WO1998040514A1

WO1998040514A1 - Clock gene and gene product

Info

Publication number: WO1998040514A1
Application number: PCT/US1998/005114
Authority: WO
Inventors: Joseph S. Takahaski; Fred Turek; Lawrence H. Pinto
Original assignee: Northwestern University
Priority date: 1997-03-13
Filing date: 1998-03-13
Publication date: 1998-09-17
Also published as: AU6558298A

Abstract

The present invention provides isolated and purified polypeptide components of the mammalian circadian clock, polynucleotides that encode those polypeptides, expression vectors containing those polynucleotides, host cells transformed with those expression vectors, a process of making the polypeptide components using those polynucleotides and vectors, and processes using those polypeptides and polynucleotides.

Description

CLOCKGENEAND GENE PRODUCT

Technical Field of the Invention

The field of the present invention is the circadian clock of mammals. More particularly, the present invention relates to mammalian genes and gene products that regulate aspects of the circadian rhythm in mammals and those processes controlled by the circadian rhythm.

Background of the Invention Circadian rhythms are a fundamental property of all eukaryotic and some prokaryotic organisms (Takahashi 1995). The underlying molecular mechanism appears similar among living systems, is cell autonomous and involves periodic macromolecular synthesis. Alterations in circadian rhythms are involved in sleep disorders such as "delayed sleep phase syndrome" which may be an alteration in the circadian period (lengthening) and the entrainment system. There is also evidence for circadian rhythm abnormalities in affective disorders. The most consistent feature of circadian rhythms observed in depressed patients is that a variety of physiological events occur earlier than normal (usually referred to as a "phase advance"). A shortened REM latency after sleep onset, which can be the manifestation of a change in the circadian coupling or organization of rhythms, appears to be a prominent characteristic of depression.

Further, a number of diagnostic tests depend on the time of day at which the test is performed. These include the dexamethasone suppression test for depression, intraocular pressure measurements for glaucoma, and plasma cortisol concentration for Addison's disease and Cushing's syndrome. .In addition, a number of clinical treatments (such as chemotherapy or alleviation of hypertension) can be optimized through the delivery of therapeutic agents at the appropriate time of day. Circadian rhythmicity appears to be deeply embedded in most aspects of the biology of organisms - indeed it is a central feature of their organization. It seems unlikely that complete understanding of most regulatory processes can be achieved without an appreciation of their circadian dimensions.

Clock genes have been described in other model systems, most notably in Drosophila and Neurospora. Three known clock genes have been characterized at the molecular and functional level. These are the period (per) and timeless (tim) genes in Drosophila, and the frequency (frq) gene in Neurospora. This work is known to the art and is described in review papers by J.S. Takahashi, Annual Review ofNeuroscience 18:531-553, 1995; and by J.C. Dunlap, Annual Review of Genetics 30:579-601, 1996. None of these three clock genes have been shown to possess a protein motif known to allow these proteins to bind DNA, rather it appears that in the case of PERIOD and TIMELESS, these proteins must interact with unidentified DNA-binding transcription factors.

The genetic approach to circadian rhythms was first described by Ron Konopka and Seymour Benzer (1971) who isolated single-gene mutations that altered circadian periodicity in Drosophila. In a chemical mutagenesis screen of the X chromosome, they found three mutants that either shortened (per s ),

lengthened (per ) or abolished (per ) circadian rhythms of eclosioπ and adult locomotor activity. In 1984, two groups at Brandeis and Rockefeller independently cloned per in a series of experiments that showed that germline transformation with DNA could rescue a complex behavioral program (reviewed in Rosbash & Hall 1989). Each of the mutant er alleles is caused by mtragemc point mutations that produce missense mutations in per S and per L ,

and a nonsense mutation mper (Bayfies et al. 1987, Yu et al. 1987). Only recently has the nature of per gene product (PER )become more clear. The Drosophila single-minded protein (SIM) (Nambu et al. 1991), the human aryl hydrocarbon receptor nuclear translocator (ARNT) (Hoffirian et al. 1991), and the aryl hydrocarbon receptor (AHR) (Burbach et al. 1992) all share with PER a domain called PAS (for PER, ARNT, SIM) (Nambu et al. 1991). The PASdomain contains about 270 amino acids of sequence similarity with two 51- amino acid direct repeats. Recent work shows that the PAS domain can function as a dimerization domain (Huang et al.1993). Because other PAS members are transcriptional regulators and PER can dimerize to them, PER could function as a transcriptional regulator either by working in concert with apartner that carries a DNA-binding domain, or by acting as a dominant- negative regulator by competing with a transcriptional regulator for dimenization or DNA binding. Consistent with this role, PER is predominantly a nuclear protein in the adult central nervous system oi Drosophila (Liu et al. 1992).

The expression of PER itself is circadian, and both er mRNA and PER protein abundance levels oscillate. Hardin et al. (1990) showed that per mRNA levels undergo a striking circadian oscillation. The per RNA rhythm persists in constant darkness and the period of the RNA rhythm is ~24 hours in per" flies and is ~20 hours in per S flies. The RNA of per 0 flies is present at a level ~50% of normal flies, but does not oscillate. In per flies that have been rescued by germline transformation with wild-type per DNA, both circadian behavior and per RNA cycles are restored. Importantly, in these transformed flies both the exogenous per RNA and the endogenous per RNA levels oscillate. In addition to a per RNA cycle, the PER protein also shows a circadian rhythm in abundance (Siwicki et al. 1988, Zerr et al. 1990, Edery et al. 1994b). The rhythm in PER protein also depends on per, because per° flies do not have a protein rhythm and because per mutants alter the PER rhythm (Zerr et al. 1990). Therefore, the circadian expression of per mRNA and protein levels both depend on an active per gene. Because per shortens the period of the

RNA cycle and because per DNA transformation rescues per RNA cycling, PER protein expression clearly regulates per RNA cycling. Hardin et al. (1990) propose that feedback of the per gene product regulates its own mRNA levels. Support for such a model has been provided by showing that transient induction of PER from a heat shock promoter/per cDNA transgene in a wild-type background can phase shift circadian activity rhythms in Drosophila (Edery et al. 1994a).

The PER protein rhythm appears to be regulated at both transcriptional and post-transcriptional levels. Hardin et al. (1992) have shown that levels of per precursor RNA cycle in concert with mature per transcripts. In addition, per promoter/CAT fusion gene constructs show that per 5' flanking sequences are sufficient to drive heterologous RNA cycles. These results suggest that circadian fluctuations in per mRNA abundance are controlled at the transcriptional level. In addition to a rhythm in per transcription and PERabundance, PER appears to undergo multiple phosphorylation events as itaccumulates each cycle (Edery et al. 1994b). The nature and functional significance of the PER phosphorylation sites, however, are not known at this time. Interestingly, the peak of the per RNA cycle precedes the peak of the PER protein cycle by about 4-6 hours. The reasons for the lag in PER accumulation are not well understood. However, the recent isolation of a second clock mutant, named timeless (tim), has provided significant insight (Sehgal et al. 1994). Tim mutants fail to express circadian rhythms in eclosion and locomotor activity, but more importantly also fail to express circadian rhythms in per mRNA abundance (Sehgal et al. 1994). Furthermore, the nuclear localization of PER is blocked in tim mutants (Vosshall et al. 1994). In 1995, tim was cloned by positional cloning and by interaction with the PAS domain of PER in a yeast two-hybrid screen (Gekakis et al. 1995, Myers et al. 1995). Like PER, TIM is a large protein without any obvious sequence homologies to other proteins. While PER dimerizes to TIM via the PAS domain, TIM is not a member of the PAS family. The expression of tim RNA levels has a striking circadian oscillation which is in phase with the per RNA rhythm. The rhythm in tim RNA levels depends on PER and is abolished in per⁰ mutants and shortened in per mutants. Thus, per and tim express a coordinate circadian rhythm that is interdependent. TIM protein also shows a circadian rhythm with a phase similar to that of PER. Formation of a PER/TIM heterodimer appears to be required for nuclear entry of the complex. In the last year, four different laboratories discovered that light exposure causes a rapid degradation of TIM protein in flies and this action of light can explain how entrainment of the circadian clock in Drosophila occurs (Hunter-Ensor et al. 1996, Lee et al. 1996, Myers et al. 1996, Zeng et al. 1996). Thus, the identification of tim and its functional interaction with per is important because it suggests that elements of a transcription-translation-nuclear transport feedback loop are central elements of the circadian mechanism in Drosophila.

In addition to the Drosophila per and tim genes, progress has been made in elucidating the molecular nature of the Neurospora frequency (frq) gene (Dunlap 1993). Like per, ύiefrq locus is defined by mutant alleles that either shorten, lengthen or disrupt circadian rhythms (Feldman & Hoyle 1973, Feldman 1982, Dunlap 1993). Cloned in 1989, the sequence of FRQ shows little resemblance to PER (except for a region containing threonine-glycine repeats) (McClung et al. 1989); however, recent molecular work shows striking functional similarities (Aronson et al. 1994). The frq gene expresses a circadian oscillation of mRNA abundance whose period is altered by frq mutations (Aronson et al. 1994). A null allele,^⁹, expresses elevated levels of frq transcript and does not show a rhythm in mRNA abundance (Aronson et al. 1994). Interestingly, no level of constitutive expression of frq in a null background can rescue overt rhthmicity, which suggests that the circadian rhythm of frq mRNA is a necessary component of the oscillator (Aronson et al. 1994). However, overexpression of a frq^* transgene does negatively autoregulate expression of the endogenous of a frq gene (Aronson et al. 1994). In addition, overexpression of frg^* transgene in a wild-type background blocks overt expression of circadian rhythms (Aronson et al. 1994). The phase of the overt circadian rhythm can be determined by a step reduction in FRQ protein expression (Aronson et al. 1994). Taken together, these experiments show that frq is likely a central component of the Neurospora circadian oscillator and that a negative autoregulatory loop regulating./rg' transcription forms the basis of the oscillation (Aronson et al. 1994). Recently a direct action of light has been found on frq expression (Crosthwaite et al. 1995). Frq transcription is rapidly induced by light exposure and this effect of light can explain photic entrainment in Neurospora in a simple and direct manner.

Although there are remarkable functional similarities between per and frq, there are also distinct differences. The phases of the mRNA rhythms are different: per peaks at night (Hardin et al. 1990), whereas^ peaks during the day (Aronson et al. 1994). While per overexpression shortens circadian period (Smith & Konopka 1982, Baylies et al. 1987), frq overexpression does not change period but rather abolishes overt rhythmicity (Aronson et al. 1994). The null allele, per°, leads to a constant level of mRNA that is about 50% of the peak level of wild-type levels in Drosophila (Hardin et al. 1990); while in o. Neurospora, frq mRNA levels are significantly elevated relative to wild-type (Aronson et al. 1994). Finally, the action of light on these two systems is opposite: light degrades TIM protein in Drosophila; whereas, it activates the transcription of frq in Neurospora. These differences can be interpreted in at least two ways: 1) the elements of each system are not fully defined and frq and per could define different elements in a conserved pathway within the oscillator feedback loop; or 2) the Drosophila and Neurospora circadian clocks could be functionally analogous rather than phylogenetically homologous. Irrespective of the interpretation, however, it appears likely that a transcription-translation autoregulatory feedback loop may be a common feature of circadian clocks.

Searching for per homologs in mammals has not been very productive despite ten years of effort by a number of laboratories. This is probably due to the relatively low level of sequence similarity of per even among the Drosophilids (Hall 1990). Putative per homologs in mammals have been reported in searches directed against the threonine-glycine (TG) repeat region of PER (Shin et al. 1985, Matsui et al. 1993) and the region of the per " mutation

(Siwicki et al. 1992). However, the TG-repeat clones show no other sequence similarity to PER, and the antigenes detected by antibodies to the per region have not been characterized molecularly. New efforts targeted against the PAS dimerization domain (Huang et al. 1993), which is moderately well-conserved among insects (Reppert et al. 1994), using either PCR-based approaches or the yeast two-hybrid system (Fields & Song 1989) could eventually succeed as more bona fide per homologs are cloned in species more closely related to insects. Alternatively, as other Drosophila clock genes are cloned in the future, some should have sequence conservation with mammals as found, for example, with genes regulating pattem formation (Krumlauf 1993) or signal transduction (Zipursky & Rubin 1994). However, at this time no confirmed orthologs of per, tim or frq have been cloned in any vertebrate.

Very little information on the genetics of mammalian circadian rhythms is available. Most work in the field has used quantitative genetic approaches such as comparisons of circadian phenotype among inbred strains of mice and rats, recombinant inbred strain analysis, or selection of natural variants (Hall 1990, Schwartz & Zimmerman 1990, Lynch & Lynch 1992). The most comprehensive analysis of inbred mouse strains was done by Schwartz & Zimmerman (1990) who compared 12 different strains and found that the most extreme strains (C57BL/6J and BALB/cByJ) had a period difference of about one hour in constant darkness. Reciprocal FI hybrid and recombinant inbred strain analysis provided no evidence of monogenic inheritance of the circadian period. Polygenic inheritance of circadian traits (or more strictly, failure to detect monogenic inheritance) has been the conclusion of every quantitative genetic analysis performed thus far. A notable exception to the general finding of polygenic control of circadian phenotype is the spontaneous mutation, tau, found in the golden hamster (Ralph & Menaker 1988). Tau is a semidommant, autosomal mutation that shortens circadian period by two hours in heterozygotes and by four hours in homozygotes. Its phenotype is remarkably similar to the Drosophila per^s allele being semidominant, changing period to the same extent, and increasing the amplitude of the phase response curve to light (Ralph & Menaker 1988, Ralph 1991). The tau mutation has been extremely useful for physiological analysis. For example, the circadian pacemaker function of the suprachiasmatic nuclei (SCN) has been definitively demonstrated by transplantation of SCN tissue derived from tau mutant hamsters to establish that the genotype of the donor SCN determines the period of the restored rhythm (Ralph et al. 1990). Furthermore, the effects of having both tau mutant and wild-type SCN tissue in the same animal show that both mutant (~20 h) and wild-type (-24 h) periodicities can be expressed simultaneously suggesting that very little interaction of the oscillators occurs under these conditions (Nogelbaum & Menaker 1992). Additional cellular interactions can also be studied by transplantation of dissociated SCΝ cells derived from tau mutant and wild-type animals (Ralph & Lehman 1991). Thus, a number of issues that could not be addressed previously have been resolved or approached by the use of the tau mutation.

Unfortunately, not much progress has been made on the genetic and molecular nature of tau. Genetic mapping and molecular cloning of tau remains difficult because of the paucity of genetic information in the golden hamster. Thus far the tau mutation has contributed substantially to physiological analysis, but it will be difficult to elucidate the nature of the tau gene product unless candidate genes become apparent or the hamster is developed as a genetic system. Brief Summary of the Invention

In one aspect, the present invention provides an isolated and purified polynucleotide comprising a nucleotide sequence consisting essentially of a nucleotide sequence selected from the group consisting of (a)(i) the sequence of SEQ ID NO: 1 from about nucleotide position 491 to about nucleotide position

2953, the sequence of SEQ ID NO: 54 from about nucleotide position 418 to about nucleotide position 2955; (b) sequences that are complementary to the sequences of (a), and (c) sequences that, when expressed, encode a polypeptide comprising an amino acid residue sequence encoded by the sequence of (a). A polynucleotide can be a DNA or RNA molecule. A preferred polynucleotide contains the nucleotide sequence from nucleotide position number 419, 416, 392, 389 or 1 to nucleotide position number 2953 of SEQ ID NO: 1. Another preferred polynucleotide contains the nucleotide sequence from nucleotide position number 490, 438, 435, 421 or 418 to nucleotide position number 2955 of SEQ ID NO: 54.

In another embodiment, a polynucleotide of the present invention is contained in an expression vector. The expression vector preferably further comprises an enhancer-promoter operatively linked to the polynucleotide. In an especially preferred embodiment, the polynucleotide contains a nucleotide sequence as set forth above. The present invention still further provides a host cell transformed with a polynucleotide or expression vector of this invention. Preferably, the host cell is a bacterial cell such as an E. coli.

In another aspect, the present invention provides an oligonucleotide of from about 15 to about 50 nucleotides containing a nucleotide sequence that is identical or complementary to a contiguous sequence of at least 15 nucleotides a polynucleotide of this invention. A preferred oligonucleotide is an antisense oligonucleotide that is complementary to a portion of the polynucleotide of SEQ ID NO: 1 or 54. In another aspect, the present invention provides a polypeptide of mammalian origin. In one embodiment, that polypeptide is an isolated and puried polypeptide of about 855 or less amino acid residues that contains the amino acid residue sequence of at least one of :

a) from residue position 35 to residue position 855 of SEQ ID NO: 2; b) from residue position 11 to residue position 855 of SEQ ID NO: 2; c) from residue position 10 to residue position 855 of SEQ ID NO: 2; d) from residue position 2 to residue position 855 of SEQ ID NO: 2; or e) from residue position 1 to residue position 855 of SEQ ID NO: 2.

In another embodiment, that polypeptide is an isolated and puried polypeptide of about 846 or less amino acid residues that contains the amino acid residue sequence of at least one of :

a) from residue position 35 to residue position 846 of SEQ ID NO: 55; b) from residue position 11 to residue position 846 of SEQ ID NO: 55; c) from residue position 10 to residue position 846 of SEQ ID NO: 55; d) from residue position 2 to residue position 846 of SEQ ID NO: 55; or e) from residue position 1 to residue position 846 of SEQ ID NO: 55.

Preferably, a polypeptide of the present invention is a recombinant human polypeptide. In another aspect, the present invention provides a process of making a polypeptide of this invention comprising transforming a host cell with an expression vector that comprises a polynucleotide of the present invention, maintaining the transformed cell for a period of time sufficient for expression of the polypeptide and recovering the polypeptide. Preferably, the host cell is an eukaryotic host cell such as a mammalian cell, or a bacterial cell. An especially preferred host cell is an E. coli. The present invention also provides a polypeptide made by a process of this invention. The present invention also provides a pharmaceutical composition comprising a polypeptide, polynucleotide, expression vector or oligonucleotide of this invention and a physiologically acceptable diluent.

In another aspect, the present invention provides uses for the polypetides, polynucleotides and oligonucleotides of the present invention.

Brief Description of the Drawings

In the drawings which form a portion of the specification:

Fig. 1 shows the location of the Clock gene locus in the mouse genome using genetic meiotic mapping.

Fig. 2 is a schematic illustration of restriction mapping of YAC and BAC clones in the Clock region.

Fig. 3 is a schematic illustration of a transcript map of the Clock region.

Fig. 4 (in two panels, 4-1 and 4-2) is a schematic illustration of the breeding strategy used to produce and rescue Clock mutants.

Fig. 5 is a schematic illustration of the breeding strategy used to produce

TG36 progeny.

Fig. 6 is a schematic illustration of the physical location of the Clock gene. Fig. 7 shows the exon structure of the Clock gene and the exon content of different cDNA clones.

Fig. 8 (in eight panels, 8-1, 8-2, 8-3, 8-4, 8-5, 8-6, 8-7 and 8-8) shows the complete nucleotide sequence of the Clock gene based upon genomic exon sequences. The nucleotide sequence of the Clock gene is designated SEQ ID

NO:l and the deduced amino acid residue sequence of the CLOCK polypeptide is designated as SEQ ID NO:2.

Fig. 9 (in five panels, 9-1, 9-2, 9-3, 9-4 and 9-5) shows the nucleotide sequence of individual exons.

Fig. 10 shows the splice acceptor and donor sequences for the exons.

Fig. 11 shows a comparison between the amino acid residue sequence of the CLOCK polypeptide with human NPAS2 and mouse NPAS2.

Fig. 12 shows the amino sequence of CLOCK with the bHLH, PAS-A, PAS-B domains of a mutant Clock gene.

Fig. 13 shows the amino acid sequence of a CLOCK variant resulting from an alternate splice.

Fig. 14 (shown in 3 panels, 14-1, 14-2 and 14-3) shows the nucleotide and deduced amino acid sequence for human CLOCK.

Fig. 15 (shown in 2 panels, 15-1 and 15-2) shows the amino acid residue alignment of the mouse and human CLOCK polypeptides.

Fig. 16 (shown in 5 panels, 16-1, 16-2, 16-3, 16-4 and 16-5) shows the nucleotide alignment of the mouse and human CLOCK genes. Detailed Description ofthe Invention

I. The Invention

The present invention provides isolated and purified polypeptide components ofthe mammalian circadian clock, polynucleotides that encode those polypeptides, expression vectors containing those polynucleotides, host cells transformed with those expression vectors, a process of making the polypeptide components using those polynucleotides and vectors, and processes using those polypeptides and polynucleotides.

π. Clock Polypeptides

In one aspect, the present invention provides a polypeptide that is an integral component ofthe mammalian circadian clock. The polypeptide serves to regulate various aspects of circadian rhythm in mammals. The polypeptide is referred to herein as the CLOCK polypeptide. The CLOCK polypeptide contains about 855 or less amino acid residues. The amino acid residue sequence of an 855 residue embodiment of CLOCK, which embodiment is the gene product ofthe Clock gene ofthe mouse, described hereinafter, is set forth in SEQ ID NO:2. Another embodiment of a CLOCK polypeptide is set forth in SEQ ID NO:55. This later embodiment shows the CLOCK polypeptide obtained from humans.

It can be seen from SEQ ED NOs:2 and 55 that both polypeptides are members member ofthe basic helix-loop-helix (bHLH)-PAS domain family of proteins. The basic region ofthe bHLH domain is known to mediate DNA binding. Thus, CLOCK likely interacts directly with DNA. The HLH and PAS domains are further known to be protein dimerization domains and indicate that CLOCK can interact with itself or with other HLH-PAS domain family members. The C-terminal portion of both polypeptides (SEQ ID NO:2 and 55) can also be seen to have a number of glutamine-rich, proline-rich and serine-rich regions that are characteristic of activation domains of transcription factors. The CLOCK polypeptide functions as a transcription factor.

There are two methionine (Met) residues in the N-terminal portion of SEQ ID NO:2 and 55, both of which can serve as the N-terminus of a CLOCK polypeptide. Those two Met residues are located at residue positions 1 and 10 of SEQ ID NO:2 and 55. Thus, a CLOCK polypeptide ofthe present invention can contain the amino acid residue sequence of SEQ ID NO:2 or 55 extending from residue number 1 or residue number 10 to the C-terminus (residue number 855 or 846). As is well known in the art, polypeptides with an N-terminal Met residue can be produced without that Met residue, which Met-minus polypeptide has the same function as the Met-positive embodiment. Thus, a CLOCK polypeptide ofthe present invention can contain the amino acid residue sequence of SEQ ID NO:2 or 55 from residue number 2 or residue number 11 to residue number 855 or 846.

As is also well know in the art, proteins having b-HLH dormans can be processed such that the polypeptide starts at the beginning of that b-HLH domain. In SEQ ID NOs:2 and 55, the b-HLH begins at amino acid residue number 35. Thus, an embodiment of a CLOCK polypeptide ofthe present invention contains a polypeptide having the amino acid residue sequence of SEQ ID NO: 2 or 55 from residue number 35 to residue number 855 or 846.

As set forth in detail hereinafter, four forms ofthe CLOCK polypeptide have been identified in the mouse. Those four forms are: (1) SEQ ID NO:2; (2) residues 1 to 513 and residues 565 to 855 of SEQ ID NO:2; (3) residues 1 to 483 and residues 514 to 855 of SEQ ID NO:2; and (4) residues 1 to 483 and residues 565 to 855 of SEQ ID NO:2.

There are also four forms ofthe human CLOCK polypeptide that have been identified. Those four forms are: (1) SEQ ID NO:55; (2) residues 1 to 513 and residues 565 to 846 of SEQ ID NO:55; (3) residues 1 to 483 and residues 514 to 846 of SEQ ID NO:55; and (4) residues 1 to 483 and residues 565 to 846 of SEQ ID NO:55.

The present invention also contemplates amino acid residue sequences that are substantially duplicative ofthe sequences set forth herein such that those sequences demonstrate like biological activity to disclosed sequences. Such contemplated sequences include those sequences characterized by a minimal change in amino acid residue sequence or type (e.g., conservatively substituted sequences) which insubstantial change does not alter the basic nature and biological activity ofthe CLOCK polypeptide.

It is well known in the art that modifications and changes can be made in the structure of a polypeptide without substantially altering the biological function of that peptide. For example, certain amino acids can be substituted for other amino acids in a given polypeptide without any appreciable loss of function. In making such changes, substitutions of like amino acid residues can be made on the basis of relative similarity of side-chain substituents, for example, their size, charge, hydrophobicity, hydrophilicity, and the like.

As detailed in United States Patent No. 4,554,101, incorporated herein by reference, the following hydrophilicity values have been assigned to amino acid residues: Arg (+3.0); Lys (+3.0); Asp (+3.0); Glu (+3.0); Ser (+0.3); Asn (+0.2); Gin (+0.2); Gly (0); Pro (-0.5); Thr (-0.4); Ala (-0.5); His (-0.5); Cys (- 1.0); Met (-1.3); Val (-1.5)- Leu (-1.8); He (-1.8)- Tyr (-2.3); Phe (-2.5); and Tφ (-3.4). It is understood that an amino acid residue can be substituted for another having a similar hydrophilicity value (e.g., within a value of plus or minus 2.0) and still obtain a biologically equivalent polypeptide. In a similar manner, substitutions can be made on the basis of similarity in hydropathic index. Each amino acid residue has been assign sd a hydropathic index on the basis of its hydrophobicity and charge characteristics. Those hydropathic index values are: He (+4.5); Nal (+4.2); Leu (+3.8); Phe (+2.8); Cys (+2.5); Met (+1.9); Ala (+1.8); Gly (-0.4); Thr (-0.7); Ser (-0.8); Tφ (-0.9); Tyr (-1.3); Pro (-1.6); His (-3.2); Glu (-3.5); Gin (-3.5); Asp (-3.5); Asn (-3.5); Lys (-3.9); and Arg (-4.5). In making a substitution based on the hydropathic index, a value of within plus or minus 2.0 is preferred.

The CLOCK polypeptide ofthe present invention contains numerous phosphorylation sites. This invention contemplates phosphorylated as well as unphosphorylated embodiments.

A CLOCK polypeptide ofthe present invention has numerous uses. By way of example, such a polypeptide can be used in a screening assay for the identification of drugs or compounds that inhibit the action of CLOCK polypeptide (e.g., DΝA binding). The CLOCK polypeptide is an integral component ofthe circadian clock of mammals. As set forth below, animals lacking the ability to produce the CLOCK polypeptide have significant dysfunctions in their circadian clock. Mutant animals producing an altered CLOCK polypeptide can be given the normal CLOCK polypeptide together with suspected agonists or antagonists and the effects of such treatment on the restoration of a normal circadian rhythm can be determined. The CLOCK polypeptide can also be used to treat animals having circadian rhythm dysfunctions as set forth hereinafter.

In addition, a CLOCK polypeptide ofthe present invention can be used to produce antibodies that immunoreact specifically with the CLOCK polypeptide or antigenic determinants thereof. Means for producing antibodies are well known in the art. An antibody directed against CLOCK polypeptide can be a polyclonal or a monoclonal antibody. Antibodies against CLOCK polypeptide can be prepared by immunizing an animal with a CLOCK polypeptide ofthe present invention or an immunogenic portion thereof. Means for immunizing animals for the production ofantibodies are well known in the art. By way of an example, a mammal can be injected with an inoculum that includes a polypeptide as described herein above. The polypeptide can be included in an inoculum alone or conjugated to a carrier protein such as keyhole limpet hemocyanin (KLH). The polypeptide can be suspended, as is well known in the art, in an adjuvant to enhance the immunogenicity ofthe polypeptide. Sera containing immunologically active antibodies are then produced from the blood of such immunized animals using standard procedures well known in the art.

The identification ofantibodies that immunoreact specifically with CLOCK polypeptide is made by exposing sera suspected of containing such antibodies to a polypeptide ofthe present invention to form in a conjugate between antibodies and the polypeptide. The existence ofthe conjugate is then determined using standard procedures well known in the art.

A CLOCK polypeptide ofthe present invention can also be used to prepare monoclonal antibodies against CLOCK polypeptide and used as a screening assay to identify such monoclonal antibodies. Monoclonal antibodies are produced from hybridomas prepared in accordance with standard techniques such as that described by Kohler et al. (Nature.256:495, 1975). Briefly, a suitable mammal (e.g., BALB/c mouse) is immunized by injection with a polypeptide ofthe present invention. After a predetermined period of time, splenocytes are removed from the mouse and suspended in a cell culture medium. The splenocytes are then fused with an immortal cell line to form a hybridoma. The formed hyridomas are grown in cell culture and screened for their ability to produce a monoclonal antibody against CLOCK polypeptide. Screening ofthe cell culture medium is made with a polypeptide ofthe present invention.

III. Clock Polynucleotides

In another aspect, the present invention provides an isolated and purified polynucleotide that encodes a CLOCK polypeptide of mammalian origin. The polynucleotide can be a DNA molecule (e.g., genomic sequence, cDNA) or an RNA molecule (e.g., mRNA). Where the polynucleotide is a genomic DNA molecule, that molecule can comprise exons and introns interspersed therein.

As set forth hereinafter in the Examples, the Clock gene contains numerous exons. One of skill in the art will readily appreciate that the entire genome including introns is contemplated by the present invention. Where the polynucleotide is a cDNA molecule, disclosed sequences include coding regions as well as 5'- and 3 '-untranslated regions.

Only coding DNA sequences are disclosed herein. The present invention also provides, however, non-coding strands that are complementary to the coding sequences as well as RNA sequences identical to or complementary to those coding sequences. One of ordinary skill will readily appreciate that corresponding RNA sequences contain uracil (U) in place of thymidine (T).

In one embodiment, a polynucleotide ofthe present invention is an isolated and purified cDNA molecule that contains a coding sequence for a CLOCK polypeptide of this invention. Exemplary and preferred such cDNA molecules are shown as SEQ ID NO:l and 54. SEQ ID NO:2 is the deduced amino acid residue sequence ofthe coding region of SEQ HD NO: 1. As set forth above, a CLOCK polypeptide ofthe present invention can be a truncated or shortened form of SEQ ID NO:2 or 55. Thus, preferred polynucleotides of this invention depend on the specific CLOCK polypeptide preferred. By way of example, where the CLOCK polypeptide contains the amino residue sequence of SEQ ID NO:2 from residue number 1 to residue ae ^— - number 855, a preferred polynucleotide contains the nucleotide sequence of SEQ HD NO: 1 from nucleotide number 389 to nucleotide number 2953. Where the CLOCK polypeptide contains the amino acid residue sequence of SEQ DD NO:2 from residue number 2 to residue number 855, a preferred polynucleotide contains the nucleotide sequence of SEQ ID NO: 1 from nucleotide number 392 to nucleotide number 2953. Where the CLOCK polypeptide contains the amino acid residue sequence of SEQ ID NO:2 from residue number 10 to residue number 855, a preferred polynucleotide contains the nucleotide sequence of SEQ ID NO: 1 from nucleotide number 416 to nucleotide number 2953. Where the CLOCK polypeptide contains the amino acid residue sequence of SEQ HD NO:2 from residue number 11 to residue number 855, a preferred polynucleotide contains the nucleotide sequence of SEQ ID NO: 1 from nucleotide number 419 to nucleotide number 2953. Where the CLOCK polypeptide contains the amino acid residue sequence of SEQ ID NO:2 from residue number 35 to residue number 855, a preferred polynucleotide contains the nucleotide sequence of SEQ ID NO: 1 from nucleotide number 491 to nucleotide number 2953. Other preferred polynucleotides such as those encoding the four distinct forms of human CLOCK, will be readily apparent to a skilled artisan by reference to the cDNA and amino acid residue sequences disclosed herein.

The present invention also contemplates DNA sequences which hybridize under stringent hybridization conditions to the DNA sequences set forth above. Stringent hybridization conditions are well known in the art and define a degree of sequence identity greater than about 70%-80%. The present invention also contemplates naturally occurring allelic variations and mutations ofthe DNA sequences set forth above so long as those variations and mutations code, on expression, for a CLOCK polypeptide of this invention as set forth hereinbefore. As is well known in the art, because ofthe degeneracy ofthe genetic code, there are numerous other DNA and RNA molecules that can code for the same polypeptides as those encoded by SEQ ID NO: 1, or portions thereof. The present invention, therefore, contemplates those other DNA and RNA molecules which, on expression, encode for a polypeptide that contains a polypeptide encoded by SEQ ID NO: 1, or portions thereof as set forth above. Having identified the amino acid residue sequence of CLOCK polypeptides, and with knowledge of all triplet codons for each particular amino acid residue, it is possible to describe all such encoding RNA and DNA sequences. DNA and RNA molecules other than those specifically disclosed herein and, which molecules are characterized simply by a change in a codon for a particular amino acid are within the scope of this invention.

A Table of codons representing particular amino acids is set forth below in Table 1.

TABLE I

First Third position Secono Position position

C5' end) (3 'end,

T U C A G

Phe Ser T)τ Cys T/U

Phe Ser Tyr Cys C

T/U Leu Ser Stop Stop A

Leu Ser Stop Tφ G

Leu Pro His Arg T/U

Leu Pro His Arg C

C Leu Pro Gin Arg A

Leu Pro Gin Arg G

lie Thr Asn Ser T/U

He Thr Asn Ser C

A He Thr Lys Arg A

Met Thr Lys Arg G

Nal Ala Asp Gly T/U

Nal Ala Asp Gly C

G Nal Ala Glu Gly A

Nal Ala Glu Gly G A simple change in a codon for the same amino acid residue within a polynucleotide will not change the structure ofthe encoded polypeptide. By way of example, it can be seen from SEQ HD NO: 1 that a TCA codon for serine exists at nucleotide positions 422-424 and at positions 512-514. It can also be seen from that same sequence, however, that serine can be encoded by a AGC codon (see e.g., nucleo.ide positions 419-421 and 617-619). Substitution ofthe latter AGC codon for serine with the TCA codon for serine, or visa versa, does not substantially alter the DNA sequence of SEQ ID NO: 1 and results in expression ofthe same polypeptide. In a similar manner, substitutions of codons for other amino acid residues can be made in a like manner without departing from the true scope ofthe present invention.

A polynucleotide ofthe present invention can also be an RNA molecule. An RNA molecule contemplated by the present invention is complementary to or hybridizes under stringent conditions to any ofthe DNA sequences set forth above. As is well known in the art, such an RNA molecule is characterized by the base uracil in place of thymidine. Exemplary and preferred RNA molecules are mRNA molecules that encode a CLOCK polypeptide of this invention.

IN. Clock Oligonucleotides

The present invention also contemplates oligonucleotides from about 15 to about 50 nucleotides in length, which oligonucleotides serve as primers and hybridization probes for the screening of DΝA libraries and the identification of DΝA or RΝA molecules that encode a CLOCK polypeptide. Such primers and probes are characterized in that they will hybridize to polynucleotide sequences encoding a CLOCK polypeptide. An oligonucleotide probe or primer contains a nucleotide sequence that is identical to or complementary to a contiguous sequence of at least 15 nucleotides of a polynucleotide ofthe present invention. Thus, where an oligonucleotide probe is 25 nucleotides in length, at least 15 of those nucleotides are identical or complementary to a sequence of contiguous nucleotides of a polynucleotide of the present invention. Exemplary polynucleotides ofthe present invention are set forth above.

A preferred oligonucleotide is an antisense oligonucleotide. The present invention provides a synthetic antisense oligonucleotide of less than about 50 nucleotides, preferably less than about 35 nucleotides, more preferably less than about 25 nucleotides and most preferably less than about 20 nucleotides. An antisense oligonucleotide ofthe present invention is directed against a DNA or RNA molecule that encodes a CLOCK polypeptide. Preferably, the antisense oligonucleotide is directed against the protein translational initiation site or the transcriptional start site. In accordance with one embodiment, an antisense molecule is directed against a region of SEQ ID NO: I from about nucleotide position 370 to about nucleotide position 410 or a portion of SEQ ID NO: 1 from about nucleotide position 400 to about nucleotide position 440. It is understood by one of ordinary skill in the art that antisense oligonucleotides can be directed either against a DNA or RNA sequence that encodes a specific target. Thus, an antisense oligonucleotide of the present invention can also be directed against polynucleotides that are complementary to those shown in SEQ ID NO: 1 or 54 as well as the equivalent RNA molecules.

Preferably, the nucleotides of an antisense oligonucleotides are linked by pseudophosphate bonds that are resistant to clevage by exonuclease or endonuclease enzymes. Preferably the pseudophosphate bonds are phosphorothioate bonds. By replacing a phosphodiester bond with one that is resistant to the action of exo-and or endonuclease, the stability ofthe nucleic acid in the presence of those enzymes is increased. As used herein, pseudophosphate bonds include, but are not limited to, methylphosphonate, phosphomoφholidate, phosphorothioate, phosphorodithioate and phosphoroselenoate bonds.

An oligonucleotide primer or probe, as well as an antisense oligonucleotide ofthe present invention can be prepared using standard procedures well known in the art. A preferred method of polynucleotide synthesis is via cyanoethyl phosphoramidite chemistry. A detailed description ofthe preparation, isolation and purification of polynucleotides encoding mammalian CLOCK is set forth below

N. Expression Vectors and Transformed Cells The present invention further provides expression vectors that contain a polynucleotide ofthe invention and host cells transformed or transfected with those polynucleotides or expression vectors.

A polynucleotide that encodes a CLOCK polypeptide is placed into an expression vector suitable for a given host cell such that the vector drives expression ofthe polynucleotide in that host cell. Vectors for use in particular cells are well known in the art and include viral vectors, phages or plasmids.

In one embodiment, a host cell is an eukaryotic host cell and an expression vector is an eukaryotic expression vector (i.e., a vector capable of directing expression in a eukaryotic cell). Such eukaryotic expression vectors are well known in the art. In another embodiment, the host cell is a bacterial cell. An especially preferred bacterial cell is an E. coli. Thus, a preferred expression vector is a vector capable of directing expression in E. coli

A polynucleotide of an expression vector ofthe present invention is preferably operatively associated or linked with an enhancer-promoter. A promoter is a region of a DΝA molecule typically within about 100 nucleotide pairs in front of (upstream of) the point at which transcription begins. That region typically contains several types of DΝA sequence elements that are located in similar relative positions in different genes. As used herein, the term "promoter" includes what is referred to in the art as an upstream promoter region or a promoter of a generalized RΝA polymerase transcription unit. Another type of transcription regulatory sequence element is an enhancer. An enhancer provides specificity of time, location and expression level for a particular encoding region (e.g., gene). A major function of an enhancer is to increase the level of transcription of a coding sequence in a cell that contains one or more transcription factors that bind to that enhancer. Unlike a promoter, an enhancer can function when located at variable distances from a transcription start site so long as the promoter is present.

As used herein, the phrase "enhancer-promoter" means a composite unit that contains both enhancer and promoter elements. An enhancer-promoter is operatively linked to a coding sequence that encodes at least one gene product. As used herein, the phrase "operatively linked" or its grammatical equivalent means that a regulatory sequence element (e.g. an enhancer-promoter or transcription terminating region) is connected to a coding sequence in such a way that the transcription of that coding sequence is controlled and regulated by that enhancer-promoter. Means for operatively linking an enhancer-promoter to a coding sequence are well known in the art.

An enhancer-promoter used in an expression vector ofthe present invention can be any enhancer-promoter that drives expression in a host cell. By employing an enhancer-promoter with well known properties, the level of expression can be optimized. For example, selection of an enhancer-promoter that is active in specific cells (e.g., cells ofthe SCN) permits tissue or cell specific expression ofthe desired product. Still further, selection of an enhancer-promoter that is regulated in response to a specific physiological signal can permit inducible expression.

A coding sequence of an expression vector is operatively linked to a transcription terminating region. RNA polymerase transcribes an encoding DNA sequence through a site where polyadenylation occurs. Typically, DNA sequences located a few hundred base pairs downstream ofthe polyadenylation site serve to terminate transcription. Those DNA sequences are referred to herein as transcription-termination regions. Those regions are required for efficient polyadenylation of transcribed messenger RNA (mRNA). Enhancer- promoters and transcription-terminating regions are well known in the art. The selection of a particular enhancer-promoter or transcription-terminating region will depend, as is also well known the art, on the cell to be transformed.

VI. Method of Making Clock Polynucleotide In another aspect, the present invention provides a process of making a

CLOCK polypeptide. In accordance with that process, a suitable host cell is transformed with a polynucleotide ofthe present invention. The transformed cell is maintained for a period of time sufficient for expression ofthe CLOCK polypeptide. The formed polypeptide is then recovered. Preferably, the polynucleotide is contained in an expression vector as set forth above.

VII. Pharmaceutical Compositions

The present invention also provides a pharmaceutical composition comprising a polypeptide, polynucleotide, oligonucleotide or expression vector of this invention and a physiologically acceptable diluent.

In a preferred embodiment, the present invention includes one or more antisense oligonucleotides, polypeptides or expression vectors, as set forth above, formulated into compositions together with one or more non-toxic physiologically tolerable or acceptable diluents, carriers, adjuvants or vehicles that are collectively refeπed to herein as diluents, for parenteral injection, for oral administration in solid or liquid form, for rectal or topical administration, or the like.

The compositions can be administered to humans and animals either orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, locally, or as a buccal or nasal spray.

Compositions suitable for parenteral administration can comprise physiologically acceptable sterile aqueous or non-aqueous solutions, dispersions, suspensions or emulsions and sterile powders for reconstitution into such sterile solutions or dispersions. Examples of suitable diluents include water, ethanol, polyols, suitable mixtures thereof, vegetable oils and injectable organic esters such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersions and by the use of surfactants.

Compositions can also contain adjuvants such as preserving, wetting, emulsifying, and dispensing agents. Prevention ofthe action of microorganisms can be insured by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride and the like. Prolonged absoφtion ofthe injectable pharmaceuticalform can be brought about by the use of agents delaying absoφtion, for example, aluminum monostearate and gelatin.

Besides such inert diluents, the composition can also include sweetening, flavoring and perfuming agents. Suspensions, in addition to the active compounds, may contain suspending agents, as for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, macrocrystalline cellulose, aluminum metahydroxide, bentonit, agar-agar and tragacanth, or mixtures of these substances, and the like. VIII. Process of Using CLOCK Polypeptides, Polynucleotides and Oligonucleotides

The present invention provides processes for using the polypeptide, polynucleotides, and oligonucleotides ofthe present invention. The compositions and methods ofthe present invention have a variety of uses. Having described the Clock gene and its expression product, the CLOCK polypeptide, it is possible to inhibit expression ofthe Clock gene using gene targeting technology as is well know in the art. Using such technology, for example, the Clock gene can be removed from the genome ofthe mouse or that gene can otherwise be mutated so as to prevent expression ofthe CLOCK polypeptide. As a result of such treatments, a mouse model is created that is characterized by having circadian clock dysfunctions. That model can then be used in screening essays to identify therapeutic agents that affect circadian rhythm or to study a variety of chemical, physiological, or behavioral activities associated with the circadian rhythm.

As set forth above, the amino acid residue sequence ofthe CLOCK polypeptide indicates that it is a transcription factor and contains a DNA binding domain. The CLOCK polypeptide, or the DNA binding domain portion thereof, can therefore be used to identify the specific DNA binding site and/or to identify agonist or antagonist substances that interfere with DNA binding ofthe CLOCK polypeptide. Means for accomplishing such screening assays are well known in the art.

Briefly, once the DNA binding site is identified, that DNA binding site, together with the DNA binding domain ofthe CLOCK polypeptide, can be exposed to a variety of agents suspected of being agonists or antagonists to DNA binding. The ability of those compounds to interfere with binding ofthe CLOCK polypeptides to its DNA binding site is indicative ofthe agonist or antagonist nature of those substances. Alternatively, the DNA binding site can be placed in an expression vector such that binding of a CLOCK polypeptide to that binding site allows for expression of a reporter gene operatively linked to the DNA binding site. The ability of compounds to inhibit or enhance expression ofthe reporter gene is indicative of agonist or antagonist activity.

The CLOCK polypeptide, or the DNA binding domain thereof, can also be used to screen DNA libraries to identify the specific binding site on a DNA molecule. Screening can be accomplished with genomic libraries in general or with specifically targeted portions of genomic DNA. As set forth above, for example, it is likely that the DNA binding domain ofthe CLOCK polypeptide binds within the promoter region ofthe Clock gene itself. Binding studies can therefore be targeted to this region ofthe Clock gene.

Once the DNA binding site ofthe CLOCK polypeptide has been determined, the three dimensional structure ofthe CLOCK polypeptide, or its DNA binding domain, bound to the target DNA site can be determined using techniques well known in the art, such as X-ray crystallography. Knowledge of the three-dimensional structure ofthe bound CLOCK polypeptide will thus allow for computer aided rational drug design for identification of agonist or antagonist compounds.

The well known yeast two-hybrid system can be used to determine whether the CLOCK polypeptide interacts with another protein (heterodimerization) or with itself (homodimerization). Briefly, yeast cells are transformed with a reporter gene operatively associated with a promoter that contains a binding site for GAL 4. That same yeast is then transformed with a polynucleotide that encodes a CLOCK polypeptide ofthe present invention, or a dimerization domain thereof. Finally, that same yeast cell is transformed a protein expression cDNA library. Transformed yeast will only survive if the CLOCK polypeptide interacts with a second protein resulting from expression ofthe protein expression cDNA library and that interaction causes GAL 4 to bind to the promoter region ofthe reporter gene and express that reporter gene. In yet another embodiment, compositions ofthe present invention can be used to screen genomic libraries in plants and animals to identify the coπesponding Clock genes in these species. Identification ofthe Clock gene in these species is important because the growth and metabolic rate of plants and animals is known to be regulated, at least in part, by the circadian rhythm. By way of example, photosynthesis in plants is known to comprise both a light and dark reaction. Manipulation ofthe circadian clock in plants, therefore, can result in alteration of those light and dark reactions. Similarly, the growth rate of animals used for feed (cattle and pigs) is known to be a function ofthe circadian rhythm. The ability to manipulate the circadian rhythm in those animals can thus result an enhanced growth of those important animals.

The expression of diurnal (i.e. 24-hr) rhythms is a fundamental property of almost all forms of life. These rhythms are regulated by an internal "biological clock" that in many organisms, including humans, can be synchronized by the light dark cycle. This internal 24-hr clock is referred to as a "circadian clock" because in the absence of any diurnal environmental cues, the period ofthe clock is rarely exactly 24 hours but is instead about 24 hrs (i.e. circa diem).

The circadian clock in mammals is known to regulate 24 hour rhythms in biochemical, cellular, metabolic and behavioral activity in most, if not all, physiological systems. The following is a list of exemplary activities controlled at least in part by the circadian clock and activities that affect that clock, which can be manipulated to restore the function of an abnormal allele ofthe Clock gene.

1 . The circadian clock is a major regulator ofthe sleep-wake cycle (Borbely, 1994; Kryger et al., 1994) and many pathologic changes in the sleep wake cycle are associated with circadian rhythm disorders (Roehrs and Roth, 1994). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and or pharmaceutical approaches for the treatment of any sleep disorders.

2. When people move rapidly across time zones, they suffer from a well-known syndrome, refeπed to as jet-lag, u: .til their biological clock and sleep-wake cycle become resynchronized to the new time zone (Graeber, 1994). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and/or pharmaceutical approaches for the treatment of j et-lag.

3. When people must be awake during the normal sleep period, and/or asleep during the normal wake period, they suffer decrements in health, performance and productivity as well as an increased rate of accidents (Monk, 1990; Monk, 1994; Smith et al., 1994; US Congress, September, 1991).

Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and/or pharmaceutical approaches for the treatment of disorders of time-keeping associated with having to be awake during the biological clock time of normal sleep and asleep during the biological clock time of normal wake. This coverage ofthe patent includes the use of Clock, and/or it's protein product for alleviating the adverse effects associated with shift work where workers are working during the time of normal sleep and sleeping during the time of normal wake.

4. The circadian clock regulates the timing of fatigue and alertness (Monk et al., 1984; Roth et al., 1994). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and/or pharmaceutical approaches for altering the cycle of fatigue and alertness, as well as for decreasing fatigue or increasing alertness by altering circadian rhythmicity.

5. Circadian rhythm disruption has been associated with many forms of altered mental states, including but not limited to depression (both unipolar and bipolar), pre-menstrual syndrome post-menopausal syndrome, and schizophrenia (Hallonquist et al., 1986; Ohta and Endo, 1985; Van Cauter and Turek, 1986; Wehr and Goodwin, 1983; Wehr et al., 1983; Wehr et al., 1979). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and/or pharmaceutical approaches for the treatment of any mental disorders.

6. Studies have shown that the human has a pronounced cycle of mood and performance (Benca, 1994; Monk et al., 1985). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and/or pharmaceutical approaches for altering the mood state or performance.

7. The circadian clock regulates the timing of many physiological and endocrine processes that when disturbed lead to various mental and physical disorders (Richter, 1979; Turek and Van Cauter, 1994; Van Cauter and Turek, 1995). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and/or pharmaceutical approaches for the treatment of any mental and physical disorders.

8. Abnormal circadian rhythm and abnormal sleep-wake cycles have been associated with various neurological diseases (Aldrich, 1994; Bliwise, 1994; Hartmann, 1994; Hineno et al., 1992; Hyde et al., 1995; Lugaresi and Montagna, 1994; Poirel, 1991; Weltzin et al., 1991). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and/or pharmaceutical approaches for the treatment of any neurological disorders.

9. The circadian clock regulates the timing of many physiological and endocrine processes associated with stress (Sapolsky, 1992; Tornatzky and Miczek, 1993; Van Cauter and Turek, 1995). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and/or pharmaceutical approaches for relieving stress or altering the stress response in humans.

10. Many components ofthe cardiovascular system show rhythmic variation, and the timing of such major insults to the cardiovascular system, such as heart attack and stroke, are known to be regulated by the circadian clock system and or be influenced by the time-of-day (Aschoff, 1992; Cohen and Muller, 1992; George, 1994; Gillis and Flemons, 1994; Maron et al., 1994;

Sano et al., 1995). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and/or pharmaceutical approaches for the treatment of any diseases ofthe cardiovascular system.

11. The circadian clock plays a central role in the regulation ofthe diurnal cycle in feeding behavior (Rusak and Zucker, 1975). Furthermore, many components ofthe system involved with feeding as well as the regulation of metabolism, body fat and weight control are regulated by the circadian clock system (Benca and Casper, 1994; de Graaf et al., 1993; Larsen et al., 1991; Orr, 1994; Van Cauter and Turek, 1995). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and/or pharmaceutical approaches for the treatment of any disorders of feeding behavior as well as attempts to regulate diet and/or food intake.

12. The circadian clock regulates the timing of many physiological and endocrine events associated with diabetes (Spallone et al., 1993; Van Cauter and Turek, 1995). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and/or pharmaceutical approaches for the treatment of diabetes and related illnesses.

13. The circadian clock regulates the timing of many components of the immune system (Calvo et al., 1995; Constantinescu, 1995; Krueger and Kamovsky, 1995). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and/or pharmaceutical approaches for the treatment of any disorders ofthe immune system.

14. For many infectious diseases, including those of viral, bacterial or parasitic origins, the circadian clock regulates the optimum time for infection to occur, as well as the response to the infection by the host organism (Walker et al., 1981). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and/or pharmaceutical approaches for the prevention, diagnosis and/or treatment of infectious diseases.

15. The circadian clock regulates the timing of many processes associated with reproduction (Turek and Van Cauter, 1994). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and/or pharmaceutical approaches for the treatment of any reproductive disorder as well as for enhancing fertility, treating infertility or for any birth control methods as well as for affecting sexual function.

16. Many ofthe physiological processes and hormones involved in pregnancy and parturition are regulated by the circadian clock (Turek and Van Cauter, 1994). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and/or pharmaceutical approaches for aiding in the maintenance of pregnancy and/or in the process of parturition.

17. The circadian clock regulates the timing of many components of the respiratory system (Douglas, 1994; Orem, 1994). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and/or phan-naccutical approaches for the treatment of any respiratory illness. 18. There are pronounced diurnal variations in the functions ofthe liver (Colantonio et al, 1989; Garcia-Pagaan et al., 1994). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and/or pharmaceutical approaches for the treatment of liver disease and or for altering liver function.

19. Many components ofthe endocrine system undergo pronounced daily changes in function (Van Cauter and Turek, 1995). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and/or pharmaceutical approaches for the treatment of any endocrine disorders, or for altering in endocrine rhythms for any puφoses.

20. The circadian clock regulates the timing ofthe pineal melatonin rhythm (Arendt, 1995). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and/or pharmaceutical approaches for using melatonin and/or melatonin related drugs in humans for therapeutic puφoses, including the use of melatonin and/or melatonin related drugs as anti-oxidants.

21. The therapeutic and toxic effects of many drugs are influenced by the time of day at which the drug is delivered and/or by the pattern of drug administration (Larsen et al., 1993; Lemmer, 1989; Walker et al., 1981). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new approaches for the use of any pharmacological agents to improve human health or welfare.

22. The therapeutic and toxic effects of many drugs are influenced by the time of day at which the drug is delivered and/or by the pattem of drug administration (Walker et al., 1981). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and/or pharmaceutical approaches in the screening of drugs for new therapeutic purposes as well as the use of Clock and its protein product for diagnostic puφoses.

23. The circadian clock regulates many physiological processes that are involved in the development or suppression of many forms of cancer (Walker et al., 1981). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and/or pharmaceutical approaches for the treatment or diagnosis of any cancer, as well as other forms of abnormal cell division.

24. The circadian clock regulates many ofthe processes associated with growth and development (Albertsson-Wlkland and Rosberg, 1988, Hokken- Koelega et al., 1990; Mirmiran et al., 1990; Van Cauter and Turek,

1995). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and/or pharmaceutical approaches for influencing growth and development.

25. The circadian clock regulates processes associated with cell division (Edmunds Jr, 1988). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and/or pharmaceutical approaches for influencing cell division and the cellular cycle.

26. There are major changes in the circadian clock system with advancing age, and age-related changes in the circadian clock system may underlie many ofthe adverse health effects associated with aging (Turek et al.,1995; Van Cauter and Turek, 1995). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and/or pharmaceutical approaches for the treatment of any age-related illnesses or age-related changes in human physiology. 27. Light may have many effects on the brain that are mediated through the transmission of neural information through the central circadian clock in mammals, the hypothalamic suprachiasmatic nucleus (SCN) (Card and Moore, 1991 ; Meijer, 1991 ; Penev et al., 1997). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and or pharmaceutical approaches for the use of light to alter neural activity in the brain.

28. The light-dark cycle is a major regulator ofthe timing of circadian rhythms that are controlled by the circadian clock of which Clock is a component (Turek, 1994; Turek and Van Reeth, 1996; Van Cauter and Turek, 1995). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and/or pharmaceutical approaches that involve the use of light or dark to shift or influence, in any way, circadian rhythm.

29. While the light-dark cycle is a major regulator ofthe timing of circadian rhythm in most humans, for many blind humans the light-dark cycle is not able to synchronize the circadian clock in a normal fashion (Sack et al.,

1992). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and/or pharmaceutical approaches for the treatment of blind people.

30. The light-dark cycle influences many functions ofthe retina including photoreceptor cells. Furthermore, the circadian clock regulates the timing of many genetic, molecular and cellular processes in the retina (Decker et al., 1995; LaVail, 1976; Young, 1980). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and/or pharmaceutical approaches for the treatment in any fashion of retinal dysfunction. 31. The circadian clock regulates a diumal rhythm in mental and physical performance in animals, including humans (Benca, 1994; Monk et al., 1985; Richter, 1979; Turek and Van Cauter, 1994; Van Cauter and Turek, 1995). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and/or pharmaceutical approaches for enhancing human mental and physical performance.

32. Increased exercise at certain times ofthe day is known to be able to shift circadian rhythms that are controlled by the circadian clock (Van Reeth et al., 1994). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and/or pharmaceutical approaches that involve the use of exercise to shift or influence in any way circadian rhythms.

33. The disruption of normal circadian rhythmicity in intensive care facilities has been associated with decreased wellness and increased morbidity

(Mann et al., 1986). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and/or pharmaceutical approaches for improving the environment of intensive care facilities or the health ofthe subjects such facilities.

34. The circadian clock plays a central role in the regulation of diumal rhythms in plant and animal species that are of commercial value to humans (1988; Reiter and Follett, 1980). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and/or pharmaceutical approaches for enhancing the growth, development, performance, productivity, or health of such species, including those involved in the production of food for human consumption, as well as animal products used in producing apparel. 35. The circadian clock plays a central role in measuring the length ofthe day, which changes on an annual basis in all regions on earth outside of those close to the equator (1988; Reiter and Follett, 1980; Turek and Van Cauter, 1994). This seasonal change in day length influences the growth, development, health, reproduction, performance and productivity of many species, including humans (1988; Reiter and Follett, 1980; Turek and Van Cauter, 1994). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and/or pharmaceutical approaches for influencing any seasonal rhythms in any species, including the use of melatonin and or melatonin related drugs to influence seasonal cyclicity.

36. The treatment of one sub-type of depression, referred to as Seasonal Affective Disorder (SAD), has been the exposure to extra bright light during the short days of winter (Penev et al., 1997; Terman, 1994; Wetterberg, 1994). Such treatment may be effective because of the effect of light on the circadian clock system. Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and/or pharmaceutical approaches for the treatment of SAD or any other disorders that are associated with the seasonal change in daylength.

37. Since Clock is the first gene to be discovered and cloned in a mammal that is a component ofthe circadian clock, it will lead to the discovery of new Clock genes that have sequence homology with Clock and its protein product. Therefore, this patent covers any use of Clock, or its protein product, to discover and clone new genes, and their protein products by sequence homology (and their commercial value).

38. Since Clock is the first gene to be discovered and cloned in a mammal that is a component ofthe circadian clock, it will lead to the discovery of new Clock genes, and their protein products, that interact with Clock or the Clock protein product. Therefore, this patent covers the use of Clock, or its protein product, to discover new genes, and their protein products that are found by determining which genes and their protein products interact with Clock, and its protein product, in a functional way.

39. Since Clock is the first gene to be discovered and cloned in a mammal that is a component ofthe circadian clock, it will lead to the discovery of new Clock genes, and their protein products, that interact with Clock or the Clock protein product. Therefore, this patent covers the use of Clock, or its protein product to screen for molecules that may have sequence similarity or functional relationships to clock or its protein product.

40. The circadian clock regulates the timing ofthe expression of many genes and the production of their protein products (Jacobshagen and Johnson, 1994; Lausson et al., 1989; Loros et al., 1989; Millar and Kay, 1991; Taylor, 1989). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and/or pharmaceutical approaches in the use of gene therapy where a particular gene and/or its protein product needs to be under or over-expressed.

41. The circadian clock is a major regulator ofthe sleep-wake cycle and many pathological changes in the sleep-wake cycle are associated with circadian rhythm disorders (Kryger et al., 1994). Therefore, this patent covers any use of Clock, or its protein product, for the development or use of new techniques and/or pharmaceutical approaches for the discovery of genes and their protein products that are involved in the regulation ofthe sleep-wake cycle. The Examples to follow illustrate particular embodiments ofthe present invention and are not limiting ofthe specification and claims in any way.

EXAMPLE 1: Isolation and phenotypic analysis of the r ouse Clock mutation

Because orthologs ofthe canonical clock genes (period, timeless, and frequency) have not been found in mammals, and because other strategies to identify mammalian clock genes have not yet been successful, a mutagenesis screening strategy to isolate clock mutations in the mouse was initiated (Takahashi et al. 1994). Circadian behavior in the mouse is precise and easily quantitated, thus it is very well suited for genetic screening. Wild type C57BL/6J strain mice, which were used for this screen, exhibit a robust circadian rhythm of wheel running activity (Pittendnigh & Daan 1976, Schwartz & Zimmerman 1990). This behavioral assay was used to screen for mice carrying mutations that cause abnormal circadian periods in constant darkness. Because most clock mutations that have been isolated in other organisms have been semidominant (Hall & Kyniacou 1990, Dunlap 1993), dominant and semidommant mutations were screened for. Analysis of about 300 G_j progeny of ENU-treated mice revealed one mouse that expressed a circadian period that was more than one hour longer than (and six standard deviations above) the normal period of 23.7 hours (Vitatema et al. 1994) . This long period phenotype was inherited as a single-locus, semidommant, autosomal mutation, which was designated Clock.

Mice homozygous for the Clock mutation expressed extremely long circadian periods of about 28 hours for the first two weeks of exposure to constant darkness, after which there was a complete loss of circadian rhythmically. The Clock gene, thus, regulates at least two fundamental properties ofthe circadian clock system: the intrinsic circadian period and the persistence of circadian rhythmicity. No anatomical defects in the SCN have been observed in association with the Clock mutation (Vitaterna et al. 1994), which suggests that the loss of circadian rhythmicity in constant darkness cannot be attributed to a gross anatomical or developmental defect.

In addition to the effects on period and persistence of circadian rhythms in Clock mutants, at least two other circadian effects ofthe mutation have been documented. The period of Clock heterozygous mice is unstable and their free- running periods tended to lengthen with time in constant darkness. In addition, the photic entrainment of Clock heterozygotes is also altered. Clock! + mice were able to entrain to 28-hour light cycles, while wild-type mice did not. Importantly, Clock/+ mice also exhibited high-amplitude phase-resetting responses to 6-hour light pulses (Type 0 resetting) as compared to wild-type mice which exhibited low amplitude (Type 1) phase resetting. Because of their loss of rhythmically in constant conditions, phase shifts in response to light pulses could not be measured in Clock/Clock homozygotes, but two findings indicate that these animals can entrain: the phase of a restored rhythm following a light pulse and the phase ofthe free-run following entrainment to a light dark cycle were both determined by the phase ofthe light signal. The increased efficacy of photic resetting stimuli and the decrease in period stability suggest that the Clock mutation may reduce circadian pacemaker amplitude in Clock heterozygotes.

To determine whether the Clock mutation affects other rhythms in mice, circadian drinking rhythms were measured. The Clock mutation affected the period and persistence of circadian drinking rhythms in a manner similar to that seen with activity suggesting that the mutation acts globally on rhythms in mice and is not restricted to locomotor activity.

The phenotype of Clock is as robust as the "best" clock mutations in Drosophila and Neurospora (Dunlap 1993). By robust is meant that the period change is on the order of 4 to 5 hours, which is followed by a complete loss of circadian rhythmicity. The magnitude ofthe period change in Clock

L 7 homozygotes was equivalent to that seen with the per and frq -.lleles that also cause periods of 28-30 hours in their respective organisms (Dunlap 1993). The loss of circadian rhythmicity seen in Clock homozygotes resemble that seen in

0 9 per and frq alleles, which are null mutations in those respective genes

(Dunlap 1993). The robustness of Clock is important for two reasons. First, mutations that have modest effects on period length (on the order of a one-hour change in period in homozygotes) could be due to secondary effects of mutations on the circadian clock system. Second, the most robust mutants in Drosophila and Neurospora are found at the per, tim and frq loci, which are genes that appear to be critical and essential elements ofthe circadian mechanism in these organisms (discussed above).

EXAMPLE 2: Antimorohic Behavior of Clock Mutation

The initial analysis ofthe Clock mutation indicated that the mutation exhibited a semidommant phenotype (Vitaterna et al. 1994). There are several possible causes of a semidominant phenotype, including the possibility that the mutation was induced in a gene that otherwise is not involved in the generation of circadian rhythms, but when mutated, interferes with the normal generation of these rhythms. To demonstrate that a particular gene is necessary for a particular biological process, one normally requires a loss-of- function allele of that gene leading to a loss ofthe phenotype in question. From genetic mapping (described below) it was found that Clock is contained within a radiation induced deletion on chromosome 5, w , that includes the Kit ( = W, Dominant

White Spotting) locus (Lyon et al. 1984). This was found by mapping the

19H l9H

SSLP content of W in (W x Mus castaneous/Eϊ) Ε _\ progeny. Multiple genetic loci, mapping both proximal from and distal of Clock, are within the 19H W deletion, indicating that Clock maps within this deletion. Access to this deletion that encompasses Clock allowed for further analysis ofthe phenotypic effect of this mutation. Muller's classic analysis of Drosophila mutations (Muller 1932), as well as more recent analysis of dominant mutations in Caenorhabditis elegans (Park & Horvitz 1986), provided a framework in which to analyze the Clock mutation. Muller described five types of mutant alleles, distinguished by manipulating the copy number ofthe mutant and wildtype alleles (via, e.g., deletions). These are hypomoφh, amoφh, hypermoφh, antimoφh, and neomoφh alleles. The circadian phenotype of W^19H heterozygous mice (hemizygous for the wild-type allele of Clock) is indistinguishable from the wild-type phenotype on a comparable strain background, indicating that the null allele of Clock is recessive to wild-type. By mating Clock/Clock mice to mice heterozygous for this deletion to generate Fi progeny, it was possible to measure the phenotype of Clock/null animals (these F_j animals are distinguishable from their Clock/ '+ litter mates by the presence of deletion-induced white coat color markings). Of particular intrest is the observation that the mean circadian period expressed by Clock/mill animals (25.6+ 0.1.5 hours) is significantly longer than that of Clock!+ animals (24.2+

0.05 hours, p<10^' ).' This indicated that the wild-type allele interacts with the Clock mutation to ameliorate the severity ofthe Clock mutant phenotype. This is the essential feature of an antimoφhic (Muller 1932), and is in contrast to what would be expected of a neomoφh mutation, in which case the wild-type allele would have no effect on the expression or severity ofthe mutant allele. Furthermore, because W 19H is large (~ 2.8 cM) and because multiple loci, both proximal from and distal of Clock, lie within the deletion, it appears unlikely that the breakpoints ofthe deletion interact directly with the Clock gene. That Clock is an antimoφh (one type of dominant negative mutation) implies that the wild-type allele function in the normal generation of circadian rhythms in the mouse. This provides strong evidence that Clock defines a gene central to the mammalian circadian system. The antimoφhic behavior ofthe Clock allele provided clues about the nature of this mutation. Antimoφhic behavior suggested that the mutant allele generates a molecule that competes with the wild-type function. This, and the observation that Clockld e on and Clock/+ have much more severe phenotypes that +/άeletiorij allows the conclusion that the Clock mutation is unlikely to be either a null mutation (amoφh), or a partial loss of function (hypomoφh). Further, because +/deletion has no phenotype different from wild-type, the Clock phenotype does not appear to be the result of haplo-insufficiency. Perhaps most important, it is likely that the mutation conferring the altered behavior in Clock mutant mice may affect the coding sequence ofthe gene, due to its ability to interfere with the function ofthe wild-type allele.

EXAMPLE 3: Genetic Mapping of Clock

The first step in the molecular identification of Clock locus was to map its location in the mouse genome. Given the extensive genetic mapping information available in the mouse (Takahashi et at. 1994), it was possible to map Clock rapidly by linkage analysis using intraspecific mapping crosses and simple sequence length polymoφhisms (SSLPs) from the MIT/Whitehead

Institute genetic map (Vitatema et al. 1994). Clock mapped to the mid portion of mouse chromosome 5 between two SSLP markers, D5MU24 and D5MU83, in a region that shows conserved synteny with human chromosome 4. The possibility of a human homolog of Clock on chromosome 4 is significant because it allows for focusing attention upon this region ofthe genome for possible linkage to circadian traits in human subjects as well as providing a candidate gene for other disorders associated with circadian rhythm dysfunctions such as delayed sleep phase syndrome (Vignau et al. 1993) and affective disorders (Wehr & Rosenthal 1989). In order to identify a more precise chromosomal region in which to focus physical mapping and molecular cloning efforts, a high-resolution genetic map ofthe Clock region was genereated using SSLPs and 1804 meioses obtained from 6 intraspecific and 2 interspecific crosses. This SSLP mapping placed Clock close to the Kit (-W, Dominant white spotting) locus (Geissler et al. 1988b). High resolution genetic mapping, with a Prull RFLP identified using a Kit cDNA probe, placed Kit 0.7 cM (7 recombinahts/988 meioses) proximal from Clock.

Using additional SSLP markers on a total of 2681 meioses, Clock has now been placed within a 0.3 cM interval, approximately 0.2 cM (5 recombinants/2681 meioses) distal of D5M 307 and 0.1 cM (1 recombinant 845meioses) proximal from D5Mit/D5Mit306 (see FIG. 1). The location of this distal recombination has been confirmed in test-cross progeny.

EXAMPLE 4: Physical Mapping of the Clock Region

Based upon the high-resolution genetic map ofthe Clock region, a physical map which spanned the critical genetic region that must contain Clock (D5MU307 - D5MU112) was constructed. To do this, yeast, artificial chromosome (YAC) clones that map to the region were isolated. Using a YAC library that has been pooled for PCR screening (Kusumi et al. 1993) , and SSLP markers, as well as sequence tagged sites (STSs), from the region surrounding Clock, over 40 YAC clones were isolated and a contig of ~4 Mb that spans the Clock region (FIG. 1) was constructed. YAC clones within the critical region were characterized by end cloning and long-range restriction mapping with pulse field gel electrophoresis (PFGE). Three nonchimeric YAC clones were identified and one of these YACs, which is 930 kb, contains both flanking markers and therefore must contain Clock. Long-range restriction mapping of the reduced genetic interval D5MU307 - D5MU112 indicated that it was about 400 kb in length (See FIG. 2). Most of this 400 kb critical region was then re- cloned in bacteria artificial chromosome (BAC) clones. BACs, which are intermediate in size (-100-200 Kb) between YACs and cosmids, have several advantages when compared to YAC clones. Although they are generally smaller than YAC clones, BACs are rarely chimeric, they are circular clones, thus they are much easier to manipulate, and they rarely suffer recombination or deletion damage (Shizuya et al. 1992). Using direct sequencing ofthe ends of the BAC clones, 12 BACs were placed on the YAC physical map using STSs. Subclone libraries from these BAC clones were placed to isolate 7 new SSLP markers. One of these markers, D5NWU1 was nonrecombinant with Clock, and a second marker, D5NWU2, defined the closest distal recombinant with Clock on the genetic and physical map. Thus the critical region containing Clock was now defined by the flanking markers D5MU307 and D5NWU2 which defined an interval less than 400 kb.

EXAMPLE 5: Transcription Unit Analysis in the Clock Region

Within the critical region containing Clock there are no known candidate genes that have previously been identified. Therefore three different approaches identifying candidate genes were initiated: 1) direct screening of SCN cDNA libraries with BAC clones as probes; 2) hybridization selection of cDNAs from SCN libraries using BAC clones as driver; and 3) shotgun sequencing random Ml 3 libraries made from BAC clones.

The first two of these methods used a pair of oligo dT primed cDNA libraries. Tissue derived from mouse SCN region was microdissected from a total of about 100 mice at four different circadian time points (circadian time

(CT) 1,7,13, and 19). For one of these libraries, poly A RNA was extracted from SCN tissue collected in constant darkness at each time point. For the other library, poly A RNA was extracted from SCN tissue collected at the same four time points: however, the animals were previously exposed to a 30 to 90 minute pulse of light. CDNA libraries were directionally cloned using the ZAP Express lambda vector (Stratagene). Primary library sizes were 1.7X10 and 1.2X pfu,

and 1x10 clones from each library were plate amplified. Average insert sizes were 2.3 and 2.2 kb and raged from 600 to 5200 bp. These cDNA libraries are important resources because the SCN is very small (about 16-20,000 neurons or -20 g protein per mouse) and is difficult and expensive to obtain high quality mRNA samples.

A. Direct screening of the cDNA libraries using whole BAC inserts. Two different BAC clones were used which together cover > V* ofthe critical region containing Clock. BAC DNA for probes was purified by restirction digest with Not I to release inserts and separation of field inversion gel electrophoresis (FIGE). BAC insert DNA was radiolabeled using random priming and the probe was preannealed with Cot-1 mouse DNA to suppress repetitive DNA sequences using methods similar to those developed for probes from entire YAC clones (Marchuk & Collins 1994). The cDNAs identified using the method were characterized in two ways. The ends ofthe clones were sequenced and these sequences were used to search the DNA and protein databases, using the Basic Local Alignment Search Tool (BLAST) (Altschul et al. 1990). Also, these cDNA clones were used as probes on a Southern blot consisting of BAC clones, restriction digested with Hindlll, that map to the critical genetic region. Using these two methods, it was possible to eliminate false positive clones by identifying clones containing repetitive sequences (e.g., LI elements) and clones that did not map to the critical genetic region (i.e., they ^' did not hybridize to the BAC clone Southern blot). This process led to the identification of fifteen cDNA clones that fell into 6 classes of cDNA clones mapping in the Clock region. These 6 classes of clones are refeπed to as "HI through H6" in (See FIG. 3). B. cDNA selection experiments.

The second method used to identify transcription unit sequences was an adaption ofthe cDNA selection protocol described by Lovett (Lovett 1994). For these experiments, SCN cDNA from lambda DNA was prepared from plate lysates from the SCN libraries described above. Lambda DNA from the cDNA library (instead of excised phagemid DNA) was used because the purification of cDNA inserts were excised by digestion with BamHI and Xhol and gel purified from lambda vector arms. cDNA was then digested with Dpnll, and BamHI adapters form the representational difference analysis (RDA) method (Lisitsyn et al. 1993) were ligated. Amp licons from the cDNA fragments were then made by PCR as described in the RDA procedure. Genomic DNA from BAC clones was released with Not I digestion and inserts were purified on pulse-field gel electrophoresis (PFGE). BAC DNA was the digested with Sau3 Al, and a different set of BamHI RDA adapters was ligated. Amplicons from the BAC DNA were then made by PCR using a biotin end-labeled oligonucleotide primer. cDNA and BAC amplicons were then hybridized in the presence of Cot-1 mouse genomic, ribosomal and vector DNA to suppress background. Hybrids were then captured with streptavidin-coated magnetic beads as described by Lovett. Two rounds of selection were performed and the efficiency was monitored with a positive control (spiked with c-fos clone), a negative control (jun-B) and Cot-1 DNA level.

Selected clones were then eluted and cloned into pBluescript vector. Clones were then picked into six 96-well plates. Replica filters were made and screened with the following probes: BAC 51 (positive probe), BAC 48 (negative probe), c-fos, and Cot- 1 DNA. Clones that were positive for BAC 51 and negative for the other three probes were analyzed. Sixty cDNA such clones were selected. These 60 selected clones were then sequenced to identify duplicates and tested for mapping back to the Clock region on Southern blots of Hindlll digested BAC clones from the critical region. Out ofthe 60 clones, 38 appeared valid by sequence, 14 had repetitive sequences and 8 were false positives (ribosomal or vector DNA). All 38 clones mapped to the Clock region BAC Southem blots. The selected cDNA fragments appeared to fall into about 13 classes. These fragments were then used to screen the SCN libraries to obtain longer cDNA clones. Eighteen cDNA clones that mapped to the region on by Southem blot were obtained (these clones are referred to as "Cl through C18" in FIG. 3), and these clones fell into 10 different classes of clones.

C. Shotgun sequencing of BAC clones. In addition to the cDNA-identifying approaches described above, random sequencing of genomic DNA were used as a third method of transcription unit analysis. With this approach: 1) a genomic scaffold (i.e., one to two-fold coverage ofthe region) could be used for sequenced-tagged site (STS) mapping and for finer mapping of cDNAs isolated by the first two techniques (as opposed to mapping by BAC Southem); 2) database searches using genomic sequence could identify cDNAs not found by direct screening and cDNA selection; and 3) genomic sequence would uncover new SSLP markers that could further diminish the region containing Clock Upon further consideration, selected BACs were sequenced to completion. Complete genomic sequence allowed precise mapping of STSs, exon mapping of cDNA clones, promoter analysis, and inteφretation of other experiments such as BAC rescue and Sout:-εm blot analysis.

Two parametes are critical for successful shotgun sequencing project: extremely pure source DNA and a high-throughput/low-cost template preparation protocol. Two independent shotgun libraries using two BACs, which together covered about 2/3 ofthe Clock critical region were constructed. BAC DNA was prepared by large-scale alkaline lysis of two-liter liquid cultures followed by a two-step CsCl gradient purification using methods adpated from the C. elegans genome project (Favello et al. 1995). The second CsCl purification of plasmid (BAC) DNA was necessary to ensure low E. coli chromosomal DNA contamination. The protocol typically yielded 5-15 μg intact BAC DNA from two liters of liquid culture. 5μg DNA were sonicated, blut-ended, and run on an agarose gel for size selection of insert DNA. The 1.3- 1.7 kb range was gel-purified and blut-end ligated into M13. Ligation products were elect: Dporated into E. coli XLl Blue MRF' and plated; 2.5-fold dilution of e ligation mixture was necessary to prevent arcing during electroporation. Clear plaques were picked into SM buffer for storage.

High-throughput Ml 3 template preparation was essential for efficient BAC sequencing. Probability theory indicates that 4X coverage of a length of DNA is necessary to achieve 98% ofthe complete sequenc. The number of templates needed to achive "n"X coverage is defined as

n*total length of DNA seque iced length per template

Knowing the length ofthe BAC DNAs (160 kb and 140 kb) and assuming 500 bases of good sequence per template, 1280 templates and 1120 templates, respectively, were needed to reach 4X coverage of each library. A magnetic bead isolation protocool adapted form Hawkins et al. (1994) in a 96- tube format was used to rapidly prepare sequence-ready M13 template. 650 μl Ml 3 cultures were grown in 96-tube racks. Cultures were centrifuged, lysated were transfeπed to new tubes, and DNA was released by heat/detergent lysis of Ml 3 protein coats. Magnetic beads and hybridization solution (2x stock: 26% PEG 8000, 20 mM MgC₁₂) were added to the tubes for selective DNA hybridization to the beads. The beads were magnetically collected and supernatant was discarded. DNA was eluted with water; the beads were magnetically collected, and the DNA was transferred to a 96-well plate for storage. This protocol typically yielded 1-2 μg sequencing template per sample; 192 templates could be prepared in about 5 hours. Fluorescence cycle sequencing was performed by an ABI PRISM Turbo 800 Molecular Biology LabStation with -21 M13 Dye Primer chemistry, and the products were run on an ABI PRISM 377 TNA sequencer. The Sequencher program (Gene Codes) removed vector sequence and low-quality sequence from each shotgun sequence and then aligned the sequences into contigs. Average sequence length was 580 b-ses. Each sequence was used to search BLAST databases (BLASTN-nr, BLASTN-dbEST, BLASTX-nr, and TBLASTX-dbEST) to identify Clock candidates by gene, EST, or protein homology. In addition, various gene finding programs were also used.

The 160 kb BAC was sequenced to 4X coverage and aligned into about

20 contigs of 4 -30 kb each. Clones that defined the ends of contigs were selected for "reverse sequencing", where the opposite end ofthe 1.5 kb inserts was sequenced by M13 Reverse Dye Primer chemistry in an attempt to join contigs. This approach reduced the number of contigs to 12, and more importantly, it provided enough information to order all contigs by STS alignment. The 140 kb BAC has been sequenced to 3X coverage so far, and its region overlapping the 160 kb BAC provided sufficient information to reduce the number of contigs in the latter to five. Extensive sequencing of these two BACs in the Clock critical region has proven to be extremely informative: all cDNAs isolated by direct screening and by cDNA selection were physically mapped, and additional Clock candidates identified by sequence homology (designated SI through S12 in FIG 3). The genomic sequence provides critical information for analysis of transcription units (such as identification of exon boundaries), inteφretation of BAC rescue experiments, and Clock mutation identification and analysis. EXAMPLE 6: Transgenic mouse expression of BAC clone and phenotypic rescue of Clock

Because the mutation was a point mutation induced by ENU, a second parallel approach using transgenic rescue to clone the Clock gene was undertaken. Transgenic mice were made by injecting BAC DNA from the clones that mapped to the Clock region. Three sets of DNA preparations were used: 1) circular full-length BAC 54 (140 kb); 2) linear Notl fragment of BAC54 (100 kb); and 3) circular full-length BAC 52 (the clone that overlaps with BAC 54 by -90 kb. Circular DNA was purified using alkaline lysis and cesium chloride gradient ultracentrifugation protocol described for the cosmid DNA purification with some modifications (Favello et al. 1995). The 100 kb linear Notl fragment of BAC 54 was gel-purified using pulse-field gel electrophoresis.

Isolated BAC DΝA was injected at a concentration of 1 μg /μl into fertilized mouse oocytes isolated from crosses between either CD1 +/+ females and (BALB/cJ X C57BL/6J) F2 Clock/Clock males or CD1 +/+ females and CD1 +/+ males as described previously (See FIG. 4) (Hogan et al. 1994). Transgenic mice were identified both by PCR and Southem blot analysis ofthe genomic DΝA prepared from tall biopsies as described (Hogan et al. 1994). Out of 64 mice bom from the BAC54 injected embryos, 6 were positive for the transgene by both methods. Four mice out of 54 were positive for the 100 kb linear fragment of BAC 54, and 2 out of 12 bom were positive for BAC 52 DΝA ( See Table 2). Table 2

Summary of BAC Transgenic Mice Lines

Trasgenic line Founder genotype DNA injected Transgene copy number Transmitance

TG14 Clock/+ BAC54 circular 2 - 3 50% DNA, 140 kb TG36 Clock/+ BAC54 circular 3 -4 50% DNA circular k-b TG55 +/+ BAC54 circular -10 50% DNA circular kb TG60 +/+ BAC54 circular 50% DNA, 140 kb TG19 +/+ BAC54 circular N/D 50% DNA, 140 kb TG48 Clock/+ BAC54 circular N/D 13% DNA, 140 kb

TG80 +/+ BAC54 100 kb 2-3 50% linear Not 1 -fragment

TG97 BAC54 100 kb 10-12 50% linear Not I -fragment TG98 +/+ BAC54 100 kb ND 50% linear Not 1 -fragment

TG91 Clock/4 BAC54 100 kb ND 10% linear Not 1 -fragment

TG121 Clock/+ BAC52 circular 50% DNA, 160 kb

TGI 26 Clock/+ BAC54 circular 4-5 50% DNA, 160 kb Mice postive for the transgene integration by both methods were crossed to either Clock/+ females (for male founders ) or Clock/Clock males (for female founders ). FI progeny from these crosses were 1) tested for the presence ofthe transgene, 2) genotyped for Clock locus by flanking SSLP markers, and 3) wheel-tested for circadian phenotype as described previously (Vitaterna et al. 1994). Results ofthe phenotypic assay are summarized in Table 3. Circadian period length from each mouse was calculated for the 20-day interval during the exposure to constant darkness by a Chi periodogram analysis.

Four lines generated from BAC 54 injections (TG14, 36, 55, 60) showed complete rescue ofthe Clock mutant phenotype both in heterozygous and homozygous Clock mutant animals. An example is pro vided for line TG36 which is representative of this group. The breeding scheme used in the experiment in shown in FIG 5. Activity records showed the phenotypic rescue with BAC 54 transgene in Clock homozygotes. As described above, the Clock mutation has been shown to lengthen circadian period by 1 hr in heterozygotes and by 4 hr in homozygotes. All transgenic animals that were genotyped as Clock/+ or Clock/Clock from these four lines showed a circadian period similar to wild type (Table 3). This result demonstrates that the Clock gene is localized within the 140 kb BAC clone.

To reduce this interval to a single gene, the transgenic functional assay was performed with a smaller DNA fragment (BAC 54 100 kb linear fragment) and an overlapping BAC clone (160 kb BAC 52 clone). Both of these genomic fragments failed to rescue the Clock mutation (Table 3). Trasgenic Line +/+ ++/tg Clock/+ Clockl+ tg Clock/Clock Clock/Clock t

TG14 N/A N/A 24.22+0.183 23.08+0.146 27.06+ 0.314 23.27+0.099 n=5 n=7 n=7 n=9

TG36 23.48±0.048 22.89±0.05 24.18+0.053 23.21±0.047 27.36+0.282 23.18±0.082 n=l l n=10 n=20 n=20 n=8 n=14

TG55 23.41±0.091 22.92+0.137 24.12±0.21 22.77±0.099 N/A N/A n=10 n=8 n=8 n=7

TG60 N/A N/A 23.91 ± 0.1 23.13±0.122 N/A N/A n=13 n=6

TG80 23.44+0.101 23.50±0.07 23.92+0.125 23.64+0.083 N/A N/A n=18 n=5 n=19 n=4

TG97 N/A N/A 23.93+0.04 26.67±0.065 N/A N/A n=4 n=7

TG121 23.50±0.142 23.66+0.125 23.99±0.11 23.96+0.032 26.83±0.4 26.87+0.161 n=4 n=2 n=13 n=5 n=2 n=4

Taken together, the results from all of these transgenic rescue ents are consistent with only a single gene in the 140 kb BAC clone we describe below.

PLE 7: mRNA Expression, Sequence and Structure of the Clock

The mRNA expression of candidate genes was screened by Northern

s in Clock mutant vs. wild-type mice. This led to the observation of

re uced mRNA expression of a candidate M13 clone with a PAS domain sequence first recognized by shotgun sequencing. This M13 genomic clone contained exons from a transcription unit that we subsequently identified as the Clock gene. There are two major transcripts from the Clock locus of -8 and -1

kb (using the cDNA clones, YZ50 or YZ54, as a probe on Northern blots).

There was a reduction in the abundance of both transcripts in the hypothalamus and eye of homozygous Clock mutants as compared to wild type mice. In

addition, there was also a diumal rhythm in the level of Clock mRNA in wild- type mice in both the hypothalamus and eye with high levels in the day and low levels at night. This rhythm in Clock mRNA is consistent with the presence of circadian oscillators in both of these tissues (i.e., the suprachiasmatic nucleus

and retina). In situ hybridization revealed that the expression of Clock mRNA is enriched in the SCN with lower levels in other regions ofthe brain. Taken together the reduced mRNA expression in Clock mutants, the diumal rhythm in

FIG. 6 shows a diagram ofthe physical extent and location ofthe Clock gene. Based on a set of 10 classes of cDNA clones from the gene, the transcribed region ofthe Clock gene spans over 90 kb of genomic sequence and contains 24 exons. Two ofthe exons (exons 1A and 1 B) are distal to the NotI site in BAC54, and thus the 100 kb fragment from BAC 54 and 160 kb clone of BAC 52 do not contain the 5' region ofthe Clock gene. Because of its substantial size, the Clock gene is the only transcription unit in BAC 54 that can account for the results ofthe transgenic rescue experiments. Based upon the physical location of this gene and the rescue experiments, we can conclude that this candidate gene encodes Clock.

The exon structure of Clock is shown in FIG. 7. Ten classes of cDNA clones have been found. There is altemative use of exons 1A and IB in clones YZ50, L8 and YZ80. In addition there is altemative splicing of exons 18 which can be seen in clone L7c, which also has a deletion of exon 19 caused by the Clock mutation (described below)

The complete nucleotide sequence of Clock based upon genomic exon sequences is shown in FIG. 8 (8-1, 8-2, 8-3). The sequences of individual exons are shown in FIG. 9 (9-1, 9-2, 9-3, 9-4). The splice donor and acceptor site sequences are shown for the intron/exon boundaries in FIG.10. There is an open reading frame of 2568 base pairs between nucleotides 389 and 2953 which encodes a 855 amino acid conceptually translated protein (called CLOCK). Following the coding sequence, which terminates with a TAG codon in Exon 23, there is a very long 3' untranslated sequence that terminates at -7500 bp (defined by a subset of cDNA clones with poly A tails at this location), and additional 3 'untranslated sequence that continues for another -2500 bp to form a second transcript of ~10kb. The 7.5 kb and 10 kb transcripts based on cDNA and genomic sequence correspond well with the -8 kb and -11 kb mRNA transcripts estimated from Northern blots. The Clock gene encodes a member ofthe basic helix-loop-helix (bHLH) - PAS domain family of proteins. A search ofthe NCBI database using BLASTN shows that the Clock nucleotide sequence is most similar to human MOP4 (68% identical), human N-PAS2 (69% identical) and mouse NPAS2 (67% identical). A search ofthe NCBI database with the conceptually translated protein sequence using BLASTX shows a similarity to these same three proteins as well as weaker similarity with a large number of bHLH-PAS proteins. An amino acid alignment of CLOCK with human NPAS2 and mouse NPAS2 is shown in FIG. 11. There is sequence similarity among the three proteins in the basic helix-loop-helix domain as well as the entire PAS domain. In addition, there are serine-rich and glutamine-rich regions that are well conserved in the midportion and C-terminal region ofthe proteins. Unlike NPAS2, however, CLOCK has a poly-glutamine stretch near the C-terminus.

In the sequence ofthe mutant Clock allele, there is a single nucleotide base substitution from A to T that alters the third position ofthe 5' (donor) splice site of exon 19. This changes the consensus sequence at this splice site from gta to gtt, which is known in the art to cause exon skipping (Krawczak, M., J. Reiss, D.N. Cooper, Human Genetics 90 :41 -54, 1992). As shown in FIG. 10, 20 out of 22 donor splice sites in the Clock gene have the consensus sequence gta and the remaining 2 sites are gtg, which is also consistent with a purine at the third position. The A to T point mutation in the mutant Clock allele is consistent with that expected from an ENU-induced mutation (Provost and Short, 1994). In the case of Clock, this leads to a deletion of exon 19. The deletion of exon 19 causes a deletion of 51 amino acids (corresponding to amino acids numbers 514 to 564 in SEQ ID NO: 2). FIG. 12 shows the amino acid sequence of CLOCK with the bHLH, PAS- A and PAS-B domains as well as the deletion in the mutant. FIG 13 shows the exon 18 alternatively spliced version of a Clock, which leads to removal of 30 amino acids (corresponding to amino acids numbers 484 to 513 in SEQ ID NO: 2). Both the wild-type and mutant versions ofthe Clock mRNA and protein, express an isoform missing exon 18. Thus, at least 4 different coding versions of CLOCK have been identified..

The deduced amino acid sequence ofthe Clock gene product provides insights about its function as a transciption factor. The basic region ofthe bHLH domain is known to mediate DNA binding and shows that CLOCK likely interacts directly with DNA. The HLH and PAS domains are each known to be protein dimenization domains and predict that CLOCK can interact directly either with itself or with other bHLH or PAS proteins. The C-terminal region of CLOCK has a number of glutamine-rich, proline-rich and serine-rich stretches that are characteristic of activation domain transcription factors.

Example 8: Human Clock Gene And Gene Product

The Clock gene regulates circadian rhythms in mice. To date, it is the only known gene with this function that has been isolated at the molecular level in a mammal. Here, we describe how we cloned the human homologue of Clock, and we disclose both the nucleotide sequence of its coding and 5'untranslated regions as well as the deduced amino acid sequence of its protein product. To achieve these ends we pursued in parallel two strategies: we sequenced several human clones identified by end-sequence in the NCBI database, and we screened a human cDNA library to isolate novel clones that hybridized with a probe ofthe mouse Clock gene.

In the course of our studies of Clock we had searched the NCBI nucleotide database and identified 4 cDNA clones (150328, 328936, 754816, 768552) whose expressed sequence tags (ESTs) indicated they were likely human homologues ofthe Clock gene. We obtained these clones from their distributor (Research Genetics, Huntsville, AL) and sequenced them. DNA sequence alignments to the mouse Clock transcript indicated that the clones fell into three classes that extended discontinuously from the middle of gene's open reading frame to its 3' end and untranslated region.

Simultaneously, we screened 10 6 clones from a commercially prepared library (Clontech) of human hypothalamic cDNAs contained the lambda gt 10 vector. The library is both oligo dT and random primed with insert sizes that range from 0.8 to 4 kb around a mean of 1.7 kb. The protocol for the screen was as follows: we random primed probe (DECAprime II, Ambion) from a phagemid clone of mouse Clock (YZ 50) cut with Sac 1 and Not 1 restriction endonucleases (NEB); we prehybridized filters for 8 hours in a buffer solution containing 6X SSC, 2X Denhardt's solution, ImM EDTA, 0.5 % SDS, and 150 g/ml of boiled sheared salmon sperm; and then hybridized the filters for a further 24 hours at 55 C in fresh hybridization solution with added probe. Following hybridization we washed the filters twice for 30 minutes at room temperature in a solution of 2X SSC/0.1% SDS; and then performed successive washes for 30 minutes each at 55 C in solutions of 2X SSC/ 0.1% SDS, IX SSC/0.1% SDS, and 0.5X SSC/0.1% SDS. With this treatment we identified on the initial round of screening 43 plaques that generated hybridization signals. We picked and plaque purified 24 of these, and then for 13 of the 24 prepared vector DNA from phage lysate. With sequencing primers flanking the vector's cloning site, we sequenced the inserts of these clones in fluorescent dye terminator reactions ran on an ABI FRJSM 377 DNA sequencer. DNA sequence alignments to the mouse Clock transcript, as well as database searches with the BLASTN algorithm, revealed that all 13 clones were derived from the human homologue of the Clock gene. We subsequently subcloned a subset of these clones into a pBluescript plasmid vector and re-confirmed their identify by sequence analysis.

Further DNA sequence alignments to the transcript ofthe mouse Clock gene revealed that the consensus sequence from the aggregate ofthe existing EST and hypothalamic clones extended through the gene's entire coding region and into much of its flanking 5' and 3' untranslated ends (Fig 16). Figure 14 records 3546 nucleotides ofthe sequence ofthe human Clock gene: the open reading frame extends for 2538 base pairs between nucleotides 418 and 2955 and is about 89% identical to the mouse orthologue. It encodes the conceptually translated protein, CLOCK, of 846 amino acids. Figure 14 records the deduced amino acid sequence ofthe gene: CLOCK is 96% identical to its mouse orthologue and it retains all the domains that originally suggested its molecular function in the mouse: HLH and PAS protein dimerization domains; a basic region adjacent to the helix loop helix domain known to mediate DNA binding; and a characteristic glutamine rich region in the C terminus, indicating that CLOCK, in humans as in mice, is likely a transcription factor (Fig. 15).

Our successful effort to isolate this the first known human circadian gene promises to provide insight into the molecular and genetic basis of normative circadian physiology. More immediately, however, the human Clock gene will become a timely candidate for the genetic analysis ofthe circadian pathophysiology implicated in disorders of sleep, affect, and endocrinology. The disclosures listed below and all other disclosures cited herein are incoφorated into the specification by reference.

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Van Cauter, E. and F.W. Turek. 1986. Depression: a disorder of timekeeping? Perspect. Biol. Med. 29:510-519. Van Cauter, E. and F.W. Turek. 1995. Endocrine and other biological rhythms. In: Endocrinology. LJ DeGroot Eds., Philadelphia, W. B. Saunders, 2487-2548.

Van Reeth, O., J. Sturis, M.M. Bryne, J.D. Blackman, M. L'Hermite-Baleriaux, R. Leproult, C. Oliner, S. Refetoff, F.W. Turek and E. Van Cauter. 1994. Nocturnal exercise phase-delays the circadian rhythms of melatonin and thyrotropin secretion in normal men. Am. J. Physiol. 266:E964-E974.

Walker, C.A., CM. Winget and K.F.A. Soliman. 1981. Chronopharmacology and Chronotherapeutics. Tallahassee, Florida, Florida A&M University Foundation, pp 417.

Wehr, T.A. and F.K. Goodwin. 1983. Circadian rhythms in psychiatry. Pacific Grove, California, Boxwood, pp

Wehr, T.A., D. Sack, N. Rosenthal, W. Duncan and J.C. Gillin. 1983. Circadian rhythm disturbances in manic-depressive illness. Federation Proc. 42:2809-2814.

Wehr, T.A., A. Wirz- Justice and F.K. Goodwin. 1979. Phase advance ofthe circadian sleep- wake cycle as an antidepressant. Science 206:710-713. Weltzin, T.E., L.K.G. Hsu, C. Pollice and W.H. Kaye. 1991. Feeding patterns in bulimia nervosa. Biol. Psychiatry 30:1093-1110. Wetterberg, L. 1994. Light and biological rhythms. J Internal Medicine 235:5-19.

Young, R.W. 1980. The chemistry ofthe retina: function, renewal, rhythms, and the nucelus. Neurochemistry 1:123-142.

SEQUENCE LISTING

(1) GENERAL INFORMATION:

(i) APPLICANT: Takahashi, Joseph S Turek, Fred W Pinto, Lawrence H

'ii) TITLE OF INVENTION: lock Gene and Gene Product

(iii) NUMBER OF SEQUENCES: 53

(iv. CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Dressier, Rockey, Milnamow & Katz

(B) STREET: Two Prudential Plaza, Suite 4700 (C CITY: Chicago

(D) STATE: Illinois

(E) COUNTRY: USA

(F) ZIP: 60601

(v) COMPUTER READABLE FORM:

(A) MEDIUM TYPE: Floppy disk

(B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS/MS-DOS

(D) SOFTWARE: Patentin Release #1.0, Version #1.30

(vi) CURRENT APPLICATION DATA:

(A) APPLICATION NUMBER: US

(B) FILING DATE:

(C) CLASSIFICATION:

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Northrup, Thomas E

(B) REGISTRATION NUMBER: 33,268

(ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 312-616-5400 '3) TELEFAX: 312-616-5460

(2) INFORMATION FOR SEQ ID NO:l:

(i) SEQUENCE CHARACTERISTICS:

(A- LENGTH: 7498 base pairs (E TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: cDNA

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 389.-2954 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:

GGGGAGGAGC GCGGCGGTAG CGGTGAATTT TGAGGGGTGG GTCGGGGGCG CGCACTCGCC 60

GCCCCTGGTG CTGCCGGCTC CCGGAGCCGT GGCGTGTCCC TGCTGTCGCC GCTCGGCTGT 120

CGCGAGCCGC CGCGGGCAGA GTCCCGGGCG GGGGAGGGAG GAAGCCGGAG CCTCAGGCAC 180

GTGAAAGAAA AGCACAAGAA GAAACTTTTA CAGGCGTTGT TGATTGGACT AGGGCAACGA 240

TTCCCAAAAT CACCAGCAAG AGTTCTGATG GTCAGTCACA CAGAAGACGG CCTTGCGTCT 300

GTGGGTGTTG GAGACTCCAT TCTAAAGATA TAAAAAGTGA AAGAGGAGAA GTACAAATGT 360

CTACCACAAG ACGAAAACAT AATGTGTT ATG GTG TTT ACC GTA AGC TGT AGT 412

Met Val Phe Thr Val Ser Cys Ser 1 5

AAA ATG AGC TCA ATT GTT GAC AGA GAT GAC AGT AGT ATT TTT GAT GGA 460 Lys Met Ser Ser lie Val Asp Arg Asp Asp Ser Ser lie Phe Asp Gly 10 15 20

TTG GTG GAA GAA GAT GAC AAG GAC AAA GCA AAA AGA GTA TCT AGA AAC 508 Leu Val Glu Glu Asp Asp Lys Asp Lys Ala Lys Arg Val Ser Arg Asn 25 30 35 40

AAA TCA GAA AAG AAA CGT AGA GAT CAG TTC AAT GTC CTC ATT AAG GAG 556 Lys Ser Glu Lys Lys Arg Arg Asp Gin Phe Asn Val Leu lie Lys Glu 45 50 55

CTG GGG TCT ATG CTT CCT GGT AAC GCG AGA AAG ATG GAC AAG TCT ACT 604 Leu Gly Ser Met Leu Pro Gly Asn Ala Arg Lys Met Asp Lys Ser Thr 60 65 70

GTT CTA CAG AAG AGC ATT GAT TTT TTG CGC AAA CAT AAA GAG ACC ACT 652 Val Leu Gin Lys Ser lie Asp Phe Leu Arg Lys His Lys Glu Thr Thr 75 80 85

GCA CAG TCA GAT GCT AGT GAG ATT CGA CAG GAC TGG AAA CCC ACA TTC 700 Ala Gin Ser Asp Ala Ser Glu lie Arg Gin Asp Trp Lys Pro Thr Phe 90 95 100

CTT AGT AAT GAA GAG TTT ACA CAG TTA ATG TTA GAG GCT CTT GAT GGT 748 Leu Ser Asn Glu Glu Phe Thr Gin Leu Met Leu Glu Ala Leu Asp Gly 105 110 115 120

TTT TTT TTA GCG ATC ATG ACA GAT GGA AGT ATA ATA TAT GTA TCT GAG 796 Phe Phe Leu Ala lie Met Thr Asp Gly Ser lie lie Tyr Val Ser Glu

125 130 135

AGT GTA ACT TCG TTA CTT GAA CAT TTA CCA TCT GAT CTT GTG GAT CAA 844 Ser Val Thr Ser Leu Leu Glu His Leu Pro Ser Asp Leu Val Asp Gin 140 145 150

AGT ATA TTT AAT TTT ATC CCA GAG GGA GAA CAT TCA GAG GTT TAT AAG 892 Ser lie Phe Asn Phe lie Pro Glu Gly Glu His Ser Glu Val Tyr Lys 155 160 165 ATA CTC TCT ACT CAT CTG CTG GAA AGT GAC TCA TTA ACC CCT GAG TAC 940 lie Leu Ser Thr His Leu Leu Glu Ser Asp Ser Leu Thr Pro Glu Tyr 170 175 180

TTA AAA TCA AAA AAT CAG TTA GAA TTC TGT TGT CAC ATG CTT CGA GGA 988 Leu Lys Ser Lys Asn Gin Leu Glu Phe Cys Cys His Met Leu Arg Gly 185 190 195 200

ACA ATA GAC CCA AAG GAG CCA TCC ACC TAT GAA TAT GTG AGA TTT ATA 1036 Thr lie Asp Pro Lys Glu Pro Ser Thr Tyr Glu Tyr Val Arg Phe lie 205 - 210 215

GGA AAT TTT AAA TCT TTA ACC AGT GTA TCA ACT TCA ACA CAC AAT GGT 1084 Gly Asn Phe Lys Ser Leu Thr Ser Val Ser Thr Ser Thr His As: Gly 220 225 230

TTT GAA GGA ACT ATA CAA CGC ACA CAT AGG CCT TCT TAT GAA GAT AGA 1132 Phe Glu Gly Thr lie Gin Arg Thr His Arg Pro Ser Tyr Glu Asp Arg 235 240 245

GTT TGT TTT GTA GCT ACT GTC AGA TTA GCT ACA CCT CAG TTC ATC AAG 1180 Val Cys Phe Val Ala Thr Val Arg Leu Ala Thr Pro Gin Phe lie Lys 250 255 260

GAA ATG TGT ACT GTT GAA GAA CCA AAT GAA GAG TTT ACA TCT AGA CAC 1228 Glu Met Cys Thr Val Glu Glu Pro Asn Glu Glu Phe Thr Ser Arg His 265 270 275 280

AGT TTA GAA TGG AAG TTT CTA TTT TTA GAT CAC AGG GCA CCA CCA ATA 1276 Ser Leu Glu Trp Lys Phe Leu Phe Leu Asp His Arg Ala Pro Pro lie 285 290 295

ATA GGC TAT TTG CCA TTT GAA GTC TTG GGA ACA TCA GGC TAT GAT TAC 1324 lie Gly Tyr Leu Pro Phe Glu Val Leu Gly Thr Ser Gly Tyr Asp Tyr 300 305 310

TAT CAT GTG GAT GAC CTA GAA AAT CTG GCA AAA TGT CAC GAG CAC TTA 1372 Tyr His Val Asp Asp Leu Glu Asn Leu Ala Lys Cys His Glu His Leu 315 320 325

ATG CAA TAT GGA AAA GGC AAA TCG TGT TAC TAT AGA TTC CTG ACC AAA 1420 Met Gin Tyr Gly Lys Gly Lys Ser Cys Tyr Tyr Arg Phe Leu Thr Lys 330 335 340

GGC CAG CAG TGG ATA TGG CTT CAG ACT CAT TAT TAT ATT ACT TAC CAT 1468 Gly Gin Gin Trp lie Trp Leu Gin Thr His Tyr Tyr lie Thr Tyr His 345 350 355 360

CAG TGG AAT TCA AGG CCA GAG TTC ATT GTT TGT ACT CAC ACT GTA GTA 1516 Gin Trp Asn Ser Arg Pro Glu Phe He Val Cys Thr His Thr Val Val 365 370 375

AGT TAT GCA GAA GTT AGG GCT GAA AGA CGG CGA GAA CTT GGC ATT GAA 1564 Ser Tyr Ala Glu Val Arg Ala Glu Arg Arg Arg Glu Leu Gly He Glu 380 385 390

GAG TCT CTT CCT GAG ACA GCT GCT GAC AAA AGC CAA GAT TCT GGG TCT 1612 Glu Ser Leu Pro Glu Thr Ala Ala Asp Lys Ser Gin Asp Ser Gly Ser 395 400 405

GAC AAT CGT ATC AAC ACA GTG AGT CTC AAG GAA GCA CTG GAA AGG TTT 1660 Asp Asn Arg He Asn Thr Val Ser Leu Lys Glu Ala Leu Glu Arg Phe 410 415 420

GAT CAC AGC CCA ACT CCT TCT GCC TCC TCT AGA AGC TCA CGA AAG TCA 1708 Asp His Ser Pro Thr Pro Ser Ala Ser Ser Arg Ser Ser Arg Lys Ser 425 430 435 440

TCT CAC ACC GCA GTC TCA GAC CCT TCC TCC ACA CCG ACA AAG ATC CCT 1756 Ser "'is Thr Ala Val Ser Asp Pro Ser Ser Thr Pro Thr Lys He Pro 445 450 455

ACT GAT ACT AGC ACT CCT CCC AGA CAG CAT TTG CCA GCT CAT GAA AAG 1804 Thr Asp Thr Ser Thr Pro Pro Arg Gin His Leu Pro Ala His Glu^' Lys 460 465 470

ATG ACA CAG CGG AGG TCG TCC TTC AGC AGT CAG TCC ATA AAC TCC CAG 1852 Met Thr Gin Arg Arg Ser Ser Phe Ser Ser Gin Ser He Asn Ser Gin 475 480 485

TCA GTT GGT CCA TCA TTA ACA CAG CCA GCG ATG TCT CAA GCT GCA AAT 1900 Ser Val Gly Pro Ser Leu Thr Gin Pro Ala Met Ser Gin Ala Ala Asn 490 495 500

TTA CCA ATT CCA CAA GGC ATG TCA CAG TTT CAG TTT TCA GCT CAG TTA 1948 Leu Pro He Pro Gin Gly Met Ser Gin Phe Gin Phe Ser Ala Gin Leu 505 510 515 520

GGA GCC ATG CAG CAT CTA AAA GAC CAG CTA GAG CAG CGG ACA CGG ATG 1996 Gly Ala Met Gin His Leu Lys Asp Gin Leu Glu Gin Arg Thr Arg Met 525 530 535

ATA GAG GCA AAT ATT CAT CGG CAG CAA GAA GAA CTA AGG AAA ATT CAA 2044 He Glu Ala Asn He His Arg Gin Gin Glu Glu Leu Arg Lys He Gin 540 545 550

GAG CAA CTT CAG ATG GTC CAT GGT CAA GGG CTA CAG ATG TTT TTG CAG 2092 Glu Gin Leu Gin Met Val His Gly Gin Gly Leu Gin Met Phe Leu Gin 555 560 565

CAA TCA AAC CCT GGA TTG AAT TTT GGT TCT GTT CAA CTT TCC TCT GGA 2140 Gin Ser Asn Pro Gly Leu Asn Phe Gly Ser Val Gin Leu Ser Ser Gly 570 575 580

AAT TCT AAT ATC CAG CAG CTC ACA CCT GTA AAT ATG CAA GGC CAG GTT 2188 Asn Ser Asn He Gin Gin Leu Thr Pro Val Asn Met Gin Gly Gin Val 585 590 595 600

GTC CCT GCT AAC CAG GTT CAG AGT GGA CAT ATC AGC ACA GGC CAG CAC 2236 Val Pro Ala Asn Gin Val Gin Ser Gly His He Ser Thr Gly Gin His 605 610 615

ATG ATA CAG CAA CAG ACT TTA CAA AGT ACA TCA ACT CAG CAG AGT CAA 2284 Met He Gin Gin Gin Thr Leu Gin Ser Thr Ser Thr Gin Gin Ser Gin 620 625 630

CAG AGT GTA ATG AGT GGA CAC AGT CAG CAG ACG TCT CTT CCA AGT CAG 2332 Gin Ser Val Met Ser Gly His Ser Gin Gin Thr Ser Leu Pro Ser Gin 635 640 645

ACA CCG AGC ACT CTC ACA GCC CCA CTG TAC AAT ACG ATG GTG ATT TCC 2380 Thr Pro Ser Thr Leu '-.hr Ala Pro Leu Tyr Asn Thr Met Val He Ser 650 655 660

CAG CCT GCA GCT GGG AGC ATG GTC CAG ATT CCA TCC AGT ATG CCA CAG 2428 Gin Pro Ala Ala Gly Ser Met Val Gin He Pro Ser Ser Met Pro Gin 665 670 675 680

AAC AGT ACC CAG AGT GCT ACA GTC ACT ACG TTC ACT CAG GAC AGA CAG 2476 Asn Ser Thr Gin Ser Ala Thr Val Thr Thr Phe Thr Gin Asp Arg Gin 685 690 695

ATA AGA TTT TCT CAA GGT CAG CAA CTT GTG ACC AAA TTA GTG ACT GCT 2524 He Arg Phe Ser Gin Gly Gin Gin Leu Val Thr Lys Leu Val Thr Ala 700 705 710

CCT GTA GCT TGT GGG GCC GTC ATG GTA CCA AGT ACC ATG CTT ATG GGT 2572 Pro Val Ala Cys Gly Ala Val Met Val Pro Ser Thr Met Leu Met Gly 715 720 725

CAG GTG GTG ACT GCC TAT CCT ACC TTC GCC ACA CAA CAG CAG CAG GCA 2620 Gin Val Val Thr Ala Tyr Pro Thr Phe Ala Thr Gin Gin Gin Gin Ala 730 735 740

CAG ACA TTA TCG GTA ACA CAA CAG CAG CAG CAG CAG CAG CAG CAG CCA 2668 Gin Thr Leu Ser Val Thr Gin Gin Gin Gin Gin Gin Gin Gin Gin Pro 745 750 755 760

CCA CAG CAA CAG CAA CAA CAA CAG CAG AGT TCC CAG GAA CAG CAG CTT 2716 Pro Gin Gin Gin Gin Gin Gin Gin Gin Ser Ser Gin Glu Gin Gin Leu

765 770 775

CCT TCA GTT CAG CAG CCA GCT CAG GCC CAG CTG GGC CAG CCA CCA CAG 2764 Pro Ser Val Gin Gin Pro Ala Gin Ala Gin Leu Gly Gin Pro Pro Gin 780 . 785 790

CAG TTC TTA CAG ACA TCT AGG TTG CTC CAC GGG AAT CCT TCG ACA CAG 2812 Gin Phe Leu Gin Thr Ser Arg Leu Leu His Gly Asn Pro Ser Thr Gin 795 800 805

CTC ATC CTC TCT GCT GCC TTT CCA CTA CAA CAG AGC ACT TTC CCT CCT 2860 Leu He Leu Ser Ala Ala Phe Pro Leu Gin Gin Ser Thr Phe Pro Pro 810 815 820

TCG CAC CAC CAG CAA CAC CAG CCT CAG CAG CAA CAG CAG CTT CCT CGG 2908 Ser His His Gin Gin His Gin Pro Gin Gin Gin Gin Gin Leu Pro Arg 825 830 835 840

CAC AGG ACT GAC AGC CTG ACT GAC CCT TCC AAG GTC CAG CCA CAG T 2954 His Arg Thr Asp Ser Leu Thr Asp Pro Ser Lys Val Gin Pro Gin 845 850 855 AGCACACACA CTTCCTCTCT GACATGCGAG AGGAAGGGGA TGGCCAGAAA GAATCGCTCA 3014

GTTGGCATGC GGTCAGAAGT TGAACAGTTT CACGAGGGTG GTCTTGAGTG TTCAGTCCCT 3074

TGATGAGACG GTAGGGAAGT GCTGCCCAGT GCTTCAGATG TCCATTAAAT ACCAGCCAGT 3134

GGGAAATGGT CATAGGGACA CAGCCAATTC TGACAGTTTC TTTGCCCAGG TATTTTTTGA 3194

TAGAAAGAGT ATATTGCCAA ATGCTAACAA GCTCAGCTAT CAACCAGATC TTTACTGAAT 3254

CCGAAGAGCA CTAACAGTGT TGGTAGCTTT AGTGGGTCTG TGCCTGCATC AAATATTACA 3314

GAGGGCACAC CACTGCCAGG GGTTTGCTTA GAATGCCATG AAGATAGTCC AGTAGTTAAT 3374

AGTCCCCACC CCAAACTCCT CTCCCTGTTC AGACAATGAT GGAACCGTGA TGACTTTGAG 3 34

AATGTTGTGC AGGTTTGAAT TCACTGTGTA CAGATGCTGT AGTGTCTCTG TGTCTGGATG 3494

GAGGAGAGAA AGCCACTTTG ATACAGAAAG CATTATCTGT CCCTCACAGG TATGAGTGCA 3554

TTT-ATTAGG TTTGACACCA TGTACAAACT GATAACAACC TCTCTTTTTT CATTTTGTTT 3614

ACAACACAGT AGTGTTCTCG TTACTTTTCC AGGGCACAAG TCTTTTTGTC CGTGCTTTGG 3674

CTGTGATGTC ACAGTTTGTT CAGTGAGGTA ACAATGTGCT GCTGGGAATG GATTTTTTTA 3734

AGGTTAAATT ATTGCTACAT TTCCACTTAC TCAGAAATAT CCCTTATTTC ATTATTTTTC 3794

AATTATGTTT GAGAGAATTG CACTGCTTTA TTATTTTAGA TGGTTGGTTG AGAGTTTAAT 3854

CACATATTTT GATATATTTC ATAGTTGGAA TATTTATGTA AATGGTTTTC AACAAGCCTG 3914

AAAGTAATTT CAAGAATGTT TCAGTTGTAA GAGTAAAGTT TGCACACAAA ACATTTTAGG 3974

CACTTTTTTA ACATTCTCAG AGGTGGGAAT TTTAACTTTT AGGATTTGTT GGAATCTTTT 4034

TATTATCTTT AAAAATTTCA ATGCTTCTTT TAGTCAGAAA TGATTCAGGG TTATTTGAGG 4094

GGAAAAAACC CATAGTGCCT TGATTTTAAT TCAGGTGATA ACTCACCATC TTGAATTCAT 4154

TGTCTGGTTT CAGTAGCAGT TTTGAAACCT TAGTACATTT TTAGCAGCAG TGTCATTCTC 4214

AAGTCCCCAT GAGGACTGCT GCGTCTCTTG GGCTGCCTGA CAGCGTCACA GCTGGGAATG 4274

GGATCCCAAA ATCGTTTCCT GTTTGCATCT TCCTCTAAAG CTAAGTAACT CTTTTAGGAA 4334

TTACCAGTAA ATACTTGCTC AGAGACAAGG GACAAGTTGT CTTTAATTTT CATTGCAGCA 4394

CTAGAATAAT GTAACTCACA TGCTTTTTAA ACATTAAGAT TTCATTTGGC AATATCATTC 4454

TCTACAGGTA ATAAACTCCA ACAAAGCTAC ATACATTTTA AAAGGCATTT TTTTAGATTT 4514

TATGGTACTA ATAATGAGTT TTTCAATTAA AGAACAAAAG ATCAGTAGGA TATAGAATAT 4574

CAAGTATTAC TGAGAAAAGG GAGGATAAGT GTGGCACATT AGAATTGACC TTAAAAGGAA 4634

AGTATGTGAT GGTGAGGTGC TAAACTGGTT TCAGCAGTGC AGATAACCTA AGGCAGAGTT 4694 GCTAGATCAG GGCTTGGGGA ACTCGGAGTC AGCTATCTGT CTCTAGCTTT GCTCTCATCA 4754

TCAGTAAGTG TGTCTTTGTT TTCCTGTTTA CCTGACTGCA ATTAAGTTAG CAAGTTAGTG 814

ATAAAAAGAA AACAACCAAA GAAAATTGGT ACCTACTCTT CTGCGTAAGA AGTGTGTCTA 4874

GATACCAGTC AGTAACTCAC ATATCACAGA AGTTCTTCTA GCTGACATTC ATACGAATAC 4934

CAGAAATAGT TGTGAGAATA CACATTTATG CAAGTTTGTG CACACGTGAC GAAATCAATG 4994

TAAGTCGAGC ACCCACATTG CTTTTCTCCC TTCCACATTG CCTTCTTCTC TTTGGCCATT 5054

CCATGTCCTC GGAGTCGGAG CTGTGCCTCG TTTATCTTTT TGCATCACAT AGCGATAAGA 5114

ATTTAGCTAC AGGAGATACA ACATGCTAGT TATGTAATGC CTGCTGTTCT TCACAGTTCA 5174

TCTCCCTGCT TAAAAGTAGC AGTTGATAAG AAACTCTAGC TGCTAAGGCT GCTGTCCACA 5234

CGGAGATGCA TGCTGGGCAA CAGTTGTCAG CACTAGCTGC CTCTTAGCTC CTTAATTCTT 5294

GGTTCCTTTG GATGGCAAAC TGTCTTTGTC TGCTCCCCAC ACGACTCCAG TATTCTGAAG 5354

AAAGTTCATC TTTTGCCTGT TCATTTCTGT AGCCAAAGCT GACTGAAACC CCAAATCTAA 5414

ATCATGAAAA GATACCAAAA AGAAACACTT CTCAGCTTCT TAGAAACCTT AACTTCTCTT 5474

GCTGTATTTC ATGGATTTGA TTTTCTTTGA AATTTTTGAT TCTGGGCAGC GCCTTTTAAT 5534

TAAGAAATTG TTAGGATGAA GGTCAAACAG GTTCTCATTG CCCTGCAGGT ACCTTGCTCT 5594

GGACTGCTTC TGTATGGGGT GACTTGGGGT TGCTGAACAC ACAGGATTAG AACAGTAAAC 5654

ACAAAGCTGC CCTTGAGGCT GGCGTTAAAC CAGAGCCTCA ATATTGAAAA TATCAAGTCC 5714

TCTTTCCTTC CTTAGAGACG AGACTGTGAG AGGAAAGCAA CTGTGGTAGG TGGGCTTGCT 5774

TGCACATGAG CACCAAGACC ATTCCCCAAG CTCTATCCTC AGGGTAGCAT TTAGAGTGCT 5834

GTGTTCTGCT GTCACATAGA CATGGCTTAG GGATGTAGCA CTAATAAAAG AATGCCCGTG 5894

CTTTTGAATA GTTGTGATAG CAAACTCTAG GCTAACTAGC AAGTGTTTGA ATTCTGTGTG 5954

CTGTATAGTA GTTGGTCATT GCCTTAAAGC AGTCTCTTGG AAGTTGGGAG CACTGAAGCA 6014

GTCCAACCAT ATATGGGCAT CACGTTGAGG GAGATGAGCC TTGTTCAAGC CTTAGAAAGG 6074

ACCCTTAGTC TACACAGGTA GATTCTTTTC ACTTGGATAT TACTGTGTTT AAAATGTTTC 6134

CACTATGTTG AGGCAGTTTT TTAAAGTGGA ACACAGATAG GATTTTTAGT ATTTCTTTTT 6194

TTGTTTCTTT GGTGATTAAA GGTTTGTTGG TAGACATTTG TGTAAAAGTT GTTCAAGCCT 6254

ATCATCTTTC CAGTACTTGT GGTCCTGTTC TTAGTACCAG AGTCCACAAT GGAAAGTGTA 6314

AACACTGGAT ATTAATATTG CTGAGGGTGC ATAGCCAGGT GTGAGCTGAC TGGAACTTCT 6374

CAGTGGTGAA GAAACAGCAC AACGGCACTT GCCATTTTCA TAGTGATTGC ATAAAGAGAC 6434 CTTCTAAGTT TGTCTGGATT GAGTGAACAC TCTTCTAAGA GGAGCTTCTC AAGTAAATGC 6494

AAAGGAAAAG AGTTGACTAT TTTTATAGCA TATTTAATAT ATTTGTATAT AACTATGAGT 6554

GTAGTAGGAA CCCTCCACAT GCCTCCCACT TTTCTAATTC CCTCCCCTTC TGCCGTAGCC 6614

CTAGTCCAGC CTCATCCGCA TGGGTAATGT GCCTACTGTC AGCCTACCTA CCAAAAGATA 6674

GTGCTGCTGC TTTCTGAGAC AGGTGAGATC AGACTCTCAT GCCTGGGGAT CCTTATGGGA 6734

GGAATAGCAC ACACTTAGAA CAACATACCA CAGTTTAAGA GCATCATTTT GAAAGGTAAT 6794

AAGCACTTTA TTGCAATTAT TCATTTAGAT AAAGTTTGTA TCTTAGGCAT TAACCGTTTT 6854

TAAAGGATCC CTAATCATCA CTTAGGTGAA ATGATAAACG ACACATTTCT GAGAAATGTT 6914

^' CAGGTCCAGT GAACCGTAGC AGGTTTATGG GAATGATTTC AAGGTAGCCA AATAAACTCT 6974

GACTTTTGTT TTGAATGTGG TGGAGTCAGG AGATTGTAGA TGTGTAGTTT GATTTAAACA 7034

CTATTGTAAA CCTATCTTGC CTATTGTGTG GACACCAAAA GAGACCAATG AGCCTGTTTA 7094

TTTTCAGAGG TCTAGGAATA TGCATCTGTC TGAGTAGATA TACAGAACTA ATCTATAAAC 7154

GGTTGGTAGT AATATTTTAG GATACAGTAA CTTAAAGAAT TATTGAGTGT TTTAAATGTG 7214 CCCTGAAATG TTGGCATGTC ATTTCAGCGT TCCCATTTGA GTTGCTCTTG TAATATTTTT 7274

GCACAAAAAG GACTGAGAAA AGACTGCTTT GGTTGAAGAA AACTATAATT TGGTCTTATT 7334

TTAATGTCTC CTGTGGAAAC ACTGGAGGTA AATTTGTTGG CATAGTTACT AATTCAGGAT 7394

ATTTAAAACA GTGTTGAACA GCTCATCAGA AATTAAGCAA ACTTATATAT TTAAAAATTA 7454

AAAATCTTTT TTTCCATGTG ACTGAAAAAA AAAAAAAAAA AAAA 7 98

(2) INFORMATION FOR SEQ ID NO:2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 855 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Met Val Phe Thr Val Ser Cys Ser Lys Met Ser Ser He Val Asp Arg 1 5 10 15

Asp Asp Ser Ser He Phe Asp Gly Leu Val Glu Glu Asp Asp Lys Asp 20 25 30

Lys Ala Lys Arg Val Ser Arg Asn Lys Ser Glu Lys Lys Arg Arg Asp 35 40 45

Gin Phe Asn Val Leu He Lys Glu Leu Gly Ser Met Leu Pro Gly Asn 50 55 60

Ala Arg Lys Met Asp Lys Ser Thr Val Leu Gin Lys Ser He Asp Phe 65 70 75 80

Leu Arg Lys His Lys Glu Thr Thr Ala Gin Ser Asp Ala Ser Glu He 85 90 95

Arg Gin Asp Trp Lys Pro Thr Phe Leu Ser Asn Glu Glu Phe Thr Gin 100 105 110

Leu Met Leu Glu Ala Leu Asp Gly Phe Phe Leu Ala He Met Thr Asp 115 120 125

Gly Ser He He Tyr Val Ser Glu Ser Val Thr Ser Leu Leu Glu His 130 135 140

Leu Pro Ser Asp Leu Val Asp Gin Ser He Phe Asn Phe He Pro Glu 145 150 155 160

Gly Glu His Ser Glu Val Tyr Lys He Leu Ser Thr His Leu Leu Glu 165 170 175

Ser Asp Ser Leu Thr Pro Glu Tyr Leu Lys Ser Lys Asn Gin Leu Glu 180 185 190

Phe Cys Cys His Met Leu Arg Gly Thr He Asp Pro Lys Glu Pro Ser

195 200 205

»

Thr Tyr Glu Tyr Val Arg Phe He Gly Asn Phe Lys Ser Leu Thr Ser 210 215 220

Val Ser Thr Ser Thr His Asn Gly Phe Glu Gly Thr He Gin Arg Thr 225 230 235 240

His Arg Pro Ser Tyr Glu Asp Arg Val Cys Phe Val Ala Thr Val Arg 245 250 255

Leu Ala Thr Pro Gin Phe He Lys Glu Met Cys Thr Val Glu Glu Pro 260 ^< 265 270

Asn Glu Glu Phe Thr Ser Arg His Ser Leu Glu Trp Lys Phe Leu Phe 275 280 285

Leu Asp His Arg Ala Pro Pro He He Gly Tyr Leu Pro Phe Glu Val 290 295 300

Leu Gly Thr Ser Gly Tyr Asp Tyr Tyr His Val Asp Asp Leu Glu Asn 305 310 315 320

Leu Ala Lys Cys His Glu His Leu Met Gin Tyr Gly Lys Gly Lys Ser 325 330 335

Cys Tyr Tyr Arg Phe Leu Thr Lys Gly Gin Gin Trp He Trp Leu Gin 340 345 350

Thr His Tyr Tyr He Thr Tyr His Gin Trp Asn Ser Arg Pro Glu Phe 355 360 365

He Val Cys Thr His Thr Val Val Ser Tyr Ala Glu Val Arg Ala Glu 370 375 380

Arg Arg Arg Glu Leu Gly He Glu Glu Ser Leu Pro Glu Thr Ala Ala 385 390 395 400

Asp Lys Ser Gin Asp Ser Gly Ser Asp Asn Arg He Asn Thr Val Ser 405 410 415

Leu Lys Glu Ala Leu Glu Arg Phe Asp His Ser Pro Thr Pro Ser Ala 420 425 430

Ser Ser Arg Ser Ser Arg Lys Ser Ser His Thr Ala Val Ser Asp Pro 435 440 445

Ser Ser Thr Pro Thr Lys He Pro Thr Asp Thr Ser Thr Pro Pro Arg 450 455 460

Gin His Leu Pro Ala His Glu Lys Met Thr Gin Arg Arg Ser Ser Phe 465 470 475 480

Ser Ser Gin Ser He Asn Ser Gin Ser Val Gly Pro Ser Leu Thr Gin 485 490 495

Pro Ala Met Ser Gin Ala Ala Asn Leu Pro He Pro Gin Gly Met Ser 500 505 510

Gin Phe Gin Phe Ser Ala Gin Leu Gly Ala Met Gin His Leu Lys Asp 515 520 525

Gin Leu Glu Gin Arg Thr Arg Met He Glu Ala Asn He His Arg Gin 530 535 540

Gin Glu Glu Leu Arg Lys He Gin Glu Gin Leu Gin Met Val His Gly 545 550 555 560

Gin Gly Leu Gin Met Phe Leu Gin Gin Ser Asn Pro Gly Leu Asn Phe 565 - 570 575

Gly Ser Val Gin Leu Ser Ser Gly Asn Ser Asn He Gin Gin Leu Thr 580 585 590

Pro Val Asn Met Gin Gly Gin Val Val Pro Ala Asn Gin Val Gin Ser 595 600 605

Gly His He Ser Thr Gly Gin His Met He Gin Gin Gin Thr Leu Gin 610 615 620

Ser Thr Ser Thr Gin Gin Ser Gin* Gin Ser Val Met Ser Gly His Ser 625 630 635 640

Gin Gin Thr Ser Leu Pro Ser Gin Thr Pro Ser Thr Leu Thr Ala Pro 645 650 655

Leu Tyr Asn Thr Met Val He Ser Gin Pro Ala Ala Gly Ser Met Val 660 665 670

Gin He Pro Ser Ser Met Pro Gin Asn Ser Thr Gin Ser Ala Thr Val 675 680 685

Thr Thr Phe Thr Gin Asp Arg Gin He Arg Phe Ser Gin Gly Gin Gin 690 695 700

Leu Val Thr Lys Leu Val Thr Ala Pro Val Ala Cys Gly Ala Val Met "05 710 715 720

Val Pro £er Thr Met Leu Met Gly Gin Val Val Thr Ala Tyr Pro Thr

725 730 735

Phe Ala Thr Gin Gin Gin Gin Ala Gin Thr Leu Ser Val Thr Gin Gin 740 745 750

Gin Gin Gin Gin Gin Gin Gin Pro Pro Gin Gin Gin Gin Gin Gin Gin 755 760 "65

Gin Ser Ser Gin Glu Gin Gin Leu Pro Ser Val Gin Gin Pro Ala Gin 770 775 780

Ala Gin --.eu Gly Gin Pro Pro Gin Gin Phe Leu Gin Thr Ser Arg Leu 785 790 795 800

Leu His Gly Asn Pro Ser Thr Gin Leu He Leu Ser Ala Ala Phe Pro 805 810 815

Leu Gin Gin Ser Thr Phe Pro Pro Ser His His Gin Gin His Gin Pro 820 825 830

Gin Gin Gin Gin Gin Leu Pro Arg His Arg Thr Asp Ser Leu Thr Asp 835 840 845

Pro Ser Lys Val Gin Pro Gin 850 855

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 70 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:

GCTGGAGAGA GGAAACCCCG GACGGCGAGA GCGCGAAGGA AATCTGGCCG CCGCCGCGCA 60

CGCGCTCCCG 70

(2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: GCGCTCCCGG TGAGTGCG 18

(2) INFORMATION FOR SEQ ID NO: 5:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid (Ci STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: TCAGGCACGG TGAGGACG 18

(2) INFORMATION FOR SEQ ID NO: 6:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: ACCAGCAAGG TAATTTCC 18

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: GTGAAAGAGG TAAAGGCG 18

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: TGTTGACAGG TATGTTTT 18

(2) INFORMATION FOR SEQ ID NO: 9:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: AGCAAAAAGG TAGTTAGC 18

(2) INFORMATION FOR SEQ ID NO: 10:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: AACATAAAGG TAAAGTGC 18

(2) INFORMATION FOR SEQ ID NO: 11:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: ATGTTAGAGG TATGTTCA 18

(2) INFORMATION FOR SEQ ID NO: 12:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: CATTTACCAG TAAGTATG 18

(2) INFORMATION FOR SEQ ID NO: 13:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

ACTTAAAATG TAAGTAGG 18

(2) INFORMATION FOR SEQ ID NO : 14 :

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14: TAACCAGTGG TAAGTTAA 18

(2) INFORMATION FOR SEQ ID NO: 15:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15: TTCATCAAGG TATGCTTC 18

(2) INFORMATION FOR SEQ ID NO: 16:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA- (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16: AGATCACAGG TAACATTA 18

(2) INFORMATION FOR SEQ ID NO: 17:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 17: ACGAGCACTG TAAGTAGC 18

(2) INFORMATION FOR SEQ ID NO: 18:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 18: TGTAGT7.AGG TAATAACT 18

(2) INFORMATION FOR SEQ ID NO: 19:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: GCTGACAAAG TATGTTTC 18

(2) INFORMATION FOR SEQ ID NO: 20:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 20: ACCCTTCCTG TGAGTGCC 18

(2) INFORMATION FOR SEQ ID NO:21:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 21: AGCAGTCAGG TACGCCTT 18

(2) INFORMATION FOR SEQ ID NO: 22:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: ATGTCACAGG TATTTTTG 18

(2) INFORMATION FOR SEQ ID NO: 23:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 ba&e pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 23:

GGGCTACAGG TAACTTAT 18

(2) INFORMATION FOR SEQ ID NO:24:

(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 18 base pairs (B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 24: TCAACTCAGG TAATTGAC 18

(2) INFORMATION FOR SEQ ID NO: 25:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:

ACAGATAAGG TAGTTGTC 18

(2) INFORMATION FOR SEQ ID NO: 26:

(i) SEQUENCE CHARACTERISTICS: • (A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: TTCTTACAGG TAACCCCC 18

(2) INFORMATION FOR SEQ ID NO: 27:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) ( i) SEQUENCE DESCRIPTION: SEQ ID NO:27: TCTGGTGTTT TCTATTGCAG TGAAAGAAA 29

(2) INFORMATION FOR SEQ ID NO: 28:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 28: CTTTTGTTTT TTTAAAACAG AGTTCTGAT 29

(2) INFORMATION FOR SEQ ID NO: 29:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: ATGTTTTCTT TTCTCACAAG GAGAAGTAC 29

(2) INFORMATION FOR SEQ ID NO: 30:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 30: CTCTGTCTTT TCTCTTGTAG AGATGACAG 29 (2 ) INFORMATION FOR SEQ ID NO : 31 :

( i) SEQUENCE CHARACTERISTICS :

(A) LENGTH: 29 base pairs

(B) TYPE : nucleic acid

(C) STRANDEDNESS : single

(D) TOPOLOGY : linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: TAATTTCTTT TTCTTCATAG AGTATCTAG 29

(2) INFORMATION FOR SEQ ID NO: 32:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 32: ACGTGTCAAT CTGTTTACAG AGACCACTG 29

(2) INFORMATION FOR SEQ ID NO: 33:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS:" single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 33: ACCATTATGT TTAATTTCAG GCTCTTGAT 29

(2) INFORMATION FOR SEQ ID NO: 34:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)

(x: SEQUENCE DESCRIPTION: SEQ ID NO: 34:

TTTTTTTTTT TATTTTTCAG TCTGATCTT 29

(2) INi :. ATION FOR SEQ ID NO:35:

(i) SEQUENCE CHARACTERISTICS: !A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

( i) SEQUENCE DESCRIPTION: SEQ ID NO: 35: CTTTTTATCA CTTATTCCAG CAAAAAATC 29

(2) INFORMATION FOR SEQ ID NO: 36:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 36: ATGTCTCCTT GCTGTTTTAG TATCAACTT 29

(2) INFORMATION FOR SEQ ID NO: 37:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 37: ACTTGTTAAT TTGTTTGTAG GAAATGTGT 29

(2) INFORMATION FOR SEQ ID NO: 38:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 38: ATTATTACTG TATAATTTAG GGCACCACC 29

(2) INFORMATION FOR SEQ ID NO: 39:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 39: TTTTATTTTT TTATTTTTAG TAATGCAAT 29

(2) INFORMATION FOR SEQ ID NO: 0:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 40:

TTGGTTCTTT CCATTTGTAG TTATTGCAG 29

(2) INFORMATION FOR SEQ ID NO: 41: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 41: TGTTCCTCTT ATCTCCTTAG AGCCAAGAT 29

(2) INFORMATION FOR SEQ ID NO: 2:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:42: TCTCTGTTGA CTGTCTTTAG CCACACCGA 29

(2) INFORMATION FOR SEQ ID NO: 3:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 43: ATCTTTTATT TTGCTTCTAG TCCATAAAC 29

(2) INFORMATION FOR SEQ ID NO: 44:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:44: CTTTCCATGT GCTGCTTCAG TTTCAGTTT 29

(2) INFORMATION FOR SEQ ID NO: 45:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid vC) STRANDEDNESS: single (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:45: TGTGATCTTT GTTTTCAAAG ATGTTTTTG 29

(2) INFORMATION FOR SEQ ID NO: 46:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 46: TTCCATACGA TCTTTTCTAG CAGAGTCAA 29

(2) INFORMATION FOR SEQ ID NO: 7:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47: TATTTTGTTT TCTCTCACAG ATTTTCTCA 29

(2) INFORMATION FOR SEQ ID NO: 8:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 29 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:48: ATCATCCTTT TTGTTTTTAG ACATCTAGG 29

(2) INFORMATION FOR SEQ ID NO:49:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

( i) SEQUENCE DESCRIPTION: SEQ ID NO: 9: GGGCTACAGG TAACTTAT 18

(2) INFORMATION FOR SEQ ID NO:50:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 18 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 50: GGGCTACAGG TTACTTAT 18

(2) INFORMATION FOR SEQ ID NO: 51: (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 747 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:

Met Asp Glu Asp Glu Lys Asp Arg Ala Lys Arg Ala Ser Arg Asn Lys 1 5 10 15

Ser Glu Lys Lys Arg Arg Asp Gin Phe Asn Val Leu He Lys Glu Leu 20 25 30

Ser Ser Met Leu Pro Gly Asn He Arg Lys Met Asp Lys He Thr Val 35 40 45

Leu Glu Lys Val He Gly Phe Leu Gin Lys His Asn Glu Val Ser Ala 50 55 60

Gin Thr Glu He Cys Asp He Gin Gin Asp Trp Lys Pro Ser Phe Leu 65 70 75 80

Ser Asn Glu Glu Phe Thr Gin Leu Met Leu Glu Ala Leu Asp Gly Phe 85 90 95

He Ala Val Thr Thr Asp Gly Ser He He Tyr Val Ser Asp Ser He 100 105 110

Thr Pro Leu Leu Gly His Leu Pro Ser Asp Val Met Asp Gin Asn Leu 115 120 125

Leu Asn Phe Leu Pro Glu Gin Glu His Ser Glu Val Tyr Lys He Leu 130 135 140

Ser Ser His Met Leu Val Thr Asp Ser Pro Ser Pro Glu Tyr Leu Lys 145 150 155 160

Ser Asp Asn Asp Leu Glu Phe Tyr Cys His Leu Leu Arg Gly Ser Leu 165 170 175

Asn Pro Lys Glu Phe Pro Thr Tyr Glu Tyr He Lys Phe Val Gly Asn 180 185 190

Phe Arg Ser Tyr Asn Asn Val Pro Ser Pro Ser Cys Asn Gly Phe Asp 195 200 205

Asn Thr Leu Ser Arg Pro Cys Arg Val Pro Leu Gly Lys Val Cys Phe 210 215 220

He Ala Thr Val Arg Leu Ala Thr Pro Gin Phe Leu Lys Glu Met Cys 225 230 235 240 Val Asp Glu Pro Leu Glu Glu Phe Thr Ser Arg His Ser Leu Glu Trp 245 250 255

Lys Phe Leu Phe Leu Asp His Arg Ala Pro Pro He He Gly Tyr Leu 260 265 270

Pro Phe Glu Val Leu Gly Thr Ser Gly Tyr Asp Tyr Tyr His He Asp 275 280 285

Asp Leu Glu Leu Leu Ala Arg Cys His Gin His Leu Met Gin Phe Gly 290 295 300

Lys Gly Lys Ser Cys Cys Tyr Arg Phe Leu Thr Lys Gly Gin Gin Trp 305 310 315 320

He Trp Leu Gin Thr His Tyr Tyr He Thr Tyr His Gin Trp Asn Ser 325 330 335

Lys Pro Glu Phe He Val Cys Thr His Ser Val Val Ser Tyr Ala Asp 340 345 350

Val Arg Val Glu Arg Arg Gin Glu Leu Ala Leu Glu Asp Pro Pro Glu 355 360 365

Ala His Ser Ala Lys Lys Asp Ser Ser Leu Glu Pro Arg Gin Phe Asn 370 375 380

Ala Leu Asp Gly Ala Ser Gly Leu Ser Pro Ser Pro Ser Ala Ser Ser 385 390 395 400

Arg Ser Ser His Lys Ser Ser His Thr Ala Met Ser Glu Pro He Ser 405 410 415

Thr Pro Thr Lys Leu Met Ala Glu Ser Thr Ala Leu Pro Arg Ala Thr 420 425 430

Leu Pro Gin Glu Leu Pro Val Gly Leu Ser Gin Ala Ala Thr Met Pro 435 440 445

Leu Ser Ser Ser Cys Asp Leu Thr Gin Gin Leu Leu Gin Pro Gin Thr 450 455 460

Leu Gin Ser Pro Ala Pro Gin Phe Ser Ala Gin Phe Ser Met Phe Gin 465 470 475 480

Thr He Lys Asp Gin Leu Glu Gin Arg Thr Arg He Leu Gin Ala Asn 485 490 495

He Arg Trp Gin Gin Glu Glu Leu His Lys He Gin Glu Gin Leu Cys 500 505 510

Leu Val Gin Asp Ser Asn Val Gin Met Phe Leu Gin Gin Pro Ala Val 515 520 525

Ser Leu Ser Phe Ser Ser He Gin Arg Pro Ala Gin Gin Gin Leu Gin 530 535 540 Gin Arg Ala Ala Gin Pro Gin Leu Val Gin Leu Gin Gly Gin He Ser 545 550 555 560

Thr Gin Val Thr Gin His Leu Leu Arg Glu Ser Ser Val He Ser Gin 565 570 575

Gly Pro Lys Pro Met Arg Ser Ser Gin Leu Ser Gly Arg Ser Ser Ser 580 585 590

Leu Ser Pro Phe Ser Ser Thr Leu Pro Pro Leu Leu Thr Thr Pro Ala 595 600 605

Ser Thr Pro Gin Asp Ser Gin Cys Gin Pro Ser Pro Asp Phe His Asp 610 615 620

Arg Gin Leu Arg Leu Leu Leu Ser Gin Pro He Gin Pro Met Met Pro 625 630 635 640

Gly Ser Cys Asp Ala Arg Gin Pro Ser Glu Val Ser Arg Thr Gly Arg 645 650 655

Gin Val Lys Tyr Ala Gin Ser Gin Phe Pro Asp His Pro Asn Ser Ser 660 665 670

Pro Val Leu Leu Met Gly Gin Ala Val Leu His Pro Ser Phe Pro Ala 675 680 685

Ser Pro Ser Pro Leu Gin Pro Ala Gin Ala Gin Gin Gin Pro Pro Pro 690 695 700

Gin Ala Pro Thr Ser Leu His Ser Glu Gin Asp Ser Leu Leu Leu Ser 705 710 715 720

Thr Phe Ser Gin Gin Pro Gly Thr Leu Gly Tyr Gin Gin Pro Gin Pro 725 730 735

Arg Pro Arg Arg Val Ser Leu Ser Glu Ser Pro 740 745

(2) INFORMATION FOR SEQ ID NO: 52:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 824 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:

Met Asp Glu Asp Glu Lys Asp Arg Ala Lys Arg Ala Ser Arg Asn Lys 1 . 5 10 15 Ser Glu Lys Lys Arg Arg Asp Gin Phe Asn Val Leu He Lys Glu Leu 20 25 30

Ser Ser Met Leu Pro Gly Asn He Arg Lys Met Asp Lys He Thr Val 35 40 45

Leu Glu Lys Val He Gly Phe Leu Gin Lys His Asn Glu Val Ser Ala 50 55 60

Gin Thr Glu He Cys Asp He Gin Gin Asp Trp Lys Pro Ser Phe Leu 65 70 75 80

Ser Asn Glu Glu Phe Thr Gin Leu Met Leu Glu Ala Leu Asp Gly Phe 85 90 95

He He Ala Val Thr Thr Asp Gly Ser He He Tyr Val Ser Asp Ser 100 105 110

He Thr Pro Leu Leu Gly His Leu Pro Ser Asp Val Met Asp Gin Asn 115 120 125

Leu Leu Asn Phe Leu Pro Glu Gin Glu His Ser Glu Val Tyr Lys He 130 135 140

Leu Ser Ser His Met Leu Val Thr Asp Ser Pro Ser Pro Glu Tyr Leu 145 150 155 160

Lys Ser Asp Ser Asp Leu Glu Phe Tyr Cys His Leu Leu Arg Gly Ser 165 170 175

Leu Asn Pro Lys Glu Phe Pro Thr Tyr Glu Tyr He Lys Phe Val Gly 180 185 190

Asn Phe Arg Ser Tyr Asn Asn Val Pro Ser Pro Ser Cys Asn Gly Phe 195 200 205

Asp Asn Thr Leu Ser Arg Pro Cys Arg Val Pro Leu Gly Lys Glu Val 210 215 220

Cys Phe He Ala Thr Val Arg Leu Ala Thr Pro Gin Phe Leu Lys Glu 225 230 235 240

Met Cys He Val Asp Glu Pro Leu Glu Glu Phe Thr Ser Arg His Ser 245 250 255

Leu Glu Trp Lys Phe Leu Phe Leu Asp His Arg Ala Pro Pro He He

260 265 270

Gly Tyr Leu Pro Phe Glu Val Leu Gly Thr Ser Gly Tyr Asp Tyr Tyr 275 280 285

His He Asp Asp Leu Glu Leu Leu Ala Arg Cys His Gin His Leu Met 290 295 300

Gin Phe Gly He Gly Lys Ser Cys Cys Tyr Arg Phe Leu Thr Lys Gly 305 310 315 320

Gin Gin Trp He Trp Leu Gin Thr His Tyr Tyr He Thr Tyr His Gin 325 330 335

Trp Asn Ser Lys Pro Glu Phe He Val Cys Thr His Ser Val Val Ser 340 345 350

Tyr Ala Asp Val Arg Val Glu Arg Arg Gin Glu Leu Ala Leu Glu Asp 355 360 365

Pro Pro Ser Glu Ala Leu His Ser Ser Ala Leu Lys Asp Lys Gly Ser 370 375 380

Ser Leu Glu Pro Arg Gin His Phe Asn Ala Leu Asp Val Gly Ala Ser 385 390 395 400

Gly Leu Asn Thr Ser His Ser Pro Ser Ala Ser Ser Arg Ser Ser His 405 410 415

Lys Ser Ser His Thr Ala Met Ser Glu Pro He Ser Thr Pro Thr Lys 420 425 430

Leu Met Ala Glu Ala Ser Thr Pro Ala Leu Pro Arg Ser Ala Thr Leu 435 440 445

Pro Gin Glu Leu Pro Val Pro Gly Leu Ser Gin Ala Ala Thr Met Pro 450 455 460

Ala Pro Leu Pro Ser Pro Leu Ser Cys Asp Leu Thr Gin Gin Leu Leu 465 470 475 480

Pro Gin Thr Val Leu Gin Ser Thr Pro Ala Pro Met Ala Gin Phe Ser 485 490 495

Ala Gin Phe Ser Met Phe Gin Thr He Lys Asp Gin Leu Glu Gin Arg 500 505 510

Thr Arg He Leu Gin Ala Asn He Arg Trp Gin Gin Glu Glu Leu His 515 520 525

Lys He Gin Glu Gin Leu Cys Leu Val Gin Asp Ser Asn Val Gin Met 530 - 535 540

Phe Leu Gin Gin Pro Ala Val Ser Leu Ser Phe Ser Ser He Gin Arg 545 550 555 560

Pro Glu Ala Gin Gin Gin Leu Gin Gin Arg Ser Ala Ala Val Thr Gin 565 570 575

Pro Gin Leu Gly Ala Gly Pro Gin Leu Pro Gly Gin He Ser Ser Ala 580 585 590

Gin Val Thr Ser Gin His Leu Leu Arg Glu Ser Ser Val He Ser Thr 595 600 605

Gin Gly Pro Lys Pro Met Arg Ser Ser Gin Leu Met Gin Ser Ser Gly 610 615 620

Arg Ser Gly Ser Ser Leu Val Ser Pro Phe Ser Ser Ala Thr Ala Ala 625 630 635 640 Leu Pro Pro Ser Leu Asn Leu Thr Thr Pro Ala Ser Thr Ser Gin Asp 645 650 655

Ala Ser Glr; Cys Gin Pro Ser Pro Asp Phe Ser His Asp Arg Gin Leu 660 665 670

Arg Leu Leu Leu Ser Gin Pro He Gin Pro Met Met Pro Gly Ser Cys 675 680 685

Asp Ala Arg Gin Pro Ser Glu Val Ser Arg Thr Gly Arg Gin Val Lys 690 695 700

Tyr Ale Gin Ser Gin Thr Val Phe Gin Asn Pro Asp Ala His Pro Ala 705 710 715 720

Asn Ser ^"ϊer Ser Ala- Pro Met Pro Val Leu Leu Met Gly Gin Ala Val 725 730 735

Leu His Pro Ser Phe Pro Ala Ser Gin Pro Ser Pro Leu Gin Pro Ala 740 745 750

Gin Ala Arg Gin Gin Pro Pro Gin His Tyr Leu Gin Val Gin Ala Pro 755 760 765

Thr Ser Leu His Ser Glu Gin Gin Asp Ser Leu Leu Leu Ser Thr Tyr 770 775 780

Ser Gin Gin Pro Gly Thr Leu Gly Tyr Pro Gin Pro Pro Pro Ala Gin 785 790 795 800

Pro Gin Pro Leu Arg Pro Pro Arg Arg Val Ser Ser Leu Ser Glu Ser 805 810 815

Ser Gly Leu Gin Gin Pro Pro Arg 820

(2) INFORMATION FOR SEQ ID NO: 53:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 816 amino acids

(B) TYPE: amino acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53:

Met Asp Glu Asp Glu Lys Asp Arg Ala Lys Arg Ala Ser Arg Asn Lys 1 5 10 15

Ser Glu Lys Lys Arg Arg Asp Gin Phe Asn Val Leu He Lys Glu Leu 20 25 30 Ser Ser Met Leu Pro Gly Asn He Arg Lys Met Asp Lys He Thr Val 35 40 45

Leu Glu Lys Val He Gly Phe Leu Gin Lys His Asn Glu Val Ser Ala 50 55 60

Gin Thr Glu He Cys Asp He Gin Gin Asp Trp Lys Pro Ser Phe Leu 65 70 75 80

Ser Asn Glu Glu Phe Thr Gin Leu Met Leu Glu Ala Leu Asp Gly Phe 85 90 95

Val He Val Val Thr Thr Asp Gly Ser He He Tyr Val Ser Asp Ser 100 105 110

Thr Thr Pro Leu Leu Gly His Leu Pro Ala Asp Val Met Asp Gin Asn 115 120 125

Leu Leu Asn Phe Leu Pro Glu Gin Glu His Ser Glu Val Tyr Lys He 130 135 140

Leu Ser Ser His Met Leu Val Thr Asp Ser Pro Ser Pro Glu Phe Leu 145 150 155 160

Lys Ser Asp Asn Asp Leu Glu Phe Tyr Cys His Leu Leu Arg Gly Ser 165 170 175

Leu Asn Pro Lys Glu Phe Pro Thr Tyr Glu Tyr He Lys Phe Val Gly 180 185 190

Asn Phe Arg Ser Tyr Asn Asn Val Pro Ser Pro Ser Cys Asn Gly Phe 195 200 205

Asp Asn Thr Leu Ser Arg Pro Cys His Val Pro Leu Gly Lys Asp Val 210 215 220

Cys Phe He Ala Thr Val Arg Leu Ala Thr Pro Gin Phe Leu Lys Glu 225 230 235 240

Met Cys Val Ala Asp Glu Pro Leu Glu Glu Phe Thr Ser Arg His Ser 245 250 255

Leu Glu Trp Lys Phe Leu Phe Leu Asp His Arg Ala Pro Pro He He 260 265 270

Gly Tyr Leu Pro Phe Glu Val Leu Gly Thr Ser Gly Tyr Asn Tyr Tyr 275 280 285

His He Asp Asp Leu Glu Leu Leu Ala Arg Cys His Gin His Leu Met 290 295 300

Gin Phe Gly Lys Gly Lys Ser Cys Cys Tyr Arg Phe Leu Thr Lys Gly 305 310 315 320

Gin Gin Trp He Trp Leu Gin Thr His Tyr Tyr He Thr Tyr His Gin 325 330 335 Trp Asn Ser Lys Pro Glu Phe He Val Cys Thr His Ser Val Val Ser 340 345 350

Tyr Ala Asp Val Arg Val Glu Arg Arg Gin Glu Leu Ala Leu Glu Asp 355 360 365

Pro Pro Thr Glu Ala Met His Pro Ser Ala Val Lys Glu Lys Asp Ser 370 375 380

Ser Leu Glu Pro Pro Gin Pro Phe Asn Ala Leu Asp Met Gly Ala Ser 385 390 395 400

Gly Leu Pro Ser Ser Pro Ser Pro Ser Ala Ser Ser Arg Ser Ser His 405 410 415

Lys Ser Ser His Thr Ala Met Ser Glu Pro He Ser Thr Pro Thr Lys 420 425 430

Leu Met Ala Glu Asn Ser Thr Thr Ala Leu Pro Arg Pro Ala Thr Leu 435 440 445

Pro Gin Glu Leu Pro Val Gin Gly Leu Ser Gin Ala Ala Thr Met Pro 450 455 460

Thr Ala Leu His Ser Ser Ala Ser Cys Asp Leu Thr Lys Gin Leu Leu 465 470 475 480

Leu Gin Ser Leu Pro Gin Thr Gly Leu Gin Ser Pro Pro Ala Pro Val 485 490 495

Thr Gin Phe Ser Ala Gin Phe Ser Met Phe Gin Thr He Lys Asp Gin 500 505 510

Leu Glu Gin Arg Thr Arg He Leu Gin Ala Asn He Arg Trp Gin Gin 515 520 525

Glu Glu Leu His Lys He Gin Glu Gin Leu Cys Leu Val Gin Asp Ser 530 535 540

Asn Val Gin Met Phe Leu Gin Gin Pro Ala Val Ser Leu Ser Phe Ser 545 550 555 560

Ser He Gin Arg Pro Ala Ala Gin Gin Gin Leu Gin Gin Arg Pro Ala 565 570 575

Ala Pro Ser Gin Pro Gin Leu Val Val Asn Thr Pro Leu Gin Gly Gin 580 585 590

He Thr Ser Thr Gin Val Thr Asn Gin His Leu Leu Arg Glu Ser Asn 595 600 605

Val He Ser Ala Gin Gly Pro Lys Pro Met Arg Ser Ser Gin Leu Leu 610 615 620

Pro Ala Ser Gly Arg Ser Leu Ser Ser Leu Pro Ser Gin Phe Ser Ser 625 630 635 640 Thr Ala Ser Val Leu Pro Pro Gly Leu Ser Leu Thr Thr He Ala Pro 645 650 655

Thr Pro Gin Asp Asp Ser Gin Cys Gin Pro Ser Pro Asp Phe Gly His 660 665 670

Asp Arg Gin Leu Arg Leu Leu Leu Ser Gin Pro He Gin Pro Met Met 675 680 685

Pro Gly Ser Cys Asp Ala Arg Gin Pro Ser Glu Val Ser Arg Thr Gly 690 695 700

Arg Gin Val Lys Tyr Ala Gin Ser Gin Val Met Phe Pro Ser Pro Asp 705 710 715 720

Ser H s Pro Thr Asn Ser Ser Ala Ser Thr Pro Val Leu Leu Met Gly 725 730 735

Gin Ala Val Leu His Pro Ser Phe Pro Ala Ser Arg Pro Ser Pro Leu 740 745 750

Gin Pro Ala Gin Ala Gin Gin Gin Pro Pro Pro Tyr Leu Gin Ala Pro 755 760 765

Thr Ser Leu His Ser Glu Gin Pro Asp Ser Leu Leu Leu Ser Thr Phe

770 775 780

Ser Gin Gin Pro Gly Thr Leu Gly Tyr Ala Ala Thr Gin Ser Thr Pro

785 790 795 800

Pro Gin Pro Pro Arg Pro Ser Arg Arg Val Ser Arg Leu Ser Glu Ser 805 810 815

(2) INFORMATION FOR SEQ ID NO: 54:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 3546 base pairs

(B) TYPE: nucleic acid

(C) STRANDEDNESS: single

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: DNA (genomic)

(ix) FEATURE:

(A) NAME/KEY: CDS

(B) LOCATION: 418..2956

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:

CATGCCTCAG GATACTCCTC AATAGCCATC GCTGTAGTAT ATCCAAAGAC AACCATCATT 60

CCCCCCCCCC GGCCCCCTGG AGCGAGAGCG CGAAGGAAAT CTGGCCGCCG CCGCCGCGAG 120

CGCTCCCGAA TTTTTACTTG TTCCTGCAAA GCTGCTGGAG CTCAGAAGCT GATTCTATCA 180

CATTGTAAGA TGCCTTTGGA TAATTCTACA GTCCTCTTAA ATGAATCTTT GAACTTGGC 240

AAGTCTCACT AGATACCTTC AATCATCATT TTGAGCTCAA AGAATTCTGA GACTTATGGT 300 TGGTCATATA GAAGAGTACC TTGAACCTAT AGTTTCCTGA AGAATCAGTT TAAAAGATCC 360

AAGGAGTACA AAAGGAGAAG TACAAATGTC TACTACAAGA CGAAAACGTA GTATGTT 17

ATG TTG TTT ACC GTA AGC TGT AGT AAA ATG AGC TCG ATT GTT GAC AGA 465 Met Leu Phe Thr Val Ser Cys Ser Lys Met Ser Ser He Val Asp Arg 1 5 10 15

GAT GAC AGT AGT ATT TTT GAT GGG TTG GTG GAA GAA GAT GAC AAG GAC 513 Asp Asp Ser Ser He Phe Asp Gly Leu Val Glu Glu Asp Asp Lys Asp 20 25 30

AAA GCG AAA AGA GTA TCT AGA AAC AAA TCT GAA AAG AAA CGT AGA GAT 561 Lys Ala Lys Arg Val Ser Arg Asn Lys Ser Glu Lys Lys Arg Arg Asp 35 40 45

CAA TTT AAT GTT CTC ATT AAA GAA CTG GGA TCC ATG CTT CCT GGT AAT 609 Gin Phe Asn Val Leu He Lys Glu Leu Gly Ser Met Leu Pro Gly Asn 50 55 60

GCT AGA AAG ATG GAC AAA TCT ACT GTT CTG CAG AAA AGC ATT GAT TTT 657 Ala Arg Lys Met Asp Lys Ser Thr Val Leu Gin Lys Ser He Asp Phe 65 70 75 80

TTA CGA AAA CAT AAA GAA ATC ACT GCA CAG TCA GAT GCT AGT GAA ATT 705 Leu Arg Lys His Lys Glu He Thr Ala Gin Ser Asp Ala Ser Glu He 85 90 95

CGA CAG GAC TGG AAA CCT ACA TTC CTT AGT AAT GAA GAG TTT ACA CAA 753 Arg Gin Asp Trp Lys Pro Thr Phe Leu Ser Asn Glu Glu Phe Thr Gin 100 105 110

TTA ATG TTA GAG GCT CTT GAT GGT TTT TTT TTA GCA ATC ATG ACA GAT 801 Leu Met Leu Glu Ala Leu Asp Gly Phe Phe Leu Ala He Met Thr Asp 115 120 125

GGA AGC ATA ATA TAT GTG TCT GAG AGT GTA ACT TCA TTA CTT GAA CAT 849 Gly Ser He He Tyr Val Ser Glu Ser Val Thr Ser Leu Leu Glu His 130 135 140

TTA CCA TCT GAT CTT GTG GAT CAA AGT ATA TTT AAT TTT ATC CCA GAA 897 Leu Pro Ser Asp Leu Val Asp Gin Ser He Phe Asn Phe He Pro Glu 145 150 155 160

GGG GAA CAT TCA GAG GTT TAT AAA ATA CTC TCT ACT CAT CTG CTG GAA 945 Gly Glu His Ser Glu Val Tyr Lys He Leu Ser Thr His Leu Leu Glu 165 170 175

AGT GAT TCA TTA ACC CCA GAA TAT TTA AAA TCA AAA AAT CAG TTA GAA 993 Ser Asp Ser Leu Thr Pro Glu Tyr Leu Lys Ser Lys Asn Gin Leu Glu 180 185 190

TTC TGT TGT CAC ATG CTG CGA GGA ACA ATA GAC CCA AAG GAG CCA TCT 1041 Phe Cys Cys His Met Leu Arg Gly Thr He Asp Pro Lys Glu Pro Ser 195 200 205

ACC TAT GAA TAT GTA AAA TTT ATA GGA AAT TTC AAA TCT TTA AAC AGT 1089 Thr Tyr Glu Tyr Val Lys Phe He Gly Asn Phe Lys Ser Leu Asn Ser 210 215 220

GTA TCC TCT TCA GCA CAC AAT GGT TTT GAA GGA ACT ATA CAA CGC ACA 1137 Val Ser Ser Ser Ala His Asn Gly Phe Glu Gly Thr He Gin Arg Thr 225 230 235 240 CAT AGG CCA TCT TAT GAA GAT AGA GTT TGT TTT GTA GCT ACT GTC AGG 1185 His Arg Pro Ser Tyr Glu Asp Arg Val Cys Phe Val Ala Thr Val Arg 245 250 255

TTA GCT ACA CCT CAG TTC ATC AAG GAA ATG TGC ACT GTT GAA GAA CCC 1233 Leu Ala Thr Pro Gin Phe He Lys Glu Met Cys Thr Val Glu Glu Pro 260 265 270

AAT GAA GAG TTT ACA TCT AGA CAT AGT TTA GAA TGG AAG TTT CTG TTT 1281 Asn Glu Glu Phe Thr Ser Arg His Ser Leu Glu Trp Lys Phe Leu Phe 275 280 285

CTA GAT CAC AGG GCA CCA CCC ATA ATA GGG TAT TTG CCA TTT GAA GTT 1329 Leu Asp His Arg Ala Pro Pro He He Gly Tyr Leu Pro Phe Glu Val 290 295 300

CTG GGA ACA TCA GGC TAT GAT TAC TAT CAT GTG GAT GAC CTA GAA AAT 1377 Le-i Gly Thr Ser Gly Tyr Asp Tyr Tyr His Val Asp Asp Leu Glu Asn 305 310 315 320

TTG GCA AAA TGT CAT GAG CAC TTA ATG CAA TAT GGG AAA GGC AAA TCA 1425 Leu Ala Lys Cys His Glu His Leu Met Gin Tyr Gly Lys Gly Lys Ser 325 330 335

TGT TAT TAT AGG TTC CTG ACT AAG GGG CAA CAG TGG ATT TGG CTT CAG 1473 Cys Tyr Tyr Arg Phe Leu Thr Lys Gly Gin Gin Trp He Trp Leu Gin 340 345 350

ACT CAT TAT TAT ATC ACT TAC CAT CAG TGG AAT TCA AGG CCA GAG TTT 1521 Thr His Tyr Tyr He Thr Tyr His Gin Trp Asn Ser Arg Pro Glu Phe 355 360 365

ATT GTT TGT ACT CAC ACT GTA GTA AGT TAT GCA GAA GTT AGG GCT GAA 1569 He Val Cys Thr His Thr Val Val Ser Tyr Ala Glu Val Arg Ala Glu 370 375 380

AGA CGA CGA GAA CTT GGC ATT GAA GAG TCT CTT CCT GAG ACA GCT GCT 1617 Arg Arg Arg Glu Leu Gly He Glu Glu Ser Leu Pro Glu Thr Ala Ala 385 390 395 400

GAC AAA AGC CAA GAT TCT GGG TCA GAT AAT CGT ATA AAC ACA GTC AGT 1665 Asp tys Ser Gin Asp Ser Gly Ser Asp Asn Arg He Asn Thr Val Ser 405 410 415

CTC AAG GAA GCA TTG GAA AGG TTT GAT CAC AGC CCA ACC CCT TCT GCC 1713 Leu Lys Glu Ala Leu Glu Arg Phe Asp His Ser Pro Thr Pro Ser Ala 420 425 430

TCT TCT CGG AGT TCA AGA AAA TCA TCT CAC ACG GCC GTC TCA GAC CCT 1761 Ser Ser Arg Ser Ser Arg Lys Ser Ser His Thr Ala Val Ser Asp Pro 435 440 445

TCC TCA ACA CCA ACC AAG ATC CCG ACG GAT ACG AGC ACT CCA CCC AGG 1809 Ser Ser Thr Pro Thr Lys He Pro Thr Asp Thr Ser Thr Pro Pro Arg 450 455 460

CAG CAT TTA CCA GCT CAT GAG AAG ATG GTG CAA AGA AGG TCA TCA TTT 1857 Gin His Leu Pro Ala His Glu Lys Met Val Gin Arg Arg Ser Ser Phe 465 470 475 480

AGT AGT CAG TCC ATA AAT TCC CAG TCT GTT GGT TCA TCA TTA ACA CAG 1905 Ser Ser Gin Ser He Asn Ser Gin Ser Val Gly Ser Ser Leu Thr Gin 485 490 495 CCA GTG ATG TCT CAA GCT ACA AAT TTA CCA ATT CCA CAA GGC ATG TCC 1953 Pro Val Met Ser Gin Ala Thr Asn Leu Pro He Pro Gin Gly Met Ser 500 505 510

CAG TTT CAG TTT TCA GCT CAA TTA GGA GCC ATG CAA CAT CTG AAA GAC 2001 Gin Phe Gin Phe Ser Ala Gin Leu Gly Ala Met Gin His Leu Lys Asp 515 520 525

CAA TTG GAA CAA CGG ACA CGC ATG ATA GAA GCA AAT ATT CAT CGG CAA 2049 Gin Leu Glu Gin Arg Thr Arg Met He Glu Ala Asn lie His Arg Gin 530 535 540

CAA GAA GAA CTA AGA AAA ATT CAA GAA CAA CTT CAG ATG GTC CAT GGT 2097 Gin Glu Glu Leu Arg Lys He Gin Glu Gin Leu Gin Met Val His Gly 545 550 555 560

CAG GGG CTG CAG ATG TTT TTG CAA CAA TCA AAT CCT GGG TTG AAT TTT 2145 Gin Gly Leu Gin Met Phe Leu Gin Gin Ser Asn Pro Gly Leu Asn Phe 565 570 575

GGT TCC GTT CAA CTT TCT TCT GGA AAT TCA TCT AAC ATC CAG CAA CTT 2193 Gly Ser Val Gin Leu Ser Ser Gly Asn Ser Ser Asn He Gin Gin Leu 580 585 590

GCA CCT ATA AAT ATG CAA GGC CAA GTT GTT CCT ACT AAC CAG ATT CAA 2241 Ala Pro He Asn Met Gin Gly Gin Val Val Pro Thr Asn Gin He Gin 595 600 605

AGT GGA ATG AAT ACT GGA CAC ATT GGC ACA ACT CAG CAC ATG ATA CAA 2289 Ser Gly Met Asn Thr Gly His He Gly Thr Thr Gin His Met He Gin 610 615 620

CAA CAG ACT TTA CAG AGT ACA TCA ACT CAG AGT CAA CAA AAT GTA CTG 2337 Gin Gin Thr Leu Gin Ser Thr Ser Thr Gin Ser Gin Gin Asn Val Leu 625 630 635 640

AGT GGG CAC AGT CAG CAA ACA TCT CTA CCC AGT CAG ACA CAG AGC ACT 2385 Ser Gly His Ser Gin Gin Thr Ser Leu Pro Ser Gin Thr Gin Ser Thr 645 650 655

CTT ACA GCC CCA CTG TAT AAC ACT ATG GTG ATT TCT CAG CCT GCA GCC 2433 Leu Thr Ala Pro Leu Tyr Asn Thr Met Val He Ser Gin Pro Ala Ala 660 665 670

GGA AGC ATG GTC CAG ATT CCA TCT AGT ATG CCA CAA AAC AGC ACC CAG 2481 Gly Ser Met Val Gin He Pro Ser Ser Met Pro Gin Asn Ser Thr Gin 675 680 685

AGT GCT GCA GTA ACT ACA TTC ACT CAG GAC AGG CAG ATA AGA TTT TCT 2529 Ser Ala Ala Val Thr Thr Phe Thr Gin Asp Arg Gin He Arg Phe Ser 690 695 700

CAA GGT CAA CAA CTT GTG ACC AAA TTA GTG ACT GCT CCT GTA GCT TGT 2577 Gin Gly Gin Gin Leu Val Thr Lys Leu Val Thr Ala Pro Val Ala Cys 705 710 715 720

GGG GCA GTC ATG GTA CCT AGT ACT ATG CTT ATG GGC CAG GTG GTG ACT 2625 Gly Ala Val Met Val Pro Ser Thr Met Leu Met Gly Gin Val Val Thr 725 730 735

GCA TAT CCT ACT TTT GCT ACA CAA CAG CAA CAG TCA CAG ACA TTG TCA 2673 Ala Tyr Pro Thr Phe Ala Thr Gin Gin Gin Gin Ser Gin Thr Leu Ser 740 745 750 GTA ACG CAG CAG CAG CAG CAG CAG AGC TCC CAG GAG CAG CAG CTC ACT 2721 Val Thr Gin Gin Gin Gin Gin Gin Ser Ser Gin Glu Gin Gin Leu Thr 755 760 765

TCA GTT CAG CAA CCA TCT CAG GCT CAG CTG ACC CAG CCA CCG CAA CAA 2769 Ser Val Gin Gin Pro Ser Gin Ala Gin Leu Thr Gin Pro Pro Gin Gin 770 775 780

TTT TTA CAG ACT TCT AGG TTG CTC CAT GGG AAT CCC TCA ACT CAA CTC 2817 Phe Leu Gin Thr Ser Arg Leu Leu His Gly Asn Pro Ser Thr Gin Leu 785 790 795 800

ATT CTC TCT GCT GCA TTT CCT CTA CAA CAG AGC ACC TTC CCT CAG TCA 2865 He Leu Ser Ala Ala Phe Pro Leu Gin Gin Ser Thr Phe Pro Gin Ser 805 810 815

CAT CAC CAG CAA CAT CAG TCT CAG CAA CAG CAG CAA CTC AGC CGG CAC 2913 His His Gin Gin His Gin Ser Gin Gin Gin Gin Gin Leu Ser Arg His 820 825 830

AGG ACT GAC AGC TTG CCC GAC CCT TCC AAG GTT CAA CCA CAG T 2956 Arg Thr Asp Ser Leu Pro Asp Pro Ser Lys Val Gin Pro Gin 835 840 845

AGCACACGTG CTTCCTCTCT TGACATCAAG GGAGGAAGGG GATGGCCCAT TAAGAGTTAC 3016

TCAGATGACC TGAGGAAAGG AGGGAAAGTT CCAGCAGTTT CATGAGATGC AGTATTGAGT 3076

GTTCTAGTTC CTGGAATTAG TTGGCAGAGA AAATGCTGCC TAGTGCTACA GATGTACATT 3136

AAATACCAGC CAGCAGGAGG TGATCATAGG GGCACAGCCA GTTCTGACAG TGTTTTAGGT 3196

GCCTGGATAT TTTTTGATGG AAAAAGAATA TATTGCCAAA TATTAAGAAG CTCAGCTATG 3256

AAATGACCTC CAGGGAATCA GAAAGGCACT AATGATGTTA GTAACTTTTA GTGGTTCTGT 3316

GCCTCTTATC AAGTGTTACA GAGGACATAC CACTGCCATG TCAGGGGTTT GCTTACAGTG 3376

ATGCCATGAA GACAGTCCAG TAGACTTGGT AGCGACCCCC TCCCCCAACC CCTCTCCCTT 3436

TTCAGATAAT GATGGAACAG TAATTACTTT CAGAATGTTG TGTGGGTTCA AATTCTCTAT 3496

GTACAGATGA TGTAAAAATA TGTATATGTC TAGATAAAAG GAGAGAAAGC 35 6

(2) INFORMATION FOR SEQ ID NO: 55:

(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 846 amino acids

(B) TYPE: amino acid (D) TOPOLOGY: linear

(ii) MOLECULE TYPE: protein

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 55:

Met Leu Phe Thr Val Ser Cys Ser Lys Met Ser Ser He Val Asp Arg 1 5 10 15

Asp Asp Ser Ser He Phe Asp Gly Leu Val Glu Glu Asp Asp Lys Asp 20 25 30

Lys Ala Lys Arg Val Ser Arg Asn Lys Ser Glu Lys Lys Arg Arg Asp 35 40 45

Gin Phe Asn Val Leu He Lys Glu Leu Gly Ser Met Leu Pro Gly Asn 50 55 60

Ala Arg Lys Met Asp Lys Ser Thr Val Leu Gin Lys Ser He Asp Phe 65 70 75 80

Leu Arg Lys His Lys Glu He Thr Ala Gin Ser Asp Ala Ser Glu He 85 90 95

Arg Gin Asp Trp Lys Pro Thr Phe Leu Ser Asn Glu Glu Phe Thr Gin 100 105 110

Leu Met Leu Glu Ala Leu Asp Gly Phe Phe Leu Ala He Met Thr Asp 115 120 125

Gly Ser He He Tyr Val Ser Glu Ser Val Thr Ser Leu Leu Glu His 130 135 140

Leu Pro Ser Asp Leu Val Asp Gin Ser He Phe Asn Phe He Pro Glu 145 150 155 160

Gly Glu His Ser Glu Val Tyr Lys He Leu Ser Thr His Leu Leu Glu 165 170 175

Ser Asp Ser Leu Thr Pro Glu Tyr Leu Lys Ser Lys Asn Gin Leu Glu 180 185 190

Phe Cys Cys His Met Leu Arg Gly Thr He Asp Pro Lys Glu Pro Ser 195 200 205

Thr Tyr Glu Tyr Val Lys Phe He Gly Asn Phe Lys Ser Leu Asn Ser 210 215 220

Val Ser Ser Ser Ala His Asn Gly Phe Glu Gly Thr He Gin Arg Thr 225 230 235 240

His Arg Pro Ser Tyr Glu Asp Arg Val Cys Phe Val Ala Thr Val Arg 245 250 255

Leu Ala Thr Pro Gin Phe He Lys Glu Met Cys Thr Val Glu Glu Pro 260 265 270

Asn Glu Glu Phe Thr Ser Arg His «Ser Leu Glu Trp Lys Phe Leu Phe 275 280 285

Leu Asp His Arg Ala Pro Pro He He Gly Tyr Leu Pro Phe Glu Val 290 295 300

Leu Gly Thr Ser Gly Tyr Asp Tyr Tyr His Val Asp Asp Leu Glu Asn 305 310 315 320

Leu Ala Lys Cys His Glu His Leu Met Gin Tyr Gly Lys Gly Lys Ser 325 330 335

Cys Tyr Tyr Arg Phe Leu Thr Lys Gly Gin Gin Trp He Trp Leu Gin 340 345 350

Thr His Tyr Tyr He Thr Tyr His Gin Trp Asn Ser Arg Pro Glu Phe 355 360 365

He Val Cys Thr His Thr Val Val Ser Tyr Ala Glu Val Arg Ala Glu 370 375 380 Arg Arg Arg Glu Leu Gly He Glu Glu Ser Leu Pro Glu Thr Ala Ala 335 390 395 400

Asp Lys Ser Gin Asp Ser Gly Ser Asp Asn Arg He Asn Thr Val Ser 405 410 415

Leu Lys Glu Ala Leu Glu Arg Phe Asp His Ser Pro Thr Pro Ser Ala 420 425 430

Ser Ser Arg Ser Ser Arg Lys Ser Ser His Thr Ala Val Ser Asp Pro 435 440 445

Ser Ser Thr Pro Thr Lys He Pro Thr Asp Thr Ser Thr Pro Pro Arg 450 455 460

Gin His Leu Pro Ala His Glu Lys Met Val Gin Arg Arg Ser Ser Phe 465 470 475 480

Ser Ser Gin Ser He Asn Ser Gin Ser Val Gly Ser Ser Leu Thr Gin 485 490 495

Pro Val Met Ser Gin Ala Thr Asn Leu Pro He Pro Gin Gly Met Ser 500 505 510

Gin Phe Gin Phe Ser Ala Gin Leu Gly Ala Met Gin His Leu Lys Asp 515 520 525

Gin Leu Glu Gin Arg Thr Arg Met He Glu Ala Asn He His Arg Gin 530 535 540

Gin Glu Glu Leu Arg Lys He Gin Glu Gin Leu Gin Met Val His Gly 545 550 555 560

Gin Gly Leu Gin Met Phe Leu Gin Gin Ser Asn Pro Gly Leu Asn Phe 565 570 575

Gly Ser Val Gin Leu Ser Ser Gly Asn Ser Ser Asn He Gin Gin Leu 580 585 590

Ala Pro He Asn Met Gin Gly Gin Val Val Pro Thr Asn Gin He Gin 595 600 605

Ser Gly Met Asn Thr Gly His He Gly Thr Thr Gin His Met He Gin 610 61S 620

Gin Gin Thr Leu Gin Ser Thr Ser Thr Gin Ser Gin Gin Asn Val Leu 625 630 635 640

Ser Gly His Ser Gin Gin Thr Ser Leu Pro Ser Gin Thr Gin Ser Thr 645 650 655

Leu Thr Ala Pro Leu Tyr Asn Thr Met Val He Ser Gin Pro Ala Ala 660 665 670

Gly Ser Met Val Gin He Pro Ser Ser Met Pro Gin Asn Ser Thr Gin 675 680 685

Ser Ala Ala Val Thr Thr Phe Thr Gin Asp Arg Gin He Arg Phe Ser 690 695 700

Gin Gly Gin Gin Leu Val Thr Lys Leu Val Thr Ala Pro Val Ala Cys 705 710 715 720 Gly Ala Val Met Val Pro Ser Thr Met Leu Met Gly Gin Val Val Thr 725 730 735

Ala Tyr Pro Thr Phe Ala Thr Gin Gin Gin Gin Ser Gin Thr Leu Ser 740 745 750

Val Thr Gin Gin Gin Gin Gin Gin Ser Ser Gin Glu Gin Gin Leu Thr 755 760 765

Ser Val Gin Gin Pro Ser Gin Ala Gin Leu Thr Gin Pro Pro Gin Gin 770 775 780

Phe Leu Gin Thr Ser Arg Leu Leu His Gly Asn Pro Ser Thr Gin Leu 785 790 795 800

He Leu Ser Ala Ala Phe Pro Leu Gin Gin Ser Thr Phe Pro Gin Ser 805 810 815

His His Gin Gin His Gin Ser Gin Gin Gin Gin Gin Leu Ser Arg His 820 825 830

Arg Thr Asp Ser Leu Pro Asp Pro Ser Lys Val Gin Pro Gin 835 840 845

Claims

WHAT IS CLAIMED IS:

1. An isolated and purified polynucleotide comprising a sequence selected from the group of nucleic acid sequences consisting of:

(a) (i) from about nucleotide 491 to about nucleotide 2953 of SEQ ID NO-1,

(ii) from about nucleotide 490 to about 2955 nucleotide of SEQ ID NO:54;

(b) sequences that are complementary to (a); and

(c) sequences that hybridize under stringent conditions to (a) and, which on expression produce a polypeptide comprising the amino acid residue sequence of SEQ ID NO:2 from residue number 35 to residue number 855 or the amino acid residue sequence of SEQ ID NO:55 from residue number 35 to residue number 846.

2. The polynucleotide of claim 1 that is a DNA molecule.

3. The polynucleotide of claim 1 that is a RNA molecule.

4. The polynucleotide of claim 1 having the sequence of SEQ ID NO: 1 from nucleotide position 419 to nucleotide 2953, the sequence of SEQ ID NO:l from nucleotide position 392 to nucleotide position 2953, the sequence of SEQ ID NO: 1 from nucleotide position 389 to nucleotide position 2953, the sequence of SEQ ID NO:l from nucleotide position 1 to nucleotide position 2953 or the sequence of SEQ ID NO: 1.

5. The polynucleotide of claim 1 having the sequence of SEQ ID NO: 54 from nucleotide position 490 to nucleotide position 2955, the sequence of SEQ ID NO:54 from nucleotide position 438 to nucleotide position 2955, the sequence of SEQ ID NO: 54 from nucleotide position 435 to nucleotide position 2955, the sequence of SEQ ID NO:54 from nucleotide position 421 to nucleotide position 2955 or the sequence of SEQ ID NO:54.

6. An expression vector comprising the polynucleotide of claim 1.

7. The expression vector of claim 6 further comprising a promoter- enhancer.

8. A host cell transformed with the polynucleotide of claim 1.

9. A host cell transformed with the expression vector of claim 6.

10. An isolated and purified oligonucleotide of at least 15 nucleotides, that is identical or complementary to a contiguous stretch of at least 15 nucleotides of SEQ ID NO:l or SEQ ID NO:54.

11. The oligonucleotide of claim 10 wherein the contiguous stretch of at least 15 nucleotides of SEQ ID NO:l is located between about nucleotide position 1 and about nucleotide position 2953 of SEQ ID NO:l.

12. The oligonucleotide of claim 10 wherein the contiguous stretch of at least 15 nucleotides of SEQ ID NO:54 is located between about nucleotide position 1 and about nucleotide position 2955 of SEQ ID NO:54.

13. The oligonucleotide of claim 10 having from about 20 to about 35 nucleotides.

14. The oligonucleotide of claim 10 that is an antisense molecule.

15. The oligonucleotide of claim 10 that is a DNA molecule.

16. The oligonucleotide of claim 10 that is an RNA molecule.

17. An isolated and purified polypeptide comprising the amino acid residue sequence of SEQ ID NO:2 from about residue number 35 to residue number 855.

18. The polypeptide of claim 17 having the amino acid residue sequence of SEQ ID NO:2 from about residue number 11 to residue number 855, the amino acid residue sequence of SEQ ID NO:2 from about residue number 10 to residue number 855, the amino acid residue sequence of SEQ ID NO:2 from about residue number 2 to residue number 855, or the amino acid residue sequence of SEQ ID NO:2.

19. An isolated and purified polypeptide comprising the amino acid residue sequence of SEQ ID NO:55 from about residue number 35 to residue number 846 .

20. The polypeptide of claim 19 having the amino acid residue sequence of SEQ ID NO:55 from about residue number 11 to residue number 846 , the amino acid residue sequence of SEQ ID NO:55 from about residue number 10 to residue number 846, the amino acid residue sequence of SEQ ID NO:55 from about residue number 2 to residue number 846, or the amino acid residue sequence of SEQ ID NO:55.

21. A process of preparing a polypeptide that regulates the circadian rhythm of a mammal comprising transforming a suitable host cell with the expression vector of claim 6 and maintaining the cell under circumstances and for a period of time sufficient for polypeptide formation.

22. The process of claim 21 wherein the polypeptide comprises the amino acid residue sequence of SEQ ID NO:2 from about residue number 35 to residue number 855, the amino acid residue sequence of SEQ ID NO:2 from about residue number 11 to residue number 855, the amino acid residue sequence of SEQ ID NO:2 from -.^'-.out residue number 10 to residue number 855, the amine acid residue sequence of SEQ ID NO:2 from about residue number 2 to residue number 855, or the amino acid residue sequence of SEQ ID NO:2.

^" 23. The process of claim 21 wherein the polypeptide comprises the amino acid residue sequence of SEQ ID NO:55 from about residue number 35 to residue number 846 , the amino acid residue sequence of SEQ ID NO:55 from about residue number 11 to residue number 846, the amino acid residue sequence of SEQ ID NO:55 from about residue number 10 to residue number 846, the amino acid residue sequence of SEQ ED NO:55 from about residue number 2 to residue number 846, or the amino acid residue sequence of SEQ ID NO:55.

24. A polypeptide prepared by the process of claim 21.

25. A pharmaceutical composition comprising the expression vector of claim 6 together with a physiologically acceptable diluent.

26. A pharmaceutical composition comprising the polynucleotide of claim 10 together with a physiologically acceptable diluent.

27. A pharmaceutical composition comprising the polypeptide of claim 19 together with a physiologically acceptable diluent.

28. A pharmaceutical composition comprising the polypeptide of claim 17 together with a physiologically acceptable diluent.