CLONING AND ANALYSIS OF THE VITAMIN K2 GENE CLUSTER

在人类和其他高等动物中，维生素K（如维生素K2或 甲萘醌）在几种代谢物的形成中充当重要的辅因子。 凝血因子 甲萘醌依赖性生化反应 凝血因子的合成是谷氨酸的γ-羧化。 另一方面，在原核细胞中，甲基萘醌的功能是 亲脂性电子载体在膜结合的电子转移链。 大多数含有甲基萘醌的细菌物种能够合成 有证据表明肠道植物群可以提供 在没有食物来源的情况下， 维生素 了解细菌甲萘醌的生物合成步骤 这种生物合成的形成和调节应该增强我们的 了解细菌与人类相互作用的这一方面。 申请人研究计划的长期目标是： (i)发现细菌生长调节的原理 甲萘醌生物合成，包括代谢和遗传水平; ii）将这些原则与甲萘醌在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 枯草芽孢杆菌是一组编码信息的基因（Men操纵子） 甲萘醌生物合成酶和相关的调控区; ii）通过限制性酶切图谱对该DNA片段进行物理分析， 亚克隆、核苷酸序列测定和转录物作图; （三）建立结构的存在、秩序和功能; 操纵子片段内的基因通过重组和互补 亚克隆序列的测量。

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.