# Patent

## Title
Clock gene BMAL2

## Abstract

The present invention provides novel clock proteins BMAL2 (Brain-Muscle-Arnt-Like protein2), which is crucial for the clock oscillation mechanism including photic-input pathway and output pathway, novel clock genes encoding the proteins, a screening method using the proteins to screen a promoter or a suppressor of the promoter transactivation, and the like.
Genes for cCLOCK, cPER2, cBMAL1 were isolated from the chicken pineal gland which is a material suitable for studying circadian clock, then cDNA encoding the novel clock protein cBMAL2 having homology with cBMAL1 was isolated and sequenced. Further, BMAL2 cDNAs in human, mouse and rat were isolated respectively from the human embryonic kidney cell line, the mouse mid brain and the rat early fibroblast, and sequences of these cDNAs were determined. BMAL2 forms a heterodimer with CLOCK or BMAL1, etc. and it also forms a homodimer.

## Background
This application is a national stage application, filed under 35 USC 371, of PCT/JP01/07197, filed Aug. 23, 2001, which claims priority to Japanese application 2001-35743, filed Feb. 13, 2001.
TECHNICAL FIELD
The present invention relates to novel proteins BMAL (Brain-Muscle-Arnt-Like protein) 2 which are involved in circadian rhythm, their genes, and their use.
BACKGROUND ART
Life activity is connaturally accompanied with various cyclic changes ranging from the behavior at the individual level to the biochemical phenomena at the cellular level. These rhythmic life activities occurring at certain cycles are called biorhythm and a periodic length of these phenomena which are repeated in cycles is often close to a periodic fluctuation of the environment such as a year, a month or a day. Sleep-wake rhythm and hormonal-secretion rhythm for such as melatonin and the adrenal cortex hormone are among those representing circadian rhythms repeated by an approximately 24-hour cycle, a daily unit. The circadian rhythms as mentioned have been observed in almost all the biological species and tissues and are regulated by the biological clock (Annu. Rev. Physiol. 55, 16–54, 1993). The suprachiasmatic nucleus (SCN) in the vertebrate central nervous system, pineal gland, specific neuronal tissues such as retina, etc. are known as tissues conforming circadian rhythm (Science 203, 1245–1247, 1979, Science 203, 656–658, 1979, Proc. Natl. Acad. Sci. USA 76, 999–1003, 1979, Brain Res. 245, 198–200, 1982, Neuron 10, 573–577, 1993, Science 272, 419–421, 1996).
As in the case of the mammalian suprachiasmatic nucleus (SCN), non-mammalian vertebrate pineal glands produce melatonin in response to circadian rhythm and light stimuli and play a central role in the physiological circadian regulation (Science 203, 1245–1247, 1979, Science 203,656–658, 1979, Proc. Natl. Acad. Sci. USA 76, 999–1003, 1979, Proc. Natl. Acad. Sci. USA 77, 2319–2322, 1980, Proc. Natl. Acad. Sci. USA 80, 6119–6121, 1983, J. Neurosci. 9, 1943–1950, 1989). The oscillation mechanism of the above-mentioned circadian rhythm is said to be characterized by the system wherein oscillation occurs at the gene level, is then amplified at the cellular level and finally reaches the individual level (Cell 96, 271–290, 1999). Oscillation at the gene level is brought by a group of genes called clock genes. Recent studies on the rodent clock genes have revealed that the circadian oscillator genes in mammals are positive and negative elements which form the transcription/translation-based negative feedback loop (Cell 96, 271–290, 1999, Annu. Rev. Neurosci. 23, 713–742, 2000). In mice, the negative elements include three period gene homologs; Perl (Cell 90, 1003–1011,1997, Nature 389,512–516,1997), Per2 (Cell 91, 1055–1064, 1997, Neuron 19, 1261–1269, 1997, Genes Cells 3, 167–176, 1998) and Per3 (EMBO J. 17, 4753–4759, 1998, Neuron, 20, 1103–1110, 1998) and two cryptochrome homologs; Cryl and Cry2 (Cell 98, 193–205, 1999, Nature 398, 627–630, 1999).
As for positive elements, BMAL1, CLOCK and the like which are basic helix-loop-helix (bHLH)-PAS (Per-Arnt-Sim) transcription elements are known. A CLOCK-BMAL1 complex is known to activate transcription through an E-box sequence (E-box: CACGTG) which is found not only in the negative element Perl (Science 280, 1564–1569, 1998) but also in clock-controleed genes such as vasopressin (Cell 96, 57–68, 1999) and in the albumin D-site binding protein gene (Genes Dev.14, 679–689, 2000). When a protein level of a negative element mentioned above is increased, its own transactivation for a promoter induced by a positive element is suppressed, the mRNA and protein levels of the negative element are down-regulated, and the molecular cycle is recommenced concomitant with the transactivation of the negative element gene. Therefore, the protein and mRNA levels of a negative element display a marked circadian oscillation. In addition to fluctuations in these clock genes, Perl and Per2 expressions are induced by light (Cell 91, 1055–1064, 1997, Neuron 19, 1261–1269, 1997, Cell 91, 1043–1053, 1997) and at least photo synchronization of an oscillatorisinducedbyPerl (J. Neurosci. 19,1115–1121,1999). Further, it has been revealed that mRNA levels of a positive element Bmall also exhibit circadian oscillation in antiphase to those of negative elements (Biochem. Biophys. Res. Commun. 250, 83–87, 1998, Biochem. Biophys. Res. Commun. 253, 199–203, 1998). Since its transcriptional rhythm is close to that of the Drosophila dClock (Science 286, 766–768, 1999), Bmall is thought to be involved in feedback loop of the negative elements (Science 286, 2460–2461, 1999, Science 288, 1013–1019, 2000).
On the other hand, the chicken (chick) pineal gland has been known that it retains the circadian oscillator as well as photic-input pathway and melatonin-output pathway in the pineal cell and that these properties can readily be retained under culturedconditions (Science 203, 1245–1247, 1979, Science 203, 656–658, 1979, Proc. Natl. Acad. Sci. USA 77, 2319–2322, 1980, Brain Res.438, 199–215, 1988, Recent Prog. Horm. Res. 45, 279–352, 1989, Nature 372, 94–97, 1994, Proc. Natl. Acad. Sci. USA 94, 304–309, 1997, Brain Res. 774, 242–245, 1997). On the basis of these observations, the chick pineal cell is thought to be a prominent model for the study of the vertebrate circadian clock systems at the cellular level (Recent Prog. Horm. Res. 45, 279–352, 1989).
It is known that the biological clock is an auto-oscillatory system which oscillates autonomically without any exogenous stimulation and which, at the same time, has a property of being reset by the exogenous light-stimulation. It is also known that the vertebrate biological clock (circadian clock) which autonomically oscillates in a period close to a day is driven by the auto-feedback-loop consisting of a negative element and a positive element. Many things, however, still remain unknown with regard to the molecular clock system and the like including photic-input and output pathways. The object of the present invention is to provide novel proteins BMAL2 (Brain-Muscle-Arnt-Like protein 2) crucial in the clock oscillation mechanism including photic-input and output pathways, genes encoding the proteins, a method for screening a promoter or a suppressor of the promoter transactivation using the proteins, and the like.
DISCLOSURE OF THE INVENTION
The present inventors have made a keen study to solve the object mentioned above, and isolated cCLOCK, cPER2 and cBMAL1 genes from the chicken pineal gland which is a material suitable for the study of circadian clock, and further isolated cDNA encoding the novel clock protein cBMAL2 which was homologous with cBMAL1 and sequenced it. The inventors have also isolated the human, mouse and rat BMAL2 cDNAs respectively from the human embryonic kidney cell line, the mouse mid brain and the rat early fibroblast and sequenced them. In the pull-down assay, these novel clock proteins BMAL2 were found to form heterodimers with CLOCK, BMAL1 or the like, and to form homodimers among themselves (BMAL2). Besides, in the luciferase assay, BMAL2 were observed not only to form heterodimers with CLOCK and activate transcription via E-box but also to form homodimers and bind to E-box to cooperatively suppress transcription. Here the present invention is accomplished.
The present invention relates to: DNA encoding a protein (a) or (b) below,

    (a) a protein comprising an amino acid sequence shown by Seq. ID No. 2, 4, 6 or 8,
    (b) a protein which comprises an amino acid sequence wherein one or a few amino acids are deleted, substituted or added in the amino acid sequence shown by Seq. ID No. 2, 4, 6 or 8 and which has the BMAL2 activity (claim 1); DNA containing a base sequence shown by Seq. ID No. 1, 3, 5 or 7 or its complementary sequence and part or whole of these sequences (claim 2); DNA which hybridizes with DNA of claim 2 under a stringent condition and which encodes a protein having the BMAL2 activity (claim 3); DNA encoding a protein (a) or (b) below,
    (a) a protein comprising an amino acid sequence shown by Seq. ID No. 10,
    (b) a protein which comprises an amino acid sequence wherein one or a few amino acids are deleted, substituted or added in the amino acid sequence shown by Seq. ID No. 10 and which has the BMAL2 activity (claim 4); DNA containing a base sequence shown by Seq. ID No. 9 or its complementary sequence and part or whole of these sequences (claim 5); DNA which hybridizes with DNA of claim 5 under a stringent condition and which encodes a protein having the BMAL2 activity (claim 6); DNA encoding a protein (a) or (b) below,
    (a) a protein comprising an amino acid sequence shown by Seq. ID No. 12 or 14,
    (b) a protein which comprises an amino acid sequence wherein one or a few amino acids are deleted, substituted or added in the amino acid sequence shown by Seq. ID No. 12 or 14 and which has the BMAL2 activity (claim 7); DNA containing a base sequence shown by Seq. ID No. 11 or 13 or its complementary sequence and part or whole of these sequences (claim 8); DNA which hybridizes with DNA of claim 8 under a stringent condition and which encodes a protein having the BMAL2 activity (claim 9); DNA encoding a protein (a) or (b) below,
    (a) a protein comprising an amino acid sequence shown by Seq. ID No. 16, 18 or 20,
    (b) a protein which comprises an amino acid sequence wherein one or a few amino acids are deleted, substituted or added in the amino acid sequence shown by Seq. ID No. 16, 18 or 20 and which has the BMAL2 activity (claim 10); DNA containing a base sequence shown by Seq. ID No. 15, 17 or 19 or its complementary sequence and part or whole of these sequences (claim 11); and DNA which hybridizes with DNA of claim 11 under a stringent condition and which encodes a protein having the BMAL2 activity (claim 12).


The present invention further relates to: a protein comprising an amino acid sequence shown by Seq. ID No. 2, 4, 6 or 8 (claim 13); a protein which comprises an amino acid sequence wherein one or a few amino acids are deleted, substituted or added in the amino acid sequence shown by Seq. ID No. 2, 4, 6 or 8 and which has the BMAL2 activity (claim 14); a protein comprising an amino acid sequence shown by Seq. ID No. 10 (claim 15); a protein which comprises an amino acid sequence wherein one or a few amino acids are deleted, substituted or added in the amino acid sequence shown by Seq. ID No. 10 and which has the BMAL2 activity (claim 16); a protein comprising an amino acid sequence shown by Seq. ID No. 12 or 14 (claim 17); a protein which comprises an amino acid sequence wherein one or a few amino acids are deleted, substituted or added in the amino acid sequence shown by Seq. ID No. 12 or 14 and which has the BMAL2 activity (claim 18); a protein comprising an amino acid sequence shown by Seq. ID No. 16, 18 or 20 (claim 19); a protein which comprises an amino acid sequence wherein one or a few amino acids are deleted, substituted or added in the amino acid sequence shown by Seq. ID No. 16, 18 or 20 and which has the BMAL2 activity (claim 20); and a peptide which comprises part of the protein of any of claims 13–20 and which has the BMAL2 activity (claim 21).
The present invention still further relates to: a fusion protein or a fusion peptide wherein the protein of any of claims 13–20 or the peptide of claim 21 is bound with a marker protein and/or a peptide tag (claim 22); an antibody which specifically binds to the protein of any of claims 13–20 or to the peptide of claim 21 (claim 23); the antibody according to claim 23, wherein the antibody is a monoclonal antibody (claim 24); a recombinant protein or peptide to which the antibody of claim 23 or 24 specifically binds and which has the BMAL2 activity (claim 25); a host cell comprising an expression system capable of expressing the protein of any of claims 13–20 or the peptide of claim 21 (claim 26); the host cell according to claim 26, wherein the host cell is further capable of expressing CLOCK and/or BMAL1 (claim 27); the host cell according to claim 26 or 27, wherein the expression system at least comprises a promoter having an E-box sequence (CACGTG) (claim 28); the host cell according to claim 28, wherein the promoter having an E-box sequence (CACGTG) is a promoter of Per gene, Tim gene, Cry gene, vasopressin gene or the albumin D-site binding protein gene (claim 29); a non-human animal which, on its chromosome, is deficient in the gene function to encode the protein of any of claims 13–20 or the peptide of claim 21 or which over-expresses the protein of any of claims 13–20 or the peptide of claim 21 (claim 30); and the non-human animal according to claim 30, wherein the non-human animal is a mouse or a rat (claim 31).
The present invention also relates to: a method for screening a promoter or a suppressor for the expression of the protein of any of claims 13–20/the peptide of claim 21 or a promoter or a suppressor of the Bmal2 activity, wherein a cell expressing the protein or peptide and a test substance are used (claim 32); the method for screening a promoter or a suppressor for the expression of the protein/peptide or a promoter or a suppressor of the Bmal2 activity according to claim 32, wherein the cell expressing the protein of any of claims 13–20 or the peptide of claim 21 is the host cell of any of claims 26–29 (claim 33); a method for screening a promoter or a suppressor for the expression of the protein of any of claims 13–20/the peptide of claim 21 or a promoter or a suppressor of the Bmal2 activity, wherein the non-human animal of claim 30 or 31 and a test substance are used (claim 34); an expression promoter of the protein of any of claims 13–20 or the peptide of claim 21, wherein the expression promoter is obtained by the screening method according to any of claims 32–34 (claim 35); an expression suppressor for the protein of any of claims 13–20 or the peptide of claim 21, wherein the expression promoter is obtained by the screening method according to any of claims 32–34 (claim 36); a promoter of the Bmal2 activity obtained by the screening method according to any of claims 32–34 (claim 37); and a suppressor for the Bmal2 activity obtained by the screening method according to any of claims 32–34 (claim 38).
The present invention further relates to: a method for screening a promoter or a suppressor for the promoter transactivation, wherein a cell which expresses the protein of any of claims 13–20 or the peptide of claim 21 and which contains a promoter having an E-box sequence (CACGTG) and a test substance are used (claim 39); the method for screening a promoter or a suppressor for the promoter transactivation according to claim 39, wherein the cell which expresses the protein of any of claims 13–20 or the peptide of claim 21 and which contains a promoter having an E-box sequence (CACGTG) is thehost cellof claim 28 or 29 (claim 40); amethodfor screening a promoter or a suppressor for the transactivation for a promoter having an E-box sequence (CACGTG) in the non-human animal of claim 30 or 31, wherein the non-human animal and a test substance are used (claim 41); a promoter of the promoter transactivation obtained by the screening method according to any of claims 39–41 (claim 42); a suppressor for the promoter transactivation obtained by the screening method according to any of claims 39–41 (claim 43); and a method for diagnosing diseases associated with the expression or the activity of BMAL2, wherein the DNA sequence encoding BMAL2 in a sample is compared with the DNA sequence encuding the protein of claim 13 or 14 (claim 44).



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows the amino acid sequence of cPER2.
FIG. 2 shows the results of the amino acid homologies in domains among cPER2 and three mouse PER proteins (mPER1–3).
FIG. 3 shows the comparison among the amino acid sequences of various BMALs.
FIG. 4 shows the results of the amino acid homologies in domains among various BMAL proteins.
FIG. 5 shows the phylogenetic tree of ARNT-BMAL proteins and their amino acid homologies with cBMAL2 or hBMAL2.
FIG. 6 shows the genomic structure of hBMAL2 gene of the present invention.
FIG. 7 shows the basic structure of mouse BMAL2 and rat BMAL2 of the present invention.
FIG. 8 shows the phylogenetic tree of the BMAL-ARNT family proteins.
FIG. 9 shows the results of the northern blotting for analyzing the expressions of cBmal2 and cBaml1 genes of the present invention.
FIG. 10 shows the results of time-course changes in mRNA levels of cBmal1, cBmal2, cPer2 and cClock in the chicken pineal glands of the individuals.
FIG. 11 shows the time-course changes in mRNA levels of cBmal1, cBmal2, cPer2 and cCLOCK in the cultured chicken pineal cells under LD or DD condition.
FIG. 12 shows the results of the daily fluctuations under LD condition in mRNA expressions of mPer2, mClock, mBmal1 and mBmal2 in the mouse suprachiasinatic nucleus.
FIG. 13 shows the results of light-dependent changes in mRNA expressions of cPer2, cBmal1 and cBmal2 in the chicken pineal glands.
FIG. 14 shows the results of the in vitro physical interactions among cBMAL2 of the present invention, cBMAL1 and cCLOCK proteins.
FIG. 15 shows the results of the binding between a E-box sequence and cBMAL1-cCLOCK or cBMAL2-cCLOCK detected by an electrophoretic mobility shift assay (EMSA).
FIG. 16 shows the results of transcriptional regulation in the 293EBNA cells induced by cBMAL1, cBMAL2 and cCLOCK.
FIG. 17 shows the cPER2 effect on transactivation mediated by E-box sequences.
FIG. 18 shows the effect of overexpression of cBMAL1 or cBMAL2 on the melatonin-rhythms of the chicken pineal cells.



BEST MODE OF CARRYUNG OUT THE INVENTION
Proteins of the present invention are exemplified by novel proteins with BMAL2 activity including: human BMAL2 shown by Seq. ID No. 2, 4, 6 or 8; chicken BMAL2 shown by Seq. ID No. 10; mouse BMAL2 shown by Seq. ID No. 12 or 14; rat BMAL2 shown by Seq. ID No. 16, 18 or 20; a protein comprising an amino acid sequence wherein one or a few amino acids are deleted, substituted or added in the amino acid sequence shown by Seq. ID No. 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20, and having BMAL2 activity; and the like. Here the BMAL2 activity is taken to mean an activity to form a heterodimer with a transcription-promoting element to promote transcription via E-box in the promoter of a clock oscillator gene, and to form a homodimer to bind to E-box to competitively suppress transcription. Any peptide comprising part of the above-mentioned proteins and having BMAL2 activity may serve as a peptide as an object of the present invention, however, a peptide having a basic helix-loop-helix (bHLH) structure or a PAS (Per-Arnt-Sim) domain is preferable. Proteins and peptides as objects of the present invention and the recombinant proteins and peptides to which the antibodies, specifically binding to these proteins and peptides, bind specifically may collectively be referred to as “the present proteins/peptides” hereinafter. The present proteins/peptides can be prepared in accordance with known methods base on their DNA sequence information or the like and there should be no limitation as to the origin of the proteins/peptides.
Any DNA may be an object of the present invention as long as the DNA encodes the present proteins/peptides mentioned above and the specific examples include DNA encoding human BMAL2 shown by Seq. ID No. 2, 4, 6 or 8, DNA encoding chicken BMAL2 shown by Seq. ID No. 10, DNA encoding mouse BMAL2 shown by Seq. ID No. 12 or 14, DNA encoding rat BMAL2 shown by Seq. ID No. 16, 18 or 20; DNA encoding a protein comprising an amino acid sequence wherein one or a few amino acids are deleted, substituted or added in the amino acid sequence shown by Seq. ID No. 2, 4, 6, 8, 10, 12, 14, 16, 18 or 20 and having BMAL2 activity; and DNA containing the base sequence shown by Seq. ID No. 1, 3, 5, 7, 9, 11, 13, 15, 17 or 19 or its complementary sequence and part or whole of these sequences. These can be prepared by known methods from, for instance, a gene library or cDNA library and the like of human, chicken, mouse, rat, etc., based on their DNA sequence information or the like.
DNA encoding a protein having BMAL2 activity of the interest which has the same effect as human BMAL2, chicken BMAL2, mouse BMAL2, rat BMAL2, etc. can be obtained by hybridization with various DNA libraries under a stringent condition by using as a probe the base sequence shown by Seq. ID No. 1, 3, 5, 7, 9, 11, 13, 15, 17 or 19 or its complementary sequence and part or whole of these sequences, and by subsequent isolation of DNA which hybridized with the probe. DNAs thus obtained are also within the scope of the present invention. One example of a hybridization condition for obtaining DNA of the present invention is hybridization at 42° C. and washing at 42° C. in a buffer solution containing 1×SSC, 0.1% SDS, and more preferable example is hybridization at 65° C. and washing at 65° C. in a buffer solution containing 0.1×SSC, 0.1% SDS. There are number of factors other than the temperature condition mentioned above that affect the hybridization stringency and those skilled in the art can actualize the same stringency as that for the hybridization referred to in the above by appropriately combining various factors.
Any fusion protein and fusion peptide may be used as a fusion protein and a fusion peptide for the present invention as long as the present proteins/peptides are bound with marker proteins and/or peptide tags. As for a marker protein, there is no limitation as long as it is a conventionally known marker protein and the specific examples include alkaline phosphatase, the Fc region of an antibody, HRP, GFP, etc. Conventionally known peptide tags including Myc tag, V5 tag, HA tag, His tag, FLAG tag, S tag, etc. are the specific examples of a peptide tag for the use in the present invention. Such fusion protein can be generated according to ordinary protocols and is useful for the following: purification of the various BHAL2 or the like by using affinity of Ni-NTA and His tag; detection of a protein which interacts with various BMAL2; quantification of an antibody against various BMAL2 or the like; and use as a laboratory reagent in this field of art.
Antibodies that specifically bind to the aforementioned proteins and peptides of the present invention can be particularly exemplified by immune-specific antibodies including monoclonal antibodies, polyclonal antibodies, chimeric antibodies, single-stranded antibodies, humanized antibodies, etc. These antibodies can be generated according to ordinary protocols by using the above-mentioned various BMAL2 proteins or the like, or part of these proteins as an antigen. However, monoclonal antibodies are more preferable than the other sorts of antibodies mentioned because of their specificity. Antibodies such as the monoclonal antibodies are useful not only for diagnosis and treatment, such as missile therapy, for the circadian rhythm sleep disorders or the like including delayed sleep phase syndrome, non-24-hour sleep-wake syndrome, advanced sleep phase syndrome, time zone change syndrome, shift work sleep disorder, etc, but for elucidating the molecular mechanism of the circadian oscillation system.
Antibodies of the present invention are created by administering to an animal (preferably non-human) the present proteins/peptides, their fragments containing epitopes, or the cells expressing the proteins/peptides on the membrane surface, according to the conventional protocols. The monoclonal antibodies can be prepared, for instance, by any optional method that provides antibodies produced by cultured materials of continuous cell line such as a hybridoma method (Nature 256, 495–497, 1975), atriomamethod, ahumanB-cellhybridomamethod (Immunology Today 4, 72, 1983), and an EBV-hybridoma method (MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp. 77–96, Alan R. Liss, Inc., 1985).
The preparation method for a single chain antibody (U.S. Pat. No. 4,946,778) can be adopted to prepare single-stranded antibodies to, the present proteins/peptides of the present invention mentioned above. Transgenic mice, other mammals, etc. can be used for expressing humanized antibodies. Clones expressing the present proteins/peptides can be isolated/identified using the antibodies mentioned above, and their polypeptides can be purified by affinity chromatography. Antibodies to the present proteins/peptides or to peptides containing their antigenic epitopes can possibly be used for diagnosis and therapy for circadian rhythm sleep disorders or the like including delayed sleep phase syndrome, non-24-hour sleep-wake syndrome, advanced sleep phase syndrome, time zone change syndrome, shift work sleep disorder, etc, and are useful for elucidating the molecular mechanism of the circadian oscillation system. Furthermore, recombinant proteins or peptides to which these antibodies specifically bind are also covered by the present proteins/peptides of the present invention as described earlier.
The functions of the present proteins/peptides can be analyzed by using, for example, antibodies such as the aforementioned monoclonal antibodies labeled with fluorescent materials including FITC (Fluorescein isothiocyanate), tetramethylrhodamine isothiocyanate, etc., radioisotopes including 125I, 32p, 14C, 35S, 3H, etc., or enzymes including alkaline phosphatase, peroxidase, β-galactosidase, phycoerythrin, etc., or fused with fluorescent proteins such as Green Fluorescent Protein (GFP), BFP, CFP, YFP, RFP, etc. to serve as fusion proteins. As for immunological detection methods using the antibodies of the present invention, RIA method, ELISA method, fluorescent-antibody method, plaque method, spot method, haemagglutination, Ouchterlony method, etc. are exemplified.
There is no particular limitation as to a host cell of the present invention as long as the host cell comprises an expression system capable of expressing the present proteins/peptides. However, a preferable host cell is such in which the genes encoding CLOCK and/or BMAL1 are incorporated so that the two proteins can be simultaneously expressed in the host cell. Even more preferably, the host cell is incorporated with a DNA fragment which at least contains a promoter having E-box sequence (CACGTG), e.g. promoters of Per gene, Tim gene, Cry gene, vasopressin gene, the albumin D-site binding protein gene, etc., or a promoter introduced with E-box sequence (CACGTG) or the like. Although there is no particular limitation as to the above-mentioned DNA fragment so far as the fragment contains a promoter having E-box sequence (CACGTG), it is preferable for readily detecting and measuring the promoter activity that the DNA fragment is linked with a reporter gene including chloramphenicol acetyltransferase (CAT) gene, luciferasegene, etc., ageneencodingafluorescent protein including a short-lived green fluorescent protein (d1EGFP), etc. or with a fusion of GFP gene and a clock oscillator gene, and the like, to the down-stream of the promoter. Further, as to a promoter introduced with E-box sequence (CACGTG), any promoter may be adopted as long as its promoter activity can be regulated by a promoting element including the present proteins/peptides, CLOCK, BMAL1, etc. or by a suppressing element including PER, TIM, CRY, etc. These promoters are exemplified by RSV promoter, trp promoter, lac promoter, recA promoter, λPL promoter, lpp promoter, SPO1 promoter, SPO2 promoter, penP promoter, PHO5 promoter, PGK promoter, GAP promoter, ADH promoter, SRα promoter, SV40 promoter, LTR promoter, CMV promoter, HSV-TK promoter, etc., but the promoters will not be limited to these exemplifications alone.
The present proteins/peptides and genes such as CLOCK and BMAL1 can be introduced into host cells by methods described in many standard laboratory manuals such as a manual of Davis et al. (BASIC METHODS IN MOLECULAR BIOLOGY, 1986), of Sambrook et al. (MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989) and the like. The methods include calcium-phosphate transfection, DEAE-dextran-mediated transfection, transvection, microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, infection, etc. The examples of host cells include bacterial prokaryotic cells such as E. coli, Streptomyces, Bacillus subtilis, Streptococcus, Staphylococcus, etc., eukaryotic cells such as yeast, aspergillus, etc., insect cells such as Drosophila S2, Spodoptera Sf9, etc., animal cells such as L cell, CHO cell, COScell, HeLacell, C127 cell, BALB/c3T3 cell (includingmutant strains deficient in dihydrofolate reductase, tymidine kinase, etc.), BHK21 cell, HEK293 cell, Bowes malignant melanoma cell, etc. and plant cells or the like.
There is no limitation to an expression system as long as the expression system is capable of expressing the present proteins/peptides described above in a host cell and the examples include chromosome-, episome- and virus-derived expression systems, for instance, vectors derived from bacterial plasmid, yeast plasmid, papovavirus such as SV40, vaccinia virus, adenovirus, fowlpox virus, pseudorabies virus and retrovirus, and vectors derived from bacteriophage, transposon and from the combination of these two, e.g. vectors derived from genetic factors of plasmid and bacteriophage such as cosmid and phagemid. Such expression system is not only for raising the expression and it may contain a regulatory sequence to regulate the expression.
Host cells comprising the above-mentioned expression systems and the present proteins/peptides obtained by culturing the cells can be used in a screening method of the present invention as described below. Further, the known methods can be adopted to collect and purify the present proteins/peptides from the cell culture, where the methods include ammonium sulfate- or ethanol-precipitation, acid extraction, anion- or cation-exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography and lectin chromatography, and the high performance liquid chromatography is preferably used. As a column especially used for affinity chromatography, for instance, columns to which antibodies to the present proteins/peptides are bound are used, and when common peptide tags are added to the present proteins/peptides mentioned above, columns to which substances having affinity with the peptide tags are bound are used, in order to obtain the present proteins/peptides. The purification methods for the present proteins/peptides mentioned above may also be employed for peptide synthesis.
In the present invention, a non-human animal whose gene function to encode the present proteins/peptides mentioned above is deficient on its chromosome means a non-human animal part or whole of whose gene on its chromosome encoding the present proteins/peptides is inactivated by gene mutations such as destruction, deletion, substitutions, etc. and thus whose function to express the present proteins/peptides is lost. Further, a non-human animal which over-expresses the present proteins/peptides is specifically represented by a non-human animal which produces larger amount of the present proteins/peptides than a wild-type non-human animal does. Specific examples of non-human animals in the present invention include non-human animals such as rodents including mice, rats, etc., osteichthyes such as zebra fish, medaka fish, etc., arthropods such as Drosophila, silkworm, etc., the non-human animals should not be limited only to these examples.
Homozygous non-human animals that are born according to Mendel's Law include the deficient type or the over-expressing type for the present proteins/peptides as well as their wild type littermates. By using the deficient type animals or the over-expressing type animals of these homozygous non-human animals together with their wild-type littermates at the same time, accurate comparative experiments can be actualized out on the individual level. Therefore in performing screening of the present invention described below, it is, preferable to use wild type non-human animals, i.e. animals of the same species as, or even better the littermates of, non-human animals whose gene function to encode the present proteins/peptides is deficient or over-expressed on their chromosomes, in parallel with the deficient or over-expressed type animals. The method of generating a non-human animal whose gene function to encode the present proteins/peptides is deficient or over-expressed on its chromosome is now explained in the following with reference to a BMAL2 knockout mouse and a BMAL2 transgenic mouse.
A mouse, for instance, whose gene function to encode BMAL2 protein is deficient on its chromosome, i.e. a BMAL2 knockout mouse is generated by the following steps. A gene encoding mouse BMAL2 is screened by using a gene fragment obtained by a method such as PCR from a mouse gene library. A gene thus screened which encodes mouse BMAL2 is subcloned with a viral vector or the like and is identified by means of DNA sequencing. Then whole or part of a gene encoding BMAL2 among this clone is substituted with a pMC1 neo gene cassette or the like and then a gene such as a diphtheria toxin A fragment (DT-A) gene, a herpes simplex virus tymidine kinase (HSV-tk) gene, etc. is introduced onto either or both of 5′- or 3′-end, and thus a targeting vector is constructed.
The targeting vectors thus constructed are linearlized and introduced into ES cells by electroporation or the like to cause homologous recombination. Among the homologous recombinants, ES cells in which homologous recombination have occurred are selected by the use of antibiotics such as G418, ganciclovir (GANC), etc. It is preferable to confirm whether the ES cells selected are the recombinants of the interest by Southern blotting or the like. A clone of the ES cells thus confirmed is microinjected into a mouse blastocyst and which blastocyst is placed back to the recipient mouse to generate a chimeric mouse. A heterozygous mouse can be obtained by intercrossing the chimeric mouse and a wild type mouse. By further intercrossing the heterozygous mice, a BMAL2 knockout mouse of the present invention can be generated. Whether the ability of expressing BMAL2 is lost in the BMAL2 knockout mouse is examined by Northern blotting upon isolating RNA from the mouse obtained by the above-described method, and by Western blotting or the like in which the BMAL2 expression in the mouse can be directly examined.
A BMAL2 transgenic mouse is created by the following steps. A promoter such as chicken β-actin, mouse neurof ilament, SV40, etc. and poly (A) such as rabbit β-globin, SV40, etc. or introns are fused with cDNA encoding BMAL2 derived from chicken, mouse, human, rat, etc., to construct a transgene. This transgene is microinjected into the pronucleus of a mouse fertilized egg. After the obtained egg cell is cultured, it is transplanted to the oviduct of the recipient mouse which was bred thereafter. Neonatal mice having the aforementioned cDNA were selected from among all the mice born and thus the transgenic mice are created. Neonatal mice having the cDNA can be selected by extracting crude DNA from the mice tails or the like and then by performing a dot hybridization method using a gene encoding the introduced BMAL2 as a probe and by PCR method or the like using a specific primer.
Genes or DNAs encoding the present proteins/peptides, the present proteins/peptides, fusion proteins in which the present proteins/peptides and marker proteins and/or peptide tags are bound, antibodies to the present proteins/peptides, host cells comprising expression systems capable of expressing the present proteins/peptides, CLOCK, BMAL1, etc., non-human animalswhose gene function to encode the present proteins/peptides is deficient on their chromosome, non-human animals which over-express the present proteins/peptides and the like make it possible to elucidate the molecular mechanism of the circadian oscillation system. In addition to that, these can be used to screen a promoter or a suppressor for expression of the present proteins/peptides, a promoter or a suppressor for the Bmal2 activity, and a promoter or a suppressor for the promoter transactivation of the clock oscillator genes or the like. Some among the substances obtained by these screening methods may possibly be used for therapy of the circadian rhythm sleep disorders or the like including delayed sleep phase syndrome, non-24-hour sleep-wake syndrome, advanced sleep phase syndrome, time zone change syndrome, shift work sleep disorder, etc.
As for a screening method for a promoter or a suppressor for expression of the present proteins/peptides, or for a promoter or a suppressor for the Bmal2 activity of the present invention, methods are exemplified that use: cells expressing the present proteins/peptides and a test substance; and a non-human animal deficient in a gene function to encode the present proteins/peptides on its chromosome or a non-human animal overexpressing the present proteins/peptides and a test substance. A screening method using cells expressing the present proteins/peptides and a test substance, as mentioned above, can be exemplified by a method wherein a test substance is made to contact or introduced into, for instance, the cells expressing the present proteins/peptides, e.g. cells obtained from wild-type non-human animals, host cells of the present invention, cells obtained from transgenic non-human animals of the present invention, etc. and wherein the Bmal2 activity and changes in the expression levels of the present proteins/peptides are measured and assessed, but the methods should not be limited to these examples alone.
As for a screening method wherein a non-human animal whose gene function to encode the aforementioned present proteins/peptides is deficient on its chromosome or a non-human animal which over-expresses the present proteins/peptides is used along with a test substance, the examples specifically include: a method wherein a non-human animal whose gene function to encode the aforementioned present proteins/peptides is deficient on its chromosome or a non-human animal which over-expresses the present proteins/peptides, as mentioned above, is administered with a test substance and subsequently the Bmal2 activity and changes in the expression levels of the present proteins/peptides in the cells obtained from the non-human animal are measured and assessed; or a method wherein a non-human animal whose gene function to encode the aforementioned present proteins/peptides is deficient on its chromosome or a non-human animal which over-expresses the present proteins/peptides mentioned above is administered with a test substance and subsequently the Bmal2 activity and changes in the expression levels of the present proteins/peptides in the non-human animal are measured and assessed.
An example of a screening method of the present invention for a promoter or a suppressor of the promoter transactivation is a method wherein a test substance and a cell expressing either the present proteins/peptides or the present proteins/peptides along with CLOCK and/or BMAL1 and containing a promoter which has E-box sequence (CACGTG), morespecifically a method in which a test substance is made to contact or introduced into the aforementioned cell and the promoter activity mediated by E-box is then measured and assessed. Another example is a method wherein a test substance is applied to a non-human animal whose gene function to encode the present proteins/peptides is deficient on its chromosome or to a non-human animal which over-expresses the present proteins/peptides to measure and assess the change in the promoter activity mediated by E-box. In addition, it is preferable to have reporter genes or the like, such as chloramphenicol acetyltransferase (CAT) gene or luciferase gene, linked to the downstream of a promoter having E-box sequence (CACGTG), in order to readily analyze the promoter activity.
The present invention also relates to a diagnostic method for diseases associated with the activity or expression of BMAL2 protein wherein the method comprises comparing the DNA sequence encoding BMAL2 protein in a sample with the DNA sequence encoding BMAL2 protein of the present invention. Mutants of DNA encoding BMAL2 protein can be detected by finding individuals with gene mutations at the DNA level, and such detection is effective for diagnosing with diseases developed by underexpression, overexpression or mutated expression of BMAL2 protein. Specific examples of samples used for the detection include cells of a subject, for example, genomic DNA obtainable biopsy of blood, urine, saliva, tissue, etc., or RNA or CDNA. The samples, however, should not be limited to these exemplifications and the amplified products of PCR or the like may also be employed in using the samples. Deletions or insertion mutations of a base sequence can be detected through the changes in size of the amplified products when compared to that of the normal gene type. Point mutation can be identified by hybridizing the amplified DNA with a gene encoding a labeled BMAL2 protein. As described in the above, the circadian rhythm sleep disorders or the like including delayed sleep phase syndrome, non-24-hour sleep-wake syndrome, advanced sleep phase syndrome, time zone change syndrome, shift work sleep disorder, etc. can be diagnosed or judged by detecting mutation in a gene which encodes BMAL2 protein.
The present invention is now further described specifically with reference to the examples, however, the scope of the invention should not be limited to these examples alone.
EXAMPLE 1
Cloning and Sequencing
1-1 (Cloning and sequencing of cClock cDNA)
cClock cDNA was amplified with the chicken pineal cDNA library (λZAPII, 5×105 pfu) as a template by PCR using LA-Taq polymerase (Takara) and a pair of primers [sense primer 1: 5′-ACTAGTCGACTTATGTTTTTTACCATAAGCACC-3′ (Seq. ID No. 21), antisense primer 1: 5′-GTCGACCTGCGCTACTGTGGCTGAGCTTTG-3′ (Seq. ID No. 22); Each of the primers has a SalI site on its 5′-terminal] which were designed on the basis of the sequences of cClock genes deposited in GenBank (GenBank accession nos. AF132531 and AF144425). The above-mentioned PCR method was performed four different times and the sequences of the five clones obtained were determined. One clone with no PCR error was selected (GenBank accession no. AF246959). The program for thermal cycles was as follows: degeneration for 1 min at 94° C. only for the first time, followed by 5 repetitive cycles each consisting of thermal degeneration for 30 sec at 94° C., annealing for 30 sec at 55° C. and extension for 3.5 min at 72° C.; followed by 15 repetitive cycles each consisting of thermal degeneration for 30 sec at 94° C., annealing fur 30 sec at 65° C. and extension for 3.5 min at 72° C.; and finally extension for 6.5 min at 72° C.
1–2 (Cloning and sequencing of cPer2 cDNA)
A 273 bp fragment of cPer2 cDNA was obtained from a chicken pineal cDNA library by PCR using Taq-Gold polymerase (PE applied biosystems) and a pair of degenerate primers [per-F, 5′-CAGCAGAT(C/G)A(A/G)CTG(C/T)IT(C/G)IGACAG(C/T)(A/G)TC(A/C)TCAG-3′ (Seq. ID No. 23) and per-R, 5′-GCT(A/G)CACTG(A/G)CTG(A/G)TG(A/C)(C/G)IGAC(A/G)CCAC(A/G)CTC-3′ (Seq. ID No. 24)] which were designed based on the nucleotide sequences of dPer and mammalian Per genes. A longer cDNA fragment (P2–5; 886bp) was amplified from a chicken pineal cDNA library by the subsequent PCR using cPer2-R1 primer [5′-TTGCTGTACCAGGCACATTACAAC-3′ (Seq. ID No. 25)] synthesized from the base sequence of the above-obtained fragment, a degenerate primer [YK-F1; 5′-(A/G)TICA(C/T)TCIGGITA(C/T)CA(A/G)GCICCI(A/C)GIATICC-3′ (Seq. ID No. 26)] and LA-Taq polymerase. This fragment was used as a hybridization probe for the screening of the chicken pineal cDNA library (λZAPII, 5×105 pfu) to isolate a clone Pa (3584 bp) encoding a larger part of cPER2 (Met1-Arg1014). This clone and the cDNA clone obtained by 3′-RACE were ligated together to generate a full-length clone for cPER2 (Met1-Thr 1344; GenBank accession no. AF246956). The result is shown in FIG. 1 in which the DNA sequence and the amino acid sequence are shown as Seq. ID Nos. 27 and 28 respectively. The bars above the sequence in FIG. 1 indicate the PAS domains (PAS-A and PAS-B) and the cytoplasmic localization domain (CLD). FIG. 2 shows the amino acid homologies in domains between cPER2 obtained as above and three mouse PER proteins (mPER1–3). The programming for thermal cycles of the above was as follows: degeneration for 1 min at 94° C. only for the first time; followed by 35 repetitive cycles each consisting of thermal degeneration for 30 sec at 94° C., annealing for 60 sec at 52° C. and extension for 1 min at 72° C.; and finally extension for 9 min at 72° C.
1–3 (Cloning and sequencing of cBmal cDNA)
cDNA clones encoding part of cBMAL1 or cBMAL2 were respectively obtained from the chicken pineal cDNA library by PCR using LA-Taq polymerase with degenerate primers [BMAL-F, 5′-GTGCT(A/C)(A/C)GGATGGC(A/T)GT(G/T)CAGC-3′ (Seq. IDNO. 29) and BMAL-R, 5′-GCG(C/T)CC(A/G)ATTGC(A/C/G)AC(A/G)AGGCAG-3′ (Seq. ID No. 30)] which were designed based on nucleotide sequences of Bmal1 of mouse, rat and human and dCycle of Drosophila. Each amplified fragment and a cDNA clone of the each amplified fragment obtained by 5′-RACE were used as probes for screening the chicken pineal cDNA library (λZAPII, 3.5×105 pfu) and cDNA clones containing the coding regions for cBMAL1b′ (GenBank accession no. AF246957) and cBMAL2 (GenBank accession no. AF246958) were respectively isolated and sequenced (FIG. 3). The bars above the sequences in FIG. 3 indicate the basic helix-loop-helix region (bHLH) and PAS domains (PAS-A and PAS-B). PCR for the above was performed using a thermal cycler (Perkin-Elmer) as follows: thermal degeneration for 1 min at 94° C. only for the first time; followed by 35 repetitive cycles each consisting of thermal degeneration for 30 sec at 94° C., annealing for 1 min at 50° C. and the extension reaction for 1 min at 72° C.; and finally extension for 9 min at 72° C.
The initiation methionine of cBMAL1b′ was predicted by comparing the cBMAL1b′ sequence mentioned above and the BMAL1 sequences of other animal species. The initiation methionine of the aforementioned cBMAL2 was predicted by the following three criteria; (i) A nonanucleotide sequence (CCGCCATGG), the 97–105 base sequence of cBmal2 shown as Seq. ID No. 9, fully matched the Kozak's translation initiation consensus sequence (Nucleic Acids Res. 12. 857–872, 1984). (ii) The above-mentioned Bmal2 cDNA clone (3.4 kb) and mRNA (3.0, 3.8 kb) were similar in size to each other. (iii) A promoter region predicted from its genomic analysis contained the upstream inframe stop codons.
Next, the amino acid homologies in domains among mBMAL1b′ and three novel BMAL proteins (cBMAL1b′, cBMAL2 and hBMAL2a) were analyzed and a phylogenetic tree of ARNT and BMAL proteins was constructed according to Neighbor-joining method using PHYLIP, v.3.572 as described in the literature (Felsenstein, J., PHYLIP, Version 3.572, University of Washington, Seattle, 1996). These results are respectively shown in FIGS. 4 and 5. In FIG. 5, since amino acids in cBMAL2 in the amino-terminal region (Met1-Arg104) and carboxy-terminal region (Gly459-Leu622) differ in number among animal species, a part corresponding to this region was omitted from each protein before calculating the amino acid homologies among the proteins, and then the phylogenetic tree was constructed. These results demonstrate that cBMAL1b′ is 93% homologous to mBMAL1b′ to show they are close to one another (FIGS. 3 and 4), while cBMAL2 (ARNT4) is not particularly close to BMAL1 (70%; FIG. 5) nor to ARNT1 (41%; FIG. 5) nor to ARNT2 (40%; FIG. 5) and hence that cBMAL2 is a novel protein having bHLH-PAS (FIG. 4).
1–4 (Cloning and sequencing of hBmal2 cDNA)
A partial sequence information of hBmal2 was obtained from two human EST clones (GenBank accession nos. AA577389 and AI218390) by in silico screening using cBmal2 as a probe (data as of October 1999). Several cDNA clones containing the 5′-untranslated region of hBmal2 gene were isolated from cDNA of 293EBNA cell (a human embryonic kidney cell line) by 5′-RACE. Then full-lengthclones were amplified by PCR using hB2F1 and hB2R1primers [hB2F1, 5′-GACCAAGTGGCTCCTGCGAT-3′ (Seq. ID No. 31) and hB2R1, 5′-GCTAGAGGGTCCACTGGATG (Seq. ID No. 32)]. To eliminate PCR errors, 17 full-length cDNA clones obtained were sequenced, and all the DNA sequences encoding hBMAL2a–d (GenBank accession nos. AF246960-AF246963), which were consistent with the human genomic sequences (GenBank accession nos. AC021737 and AC016008), were determined. The programming for the PCR thermal cycles mentioned above was as follows: degeneration for 1 min at 94° C. only for the first time; followed by 20 repetitive cycles each consisting of thermal degeneration for 30 sec at 94° C., annealing for 60 sec at 60° C. and extension for 2 min at 72° C.; and finally extension for 8 min at 72° C. These results are shown in FIGS. 3, 4 and 5. The arrowheads below the sequences in FIG. 3 indicate the insertion sites of introns in hBmal2 gene.
cDNA sequences encoding 4 variants of hBAML2 (hBMAL2a–d) and obtained from 293EBNA cells as described above, were compared with the genome sequences registered at GenBank (accession nos. AC021737 and AC016008). Then the cDNA sequences were divided into 17 exons as in the case of mBmal1 (Biochem. Biophys. Res. Commun. 260, 760–767, 1999) to examine the genomic organization of hBmal2. The results are shown in FIG. 6. Bars with GenBank accession numbers in FIG. 6 represent genomic and CDNA clone regions and shaded parts are the spliced regions in the isolated mutants. These results show that the cDNA clone of hBMAL2b is devoid of Exon 4 (corresponding to Val96-Arg109 in hBMAL2a) and that of hBMAL2c is devoid of both Exons 3 and 4 (corresponding to Gln75-Arg109 in hBMAL2a) and having Exon 1 to which DNA encoding the amino acid sequence of 11 amino acid residues (GEVAGGEATAP) added in-between Gly10 and Gly11 in hBMAL2a is extended. hBMAL2d was revealed to be the shortest mutant which is devoid of both Exon 1 (as in hBMAL2a/b) and Exons 3/4 (as in hBMAL2c) in cDNA.
1–5 (Cloning and sequencing of mBmal2 cDNA)
To identify the mouse Bmal2 ortholog (mBmal2) expressed in the suprachiasmatic nuclei (SCN), a 629 bp fragment CDNA was obtained by RT-PCR for total RNA extracted from the mouse mid-brain, by using LA-Taq polymerase (Takara) and two primers synthesized according to the hBmal2 sequence; hBMAL2-F4: 5′-GTGCTGGTAGTATTGGAACAGATATTG-3′ (Seq. ID No. 33) and hBMAL2-R1: 5′-GCTAGAGGGTCCACTGGATG-3′ (Seq. IDNo. 34). Subsequently, several cDNA clones were isolated which contain 5′- or 3′-untranslated region of mBmal2 cDNA by the method of 5′- and 3′-rapid amplification of cDNA ends. Based on these sequence information, two primers [mBMAL2-F1: 5′-GGTCGACCACCATGGAGTTTTCCAAGGAAACG-3′ (Seq. ID No. 35), mBMAL2-R1: 5′-GCTAGAGTGCCCACTGGATGTCAC-3′ (Seq. ID No. 36)] were designed that were capable of amplifying full-length clones covering the total coding sequence of mBMAL2a or mBMAL2b (FIG. 7; GenBank accession nos. AY005163 and AY014836). Another RT-PCR was performed using these primers and LA-Taq polymerase to obtain mBMAL2a comprising 579 amino acid residues. This amino acid sequence contained bHLH, PAS-A and PAS-B domains and was homologous to hBMAL2 by 74%, cBMAL2 by 63% and zBMAL2 by 48%. On the contrary, mBMAL2b consists of amino acid residues that are about one third of those of mBMAL2a (199 amino acid residues) and is devoid of PAS-B domain (FIG. 7). Although this form of mutation is similar to that previously found in hBMAL1c (a BMAL1 mutant devoid of the C-terminal half in the BMAL1 comprising a long chain; Biochem. Biophys. Res. Commun. 233, 258–264, 1997), its physiological meaning is yet unknown.
1–6 (Cloning and sequencing of rBmal2 cDNA)
Next, cDNA clone of rat Bmal2 (rBmal2) covering almost a total coding region was isolated from the rat early fibroblast rat-1 cells by RT-PCR using two primers [mBMAL2-F1 and mBMAL2-R1]. Three clones isolated, rBMAL2a–c, were determined for the amino acid sequences (FIG. 7; respectively registered to GenBank under GenBank accession nos. AF327071, AY014837, AY014838). The amino acid sequence at the amino-terminal in rBmal2 (corresponding to the position of mBMAL2-F1 primer) was obtained from the in silico screening (GenBank accession no. AA944306). These results demonstrate that rBMAL2a comprising the longest sequence among the clones obtained is most similar to mBMAL2a in its structure. In FIG. 7, dots at the end of the rBMAL2 sequence indicate the position corresponding to mBMAL2-R1, a PCR primer. The asterisk indicates the position of the in-frame stop codon of mBMAL2b and the number at the end of each line (with “+” on the right shoulder) indicates the number of amino acid residues for rBMAL2a.
Next, the phylogenetic tree for the BMAL-ARNT family was constructed according to the amino acid homologies among various proteins (FIG. 8). Before constructing the phylogenetic tree, several amino acid sequences of BMAL-ARNT proteins that were obtained from GenBank were aligned with Gene Works (Ver.2.55, clustal V), then some regions with gaps were omitted. Since the length of amino acids in amino- and carboxyl-terminal regions (corresponding to the 1–59 amino acid sequence and the 413–579 amino acid sequence of mBMAL2a) differ among mutants, these regions were also omitted. Then the Neighbor-joining tree was constructed using a PHYLIP 3.572 software package (Felsenstein, J., PHYLIP, Version 3.572, University of Washington, Seattle, 1996) (FIG. 8), and the topology of the phylogenetic tree obtained as above was analyzed by PROTML 2.3 program which adopts a local rearrangement method for the maximum likelihood analysis and JTT-F model for the amino acid substitution (Adachi, J. and Hasegawa, M., MOLPHY: Programs for molecular phylogenetic based on maximum likelihood, Version 2.3, Institute of Statistical Mathematics, Tokyo, 1996). Further, in order to assess the reliability of that phylogenetic tree, a boot strap test was carried out and the boot strap probabilities of over 70% were respectively shown near the diversion points in FIG. 8. The diversion points shown by closed circles indicate the divergence of species and those shown by open circles indicate gene duplications in FIG. 8.
When the above result is taken into account together with the fact that there is only a single copy of dCyc gene, a Bmal1/2-like gene, in the Drosophila genome, Bmall and Bmal2 genes are likely to be generated from the gene duplication occurred in their ancestral vertebrates (Diversion point b in FIG. 8). Besides, branches at the divergence among the members in the BMAL2 cluster are much longer than those of BMAL1, meaning that the phylogenetic tree topology in the BMAL2 cluster reflects the phylogenetic development of vertebrates. It can therefore be concluded from these facts that these Bmal2 genes are in orthologous relationships with each other and have developed from a highly frequent amino acid substitution. This conclusion can also be supported by the fact that no m/r/c/z Bmal2 orthologs other than hBmal2 can be found in the human gene data base (the htgs database was searched on 9th Dec., 2000). Diversion point a in FIG. 8 probably indicates divergence between ancestors of vertebrates and arthropods and diversion points c–f indicate divergence among vertebrate species. Besides, the above-mentioned phylogenetic tree had the same topology as phylogenetic trees obtained by Parsimony and Neighbor-joining methods.
Comparison of substitution rates in amino acids among the members of BMAL1/2 clusters revealed that the amino acid substation rate of BMAL2 is higher than that of BMAL1 by about 20-fold. This shows that the selective pressure in BMAL2 after gene duplication is lower than that in BMAL1. What is important is that there is no any specific region in which the total amino acid homology among BMAL2 proteins is decreased. Highly conserved structure of BMAL1 protein which has a higher selective pressure is thought to include some unrecognized function which has been lost in BMAL2. BMAL1 is thought to interact with several essential regulatory factors that have not yet been characterized, because both BMAL proteins interact with CLOCK which is a functional heterodimer partner with BMAL proteins (Science 280, 1564–1569, 1998, Proc. Natl. Acad. Sci. USA 97, 4339–4344, 2000, J. Neurosci. 20, RC83, 2000, J. Biol. Chem. 275, 36847–36851, 2000, Proc. Natl. Acad. Sci. USA 95, 5474–5479, 1998, Biochem. Biophys. Res. Commun. 248, 789–794, 1998), with a neuron PAS domain protein 2 (NPAS2 or MOP4) (J. Neurosci. 20, RC83, 2000, Proc. Natl. Acad. Sci. USA 95, 5474–5479, 1998), with a hypoxia-inducing factor 1α(HIF1α) (J. Neurosci. 20, RC83, 2000, Proc. Natl. Acad. Sci. USA 95, 5474–5479, 1998, Biochem. Biophys. Res. Commun. 248, 789–794, 1998),or with HIF2α(HLF or EPAS1) and with the like. Therefore, analyzing the differences between the functions of BMAL1 and BMAL2 is thought to contribute to uncover their unique evolution processes.
EXAMPLE 2
Northern Blot Analysis
Total RNA (7.5 μg) of each tissue from one-week-old chicks (pineal gland, retina, cerebrum, heart, kidney and skeletal muscle) was analyzed by Northern blotting in a manner as described in J. Neurochem. 70, 908–913, 1998. These tissues were harvested at 0, 6, 12 and 18 hr in Zeitgeber time (ZT), frozen with liquid nitrogen and mixed before extracting RNA. Each of total RNA was separated by an agarose gel electrophoresis and blotted on a nitrocellulose membrane. The blotting membrane was hybridized with a cBmal1 probe or a cBmal2 probe and washed (10 min×3 times) in 0.1×SSC at 50° C., then analyzed using a FLA2000 bioimage analyzer (FUJI PHOTO FILM). The membrane was subsequently hybridized with a chicken histone H4cDNA probe and analyzed. The chicken histone H4cDNA probe used was prepared by amplification by PCR with a primer [sense primer 2; 5′-CATGTCTGGCAGAGGCAAG-3′ (Seq. ID No. 37) and antisenseprimer2; 5′-TTAGCCGCCGAAGCCGTAG-3′ (Seq. ID No.38)], which was designed from the chicken pineal cDNA based on the sequence (accession no. M74533) deposited in GenBank, and by the subsequent cloning. The results are shown in FIG. 9. These results demonstrate that two cBmal2 genes (3.8 Kb and 3.0 Kb, indicated by arrows) and cBmal1 gene (3.3 Kb) are expressed in all the tissues examined at various intensities. It was confirmed as a result of normalization to histone H4 that heart and kidney exhibited low transcriptional levels of cBmal1 and that no apparent difference was observed in the transcriptional levels of cBmal2 among the tissues examined.
EXAMPLE 3
Expression of Chicken Clock Genes in the Pineal Gland
One-day-old chicks were entrained to LD cycle (12 hr with light/12 hr in the dark) for 3 weeks, then placed in DD (constant darkness) condition for 2 days, and the pineal glands were collected every 4 hr over the last 3 days. Total RNA from each pineal gland was analyzed by Northern blotting to detect expression of chicken Clock genes (cBmal1, cBmal2, cPer2 and cClock) in the pineal gland. Total RNA (6 μg) obtained from each pineal gland mentioned above was separated by an agarose gel electrophoresis, blotted on a nitrocellulose membrane. Two such blotting membranes were prepared. A blot was first hybridized with a cBmal2 probe or a cPer2 probe and the blotting membrane was washed in 0.1×SSC at 50° C. (10 min×3 times), which was then analyzed using a FLA2000 bioimage analyzer (FUJI PHOTO FILM). Next, the blot was hybridized with the histone H4cDNA and analyzed in the same way. The aforementioned cDNA fragment P2–5wasusedas the cPer2 probe. For another blotting membrane, the blot was first hybridized with a cBmal1 probe as in the above, then with a histone H4cDNA and finally with a cClock probe, and was analyzed with a FLA2000 bioimage analyzer. These results are shown in FIG. 10 (bottom lane). Signals for cBmal1 (open circles) and cBmal2 (closed circles) were quantified by MacBAS software (FUJI PHOTO FILM), normalized to those for the histone H4 cDNA, and the mean value was set as 1 in each case to analyze the time-course changes in transcriptional levels of the chicken Clock genes. The results are shown in FIG. 10 (top lane). A cross bar above the Northern blotting results in FIG. 10 indicates light and bright cycles. An open region indicates a light cycle, closed regions indicate (subjective) dark cycles and shaded regions indicate subjective light cycles. Three cPer2 transcripts (9.7 Kb, 7.5 Kb and 4.1 Kb) and a single cClock transcript (8.5 Kb) were confirmed by these results.
EXAMPLE 4
Expression of Chicken Clock Genes in the Pineal Cell Culture
The time course changes in the transcription amounts of chicken clock genes [cBmal1 (open circle), cBmal2 (closed circle), cPer2 (open triangle) and cClock (open square)] in the pineal cell culture were analyzed by a quantitative RT-PCR method and the results were compared to those in Example 3 above (FIG. 11). Pineal cells from one-day-old chicks were plated on 35 mm dishes (cells from 8 pineal glands per a dish) and cultured for 5 days under LD cycles in Medium 199 (Life Technologies) supplemented with 10% fetal bovine serum. On day 6, part of the cultured cells was shifted to culture under constant darkness (DD, right in FIG. 11). The rest of the cultured cells remained in the culture under the LD condition and subjected to a further culture on day 7 under constant darkness (LD, left in FIG. 11). Then each pineal cell was harvested every 4 hours. The pineal cells harvested were suspended in TRIzol reagent (Life Technologies) and stored at −80° C. until total RNA was isolated. 1 μg each of the total RNA was reverse-transcribed by the SuperScriptII (Life Technologies) reverse transcriptase and a portion of the reaction product was used for PCR analysis. First, an optimal number of PCR cycle was determined for each primer to give linear relationships between the amounts of the template cDNA and amplification products and PCR was carried out under such condition. The PCR products obtained were separated by a 7.5% polyacrylamide gel electrophoresis, stained with SYBR Green I (Molecular Probes), and the transcriptional level of each chicken clock gene was quantified with a FLA2000 bioimage analyzer (FUJI PHOTO FILM). Change in the transcriptional level of GAPDH, as a control, was measured in a similar manner as the above. Intensity of each signal was normalized to that of GAPDH, and the mean value for each gene on day 6 was set to 1. Then all the values (mRNA levels) were obtained from three different culture samples, which were shown by mean±SEM.
The primers and number of PCR cycles mentioned above were set up as follows. For cBmal1, cB1F1600 primer; 5′-TCCAGACATTTCTTCAGCTGG-3′ (Seq. ID No. 39) and cBIREND-primer; 5′-GGATGTTGAAGCAAGGTGC-3′ (Seq. ID No. 40) were used and 23 cycles were practiced. For cBmal2, cB2F1270-primer; 5′-ACGAGTACTGCCATCAAGATG-3′ (Seq. ID No. 41) and cB2REND-primer; 5′-GAGAGCCCATTGGATGTCAC-3′ (Seq. ID No. 42) were used and 23 cycles were practiced. For cClock, cqCF862-primer; 5′-TTCTTGGATCACAGGGCAC-3′ (Seq. ID No. 43) and cqCR1364-primer; 5′-GGAGTGCTAGTGTCCACTGTCA-3′ (Seq. ID No. 44) were used and 25 cycles were practiced. For cPer2, cP2RTF primer; 5′-GGAAGTCCTTGCAGTGCATAC-3′ (Seq. ID No. 45) and cP2RTR-primer; 5′-ACAGGAAGCGGATATGCAG-3′ (Seq. ID No. 46) were used and 24 cycles were practiced. For GAPDH (GenBank accession no. K01458), cGAF-primer; 5′-ACCACTGTCCATGCCATCAC-3′ (Seq. ID No. 47) and cGAR-primer; 5′-TCCACAACACGGTTGCTGTA-3′ (Seq. ID No. 48) were used and 15 cycles were practiced. Taq-Gold was used as polymerase. The program of PCR thermal cycler for each clock gene was as follows: degeneration for 9 min at 95° C. only for the first time; followed by repetitive cycles each consisting of thermal degeneration for 15 sec at 94° C., annealing for 30 sec at 55° C. and extension for 30 sec at 72° C.; and finally the extension reaction for 7 min at 72° C.
FIG. 11 shows the results of the above. It was confirmed by the result that all four kinds of transcripts that were expressed in the chick pineal cells displayed daily fluctuations in abundance with diverged phases and amplitudes in LD cycles and under DD condition. The fluctuation profiles in vivo in Example 3 (FIG. 10) are very similar to those in vitro in Example 4 (FIG. 11), where the cPer2 mRNA levels had a peak early in the morning and a trough early at night. This result was similar to the fluctuation profile of mPer1 in the mouse SCN (Cell 90, 1003–1011, 1997, Nature 389, 512–516, 1997). A high level expression of cPer2 sustained at the early light phase (Zeitgeber time (ZT) 2–6) under LD condition, as compared with a rapid decline in cPer2 expression at circadian time (CT) 2–6 under DD condition, indicated that the pineal photoreception plays a role in keeping the high level expression of cPer2 in the morning. The mRNA levels of cBmal1 and cBmal2 also exhibited clear oscillations and their phases were opposite to that of cPer2 (FIG. 11). Peak time in the cBmal2 mRNA level was delayed by about 4 hr compared to that in the in vitro cBmal1 mRNA level. This tendency was also observed in the in vivo fluctuation profile. In contrast, the cClock mRNA level showed a relatively low amplitude with a broad peak at ZT 10–18 or CT 10–18, and the peak seems to cover the peaks in expression levels of the two Bmal genes. A similar oscillation of cClock mRNA is observed in the chicken retina (Mol. Brain Res. 70, 253–263, 1999).
EXAMPLE 5
Expression of the Mouse Clock Genes in the Suprachiasmatic Nuclei
mRNA levels of mBmal2 and known clock genes (mPer2, mClock and mBmal1) of the mouse suprachiasmatic nuclei under LD cycles were studied as follows. 5-week-old male C57BL/6 mice were subjected to LD cycles at 23° C.±1° C. (about 200 lux of bright cycle under a fluorescent lamp) and bred with free access to feed and water. 3 weeks thereafter, the mice were decapitated and the brains were rapidly isolated, frozen, and sectioned into thin strips with 700 μm thickness. Small tissue sections including SCN on both sides were taken out from the sections by using a 20-gauge needle, and the mRNA expression levels in mBmal2, mPer2, mClock, mBmal1, etc. in the tissue sections were quantified by a quantitative RT-PCR. Three independent RNA samples prepared from six mice (n=3) were respectively quantified and each signal intensity thus obtained was normalized to the signals for mGAPDH and the mean of the three values (mean±SEM) were calculated. p values in FIG. 12 were determined by using Student's t test.
The above-mentioned primers and number of PCR cycle were determined to give linear relationships between the amounts of the template cDNA and amplification products. For mBmal2, mBMAL2-F2 primer; 5′-TGGTTGGATGCGAAAGAGG-3′ (Seq. ID No. 49) and mBMAL2-R4 primer; 5′-AGGTTTCTCTCTTGGTGAACC-3′ (Seq. ID No. 50) were used and 28 cycles were practiced. For mBmal1 (GenBank accession no. AB012600), rmBmal1-F1 primer; 5′-TGGTACCAACATGCAATGC-3′ (Seq. ID No. 51) and rmBmal1-R1 primer; 5′-AGTGTCCGAGGAAGATAGCTG-3′ (Seq. ID No. 52) are used and 28 cycles were practiced. For mPer2 (GenBank accession no. AB016532), rmPer2-F1 primer; 5′-GCTCACTGCCAGAACTATCTCC-3′ (Seq. ID No. 53) and rmPer2-R1 primer; 5′-CCTCTAGCTGAAGCAGGTTAAG-3′ (Seq. ID No. 54) are used and 30 cycles were practiced. For mClock (GenBank accession no. AB019258), rmClock-F1 primer; 5′-CAAGGTCAGCAACTTGTGACC-3′ (Seq. ID No. 55) and rmClock-R1 primer; 5′-AGGATGAGCTGTGTCGAAGG-3′ (Seq. ID No. 56) were used and 28 cycles were practiced. For mGAPDH (GenBank accession no. X02231), rmGAPDH-F1 primer; 5′-CATCACCATCTTCCAGGAGC-3′ (Seq. ID No. 57) and rmGAPDH-R1 primer; 5′-ATTGAGAGCAATGCCAGCC-3′ (Seq. ID No. 58) were used and 21 cycles were practiced. Programming for the PCR thermal cycler for each clock gene was carried out under the condition described in Example 4.
The results of the above are shown in FIG. 12. In these results, the mPer2 mRNA level displayed daily fluctuations in abundance in the SCN region (FIG. 12A) as are reported in the literatures (Genes Cell 3, 167–176, 1998, Science 288, 1013–1019, 2000). Besides, the mBmall mRNA level showeda faint oscillation in almost antipahse to mPer2 which is in LD cycles (FIG. 12C). On the contrary, mRNA level of mBmal2 was almost constant all day long which was similar to the case of mClock (FIG. 12B, D), suggesting the difference in transcriptional regulation between mBmal1 and mBmal2 genes.
EXAMPLE 6
Changes in the Photo-Dependency of mRNA Levels in cPer2, cBmal1 and cBmal2 in the Chick Pineal Glands
Since the expression level of cBmal1/2 in the early morning was low (FIG. 12), a possible light-dependent down-regulation of cBmal1/2 transcriptions was tested. Chicks were exposed to light for a time period when both cBmal1/2 expression levels were high in the dark (CT14-CT15), as is seen from the results of Example 4, and changes in mRNA levels were evaluated at CT15.5 and CT17. One-day-old chicks were entrained to LD cycle for a week and then placed in DD condition. The chick pineal glands that were exposed to a 1-hr light-pulse (350 lux) (CT14–CT15) on the first day of DD condition (FIG. 13A, below) and the chick pineal glands without exposure to light-pulse (FIG. 13A, top) were respectively isolated at CT15.5 or CT17 and the total RNA (8 μg) obtained from each of the pineal glands were respectively separated by an agarose gel electrophoresis and blotted onto a nitrocellulose membrane.
Each blotting membrane as aforementioned was cut into two pieces and one (containing RNA longer than 2.4 Kb) was hybridized with a cBmal1, cBmal2 or cPer2 probe and another with a histone H4 probe. Then the signals for cBmal1 (FIG. 13B), cBmal2 (FIG. 13C), cPer2 (FIG. 13D) and histone H4 were quantified by MacBAS software (FUJI PHOTO FILM) and the intensity of all the signals were normalized to those for histone H4. The mean value of each gene at CT14 was set to 1 and the mRNA levels were determined. The values were determined from triplicate experiments practiced in a similar way as in the above and shown as mean±SEM. FIG. 13 shows the results. In FIG. 13, “an asterisk” and “double asterisks” mean p <0.05 and p <0.02, respectively. p values were determined using Student's t test. These results demonstrate that mRNA levels of cBmal1 and cBmal2 observed in the pineal glands of chicks exposed to light at CT15.5 were substantially lower than those of the control animals without exposure to light. On the contrary, the light-induced cPer2 expression was confirmed at CT17, two hours after the exposure to light, as was observed for mPer1 and mPer2 in the SCN of the mice exposed to light (Cell 91, 1055–1064, 1997, Neuron 19, 1261–1269, 1997, Genes Cells 3, 167–176, 1998).
EXAMPLE 7
Functional Property of cBMAL2; Pull-Down Assay
A close kinship between BMAL1 and BMAL2 among ARNT-(aryl hydrocarbon receptor nuclear translocator) related proteins (FIG. 5) seems to indicate their functional similarity. Therefore, relationships among cBMAL1, cBMAL2 and cCLOCK were tested by a glutathione-S-transferase (GST) pull-down assay using three kinds of bacterially expressed GST-fusion proteins [GST-cCLOCKΔ (a fusion of GST and Met1-Ser466cCLOCK truncated at the carboxy-terminal region), GST-cBMAL1 and GST-cBMAL2], together with [35S]-labeled cBMAL1Δ (Met1-Ser449) or [35S]-labeled cBMAL2Δ (Met1-Leu458) that were transcribed and translated in vitro. Because GST-cCLOCK (a fusion protein composed of GST and the full-length cCLOCK) was not solubilized by 2% Triton X-100, GST-cCLOCKA mentioned above was used instead.
A DNA fragment encoding GST-cCLOCKΔ, GST-BMAL1, GST-BMAL2 or GST, mentioned above, was introduced into a pGEX5X-1 expression vector and expressed in BL21 E. coli strain. Each E. coli was subjected to lysis in buffer A [10 mM Na-phosphate (pH 7.9), 140 mM NaCl, 1 mM MgCl2, 10 mM EDTA, 5 mM 2-mercaptoethanol, 2 mM PMSF and one tablet of Complete EDTA-free protease inhibitor (Roche Diagnostics) per 50 mL], then each of solubilized fusion proteins or GST was purified by glutathione-Sepharose column (Amersham Pharmacia Biotech). On the other hand, [35S]-labeled cBMAL1Δ (Met1-Ser449) and [35S]-labeled cBMAL2Δ (Met1-Leu458) mentioned above were prepared by the in vitro transcription and translation of an expression plasmid containing CDNA fragment of cBMAL1Δ (Met1-Ser449) or cBMAL2Δ (Met1-Leu458) in the presence of [35S] methionine and with the aid of TNT-T7 Quick Coupled Transcription/Translation System (Promega). [35S]-labeled luciferase as a control was similarly transcribed and translated in vitro.
8 μL each of the [35S]-labeled protein (cBMAL1Δ, cBMAL2Δ or luciferase protein) solutions was mixed with 40 μL of glutathione-sepharose beads, to which GST-cCLOCKA (0.1 μg), GST-cBMAL1 (1.1 μg), GST-cBMAL2 (3.3 μg) or GST (5.6 μg) had been bound. Then the mixtures were incubated in 140 μL of buffer B [20 mM Hepes-NaOH (pH 7.9), 20% (w/v) glycerol, 15 mM KCl, 0.2% Triton X-100, 2.5% skim milk, one tablet of Complete EDTA-free protease inhibitor per 50 mL] on ice for 1 hr with gentle rotation. After the incubation the mixtures were washed four times with buffer C [10 mM Tris-HCl (pH 7.5), 0.2% Triton X-100, 150 mM NaCl, 2 mM EDTA, 1 mM PMSF, one tablet of Complete EDTA-free protease inhibitor] and were separated by a SDS-polyacrylamide (10%) gel electrophoresis, then the gel was analyzed for autoradiograph by using a FLA2000 bioimage analyzer (FUJI PHOTO FILM).
The results of the above are shown in FIG. 14. Lanes 16–18 is the results of electrophoresis for [35S]-labeled cBMAL1Δ, cBMAL2Δ or luciferase (2.5% each of the inputs). A faint signal observed in lane 17 (the upper band) is due to the migration of luciferase from lane 18. These results revealed that GST-cCLOCKΔ specifically bound not only with cBMAL1Δ but also with cBMAL2Δ in vitro (FIG. 14, lanes 1, 2). Interestingly, GST-cBMAL2 bound with both cBMAL proteins (FIG. 14, lanes 4, 5), and GST-cBMAL1 also showed similar binding profiles (FIG. 14, lanes 7, 8), indicating potential activity of cBMAL proteins to form a homodimer as well as a cBMAL1-cBMAL2 heterodimer. It was also demonstrated that a CBMAL protein deficient in the C-terminal bound more efficiently with a GST-fusion protein than with a full-length CBMAL protein.
EXAMPLE 8
an Electrophoretic Mobility Shift Assay Using a cPer2 E-box-Containing Probe
A binding of cBMAL1-cCLOCK or cBMAL2-cCLOCK to the E-box sequence was examined by an electrophoretic mobility shift assay (EMSA) in which an E-box (CACGTG)-containing sequence present in a promoter region of cPer2 gene was used as a probe. For preparation of the probe, oligonucleotides [cP2E1-S: 5′-GTGTCACACGTGAGGCTTA-3′ (Seq. ID No. 59) and cP2E1-AS: 5′-TAAGCCTCACGTGTGACAC-3′ (Seq. ID No. 60)] were synthesized that correspond to the E-box sequence and its flanking sequences within a putative promoter/enhancer region of cPer2 gene. These oligonucleotides synthesized were annealed together and subcloned into a pCR2.1 vector using TOPO-TA cloning kit (Invitrogen, Calif.), from which a 39 bp fragment was excised with a restriction enzyme EcoRI and used. The above-mentioned cBMAL1, cBMAL2 and cCLOCK were prepared by being transcribed and translated in vitro from an expression plasmid containing the cDNA of cBmal1, cBmal2 or cClock with the aid of TNT-T7 Quick Coupled Transcription/Translation System (Promega). A pcDNA3.1/V5/His empty vector, an expression vector, alone was transcribed and translated similarly as in the above and used as a control.
5 μL each of the protein mixtures thus obtained (BMAL1+BMAL2, BMAL1+CLOCK, BMAL2+CLOCK) was added with 32 μL of buffer [25 mM Hepes-KOH (pH 7.6), 100 mM KCl, 0.1 mM EDTA, 10% (v/v) glycerol, 7.5 mM MgCl2, 1 mM DTT and 1 μg denatured salmon sperm DNA] containing a 32P-labeled probe (33 fmoles, 1.3×105 cpm) andwas incubated for 20 min at 23° C. After the incubation, each mixture was separated by a 6% polyacrylamide gel electrophoresis and analyzed similarly as in Example 7 using a FLA2000 bioimage analyzer (FUJI PHOTO FILM). FIG. 15 shows the results. In FIG. 15, lane 1 is the result of the labeled probe alone, lanes 2–5 are the results of the reactions between each translation product (control, BMAL1, BMAL2 or CLOCK) and the labeled probe. In the figure, the asterisk denotes the position of the free probe, closed arrowheads represent specific complexes with the bHLH-PAS proteins, and open arrowheads indicate background. It was confirmed from these results that in the presence of cCLOCK, cBMAL2 and cBMAL1 had respectively formed two or three complexes (closed arrowheads in lanes 7 and 8 in FIG. 15). It is unlikely that these complexes represent homodimers of any of the PAS proteins examined (cCLOCK, cBMAL1 or cBMAL2), because no specific bands were observed when cCLOCK, cBMAL1 or cBMAL2 alone was reacted with the probe (lanes 3–6 in FIG. 15). These results suggest that the cPer2 E-box is one of the in vivo targets of cCLOCK-cBMAL1/2 heteromer.
EXAMPLE 9
Transcriptional Regulation by cBMAL1, cBMAL2 and cCLOCK in 293EBNA Cells
Abilities for the transcriptional activation and suppression of cBMAL1, cBMAL2 and cCLOCK were tested with the mPer2 E-box or the mPer1 promoter as a role model in the feed-back-loop and the vasopressin gene E-box as a role model in output pathways. Human embryonic kidney 293EBNA cells (Invitrogen) were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Life Technologies) which cultured cells were then plated at 3+105 cells per well on six-well plates and transfected by using a total of 1.0 μg of various expression plasmids [an expression vector, plasmids containing reporter genes, 0.25 ng of Renilla luciferase reporter (pRL-CMV; Promega), and plasmids containing cDNAof each clock gene (cBmal1, cBmal2, cClock) with the amount indicated in FIG. 16] together with Lipofectamine plus (Life Technologies).
As for the expression vector mentioned above, pcDNA3.1/V5/His empty vector (Invitrogen) was used. As for the reporter genes mentioned above, 25 ng of the firefly luciferase reporter (cPer2 E-box-luc; a derivative of pGL3-Promoter; Promega) containing mPer2 E-box, 25 ng of cPer2 mut.E-box-luc, 50 ng of the firefly luciferase reporter containing mPer1 promoter (mPer1-luc; a derivative of pGL3-Basic; Promega) 25 ng of the firefly luciferase reporter containing the mouse vasopressin E-box (AVP E-box-luc; a derivative of pGL3-Promoter; Promega), 25 ng of AVP mut. E-box-luc, or 25 ng of TRE-luc were used. Two days after the transfection, cell extracts were subjected to dual-luciferase assays by luminometry (Promega) according to the manufacturer's protocol. For each extract, the firefly luciferase activity was normalized by the Renilla luciferase activity and the mean value (means±SEM) was determined from the values of three independent culture extracts.
The aforementioned plasmids containing reporter genes were prepared as follows. The E-box sequence, CACGTG and its flanking sequences within the promoter/enhancer region of cPer2 gene were linked in tandem (5′-GTGTCACACGTGAGGCTTAGTGTCACACGTGAGGCTTAGTGTCACACGTGAGGCTTA-3′), which was then inserted into a luciferase reporter containing SV-40 (pGL3-Promoter, Promega) and thus the cPer2 E-box-luc was constructed. The cPer2 mut.E-box as a reporter plasmid in the control experiment was constructed by mutating the E-box sequences into GGACCT in a similar way as previously reported (Cell 96, 57–68, 1999). mper1-luc was constructed as follows; a 2.2 Kb upstream fragment of mPer1 was amplified by PCR using the DNA templates from the mouse genome [sense primer 3; 5′-TCGAGCTCTTTGGTACCTGGCCAGCAACC-3′ (Seq. ID No. 61) and anti-senseprimer3; 5′-TCACGACACCTGGCCGTTCGAGG-3′ (Seq. IDNo. 62)] and LA-Taq polymerase, base sequences for the six clones individually obtained by PCR were determined, and then one clone without PCR error among the six clones was linked to a luciferase reporter (pGL3-Basic, Promega). AVP E-box-luc was constructed by linking E-box sequence (CACGTG) in the promoter/enhancer region in the mouse vasopressin gene and its flanking sequences, and then by inserting the resulting sequence (5′-TCAGGCCCACGTGTCCCA-3′) into the luciferase reporter containing SV-40 promoter (pGL3-Promoter, Promega). Further, the AVP mut. E-box-luc (a reporter with a E-box mutation) which is a reporter plasmid for the control experiment was prepared in a way previously described (Cell 96, 57–68, 1999). TRE-luc was prepared as follows; the phorbol ester-responsive element (TRE) and its flanking sequences within human collagenase gene were linked in tandem [5′-CGGCTGACTCATCAAGCTGACTCATCAAGCTGACTCATCAA-3′ (Seq. ID No. 63)], which was then inserted into the BglII site in a luciferase reporter in which a BglII-HindIII fragment of pRL-TK vector (Promega) was ligated to a pGL3-Basic vector (pGL3-TK-promoter vector).
These results are shown in FIG. 16. The results show that cCLOCK binds to not only cBMAL1 but cBMAL2 and promotes the transactivation which is medidted by the cPer2 E-box (FIG. 16A). Similar results were obtained by using a 2.2-kb mPer1 promoter harboring three E-box sequences (CACGTG) (FIG. 16B). Interestingly, the transactivation elicited by cBMAL2-cCLOCK showed a clear peak when a relatively low dose (20 ng) of a cBmal2 expression plasmid and cClock plasmid (250 ng) were coexpressed, and a higher dose than the above of cBmal2 plasmid suppressed the transactivation in FIG. 16B (see the left of the figure) and FIG. 16C (see 10th-16th bars from the left of the figure). cBmal1, however, seems to have no such inhibitory effect. Endogenous transactivation neither from the TPA-responsive element (TRE, FIG. 16B) nor from the SV40-promoter was suppressed by application of a high dose (160 ng) of cBmal2, which fact suggests that the suppression is due to the specific effect on E-box or E-box-binding component(s).
Since cBmal1 and cBmal2 had the slightly shifted expression profiles as can be seen in FIGS. 10 and 11, a cooperative effect of cBMAL1 and cBMAL2 on the transcriptional regulation was tested. In the case of a vasopressin gene E-box as a reporter (FIG. 16C), a low level expression (10ng) of cBmal2 notably enhanced cBMAL1-cCLOCK transactivation (see 17th–23rd bars from the left in FIG. 16C). A similar or more pronounced cooperative effect was observed with a low dose of cBmal1 plasmid (10 ng) for cBMAL2-cCLOCK transactivation (see 24th–30th bars from the left in FIG. 16C). Besides, the cooperative activation was considerably suppressed by the application of larger amounts of cBmal2 (80–160 ng) or cBmal1 (40–160 ng). Similar results were also observed in the cases when a cPer2 E-box or a mPer1 promoter was used, albeit with less degrees (FIG. 16B).
EXAMPLE 10
Effect of cPER2 on Transactivation Mediated by E-box Sequences
Next, whether cPER2 negatively acted on the transactivation elicited by the transactivator cBMAL-cCLOCK was examined. The experiment described in this Example 10 was performed in a similar way as in Example 9 except that plasmids containing a cPer2 cDNA were transfected, with the amounts shown in FIGS. 17A and 17B, to the expression plasmids which were to be transfected to the human embryonic kidney293EBNA cells. The results are shown in FIGS. 17A and 17B. The results show that coexpression of cPer2 plasmid (250 ng) in 293EBNA cells inhibited the cBMAL2-cCLOCK-dependent transactivation mediated by cPer2 E-box, and the degree of the inhibitory effect was stronger than that on cBMAL1-cCLOCK-dependent transactivation under the same conditions (FIG. 17A). Similar tendency was also observed in the case of cBMAL-cCLOCK-dependent transactivation mediated by the vasopressin E-box (FIG. 17B), and the higher degree of inhibitory effect was observed with the increase in the cPER2 amount.
Then, intrinsic properties of the cPer2 E-box mediated transactivation were studied in the cultured chick pineal cells. The pineal cells prepared from one-day-old chicks were plated at 4×105 cells per well on 24-well plates and cultured under LD cycle. At ZT9 on Day 3 of the culture, the pineal cells were transfected with 500 ng of either the aforementioned cPer2 expression plasmid or pcDNA3.1/V5/His (control), 250 ng of either the cPer2 E-box-luc or the cPer2 mut.E-box-luc, and 5 ng of pRL-CMV (Promega) by using Lipofectamine plus. At ZT6 on the next day of the transfection, the cell extracts were subjected to a dual-luciferase assay and the results are shown in FIG. 17C. The results demonstrated that the endogenous transactivation mediated by cPer2 E-box was markedly decreased as a result of mutating the E-box sequence and that the inhibitory effect on transactivation induced by forced expression of cPER2 was also E-box-dependent. These facts suggest that the chicken pineal cells express a positive factor acting on the cPer2 E-box and that this factor exhibits an effect on the negative regulation by cPER2.
EXAMPLE 11
Ablation of Melatonin Rhythm by the Overexpression of cBMAL1 and cBMAL2
cBMAL1 or cBMAL2 was overexpressed in the cultured chick pineal cells and its effect on the melatonin rhythm was examined to evaluate the roles of the two PAS proteins in maintenance of the rhythmicity. The chick pineal cells were cultured in 24-well cloning plates (Greiner Labortechnik, Frickenhausen, Germany) for 2 days and transfected with 500 ng of either cBMAL1 or cBMAL2 expression plasmid mentioned above or pcDNA3. 1/V5/His (control) by using a combination of Lipofectamine plus (Life Technologies) and Genefector (VennNova LLc, FL). 2 days after the transfection, the cells were subjected to a 4-day culture in the media containing 200 mg/L G418 (Life Technologies) to select the transfected cells and the cells selected were further cultured in the media containing 50 mg/L G418. The culture media were collected every 4 hours to quantify the released melatonin by the previously described method (Neurosci 20, 986–991, 2000). FIG. 18 shows the results. Four data in each panel are the results obtained from the individual cultures where each value was determined by setting the average of melatonin production levels during the LD cycles to 1. The bar at the bottom of FIG. 18 represents lighting conditions.
A slight phase-delaying was observed upon studying the melatonin rhythm in the pineal gland of each cell. This change was also observed in the untransfected pineal cells, and such clock oscillation was also observed after culturing control cells (FIG. 9A) and cells overexpressing proteins unrelated to clock proteins such as a ml or m2 acetylcholine receptor, under DD condition for several days. In contrast to these control cells, cBMAL1- or cBMAL2-overexpressing cells displayed only a single oscillation in melatonin production under DD condition, which was thereafter kept at a constant level (FIGS. 18B and 18C). Under the LD cycles, daily melatonin fluctuations in cBMAL1- or cBMAL2-overexpressing cells were quite similar to those of control cells, indicating that cellular mechanisms for light-dependent melatonin production were stably maintained by the overexpressed cBMAL proteins. In spite of this, the ablation of rhythm under DD condition strongly suggests that cBMAL1 and cBMAL2 are both indispensable factors for rhythmic oscillation.
INDUSTRIAL APPLICABILITY
The present invention makes it possible to provide novel clock proteins having the novel BMAL2 activity crucial for the clock oscillation mechanism including photic-input pathway and output pathway, and the gene DNAs encoding the proteins. Further, with the use of these proteins and the gene DNAs, substances useful for prevention and therapy of the circadian rhythm sleep disorders or the like including delayed sleep phase syndrome, non-24-hour sleep-wake syndrome, advanced sleep phase syndrome, time zone change syndrome, shift work sleep disorder, etc. can be screened, in addition to which a molecular mechanism of the circadian oscillation system can also be elucidated. Still further, the proteins of the present invention having the BMAL2 activity have functions both for promoting and suppressing transcription and are thought to be involved in diverse biological functions by binding with partners other than CLOCK. The proteins are therefore expected to be applied to specifically inhibit a group of functions in the transcriptional regulatory regions including that of period genes by gene-introduction of BMAL2 or the BMAL2-dominant negative mutants in an excessive amount from the outside.

## Claims

1. DNA encoding a protein comprising an amino acid sequence shown by Seq. ID No. 10.

2. DNA containing a base sequence shown by Seq. ID No. 9 or its complementary sequence.

3. DNA which hybridizes with DNA of claim 2 under a stringent condition comprising hybridization at 65° C. and washing at 65° C. in a buffer solution containing 0.1 X SSC, 0.1% SDS, and which encodes a protein having the BMAL2 activity.

4. A host cell comprising an expression system capable of expressing a protein encoded by DNA according to any one of claims 1–3.

5. The host cell according to claim 4, wherein the host cell is further capable of expressing CLOCK and/or BMAL1.

6. The host cell according to claim 4, wherein the expression system at least comprises a promoter having an E-box sequence (CACGTG).

7. The host cell according to claim 6, wherein the promoter having an E-box sequence (CACGTG) is a promoter of Per gene, Tim gene, Cry gene, vasopressin gene or the albumin D-site binding protein gene.