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.2000 Apr 11;97(8):4339-44.
doi: 10.1073/pnas.97.8.4339.

Asynchronous oscillations of two zebrafish CLOCK partners reveal differential clock control and function

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Asynchronous oscillations of two zebrafish CLOCK partners reveal differential clock control and function

N Cermakian et al. Proc Natl Acad Sci U S A..

Abstract

Most clock genes encode transcription factors that interact to elicit cooperative control of clock function. Using a two-hybrid system approach, we have isolated two different partners of zebrafish (zf) CLOCK, which are similar to the mammalian BMAL1 (brain and muscle arylhydrocarbon receptor nuclear translocator-like protein 1). The two homologs, zfBMAL1 and zfBMAL2, contain conserved basic helix-loop-helix-PAS (Period-Arylhydrocarbon receptor-Singleminded) domains but diverge in the carboxyl termini, thus bearing different transcriptional activation potential. As for zfClock, the expression of both zfBmals oscillates in most tissues in the animal. However, in many tissues, the peak, levels, and kinetics of expression are different between the two genes and for the same gene from tissue to tissue. These results support the existence of independent peripheral oscillators and suggest that zfBMAL1 and zfBMAL2 may exert distinct circadian functions, interacting differentially with zfCLOCK at various times in different tissues. Our findings also indicate that multiple controls may be exerted by the central clock and/or that peripheral oscillators can differentially interpret central clock signals.

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Figures

Figure 1
Figure 1
Cloning and sequence analysis ofzfBmal cDNAs. (A) Yeast growth is supported on selective medium in two-hybrid assays including both CLOCK and either of the zfBMALs (clones 40–1 and 40–2). (B) DNA sequence identity between theBmal cDNAs. The hatched bar in 40–1/6 represents the region differing between the two clones. The ORF is denoted by a thicker box. (C) Alignment of human and zebrafish BMALs. Except for their amino terminus, human isoforms a and b are identical. Identical (inverted) and similar (gray background) residues are indicated. Hatched and solid underlined sequences highlight the bHLH domain and the PAS domain repeats, respectively. (D) Phylogenetic analysis of BMAL proteins. The result of 100 bootstrap resamplings is shown. mARNT1, mARNT2, and mCLOCK were used to root the tree (seeMaterials and Methods for details).
Figure 2
Figure 2
Interaction of BMALs with CLOCK. (A) Yeast two-hybrid assays using various constructions in conjunction with GAL4 DBD-CLOCK. (Left) Representation of the different BMAL-based constructions. Hatched box denotes the GAL4 AD. (Right) β-Galactosidase activity, expressed in standard Miller units. The BMAL1 and BMAL2 constructions shown with a separated GAL4 AD (fourth and eighth from the top) correspond to the original clones isolated in the screen (40–1 and 40–2). (B) GST pull-down assay. Equivalent amounts of35S-labeled complete or partial BMAL1 and BMAL2 proteins were prepared and analyzed by SDS/PAGE (input). (Lower) SDS/PAGE analysis of the proteins binding to GST or GST-CLOCK beads. Luciferase was used as a input negative control. (C) Different association affinities of zfBMAL proteins with CLOCK.35S-labeled BMAL1 and BMAL2 were produced byin vitro transcription/translation, and radioactivity was quantified by trichloroacetic acid precipitation and scintillation counting. GST pull-down was carried out as described inMaterials and Methods, with equivalent amounts of GST-CLOCK and increasing amounts of BMALs. After washes, the radioactivity remaining on the resin was quantified by scintillation counting.
Figure 3
Figure 3
Rhythmic expression of theBmal genes in the zebrafish brain, eyes, and pineal gland. (A,C, andD) RPA of the expression of theBmal transcripts in the brain, eyes, and pineal gland under LD (A andD) or constant darkness (C) conditions. Equivalent amounts of RNA were used in each case, as assayed by ethidium staining and CREB (cAMP responsive binding protein) RPA control. “t” is the negative control with only tRNA. Numbers above each lane correspond to the ZT points at which RNA samples were prepared. (A andD) White/black bars represent the duration of the LD periods. (C) Gray/black bars show the extent of subjective day/night periods in dark-dark (DD) conditions. (B)In situ hybridization of zebrafish brain sections at ZT3 or ZT12 (in LD). For each time point, two successive sections were analyzed withBmal1 andBmal2 probes.
Figure 4
Figure 4
Differential expression patterns ofBmal1 andBmal2 in different zebrafish peripheral organs. RPA analysis ofBmal expression in adult zebrafish heart, kidney, liver, spleen, and testis at the indicated ZT points under LD conditions. “t” corresponds to a tRNA negative control, and white/black bars represent the day/night periods.
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