ランソウの熱ショックタンパク質(Hsp)遺伝子の発現調節機構の解明

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1. A novel heat shock gene, orf7.5, was cloned from the cyanobacterium Synechococcus sp. PCC 7942. An interruption in the coding region led to a marked decrease in the basal thermotolerance at 50℃ in the transformant, indicating that the gene plays an essential role in thermal stress management. The orf7.5 mutant could be complemented with a return to the wild-type phenotype by a DNA fragment containing orf7.5, but not by mutated orf7.5s in which a nonsense mutation was generated by introducing a frameshift or a point mutation within the orf7.5 coding region. Thus, thermotolerance requires an appropriate translation product, not simply a transcript.Accumulation of the groESL or htpG transcript in the temperature-sensitive mutants was strongly reduced, suggesting that the orf7.5 gene be involved in the expression of these genes as a positive regulator.2. In order to elucidate regulation of the heat-shock gene expression in cyanobacteria, a reporter-assay system was employed. 5'-upstream … More regulatory region of the groESL operon from Synechococcus sp. PCC 7942 was fused with the promoter-less lacZ gene. The fused gene was expressed in E. coli constitutively at both 30 and 42 ℃. In cyanobacteria however the same groESL-lacZ gene, which was introduced into a neutral region of Synechococcus sp. PCC 7942 chromosome was expressed upon heat shock. These results indicate the presence of a mechanism, which represses expression of the groESL operon in cyanobacteria. Although the hspA gene encoding a small Hsp homologue from Synechococcus vulcanus was transcribed in E.coli constitutively like the groESL operon, its translation in E.coli was strongly inhibited at 30 ℃. The result indicates the presence of a post-transcriptional regulation of the cyanobacterial Hsp gene.3. We cloned a gene encoding a homologue of HtpG from the cyanobacterium Synechococcus sp. PCC 7942. In order to elucidate the role of HtpG in vivo, we inactivated the gene by targeted mutagenesis. The mutant cells were unable to grow at 45 ℃ and lost both the basal and acquired thermotolerances, indicating that HtpG plays an essential role tor the thermal stress management in cyanobacteria, the first such an example for a prokaryotic organism. Studies with the htpG mutant demonstrate that the HtpG protein contributes significantly to the ability of cyanobacteria to acclimate to low temperatures. We constructed a cyanobacterial strain, which expresses HspA constitutively. This is the first attempt to constitutively express any kind of Hsp in cyanobacteria. The transformant displayed improved viability compared with the reference strain upon transfer fro 30 to 50 ℃ in the light. Expression of HspA increased thermal stress resistance to photosystem II and protected the light-harvesting phycobilisomes from heat-induced photobleaching Less