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Regulation of gene expression by visible light

可见光调控基因表达

基本信息

批准号：
04454610
负责人：
INOKUCHI Hachiro
金额：
$ 3.97万
依托单位：
KYOTO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1992
资助国家：
日本
起止时间：
1992 至 1993
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04454610/
关键词：
light-sensitive mutant of E.coli biosynthesis of heme biosynthesis of chlorophyll hemK protoporphyrin IX active species of oxygen ferrochelatase cDNA clones of the hemA and hemH from barley and cucumber 光感受性変異株 ΔvisA(=ΔhemH)hemE hem

项目摘要

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.

生物合成途径中的一些中间产物在可见光照射下通过光化学反应产生活性氧。例如，已知血红素生物合成的中间体原卟啉IX就是这样的光敏剂。近年来，本实验室分离并鉴定了几株大肠杆菌的光敏感突变株。编码亚铁螯合酶（催化血红素生物合成的最后一步的酶）的visA（hemH）基因突变的细胞被可见光照射杀死。已从visA（hemH）缺失菌株中分离出光抗性突变体。由于未发现visA的真回复突变体（hemH），因此回复突变体应该是visA基因和亚铁螯合酶催化反应前一步血红素生物合成途径相关基因的双突变体。本研究项目结合细胞的光反应，对血红素生物合成相关基因进行了研究。(1)利用分离的突变株，克隆了大肠杆菌hemE和hemG基因，并进行了序列测定。(2)鉴定了两个参与血红素生物合成的新基因。一个新的基因，命名为hemK，组成一个hemA-prfA-hemK操纵子在27分钟的likage地图上。hemK基因编码225个氨基酸的蛋白质。由于突变细胞积累原卟啉原，缺陷可能位于与hemG基因功能等同的基因中。(3)用大肠杆菌的visA缺失或hemA缺失突变体互补的方法从大麦和黄瓜中分离hemA和hemH的cDNA克隆。(4)构建了铁螯合酶的高效表达系统，并大量制备了铁螯合酶。对纯化后的酶的理化性质进行了研究。这种酶的晶体学研究正在进行中。