MOLECULAR GENETICS OF BACILLUS SUBTILIS RNA POLYMERASE

枯草芽孢杆菌的产孢过程是一个模型系统， 以及异常的细胞发育和分化， 主要受转录调控。 虽然复杂 生物体的转录装置被很好地表征 从生物化学上讲，需要进行遗传分析来建立生理学上的 作用、功能相互作用和各种成分的调节。 B。枯草杆菌RNA聚合酶存在于至少五种细胞形式中， 共享一个共同的多亚基核心酶，每个亚基与一个 不同的sigma因子赋予特征性启动子识别 的特异性 这项建议的具体目标是表征四个基因， 编码（1）聚合酶的主要43，000道尔顿σ因子;（2） 一种小的，37，000道尔顿的σ因子，可能控制基因表达的早期， 孢子形成过程;（3）α，核心亚基;和（4）δ， 核心相关亚基，可能影响转录特异性。 对于所有四种基因，方法将是相同的：（1）从一个或多个细胞中分离基因。 使用抗体或寡核苷酸探针的λ表达载体库;（2） 通过独立的生物化学方法确认基因产物的身份; (3)确定每个克隆区域的分子遗传组织， 大细胞分析、DNA序列分析和S1内切酶图谱分析， 体内转录本;（4）将克隆的基因定位在B上。枯草 使用整合作图质粒定位染色体;和（5）定位已知的 在克隆的DNA上的区域中产生突变，或者在克隆的DNA上的区域中产生新的突变。 通过体外诱变克隆基因，以建立功能结构域 以及四种基因产物的生理作用。 这项研究将 有助于理解基因调控的全球机制 在B中表达。subtilis并提出控制发展的逻辑 系统. 这种认识在发展B中有实际应用。 枯草杆菌作为表达和分泌克隆外源基因的宿主 对人类治疗有益的产品。 在基本层面上，比较B。 subtilis和E.大肠杆菌基因组织和调控机制， 表明原核生物结构的哪些特征是基本的。

The sporulation process of Bacillus subtilis is a model system for normal and abnormal cell development and differentiation that appears to be chiefly regulated by transcriptional control. Although the complex transcriptional apparatus of the organism is well characterized biochemically, a genetic analysis is needed to establish the physiological roles, functional interactions, and regulation of the various components. B. subtilis RNA polymerase exists in at least five cellular forms, each sharing a common, multi-subunit core enzyme and each associated with a different sigma factor that confers a characteristic promoter recognition specificity. This proposal specifically aims to characterize four genes, encoding (1) the major, 43,000 dalton sigma factor of the polymerase; (2) a minor, 37,000 dalton sigma factor that may control gene expression early in the sporulation process; (3) alpha, a core subunit; and (4) delta, a core-associated subunit that may influence transcriptional specificity. Methodology will be the same for all four genes: (1) isolate genes from a lambda expression vector bank using antibody or oligonucleotide probes; (2) confirm the identity of the gene products by independent biochemical means; (3) determine the molecular genetic organization of each cloned region by maxicell analysis, DNA sequence analysis, and S1 endonulease mapping of in vivo transcripts; (4) map the locus of the cloned gene on the B. subtilis chromosome using an integrative mapping plasmid; and (5) locate known mutations in the region on the cloned DNA, or make new mutations in the cloned genes by in vitro mutagenesis, to establish the functional domains and physiological roles of the four gene products. This research will contribute to understanding the global mechanisms regulating gene expression in B. subtilis and suggest the logic controlling developmental systems. Such understanding has a practical application in developing B. subtilis as a host for expressing and excreting cloned foreign gene products beneficial for human therapy. And on a basic level, comparing B. subtilis and E. coli gene organization and regulatory mechanisms can suggest which features of prokaryotic architecture are fundamental.