Mammalian Timeless And Methods Of Use Thereof

MAMMALIAN TIMELESS AND METHODS OF USE THEREOF

GOVERNMENTAL SUPPORT

The research leading to the present invention was supported, at least in part, by a grant from the NSF Center for Biological Timing and NIH Grant GM 54339. Accordingly, the Government may have certain rights in the invention.

FIELD OF THE INVENTION

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.

BACKGROUND OF THE INVENTION

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)]. Molecular components that comprise these pacemakers have been identified in a diverse set of organisms including the fruit fly, mouse and fungi [Dunlap, Science 280:1548-1549 (1998)]. However, until recently, it has remained unclear whether the circadian pacemakers of these various organisms share a common molecular mechanism.

Perhaps the best-characterized circadian system at the molecular genetic level is Drosophila melanogaster [Rosato et al, Bioessays 19:1075-1082, (1997); Rosato et al, Nucleic Acids Res. 25:455-458 (1997); Young, Annul. Reviews in Biochemistry 67:135-152 (1998)]. Two oscillator components, period (per) and timeless (tim), undergo rhythms in messenger RNA and protein abundance [Edery et al, Proc. Natl. Acad. Sci. U.S.A. 91:2260-2264 (1994); Hardin, et al, Nature 343:536-540 (1990); Myers et al., See Comments, Science 271:1736- 1740 (1996); Sehgal et al, Science 270: 808-810 (1995)]. Immunocytochemical experiments demonstrated that PER is a nuclear protein in a variety of Drosophila tissues [Konopka and Benzer Proc. Natl. Acad. Sci. U.S.A. 68:2112 (1971); Baylies et al in Molecular Genetics of Biological Rhythms, pp. 123-153, M. W. Young, Ed. (Dekker, New York, 1993)]. In cells of the adult fly visual and nervous systems, the amount of 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,

Nature, 343:536-540 (1990)]. Three mutant alleles - per0, r5, andper\ - cause arrhythmic behavior or shorten or lengthen periods, respectively [Konopka and Benzer Proc. Nat Acad. Sci. U.S.A. 68:2112 (1971)]. These mutations also produce corresponding changes in the rhythms of per RNA and protein amounts [Edery et al Proc. Natl. Acad. Sci. U.S.A 91:2260 (1994); Hardin et al Nature 343:536 (1990); Proc. Natl. Acad. Sci. U.S.A. 89:11711 (1992); Sehgal et al Science 263:1603 (1994)] and PER immunoreactivity in nuclei [Sewicki et al Neuron 1:141 (1988); Saez and Young Moi. Cell. Biol. 8:5378 (1988); Zerr et al J. Neurosci. 10:2749 (1990)]. This suggested a role for molecular oscillations of per in the establishment of behavioral rhythms [Hardin et al, Proc. Natl. Acad. Sci. U.S.A. 89:11711 (1992)].

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)]. Given the homologies to sim and the aryl hydrocarbon receptor (which are thought to regulate transcription), the effects of per on behavioral rhythms had early on been suggested to depend on circadian regulation of gene expression, including that of per itself [Hardin et al, Nature 343:536 (1990); Hardin et al, Proc. Natl. Acad. Sci. U.S.A. 89:11711 (1992)].

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. In particular, in Drosophila, the gene products are present in the cytoplasm late in the day when a sleeping cycle is induced, whereas when the gene products are in the nucleus late at night a waking cycle follows. Nuclear entry of dTIM and dPER is mediated by the physical interaction of the two proteins through known binding domains [Gekakis, et al, Science 270:811-815 (1995); Saez, et al.., Neuron 17:911-920 (1996)].

In Drosophila 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.

Mutations in the per and/or tim genes affect both overt circadian rhythms of eclosion and locomotor activity [Konopka et al, Pro. Natl. Acad. Sci. U.S.A. 68:2112-2116 (1971);

Sehgal et al, Science 263:1603-1606 (1994)] as well as molecular oscillations of per and tim gene products [Hardin. et al. Nature 343:536-540 (1990); Sehgal et al. Science 270:808-810 (1995)]. These two genes are involved in a negative autoregulatory feedback loop that underlies overt rhythm generation [Hardin, Nature 343:536-540 (1990); Sehgal et al. Science 270:808-810 (1995); Zeng, et al, EMBO J. 13:3590 (1994)]. 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)].

Null mutations in either of two genes, period (per) and timeless (tim), abolish behavioral rhythmicity, while alleles encoding proteins with missense mutations have been recovered at both loci and show either short- or long-period behavioral rhythms [Konopka and Benzer, Proc. Natl. Acad. Sci USA, 68:2112-2116 (1971); Sehgal et al, Science, 263:1603-1606 (1994); Rutila et al, Neuron, 17:921-929 (1996)]. The RNA and protein products of the genes oscillate with a circadian rhythm in wild-type flies. These molecular rhythms are abolished by null mutations of either gene, and the periods of all molecular rhythms are correspondingly altered in each long- and short-period mutant indicating a regulatory interaction between these genes (Hardin et al, Nature, 343:536-540 (1990); Edery et al, Proc. Natl. Acad. Sci USA, 91:2260-2264 (1994); Sehgal et al, Science, 263:1603-1606 (1994); Vosshall et al, Science, 263:1606-1609 (1994); Seghal et al, Science, 270:808-810 (1995); Price et al., EMBO J., 14:4044-4049 (1995); Hunter-Ensor et al., Cell, 84:677-685 (1996); Myers et al, Science, 271:1736-1740 (1996); Zeng et al, Nature, 380:129-135 (1996)].

Production of these molecular cycles appears to depend on the rhythmic formation and nuclear localization of a complex containing the PER and TIM proteins [Seghal et al, Science, 270:808-810 (1995); Gekakis et al, Science, 270:811-815 (1995); Lee et al., Science, 271:1740-1744 (1996); Saez and Young, Neuron, 17:911-920 (1996); Saez and Young, Neuron, 17:911-920 (1996)]. 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. Cell, 84:677-685 (1996); Lee et al, Science, 271:1740-1744 (1996); Myers et al, Science, 271:1736-1740 (1996); Saez and Young, Neuron, 17:911-920 (1996);. Zheng et al., Nature, 380:129-135 (1996)]. Studies of per1-, a mutation that lengthens the period of behavioral rhythms [Konopka and Benzer, Proc. Natl. Acad. Sci. USA, 68:2112-2116 (1971)] and delays nuclear localization of PER protein [Curtin et al, Neuron, 14:365-372 (1995)], have shown that the PERL protein has reduced affinity for TIM [Gekakis et al.. Science, 270:811-815 (1995). This suggests that rates of PER/TIM association influence the period of the molecular cycle in mutant and wild type flies.

Seghal et al.,[ Science, 270:808-810 (1995)] proposed a model for the Drosophila clock in which delayed formation of PER/TIM complexes ensures separate phases of pen Him transcription and nuclear function of the encoded proteins. Recent mathematical treatments of the Drosophila data are consistent with this model [Leloup and Goldbeter, J. Biol. Rhythms, 13:70-87 (1998)]. Entrainment of this oscillator is regulated through the TIM protein, which is rapidly eliminated from the nucleus and cytoplasm of pacemaker cells when Drosophila are exposed to daylight [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); Zheng et al, Nature, 380:129-135 (1996)]. Studies of transgenic Drosophila have shown that adult behavioral rhythms can be linked to per and tim expression in a small group of central brain cells, the lateral neurons [Ewer ef al, (1992); Frisch et al, Neuron, 12:555-570 (1994);

Vosshall and Young, Neuron, 15:345-360 (1995)]. per and tim are also expressed in larval brain cells that are most likely the larval LNs [Kaneko et al, Neurosci., 17:6745-6760 (1997)] , suggesting a basis for larval entrainment to light/dark cycles [Sehgal et al, Proc. Natl acad. Sci. USA, 89:1423-1427 (1992)]. Oscillations of per and tim RNA, and PER and TIM proteins have been found outside of the head in a variety of tissues [Giebultowicz and Hege, Nature, 386:664 (1997); Emery et al, Proc. Natl. Acad. Sci. USA, 94:4092-4096 (1997); Plautz et al, Science, 278:1632-1635 (1997)]. Some of the latter oscillations were observed in vitro with isolated tissues, further indicating a cell autonomous mechanism [Giebultowicz and Hege, Nature, 386:664 (1997); Emery et al, Proc. Natl Acad. Sci. USA, 94:4092-4096 (1997); Plautz et al, Science, 278:1632-1635 (1997)].

In summary, 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. Because PER/TIM heterodimers are only observed when high levels of per and tim RNA have accumulated, self-sustained oscillations are produced in the feedback loop [Gekakis et al, Science, 270:811-815 (1995); Hunter-Ensor et al, Cell, 84:677-685 (1996); Myers et al, Science, 271: 1736-1740 (1996); Saez and Young, Neuron, 17:911-920 (1996); Sehgal et al, Science, 270:808-810 (1995) and Zeng et al, Nature, 380:129-135 (1996)]. Although molecular oscillations are maintained in constant darkness for per and tim RNA and for PER and TIM proteins, light can entrain the phases of these rhythms through rapid degradation of the light-sensitive TIM protein [Hunter-Ensor et al, Cell, 84:677-685 (1996); Myers et al., Science, 271:1736-1740 (1996) and Zeng et al, Nature, 380:129-135 (1996)]. Circadian oscillations of PER and TIM phosphorylation have also been described [Edery et al, PNAS, USA, 91:2260-2264 (1994) and Zeng et al, Nature, 380:129-135 (1996)].

The recent identification of several PER homologues from mammals [Albrecht et al. Cell, 91:1055-1064 (1997); Shearman et al, Neuron, 19:1261-1269 (1997); Shigeyoshi et al, Cell, 19:1043-1053 (1997); Sun et al, Cell, 90:10031011 (1997) and Tei et al, Nature, 389:512- 516 (1997)] suggests that, like many other biological processes, key molecules and mechanisms involved in circadian rhythms may be evolutionarily conserved between flies and mammals. A related mechanism has also been well defined in Neurospora [cf. Crosthwaite et al, Science, 276:763-769 (1997); Dunlap, Ann. Rev. of Gen, 30:579-601 (1996) and Garceau et al, Cell, 89:469-476 (1997)] and additional genes and proteins are known to play roles in the mouse [Antoch et al, (1997); King et al, Cell, 89:641-653 (1997) and Vitaterna et al, Science, 264:719-725 (1994)] the hamster [Ralph and Menaker, Science, 241:1225-1227 (1988)] and Arabidopsis [Millar et al, Science, 267:1161-1163 (1995)].

Indeed, recent studies have demonstrated a striking similarity between the mammalian and Drosophila circadian systems. In mice, the Clock gene regulates the period and persistence of circadian rhythms [Vitaterna et al, Science 264:719-725 (1994)]. The molecular identification of Clock was consistent with the conservation of the circadian system between flies and mammals since it encodes a novel basic-helix-loop-helix (bHLH)-PAS (PER-ARNT-SIM) transcription factor [King et al, Cell, 89:641-653 (1997); Antoch et al.. Cell, 89:655-667 (1997)]. In addition, three mammalian per homologs, mPerl, mPerl and mPer3, have been identified. All three genes show oscillations in mRNA abundance in the SCN and retina albeit with slightly different circadian profiles and light responsivity

[Albrecht et al.Cell 91:1055-1064 (1997); Shearman et al., Neuron 19:1261-1269 (1997); Shigeyoshi et al.Cell 91:1043-1053 (1997); Sun, et al. Cell 90:1003-1011(1997); Takumi, et al, Genes Cells 3: 167-176 (1998); Takumi, et al, Embo J. 17:4753-4759 (1998); Tei et al, Nature 389:512-516 (1997); Zylka, ef al. Neuron 20:1103-1110 (1998)]. Recent work has also shown that CLOCK heterodimerizes with a bHLH-PAS partner known as BMAL1 or MOP3 [Gekakis et al. Science 280:1564-1569 (1998); Hogenesch ef al,Proc. Natl. Acad. Sci. U.S.A. 95, 5474-5479 (1998)]. 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)]. Concomitantly, the corresponding genes in Drosophila were discovered with the identification of dClock, a homolog of mouse Clock, and cycle/dbmal, a homolog of mammalian BMAL1 [Allada et al, Cell 93:791-804 (1998); Darlington ef al, Science 280:1599-603 (1998); Rutila ef al. Cell 93:805-814 (1998)]. Mutations in these two genes cause arrythmicity in fly circadian behavior and abolish per and tim molecular oscillations [Allada et al.,Cell 93:791-804 (1998); Rutila ef al.,Cell 93:805-814 (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)]. Indeed, 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)]. In addition, dPER and dTIM appear to feedback negatively on their own promoters via the dCLOCK-dBMAL complex [Darlington ef al, Science 280:1599-1603 (1998)]. Thus the identification of 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. However, heretofore, the identification of all of the corresponding factors involved in circadian rhythm in mammals has remained elusive.

Therefore there is a need to identify mammalian factors involved in circadian rhythms. More specifically, there is a need to identify mammalian orthologs of Drosophila proteins that are involved in circadian rhythms and to ascertain their role in mammalian systems. Furthermore, there is a need to use such proteins to identify agents that can aid in the regulation of biological clocks, including as an aid in overcoming such maladies as jet lag.

The citation of any reference herein should not be construed as an admission that such reference is available as "Prior Art" to the instant application.

SUMMARY OF THE INVENTION

The present invention discloses a mammalian protein TIMELESS (TIM), which is involved in circadian rhythms. 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.

More particularly, 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.

One aspect of the present invention includes a nucleic acid that comprises a nucleotide sequence that encodes a mammalian TIM. In one such embodiment the nucleic acid comprises a nucleotide sequence that encodes a murine TIM. In another embodiment, the nucleic acid comprises a nucleotide sequence that encodes a bovine TIM. In a preferred embodiment the mammalian TIM is a human TIM.

Accordingly 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. In a preferred embodiment the orthologue has at least 50% identity with either and preferably both SEQ ID NO:2 and/or SEQ ID NO:20. In a more preferred embodiment the orthologue has at least 70% identity with either or preferably both SEQ ID NO:2 and/or SEQ ID NO:20. In an even more preferred embodiment the orthologue has at least 80% identity with either or preferably both SEQ ID NO:2 and/or SEQ ID NO:20. In a particular embodiment the ortholog has at least 90% and preferably 95% identity with either SEQ ID NO:2 or SEQ ID NO:20.

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 the Drosophila PER protein into the nucleus. Similarly, it is preferable that when the expression of a nucleic acid encoding a mammalian TIM of the present invention is performed in conjunction with the expression of murine perl in a NIH-3T3 cell, the resulting co-expression of TIM and PER can specifically inhibit CLOCK-BMAL-induced transactivation of the murine perl promoter.

In a particular embodiment, a nucleic acid of the present invention that encodes a mammalian TIM protein binds the Drosophila PERIOD (PER) protein in vitro. In yet another embodiment 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). Similarly, 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). In a particular embodiment 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. In one such embodiment the nucleic acid encodes a human TIM protein having the amino acid sequence of SEQ ID NO:2. In other embodiments, 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. In related embodiments 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.

In yet another embodiment the nucleotide sequence has a nucleotide sequence of SEQ ID NO:l. In still other embodiments the 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. In one such embodiment the nucleic acid encodes a variant that has an amino acid sequence of SEQ ID NO:20. In other embodiments, 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. In related embodiments 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. In still other embodiments 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. In addition, 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. In addition, 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. In a related embodiment 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. In a related embodiment the nucleic acid that encodes a mammalian TIM hybridizes to the nucleotide sequence of SEQ ID NO: 1.

In still another embodiment 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. In a related embodiment the nucleic acid hybridizes to a nucleotide sequence that encodes the amino acid sequence of SEQ ID NO:20 comprising a conservative substitution. In a related embodiment the nucleic acid encodes a mammalian TIM and hybridizes to the nucleotide sequence of SEQ ID NO: 19.

In an alternative embodiment the nucleic acid comprises a nucleotide sequence that encodes a modified mammalian TIM. In one such embodiment 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. In another embodiment the nucleic acid comprises a nucleotide sequence that encodes a modified mammalian TIM that compπses a TIM having an amino acid sequence of SEQ ID NO 2 compπsing a conservative substitution, or a fragment thereof In yet another embodiment the 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 In still another embodiment the nucleic acid compπses a nucleotide sequence that encodes a modified mammalian TIM that compπses a TIM having an amino acid sequence of SEQ ID NO 20 compπsing a conservative substitution, or a fragment thereof

All of the 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 In addition methods of expressing the recombinant DNA molecules for making the corresponding recombinant proteins and peptides are also provided Thus, 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 cultuπng the cell in an appropriate cell culture medium under conditions that provide for expression of the mammalian TIM protein by the cell In one such embodiment, a recombinant mammalian TIM protein is expressed in a cell containing an expression vector of the piesent invention by cultuπng the cell in an appropriate cell culture medium under conditions that provide for expression of the mammalian TIM protein by the cell In a prefeπed embodiment of this type, the method further comprises the step of purifying the recombinant mammalian TIM The present invention also includes the recombinant mammalian TIM proteins and TIM fragments made and or puπfied by such methods

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 In a preferred embodiment the ortholog has at least 50% identity with either or preferably both SEQ ID NO 2 and/or SEQ ID NO 20 In a more preferred embodiment the ortholog has at least 70% identity with either or preferably both SEQ ID NO 2 and/or SEQ ID NO 20 In an even more prefeπed embodiment the ortholog has at least 80% identity with either or preferably both SEQ ID NO:2 and/or SEQ ID NO:20. In a particular embodiment the ortholog has at least 90% and preferably 95% identity with either SEQ ID NO:2 or SEQ ID NO:20.

As is true for the nucleic acids of the present invention, the corresponding proteins can be identified and/or defined by the same or analogous criteria. Thus, 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. Similarly, it is preferable that when the expression of a nucleic acid encoding a mammalian TIM of the present invention is performed in conjunction with the expression of murine PERI in a NIH- 3T3 cell, the resulting co-expression of TIM and PER can specifically inhibit CLOCK- BMAL-induced transactivation of the murine per7 promoter.

In a particular embodiment, a mammalian TIM protein of the present invention binds Drosophila PERIOD (PER) protein in vitro. In yet another embodiment 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). Similarly, 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). In a particular embodiment 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 coπesponding 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. In one such embodiment the human TIM protein has the amino acid sequence of SEQ ID NO:2. In other embodiments, 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. In related embodiments 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. In one such embodiment the murine variant has an amino acid sequence of SEQ ID NO:20. In other embodiments, the murine TIM protein has the amino acid sequence of SEQ ID NO:22, or SEQ ID NO:24, or SEQ ID NO:26. In related embodiments 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. Another such embodiment is 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.

In a particular embodiment a fragment of the mammalian TIM protein of the present invention binds to PER. In another embodiment the fragment of the mammalian TIM protein when bound to PER can specifically inhibit CLOCK-BMAL-induced transactivation of the murine perl promoter. In another embodiment the fragment of the mammalian TIM protein promotes nuclear entry of Drosophila PER in Drosophila (S2) cells. In still another embodiment a fragment of the mammalian TIM protein comprises a cytoplasmic localization domain (CLD). In a prefeπed embodiment of this type the CLD comprises an acidic tetrapeptide at or near the C-Terminal end of the CLD.

As further disclosed below, all of the mammalian TIM proteins and fragments of mammalian TIM proteins of the present invention can be combined in a fusion proteins. In addition, 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. In one such embodiment the modified mammalian TIM comprises a TIM having an amino acid sequence of SEQ ID NO:2 or a fragment thereof. In another embodiment the modified mammalian TIM comprises a TIM having an amino acid sequence of SEQ ID NO:20, or a fragment thereof. In still another embodiment 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. In yet another embodiment 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.

More particularly the present invention provides fusion proteins comprising the proteins, peptides and fragments thereof of the present invention. Thus 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. In a particular embodiment 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. In one such embodiment the antibody is a polyclonal antibody. In another embodiment the antibody is a monoclonal antibody. In a particular embodiment of this type 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. In addition, the present invention provides animals that have one or both alleles which encode the timeless gene being defective or deleted. In a particular embodiment of this type, the present invention provides a knockout mouse. Such a knockout mouse comprises a first and a second allele which naturally encode and express a muπne timeless gene, but both the first and second allele each contain a defect which prevents the knockout mouse from expressing the timeless gene In a particular embodiment of this type, 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 coπesponding mRNAs One such embodiment compπses 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 occuπed The detection of the binding indicates the presence or activity of the protein or the mRNA in the biological sample In a particular embodiment of this type, the binding partner is a nucleotide probe having specificity tor an mRNA encoding the mammalian TIM In another embodiment the binding partner is a PER protein or fragment of a PER protein that binds mammalian TIM In still another embodiment 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 One such embodiment comprises contacting 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 Preferably 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 Preferably 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 protein that binds to the mammalian TIM Both the PER protein fragment and the TIM protein fragment should bind to TIM and PER respectively in the absence of the agent In a prefeπed embodiment the PER protein fragment and the TIM protein fragment bind to each other in the absence of the agent When an increase in the binding of the mammalian TIM protein to the PER protein is determined, the agent is identified as an agonist of the binding of the mammalian TIM protein to the PER protein; whereas when a decrease in the binding of the mammalian TIM protein to the PER protein is determined, the agent is identified as an antagonist of the binding of the mammalian TIM protein to the PER protein.

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. When an increase in the entry of the PER protein to the nucleus is determined, the agent is identified as an agonist; whereas when a decrease in the entry of the PER protein to the nucleus is determined, the agent is identified as an antagonist.

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. 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. In a particular embodiment of this type the nucleic acids encoding the mammalian TIM protein and the PER protein are expressed transiently whereas the coπesponding expression of CLOCK and BMALl is constitutive. In a prefeπed embodiment of this type 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. When the determination in the absence of the PER protein and the mammalian TIM protein indicates no significant change in the amount of expression of the reporter gene in the cell, 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.

The present invention also provides kits for performing the methods of the present invention. One particular kit can be used in detecting the presence of mammalian TIM protein or mRNA in a cellular sample. In one such embodiment the kit comprises a predetermined amount of a detectably labeled binding partner of the mammalian TIM protein or mRNA. In a particular embodiment of this type the binding partner is antibody to the mammalian TIM protein. In a prefeπed embodiment of this type, the kit also contains a separate sample of mammalian TIM to use as a standard. In another embodiment, 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. Once the coding region of the nucleotide sequence is identified, the coπesponding amino acid sequence can be readily determined using the genetic code, preferably with the aid of a computer. Preferably the full-length nucleotide sequence of the coding region of an ortholog to the human and murine timeless gene is identified. It is also preferable that 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. In a particular embodiment 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 coπesponding suprachiasmatic nucleus (SCN), i.e., if the putative ortholog is a bovine ortholog, the SCN of a bovine is tested.

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

It is a further object of the present invention to provide antibodies to the mammalian TIMs, and methods for their preparation, including by recombinant means

It is a still further object of the present invention to provide nucleic acids encoding mammalian TIM proteins and the corresponding nucleotide sequences

It is a further object of the present invention to provide a method for detecting the presence of the TIM proteins and mRNA in isolated mammalian cells and mammalian tissue samples

It is a further object of the present invention to provide a method and associated assay system for screening substances such as drugs, agents and the like, that are potentially effective in either mimicking the activity, or alternatively combating the adverse effects of the TIM proteins and mRNAs in mammals

It is a still further object of the present invention to provide a method for the treatment of mammals to control depression, jet lag and/or narcolepsy

It is a still further object of the present invention to provide pharmaceutical 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

It is a still further object of the present invention to provide a method of obtaining mammalian orthologs to the human and murine TIMs

It is a still further object of the present invention to provide cells that express mammalian TIM including cells that express mutant forms of mammalian TIM

It is a still further object of the present invention to provide transgemc animals that express mutant forms mammalian TIM including knockout animals These and other aspects of the present invention will be better appreciated by reference to the following drawings and Detailed Description.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A-1B shows the Cloning of human tim (Fig. lA) and murine tim (Fig.lB). The clones were obtained from the EST database (naπow 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.

Figure 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.

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 timSL and tim0 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 caπied out on 3 independent RNA samples, each run in duplicate. Eπor 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. 35S-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, 35S-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. Transcriptional activation in mouse NIH-3T3 cells of a luciferase reporter gene from 5' flanking sequences derived from the mPerl gene (Figs. 11 A-l 1C) or the muscle creatine kinase (mck ) gene (Fig.11 D). 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. + or - denotes an expression plasmid with or without, respectively, the indicated full-length cDNA insert. Shown are the mean and S.E.M. of n=6 independent experiments in each case. Some of the standard eπor bars are too small to be seen at this scale.

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, heterodimeπzation 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

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

DETAILED DESCRIPTION OF THE INVENTION

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 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 Moreover, 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

Accordingly, 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 vaπants of these orthologs are also provided In one such embodiment, the mammalian TIM proteins and nucleic acids are munne proteins and nucleic acids In a prefeπed embodiment, 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. In one such embodiment a mammalian TIM protein can bind to a PERIOD (PER) protein. In another embodiment, the expression of mammalian TIM expression in situ and/or in vivo promotes nuclear entry of the PER. In still another embodiment, expression of mammalian TIM and mammalian PERI specifically inhibit CLOCK-BMALl-induced transactivation of the mammalian perl promoter.

Therefore, if appearing herein, the following terms shall have the definitions set out below:

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). As exemplified herein, 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. As exemplified herein, 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.

As used herein the term "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)].

As used herein a "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. As used herein 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. Preferably, the tetrapeptide is DEDD, SEQ ID NO:27.

As used herein 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 refeπed to as a "glutamate-rich sequence."

As used herein the term "approximately" is used to signify that a value is within ten percent of the indicated value i.e., a protein containing "approximately" 1208 amino acid residues can contain between 1087 and 1329 amino acid residues.

As used herein the term "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. In another such embodiment the 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. In still another embodiment the 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. Thus, 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-occuπing mutational events do not give rise to a heterologous region of DNA as defined herein.

A "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. When referring to a nucleic acid that is DNA, and more specifically a DNA having a particular nucleotide sequence, i.e., SEQ ID NO:, both the "sense" strand and the complementary "antisense" strand are intended to be included. Thus a nucleic acid that is hybridizable to SEQ ID NO:l, for example, can be either hybridizable to the "sense" strand of SEQ ID NO: 1, which is particularly listed in the SEQUENCE LISTING, or to the "antisense" strand which can be readily determined from that SEQUENCE LISTING. The term nucleic acid molecule, and in particular DNA or RNA molecule, refers only to the primary and secondary structure of the molecule, and does not limit it to any particular tertiary forms. Thus, this term includes double-stranded DNA found, inter alia, in linear or circular DNA molecules (e.g., restriction fragments), plasmids, and chromosomes. In discussing the structure of particular double-stranded DNA molecules, 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. For preliminary screening for homologous nucleic acids, low stringency hybridization conditions, corresponding to a Tm 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 coπespond to a higher Tm, e.g., 40% formamide, with 5x or 6x SSC. High stringency hybridization conditions coπespond to the highest Tm, 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 Tm for hybrids of nucleic acids having those sequences. The relative stability (coπesponding to higher Tm) of nucleic acid hybridizations decreases in the following order: RNA:RNA, DNA:RNA, DNA:DNA. For hybrids of greater than 100 nucleotides in length, equations for calculating Tm have been derived (see Sambrook ef al, supra, 9.50-0.51). For hybridization with shorter nucleic acids, i.e., oligonucleotides, 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). Preferably 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. In a specific embodiment, the term "standard hybridization conditions" refers to a Tm of

55 °C, and utilizes conditions as set forth above e.g., 5X SSC. In a prefeπed embodiment, the

Tm is 60°C; in a more prefeπed embodiment, the Tm is 65 °C.

"Homologous recombination" refers to the insertion of a foreign DNA sequence of a vector in a chromosome. Preferably, the vector targets a specific chromosomal site for homologous recombination. For specific 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. In eukaryotic cells, 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. For purposes of defining the present invention, 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. Within the promoter sequence will be found 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.

As used herein, the term "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).

As used herein, the term "ortholog" refers to the relationship between proteins that have a common evolutionary origin and differ because they originate from different species. For example, Drosophila TIMELESS is a ortholog of human TIMELESS.

The term "sequence similarity" in all its grammatical forms refers to the degree of identity or coπespondence between nucleic acid or amino acid sequences of proteins that do not share a common evolutionary origin (see Reeck ef al, supra). However, in common usage and in the instant application, the term "homologous," when modified with an adverb such as "highly," may refer to sequence similarity and not necessarily a common evolutionary origin.

In a specific embodiment, 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. In a particular embodiment 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. Alternatively, 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.

As used herein an 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.

As used herein, 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.

The term "coπesponding 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. Thus, the term "coπesponding to" refers to the sequence similarity, and not the numbering of the amino acid residues or nucleotide bases.

TIM Proteins and Fragments

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. Structurally, 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). These include 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.

In one embodiment the mammalian TIM is a murine protein. In a preferred embodiment the TIM is a human protein. In another embodiment 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. In still other embodiments 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. In yet other embodiments 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: The present invention also provides "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. In a particular embodiment of this type, 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. In another embodiment of this type, 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. In prefeπed embodiments 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. In a specific embodiment, 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 refeπed to above.

In one such embodiment, 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. Expression of a TIM as a fusion protein can facilitate stable expression, or allow for purification based on the properties of the fusion partner. For example, GST binds glutathione conjugated to a solid support matrix, MBP binds to a maltose matrix, and 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. Alternatively the chimeric TIM protein may contain the green fluorescent protein, and be used to determine the intracellular localization of the TIM in the cell.

Genes Encoding TIM Proteins

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. Similarly such 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. As used herein, 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.

In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (herein "Sambrook ef al, 1989"); DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover ed. 1985); Oligonucleotide Synthesis (M.J. Gait ed. 1984); Nucleic Acid Hybridization [B.D. Hames & S.J. Higgins eds. (1985)]; Transcription And Translation [B.D. Hames & S.J. Higgins, eds. (1984)]; Animal Cell Culture [R.I. Freshney, ed. (1986)]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984); F.M. Ausubel et al (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

A gene encoding TIM, whether genomic DNA or cDNA, can be isolated from any source, particularly from a human cDNA or genomic library. In view and in conjunction with the present teachings, 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).

Accordingly, 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.

In the molecular cloning of the gene from genomic DNA, DNA fragments are generated, some of which will encode the desired gene. The DNA may be cleaved at specific sites using various restriction enzymes. Alternatively, one may use 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.

Once the DNA fragments are generated, identification of the specific DNA fragment containing the desired 77 gene may be accomplished in a number of ways. For example, 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). For example, a set of oligonucleotides coπesponding 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). Preferably, 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.

Further selection can be caπied out on the basis of the properties of the gene, e.g., if the gene encodes a protein product having the isoelectric, electrophoretic, amino acid composition, or partial amino acid sequence of the TIM as disclosed herein. Thus, the presence of the gene may be detected by assays based on the physical, chemical, or immunological properties of its expressed product. For example, 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. In this procedure, nucleotide fragments are used to isolate complementary mRNAs by hybridization. Such 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) of 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. In addition, 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. In a specific embodiment, 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. Preferably, derivatives are made that have enhanced or increased functional activity or greater specificity.

Due to the degeneracy of nucleotide coding sequences, other DNA 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. Likewise, 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. For example, 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. For example, 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.

Particularly prefeπed conservative substitutions are: - Lys for Arg and vice versa such that a positive charge may be maintained;

- Glu for Asp and vice versa such that a negative charge may be maintained;

- Ser for Thr such that a free -OH can be maintained; and

- Gin for Asn such that a free NH2 can be maintained.

Amino acid substitutions may also be introduced to substitute an amino acid with a particularly preferable property. For example, 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 β-turns in the protein's structure.

The genes encoding 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. For example, 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. In the production of the gene encoding a derivative or analog of TIM, care should be taken to ensure that the modified gene remains within the same translational reading frame as the TIM gene, uninteπupted by translational stop signals, in the gene region where the desired activity is encoded.

Additionally, 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. Preferably, such 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. 253:6551; Zoller and Smith, 1984, DNA 3:479-488; Oliphant ef al, 1986, Gene 44: 177; Hutchinson ef al, 1986, Proc. Natl. Acad. Sci. U.S.A. 83:710), use of TAB® linkers (Pharmacia), etc. PCR techniques are prefeπed for site directed mutagenesis (see Higuchi, 1989, "Using PCR to Engineer DNA", in PCR Technology: Principles and Applications for DNA Amplification, H. Erlich, ed., Stockton Press, Chapter 6, pp. 61-70) or as described in the Example below..

The identified and isolated gene can then be inserted into an appropriate cloning vector. A large number of 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. However, if the complementary restriction sites used to fragment the DNA are not present in the cloning vector, the ends of the DNA molecules may be enzymatically modified. Alternatively, 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. Preferably, the cloned gene is contained on a shuttle vector plasmid, which provides for expansion in a cloning cell, e.g., E. coli, and facile purification for subsequent insertion into an appropriate expression cell line, if such is desired. For example, 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 2μ plasmid.

In an alternative method, 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.

The matched EST can then be fully sequenced. The Example below describes one particular procedure, though many equivalent systems and variations are known and practiced in the art. In one such alternative procedure, 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 coπesponding 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.

Alternatively, plasmids containing matched EST ortholog fragments can be used to transform competent bacteria (e.g, from Gibco BRL, Gaithersburg Md). Bacteria can be streaked, then grown up overnight. 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 coπesponding 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.

Expression of TIM Proteins

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." Thus, 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. One particular use for such expression vectors is to express 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. The expression elements of vectors vary in their strengths and specificities. Depending on the host- vector system utilized, any one of a number of suitable transcription and translation elements may be used.

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. In this regard, 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).

Expression of 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. 78:1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al, 1982, Nature 296:39-42); prokaryotic expression vectors such as the β-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. 80:21-25); see also "Useful proteins from recombinant bacteria" in Scientific American, 1980, 242:74-94; promoter elements from yeast or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter; and the animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift ef al, 1984, Cell 38:639-646; Ornitz ef al, 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald, 1987, Hepatology 7:425-515); insulin gene control region which is active in pancreatic beta cells (Hanahan, 1985, Nature 315:115- 122), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al, 1984, Cell 38:647-658; Adames ef β/., 1985, Nature 318:533-538; Alexander et al, 1987, Moi. Cell. Biol. 7: 1436-1444), 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. 1 : 161- 171), 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. In the first approach, the nucleic acids can be amplified by PCR to provide for detection of the amplified product. In the second approach, 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. In the third approach, the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "selection marker" gene functions (e.g., β-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. In another example, if the nucleic acid encoding TIM is inserted within the "selection marker" gene sequence of the vector, recombinants containing the TIM insert can be identified by the absence of the selection marker gene function. In the fourth approach, 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, for example, 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 λ, e.g., NM989, and other phage DNA, e.g., M13 and filamentous single stranded phage DNA; yeast plasmids such as the 2μ 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.

For example, in a baculovirus expression systems, both non-fusion transfer vectors, such as but not limited to pVL941 (BanϊΑl cloning site; Summers), pVL1393 (BamHl, Smal, Xbal, EcoRl, Notl, Xmalll, Bglll, and Pstl cloning site; Invitrogen), pVL1392 (Bglll, Pstl, Notl, X αlll, 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 with the initiation codon; Summers), pAc701 and pAc702 (same as pAc700, with different reading frames), pAc360 (BamHl cloning site 36 base pairs downstream of a polyhedrin initiation codon; Invitrogen(195)), and pBlueBacHisA, B, C (three different reading frames, with BamHl, Bglll, Pstl, Ncol, and Hindlll cloning site, an N-terminal peptide for ProBond purification, and blue/white recombinant screening of plaques; Invitrogen (220)) can be used.

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). Alternatively, 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). In another embodiment, a vector that directs episomal expression under control of Epstein Barr Virus (EBV) 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, Xhol, Noil, Hindlll, Nhel, and Kpnl cloning site, RSV-LTR promoter, histidinol selectable marker; Invitrogen), pREP9 (Kpnl, Nhel, Hindlll, Notl, Xhol, Sfil, and BamHl cloning site, RSV-LTR promoter, G418 selectable marker; Invitrogen), and pEBVHis (RSV- LTR promoter, hygromycin selectable marker, N-terminal peptide purifiable via ProBond resin and cleaved by enterokinase; Invitrogen). 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 (see, Kaufman, 1991, supra) for use according to the invention include but are not limited to pSCl 1 (Smal cloning site, TK- and β-gal selection), pMJ601 (Sail, Smal, A/71, Narl, BspMll, BamHl, Apal, Nhel, Sacϊl, Kpnl, and Hindlll cloning site; TK- and β-gal selection), and pTKgptFIS (EcoRl, Pstl, Sail, Accl, Hindll, Sbal, BamHl, and Hpa cloning site, TK or XPRT selection).

Yeast expression systems can also be used according to the invention to express the TIM protein. For example, the non-fusion pYES2 vector (Xbal, Sphl, Shol, Notl, GstXl, £cøRI, 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. Once a particular recombinant DNA molecule is identified and isolated, several methods known in the art may be used to propagate it. Once a suitable host system and growth conditions are established, recombinant expression vectors can be propagated and prepared in quantity. As previously explained, 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.

In addition, 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. For example, expression in a bacterial system can be used to produce an nonglycosylated core protein product. However, 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. Moreover, expression in mammalian cells can provide a tool for reconstituting, or constituting, TIM activity. Furthermore, 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. In one embodiment the cells are mammalian cells. In another embodiment the cells are murine cells. In prefeπed embodiments the cells are human cells. General Protein Purification Procedures:

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. For example, 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. In addition, high speed ultracentrifugation may be used either alone or in conjunction with other extraction techniques.

Generally good secondary isolation or purification steps include solid phase absorption using calcium phosphate gel or hydroxyapatite; or solid phase binding. 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. Alternative means of 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), including such related techniques as FPLC, is presently the most common means of performing liquid chromatography. Size exclusion techniques may also be accomplished with the aid of low speed centrifugation.

In addition size permeation techniques such as gel electrophoretic techniques may be employed. These techniques are generally performed in tubes, slabs or by capillary electrophoresis.

Almost all steps involving protein purification employ a buffered solution. Unless otherwise specified, generally 25-100 mM concentrations are used. Low concentration buffers generally infer 5-25 mM concentrations. High concentration buffers generally infer concentrations of the buffering agent of between 0.1-2M concentrations. 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.

Materials to perform all of these techniques are available from a variety of sources such as Sigma Chemical Company in St. Louis, Missouri.

Antibodies to TIM Proteins

According to the invention, 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. Alternatively, an antibody of the invention may be specific for a single form of the TIM, such as human TIM. Various procedures known in the art may be used for the production of polyclonal antibodies to a TIM of the present invention or derivative or analog thereof. For the production of antibody, 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. In one embodiment, a TIM or fragment thereof can be conjugated to an immunogenic carrier, e.g., bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH). Various 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.

For preparation of monoclonal antibodies directed toward a TIM of the present invention, or fragment, analog, or derivative thereof, 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. 80:2026-2030 (1983)], and the EBV-hybridoma technique to produce human monoclonal antibodies [Cole ef al, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985)]. In an additional embodiment of the invention, monoclonal antibodies can be produced in germ-free animals utilizing recent technology [PCT/US90/02545]. In fact, according to the invention, techniques developed for the production of "chimeric antibodies" [Morrison et al, J. Bacteriol. 159:870 (1984); Neuberger ef al, Nature 312:604-608 (1984); Takeda ef al, Nature 314:452-454 (1985)] by splicing the genes from a rabbit antibody molecule specific for a murine TIM, for example, together with genes from a human antibody molecule of appropriate biological activity can be used; such antibodies are within the scope of this invention. Such human or humanized chimeric antibodies are prefeπed for use in therapy of human diseases or disorders (described infra), since the human or humanized antibodies are much less likely than xenogenic antibodies to induce an immune response, in particular an allergic response, themselves. According to the invention, techniques described for the production of single chain antibodies [U.S. Patent Nos. 5,476,786 and 5,132,405 to Huston; 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. For example, 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.

In the production of antibodies, 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. In one embodiment, antibody binding is detected by detecting a label on the primary antibody. In another embodiment, the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody. In a further embodiment, 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. 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.

In a specific embodiment, antibodies that agonize or antagonize the activity of a mammalian TIM can be generated. Such antibodies can be tested using the assays described herein.

Assays for Identifying Agonists and Antagonists of TIM

Identification of the TIM protein provides a basis for screening for drugs capable of specific interaction with the functionally relevant aspects of the protein. For example, 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.

Thus 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.

Knowledge of the primary sequence of a mammalian TIM protein of the present invention, and the similarity of that sequence with proteins of known function, can provide an initial clue as the agonists or antagonists of the protein. Identification and screening of antagonists is further facilitated by determining structural features of the mammalian TIM protein, e.g., using X-ray crystallography, neutron diffraction, nuclear magnetic resonance spectrometry, and other techniques for structure determination. These techniques provide for the rational design or identification of agonists and antagonists. 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. Sci., 87:6378-6382 (1990); Devlin et al, Science, 249:404-406 (1990)], very large libraries can be constructed (106-108 chemical entities). A second approach uses primarily chemical methods, of which the Geysen method [Geysen ef al. , Molecular

Immunology 23:709-715 (1986); Geysen et al. J. Immunologic Method 102:259-274 (1987)] and the method of Fodor et al. [Science 251:767-773 (1991)] are examples. Furka ef al. [14th International Congress of Biochemistry, Volume 5, Abstract FR:013 (1988); Furka, Int. J. Peptide Protein Res. 37:487-493 (1991)], Houghton [U.S. Patent No. 4,631,211, issued December 1986] and Rutter ef al. [U.S. Patent No. 5,010,175, issued April 23, 1991] describe methods to produce a mixture of peptides that can be tested as agonists or antagonists.

In another aspect, synthetic libraries [Needels et al, Proc. Natl. Acad. Sci. USA 90:10700-4 (1993); Ohlmeyer ef al, Proc. Natl. Acad. Sci. USA 90: 10922-10926 (1993); Lam et al, International Patent Publication No. WO 92/00252; Kocis et al, International Patent Publication No. WO 9428028, each of which is incorporated herein by reference in its entirety], and the like can be used to screen for binding partners of the mammalian TIM protein.

The screening can be performed directly using peptides coπesponding to the CLD domain or PER binding domain of mammalian TIM. Alternatively, 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. For example, 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 [Pataπoyo, 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. In one embodiment, the compound may be directly labeled including as part of a fusion protein, e.g., with green fluorescent protein. In another embodiment, 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. Other 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 Eu3+, to name a few fluorophores), 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. For example, if it is desired to evaluate a compound as an agonist for 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) is then measured (photometrically in this example), and dose response curves are obtained and compared to those in which the agonist is not included in the assay. 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 refeπed. 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.

Labels

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 Eu3+, to name a few fluorophores), chromophores, radioisotopes, chelating agents, dyes, colloidal gold, latex particles, ligands (e.g., biotin), and chemiluminescent agents. When a control marker is employed, the same or different labels may be used for the receptor and control marker.

In the instance where a radioactive label, such as the isotopes 3H, 14C, 32P, 35S, 36C1, 51Cr, 57Co, 58Co, 59Fe, 90Y, 125I, 131I, and 186Re are used, known cuπently available counting procedures may be utilized. In the instance where the label is an enzyme, 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. Among examples of colored labels, which can be used according to the present invention, 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. (WO 88/08534); dyed latex such as described by May, supra, Snyder (EP-A 0 280 559 and 0 281 327); or dyes encapsulated in liposomes as described by Campbell et al. (U.S. Patent 4,703,017). Other direct labels include a radionucleotide, a fluorescent moiety or a luminescent moiety. In addition to these direct labelling devices, indirect labels comprising enzymes can also be used according to the present invention. Various types of 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.

Other labels for use in the invention include magnetic beads or magnetic resonance imaging labels.

In another embodiment, a phosphorylation site can be created on an antibody of the invention for labeling with 32P, 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 [35S]-methionine (as described below in the Example) or [32P]- orthophosphate. In addition to metabolic (or biosynthetic) labeling with [35S]-methionine, the invention further contemplates labeling with [14C]-amino acids and [3H]-amino acids (with the tritium substituted at non-labile positions).

Solid Supports

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. In another embodiment, 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). As used herein, a solid phase support is not limited to a specific type of support. Rather a large number of supports are available and are known to any person having skill in the art. 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. For example, 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). In synthesis of oligonucleotides, a silica based solid phase support may be prefeπed. Silica based solid phase supports are commercially available (e.g., from Peninsula Laboratories, Inc.; and Applied Biosystems, Inc.)

Antisense. Gene Targeting and Ribozymes

The functional activity of the mammalian TIM protein can be evaluated transgenically. In one embodiment of this type, 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) coπesponding 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)].

Alternatively, 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 coπesponding 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)]. Preferably, 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. The cell does not translate an mRNA in this double-stranded form. Therefore, antisense nucleic acids interfere with the expression of mRNA into protein. Preferably a DNA antisense nucleic acid is employed since such an RNA DNA duplex is a prefeπed 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. U.S.A. 85:4010-4014 (1988)] and in situ [Arima ef al, Antisense Nucl. Acid Drug Dev. 8:319-327 (1998); Hou ef al. Antisense Nucl. Acid Drug Dev. 8:295-308 (1998)]

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. Investigators have identified two types of ribozymes, Tetrahymena-type and "hammerhead"-type [Haselhoff and Gerlach, Nature 334:585-591 (1988)]. 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.

Therefore, 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.

Gene Therapy and Transgenic Vectors

In one embodiment of the present invention, a gene encoding a mammalian TIM protein is introduced in vivo in a viral vector. Such 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. Defective viruses, which entirely or almost entirely lack viral genes, are prefeπed. 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. Examples of particular vectors include, but are not limited to, a defective herpes virus 1 (HSV1) vector [Kaplitt ef al, Molec. Cell. Neurosci. 2:320-330 (1991)], an attenuated adenovirus vector, such as the vector described by Stratford-Perricaudet ef al. [J. Clin. Invest. 90:626-630 (1992)], and a defective adeno-associated virus vector [Samulski et al, J. Virol. 61:3096-3101 (1987); Samulski ef al, J. Virol 63:3822-3828 (1989)].

For in vitro administration, 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. For example, immunosuppressive cytokines, such as interleukin-12 (IL-12), interferon-γ (IFN-γ), or anti-CD4 antibody, can be administered to block humoral or cellular immune responses to the viral vectors [see, e.g., Wilson, Nature Medicine (1995)]. In addition, it is advantageous to employ a viral vector that is engineered to express a minimal number of antigens.

In another embodiment 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)].

Targeted gene delivery is described in International Patent Publication WO 95/28494, published October 1995.

Alternatively, the vector can be introduced in vivo by lipofection. For the past decade, there has been increasing use of 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)]. The use of 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)]. The use of 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.

It is also possible to introduce the vector in vivo as a naked DNA plasmid. 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].

Administration

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 caπier 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.

Also, 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. For example, 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. Likewise, small molecules that mimic or antagonize the activity(ies) of mammalian TIM may be identified (see above) or synthesized, and may be used in diagnostic and/or therapeutic protocols. The present invention further contemplates therapeutic compositions useful in practicing the therapeutic methods of this invention. 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. In a prefeπed embodiment, the composition comprises an antigen capable of modulating the specific introduction of TIM into a target cell.

The preparation of therapeutic compositions which contain polypeptides, analogs or active fragments as active ingredients is well understood in the art. Typically, 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. In addition, if desired, 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. The term "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 prefeπed embodiments of the invention. It should in no way be construed, however, as limiting the broad scope of the invention.

EXAMPLE

Mammalian Circadian Autoregulatory Loop: A TIMELESS Ortholog and mPerl Interact and Negatively Regulate CLOCK-BMALl -Induced Transcription

Summary A highly conserved picture of the Drosophila and mammalian circadian systems has recently emerged. However the identity and characterization of a mammalian Ortholog of the

Drosophila timeless gene was needed in order to complete the picture. A database search was initiated for mammalian ESTs that coπespond 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. Furthermore, hTIM and mPERl are shown to negatively regulate transcription of the mPerl promoter; thus, closing the mammalian circadian loop.

Experimental Procedures 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 coπesponding 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 caπied 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. Positive subwell stocks were plated out onto LB-Ampicillin (lOOug/ml) plates and colonies were screened to identify hTim and mTim cDNA clones. One clone, M11H7CH06, was obtained from the mouse library screen; however, it was a partial clone and was chimeric, containing an actin cDNA fragment at its 3' end. One full-length human clone, H5E11CH06, was obtained from the placental library screen. RACE PCRs were performed using Clontech Marathon cDNAs (mouse muscle and brain, human thymus and hypothalamus) as template following the manuf cturer's protocol with the following modifications. Conditions for PCR were as follows:

94°C 30 seconds, then 5 cycles 94°C 5 seconds, 72°C 6 minutes, 5 cycles 94°C 5 seconds, 70°C 6 minutes, 25 cycles 94°C 5 seconds, 68°C 6 minutes. Nested primers were used to increase specificity of amplified products for clones coπesponding to hTim and mTim.

Four sets of mouse 5' and 3' RACE primers were used:

Mtitn5gsp5 : 5 ' -tccaacattttgaggaagaggtggg-3 '

Mtim5nsp5 : 5 ' -gaaagagcgccaggaatagttctcg-3 ' Mtim5gsp3: 5'-ctggccacggtgaacgagatg-3'

Mtim5nsp3: 5'-atgaggctgttagggagagcagtcg-3'

Mtim3gsp5 : 5 ' -atccttgtgggcgaggtatagttcc-3 '

Mtim3nsp5 : 5 ' -ctgaagctgggcctcttcctcagg-3 '

Mtim3gsp3 : 5 ' -ccgtgaaccagaaagcgtttgtgg-3 ' Mtim3nsp3: 5'-ggagctgctgttctggaagaacacc-3'

One primer set for human 5' RACE PCR was used:

HstimGSP5 : 5 ' -agctaagcgtccctgccctactcc-3 '

HstimNSP5 : 5 ' -gggttctggtcacgaaacataaggg-3 ' .

Appropriate bands were gel-purified and subcloned into pGEM-T Easy for further analysis. Two 5' mouse clones, mTinό'Al and mTim5,A2, were isolated by RACE PCR initiated from the 5' of 534423 sequence. mTim5,A2 contains an intron near its 3' end and 70bp of mitochondrial DNA at the very 5' end. One 3' mouse clone, m77 3'AlC, was isolated from RACE initiated from the 534423 sequence; however, it contained a ~lkb intron and was internally primed.

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.

DNA Sequencing and Sequence Analysis: All sequencing was carried out on an ABI 377XL fluorescence-based automated sequencer. Sequencing reactions were carried out using the big Dye Terminator kit at to 1/4 chemistry depending on the template. Sequences were edited and assembled using Sequencher 3.0 (Genecodes, Ann Arbor, MI). Further protein and DNA sequence analysis was caπied out using MacVector 6.0.1, Oxford Molecular Group PLC (1996) using the Clustal W algorithm and alignment default parameters with default parameters for identity and similarity.

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.

Mapping: PCR amplification from genomic DNA of various strains of mice revealed an intron at nucleotide position 1543 of the mTim cDNA. This intron was 107 bp long in C3H/cJ mice as compared to 118 bp in C57BL/6J. This length polymorphism was used to map mTim. Primer sequences for DlONwul-Tim were:

5'-atgaggctgttagggagagcag-3' and 5'-aactttcgaaagagcgccag-3'.

60 [(C3H/cJ X C57BL/6J) FI X C57BL/6J] N2 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). Primers used for the hTim STS were as follows: HstimRHfor 5'-cagcatgatgagacctattatatgtgg-3' and HstimRHrev: 5'-actgagggtctcagaaaccagg-3'

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. 18 cycles of 95°C for 30 seconds, 55°C for 1 minute, and 68°C for 23.5 minutes was used. Successfully mutated clones were identified by colony PCR and the intron loopout was confirmed by sequencing. SDEQuikTimS: 5'-gttggcatcctccatcttgccaaatggagcggagtccctg-3' SDEQuikTimAS : 5 ' -cagggactccgctccatttggcaagatggaggatgccaac-3 '

Animals: CD-I 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.

In Situ Hybridization: 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 5X107 cpm/ml of the relevant 33P-labeled probe. mPerl and two mTim probes ( Ti -P-1 and mTim-R-2) 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.

TaqMan Real Time Quantitative RT-PCR Assay: Total 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. For the 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.

For the mTim-P-3 assay and GAPDH control, separate tubes were used. 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. mPerl -FOR: 5 ' -accttggccacactgcagta-3' mPerl -REV: 5'-ctccagactccactgctggtaa-3' mPerl -PROBE: 5'-attcctggttagcctgaacctgcttgaca-3' mT m-P-3FOR : 5'-gcccagcttcaggaactatacct-3' mTfm-P-3REV: 5'-aggtgcgccaatatggtttc-3' mTi -P-3PROBE: 5'-cactacatcttgaccttccacatccttgtgg-3' mTim-P-4FOR : 5'-ggagaaggatgtcgtctttcaca -3' m77 -P-4REV: 5'-ctgggctgctttcccagat -3' mTι'm-P-4PROBE: 5'-aggccttcacaatctccagaactacagctca -3' mPerl and mTim relative mRNA abundance was calculated using the comparative Ct method.

Biochemical Interaction: hTIM, mPERl, and mPER2 polypeptide fragments labeled with 35S-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. 35S-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.

Nuclear Localization Assay: 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)]. To induce the expression of the transfected genes 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. Cells were incubated overnight at room temperature with anti-dPER antibodies (1:5000) diluted in blocking solution, washed with PBS, and incubated with fluorescence-conjugated goat anti-rabbit IgG for 1 hour. Coverslips with stained cells were washed with PBS, incubated with 0.01% Hoechst (in PBS) for 10 minutes, washed in PBS and mounted with Gel/mount (Biomeda).

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 μg (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)]. Expression of full-length cDNAs was 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.

Results Molecular Cloning of Human and Mouse Timeless Orthologs: An EST database was searched to determine whether any mammalian timeless homologues could be identified. A search in September 1997 revealed one human EST, IMAGE #746219, whose 5' end sequence was recovered in a query with the full-length Drosophila TIM sequence (Accession # AF032401) with a P value of 0.16 in a TBLASTN search of the EST database. While the score of the alignment was marginal, the conservation of a stretch of 12 amino acids was nonetheless intriguing. Analysis of the 3' end sequence of this clone indicated that the similarity to dTIM extended to both ends of the EST. The clone was obtained and sequenced in its entirety. The sequence was then used to search iteratively for additional ESTs sharing identity with 746219. This search revealed several EST sequences coπesponding 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. 25:4710-4714 (1997); Ousley, et al.,Genetics 248:815-825 (1998); Rosato, ef al, Bioessays 19:1075-1082 (1997); Rosato, et al, Nucleic Acids Res. 25:455-458 (1997)]. To isolate a full-length clone an aπayed human placenta cDNA library was screened (Origene Technologies) using primers directed to the 5' end and midportion of the hTim coding sequence. A single clone, H5E11CA03, out of 5 X 105 clones screened was isolated and sequenced. This clone contained the complete ORF of hTim; however, it also contained a retained intron at nucleotide position 3007. All clones isolated in these experiments are shown in Figures 1A-1B. The complete hTim cDNA sequence is 4414 base pairs in length. The mouse Tim cDNA was cloned in much the same fashion as hTim and obtained a cDNA sequence 4438 base pairs in size (see Figure IB and Experimental Methods, above).

Translation of the hTim ORF predicts a protein of 1208 amino acids (when an AAG splice variant is included) and translation of the mTim ORF predicts a protein of 1197 amino acids (when two CAG splice variants are included) (Table 1). The human and mouse Tim coding sequences are 82% identical at the nucleotide and 84% identical at the amino acid level (Figure 2). hTIM and mTIM share four regions of sequence conservation with D. melanogaster and D. virilis TIM, and these are designated Tim Homology (TH) 1-4 (Figure 3). 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)]. Because TH2 and TH3 are separated in the mammalian TIM proteins, it appears likely that 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. In particular, the region containing the timSL 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). Finally the cytoplasmic localization domain (CLD) in dTIM (amino acids 1228-1389) 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. The C-termini of the hTIM and mTIM proteins contain no other discernable sequence similarity to the dTIM CLD.

TABLE 1

Besides Drosophila TIM, there are no closely related proteins to mammalian TIM in animals. The only other vertebrate example is a partial peptide purified and sequenced from bovine pituitaries (Accession # AF041856) which could represents the bovine ortholog of tim. mTIM and hTIM share some homology with a hypothetical yeast ORF of unknown function (Accession # P53840). Finally, 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).

Splice Variants and Polymorphisms in Mammalian Tim: In the course of cloning hTim and mTim, several splice variants and polymorphisms were found in the two genes. In mTim two 5' splice variants were found. mTim5'A2 contains a different 5' UTR sequence from TMBGTAE03, nucleotides 1-65 of TMBGTAE03 are replaced by 86 nucleotides. Also, mouse clones that contained insertions of a CAG trinucleotide at two positions in the cDNA (at nucleotide positions 2988 and 3664) resulting in insertions of glutamine and alanine residues, respectively were also found. These insertions appeared likely to be due to alternative splice acceptor usage at intron-exon boundaries. Genomic sequence confirmed that this was indeed the case: both insertions occur at defined intron-exon boundaries and the sequence at both splice acceptor sites was CAGCAG. If the first AG is used, the splice would include the CAG trinucleotide. These two splicing events most likely occur independently as clones of all four classes (+/- insertion at 2 positions) were identified (Figure 1A-1B).

A similar splice variant was found in the 5' end of the human TIM gene. An AAG insertion was detected at nucleotide 673 resulting in a lysine insertion after amino acid 177. This could occur because of an ambiguous splice acceptor as seen with the two insertions present in the mouse cDNA sequences. In addition, four single-nucleotide polymorphisms (SNPs) were identified by comparison of cDNA clone H5E11CA03 to sequence of the 746219 and 417249 ESTs. The first SNP, an A to G change, is a silent polymorphism occurring at nucleotide position 765. 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. These SNPs could prove useful in examination of the genetic basis of circadian rhythm dysfunction in humans through association studies.

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. This polymorphic marker was used to map mTim in a backcross panel and it was found that mTim maps to the distal portion of mouse chromosome 10 approximately 1.7 centimorgans distal to D10MU87 (1 recombinant out of 59 meioses, LOD score = 15.6). This marker was named DlONwul-Tim. No informative SSLP marker was located distal to DlONwul-Tim in this cross.

Human TIM was mapped by radiation hybrid analysis on the Stanford G3 panel using a sequence tagged site (STS) to nucleotide positions 1253-1351. WI-7760 is the closest linked STS marker to hTim that was found, having with a LOD score of 11.05. This places hTim on human chromosome 12 in a region of conserved syntony with mouse chromosome 10.

Unlike the mammalian per gene family, which consists of at least three members (mPerl, mPerl, and mPer3), there are no obvious paralogs of the mammalian timeless gene. Southern blot analysis in the mouse reveals only a single band when probed with an mTim cDNA probe. In addition, database searching with the full-length mTIM and hTIM protein sequences produces no ESTs or known mammalian genes with even marginal sequence similarity. While the 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 coπesponding 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. During mouse development, mTim mRNA was highest at embryonic day 11 and then gradually decreased (Figure 4D). Because 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. To determine whether a circadian rhythm in mTim mRNA occurs, SCN expression was examined on the third circadian cycle in constant conditions (every four hours starting at 54 hours into constant darkness). Experiments were performed using the two different mTim riboprobes, and no evidence of mTim mRNA cycling with either probe was found (Figure 6A). As a positive control, mPerl mRNA levels in adjacent brain sections were measured, and as reported previously, a high amplitude mPerl rhythm that peaked at 66 hours in constant darkness [approximately circadian time (CT) 6] was clearly seen (Figs. 5A,5B, and 6A). As indicated in Fig. 6A, however, the mTim in situ hybridization signal was low relative to that seen with mPerl so that it is possible that an mTim mRNA cycle could not be reliably detected in these experiments. In the retina, in situ hybridization with mTim probes revealed expression in the outer nuclear layer, inner nuclear layer, and ganglion cell layer. The pattern of mTim mRNA expression in the retina was identical to that seen with mPerl, Clock and BMALl [Gekakis ef al, Science 280:1564-1569 (1998)]. To determine whether the mouse retina exhibits circadian oscillations of mTim mRNA, TaqMan quantitative RT-PCR methods were used to measure both mTim and mPerl mRNA levels on the third circadian cycle in constant conditions (every four hours starting at 54 hours into constant darkness). As reported previously, mPerl mRNA levels in the retina were circadian with a peak between 66-70 hours in constant darkness (approximately CT6-CT10). Using two different mTim TaqMan probes, low but clearly detectable levels of mTim mRNA in the retina were found, but similar to that seen in the SCN there was no significant circadian rhythm. These two sets of experiments are consistent with at least five independent attempts to measure circadian rhythms of mTim mRNA in mice. Therefore, no circadian rhythms of mTim mRNA levels can be detected in the SCN and retina of mice.

Biochemical Interaction of hTIM with Drosophila and Mammalian PER: In Drosophila, dPER and dTIM heterodimerize both in vivo and in vitro [Gekakis ef al., Science 270:811- 815 (1995); Lee, ef al, Science 271:1740-4 (1996); Saez, ef al, Neuron 17:911-920 (1996); Zeng, et al, Nature 380:129-35 (1996)]. Thus one functional test of the mammalian TIM proteins is their ability to interact with Drosophila and mammalian PER proteins. Direct association of hTIM and dPER polypeptide fragments in vitro were therefore tested (Figs.7A- 7C). 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, 35S-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. These experiments demonstrate that hTIM and dPER polypeptides directly associate in vitro. The pattern of dPER fragments that associate with hTIM is identical to that seen with Drosophila TIM polypeptide fragments (Figure 8). These protein-protein interaction experiments are consistent with human TIM being an ortholog of Drosophila tim.

To determine whether the mammalian per homologs could interact with hTIM and mTIM, 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, 35S-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. These experiments provide direct evidence for protein-protein interaction of mammalian TIM with either mPERl or mPER2. By analogy to that seen in Drosophila, these results indicate that TIM dimerizes with PER in mammals and that this interaction could be important for the function of these mammalian 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). Although 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.

Inhibition of CLOCK-BMALl Transactivation by mPERl and hTIM: An additional important functional criterion for the coπect identification of a putative mammalian ortholog of dTIM (or dPER) is the ability to engage in inhibition of Per gene transactivation by the CLOCK-BMALl heterodimer, a central feature of the negative feedback model of Drosophila and mammalian circadian clocks [Allada ef al.,Cell 93:791-804 (1998); Darlington ef al., Science 280:1599-6031 (1998); Gekakis, ef al., Science 280:1564-1569 (1998); Rutila ef al.,Cell 93:805-814 (1998)] that has received direct experimental support in the case of Drosophila PER and TIM [Darlington ef al, Science 280: 1599-1603 (1998)]. To test this prediction with regard to hTIM and mPERl, luciferase reporter assays were caπied 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.

Expression of CLOCK and BMALl together resulted in a robust activation of transcription from a 2.0-kb 5' flanking fragment from the mPerl gene that included all three E-boxes (Figure 11A), as seen previously [Gekakis, N. ef al, Science 280, 1564-1569 (1998)]. Additional expression of either mPERl or hTIM resulted in a significant inhibition of this transcriptional activation (45% and 42% inhibition, respectively; P < 0.0005 for each). Expression of mPERl and hTIM together resulted in a level of inhibition only marginally greater than that seen with the expression of either one alone (52% inhibition). Expression of mPERl or hTIM in the absence of CLOCK and BMALl expression resulted in no detectable inhibition of the basal transcriptional activity. An essentially identical pattern of results was obtained using a 54-bp fragment in which the three mPerl E-boxes and their immediate flanking sequences were linked together (Figure 11B).

The specificity of the inhibitory effects of hTIM and mPERl on transactivation from E-box sites was examined next. Once again, expression of either mPERl or hTIM significantly inhibited transactivation by the CLOCK-BMALl heterodimer from mPerl E-box sites (51% and 57% inhibition, respectively); but expression of Id, a known inhibitor of the MyoD-E12 heterodimer and related bHLH proteins [Benezra, ef al, Cell 61:49-59 (1990)], caused only a small inhibition 24%) that was not statistically significant (Figure 11C). Conversely, expression of either mPERl or hTIM produced little or no inhibition of transactivation by the MyoD-E12 heterodimer from a muscle creatine kinase (mck) gene E-box site (4% and 19% inhibition, respectively); whereas, expression of Id resulted in a large and highly significant inhibition (69% inhibition) as anticipated (Figure 11D).

These results indicate that both hTIM and mPERl can inhibit CLOCK-BMALl transactivation from an mPerl gene regulatory element, as predicted if they represent mammalian circadian clock components. These experiments do not necessarily indicate that hTIM or mPERl can act alone to inhibit CLOCK-BMALl transactivation. Northern analysis revealed that untransfected NIH-3T3 cells expressed both the 4.7-kb mPerl and 4.5-kb mTim transcripts. Expression of these likely clock components in a mouse fibroblast cell line is not surprising given that cultured rat fibroblasts have recently been shown to express perl and per2 and to contain a functional circadian clock [Balsalobre ef al.,Cell 93:929-937 (1998)]. The most likely interpretation of these results is that exogenous hTIM or mPERl interacted with endogenous mPERl and mTIM, respectively, to inhibit CLOCK-BMALl transactivation. This interpretation is supported by the demonstration of a direct protein-protein interaction between mTIM and mPERl (Fig. 8). That coexpression of exogenous mPERl and hTIM did not lead to significantly greater inhibition than either alone could be a limitation imposed by the transfection conditions or it could reflect a requirement for an endogenous factor other than mPERl or mTIM that is limiting for the inhibition.

Discussion The identification of a mammalian TIMELESS ortholog completes the description of a basic four-component model of the circadian autoregulatory loop in mammals involving Clock, BMALl, the mPer paralogs, and Tim. Recent work has lead to a similar model in Drosophila (Figure 12 A) in which PER and TIM are co-regulated in a circadian manner and interact to inhibit their own transcription mediated by dCLOCK-dBMAL transactivation via E box elements in the per and tim promoters [Darlington et al, Science 280:1548-1549 (1998)]. While the same set of genes appears to be involved in both Drosophila and mammals, there are interesting similarities and differences apparent already. At the level of the core circadian mechanism, the positive elements of the system, CLOCK and BMAL, are remarkably well conserved in Drosophila and mammals (Figure 12A-12B). In both systems, the CLOCK-BMAL heterodimeric complex activates transcription of their respective period gene promoters via identical E box regulatory elements. In mammals such an analysis has been demonstrated for the mPerl promoter, but has not yet been reported for mPer2 or mPer3. However, because all three mammalian per genes express circadian rhythms at the mRNA level, it appears likely that all three will be regulated by CLOCK-BMAL. An important variable both among the various per paralogs and among the various tissues in which they are expressed, are the differences in the phases of the per mRNA rhythms in mammals. While the mRNA rhythms of mPerl, and to a lessor extent mPerl and mPer3, peak in the day time in the SCN, the coπesponding per rhythms in the rest of the body peak much later, near dusk. Thus the phases of the per mRNA rhythms in the SCN clearly differ from that seen in Drosophila; however the rest of the body tissues in mammals express per mRNA rhythms very similar to Drosophila. The expression of mTim mRNA levels in mice is apparently different from Drosophila and does not appear to oscillate. The lack of an mTim mRNA rhythm suggests that unlike in Drosophila, CLOCK-BMAL may not positively transactivate the mTim promoter - in this case the lack of a rhythm would be due to the absence of CLOCK-BMAL as a target of negative feedback by PER-TIM. Alternatively, the lack of an mTim mRNA rhythm could be due to something as trivial as a long mRNA half life - in this case mTim transcription would still be circadian, however, the steady state mRNA abundance of mTim would be constant. Such issues can be addressed directly due to the cloning of mTim of the present invention.

In considering the four-component autoregulatory loop in mammals disclosed herein, it is not a requirement for both the PER and TIM proteins to oscillate as long as the oscillating proteins (in this case, the PERs) are rate limiting within their dynamic range. In this case, the mammalian PER proteins would be the dominant "state variables" of the oscillator system and TIM would become an essential permissive factor. The presence of three different Per genes in mammals is also consistent with the apparent constitutive mammalian TIM expression. Because the three per genes have different phased mRNA rhythms, it appears to be optimal to have constant TIM expression since there appears to be only one Tim gene in mammals. A second and perhaps more important reason to have only one TIM when there are multiple PERs is to integrate this circadian pathway. 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. Thus one can already envisage a number of subtleties in the circadian transcription-translation feedback loop oscillator system by comparing the Drosophila and mouse systems.

At the input level, there are already a number of interesting differences in the four-component model of the circadian loop in Drosophila and mammals. The most obvious is the site at which light acts in the mammalian SCN as compared to Drosophila (Figure 12A and 12B). In mice, recent work has shown that light exposure rapidly induces mPerl and mPer2 mRNA levels in the SCN [Albrecht ef al, Cell 91:1055-1064 (1997); Shearman, ef al., Neuron 19: 1261-1269 (1997); Shigeyoshi et al.,Cell 91: 1043-1053 (1997); Takumi et al. Genes Cells 3:167-176 (1998); Takumi ef al, Embo. J. 17:4753-4759 (1998); Zylka ef al., Neuron 20:1103-1110 (1998)]. Interestingly, mPer3 is not responsive to acute light stimulation [Takumi et al, Genes Cells 3:167-176 (1998); Takumi, et al, Embo. J. 17:4753- 4759 (1998); Zylka et al, Neuron 20, 1103-1110 (1998)]. In contrast in Drosophila, light does not directly affect PER, but rather causes a rapid degradation of TIM protein [Hunter-Ensor, ef al, Cell 84:677-685 (1996); Lee, ef al, Science 271: 1740-1744 (1996); Myers, ef al, See Comments, Science 271, 1736-1740 (1996); Zeng, et al, Nature 380:129- 135 (1996)]. As discussed previously [Shigeyoshi ef al, Cell 91:1043-1053 (1997)], these differences (increased transcription vs. decreased protein level) make functional sense depending on whether the clock component (protein) peaks in the day or in the night. 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. By contrast, 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. Because the peaks of mammalian per mRNA rhythms occur at dusk in most peripheral tissues, 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. Thus, it is possible that a Drosophila-lϊke model may apply more directly to peripheral tissues in mammals.

With clones of both mammalian per and tim now in hand it is also possible to examine postranscriptional steps in the regulation of mammalian PER and TIM protein accumulation. These appear to be central to forming a circadian clock from a central autoregulatory loop in Drosophila [Dembinska, et al, J. Biol. Rhythms 12: 157-172 (1997); Edery, et al, Proc. Natl. Acad. Sci. U.S.A. 91:2260-2264 (1994); Kloss ef al, Cell 94:97-107 (1998); Price et al, Cell 94:83-95 (1998); So et al, Embo. J. 16:7146-7155 (1997)]. Given that entirely different but analogous sets of genes form circadian autoregulatory feedback loops of transcription and translation in Neurospora and cyanobacteria (Dunlap, Annul. Rev. Genet. 30: 579-601 (1996)], the extent to which higher order levels of regulation of the circadian mechanism are conserved can be compared.

Therefore, a four-component 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.

The present invention is not to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description and the accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

It is further to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for description.

Various publications are cited herein, the disclosures of which are hereby incorporated by reference herein in their entireties.

WHAT IS CLAIMED IS:

1. An isolated nucleic acid encoding a mammalian ortholog of the Drosophila TIMELESS (TIM) protein, wherein:

(a) expression of said nucleic acid in Drosophila (S2) cells promotes nuclear entry of Drosophila PER protein;

(b) expression of the nucleic acid and a second nucleic acid encoding murine PERI specifically inhibit CLOCK-BMALl -induced transactivation of the murine perl promoter; and

(c) the amino acid sequence of the ortholog has at least 50% identity with both SEQ ID NO:2 and SEQ ID NO:20.

2. The isolated nucleic acid of Claim 1 wherein said mammalian TIM protein binds Drosophila PERIOD (PER) protein and mouse PERI and PER2 in vitro.

3. The isolated nucleic acid of Claim 1 wherein said mammalian TIM protein comprises: (a) a PER protein binding domain; and (b) a cytoplasmic localization domain (CLD); wherein the CLD comprises an acidic tetrapeptide at or near the C-Terminal end of the CLD.

4. The isolated nucleic acid of Claim 3 wherein the acidic tetrapeptide is DEDD (SEQ ID

NO:27).

5. The isolated nucleic acid of Claim 3 further comprising a GLU-ASP rich region.

6. An isolated nucleic acid encoding a variant of the human TIMELESS protein.

7. The isolated nucleic acid of Claim 6 wherein the variant has an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6,

SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, and SEQ ID NO:16.

8. The isolated nucleic acid of Claim 7 wherein the nucleic acid has a nucleotide selected from the group consisting of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: l l, SEQ ID NO:13, SEQ ID NO: 15, SEQ ID NO: 17, and SEQ ID NO:18.

9. The isolated nucleic acid of Claim 6 wherein the variant has an amino acid selected from the group consisting of SEQ ID NO:2 comprising a conservative amino acid substitution, SEQ ID NO:4 comprising a conservative amino acid substitution, SEQ ID NO:6 comprising a conservative amino acid substitution, SEQ ID NO: 8 comprising a conservative amino acid substitution, SEQ ID NO: 10 comprising a conservative amino acid substitution, SEQ ID NO: 12 comprising a conservative amino acid substitution, SEQ ID NO: 14 comprising a conservative amino acid substitution, and SEQ ID NO: 16 comprising a conservative amino acid substitution.

10. An isolated nucleic acid encoding a variant of the murine TIMELESS protein.

11. The isolated nucleic acid of Claim 10 wherein the variant has an amino acid sequence selected from the group consisting of SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:26.

12. The isolated nucleic acid of Claim 11 wherein the nucleic acid has a nucleotide sequence selected from the group consisting of SEQ ID NO: 19, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:26.

13. The isolated nucleic acid of Claim 10 wherein the variant has an amino acid sequence selected from the group consisting of SEQ ID NO:20 comprising a conservative amino acid substitution, SEQ ID NO:22 comprising a conservative amino acid substitution, SEQ ID NO:24 comprising a conservative amino acid substitution, and SEQ ID NO:26 comprising a conservative amino acid substitution.

14. A recombinant DNA molecule that is operatively linked to an expression control sequence, wherein the recombinant DNA molecule comprises the isolated nucleic acid of Claim 1.

15 . An expression vector containing the recombinant DNA molecule of Claim

14.

16. A method of expressing a recombinant TIMELESS protein in a cell containing the expression vector of Claim 15 comprising culturing the cell in an appropriate cell culture medium under conditions that provide for expression of the TIMELESS protein by the cell.

17. The method of Claim 16 further comprising the step of purifying the recombinant TIMELESS.

18. The recombinant TIMELESS protein purified by the method of Claim 17.

19. An isolated nucleic acid consisting of at least 24 consecutive nucleotides of a nucleotide sequence that encodes a TIMELESS protein having an amino acid sequence selected from the group consisting of SEQ ID NO:2, and SEQ ID NO:2 comprising a conservative substitution thereof.

20. The isolated nucleic acid of Claim 19 further comprising a heterologous nucleotide sequence.

21. An isolated nucleic acid consisting of at least 24 consecutive nucleotides of a nucleotide sequence that encodes a TIMELESS protein having an amino acid sequence selected from the group consisting of SEQ ID NO:20, and SEQ ID NO:20 comprising a conservative substitution thereof.

22. The isolated nucleic acid of Claim 21 further comprising a heterologous nucleotide sequence.

23. A nucleotide probe for a nucleic acid, wherein the nucleic acid has a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:26.

24. A nucleic acid that hybridizes under standard conditions to the nucleic acid of SEQ ID NO: 1.

25. An isolated TIM protein that is a mammalian ortholog of the Drosophila

TIMELESS (TIM) protein, wherein:

(a) expression of a nucleic acid encoding the mammalian TIM protein in Drosophila (S2) cells promotes nuclear entry of Drosophila PER protein;

(b) expression of the nucleic acid and a second nucleic acid encoding murine PERI specifically inhibit CLOCK-BMALl -induced transactivation of the murine perl promoter; and

(c) the amino acid sequence of the ortholog has at least 50% identity with both SEQ ID NO:2 and SEQ ID NO:20.

26. The isolated mammalian TIM protein of Claim 25 wherein said mammalian TIM protein binds Drosophila PERIOD (PER) protein and mouse PERI and PER2 in vitro.

27. The isolated mammalian TIM protein of Claim 25 wherein said mammalian TIM protein comprises:

(a) a PER protein binding domain; and

(b) a cytoplasmic localization domain (CLD); wherein the CLD comprises an acidic tetrapeptide at or near the C-Terminal end of the CLD.

28. The isolated mammalian TIM protein of Claim 27 wherein the acidic tetrapeptide is DEDD (SEQ ID NO:27).

29. The isolated mammalian TIM protein of Claim 27 further comprising a GLU-ASP rich region.

30. An isolated mammalian TIM protein that is a variant of the human TIMELESS protein.

31. The isolated mammalian TIM protein of Claim 30 wherein the variant has an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, and SEQ ID NO:16.

32. The isolated mammalian TIM protein of Claim 31 wherein the variant has an amino acid sequence selected from the group consisting of SEQ ID NO:2 comprising a conservative amino acid substitution, SEQ ID NO:4 comprising a conservative amino acid substitution, SEQ ID NO:6 comprising a conservative amino acid substitution, SEQ ID NO: 8 comprising a conservative amino acid substitution, SEQ ID NO: 10 comprising a conservative amino acid substitution, SEQ ID NO: 12 comprising a conservative amino acid substitution, SEQ ID NO: 14 comprising a conservative amino acid substitution, and SEQ ID NO: 16 comprising a conservative amino acid substitution.

33. An isolated mammalian TIM protein that is a variant of the murine

TIMELESS protein.

34. The isolated mammalian TIM protein of Claim 33 wherein the variant has an amino acid sequence selected from the group consisting of SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, and SEQ ID NO:26.

35. The isolated mammalian TIM protein of Claim 33 wherein the variant has an amino acid sequence selected from the group consisting of SEQ ID NO:20 comprising a conservative amino acid substitution, SEQ ID NO:22 comprising a conservative amino acid substitution, SEQ ID NO:24 comprising a conservative amino acid substitution, and SEQ ID NO:26 comprising a conservative amino acid substitution.

36. A fragment of the mammalian TIM protein of Claim 25, wherein the fragment binds to PER protein.

37. The fragment of the mammalian TIM protein of Claim 36, wherein the fragment when bound to PER protein can specifically inhibit CLOCK-BMALl -induced transactivation of the murine perl promoter.

38. The fragment of the mammalian TIM protein of Claim 36, wherein the fragment promotes nuclear entry of Drosophila PER protein in Drosophila (S2) cells.

39. A fusion protein comprising the fragment of Claim 36.

40. A fusion protein comprising the mammalian TIM protein of Claim 25.

41. A fragment of the mammalian TIM protein of Claim 25 comprising a cytoplasmic localization domain (CLD); wherein the CLD comprises an acidic tetrapeptide at or near the C-Terminal end of the CLD.

42. A fusion protein comprising the fragment of Claim 41.

43. A proteolytic fragment of the mammalian TIM protein of Claim 25.

44. An antibody to the mammalian TIM protein of Claim 25.

45. The antibody of Claim 44 which is a polyclonal antibody.

46. The antibody of Claim 44 which is a monoclonal antibody.

47. An immortal cell line that produced a monoclonal antibody according to Claim 46.

48. A mammalian cell that naturally encodes the timeless gene but has been manipulated so as to be incapable of expressing the timeless gene.

49. A knockout mouse comprising a first and a second allele which naturally encode and express a murine timeless gene, wherein the first allele and the second allele each contain a defect which prevents the knockout mouse from expressing the timeless gene.

50. The knockout mouse of Claim 49 wherein the mouse has an abnormal circadian rhythm.

51. A method for detecting the presence of the mammalian TIM protein of Claim 25 or an mRNA encoding the mammalian TIM protein comprising:

(a) contacting a biological sample from a mammal in which the presence or activity of said protein is suspected with a binding partner of said protein or mRNA under conditions that allow binding of said protein or mRNA to said binding partner to occur; and

(b) detecting whether binding has occuπed between said protein or mRNA in said sample and the binding partner; wherein the detection of binding indicates that presence or activity of said protein or mRNA in said sample.

52. The method of Claim 51 wherein the binding partner is selected from the group consisting of a nucleotide probe for an mRNA encoding the mammalian TIM, a PER protein, and an antibody to the mammalian TIM protein.

53. A method of identifying an agent that can modulate the binding of a mammalian TIM protein to a PER protein comprising:

(a) contacting a mammalian TIM protein or a mammalian TIM fragment with a PER protein or a PER protein fragment, in the presence of the agent; wherein the mammalian

TIM fragment comprises a fragment of a mammalian TIM protein that contains a PER protein binding domain (PBD) and the PER protein fragment comprises a fragment of a PER protein that binds to the mammalian TIM in the absence of the agent; and

(b) determining the binding of the mammalian TIM protein or the mammalian TIM fragment with the PER protein or the PER protein fragment; wherein an agent is identified as modulating the binding of the mammalian TIM protein to the PER protein when said determining is indicative of a change in the binding relative to that in the absence of the agent.

54. The method of Claim 53 wherein when said determining indicates an increase in the binding of the mammalian TIM protein to the PER protein, the agent is identified as an agonist of the binding of the mammalian TIM protein to the PER protein; and wherein when said determining indicates a decrease in the binding of the mammalian TIM protein to the PER protein, the agent is identified as an antagonist of the binding of the mammalian TIM protein to the PER protein.

55. 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 comprising:

(a) 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; wherein said mammalian TIM protein promotes the entry of the PER protein into the nucleus of the cell in absence of the agent; and

(b) determining the amount of PER protein in the nucleus; wherein 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 said determining is indicative of a change in the amount of PER protein in the nucleus relative to that in the absence of the agent.

56. The method of Claim 55 wherein when said determining indicates an increase in the entry of the PER protein to the nucleus, the agent is identified as an agonist; and wherein when said determining indicates a decrease in the entry of the PER protein to the nucleus, the agent is identified as an antagonist.

57. A method of identifying an agent that can modulate the effect of a PER protein and a mammalian TIM protein on the transactivation of the CLOCK-BMALl heterodimer comprising:

(a) transiently expressing mammalian timeless and period in a cell, wherein the cell: (i) comprises a reporter gene operatively linked to an expression control sequence that is transactivated by the CLOCK-BMALl heterodimer; and (ii) expresses nucleic acids encoding CLOCK and BMALl constitutively;

(b) contacting a potential agent with the cell; and (c) determining the amount of the reporter gene expressed in the cell; wherein 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-BMALl heterodimer when said determining indicates a change in the amount of expression of the reporter gene in the cell relative to that in the absence of the agent.

58. The method of Claim 57 wherein when said determining 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 PER protein and mammalian TIM on the transactivation of the CLOCK-BMALl heterodimer; and wherein when said determining 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 PER protein and mammalian TIM on the transactivation of the CLOCK- BMALl heterodimer.

59. The method of Claim 57 further comprising:

(d) contacting the candidate agent with the cell in the absence of the expression of period and mammalian timeless; and

(e) determining the amount of the reporter gene expressed in the cell; wherein a 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-BMALl heterodimer when said determining indicates a no apparent change in the amount of expression of the reporter gene in the cell in the absence PER protein and mammalian TIM expression.

60. A test kit for the demonstration of the mammalian TIM protein of Claim 25 in a cellular sample comprising a predetermined amount of a detectably labeled binding partner of the protein.

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