CLONING AND ANALYSIS OF THE VITAMIN K2 GENE CLUSTER

维生素K2基因簇的克隆与分析

• 批准号: 3286860
• 负责人: HARRY W TABER
• 金额: $ 7.82万
• 依托单位: ALBANY MEDICAL COLLEGE
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-09-30 至 1988-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3286860
• 关键词: Bacillus subtilis; bacterial genetics; endonuclease; eukaryote; gel electrophoresis; gene complementation; genetic manipulation; genetic mapping; genetic recombination; genetic transcription; menadione; microorganism metabolism; molecular cloning; nucleic acid sequence; operon; radiotracer; regulatory gene; structural genes; vitamin K; vitamin biosynthesis

In humans and other higher animals, vitamin K (as vitamin K2 or menaquinone) serves as an essential cofactor in the formation of several clotting factors. The menaquinone-dependent biochemical reaction in clotting factor synthesis is the Gamma-carboxylation of glutamic acid. Among prokaryotic cells, on the other hand, menaquinone functions as a lipophilic electron carrier in membrane-bound electron transfer chains. Most bacterial species containing menaquinone are capable of synthesizing it de novo, and there is some evidence that intestinal flora can supply their animal hosts with menaquinone in the absence of dietary sources of the vitamin. Knowledge of the biosynthetic steps in bacterial menaquinone formation and the regulation of this biosynthesis should enhance our understanding of this aspect of bacterial-human interaction. The long-term objectives of the applicant's research program are: (i) to discover the principles governing regulations of bacterial menaquinone biosynthesis, both at the metabolic and the genetic levels; ii) to relate these principles to the functioning of menaquinone in the biology of prokaryotic cells. The specific aims of this research proposal are: i) the use of molecular cloning techniques to isolate from the bacterium Bacillus subtilis a cluster of genes (the men operon) encoding information for the menaquinone biosynthetic enzymes and associated regulatory regions; ii) the physical analysis of this DNA fragment by restriction mapping, subcloning, nucleotide sequence determination, and transcript mapping; iii) establishment of the presence, order, and functionality of structural genes within the operon fragment by recombination and complementation measurements of subcloned sequences.

在人类和其他高等动物中，维生素K(如维生素K2或 菜籽酮)是形成几种 凝血因子。大鼠脑脊液中依赖于菜籽酮的生化反应 凝血因子的合成是谷氨酸的伽玛羧化反应。 另一方面，在原核细胞中，菜籽酮的功能是 膜结合电子转移链中的亲脂电子载体。 大多数含有孟喹酮的细菌都能合成 它是从头开始的，有一些证据表明肠道菌群可以提供 在缺乏饲料来源的情况下，它们的动物宿主会使用孟喹酮 维他命。细菌孟喹酮生物合成步骤的认识 这种生物合成的形成和调节应该会增强我们的 了解细菌与人类相互作用的这一方面。 申请者研究计划的长期目标是： (I)发现管理细菌的规则的原则 在代谢和遗传水平上的尿喹酮生物合成； 二)将这些原则与脑啡酮在人体内的功能联系起来 原核细胞生物学。 这项研究建议的具体目标是： I)使用分子克隆技术从细菌中分离 枯草杆菌是一组编码信息的基因(人类操纵子) 用于菜籽酮生物合成酶和相关的调节区； Ii)通过限制图谱对该DNA片段进行物理分析， 亚克隆、核苷酸序列测定和转录本定位； 三)建立结构的存在、秩序和功能 操纵子片段中的基因通过重组和互补 亚克隆序列的测量。