RUI: Genetic and Molecular Characterization of a Novel Transcription Unit in Bacillus subtilis

该启动基金的主要目标是：1)鉴定和表征sigD操纵子转录启动子序列的表达模式；2)获得形成该转录单元3'端的基因序列。枯草芽孢杆菌的sigmaD因子由sigD结构基因编码，是鞭毛蛋白、运动性和趋化性基因转录所必需的。有趣的是，sigD是一个非常大的操纵子（26kb）中最后一个完整的开放读取，包含编码形成钩基体复合物的结构蛋白的基因，将鞭毛连接到细菌细胞的多亚基结构，以及趋化所需的调节蛋白。该操纵子的转录至少部分由sigmaD结构基因上游约24千碱基的启动子元件启动。该启动子元件已被克隆为质粒pAZ210中5.7 kb插入的一部分，并且已知位于3kb的限制性片段内。获得该片段的序列是为了根据其与枯草芽孢杆菌中已知的启动子一致序列的同源性来鉴定候选启动子元件。所获得的信息将允许合成适当的引物延伸探针。这些探针将用于引物延伸反应，以确定哪些候选启动子序列在体内被用来启动sigD操纵子的转录。引物延伸分析也将用于准确表示该启动子元件的表达在整个发育过程中是如何被调节的。所获得的表达模式可能为参与调节sigD操纵子转录的分子机制提供线索。在平行研究中，sigD转录单元的3'端被获得并被表征。如上所述，sigD是h被克隆和测序的DNA区域中最后一个完整的开放阅读框。在该区域内没有转录终止子的特征序列，并且在sigD的翻译终止子下游发现了部分开放阅读框，这表明该转录单元的长度大于26 kb。为了获得该转录单元的3‘端，将用sigD基因3’端的限制性片段探测枯草芽孢杆菌基因组文库。将对得到的克隆进行测序，以确定潜在的转录终止子和任何额外的开放阅读框。利用这些信息，将准备S1探针，通过S1分析来绘制sigD操纵子的3‘端，并通过这种方式验证已经获得了sigD转录单元的3’端。这项研究产生的信息将为一个非常大而重要的细菌基因群的遗传组织和调控提供基本信息。＊＊＊

9500398 Marquez The primary objectives of this starter grant are to 1) identify, and characterize the pattern of expression from the promoter sequence that initiates transcription of the sigD operon, and 2) obtain the genetic sequence that forms the 3' end of this transcription unit. The sigmaD factor of the bacterium Bacillus subtilis is encoded by the sigD structural gene, and is required for the transcription of flagellin, motility, and chemotaxis genes. Interestingly, sigD is the last complete open reading from in a very large operon (26 kb), containing genes which encode structural proteins that form the hook-basal body complex, the multi-subunit structure that tethers the flagellum to the bacterial cell, as well as regulatory proteins required for chemotaxis. Transcription of this operon is initiated, at least in part, by a promoter element about 24 kilobases upstream of the structural gene for sigmaD. This promoter element has been cloned as part of a 5.7 kb insert in the plasmid pAZ210 and is known to reside within a 3 kb restriction fragment. The sequence of this fragment is being obtained in order to identify candidate promoter elements based on their homology to known promoter consensus sequences in B. subtilis. The information obtained will allow for the synthesis of the appropriate primer extension probes. These probes will be used in a primer extension reaction to determine which of the candidate promoter sequences is utilized in vivo to initiate transcription of the sigD operon. Primer extension analysis will also be used to generate an accurate representation of how expression from this promoter element is regulated throughout development. The pattern of expression obtained may provide clues as to the molecular mechanisms involved in regulating transcription of the sigD operon. In parallel studies the 3' end of the sigD transcription unit is being obtained and characterized. As indicated above, sigD is the last complete open reading frame in the region of DNA that h as been cloned and sequenced. There are no sequences within this region which are characteristic of transcriptional terminators, and a partial open reading frame has been identified immediately downstream of the translational stop for sigD, suggesting that this transcription unit is greater than 26 kb in length. In order to obtain the 3' end of this transcription unit, a B. subtilis genomic library will be probed with a restriction fragment from the 3' end of the sigD gene. The resultant clone(s) will be sequenced in order to identify potential transcription terminators and any additional open reading frames. Utilizing this information an S1 probe will be prepared to map the 3' end of the sigD operon by S1 analysis and in this way verify that the 3' end of sigD transcription unit has been obtained. %%% The information generated by this study will provide basic information as to the genetic organization and regulation of a very large and important group of bacterial genes. ***