Regulation of gene expression by visible light

生物合成途径中的一些中间体在可见光照射下通过光化学反应产生一种活性氧。例如，已知的原卟啉IX，一种血红素生物合成的中间体，就是这样的光敏剂。最近，在我的实验室里已经分离到了几个大肠杆菌的光敏突变体。编码铁络合酶的VISA(HemH)基因突变的细胞在可见光照射下被杀死，该酶催化血红素生物合成的最后一步。VISA(HemH)缺失菌株的耐光性突变体已被分离。由于预期的是VISA的真正回复突变(HemH)，回复突变应该是VISA基因和参与血红素生物合成途径的相关基因在铁络合酶催化反应之前的一个步骤中的双重突变。本研究结合细胞光反应，对参与血红素生物合成的基因进行了研究。(1)利用分离的突变株，克隆并测序了大肠杆菌的血红素和血红素G基因。(2)鉴定了两个新的参与血红素生物合成的基因。一个新基因，命名为hemk，在相似图上27分钟组成一个HEMA-PRFA-hemk操纵子。大麻编码225个氨基酸的蛋白质。由于突变细胞积累了原卟啉原，缺陷可能位于与hemG基因在功能上等同的基因上。(3)通过与缺失VISA的突变体和缺失突变体互补的方法，从大麦和黄瓜中分离得到了HEMA和hemH的cDNA克隆。(4)构建了Fe3+螯合酶的高效表达系统，并制备了大量的该酶。对纯化后的酶进行了物理和生化性质的研究。这种酶的结晶学研究正在进行中。

Some intermediates in the biosynthetic pathway generate an active pecies of oxygen via photochemical reactions upon illumination by visible light. For example, it is known that protoporphyrin IX, a intermediate in the biosynthesis of heme, is such a photosensitizer. Recently, Several light-sensitive mutant of E.coli have been isolated and characterized in my laboratory. The cells with mutantions in the visA (hemH) gene encoding ferrochelatase, the enzyme that catalyzes the final step in the biosynthesis of heme, are killed by illumination with visible light. The photoresistant mutants from the visA (hemH)-deleted strain have been isolated. Since on true revertant of visA (hemH) were expected, the revertats should be double mutants in the visA gene and in related genes that is involved in the heme biosynthetic pathway at a step before the reaction catayzed by ferrochelatase. In this research project, the studies on the genes involved in the biosynethsis of heme have been done in connection with light-responses of the cells.(1) Using mutants isolated, the hemE and hemG gene of E.coli were cloned and sequenced.(2) Two new genes involved in the biosynthesis of heme were identified. A new gene, designated hemK, composes a hemA-prfA-hemK operon at 27 min on the likage map. The hemK encodes 225 amino acids protein. Because the mutant cells accumulate protoporphyrinogen, the defect may locate in a gene functionally equvalent with the hemG gene.(3) The cDNA clones of the hemA and hemH from barley and cucumber were isolated by a method of complementation with the visA-deleted or the hemA-deleted mutant of E.coli.(4) A high expression system of the enzyme ferrochelatase was constructed and a large amount of the enzyme were prepared. The physical and biochemical natures of the purified enzyme were investigated. The crystallographic studies of this enzyme are under way.