Clock gene and methods of use thereof

Details Clock gene and methods of use thereof Clock gene and methods of use thereof

2000-04-20

2002-11-05

Guzo, David (Department: 1636)

Chemistry: natural resins or derivatives; peptides or proteins;

Peptides of 3 to 100 amino acid residues

25 or more amino acid residues in defined sequence

C530S350000, C530S325000, C530S326000, C530S327000, C530S328000, C530S329000

Reexamination Certificate

active

06476188

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to a protein involved in the nuclear localization of proteins, and more specifically to a protein involved in maintaining circadian rhythms. The invention also relates to mutants of the protein, nucleic acid and amino acid sequences encoding the protein itself, as well as methods of using the protein including in drug assays.
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 appear to be a universal component of animal behavior [Pittendrigh, C. S.,
Proc. Natl. Acad. Sci USA,
58:1762-1767 (1967); Pittendrigh, C. S.,
Neurosciences,
437-458 (1974)]. Indeed, circadian physiological rhythms are not limited to the animal kingdom, and genetic screens have identified clock genes in Drosophila [Konopka and Benzer,
Proc. Natl. Acad. Sci. USA,
68:2112-2116 (1971); and Sehgal et al.,
Science,
263:1603-1606 (1994)], Chlamydomonas [Bruce, V. G.
Genetics,
70:537-548 (1972)], Neurospora [Feldman and Hoyle,
Neurospora crassa Genetics,
75:605-613 (1973); Crosthwaite et al.,
Science,
276:763-769 (1997)], Cyanobacteria [Kondo et al.,
Science,
266:1233-1236 (1994)], Arabidopsis [Millar et al.,
Science,
267:1161-1163 (1995)], hamster [Ralph and Menaker,
Science,
241:1225-1127(1988)], and mouse [Vitaterna et al.,
Science,
264:719-725 (1994)].
Fruit flies show circadian regulation of several behaviors [Pittendrigh in
The Neurosciences Third Study Program,
Chap. 38, F. O. Schmitt and F. G. Worden, Eds. (MIT Press, Cambridge Mass., 1974); Jackson, in
Molecular Genetics of Biological Rhythms,
pp. 91-121, M. W. Young, Ed. (Dekker, New York, 1993)]. When populations of Drosophila are entrained to 12 hours of light followed by 12 hours of darkness (LD 12:12), adults emerge from pupae (eclose) rhythmically, with peak eclosion recurring every morning. The eclosion rhythm persists when the entraining cues are removed and behavior is monitored in constant darkness, thus indicating the existence of an endogenous clock. Adult locomotor activity is also controlled by an endogenous clock and recurs rhythmically with a 24-hour period.
In the fruit fly
Drosophila melanogaster,
two genes are essential components of the circadian clock, period and timeless [Sehgal et al.
Science
263:1603 (1994)]. Mutations in either of these genes can produce arrhythmicity or change the period of the rhythm by several hours [Konopka and Benzer
Proc. Natl. Acad. Sci. U.S.A.
68:2112 (1971); Sehgal et al
Science
263:1603 (1994)]. Molecular studies [Bargiello et al
Proc. Natl. Acad. Sci. U.S.A.
81:2142 (1984); Reddy et al
Cell
38:701 (1984); Myers
Science
270:805 (1995); Hardin et al.
Nature
343:536 (1990); Sehgal et al.,
Science,
270:808-810 (1995)] have shown that per and tim are transcribed with indistinguishable circadian rhythms that are influenced by an interaction of the TIM and PER proteins [Sehgal et al.
Science
263:1603 (1994); Gekakiset et al
Science,
270:811 (1995)]. A physical association of the two proteins appears to be required for accumulation and nuclear localization of PER [Sehgal et al
Science
263:1603 (1994); Gekakiset et al. (1995); Price et al.
EMBO J.,
14:4044 (1995)]. It is likely that nuclear localization leads to suppression of per and tim transcription [Hardin et al.
Nature,
343:536 (1990); Sehgal et al.,
Science,
270:808-810(1995)]. Cycles of gene expression are thought to be sustained by 5 hour differences in the phases of RNA and protein accumulation. The observed delays in PER accumulation may result, in part, from a requirement for TIM to stabilize PER [Sehgal et al
Science
263:1603 (1994); Sehgal et al.
Science,
270:808-810(1995); Price et al.
EMBO J.,
14:4044-4049 (1995)].
More specifically, mutations in the Drosophila period (per) gene, for example, disrupt circadian rhythms of pupal eclosion and adult locomotor behavior [Konopka and Benzer
Proc. Natl. Acad. Sci. U.S.A.
68:2112 (1971)]. Although per has been cloned and sequenced and its pattern of expression has been analyzed [Baylies et al. in
Molecular Genetics of Biological Rhythms,
pp. 123-153, M. W. Young, Ed. (Dekker, New York, 1993); Rosbash and Hall
Neuron
3:387 (1989)], the biochemical function of the PER protein is unknown. PER shares some homology with a family of transcription factors [Crews et al
Cell
52:143 (1988); Nambu et al
Cell
67:1157 (1991); Reisz-Porszasz et al
Science
256:1193 (1992); Hoffman et al
Cell
252:954 (1991); Burbach et al
Proc. Natl. Acad. Sci. U.S.A.
89:8185 (1992)] that possess a common sequence motif called the PAS domain.
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, N.Y., 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
Mol. 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 [Konopka and Benzer
Proc. Natl. Acad. Sci. U.S.A.
68:2112 (1971)]. Three mutant alleles—per
O
, per
S
, and per
L
,—cause arrhythmic behavior or shorten or lengthen periods, respectively [Konopka and Benzer
Proc. Natl. 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
Mol. Cell. Biol.
8:5378 (1988); Zerr et al
J. Neurosci.
10:2749 (1990)]. This suggests a possible 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)].
Several mutations that affect eclosion and locomotor activity have been isolated in behavioral screens [Jackson, in
Molecular Genetics of Biological Rhythms,
pp. 91-121, M. W. Young, Ed. (Dekker, N.Y., 1993); Konopka and Benzer
Proc. Natl. Acad. Sci. U.S.A.
68:2112 (1971); Rosbash and Hall
Neuron
3:387 (1989); Baylies et al in
Molecular Genetics of Biological Rhythms,
pp. 123-153, M. W. Young, Ed. (Dekker, N.Y., 1993); Jackson,
J. Neurogenet
1:3 (1983); Dushay et al
J. Biol. Rhythms
4:1 (1989); Dushay et al
Genetics
125:557 (1990); Konopka et al.,
Proc. Natl. Acad. Sci. U.S.A.
68:2112 (1991)]. The b

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