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. ***
