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首页> 外文学位 >Characterization of the ARC1 gene of Saccharomyces cerevisiae and development of a high-yield expression system for the production of the cyanobacterial phytochrome Cph1 in Escherichia coli.
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Characterization of the ARC1 gene of Saccharomyces cerevisiae and development of a high-yield expression system for the production of the cyanobacterial phytochrome Cph1 in Escherichia coli.

机译：酿酒酵母的ARC1基因的表征和高产表达系统的发展，用于在大肠杆菌中生产蓝细菌植物色素Cph1。

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摘要

This dissertation is presented in two parts. The first section consists of work done in the laboratory of Professor Harry Matthews on the putative yeast histidine kinase Arc1p. The second deals with research conducted in the laboratory of Professor J. Clark Lagarias on the plant photoreceptor phytochrome.; In the Matthews lab, a protein kinase that phosphorylates histidine-75 of histone H4 had been purified from Saccharomyces cerevisiae. Partial sequence data which had been obtained for the enzyme was used to extract the complete amino acid sequence from the GenBank database. This protein, then known as G4p1 and now called Arc1p for a&barbelow;minoacyl-tR&barbelow;NA synthetase c&barbelow;ofactor 1&barbelow;, had been previously cloned by another group. Cell extracts of cultures, in which expression of the complete ARC1 gene and of a portion of the gene encoding the C-terminal region of Arc1p was induced, had histidine kinase activity with histone H4 as a substrate, while those in which expression was not induced had only background activity. The activity in the cell extracts was sensitive to the kinase inhibitor genistein, as was the kinase purified from yeast. Phosphoamino acid analysis confirmed that H4 was phosphorylated on histidine. Experiments comparing histidine kinase activity in cell extracts from yeast containing an ARC1 gene disruption indicated that yeast lacking Arc1p have lower kinase activity than wild-type yeast. Replacing the ARC1 gene using an expression vector leads to an increase in kinase activity to levels higher than those in wild-type yeast.; In the Lagarias lab, my work contributed to the study of the mechanism of the photoisomerization reaction of the plant photoreceptor phytochrome via resonance Raman spectroscopy. Several systems for the production of the Cyanobacterial phytochrome Cph1 in Escherichia coli were tested to determine which would allow for the purification of Cph1 in the substantial quantities required for these experiments. In addition, differentially-labelled bilin chromophores were converted from the dimethyl ester forms to the carboxylic acid forms necessary for the resonance Raman studies. These analogs will be inserted into apophytochrome and used to address the structure of the Pr and Pfr chromophores in situ.
机译：本文分为两个部分。第一部分包括在哈里·马修斯教授的实验室中完成的关于假定的酵母组氨酸激酶Arc1p的工作。第二项研究是在J. Clark Lagarias教授的实验室中进行的有关植物光感受器植物色素的研究。在马修斯实验室中，已经从酿酒酵母中纯化了磷酸化组蛋白H4的组氨酸75的蛋白激酶。已为该酶获得的部分序列数据用于从GenBank数据库中提取完整的氨基酸序列。这种蛋白质，后来被称为G4p1，现在被称为Arc1p，是一个氨基酰基-tR＆NA合成酶c＆ofactor 1＆barbelow ;，先前已被另一小组克隆。诱导完整ARC1基因和编码Arc1p C端区域的基因一部分表达的培养物细胞提取物具有组氨酸激酶活性，以组蛋白H4为底物，而未诱导表达仅有背景活动。细胞提取物中的活性对激酶抑制剂金雀异黄素敏感，从酵母中纯化的激酶也是如此。磷酸氨基酸分析证实，H4在组氨酸上被磷酸化。比较含有ARC1基因破坏的酵母细胞提取物中组氨酸激酶活性的实验表明，缺少Arc1p的酵母的激酶活性低于野生型酵母。用表达载体替换ARC1基因导致激酶活性增加到比野生型酵母更高的水平。在拉加里亚斯实验室中，我的工作通过共振拉曼光谱研究了植物光感受器植物色素的光异构化反应机理。测试了几种在大肠杆菌中生产蓝藻细胞色素Cph1的系统，以确定哪种系统可以纯化这些实验所需的大量Cph1。另外，将差异标记的联蛋白生色团从二甲基酯形式转化为共振拉曼研究所必需的羧酸形式。这些类似物将被插入到脱附生物色素中，并用于原位处理Pr和Pfr发色团的结构。

著录项

  • 作者

    Santos, Jennifer Lynn.;

  • 作者单位

    University of California, Davis.;

  • 授予单位 University of California, Davis.;
  • 学科 Chemistry Biochemistry.; Biology Molecular.
  • 学位 Ph.D.
  • 年度 2002
  • 页码 181 p.
  • 总页数 181
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 生物化学;分子遗传学;
  • 关键词

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