invention discloses a mammalian protein TIMELESS (TIM), which is involved in circadian rhythms.
  • TIM mammalian protein
  • This protein is a mammalian orthologue of the Drosophila TIMELESS protein described in U.S. Patent Application Nos: 08/408,518, filed March 20, 1995 (now abandoned), 08/442,214, filed May 16, 1995 (now abandoned), 08/552,354, filed November 2, 1995 (now abandoned) and 08/619,198, Filed March 21, 1996 (co-pending) which are all hereby incorporated by reference in their entireties.
  • mammalian TIM plays a role in the nuclear transport of the gene product, PERIOD (PER) of the important clock gene, period ⁇ per). Therefore the present invention provides isolated nucleic acids and/or recombinant DNA molecules that encode mammalian TIM proteins and TIM fragments including nucleic acids encoding TIM chimeric and fusion peptides and proteins of the present invention. The present invention further provides the isolated and/or recombinant mammalian TIM proteins and TIM fragments, including chimeric and fusion peptides and proteins of mammalian TIM proteins and fragments. In addition, the present invention provides antibodies to mammalian TIM proteins and TIM fragments. Methods of using these nucleic acids, proteins, protein fragments, and antibodies, including as reagents for drug screening and therapeutics, are also provided.
  • nucleic acid that comprises a nucleotide sequence that encodes a mammalian TIM.
  • the nucleic acid comprises a nucleotide sequence that encodes a murine TIM.
  • the nucleic acid comprises a nucleotide sequence that encodes a bovine TIM.
  • the mammalian TIM is a human TIM.
  • the present invention provides an isolated nucleic acid encoding a mammalian orthologue of the Drosophila TIMELESS (TIM) protein in which the amino acid sequence of the orthologue has at least 30% identity with either or preferably both SEQ ID NO:2 and/or SEQ ID NO:20.
  • the orthologue has at least 50% identity with either and preferably both SEQ ID NO:2 and/or SEQ ID NO:20.
  • the orthologue has at least 70% identity with either or preferably both SEQ ID NO:2 and/or SEQ ID NO:20.
  • the orthologue has at least 80% identity with either or preferably both SEQ ID NO:2 and/or SEQ ID NO:20.
  • the ortholog has at least 90% and preferably 95% identity with either SEQ ID NO:2 or SEQ ID NO:20.
  • a nucleic acid of the present invention that encodes a mammalian TIM when performed in Drosophila (S2) cells preferably can promote the entry of the Drosophila PER protein into the nucleus.
  • S2 Drosophila
  • a nucleic acid of the present invention that encodes a mammalian TIM protein binds the Drosophila PERIOD (PER) protein in vitro.
  • a nucleic acid of the present invention that encodes a mammalian TIM protein binds mouse PERI and PER2 in vitro.
  • a nucleic acid of the present invention also preferably encodes a mammalian TIM protein that comprises a PER binding domain and a cytoplasmic localization domain (CLD). More preferably the CLD comprises an acidic tetrapeptide at or near the C-Terminal end of the CLD. Even more preferably the acidic tetrapeptide is DEDD (SEQ ID NO:27).
  • a nucleic acid of the present invention preferably encodes a mammalian TIM protein that comprises a GLU-ASP rich region. More preferably the GLU-ASP rich region contains thirteen consecutive glutamic acid residues.
  • a nucleic acid of the present invention also preferably encodes a mammalian TIM protein that comprises a nuclear localization sequence (NLS).
  • the nucleic acid encodes a mammalian TIM protein that comprises at least one, preferably two, more preferably three, and most preferably four regions which correspond to the TIM Homology regions (TH1, TH2, TH3, and TH4 as depicted in Figure 2) and which have at least 30%, preferably 50%, more preferably 70%, and even more preferably 80% identity with its corresponding region of SEQ ID NO:2.
  • the present invention further provides isolated and recombinant nucleic acids that encode all of the variants of the mammalian TIM proteins. Accordingly the present invention includes nucleic acids that encode variants of the human TIM protein.
  • the nucleic acid encodes a human TIM protein having the amino acid sequence of SEQ ID NO:2.
  • the nucleic acid encodes a human TIM protein having the amino acid sequence of SEQ ID NO:4, or SEQ ID NO:6, or SEQ ID NO:8, or SEQ ID NO: 10, or SEQ ID NO: 12, or SEQ ID NO: 14, or SEQ ID NO: 16.
  • the nucleic acid encodes a human TIM protein having the amino acid sequence of SEQ ID NO:2, or SEQ ID NO:4, or SEQ ID NO:6, or SEQ ID NO:8, or SEQ ID NO: 10, or SEQ ID NO: 12, or SEQ ID NO: 14, or SEQ ID NO: 16 that comprises a conservative amino acid substitution.
  • nucleotide sequence has a nucleotide sequence of SEQ ID NO:l.
  • nucleotide sequence has a nucleotide sequence of SEQ ID NO:3, or SEQ ID NO:5, or SEQ ID NO:7, or SEQ ID NO:9, or SEQ ID NO: 11, or SEQ ID NO:13, or SEQ ID NO:15, or SEQ ID NO:17, or SEQ ID NO:18.
  • the present invention further includes nucleic acids that encode variants of the murine TIM protein.
  • the nucleic acid encodes a variant that has an amino acid sequence of SEQ ID NO:20.
  • the nucleic acid encodes a murine TIM protein having the amino acid sequence of SEQ ID NO:22, or SEQ ID NO:24, or SEQ ID NO:26.
  • the nucleic acid encodes a murine TIM protein having the amino acid sequence of SEQ ID NO:20, or SEQ ID NO:22, or SEQ ID NO:24, or SEQ ID NO:26 that comprises a conservative amino acid substitution.
  • the nucleotide sequence has a nucleotide sequence of SEQ ID NO: 19, or SEQ ID NO:21, or SEQ ID NO:23, or SEQ ID NO:25.
  • the present invention further provides a nucleic acid consisting of at least 18, preferably at least 24, and more preferably at least 36 consecutive nucleotides of a nucleotide sequence that encodes a TIMELESS protein having an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:20.
  • Another such embodiment is a nucleic acid consisting of at least 18, preferably at least 24, and more preferably at least 36 consecutive nucleotides of a nucleotide sequence that encodes a TIMELESS protein having an amino acid sequence of SEQ ID NO:2 comprising a conservative substitution, or SEQ ID NO:20 comprising a conservative substitution.
  • the present invention provides nucleotide probes and primers of at least 12 preferably at least 18, and more preferably at least 36 nucleotides of a nucleotide sequence having the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 19.
  • specific probes and primers that can be used to distinguish specific variants of the nucleic acids encoding the mammalian TIMs are also part of the present invention.
  • the present invention further provides a nucleic acid that encodes a mammalian TIM and hybridizes to a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:2.
  • the nucleic acid encodes a mammalian TIM and hybridizes to a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:2 comprising a conservative substitution.
  • the nucleic acid that encodes a mammalian TIM hybridizes to the nucleotide sequence of SEQ ID NO: 1.
  • the nucleic acid comprises a nucleotide sequence which encodes a mammalian TIM and hybridizes to a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:20.
  • the nucleic acid hybridizes to a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:20 comprising a conservative substitution.
  • the nucleic acid encodes a mammalian TIM and hybridizes to the nucleotide sequence of SEQ ID NO: 19.
  • the nucleic acid comprises a nucleotide sequence that encodes a modified mammalian TIM.
  • the nucleic acid comprises a nucleotide sequence that encodes a modified mammalian TIM that comprises a TIM having an amino acid sequence of SEQ ID NO:2 or a fragment thereof.
  • nucleic acid comprises a nucleotide sequence that encodes a modified mammalian TIM that comp ⁇ ses a TIM having an amino acid sequence of SEQ ID NO 2 comp ⁇ sing a conservative substitution, or a fragment thereof
  • nucleic acid compnses a nucleotide sequence that encodes a modified mammalian TIM that comprises a TIM having an amino acid sequence of SEQ ID NO 20, or fragment thereof
  • nucleic acid comp ⁇ ses a nucleotide sequence that encodes a modified mammalian TIM that comp ⁇ ses a TIM having an amino acid sequence of SEQ ID NO 20 comp ⁇ sing a conservative substitution, or a fragment thereof
  • nucleic acids of the present invention can further comprise a heterologous nucleotide sequence Furthermore, all of the nucleic acids of the present invention can be constructed into recombinant DNA molecules Such recombinant DNA molecules can be opeiatively linked to an expression control sequence
  • the expression vectors containing the recombinant DNA molecules of the present invention are also provided by the present invention
  • the recombinant mammalian TIM proteins and TIM fragments can be expressed in a cell (either a prokaryotic cell or a eukaryotic cell) containing an expression vector of the present invention by cultu ⁇ ng the cell in an appropriate cell culture medium under conditions that provide for expression of the mammalian TIM protein by the cell
  • a recombinant mammalian TIM protein is expressed in a cell containing an expression vector of the piesent invention by
  • Another aspect of the present invention provides an isolated mammalian ortholog of the Drosophila TIMELESS (TIM) protein in which the amino acid sequence of the ortholog has at least 30% identity with either or preferably both SEQ ID NO 2 and/or SEQ ID NO 20
  • the ortholog has at least 50% identity with either or preferably both SEQ ID NO 2 and/or SEQ ID NO 20
  • the ortholog has at least 70% identity with either or preferably both SEQ ID NO 2 and/or SEQ ID NO 20
  • the ortholog has at least 80% identity with either or preferably both SEQ ID NO:2 and/or SEQ ID NO:20.
  • the ortholog has at least 90% and preferably 95% identity with either SEQ ID NO:2 or SEQ ID NO:20.
  • the corresponding proteins can be identified and/or defined by the same or analogous criteria.
  • the expression of a nucleic acid of the present invention that encodes a mammalian TIM when performed in Drosophila (S2) cells, preferably can promote the entry of Drosophila PER into the nucleus.
  • S2 Drosophila
  • a mammalian TIM protein of the present invention binds Drosophila PERIOD (PER) protein in vitro.
  • a mammalian TIM protein of the present invention binds mouse PERI and PER2 in vitro.
  • a mammalian TIM protein of the present invention preferably comprises a PER binding domain and a cytoplasmic localization domain (CLD). More preferably the CLD comprises an acidic tetrapeptide at or near the C-Terminal end of the CLD. Even more preferably the acidic tetrapeptide is DEDD (SEQ ID NO:27).
  • a mammalian TIM protein of the present invention preferably comprises a GLU-ASP rich region. More preferably the GLU- ASP rich region contains thirteen consecutive glutamic acid residues.
  • a mammalian TIM protein of the present invention preferably comprises a nuclear localization sequence (NLS).
  • a mammalian TIM protein comprises at least one, preferably two, more preferably three, and most preferably four regions which correspond to the TIM Homology regions (TH1, TH2, TH3, and TH4 as depicted in Figure 2) and which have at least 30%, preferably 50%, more preferably 70%, and even more preferably 80% identity with its co ⁇ esponding region of SEQ ID NO:2.
  • the present invention further provides isolated and recombinant variants of the mammalian TIM proteins. Accordingly the present invention includes all variants of the human TIM protein.
  • the human TIM protein has the amino acid sequence of SEQ ID NO:2.
  • the human TIM protein has the amino acid sequence of SEQ ID NO:4, or SEQ ID NO:6, or SEQ ID NO:8, or SEQ ID NO: 10, or SEQ ID NO: 12, or SEQ ID NO: 14, or SEQ ID NO: 16.
  • the human TIM protein has the amino acid sequence of SEQ ID NO:2, or SEQ ID NO:4, or SEQ ID NO:6, or SEQ ID NO:8, or SEQ ID NO: 10, or SEQ ID NO: 12, or SEQ ID NO: 14, or SEQ ID NO: 16 that comprises a conservative amino acid substitution.
  • the present invention further includes all of the variants of the murine TIM protein.
  • the murine variant has an amino acid sequence of SEQ ID NO:20.
  • the murine TIM protein has the amino acid sequence of SEQ ID NO:22, or SEQ ID NO:24, or SEQ ID NO:26.
  • the murine TIM protein has the amino acid sequence of SEQ ID NO:20, or SEQ ID NO:22, or SEQ ID NO:24, or SEQ ID NO:26 that comprises a conservative amino acid substitution.
  • the present invention further provides a mammalian TIM protein or TIM fragment consisting of at least 6, preferably at least 8, and more preferably at least 12 consecutive amino acid residues of a mammalian TIMELESS protein having an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:20.
  • a mammalian TIM protein or TIM fragment consisting of at least 6, preferably at least 8, and more preferably at least 12 consecutive amino acid residues of a mammalian a TIMELESS protein having an amino acid sequence of SEQ ID NO:2 comprising a conservative substitution or SEQ ID NO:20 comprising a conservative substitution.
  • a fragment of the mammalian TIM protein of the present invention binds to PER.
  • the fragment of the mammalian TIM protein when bound to PER can specifically inhibit CLOCK-BMAL-induced transactivation of the murine perl promoter.
  • the fragment of the mammalian TIM protein promotes nuclear entry of Drosophila PER in Drosophila (S2) cells.
  • a fragment of the mammalian TIM protein comprises a cytoplasmic localization domain (CLD).
  • the CLD comprises an acidic tetrapeptide at or near the C-Terminal end of the CLD.
  • all of the mammalian TIM proteins and fragments of mammalian TIM proteins of the present invention can be combined in a fusion proteins.
  • the present invention provides proteolytic fragments of all of the mammalian TIM proteins of the present invention.
  • the present invention further provides modified mammalian TIM proteins.
  • the modified mammalian TIM comprises a TIM having an amino acid sequence of SEQ ID NO:2 or a fragment thereof.
  • the modified mammalian TIM comprises a TIM having an amino acid sequence of SEQ ID NO:20, or a fragment thereof.
  • the modified mammalian TIM comprises a TIM having an amino acid sequence of SEQ ID SEQ ID NO:2 comprising a conservative substitution, or a fragment thereof.
  • the modified mammalian TIM comprises a TIM having an amino acid sequence of SEQ ID NO:20 comprising a conservative substitution, or a fragment thereof.
  • the present invention provides fusion proteins comprising the proteins, peptides and fragments thereof of the present invention.
  • all of the mammalian TIMs and fragments thereof of the present invention can be "modified” i.e., placed in a fusion of chimeric peptide or protein, or labeled, e.g., to have an N-terminal FLAG-tag.
  • a TIM can be modified to contain a marker protein such as green fluorescent protein as described in U.S. Patent No. 5,625,048 filed April 29, 1997 and WO 97/26333, published July 24, 1997 (each of which are hereby incorporated by reference herein in their entireties).
  • Still another aspect of the present invention provides an antibody to a mammalian TIM protein of the present invention.
  • the antibody is a polyclonal antibody.
  • the antibody is a monoclonal antibody.
  • the monoclonal antibody is a chimeric antibody.
  • the present invention further provides immortal cell lines that produce the monoclonal antibodies of the present invention.
  • the present invention further provides a mammalian cell that naturally encodes the timeless gene but has been manipulated so as to be incapable of expressing the gene.
  • the present invention provides animals that have one or both alleles which encode the timeless gene being defective or deleted.
  • the present invention provides a knockout mouse.
  • Such a knockout mouse comprises a first and a second allele which naturally encode and express a mu ⁇ ne timeless gene, but both the first and second allele each contain a defect which prevents the knockout mouse from expressing the timeless gene
  • the knockout mouse has an abnormal circadian rhythm
  • Another aspect of the present invention provides methods for detecting the presence or activity of the mammalian TIM proteins of the present invention or their co ⁇ esponding mRNAs
  • One such embodiment comp ⁇ ses contacting a biological sample trom a mammal in which the presence or activity of the protein is suspected with a binding partner of the protein or a binding partner of the mRNA under conditions that allow binding of the protein or the mRNA with their respective binding partners to occur, and then detecting whether the binding has occu ⁇ ed
  • the detection of the binding indicates the presence or activity of the protein or the mRNA in the biological sample
  • the binding partner is a nucleotide probe having specificity tor an mRNA encoding the mammalian TIM
  • the binding partner is a PER protein or fragment of a PER protein that binds mammalian TIM
  • the binding partner is an antibody to the mammalian TIM protein
  • the present invention also provides a method of identifying an agent that can modulate the binding of a mammalian TIM protein to a PER protein
  • a mammalian TIM protein or a mammalian TIM fragment with a PER protein or a PER protein fragment, m the presence of the agent
  • the binding of the mammalian TIM protein or the mammalian TIM fragment to the PER protein or the PER protein fragment is then determined
  • An agent is identified as a modulator of the binding of the mammalian TIM protein to the PER protein when the determination is indicative of a change in the binding relative to that in the absence of the agent
  • standard statistical analyzes are performed on the binding determinations of the method and only statistically relevant changes are used to identify the agent as a modulator
  • the mammalian TIM fragment used in this method comprises a fragment of a mammalian TIM protein that contains a PER binding domain (PBD), whereas the PER protein fragment preferably comprises a fragment of a PER
  • the present invention further provides a method of identifying an agent that can modulate the effect of a mammalian TIM protein to promote the nuclear entry of PER protein in a cell.
  • One such embodiment comprises contacting a cell with an agent, wherein the cell expresses a nucleic acid encoding a mammalian TIM protein and a nucleic acid encoding a PER protein and the mammalian TIM protein promotes the entry of the PER protein into the nucleus of the cell in absence of the agent. The amount of PER protein in the nucleus is then determined.
  • An agent is identified as a modulator of the effect of a mammalian TIM protein to promote the entry of the PER protein to the nucleus when the determination is indicative of a change in the amount of PER protein in the nucleus relative to that in the absence of the agent.
  • Preferably standard statistical analyzes are performed on the determinations of the amount of PER protein in the nucleus, and only statistically relevant changes are used to identify the agent as a modulator.
  • the present invention further provides a method for identifying an agent that can modulate the effect of a PER protein and a mammalian TIM protein on the transactivation of the CLOCK-BMAL1 heterodimer.
  • One such embodiment comprises expressing mammalian Tim and PER in a cell, wherein the cell comprises a reporter gene operatively linked to an expression control sequence that is transactivated by the CLOCK-BMAL1 heterodimer and the cell expresses nucleic acids encoding CLOCK and BMALl.
  • a potential agent is then contacted with the cell and the amount of the reporter gene expressed in the cell is determined.
  • a potential agent is identified as a candidate agent that modulates the effect of the mammalian TIM protein and the PER protein on the transactivation of the CLOCK- BAM 1 heterodimer when the determination indicates a change in the amount of expression of the reporter gene in the cell relative to that in the absence of the agent.
  • Preferably standard statistical analyses are performed on the determinations of the amount of reported gene expressed, and only statistically relevant changes are used to identify the agent as a modulator.
  • the candidate agent When the determination indicates an increase in the expression of the reporter gene in the cell, the candidate agent is identified as an antagonist of the effect of the PER protein and mammalian TIM protein on the transactivation of the CLOCK-BMAL1 heterodimer; whereas when the determination indicates a decrease in the expression of the reporter gene in the cell, the candidate agent is identified as an agonist of the effect of the PER protein and mammalian TIM protein on the transactivation of the CLOCK-BMAL1 heterodimer.
  • the nucleic acids encoding the mammalian TIM protein and the PER protein are expressed transiently whereas the co ⁇ esponding expression of CLOCK and BMALl is constitutive.
  • the method further comprises contacting the candidate agent with the cell in the absence of the expression of the PER protein and mammalian TIM protein.
  • the amount of the reporter gene expressed in the cell is then determined.
  • the candidate agent is identified as an agent that modulates the effect of the mammalian TIM protein and the PER protein on the transactivation of the CLOCK-BMAL1 heterodimer.
  • kits for performing the methods of the present invention can be used in detecting the presence of mammalian TIM protein or mRNA in a cellular sample.
  • the kit comprises a predetermined amount of a detectably labeled binding partner of the mammalian TIM protein or mRNA.
  • the binding partner is antibody to the mammalian TIM protein.
  • the kit also contains a separate sample of mammalian TIM to use as a standard.
  • the kit comprises a nucleic acid probe that has specificity for an mRNA encoding the TIM protein.
  • the kits can also comprise other reagents and written protocols.
  • Other kits can include vectors that encode the mammalian TIM proteins and/or cells that contain such vectors.
  • Yet another aspect of the present invention comprises methods of identifying the nucleotide and amino acid sequences of other orthologs of the human and murine timeless gene.
  • the co ⁇ esponding amino acid sequence can be readily determined using the genetic code, preferably with the aid of a computer.
  • the full-length nucleotide sequence of the coding region of an ortholog to the human and murine timeless gene is identified.
  • the non-human and non-murine gene is a mammalian gene. Recombinant DNA molecules and the recombinant mammalian TIM proteins obtained by these methods are also part of the present invention.
  • One method of identifying a nucleotide sequence of the coding region of an ortholog to the human and murine timeless gene comprises comparing SEQ ID NO:2 and/or SEQ ID NO:20 with the amino acid sequences encoded by nucleic acids that are obtained from a library of nucleic acids containing partial nucleotide sequences of the coding regions from non-human and non-murine genes. Preferably this determination is aided by computer analysis. A nucleic acid containing a partial nucleotide sequence of a coding region from a non-human and non-murine gene that is highly homologous to SEQ ID NO:2 ⁇ e.g., 30%, or 50%, or 70%, or 80% or more amino acid sequence identity) can then be selected. Methods of ascertaining which nucleic acid and amino acid sequences are highly homologous or have a particular percent amino acid sequence identity are described below.
  • the full-length sequence of the coding region of the non-human and non-murine gene is preferably determined.
  • the sequence is identified as being that of the ortholog of the human and murine timeless gene when it is highly homologous to SEQ ID NO:2 as discussed above.
  • this method further comprises determining whether the nucleotide sequence that contains a coding region for an amino acid sequence that is highly homologous to SEQ ID NO:2 is also expressed in the co ⁇ esponding suprachiasmatic nucleus (SCN), i.e., if the putative ortholog is a bovine ortholog, the SCN of a bovine is tested.
  • SCN co ⁇ esponding suprachiasmatic nucleus
  • the present invention also provides methods of preventing and/or treating disorders of a circadian rhythm which include depression, narcolepsy and jet lag. Such methods rely on temporary antagonisms to transiently inhibit the natural clock, and then supplying agonists to subsequently reset it e.g., for the treatment of jet lag.
  • One such embodiment comprises administering to an animal a therapeutically effective amount of a mammalian TIM protein.
  • Another such embodiment comprises administering to a mammal a therapeutically effective amount of an agent capable of promoting the production and/or activity of a mammalian TIM.
  • Yet another such embodiment comprises a mixtures of such agents.
  • Still another embodiment comprises administering to an animal a therapeutically effective amount of an agent capable of inhibiting the activity of the mammalian TIM. Accordingly, it is a principal object of the present invention to provide mammalian TIM proteins in a purified form that exhibit activities associated with circadian rhythms
  • compositions for use in therapeutic methods which compnse or are based upon mammalian TIM or upon agents or drugs that control the production, or that mimic or antagonize the activities of mammalian TIM
  • Figures 1A-1B shows the Cloning of human tim (Fig. lA) and murine tim (Fig.lB).
  • the clones were obtained from the EST database (na ⁇ ow solid black line), library screening (green vertical lines), RACE (blue slanted lines) and PCR (wide solid black lines) experiments are indicated for both mtim and htim.
  • Introns are indicated by triangles below clones containing them. Insertions are delineated by small triangles above the clone.
  • Internal priming sites (A-rich sequence) are identified by filled squares. Dashed lines indicate ESTs whose clones could not be recovered (likely misaddressed).
  • the loopout clone derived by site-directed mutagenesis from H5E11CA03 which represents the full-length htim cDNA is also indicated.
  • Two clones were chimeric (mtDNA - mitochondrial DNA and Actin- Actin cDNA fragment).
  • TMBGTAE03 and mtim 5' A2 show splice variation at the 5' end indicated by the slanted segments at their 5' end.
  • FIG. 2 shows the sequence alignment of mammalian Tims, the ClustalW Alignment of the hTIM and mTIM proteins. Highlighted are the Tim Homology domains, designated TH1-TH4. The putative nuclear localization signal (NLS) sequences are underlined as is the glutamate rich sequence. The DEDD sequence which is conserved in the D. melanogaster cytoplasmic localization domain (CLD) is also indicated.
  • NLS nuclear localization signal
  • Figure 3 shows the four homology domains of the human, mouse, D. melanogaster, D. virilis and D. hydei TIM proteins. The percent identity and similarity between mouse TIM and D. melanogaster TIM in each of the four domains is indicated. Locations of functional sites of reference in the D. melanogaster TIM sequence are noted including the tim SL and tim 0 mutation sites, the PER-binding regions (PB1 and PB2), CLD and NLS. Putative NLS's in the other proteins are also indicated.
  • Figures 4A-4D show the tissue distribution of mTim and HTim mRNA expression
  • Figs. 4A-4B show human multiple tissue RNA blots.
  • Figs. 4C-4D show mouse multiple tissue and mouse embryonic tissue RNA blots. 2ug of poly-A(+) RNA of the indicated tissues was loaded on each lane. Primary transcript of 4.5 kb is evident in both mouse and human tissues. Blots were normalized with actin. (M.L. - mucosal lining)
  • Figures 5A-5D show the mRNA expression in SCN and retina by in situ hybridization.
  • Figs. 5A-5D show coronal sections of mouse brains at CT 6 (Figs. 5 A and 5C) and CT 18 (Figs. 5B and 5D) showing hybridization with mPerl (Figs. 5A and 5B) and mTim (Figs. 5C and 5D).
  • mPerl clearly demonstrates a circadian variation in abundance whereas there is no apparent variation in mTim expression.
  • Figures 6A-6B show the mRNA expression in SCN and retina by quantitative RT-PCR (TaqMan).
  • Fig. 6A shows the expression of mPerl and mTim mRNA levels in the mouse SCN.
  • mPerl and mTim two probes PI and P2
  • mRNA levels in the SCN were determined from adjacent sections of mouse brains obtained from 3 animals per time point indicated. (N-2 per probe).
  • Fig. 6B shows the expression of mPerl and mTim mRNA in the retina by quantitative RT-PCR.
  • TaqMan RT-PCR assays were ca ⁇ ied out on 3 independent RNA samples, each run in duplicate. E ⁇ or bars indicated S.E.M. Error bars for the mTim quantitation are too small to be seen at this scale.
  • Figures 7A-7C depict the interaction of hTIM with dPER, mPERl and mPER2 in vitro, Interactions of hTIM and dPER proteins.
  • 35 S-labeled hTIM (input) was incubated with GST alone and with GST-PER fusion proteins and analyzed for binding by SDS-PAGE and autoradiography as described in experimental procedures.
  • the top panel shows a Coomassie-stained SDS-PAGE of the GST (Fig.7A) and GST-dPER fusion proteins used in the binding assay.
  • Bottom panels show differential binding of in vitro translated, hTIM 1-1207 (Fig.7B) and hTIM 1-560 (Fig. 7C) respectively to the indicated fusion proteins.
  • the input lane shows the in vitro translated product before the binding reaction.
  • Figure 8 is a schematic representation and summary of the interaction of dPER with hTIM and dTIM. Positions of the dPER NLS, PAS, and CLD domains are indicated at top [Saez et al, Neuron 17:911-920 (1996)].
  • the dPER polypeptide fragments in the fusion proteins are indicated with respect to amino acid numbering of the full-length Canton-S, D. melanogaster protein [Myers, et al, Nucleic Acids Res. 25:4710-4714 (1997)].
  • the numbering of dTIM refers to amino acid sequence of [Myers, ef al, Nucleic Acids Res. 25:4710-4714 (1997)].
  • Figure 9A-9C show that hTIM interacts with the mouse PERI and PER2 proteins.
  • Full-length mouse PERI and PER2 were in vitro translated, 35 S-labeled and incubated with GST-hTIM, GST-mTIM, and GST alone as described.
  • Figure 9A shows the Coomassie-stained SDS-PAGE gel of the GST fusions utilized in the binding assay.
  • Figs. 9B-9C show autoradiographs of the in vitro translated mPERl (Fig. 9B) and mPER2 (Fig. 9C) bound to the indicated GST-hTIM fusion proteins.
  • the input lanes show the indicated in vitro translation products before the binding reaction. Molecular sizes are in kilodaltons.
  • Figure 10A-10F show that hTIM promotes nuclear entry of dPER S2 cells transfected hs-per (Figs.lOA and 10D) and co-transfected with hs-per (Figs.lOB and 10E) and hs-htim and (Figs. IOC and 10F) were induced by heat shock, fixed 4 hours later and immunostained with anti-PER antibodies.
  • the antibody-antigen complex was detected with rhodamine-conjugated anti-rabbit IgG (red).
  • the cells of Figs. 10A-10C were also stained with Hoechst (blue) for detection of DNA in nuclei in Figs. 10D-10F.
  • Figures 11A-1 ID show that hTIM and mPERl inhibit mPerl gene transactivation by the CLOCK-BMALl heterodimer.
  • Fig 11 A shows the effect of hTIM, mPERl, or both on transactivation by the CLOCK-BMALl heterodimer from a 2.0-kb mPerl promoter fragment including all three E-boxes (cacgtg).
  • Figure 1 IB shows the effect of hTIM, mPERl, or both on transactivation by the CLOCK-BMALl heterodimer from a 54-bp construct consisting of the three mPerl E-boxes and their immediate flanking sequences linked together.
  • Figure 11C shows the effect of hTIM, mPERl, or Id protein on transactivation by the CLOCK-BMALl heterodimer from the 54-bp mPerl construct.
  • Figure 1 ID shows the effect of hTIM, mPERl, or Id protein on transactivation by the MyoD-E12 heterodimer from a 60-bp construct including four copies of the mck gene-specific E-box (caggtg) and immediate flanking sequences.
  • Figure 12A-12B depicts the summary of Drosophila and Mammalian Circadian autoregulatory Loops: Fig.l2A shows the Drosophila circadian feedback loop.
  • dCLOCK-dBMAL start the cycle by activating transcnption of the per and tim genes Once sufficient levels of PER and TIM are attained, heterodime ⁇ zation can occur, thus allowing nuclear translocation The cycle is closed with the inhibition of dCLOCK-dBMAL by nuclear PER-TIM complex DOUBLETIME (DBT, Kloss et al , 1998, Pnce et al , 1998, and the subject matter of co-pending U S Patent Application No 09/100,664 filed on June 19, 1998, which is hereby incorporated by reference in its entirety) is required for proper phosphorylation and turnover of PER
  • DOUBLETIME DOUBLETIME
  • Fig 12B shows the mammalian circadian autoregulatory loop showing identified roles of various components (1) direct association of mPERl and hTIM, (2) ability of hTIM to allow nuclear entry of PER, (3) Inhibition of CLOCK-BMALl induced activity of the mPerl promoter LRE- light responsive element
  • the present invention provides mammalian factors involved in circadian rhythms More specifically, the present invention identifies mammalian orthologs of the Drosophila protein, TIMELESS (TIM), that is involved in circadian rhythms
  • TIMELESS TIM
  • the present invention further substantiates the role of TIM in mammalian systems
  • Mammalian TIM proteins can be used as agents themselves or alternatively, to identify other agents that can aid in the regulation of biological clocks in mammals, including in humans
  • the identification of mammalian TIM completes the elucidation of the basic four-component circadian autoregulatory loop in mammals, i e , TIM, the PERIOD (PER) protein, the CLOCK protein and the BMALl or MOP3 protein
  • the present invention provides nucleic acids and proteins of mammalian orthologs of the Dtosophila timeless (dTim) gene
  • the nucleotide and amino acid sequences of these mammalian orthologs along with va ⁇ ants of these orthologs are also provided
  • the mammalian TIM proteins and nucleic acids are munne proteins and nucleic acids
  • the mammalian TIM proteins and nucleic acids are human proteins and nucleic acids
  • the present invention also provides the genomic mapping of the genes encoding the mammalian TIM proteins
  • the mammalian TIMELESS (TIM) proteins disclosed herein, preferably function in a manner that is analogous to the Drosophila TIM protein in at least one or more different ways.
  • a mammalian TIM protein can bind to a PERIOD (PER) protein.
  • PER PER
  • the expression of mammalian TIM expression in situ and/or in vivo promotes nuclear entry of the PER.
  • expression of mammalian TIM and mammalian PERI specifically inhibit CLOCK-BMALl-induced transactivation of the mammalian perl promoter.
  • TIMELESS As used herein "TIMELESS”, “TIM”, “TIMELESS PROTEIN” and “TIM protein” are interchangeable names for a protein first identified in Drosophila and described in U.S. Patent Application Nos: 08/408,518, filed March 20, 1995 (now abandoned), 08/442,214, filed May 16, 1995 (now abandoned), 08/552,354, filed November 2, 1995 (now abandoned) and 08/619,198, Filed March 21, 1996 (co-pending) which are all hereby incorporated by reference in their entireties. More particularly, mammalian TIM plays a role in the nuclear transport of the gene product PERIOD (PER) of the important clock gene, period ⁇ per).
  • PER gene product of the important clock gene
  • human Tim and variants thereof have the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 14, and 16, and the nucleic acid sequences consisting of the coding regions of SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 17, and 18.
  • murine Tim and variants thereof have the amino acid sequences of SEQ ID NOs: 20, 22, 24, and 26 and the nucleic acid sequences consisting of the coding regions of SEQ ID NOs: 19, 21, 23, and 25.
  • PER binding domain or "PD”, or “PBD” or “PB” are used interchangeably and denote a domain of a TIM protein that is involved in the binding of the PER protein to form a heterodimer [Saez and Young, Neuron, 17:911-920 (1996)].
  • cytoplasmic localization domain or “CLD” is the portion of a TIM protein required for retention of the protein in the cytoplasm (as opposed to in the nucleus), as further defined by Saez and Young [Neuron 17:911-920 (1996)] and as depicted schematically in Figure 3.
  • an "acidic tetrapeptide” is a portion of a TIM protein comprising four consecutive amino acid residues which are either glutamic acid or aspartic acid.
  • a particular example of such an acidic tetrapeptide is DEDE, SEQ ID NO:28.
  • the tetrapeptide is DEDD, SEQ ID NO:27.
  • the "GLU-ASP rich region” is a portion of a TIM protein comprising thirteen consecutive amino acid residues in which at least 11, preferably 12, and more preferably 13 are either glutamic acid or aspartic acid. In a particular embodiment of this type the GLU- ASP rich region comprises 13 consecutive glutamic acid residues. When a majority of the acidic residues in a GLU-ASP rich region are glutamic acid residues, the GLU-ASP rich region can also be refe ⁇ ed to as a "glutamate-rich sequence.”
  • a protein containing "approximately” 1208 amino acid residues can contain between 1087 and 1329 amino acid residues.
  • binds to is meant to include all such specific interactions that result in two or more molecules showing a preference for one another relative to some third molecule. This includes processes such as covalent, ionic, hydrophobic and hydrogen bonding but does not include non-specific associations such as solvent preferences.
  • a “vector” is a replicon, such as a plasmid, phage or cosmid, to which another DNA segment may be attached so as to bring about the replication of the attached segment.
  • a “replicon” is any genetic element (e.g., plasmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., capable of replication under its own control.
  • a "cassette” refers to a segment of DNA that can be inserted into a vector at specific restriction sites.
  • the segment of DNA can encode a polypeptide of interest, and the cassette and restriction sites are designed to ensure insertion of the cassette in the proper reading frame for transcription and translation.
  • a cell has been "transfected” by exogenous or heterologous DNA when such DNA has been introduced inside the cell.
  • a cell has been “transduced” by exogenous or heterologous DNA when the exogenous or heterologous DNA is introduced by a viral vector.
  • a "heterologous nucleotide sequence” as used herein is a nucleotide sequence that is added to a nucleotide sequence of the present invention by recombinant methods to form a nucleic acid which is not naturally formed in nature. Such nucleic acids can encode chimeric and/or fusion proteins. Thus the heterologous nucleotide sequence can encode peptides and/or proteins which contain regulatory and/or structural properties.
  • heterologous nucleotide can encode a protein or peptide that functions as a means of detecting the protein or peptide encoded by the nucleotide sequence of the present invention after the recombinant nucleic acid is expressed.
  • heterologous nucleotide can function as a means of detecting a nucleotide sequence of the present invention.
  • a heterologous nucleotide sequence can comprise non-coding sequences including restriction sites, regulatory sites, promoters and the like.
  • a "heterologous" region of the DNA construct is an identifiable segment of DNA within a larger DNA molecule that is not found in association with the larger molecule in nature.
  • the gene when the heterologous region encodes a mammalian gene, the gene will usually be flanked by DNA that does not flank the mammalian genomic DNA in the genome of the source organism.
  • Another example of a heterologous coding sequence is a construct where the coding sequence itself is not found in nature (e.g., a cDNA where the genomic coding sequence contains introns, or synthetic sequences having codons different than the negative gene). Allelic variations or naturally-occu ⁇ ing mutational events do not give rise to a heterologous region of DNA as defined herein.
  • nucleic acid molecule refers to the phosphate ester polymeric form of ribonucleosides (adenosine, guanosine, uridine or cytidine; "RNA molecules”) or deoxyribonucleosides (deoxyadenosine, deoxyguanosine, deoxythymidine, or deoxycytidine; "DNA molecules”), or any phosphoester analogs thereof, such as phosphorothioates and thioesters, in either single stranded form, or a double-stranded helix. Double stranded DNA-DNA, DNA-RNA and RNA-RNA helices are possible.
  • nucleic acid that is DNA and more specifically a DNA having a particular nucleotide sequence, i.e., SEQ ID NO:
  • SEQ ID NO:l a nucleic acid that is hybridizable to SEQ ID NO:l
  • nucleic acid molecule refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms.
  • this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes.
  • sequences may be described herein according to the normal convention of giving only the sequence in the 5' to 3' direction along the nontranscribed strand of DNA (i.e., the strand having a sequence homologous to the mRNA).
  • a "recombinant DNA molecule” is a DNA molecule that has undergone a molecular biological manipulation.
  • a nucleic acid molecule is "hybridizable" to another nucleic acid molecule, such as a cDNA, genomic DNA, or RNA, when a single stranded form of the nucleic acid molecule can anneal to the other nucleic acid molecule under the appropriate conditions of temperature and solution ionic strength (see Sambrook ef al, supra).
  • the conditions of temperature and ionic strength determine the "stringency" of the hybridization.
  • low stringency hybridization conditions corresponding to a T m of 55 °, can be used, e.g., 5x SSC, 0.1% SDS, 0.25% milk, and no formamide; or 30% formamide, 5x SSC, 0.5% SDS).
  • Moderate stringency hybridization conditions co ⁇ espond to a higher T m , e.g., 40% formamide, with 5x or 6x SSC.
  • High stringency hybridization conditions co ⁇ espond to the highest T m , e.g., 50% formamide, 5x or 6x SSC.
  • Hybridization requires that the two nucleic acids contain complementary sequences, although depending on the stringency of the hybridization, mismatches between bases are possible.
  • the appropriate stringency for hybridizing nucleic acids depends on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of similarity or homology between two nucleotide sequences, the greater the value of T m for hybrids of nucleic acids having those sequences.
  • RNA:RNA, DNA:RNA, DNA:DNA The relative stability (co ⁇ esponding to higher T m ) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA.
  • equations for calculating T m have been derived (see Sambrook ef al, supra, 9.50-0.51).
  • the position of mismatches becomes more important, and the length of the oligonucleotide determines its specificity (see Sambrook et al, supra, 11.7-11.8).
  • a minimum length for a hybridizable nucleic acid is at least about 12 nucleotides; preferably at least about 18 nucleotides; and more preferably the length is at least about 27 nucleotides; and most preferably at least about 36 nucleotides.
  • standard hybridization conditions refers to a T m of
  • T m is 60°C; in a more prefe ⁇ ed embodiment, the T m is 65 °C.
  • Homologous recombination refers to the insertion of a foreign DNA sequence of a vector in a chromosome.
  • the vector targets a specific chromosomal site for homologous recombination.
  • the vector will contain sufficiently long regions of homology to sequences of the chromosome to allow complementary binding and incorporation of the vector into the chromosome. Longer regions of homology, and greater degrees of sequence similarity, may increase the efficiency of homologous recombination.
  • a DNA "coding sequence” is a double-stranded DNA sequence which is transcribed and translated into a polypeptide in a cell in vitro or in vivo when placed under the control of appropriate regulatory sequences. The boundaries of the coding sequence are determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxyl) terminus.
  • a coding sequence can include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. If the coding sequence is intended for expression in a eukaryotic cell, a polyadenylation signal and transcription termination sequence will usually be located 3' to the coding sequence.
  • Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • polyadenylation signals are control sequences.
  • a "promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter sequence is bounded at its 3 ' terminus by the transcription initiation site and extends upstream (5 ' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined for example, by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • a coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then trans-RNA spliced and translated into the protein encoded by the coding sequence.
  • a “signal sequence” is included at the beginning of the coding sequence of a protein to be expressed on the surface of a cell. This sequence encodes a signal peptide, N-terminal to the mature polypeptide, that directs the host cell to translocate the polypeptide.
  • the term "translocation signal sequence” is used herein to refer to this sort of signal sequence. Translocation signal sequences can be found associated with a variety of proteins native to eucaryotes and prokaryotes, and are often functional in both types of organisms.
  • sequence homology in all its grammatical forms refers to the relationship between proteins that possess a "common evolutionary origin.” including proteins from superfamilies (e.g., the immunoglobulin superfamily) and homologous proteins from different species (e.g., myosin light chain, etc.) (Reeck ef al, 1987, Cell 50:667).
  • ortholog refers to the relationship between proteins that have a common evolutionary origin and differ because they originate from different species.
  • Drosophila TIMELESS is a ortholog of human TIMELESS.
  • sequence similarity in all its grammatical forms refers to the degree of identity or co ⁇ espondence between nucleic acid or amino acid sequences of proteins that do not share a common evolutionary origin (see Reeck ef al, supra).
  • sequence similarity when modified with an adverb such as “highly,” may refer to sequence similarity and not necessarily a common evolutionary origin.
  • two highly homologous DNA sequences can be identified by the homology of the amino acids they encode. Such comparison of the sequences can be performed using standard software available in sequence data banks.
  • two highly homologous DNA sequences encode amino acid sequences having 30%, preferably 50%, more preferably 70% and even more preferably 80% identity. More particularly, two highly homologous amino acid sequences have 30%, preferably 50%, more preferably 70% and even more preferably 80% identity.
  • two highly homologous DNA sequences can be identified by Southern hybridization experiment under, for example, stringent conditions as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Maniatis ef al, supra; DNA Cloning, Vols. I & II, supra; Nucleic Acid Hybridization, supra.
  • amino acid sequence is 100% "homologous" to a second amino acid sequence if the two amino acid sequences are identical, and/or differ only by neutral or conservative substitutions as defined below. Accordingly, an amino acid sequence is 50% "homologous" to a second amino acid sequence if 50% of the two amino acid sequences are identical, and/or differ only by neutral or conservative substitutions.
  • DNA and protein sequence percent identity can be determined using MacVector 6.0.1, Oxford Molecular Group PLC (1996) and the Clustal W algorithm with the alignment default parameters, and default parameters for identity. These commercially available programs can also be used to determine sequence similarity using the same or analogous default parameters.
  • co ⁇ esponding to is used herein to refer similar or homologous sequences, whether the exact position is identical or different from the molecule to which the similarity or homology is measured.
  • co ⁇ esponding to refers to the sequence similarity, and not the numbering of the amino acid residues or nucleotide bases.
  • the present invention provides isolated and/or recombinant mammalian TIM proteins and fragments.
  • TIM plays an essential role in circadian rhythms in Drosophila and as disclosed herein, in mammals as well.
  • Mammalian TIM has been shown herein to be capable of binding to PER in vitro, and in vivo.
  • Structural and functional analyzes show that human TIM (hTIM) and murine TIM (mTIM) are mammalian orthologs of the Drosophila circadian protein, TIMELESS.
  • hTIM and mTim show highest sequence similarity to the Drosophila TIM proteins and to no other known proteins. Comparison of the TIM proteins reveals four regions of similarity among the insect and mammalian proteins (TH1-TH4).
  • hTIM regions of the Drosophila TIM protein involved in nuclear localization, protein-protein interaction with PER, and cytoplasmic localization. These structural similarities were tested for functional similarities and indeed hTIM was found to associate physically with Drosophila and mouse PER proteins in vitro, to promote nuclear entry of Drosophila PER in S2 cells, and to negatively regulate CLOCK-BMALl driven transactivation of the mPerl promoter in NIH-3T3 mouse fibroblasts. Taken together the results presented herein demonstrate that hTIM and mTIM are mammalian orthologs of the Drosophila circadian gene, timeless.
  • the mammalian TIM is a murine protein.
  • the TIM is a human protein.
  • the TIM is a protein encoded by a nucleotide sequence that is hybridizable with the complementary strand of the coding sequence of SEQ ID NO: l under standard, and/or stringent conditions.
  • a human TIM protein is encoded by a nucleotide sequence having the coding sequence of SEQ ID NOs: 1, 3, 5, 7,9,11,13,15,17, or 18.
  • the human TIM has an amino acid sequence of SEQ ID NOs:2, 6, 8, 10, 14 or 16 comprising one or more conservative substitutions.
  • the TIM proteins of the present invention may be used for many purposes including in assays to identify novel drugs, and the like, and in protein structure and mechanistic studies.
  • Modified TIMs i.e., TIMs that are tagged proteins, labeled proteins, fusion proteins and the like. Such TIMs may be used for example as antigens or for marker purposes.
  • the fusion protein comprises an TIM protein or TIM fragment having an amino acid sequence of SEQ ID NO:20 or SEQ ID NO:20 comprising a conservative substitution.
  • the fusion protein comprises an TIM protein or TIM fragment having an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:2 comprising a conservative substitution
  • Such TIM proteins and fragments preferably retain their ability to bind PER.
  • One particular use of the TIM fusion proteins of the present invention is for the production of the TIM- antibodies of the present invention.
  • a TIM fusion protein comprises at least a portion of a non-TIM protein joined via a peptide bond to at least a portion of a TIM polypeptide.
  • the portion of the TIM is functional.
  • the non-TIM sequences can be amino- or carboxy-terminal to the TIM sequences. More preferably, for stable expression of a TIM fusion protein, the portion of the non-TIM fusion protein is joined via a peptide bond to the amino terminus of the TIM protein.
  • a recombinant DNA molecule encoding such a fusion protein comprises a sequence encoding at least a portion of a non-TIM protein joined in-frame to the TIM coding sequence, and can encodes a cleavage site for a specific protease, e.g., thrombin or Factor Xa, preferably at the TIM-non-TIM juncture.
  • the fusion protein is expressed in Escherichia coli.
  • Such a fusion protein can be used to isolate the TIMs of the present invention, through the use of an affinity column which is specific for the protein fused to the TIM. The purified TIM may then be released from the fusion protein through the use of a proteolytic enzyme and the cleavage site such as has been refe ⁇ ed to above.
  • a chimeric TIM can be prepared, e.g., a glutathione-S-transferase (GST) fusion protein, a maltose-binding (MBP) protein fusion protein, or a poly-histidine- tagged fusion protein, for expression in a eukaryotic cell.
  • GST glutathione-S-transferase
  • MBP maltose-binding
  • poly-histidine- tagged fusion protein for expression in a eukaryotic cell.
  • Expression of a TIM as a fusion protein can facilitate stable expression, or allow for purification based on the properties of the fusion partner.
  • GST binds glutathione conjugated to a solid support matrix
  • MBP binds to a maltose matrix
  • poly-histidine chelates to a Ni-chelation support matrix.
  • the fusion protein can be eluted from the specific matrix with appropriate buffers, or by treating with a protease specific for a cleavage site usually engineered between the TIM and the fusion partner (e.g., GST, MBP, or poly-His) as described above.
  • a protease specific for a cleavage site usually engineered between the TIM and the fusion partner (e.g., GST, MBP, or poly-His) as described above.
  • the chimeric TIM protein may contain the green fluorescent protein, and be used to determine the intracellular localization of the TIM in the cell.
  • the present invention contemplates isolation of a gene encoding a TIM of the present invention, including a full length, or naturally occurring form of TIM, and antigenic fragments thereof from any animal, particularly mammalian, and more particularly human, source.
  • Such nucleic acids may be used for designing primers for RT-PCR, and for making probes that are useful for determining the expression of TIM messenger RNA in tissues and tumors as described in the Example below.
  • nucleic acids can be used to determine the expression of TIM messenger RNA in normal tissues and tumors by Northern Blot analysis, RNA protection assays and the like.
  • the term "gene” refers to an assembly of nucleotides that encode a polypeptide, and includes cDNA and genomic DNA nucleic acids. Therefore, the present invention provides the primary structure of genes encoding a murine TIM protein and a human TIM protein.
  • a gene encoding TIM can be isolated from any source, particularly from a human cDNA or genomic library.
  • methods well known in the art, as described above can be used for obtaining 77M genes from any source (see, e.g., Sambrook et al, 1989, supra).
  • any animal cell or transformed animal cell line potentially can serve as the nucleic acid source for the molecular cloning of a TIM gene.
  • the DNA may be obtained by standard procedures known in the art from cloned DNA (e.g., a DNA "library”), and preferably is obtained from a cDNA library prepared from tissues with high level expression of the protein, by chemical synthesis, by cDNA cloning, or by the cloning of genomic DNA, or fragments thereof, purified from the desired cell (See, for example, Sambrook et al, 1989, supra; Glover, D.M. (ed.), 1985, DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I, II).
  • Clones derived from genomic DNA may contain regulatory and intron DNA regions in addition to coding regions; clones derived from cDNA will not contain intron sequences. Whatever the source, the gene should be molecularly cloned into a suitable vector for propagation of the gene.
  • DNA fragments are generated, some of which will encode the desired gene.
  • the DNA may be cleaved at specific sites using various restriction enzymes.
  • DNAse in the presence of manganese to fragment the DNA, or the DNA can be physically sheared, as for example, by sonication.
  • the linear DNA fragments can then be separated according to size by standard techniques, including but not limited to, agarose and polyacrylamide gel electrophoresis and column chromatography.
  • the generated DNA fragments may be screened by nucleic acid hybridization to a labeled probe of the present invention (Benton and Davis, 1977, Science 196: 180; Grunstein and Hogness, 1975, Proc. Natl. Acad. Sci. U.S.A. 72:3961).
  • a set of oligonucleotides co ⁇ esponding to the sequence information provided by the present invention can be prepared and used as probes for DNA encoding TIM (e.g., in combination with a poly-T primer for RT-PCR).
  • a probe is selected that is highly unique to the TIM of the invention. Those DNA fragments with substantial homology to the probe will hybridize. As noted above, the greater the degree of homology, the more stringent hybridization conditions can be used.
  • cDNA clones, or DNA clones which hybrid-select the proper mRNAs can be selected which produce a protein that, e.g., has similar or identical electrophoretic migration, isoelectric focusing or non-equilibrium pH gel electrophoresis behavior, proteolytic digestion maps, or antigenic properties as known for TIM.
  • a TIM gene of the invention can also be identified by mRNA selection, i.e., by nucleic acid hybridization followed by in vitro translation.
  • nucleotide fragments are used to isolate complementary mRNAs by hybridization.
  • DNA fragments may represent available, purified TIM DNA, or may be synthetic oligonucleotides designed from the partial amino acid sequence information.
  • Immunoprecipitation analysis or functional assays e.g., ability to promote the nuclear entry of PER
  • the in vitro translation products of the products of the isolated mRNAs identifies the mRNA and, therefore, the complementary DNA fragments, that contain the desired sequences.
  • specific mRNAs may be selected by adsorption of polysomes isolated from cells to immobilized antibodies specifically directed against TIM.
  • a radiolabeled TIM cDNA can be synthesized using the selected mRNA (from the adsorbed polysomes) as a template.
  • the radiolabeled mRNA or cDNA may then be used as a probe to identify homologous TIM DNA fragments from among other genomic DNA fragments.
  • the present invention also relates to cloning vectors containing genes encoding analogs and derivatives of TIM of the invention, that have the same or homologous functional activity as TIM, and in particular orthologs thereof from other species.
  • the production and use of derivatives and analogs related to TIM are within the scope of the present invention.
  • the derivative or analog is functionally active, i.e., promoting the nuclear entry of PER.
  • TIM derivatives can be made by altering encoding nucleic acid sequences by substitutions, additions or deletions that provide for functionally equivalent molecules.
  • derivatives are made that have enhanced or increased functional activity or greater specificity.
  • nucleotide coding sequences which encode substantially the same amino acid sequence as a TIM gene may be used in the practice of the present invention. These include but are not limited to allelic genes, homologous genes from other species, and nucleotide sequences comprising all or portions of 77 genes which are altered by the substitution of different codons that encode the same amino acid residue within the sequence, thus producing a silent change.
  • the TIM derivatives of the invention include, but are not limited to, those containing, as a primary amino acid sequence, all or part of the amino acid sequence of a TIM protein including altered sequences in which functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a conservative amino acid substitution.
  • Such alterations define the term "a conservative substitution" as used herein.
  • one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity, which acts as a functional equivalent, resulting in a silent alteration.
  • Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs.
  • the nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine.
  • Amino acids containing aromatic ring structures are phenylalanine, tryptophan, and tyrosine.
  • the polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine.
  • the positively charged (basic) amino acids include arginine, lysine and histidine.
  • the negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such alterations will not be expected to affect apparent molecular weight as determined by polyacrylamide gel electrophoresis, or isoelectric point.
  • Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property.
  • a Cys may be introduced at a potential site for disulfide bridges with another Cys.
  • Pro may be introduced because of its particularly planar structure, which induces ⁇ -turns in the protein's structure.
  • TIM derivatives and analogs of the invention can be produced by various methods known in the art. The manipulations which result in their production can occur at the gene or protein level.
  • the cloned TIM gene sequence can be modified by any of numerous strategies known in the art (Sambrook ef al, 1989, supra). The sequence can be cleaved at appropriate sites with restriction endonuclease(s), followed by further enzymatic modification if desired, isolated, and ligated in vitro.
  • the TIM-encoding nucleic acid sequence can be mutated in vitro or in vivo, to create and/or destroy translation, initiation, and/or termination sequences, or to create variations in coding regions and/or form new restriction endonuclease sites or destroy preexisting ones, to facilitate further in vitro modification.
  • mutations enhance the functional activity of the mutated TIM gene product.
  • Any technique for mutagenesis known in the art can be used, including but not limited to, in vitro site-directed mutagenesis (Hutchinson, C, et al, 1978, J. Biol. Chem.
  • the identified and isolated gene can then be inserted into an appropriate cloning vector.
  • vector-host systems known in the art may be used. Possible vectors include, but are not limited to, plasmids or modified viruses, but the vector system must be compatible with the host cell used. Examples of vectors include, but are not limited to, E. coli, bacteriophages such as lambda derivatives, or plasmids such as pBR322 derivatives or pUC plasmid derivatives, e.g., pGEX vectors, pmal-c, pFLAG, etc.
  • the insertion into a cloning vector can, for example, be accomplished by ligating the DNA fragment into a cloning vector which has complementary cohesive termini.
  • the ends of the DNA molecules may be enzymatically modified.
  • any site desired may be produced by ligating nucleotide sequences (linkers) onto the DNA termini; these ligated linkers may comprise specific chemically synthesized oligonucleotides encoding restriction endonuclease recognition sequences.
  • Recombinant molecules can be introduced into host cells via transduction, transformation, transfection, infection, electroporation, etc., so that many copies of the gene sequence are generated.
  • the cloned gene is contained on a shuttle vector plasmid, which provides for expansion in a cloning cell, e.g., E.
  • a shuttle vector which is a vector that can replicate in more than one type of organism, can be prepared for replication in both E. coli and Saccharomyces cerevisiae by linking sequences from an E. coli plasmid with sequences from the yeast 2 ⁇ plasmid.
  • the desired gene may be identified and isolated after insertion into a suitable cloning vector in a "shot gun" approach. Enrichment for the desired gene, for example, by size fractionation, can be done before insertion into the cloning vector.
  • the nucleotide sequence of the human timeless, e.g., SEQ ID NO: 1 or more preferably the amino acid sequence e.g., SEQ ID NO:2, can also be used to search for highly homologous genes from other species, including lower vertebrate species using computer data bases containing partial nucleic acid sequences. Bovine ESTs, for example, can be searched.
  • the human TIMELESS amino acid sequence for example, can be compared with computer translated bovine EST sequences, e.g., in GenBank , using GCG software and the blast search program for example. Matches with highly homologous EST sequences can then be obtained.
  • DNA sequencing reactions can be assembled on a Beckman Biomek robotic system using standard dye-terminator chemistry, Taq polymerase and thermal cycling conditions described by the vendor (Perking Elmer/ Applied Biosystems Division (PE/AB)). Preferably sequencing is performed multiple times to insure accuracy. Reaction products can be resolved on PE/ABD model 373 and 377 automated DNA sequencers. Contig assembly can be performed using any number of programs (e.g., Gap4) and a consensus sequence can be further analyzed using the GCG suite of applications. The resulting sequence can then be used in place of, and/or in conjunction with SEQ ID NO:l, for example, to identify other ESTs which contain coding regions of the bovine homologue to TIM.
  • Plasmids containing the matched ESTs can be digested with restriction enzymes in order to release the cDNA inserts. If the plasmid does not contain the full length ortholog, the digests can be purified, e.g., run on an agarose gel and the bands co ⁇ esponding to the inserts can be cut from the gel and purified (Qiagen Gel Extraction kit). Such purified inserts are likely to contain overlapping regions which can be combined as templates of a PCR reaction using primers which are preferably located outside of the bovine TIM open reading frame.
  • the PCR reaction can be performed by RACE PCR as described in the Example below, or by using ELONGASE (and its standard amplification system) supplied by Gibco-BRL, Gaithersburg, Md, under the following standard conditions: 5 minutes at 94°C; followed by 25 cycles of : 30 seconds at 94°C, 30 seconds at 50°C, and 3.5 minutes at 72°C; followed by 10 minutes at 72°C.
  • Amplification should yield the expected product which can be ligated into a vector and used to transform an E coli derivative via TA cloning (Invitrogen) for example.
  • the resulting full-length bovine TIM for example, can be placed into an expression vector and the expressed recombinant TIM can then be assayed for PER binding activity.
  • Plasmid preps can be performed (e.g., Quiagen Corp, Santa Clarita CA) and the plasmids can be digested by simultaneous restriction digest. Products of the digest can be separated by size on an agarose gel, for example, and purified. The co ⁇ esponding bands cut from these gels can be ligated to form a full-length 77 cDNA and used to transform competent bacteria (DHFalpha) and the resulting plasmid can be purified.
  • DHFalpha transform competent bacteria
  • the present invention provides for expressing the nucleic acids which encode the TIM proteins and TIM fragments, derivatives or analogs, or a functionally active derivative, including a chimeric protein, thereof, that has been inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence. Such elements are termed herein a "promoter.”
  • an appropriate expression vector i.e., a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence.
  • promoter a vector which contains the necessary elements for the transcription and translation of the inserted protein-coding sequence.
  • the nucleic acid encoding a mammalian TIM of the present invention is operationally associated with a promoter in an expression vector of the invention (see Example, below). Both cDNA and genomic sequences can be cloned and expressed under control of such regulatory sequences.
  • An expression vector also preferably includes a replication origin.
  • a TIM protein in large quantities that can be used for functional and structural studies of the purified protein.
  • the necessary transcriptional and translational signals can be provided on a recombinant expression vector, or they may be supplied by the native gene encoding TIM and/or its flanking regions.
  • Potential chimeric partners for the TIM of the present invention include green fluorescent protein which may be useful in monitoring the cellular localization of the TIM or glutathione-S-transferase (GST) as described in the Example, below.
  • Potential host-vector systems include but are not limited to mammalian cell systems, infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors; or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA.
  • virus e.g., vaccinia virus, adenovirus, etc.
  • insect cell systems infected with virus e.g., baculovirus
  • microorganisms such as yeast containing yeast vectors
  • the expression elements of vectors vary
  • a recombinant TIM protein of the invention, or functional fragment, derivative, chimeric construct, or analog thereof, may be expressed chromosomally, after integration of the coding sequence by recombination.
  • any of a number of amplification systems may be used to achieve high levels of stable gene expression (See Sambrook ef al, 1989, supra).
  • the cell containing the recombinant vector comprising the nucleic acid encoding TIM is cultured in an appropriate cell culture medium under conditions that provide for expression of TIM by fhe cell.
  • any of the methods previously described, or described in the Example below, for the insertion of DNA fragments into a cloning vector may be used to construct expression vectors containing a gene consisting of appropriate transcriptional/translational control signals and the protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombination (genetic recombination).
  • TIM may be controlled by any promoter/enhancer element known in the art, but these regulatory elements must be functional in the host selected for expression.
  • Promoters which may be used to control TIM gene expression include, the SV40 early promoter region (Benoist and Chambon, 1981, Nature 290:304-310), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al, 1980, Cell 22:787- 797), the herpes thymidine kinase promoter (Wagner et al, 1981, Proc. Natl. Acad. Sci. U.S.A.
  • prokaryotic expression vectors such as the ⁇ -lactamase promoter (Villa-Kamaroff, et al, 1978, Proc. Natl. Acad. Sci. U.S.A. 75:3727-3731), or the tac promoter (DeBoer, et al, 1983, Proc. Natl. Acad. Sci. U.S.A.
  • mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al, 1986, Cell 45:485-495), albumin gene control region which is active in liver (Pinkert ef al, 1987, Genes and Devel. 1:268- 276), alpha-fetoprotein gene control region which is active in liver (Krumlauf ef al, 1985, Moi. Cell. Biol. 5:1639-1648; Hammer ef al, 1987, Science 235:53-58), alpha 1-antitrypsin gene control region which is active in the liver (Kelsey ef al, 1987, Genes and Devel.
  • beta-globin gene control region which is active in myeloid cells (Mogram ef al, 1985, Nature 315:338-340; Kollias et al, 1986, Cell 46:89-94), myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead ef al, 1987, Cell 48:703-712), myosin light chain-2 gene control region which is active in skeletal muscle (Sani, 1985, Nature 314:283-286), and gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason ef al, 1986, Science 234: 1372-1378).
  • Expression vectors containing a nucleic acid encoding a TIM of the invention can be identified by four general approaches: (a) PCR amplification of the desired plasmid DNA or specific mRNA, (b) nucleic acid hybridization, (c) presence or absence of selection marker gene functions, and (d) expression of inserted sequences.
  • the nucleic acids can be amplified by PCR to provide for detection of the amplified product.
  • the presence of a foreign gene inserted in an expression vector can be detected by nucleic acid hybridization using probes comprising sequences that are homologous to an inserted marker gene.
  • the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "selection marker" gene functions (e.g., ⁇ -galactosidase activity, thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.) caused by the insertion of foreign genes in the vector.
  • selection marker e.g., ⁇ -galactosidase activity, thymidine kinase activity, resistance to antibiotics, transformation phenotype, occlusion body formation in baculovirus, etc.
  • recombinant expression vectors can be identified by assaying for the activity, biochemical, or immunological characteristics of the gene product expressed by the recombinant, provided that the expressed protein assumes a functionally active conformation. For example, the binding activity of TIM for PER can be tested.
  • a wide variety of host/expression vector combinations may be employed in expressing the DNA sequences of this invention.
  • Useful expression vectors may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences.
  • Suitable vectors include derivatives of SV40 and known bacterial plasmids, e.g., E.
  • coli plasmids col El, pCRl, pBR322, pMal-C2, pET, pGEX (Smith ef al, 1988, Gene 67:31-40), pMB9 and their derivatives, plasmids such as RP4; phage DNAS, e.g., the numerous derivatives of phage ⁇ , e.g., NM989, and other phage DNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2 ⁇ plasmid or derivatives thereof; vectors useful in eukaryotic cells, such as vectors useful in insect or mammalian cells; vectors derived from combinations of plasmids and phage DNAs, such as plasmids that have been modified to employ phage DNA or other expression control sequences; and the like.
  • phage DNAS e.g., the numerous derivatives of phage ⁇ , e.g.
  • both non-fusion transfer vectors such as but not limited to pVL941 (Ban ⁇ l cloning site; Summers), pVL1393 (BamHl, Smal, Xbal, EcoRl, Notl, Xmalll, Bglll, and Pstl cloning site; Invitrogen), pVL1392 (Bglll, Pstl, Notl, X ⁇ lll, EcoRl, Xbal, Smal, and BamHl cloning site; Summers and Invitrogen), and pBlueZtacIII (BamHl, Bglll, Pstl, Ncol, and Hindlll cloning site, with blue/white recombinant screening possible; Invitrogen), and fusion transfer vectors, such as but not limited to pAc700 (BamHl and Kp cloning site, in which the BamHl recognition site begins
  • Mammalian expression vectors contemplated for use in the invention include vectors with inducible promoters, such as the dihydrofolate reductase (DHFR) promoter, e.g., any expression vector with a DHFR expression vector, or a D/ R/methotrexate co-amplification vector, such as pED (Pstl, Sail, Sbal, Smal, and EcoRl cloning site, with the vector expressing both the cloned gene and DHFR; see Kaufman, Current Protocols in Molecular Biology, 16.12 (1991).
  • DHFR dihydrofolate reductase
  • a glutamine synthetase/methionine sulfoximine co- amplification vector such as pEE14 (Hindlll, Xbal, Smal, Sbal, EcoRl, and Bell cloning site, in which the vector expresses glutamine synthase and the cloned gene; Celltech).
  • a vector that directs episomal expression under control of Epstein Barr Virus can be used, such as pREP4 (BamHl, Sfil, Xhol, Notl, Nhel, Hindlll, Nhel, Pvull, and Kpnl cloning site, constitutive RSV-LTR promoter, hygromycin selectable marker; Invitrogen), pCEP4 (BamHl, Sfil, Xhol, Notl, Nhel, Hindlll, Nhel, Pvull, and Kpnl cloning site, constitutive hCMV immediate early gene, hygromycin selectable marker; Invitrogen), pMEP4 (Kpnl, Pvul, Nhel, Hindlll, Notl, Xhol, Sfil, BamHl cloning site, inducible methallothionein Ha gene promoter, hygromycin selectable marker: Invitrogen), pREP8 (BamHl, Sfil
  • Selectable mammalian expression vectors for use in the invention include pRc/CMV (Hindlll, BstXl, Notl, Sbal, and Apal cloning site, G418 selection; Invitrogen), pRc/RSV (Hindlll, Spel, BstXl, Notl, Xbal cloning site, G418 selection; Invitrogen), and others.
  • Vaccinia virus mammalian expression vectors for use according to the invention include but are not limited to pSCl 1 (Smal cloning site, TK- and ⁇ -gal selection), pMJ601 (Sail, Smal, A/71, Narl, BspMll, BamHl, Apal, Nhel, Sac ⁇ l, Kpnl, and Hindlll cloning site; TK- and ⁇ -gal selection), and pTKgptFIS (EcoRl, Pstl, Sail, Accl, Hindll, Sbal, BamHl, and Hpa cloning site, TK or XPRT selection).
  • pSCl 1 Mal cloning site, TK- and ⁇ -gal selection
  • pMJ601 Smal, A/71, Narl, BspMll, BamHl, Apal, Nhel, Sac ⁇ l, Kpnl, and Hindlll cloning site
  • Yeast expression systems can also be used according to the invention to express the TIM protein.
  • the non-fusion pYES2 vector (Xbal, Sphl, Shol, Notl, GstXl, £c ⁇ RI, BstXl, BamHl, Sacl, Kpnl, and Hindlll cloning sit; Invitrogen) or the fusion pYESHisA, B, C (Xbal, Sphl, Shol, Notl, BstXl, EcoRl, BamHl, Sacl, Kpnl, and Hindlll cloning site, N- terminal peptide purified with ProBond resin and cleaved with enterokinase; Invitrogen), to mention just two, can be employed according to the invention.
  • the expression vectors which can be used include, but are not limited to, the following vectors or their derivatives: human or animal viruses such as vaccinia virus or adenovirus; insect viruses such as baculovirus; yeast vectors; bacteriophage vectors (e.g., lambda), and plasmid and cosmid DNA vectors, to name but a few.
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired.
  • Different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation, cleavage [e.g., of signal sequence]) of proteins.
  • Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the foreign protein expressed.
  • expression in a bacterial system can be used to produce an nonglycosylated core protein product.
  • TIM expressed in bacteria may not be properly folded.
  • Expression in yeast can produce a glycosylated product.
  • Expression in eukaryotic cells can increase the likelihood of "native" glycosylation and folding of a heterologous protein.
  • expression in mammalian cells can provide a tool for reconstituting, or constituting, TIM activity.
  • different vector/host expression systems may affect processing reactions, such as proteolytic cleavages, to a different extent.
  • Vectors are introduced into the desired host cells by methods known in the art, e.g., transfection, transduction, electroporation, microinjection, cell fusion, DEAE dextran, calcium phosphate precipitation, lipofection (lysosome fusion), use of a gene gun, or a DNA vector transporter (see, e.g., Wu et al, 1992, J. Biol. Chem. 267:963-967; Wu and Wu, 1988, J. Biol. Chem. 263: 14621-14624; Hartmut et al, Canadian Patent Application No. 2,012,311, filed March 15, 1990).
  • the present invention also provides cell lines made from cells transfected or transduced with the TIMs of the present invention.
  • the cells are mammalian cells.
  • the cells are murine cells.
  • prefe ⁇ ed embodiments the cells are human cells.
  • the initial step for purifying the TIMs of the present invention, TIM fragments and related tagged or fusion proteins generally consists of lysing the cells containing the TIMs.
  • Cell lysis can be achieved by a number of methods including through the use of a physical means such as a French press, a sonicator, or a blender; or through chemical means including enzymatic extractions (with for example, lysozyme or pancreatin), and/or organic extractions or solubilizations with detergents, such as sodium dodecyl sulfate (SDS), Triton X-100, nonidet P-40 (NP-40), digoxin, sodium deoxycholate, and the like, including mixtures thereof; or through a combination of chemical and physical means.
  • SDS sodium dodecyl sulfate
  • NP-40 nonidet P-40
  • solubilization can be enhanced by sonication of the suspension.
  • Subsequent steps of purification include salting in or salting out, such as in ammonium sulfate fractionations; solvent exclusion fractionations, e.g., an ethanol precipitation; detergent extractions to free the membrane bound TIMs (if any) of the present invention using such detergents as Triton X-100, Tween-20 etc.; or high salt extractions.
  • Solubilization of proteins may also be achieved using aprotic solvents such as dimethyl sulfoxide and hexamethylphosphoramide.
  • high speed ultracentrifugation may be used either alone or in conjunction with other extraction techniques.
  • Solid phase binding may be performed through ionic bonding, with either an anion exchanger, such as diethylaminoethyl (DEAE), or diethyl [2-hydroxypropyl] aminoethyl (QAE) SEPHADEX or cellulose; or with a cation exchanger such as carboxymethyl (CM) or sulfopropyl (SP) SEPHADEX or cellulose.
  • an anion exchanger such as diethylaminoethyl (DEAE), or diethyl [2-hydroxypropyl] aminoethyl (QAE) SEPHADEX or cellulose
  • a cation exchanger such as carboxymethyl (CM) or sulfopropyl (SP) SEPHADEX or cellulose.
  • Solid phase binding includes the exploitation of hydrophobic interactions e.g., the using of a solid support such as PHENYLSEPHAROSE and a high salt buffer; affinity-binding, using, e.g., placing a nucleoside or nucleoside analog on to an activated support; immuno-binding, using e.g., an antibody to a TIM of the present invention bound to an activated support; as well as other solid phase supports including those that contain specific dyes or lectins etc.
  • a further solid phase support technique that is often used at the end of the purification procedure relies on size exclusion, such as SEPHADEX and SEPHAROSE gels, or pressurized or centrifugal membrane techniques, using size exclusion membrane filters.
  • Solid phase support separations are generally performed batch-wise with low-speed centrifugations or by column chromatography.
  • High performance liquid chromatography HPLC
  • FPLC FPLC
  • Size exclusion techniques may also be accomplished with the aid of low speed centrifugation.
  • size permeation techniques such as gel electrophoretic techniques may be employed. These techniques are generally performed in tubes, slabs or by capillary electrophoresis.
  • Typical buffers can be purchased from most biochemical catalogues and include the classical buffers such as Tris, pyrophosphate, monophosphate and diphosphate.
  • the Good buffers [Good, et al, Biochemistry, 5:467 (1966); Good ef al. Meth. EnzymoL, 24: Part B, 53 (1972) ; and
  • Fergunson et. al Anal. Biochem. 104:300,(1980)] such as Mes, Hepes, Mops, tricine and Ches.
  • a mammalian TIM protein obtained from a natural source or produced recombinantly or by chemical synthesis, and fragments or other derivatives or analogs thereof, including fusion proteins may be used as an immunogen to generate antibodies that recognize the mammalian TIM polypeptide.
  • Such antibodies include but are not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, and an Fab expression library.
  • the anti-TIM antibodies of the invention may be cross reactive, e.g., they may recognize a TIM from different species. Polyclonal antibodies have greater likelihood of cross reactivity.
  • an antibody of the invention may be specific for a single form of the TIM, such as human TIM.
  • a TIM or a derivative e.g., fragment or fusion protein
  • various host animals can be immunized by injection with a TIM or a derivative (e.g., fragment or fusion protein) thereof, including but not limited to rabbits, mice, rats, sheep, goats, etc.
  • a TIM or fragment thereof can be conjugated to an immunogenic carrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH).
  • BSA bovine serum albumin
  • KLH keyhole limpet hemocyanin
  • adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
  • BCG Bacille Calmette-Guerin
  • Corynebacterium parvum bacille Calmette-Guerin
  • any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. These include but are not limited to the hybridoma technique originally developed by Kohler and Milstein [Nature 256:495-497 (1975)], as well as the trioma technique, the human B-cell hybridoma technique [Kozbor et al, Immunology Today 4:72 1983); Cote et al, Proc. Natl. Acad. Sci. U.S.A.
  • monoclonal antibodies can be produced in germ-free animals utilizing recent technology [PCT/US90/02545].
  • techniques developed for the production of "chimeric antibodies" [Morrison et al, J. Bacteriol.
  • U.S. Patent 4,946,778 can be adapted to produce TIM-specific single chain antibodies.
  • An additional embodiment of the invention utilizes the techniques described for the construction of Fab expression libraries [Huse et al, Science 246: 1275-1281 (1989)] to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for a TIM or its derivatives, or analogs.
  • Antibody fragments which contain the idiotype of the antibody molecule can be generated by known techniques.
  • such fragments include but are not limited to: the F(ab') 2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab') 2 fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.
  • screening for the desired antibody can be accomplished by techniques known in the art, e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), Western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.
  • radioimmunoassay e.g., ELISA (enzyme-linked immunosorbant assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitin reactions, immunodiffusion assays, in situ immunoa
  • antibody binding is detected by detecting a label on the primary antibody.
  • the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
  • the secondary antibody is labeled.
  • Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention. For example, to select antibodies which recognize a specific epitope of a TIM, for example the kinase catalytic site, one may assay generated hybridomas for a product which binds to a TIM fragment containing such an epitope.
  • an antibody specific to a TIM protein For selection of an antibody specific to a TIM protein from a particular species of animal, one can select on the basis of positive binding with a mammalian TIM expressed by or isolated from cells of that species of animal.
  • the foregoing antibodies can be used in methods known in the art relating to the localization and activity of the TIM, e.g., for Western blotting, imaging TIM in situ, measuring levels thereof in appropriate physiological samples, etc. using any of the detection techniques mentioned above or known in the art. More particularly, the antibodies of the present invention can be used in flow cytometry studies, in immunohistochemical staining, and in immunoprecipitation which serves to aid the determination of the level of expression of a TIM in a tumor or normal cell or tissue.
  • antibodies that agonize or antagonize the activity of a mammalian TIM can be generated. Such antibodies can be tested using the assays described herein.
  • Identification of the TIM protein provides a basis for screening for drugs capable of specific interaction with the functionally relevant aspects of the protein.
  • an agonist or antagonist can be identified that stimulate or inhibit the promoting of nuclear entry of PER by the TIM protein. Since TIM plays an important role in circadian rhythms such agonists or antagonists can be used in treating disorders related to biological clocks. Accordingly, in addition to rational design of compounds that bind to mammalian TIM, the present invention contemplates an alternative method for identifying specific agents that bind to TIM using the various screening assays known in the art.
  • any screening technique known in the art can be used to screen for agonists or antagonists to the mammalian TIM protein.
  • the present invention contemplates screens for small molecule ligands or ligand analogs and mimics, as well as screens for natural ligands that bind to and antagonize TIM in vivo.
  • Another approach uses recombinant bacteriophage to produce large libraries. Using the "phage method" [Scott and Smith, 1990, Science 249:386-390 (1990); Cwirla, ef al, Proc. Natl. Acad.
  • the screening can be performed directly using peptides co ⁇ esponding to the CLD domain or PER binding domain of mammalian TIM.
  • chimeric proteins which contain the PER binding domain of mammalian TIM may be used, as such proteins will contain one element under investigation.
  • Screening can be performed with recombinant cells that express the mammalian TIM protein, or alternatively, using purified protein, and/or specific structural/functional domains of the mammalian TIM protein e.g., produced recombinantly, as described above.
  • a labeled mammalian TIM protein can be used to screen libraries, as described in the foregoing references for small molecules that will inhibit the PER binding activity of the mammalian TIM protein.
  • the effective peptide(s) can be synthesized in large quantities for use in in vivo models and eventually in humans to modulate the TIM protein. It should be emphasized that synthetic peptide production is relatively non-labor intensive, easily manufactured, quality controlled and thus, large quantities of the desired product can be produced quite cheaply. Similar combinations of mass produced synthetic peptides have recently been used with great success [Pata ⁇ oyo, Vaccine 10:175-178 (1990)].
  • the reagents that contain the mammalian TIM protein or TIM fragments can be labeled for use in the screening assays.
  • the compound may be directly labeled including as part of a fusion protein, e.g., with green fluorescent protein.
  • a labeled secondary reagent may be used to detect binding of the compound to a solid phase support containing a binding molecule of interest. Binding may be detected by in situ formation of a chromophore by an enzyme label. Suitable enzymes include, but are not limited to, alkaline phosphatase and horseradish peroxidase.
  • labels for use in the invention include colored latex beads, magnetic beads, fluorescent labels (e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE), Texas red (TR), rhodamine, Lucifer Yellow, AMCA blue, free or chelated lanthanide series salts, especially Eu 3+ , to name a few fluorophores), chemiluminescent molecules, radio-isotopes, or magnetic resonance imaging labels.
  • fluorescent labels e.g., fluorescein isothiocyanate (FITC), phycoerythrin (PE), Texas red (TR), rhodamine, Lucifer Yellow, AMCA blue, free or chelated lanthanide series salts, especially Eu 3+ , to name a few fluorophores
  • chemiluminescent molecules chemiluminescent molecules
  • radio-isotopes or magnetic resonance imaging labels.
  • an assay useful and contemplated in accordance with the present invention is known as a "cis/trans” assay. Briefly, this assay employs one or more constructs, which encode: a PER and a mammalian TIM and a reporter gene that is under the positive control of the heterodimeric transcription factor CLOCK-BMALl, which is expressed by the test cell line. (One such system which uses the three E-boxes of the 5' flanking region of the mPerl gene is included in the Example, below).
  • the contruct(s) are transfected into an appropriate cell line (e.g., NIH 3T3 cells, 293 cells, COS cells, Drosphila 52 cells) and the expression of the reporter (such as luciferase, or green fluorescent protein) can be monitored.
  • This assay may be performed to identify antagonists and agonists to the mammalian TIM- PER heterodimer.
  • a constuct is used that possesses a promoter linked to the reporter gene (e.g., luciferase) in which a response element is inserted that is ultimately under the control of the PER-Tim dimer (e.g., a response element under the control of the CLOCK-BMALl heterodimeric transcription factor as described in the Example, below).
  • the resulting signal chemiluminescence in this example
  • dose response curves are obtained and compared to those in which the agonist is not included in the assay.
  • TIM-PER heterodimer Since the mammalian TIM protein in conjunction with PER serves to inhibit the CLOCK-BMALl heterodimeric transcription factor, and therefore inhibit transcription of the reporter gene, an agonist for the TIM-PER heterodimer should cause a decrease in the transcription of a reporter gene. Similarly an antagonist of the TIM-PER heterodimer should cause a increase in the transcription of the reporter gene. Protocols somewhat analogous to the one presented above can be found U.S. Patent No. 4,981,784 and PCT International Publication No. WO 88/03168, for which purpose the artisan is refe ⁇ ed. Additional assays that can be used to test potential agents/drugs can be readily adapted from the assays described below in the Examples and above in the Brief Description of the Drawings.
  • Suitable labels include enzymes and proteins such as green fluorescent protein, fluorophores (e.g., fluorescene isothiocyanate (FITC), phycoerythrin (PE), Texas red (TR), rhodamine, free or chelated lanthanide series salts, especially Eu 3+ , to name a few fluorophores), chromophores, radioisotopes, chelating agents, dyes, colloidal gold, latex particles, ligands (e.g., biotin), and chemiluminescent agents.
  • fluorophores e.g., fluorescene isothiocyanate (FITC), phycoerythrin (PE), Texas red (TR), rhodamine, free or chelated lanthanide series salts, especially Eu 3+ , to name a few fluorophores
  • chromophores e.g., radioisotopes, chelating agents, dyes, colloidal gold, latex particles,
  • radioactive label such as the isotopes 3 H, 14 C, 32 P, 35 S, 36 C1, 51 Cr, 57 Co, 58 Co, 59 Fe, 90 Y, 125 I, 131 I, and 186 Re
  • known cu ⁇ ently available counting procedures may be utilized.
  • detection may be accomplished by any of the presently utilized colorimetric, spectrophotometric, fluorospectrophotometric, amperometric or gasometric techniques known in the art.
  • Direct labels are one example of labels which can be used according to the present invention.
  • a direct label has been defined as an entity, which in its natural state, is readily visible, either to the naked eye, or with the aid of an optical filter and/or applied stimulation, e.g. U.V. light to promote fluorescence.
  • colored labels include metallic sol particles, for example, gold sol particles such as those described by Leuvering (U.S. Patent 4,313,734); dye sole particles such as described by Gribnau et al. (U.S. Patent 4,373,932) and May et al.
  • direct labels include a radionucleotide, a fluorescent moiety or a luminescent moiety.
  • indirect labels comprising enzymes can also be used according to the present invention.
  • enzyme linked immunoassays are well known in the art, for example, alkaline phosphatase and horseradish peroxidase, lysozyme, glucose-6- phosphate dehydrogenase, lactate dehydrogenase, urease, these and others have been discussed in detail by Eva Engvall in Enzyme Immunoassay ELISA and EMIT in Methods in Enzymology, 70. 419-439, 1980 and in U.S. Patent 4,857,453.
  • Suitable enzymes include, but are not limited to, alkaline phosphatase and horseradish peroxidase.
  • labels for use in the invention include magnetic beads or magnetic resonance imaging labels.
  • a phosphorylation site can be created on an antibody of the invention for labeling with 32 P, e.g., as described in European Patent No. 0372707 (application No. 89311108.8) by Sidney Pestka, or U.S. Patent No. 5,459,240, issued October 17, 1995 to Foxwell ef al.
  • Proteins including the mammalian TIMs of the present invention and antibodies thereto, can be labeled by metabolic labeling.
  • Metabolic labeling occurs during in vitro incubation of the cells that express the protein in the presence of culture medium supplemented with a metabolic label, such as [ 35 S]-methionine (as described below in the Example) or [ 32 P]- orthophosphate.
  • a metabolic label such as [ 35 S]-methionine (as described below in the Example) or [ 32 P]- orthophosphate.
  • the invention further contemplates labeling with [ 14 C]-amino acids and [ 3 H]-amino acids (with the tritium substituted at non-labile positions).
  • a solid phase support for use in the present invention will be inert to the reaction conditions for binding.
  • a solid phase support for use in the present invention must have reactive groups in order to attach a binding partner, such as an oligonucleotide encoding a mammalian TIM, a mammalian TIM, or an antibody to a mammalian TIM, or for attaching a linker or handle which can serve as the initial binding point for any of the foregoing.
  • the solid phase support may be a useful chromatographic support, such as the carbohydrate polymers SEPHAROSE, SEPHADEX, agarose and agarose beads (as described in the Example below).
  • a solid phase support is not limited to a specific type of support.
  • Solid phase supports include silica gels, resins, derivatized plastic films, glass beads, cotton, plastic beads, alumina gels, magnetic beads, membranes (including but not limited to nitrocellulose, cellulose, nylon, and glass wool), plastic and glass dishes or wells, etc.
  • solid phase supports used for peptide or oligonucleotide synthesis can be used, such as polystyrene resin (e.g., PAM-resin obtained from Bachem Inc., Peninsula Laboratories, etc.), POLYHIPE® resin (obtained from Aminotech, Canada), polyamide resin (obtained from Peninsula Laboratories), polystyrene resin grafted with polyethylene glycol (TentaGel®, Rapp Polymere, Tubingen, Germany) or polydimethylacrylamide resin (obtained from Milligen Biosearch, California).
  • a silica based solid phase support may be prefe ⁇ ed.
  • Silica based solid phase supports are commercially available (e.g., from Peninsula Laboratories, Inc.; and Applied Biosystems, Inc.)
  • the functional activity of the mammalian TIM protein can be evaluated transgenically.
  • a transgenic mouse model is used.
  • a mammalian TIM gene for example, can be used in complementation studies employing transgenic mice.
  • Transgenic vectors including viral vectors, or cosmid clones (or phage clones) co ⁇ esponding to the wild type locus of candidate gene, can be constructed using the isolated TIM gene. Cosmids may be introduced into transgenic mice using published procedures [Jaenisch, Science, 240:1468-1474 (1988)].
  • a transgenic animal model can be prepared in which expression of the mammalian tim gene is either prevented or altered due to a disruption in its co ⁇ esponding gene.
  • Such alterations can be made in any non-human animal including mice, and therefore such animals with altered 77M alleles are also part of the present invention. Altering a single allele may be preferable in certain cases since a single alteration can be dominant, and disruption of both alleles could potentially be lethal.
  • Gene expression is disrupted, according to the invention, when no functional protein is expressed. Knock-out technology to delete a gene is described in U.S. Patents 5,464,764, Issued 11/7/95; and 5,777,195, Issued July 7, 1998 (both of which are hereby incorporated by reference herein in their entireties.)
  • the present invention also extends to the preparation of antisense nucleotides and ribozymes that may be used to interfere with the expression of the mammalian tim gene.
  • This approach utilizes antisense nucleic acid and ribozymes to block translation of a specific mRNA, either by masking that mRNA with an antisense nucleic acid or cleaving it with a ribozyme.
  • Antisense nucleic acids are DNA or RNA molecules that are complementary to at least a portion of a specific mRNA molecule [See Weintraub, Sci. Amer. 262:40-46 (1990); Marcus-Sekura, Nucl. Acid Res, 15: 5749-5763 (1987); Marcus-Sekura Anal.Biochem., 172:289-295 (1988); Brysch et al, Cell Moi Neurobiol, 14:557-568 (1994)].
  • the antisense molecule employed is complementary to a substantial portion of the mRNA. In the cell, the antisense molecule hybridizes to that mRNA, forming a double stranded molecule.
  • RNA DNA duplex is a prefe ⁇ ed substrate for RNase H. Oligomers of greater than about fifteen nucleotides and molecules that hybridize to the AUG initiation codon will be particularly efficient, though larger molecules that are essentially complementary to the entire mRNA are more likely to be effective. Antisense methods have been used to inhibit the expression of many genes in vitro [Marcus-Sekura, Anal.Biochem., 172:289-295 (1988); Hambor ef al, Proc. Natl Acad. Sci.
  • Ribozymes are RNA molecules possessing the ability to specifically cleave other single stranded RNA molecules in a manner somewhat analogous to DNA restriction endonucleases. Ribozymes were discovered from the observation that certain mRNAs have the ability to excise their own introns. By modifying the nucleotide sequence of these ribozymes, researchers have been able to engineer molecules that recognize specific nucleotide sequences in an RNA molecule and cleave it [Cech, JAMA, 260:3030-3034 (1988); Cech, Biochem. Intl, 18:7-14 (1989)] . Because they are sequence-specific, only mRNAs with particular sequences are inactivated.
  • Tetrahymena-type ribozymes recognize four-base sequences, while "hammerhead”-type recognize eleven- to eigh teen-base sequences. The longer the recognition sequence, the more likely it is to occur exclusively in the target mRNA species. Therefore, hammerhead-type ribozymes are preferable to Tetrahymena-type ribozymes for inactivating a specific mRNA species, and eighteen base recognition sequences are preferable to shorter recognition sequences.
  • antisense molecules can be prepared against the DNA sequences of mammalian TIM described herein, and in addition, ribozymes that cleave mRNAs encoding the mammalian TIM proteins of the present invention can readily constructed.
  • a gene encoding a mammalian TIM protein is introduced in vivo in a viral vector.
  • viral vectors include an attenuated or defective DNA virus, such as but not limited to herpes simplex virus (HSV), papillomavirus, Epstein Ban virus (EBV), adenovirus, adeno-associated virus (AAV), or a defective retrovirus such as HIV.
  • HSV herpes simplex virus
  • EBV Epstein Ban virus
  • AAV adeno-associated virus
  • Defective viruses which entirely or almost entirely lack viral genes, are prefe ⁇ ed. Defective virus is not infective after introduction into a cell.
  • Use of defective viral vectors allows for administration to cells in a specific, localized area, without concern that the vector can infect other cells. Thus, any tissue can be specifically targeted.
  • HSV1 vector herpes virus 1
  • attenuated adenovirus vector such as the vector described by Stratford-Perricaudet ef al. [J. Clin. Invest. 90:626-630 (1992)]
  • a defective adeno-associated virus vector such as the vector described by Stratford-Perricaudet ef al. [J. Clin. Invest. 90:626-630 (1992)
  • a defective adeno-associated virus vector Samulski et al, J. Virol. 61:3096-3101 (1987); Samulski ef al, J. Virol 63:3822-3828 (1989)].
  • an appropriate immunosuppressive treatment may be employed in conjunction with the viral vector, e.g., adenovirus vector, to avoid immuno-deactivation of the viral vector and transfected cells.
  • the viral vector e.g., adenovirus vector
  • immunosuppressive cytokines such as interleukin-12 (IL-12), interferon- ⁇ (IFN- ⁇ ), or anti-CD4 antibody
  • IL-12 interleukin-12
  • IFN- ⁇ interferon- ⁇
  • anti-CD4 antibody can be administered to block humoral or cellular immune responses to the viral vectors [see, e.g., Wilson, Nature Medicine (1995)].
  • the gene can be introduced in a retroviral vector, e.g., as described in Anderson et al, U.S. Patent No. 5,399,346; Mann et al, Cell 33:153 (1983); Temin ef al, U.S. Patent No. 4,650,764; Temin ef al, U.S. Patent No. 4,980,289; Markowitz et al, J. Virol. 62: 1120 (1988); Temin et al, U.S. Patent No. 5,124,263; Dougherty et al, International Patent Publication No. WO 95/07358, published March 16, 1995; and Kuo ef al, Blood 82:845 (1993)].
  • a retroviral vector e.g., as described in Anderson et al, U.S. Patent No. 5,399,346; Mann et al, Cell 33:153 (1983); Temin ef al, U.S. Patent No. 4,650,764; Temin
  • the vector can be introduced in vivo by lipofection.
  • liposomes for encapsulation and transfection of nucleic acids in vitro.
  • Synthetic cationic lipids designed to limit the difficulties and dangers encountered with liposome mediated transfection can be used to prepare liposomes for in vivo transfection of a gene encoding a marker [Feigner, ef. al, Proc. Natl Acad. Sci. U.S.A. 84:7413-7417 (1987); see Mackey, ef al, Proc. Natl. Acad. Sci. U.S.A. 85:8027-8031 (1988)].
  • cationic lipids may promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged cell membranes [Feigner and Ringold, Science 337:387-388 (1989)].
  • lipofection to introduce exogenous genes into the specific organs in vivo has certain practical advantages. Molecular targeting of liposomes to specific cells represents one area of benefit. It is clear that directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as pancreas, liver, kidney, and the brain.
  • Lipids may be chemically coupled to other molecules for the purpose of targeting [see Mackey, ef al, Proc. Natl. Acad. Sci. U.S.A. 85:8027-8031 (1988)].
  • Targeted peptides e.g., hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.
  • naked DNA vectors for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun, or use of a DNA vector transporter [see, e.g., Wu ef al, J. Biol. Chem. 267:963-967 (1992); Wu and Wu, /. Biol. Chem. 263:14621-14624 (1988); Hartmut et al, Canadian Patent Application No. 2,012,311, filed March 15, 1990].
  • the nucleic acids encoding mammalian TIM and mammalian TIM fragments and the proteins and fragments encoded thereby can be used in the treatment of numerous sleep-related disorders, including depression, narcolepsy and other mental disorders linked to the sleep- wake cycle. These proteins and nucleic acids can also be used in the treatment of jet lag. Thus, in instances where it is desired to increase the transcription of per an appropriate inhibitor of TIM binding to PER could be introduced to thereby aid in the CLOCK-BMALl - dependent transcription of the per gene.
  • Mammalian TIM or a binding partner or agents exhibiting either mimicry or antagonism to mammalian TIM, or control over its production may be prepared in pharmaceutical compositions, with a suitable ca ⁇ ier and at a strength effective for administration by various means to a patient experiencing an adverse medical condition associated with PER- TIM, CLOCK-BMALl system.
  • a variety of administrative techniques may be utilized, among them parenteral techniques such as subcutaneous, intravenous and intraperitoneal injections, catheterizations and the like. The precise doses used should be based upon the recommendations and prescription of a qualified physician or veterinarian.
  • antibodies including both polyclonal and monoclonal antibodies, and drugs that modulate the production or activity of the mammalian TIM protein may possess certain diagnostic applications and may for example, be utilized for the purpose of detecting and/or measuring conditions such as to classify groups of individuals with sleep-related disorders, in order to better treat the disorders.
  • mammalian TIM may be used to produce both polyclonal and monoclonal antibodies in a variety of cellular media, by known techniques such as the hybridoma technique utilizing, for example, fused mouse spleen lymphocytes and myeloma cells.
  • a subject therapeutic composition includes, in admixture, a pharmaceutically acceptable excipient (carrier) and mammalian TIM, a polypeptide analog thereof or fragments thereof, as described herein as an active ingredient.
  • the composition comprises an antigen capable of modulating the specific introduction of TIM into a target cell.
  • compositions which contain polypeptides, analogs or active fragments as active ingredients are well understood in the art.
  • such compositions are prepared as injectables, either as liquid solutions or suspensions, however, solid forms suitable for solution in, or suspension in, liquid prior to injection can also be prepared.
  • the preparation can also be emulsified.
  • the active therapeutic ingredient is often mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient. Suitable excipients are, for example, water, saline, dextrose, glycerol, ethanol, or the like and combinations thereof.
  • the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents which enhance the effectiveness of the active ingredient.
  • a polypeptide, analog or protein fragment can be formulated into the therapeutic composition as neutralized pharmaceutically acceptable salt forms.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed from the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropyl amine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.
  • the therapeutic polypeptide-, analog- or active fragment-containing compositions are may be administered intravenously, as by injection of a unit dose, for example.
  • unit dose when used in reference to a therapeutic composition of the present invention refers to physically discrete units suitable as unitary dosage for humans, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required diluent; i.e., carrier, or vehicle.
  • the present invention may be better understood by reference to the following non-limiting Example, which is provided as exemplary of the invention. The following example is presented in order to more fully illustrate the prefe ⁇ ed embodiments of the invention. It should in no way be construed, however, as limiting the broad scope of the invention.
  • Drosophila timeless gene was needed in order to complete the picture.
  • a database search was initiated for mammalian ESTs that co ⁇ espond to such a homolog.
  • the identification and subsequent cloning of the mouse and human genes, mTim and hTim, that share extensive sequence homology with dTIM was performed. Functional evidence that these mammalian Tim genes encode orthologs of the Drosophila timeless gene was obtained.
  • hTIM and mPERl are shown to negatively regulate transcription of the mPerl promoter; thus, closing the mammalian circadian loop.
  • EST Clones Drosophila melanogaster TIM protein sequence (GenBank Accession #AF032401) was used to search the EST database using the TBLASTN algorithm. Clones of interest were ordered from Research Genetics and sequenced. Full insert EST sequences were used as queries to search the EST database using the BLASTN and TBLASTX algorithms in order to identify overlapping EST clones. This process was repeated with each identified clone until all EST clones co ⁇ esponding to the mTim and hTim genes were identified.
  • cDNA library Screening Genetrapper library screen. - The Gibco-BRL Genetrapper Positive Selection System was used to screen a mouse brain plasmid library (Life Technologies). cDNA capture and second strand repair were carried out using the primer 5ttrapl: 5'-ctacagctcagatctgggaaagc-3 ⁇ an oligonucleotide designed from sequence of mTim5' A2. Screening was ca ⁇ ied out as described in the protocol supplied by Gibco-BRL. This yielded one clone, TMBGTAE03. This clone was primed from the same internal priming sequence from which 534423 originates.
  • Phage library screens A Unizap XR Human retinal cDNA library (Stratagene) was screened using 746219 insert as a probe by hybridization in 50% formamide, 10% Dextran sulphate, IM NaCl, 1% SDS and lOOmg/ml sheared salmon sperm DNA and washing in 2X SSC 0.1% SDS for 30 minutes at room temperature, then three times in 0.2X SSC 0.1% SDS at 65°C for 45 minutes.
  • a 1-gtl 1 human hypothalamic cDNA library was also screened with a dual probe from the 746219 and 417249 inserts in the same manner. Two independent clones from the human retinal library were identified using the 746219 insert as the probe. These clones were no larger than the 746219 clone and were also internally primed from the A-rich sequence. The human hypothalamic cDNA library yielded one short clone that contained no additional sequence.
  • Arrayed cDNA library screens Master plates of the human placenta cDNA library and mouse embryonic cDNA library from Origene Technologies were screened by PCR. Mouse library screening was performed with primers mTim3GSP3 and mTim3GSP5 (sequence below). Primers for the human library screen were as follows: 417249.4: 5'- tggagctgttgttctggaag-3', 417249.5.1: 5'-atatgacccaggacatcatctga-3'. Positive subplates (subplate number 5E for the human library and subplate number 1 IH for the mouse library) were ordered and screened by PCR to identify positive subwells.
  • Nested primers were used to increase specificity of amplified products for clones co ⁇ esponding to hTim and mTim.
  • Mtim5nsp5 5 ' -gaaagagcgccaggaatagttctcg-3 '
  • Mtim5gsp3 5'-ctggccacggtgaacgagatg-3'
  • Mtim3gsp3 5 ' -ccgtgaaccagaaagcgtttgtgg-3 ' Mtim3nsp3: 5'-ggagctgctgttctggaagaacacc-3'
  • HstimGSP5 5 ' -agctaagcgtccctgccctactcc-3 '
  • HstimNSP5 5 ' -gggttctggtcacgaaacataaggg-3 ' .
  • 3' RACE was carried out originating from 315895 sequence using gene specific primers m7 3GSP3 and m7 m3NSP3 and six clones were isolated. Four clones appeared to be full-length with 2 CAG splice variations alternatively present or absent (see text). A lkb gap in the mouse cDNA was filled by PCR with primers w7»»3gsp5 and m77m5gsp3 to bridge this gap.
  • Northern blot Analysis Multiple tissue northern blots were purchased from Clontech. A probe for the human MTN blots was generated by random priming the original IMAGE clone 746219 insert using Pharmacia Ready-to-Go DNA labeling beads. A probe for the mouse embryonic and multiple tissue blots was generated from the mTim 5' Al clone. The blots were hybridized in Express Hybridization Solution (Clontech) and were washed according to manufacturer's protocols.
  • mice 60 [(C3H/cJ X C57BL/6J) FI X C57BL/6J] N 2 mice were used as a mapping panel.
  • 72 SSLP markers between the C57BL/6J and C3H/cJ strains (identified from the MIT Whitehead database and obtained through Research Genetics) were tested for linkage to DlONwul-Tim. Once linkage was established to the distal arm of mouse chromosome 10, additional markers were scored to fine-map the mTim locus.
  • the hTim gene was mapped by STS screening of the Stanford G3 panel of 83 radiation hybrid cell lines (Research Genetics).
  • Site-Directed Mutagenesis In order to generate a full-length cDNA clone of hTim for further functional studies, site-directed mutagenesis was used to delete the 216 base pair intron from the H5E11CA03 clone. Mutagenesis was performed with the Quik Change Site Directed Mutagenesis kit (Stratagene) using two oligonucleotides, SDEQuikTimS and SDEQuikTimAS (see below), designed with the 216bp intron sequence deleted per the manufacturer's directions with the following modifications. 200ng of the starting dsDNA template (H5E11CA03) was used with 375ng of each primer and 3ul of the dNTP mix in a final 50ul reaction volume.
  • mice were housed in 12 hours of light and 12 hours dark (LD 12:12) for at least 1 week and then released into constant darkness (DD). Three mice were sacrificed in total darkness every four hours beginning at 54 hours in DD for one 24-hour cycle (7 time points). Optic nerves were severed under infrared light (15 W Kodak Safelight with #11 filter). Brains were removed under dim red light (Kodak filter no. 1 A) and frozen on dry ice. In a separate experiment, (BALB/cJ X C57BL/6J) F5 intercross albino mice were housed in similar conditions and 3 animals were sacrificed every four hours beginning at 58 hours in DD over 6 time points. Eyeballs were collected and frozen immediately in tubes on dry ice.
  • Coronal sections encompassing the SCN of 20um thickness were collected from each brain and thaw-mounted on gelatin-coated slides. Sections were fixed for 5 min in 4% paraformaldehyde in PBS and treated for 10 min in 0.1M triethanolamine/acetic anhydride then dehydrated through an ethanol series. Slides were hybridized overnight at 47°C in hybridization solution composed of 50% formamide, 300 mM NaCl, 10 mM Tris HCI pH 8.0, lmM EDTA, IX Denhardt's, 10% dextran sulfate, lOmM DTT and containing 5X10 7 cpm/ml of the relevant 33 P-labeled probe.
  • mPerl and two mTim probes were prepared using the Ambion MaxiScript in vitro transcription kit from templates containing nucleotides 468 to 821 of mPerl (GenBank Accession # AF022992), 2392 to 2633 for mTim-P-1, and 764 to 1593 for mTim-R-2.
  • RNA from eyeballs was extracted using TRIZOL reagent (Life Technologies) according to the manufacturer's protocols. lOOng of total RNA from each sample was used in duplicate RT-PCR reactions consisting of IX TaqMan EZ buffer, 3mM manganese acetate, 300mM each of dATP,dCTP,dGTP, 600mM dUTP and appropriate primers and probe.
  • mPerl and Tim-P-4 assays mPerl primers and probe and GAPDH control primers (Rodent GAPDH control Kit, ABI) and probe were used in a single-tube assay. Probes were labeled with 6carboxy-fluoroscein (6-FAM) on the 5' end and with 6-carboxy-tetramethyl rhodamine (TAMRA) on the 3' end.
  • 6-FAM 6carboxy-fluoroscein
  • TAMRA 6-carboxy-tetramethyl rhodamine
  • RT-PCR reactions were carried out in a Perkin Elmer ABI 7700 machine using the following thermal cycling parameters: 50°C for 2 minutes, 60°C for 30 minutes, then 95°C for 5 minutes followed by 40 two-step cycles of 94°C for 20 seconds 62°C for 1 minute.
  • Biochemical Interaction hTIM, mPERl, and mPER2 polypeptide fragments labeled with 35 S-methionine were synthesized by coupled transcription-translation in vitro (TNT Lysate System, Promega).
  • GST, GST-dPER, GST-hTIM and GST-mTIM fusion proteins were produced in E. coli using the pGEX vector (Pharmacia) and purified using glutathione-agarose beads. 35 S-labeled proteins were incubated with control (GST) or GST fusion beads for 30 minutes. The beads were washed with a buffer containing 0.5% NP-40 and 200mM KC1. The proteins were denatured in Laemmli loading buffer and resolved by SDS-PAGE.
  • a Schneider 2 (S2) cell line was transiently transfected with hs-hTim and hs-dPer as described [Saez ef al, Neuron 17:911-920 (1996)].
  • cell lines were incubated for 30 minutes in a 37°C water bath and allowed to recover at room temperature for 4 hours.
  • Heat shock-induced S2 cells were allowed to attach to a glass coverslip for 15 minutes and were fixed with 4% paraformaldehyde in PBS for 15 minutes. Fixed cells were washed with PBS and incubated with blocking solution containing 5% goat serum, 0.1% Triton in PBS.
  • Transfection and Luciferase Reporter Gene Assays Transfection of NIH 3T3 cells with luciferase reporter and cDNA expression plasmids and assays of luciferase activity were performed essentially as described [Gekakis, et al, Science 280: 1564-1569 (1998)]. Cells were transfected (Lipofectamine-Plus, Gibco-BRL) in 6-well plates at 25-50% confluence with 10 ng of the firefly luciferase reporter plasmid, 1 ⁇ g (total) of expression plasmids, and 0.5 ng of the internal control Renilla Luciferase plasmid.
  • Luciferase reporters were constructed in pGL3-promoter (Promega) with the following inserts: mPerl , 2.0-kb promoter fragment [Gekakis ef al, Science 280:1564-1569 (1998)] or a 54-bp fragment containing the three E-boxes and immediate flanking sequences linked together in their native 5'-to-3' order [Gekakis, ef al, Science 280:1564-1569 (1998)]; mck, a 60-bp fragment consisting of four iterations of the muscle creatine kinase right E-box plus immediate flanking sequences [Skapek, ef al, Moi Cell Biol. 16:7043-7053 (1996)].
  • cDNAs were driven by the cytomegalovirus immediate early promoter using the following expression plasmids: mouse Clock and hamster BMALl inserts were in pcDNA3 (Invitrogen), hTim insert was in pCMV6-XL3 (Origene), Per7 insert was in pCMV-SPORT2 (Gibco-BRL), and MyoD, E12, and Id inserts were in pCS2 [Skapek, ef al, Moi Cell Biol. 16:7043-7053 (1996)]. The total amount of each type of expression plasmid (250 ng each) was kept constant in any given experiment by including nonrecombinant expression plasmids in transfections, as necessary.
  • the sequence was then used to search iteratively for additional ESTs sharing identity with 746219.
  • This search revealed several EST sequences co ⁇ esponding to human EST clones 417249, 531927 and one mouse EST clone 534423. These clones were obtained and sequenced and analysis revealed that the previously unsequenced region of 417249 shared additional homology with Drosophila TIM.
  • the cDNA contig generated from the 746219 and 417249 sequence revealed an uninterrupted open reading frame (ORF) of 2.5kb. This ORF was incomplete as no consensus start of translation or stop codons were present in this sequence.
  • the complete human cDNA sequence of hTim was obtained by screening several libraries as well as using RACE PCR. 5' RACE was used on human thymus cDNA to identify clones containing additional 5' sequence. One major 1 kb 5'RACE product was isolated, subcloned and sequenced. From this clone the start of translation was identified by comparison with the Drosophila virilis and D. melanogaster TIM sequences [Myers, ef al, Nucleic Acids Res.
  • Drosophila TIM and mouse TIM share 30% identity and 55% similarity in TH1, 23% identity and 47% similarity in TH2, 22% identity and 42% similarity in TH3, and 35% identity and 55% similarity in TH4 (Figure 3).
  • This degree of sequence similarity is comparable to or greater than that seen with dPER and each of the mPERs. hTIM and mTIM share a greater length of similarity with D. virilis TIM than D. melanogaster TIM in THl and TH2.
  • the TH2 and TH3 domains in Drosophila span a stretch of amino acids implicated in dPER binding (PB2) [Saez ef al, Neuron 17:911-920 (1996)].
  • TH3 contains the functional dPER-binding domain since it shares a larger overlap with PB2.
  • Other functional domains identified in dTIM are also conserved in hTIM and mTIM.
  • the PB1 domain contains the dTIM nuclear localization signal (NLS) sequence that is present in hTIM and mTIM; however, the rest of the domain is not conserved.
  • NLS nuclear localization signal
  • the region containing the tim SL mutation [Rutila, ef al, Neuron 17:921-929 (1996)], which is within the PB1 domain, is not well conserved.
  • the glutamate-rich sequence found in dTIM is also present in hTIM and mTIM as repeats of 13 and 11 glutamate residues at amino acid positions 665 and 662, respectively.
  • the mammalian proteins also carry several other short stretches of glutamate-rich sequence that are not present in dTIM ( Figures 2 and 3).
  • the cytoplasmic localization domain (CLD) in dTIM contains a tetrapeptide DEDD (in D. virilis the sequence is DEDE) that is present at the extreme C termini of the hTIM and mTIM sequences.
  • DEDD tetrapeptide
  • the C-termini of the hTIM and mTIM proteins contain no other discernable sequence similarity to the dTIM CLD.
  • TIM Drosophila TIM
  • mTIM and hTIM share some homology with a hypothetical yeast ORF of unknown function (Accession # P53840).
  • mTIM and hTIM show some weak sequence similarity with a C. elegans EST (Accession # C43225). This could represent a nematode timeless homolog, but so far this is the only sequence similarity with a circadian gene in this species (there are no obvious hits with per, Clock or BMALl).
  • SNPs single-nucleotide polymorphisms
  • the second is a T to A, which changes the coding sequence from a leucine to an isoleucine at amino acid 455.
  • the third SNP is a G to A, which alters the coding sequence from a valine to a methionine at amino acid 592.
  • the fourth SNP that was detected was an A to G resulting in a glutamine to arginine alteration at amino acid 831.
  • mapping of mTim and hTim In order to map mTim genomic DNA, was amplified using primers designed within the 534423 EST sequence from various strains of mice to search for allelic variation in length. A length polymorphism was found between C3H/HeJ and C57BL6/J mice in the intronic sequence contained in the amplified PCR products. The C3H/HeJ allele is 11 base pairs shorter than the corresponding C57BL6/J allele.
  • EST database may represent only half of all genes, the fact that every known mammalian circadian gene has multiple ESTs covering it (and more importantly, that each has at least two EST clones containing coding sequence for the co ⁇ esponding gene) provides an additional reason to believe that there are no other mammalian Tim paralogs.
  • mRNA Expression of mTim and hTim To examine the mRNA expression of hTim, Northern blot analysis was performed on multiple tissue blots using EST clone 746219 as probe (Figs. 4A and 4B). A single hTim transcript of 4.5-kb was found in all human tissues analyzed. hTim mRNA was widely expressed with highest levels in the placenta, pancreas, thymus and testis. In the mouse, a 4.5-kb mTim transcript was expressed in the heart, brain, spleen, liver and testes with lower expression in the lung and kidney (Figure 4C). A minor 3-kb transcript was also seen in heart, brain and liver.
  • Mouse skeletal muscle contained two transcripts of 6-kb and 2.5-kb.
  • mTim mRNA was highest at embryonic day 11 and then gradually decreased ( Figure 4D).
  • Drosophila timeless exhibits circadian oscillations in both mRNA and protein and because the mammalian per genes also have circadian rhythms in mRNA levels
  • mTim mRNA levels in either the SCN or retina were tested to see if they were cyclic.
  • In situ hybridization studies demonstrated that mTim is expressed in the mouse SCN at low but detectable levels using two different riboprobes from the mTim cDNA (Figs. 5C and 5D).
  • Sense control probes for mTim were negative relative to antisense probes in the SCN.
  • Glutathione-S-transferase (GST)-dPER fusion proteins or GST alone were expressed in bacteria, purified using glutathione-agarose beads, and incubated with in vftro-translated, 35 S-labeled hTIM fragments (hTIM 1-1207 and hTIM 1-560).
  • SDS-PAGE analysis demonstrated that full-length hTIM binds to GST-dPER (1-640), GST-dPER (1-365), GST-dPER (1-118), GST-dPER (368-448), GST-dPER (448-512), and GST-dPER (819-1186), but not detectably with GST-dPER (530-640) or GST alone (Figure 7A-7C).
  • the hTIM 1-560 fails to bind GST-dPER (368-448) and GST-dPER (448-512) but recapitulates full-length hTIM interaction with the other dPER fragments.
  • mPERl and mPER2 were also tested for direct association in vitro (Figs. 9A-9C).
  • GST-hTIM and GST-mTIM fusion proteins were expressed and purified as described above and incubated with in vr ' fro-translated, 35 S-labeled mPERl and mPER2 full-length proteins.
  • SDS-PAGE analysis reveals that both hTIM and mTIM polypeptides are able to interact with mPERl or mPER2 proteins.
  • dPER Nuclear Localization by hTIM To determine whether mammalian TIM could mimic dTIM in a cellular context, the ability of mammalian TIM to facilitate nuclear entry of dPER was examined. An assay has previously been described in S2 cells that demonstrates that coexpression of dPER and dTIM are required for nuclear localization of either protein [Saez et al, Neuron 17:911-920 (1996)]. As shown earlier, expression of dPer alone in S2 cells results in cytoplasmic localization of the dPER protein (Fig. 10A and 10D). When hTIM is coexpressed with dPer in S2 cells, dPER translocates to the nucleus (Figs. 10B, 10C, 10E, and 10F).
  • hTIM expression is sufficient to promote nuclear localization of dPER, it was not determined whether hTIM is also translocated to the nucleus. hTIM and mTim have several putative nuclear localization signals that suggest that they are indeed nuclear proteins. In any case, the efficiency of hTIM in promoting nuclear entry of dPER is equivalent to that seen with Drosophila TIM. Thus, the functional similarity between dTIM and hTIM in this nuclear localization assay provides further evidence that human TIM is an ortholog of Drosophila tim.
  • luciferase reporter assays were ca ⁇ ied out in cultured NIH-3T3 mouse fibroblast cells into which expression plasmids for full-length CLOCK, BMALl, hTIM, and mPERl had been transfected in various combinations.
  • the three per genes would be loosely coupled and would share regulation of the circadian autoregulatory feedback loop.
  • the circadian transcription-translation feedback loop oscillator system By virtue of sharing a common dimerization partner, the three per genes would be loosely coupled and would share regulation of the circadian autoregulatory feedback loop.
  • a day peaking protein is low at night, therefore to reset the rhythm, light must increase the level of the protein, which is most efficiently accomplished by induction of transcription.
  • a night peaking protein (such as Drosophila TIMELESS) is elevated at night, therefore to reset the rhythm, light must decrease the level of the protein, which is best accomplished by protein degradation.
  • the mammalian PER proteins may in fact be reaching peak levels at night. If true, one would predict that light-induced TIM or PER protein degradation would be expected to take place in these tissues.
  • a Drosophila-l ⁇ ke model may apply more directly to peripheral tissues in mammals.
  • circadian autoregulatory loop involving the positive elements, CLOCK and BMALl, and the negative elements, PER and TIM, underlies the generation of circadian oscillations in mammalian cells.
  • the recent demonstration of circadian rhythms in peripheral mammalian tissues and cell lines [Balsalobre ef al, Cell 93:929-937 (1998); Zylka ef al, Neuron 20: 1103-1110 (1998)] underscores the significance of circadian rhythmicity in cells throughout the body.

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Abstract

La présente invention concerne des acides nucléiques isolés et/ou des molécules d'ADN recombinant codant pour la protéine mammifère TIMELESS. La présente invention concerne également une protéine mammifère TIMELESS isolée et/ou recombinante, ainsi que des anticorps de cette protéine mammifère TIMELESS. Cette invention concerne enfin des procédés d'utilisation des acides nucléiques, des protéines, et des anticorps susmentionnés, notamment comme agents thérapeutiques.
PCT/US1999/022777 1998-10-02 1999-09-30 Proteine mammifere timeless et ses procedes d'utilisation WO2000020585A1 (fr)

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CN110305956A (zh) * 2019-08-09 2019-10-08 北京大学 影响抑郁行为或抗抑郁行为的主效标记及其应用

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104017053A (zh) * 2014-06-25 2014-09-03 苏州普罗达生物科技有限公司 一种per2蛋白激动剂多肽及其应用
CN104017053B (zh) * 2014-06-25 2016-09-28 江晨 一种per2蛋白激动剂多肽及其应用
CN110305956A (zh) * 2019-08-09 2019-10-08 北京大学 影响抑郁行为或抗抑郁行为的主效标记及其应用

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