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

We cloned the pS1K1 plasmid in the process of apparently “complementing” a circadian clock mutant of cyanobacterium Synechococcus 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 the sp22 mutation was later found in a recently cloned new clock gene, kaiC. Therefore, the period-extender gene pex that was carried on pS1K1 was a suppressor gene for the sp22 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.

我们在表观“互补”蓝细菌聚球藻属（Synechococcus sp.）菌株PCC 7942的SP22昼夜节律突变株的过程中，克隆了pS1K1质粒（pS1K1 plasmid）。该突变株的节律周期为22小时（T. Kondo、N. F. Tsinoremas、S. S. Golden、C. H. Johnson、S. Kutsuna与M. Ishiura，《科学》，266:1233–1236，1994年）。序列分析结果显示，SP22在pS1K1质粒所携带的基因组DNA片段上并未发生突变；后续研究发现，sp22突变位点位于新近克隆的节律新基因kaiC（kaiC）中。因此，pS1K1质粒所携带的延周期基因pex（period-extender gene, pex）是sp22突变的抑制基因。pex基因编码一条含148个氨基酸残基的蛋白质。在包括集胞藻（Synechocystis）基因组数据库在内的DNA与蛋白质数据库中，均未检索到具有显著同源性的序列。pex基因的转录起始位点位于翻译起始密码子上游129 bp与164 bp处，转录产物为长度0.6 kb的mRNA转录本。通过使用c-Myc表位标签（c-Myc epitope tag）的表位标记融合技术，可检测到分子量为15 kDa的Pex（Pex）融合蛋白。在野生型细胞中敲除pex基因会使节律周期缩短1小时，且不影响节律的其他特性；而过表达pex基因则会使节律周期延长3小时，并伴随节律振幅的降低。在本次研究所检测的多种节律突变株中，过表达pex会导致细胞呈现无节律性。综上，Pex极有可能作为聚球藻（Synechococcus）昼夜节律的调控修饰因子发挥功能。