A Period-Extender Gene, pex , That Extends the Period of the Circadian Clock in the Cyanobacterium Synechococcus sp. Strain PCC 7942

Abstract

We cloned the pS1K1 plasmid in the process of apparently “complementing” a circadian clock mutant of cyanobacteriumSynechococcus sp. strain PCC 7942, SP22, which has a 22-h period (T. Kondo, N. F. Tsinoremas, S. S. Golden, C. H. Johnson, S. Kutsuna, and M. Ishiura, Science 266:1233–1236, 1994). Sequence analysis revealed that SP22 did not have a mutation in the genomic DNA segment carried on pS1K1, and thesp22 mutation was later found in a recently cloned new clock gene, kaiC. Therefore, the period-extender genepex that was carried on pS1K1 was a suppressor gene for thesp22 mutation. The pex gene encoded a protein of 148 amino acid residues. No meaningful homologs were found in DNA or protein databases including the Synechocystis genome database. The pex gene was transcribed from 129 and 164 bp upstream of the translation initiation codon as 0.6-kb transcripts. The Pex protein was detected as a fusion protein with a molecular mass of 15 kDa by the epitope tag fusion method using a c-Myc epitope tag. Disruption of the pex gene in wild-type cells shortened the period of the rhythms by 1 h, although it did not affect other properties of the rhythms, whereas its overexpression extended the period by 3 h with a concomitant reduction in the amplitude of the rhythms. In various clock mutants examined, overexpression caused arrhythmicity. Thus, Pex is likely to function as a modifier of the circadian clock in Synechococcus.

Physical interactions among circadian clock proteins KaiA, KaiB and KaiC in cyanobacteria

“…To test the antigen specificity of the antisera, AMC149 and NUC39 were grown in BG‐11 liquid medium in continuous light of 46 μE/m 2 /s from white fluorescent lamps at 30°C (standard conditions) until the cell density reached 1.0 OD 730 . Cell pellets from the 30 ml culture were resuspended in 500 μl of phosphate‐buffered saline (PBS) and disrupted using the Mini Bead‐beater (Biospec Products) with 1 ml of zirconium beads (0.1 mm in diameter, Biospec Products) as described previously (Kutsuna et al ., 1998). The suspension was centrifuged at 15 000 r.p.m.…”

“…Nco I– Bam HI segments of pSPK–CI and pSPK–CII that contained the whole CI‐ and CII‐encoding sequences were ligated with Nco I‐ and Bam HI‐digested p322P trc (Kutsuna et al ., 1998) to obtain p322P trc :: CI and p322P trc ::CII , respectively. The smaller Bgl II‐fragments from p322P trc :: CI and p322P trc ::CII were inserted into the Bam HI site of the pTS2KC (Kutsuna et al ., 1998) to obtain pTS2C‐P trc ::CI and pTS2C‐P trc ::CII , respectively.…”

“…Nco I– Bam HI segments of pSPK–CI and pSPK–CII that contained the whole CI‐ and CII‐encoding sequences were ligated with Nco I‐ and Bam HI‐digested p322P trc (Kutsuna et al ., 1998) to obtain p322P trc :: CI and p322P trc ::CII , respectively. The smaller Bgl II‐fragments from p322P trc :: CI and p322P trc ::CII were inserted into the Bam HI site of the pTS2KC (Kutsuna et al ., 1998) to obtain pTS2C‐P trc ::CI and pTS2C‐P trc ::CII , respectively. The P kaiBC ‐reporter strain NUC38 was transformed with each plasmid as described previously (Kutsuna et al ., 1998) to harbor the expression construct at a specific genomic targeting site of the chromosome (NSII; GenBank/EMBL/DDBJ accession No.…”

“…The smaller Bgl II‐fragments from p322P trc :: CI and p322P trc ::CII were inserted into the Bam HI site of the pTS2KC (Kutsuna et al ., 1998) to obtain pTS2C‐P trc ::CI and pTS2C‐P trc ::CII , respectively. The P kaiBC ‐reporter strain NUC38 was transformed with each plasmid as described previously (Kutsuna et al ., 1998) to harbor the expression construct at a specific genomic targeting site of the chromosome (NSII; GenBank/EMBL/DDBJ accession No. U44761).…”

The ups and downs of daily life: Profiling circadian gene expression in Drosophila

“…Like any good clock, circadian pacemakers are insensitive to temperature fluctuations and can be reset by various external stimuli. (6) Individual cells show circadian oscillations that arise from an intracellular clock; (3) oscillations in groups of cells must be synchronized or maintained at specific phase differences through intercellular signaling. This is particularly evident for behavioral rhythms.…”