Characterization by deletion mutagenesis in vitro of the promoter region of ompF, a positively regulated gene of Escherichia coli

The ompF gene codes for a major outer membrane protein whose expression is positively regulated by the ompR and envZ genes. Two sets of promoter deletions, upstream deletions and downstream deletions, were generated in vitro, and the promoter function was studied by connecting them with the tet genes. One of the hybrid genes thus constructed had a functioning ompF-tet hybrid promoter. The 107 base-pair fragment was found to be functioning as the ompF promoter, 90 nucleotides upstream and 17 nucleotides downstream of the mRNA start site that was also determined in this study. The start site was preceded by a convenient Pribnow box. Although the sequence at the -35 region had a low degree of homology to the consensus sequence, analyses of the hybrid promoter suggested that this region is involved in the promoter function in relation to the Pribnow box. They also indicated that the domain responsible for regulation by the ompR gene is located within the -35 region and its upstream region.     

Identification of basal and cyclic AMP regulatory elements in the promoter of the phosphoenolpyruvate carboxykinase gene.

Promoter elements important for basal and cyclic AMP (cAMP)-regulated expression of the phosphoenolpyruvate carboxykinase (PEPCK) gene have been identified by analysis of a series of PEPCK promoter mutations in transfection experiments. Fusion genes containing wild-type and mutated PEPCK promoter sequences from -600 to +69 base pairs (bp) fused to the coding sequence for chloramphenicol acetyltransferase were studied. Internal deletion mutations that replaced specific bases with a 10-bp linker within the region from -129 bp to -18 bp of the PEPCK promoter were examined. In addition, wild-type and mutated DNA templates were used as probes in DNase I protection experiments to determine sites of protein-DNA interaction. The PEPCK promoter contains a binding site for nuclear factor 1-CAAT. Deletion of the 5' end of this binding site reduced the size of the DNase I footprint in this region but had no effect on promoter activity. In contrast, deletion or disruption of the 3' end of this binding site completely eliminated protein binding and reduced promoter activity by 50%. Deletion of core sequences of the cAMP regulatory element (CRE) resulted in loss of cAMP responsiveness and an 85% decrease in basal promoter activity, indicating that the CRE also functions as a basal stimulatory element. Mutation of the core sequence of the CRE resulted in loss of the DNase I footprint over the CRE. Internal deletions flanking the CRE showed no loss of induction by cAMP but did have reduced promoter activity. This delimits the CRE to an 18-bp region between nucleotides -100 and -82. Analysis of mutations that disrupted bases between the CRE and the initiation site identified a basal inhibitory element adjacent to a basal stimulatory element, both located just 3' of the CRE, as well as a basal stimulatory element coincident with the TATA consensus sequence centered at -27. These data demonstrate that several cis-acting elements are located within 130 nucleotides of the initiation site of the PEPCK gene and that the CRE is essential for both basal promoter activity and cAMP-regulated expression of this gene.     

家蚕核多角体病毒解旋酶基因启动子功能区域缺失分析

杆状病毒DNA解旋酶是病毒复制所必需的。瞬时表达分析显示 ,家蚕核多角体病毒解旋酶基因启动子属于延迟早期基因启动子。通过PCR技术在该启动子区产生的一系列缺失分析表明 ,解旋酶基因启动子的基础转录调控区主要位于ATG上游 - 5 1 0～ - 4 1 0bp之间。当只保留ATG上游 98bp区段时 ,仍可测到该启动子的基础活性。在病毒因子存在下 ,将启动子区域删除到ATG上游 - 4 1 0bp时 ,对启动子活性影响不大 ;若继续删除 ,则其活性显著下降。据此推测对病毒因子响应的启动子区段应主要位于ATG上游 - 4 1 0～ - 30 9bp之间     

Regulation of BN115, a low-temperature-responsive gene from winter Brassica napus.

The genomic clone for BN115, a low-temperature-responsive gene, was isolated from winter Brassica napus and its sequence was determined. A 1.2-kb fragment of the 5' regulatory region (from bp -1107 to +100) was fused to the beta-glucuronidase (GUS) reporter gene and BN115-promoted GUS expression was observed in green tissues of transgenic B. napus plants only after incubation at 2 degrees C. No expression was observed after incubation at 22 degrees C, either in the presence or the absence of ABA. Microprojectile bombardment of winter B. napus leaves with a BN115 promoter/GUS construct yielded similar results and was used to analyze a series of deletions from the 5' end of the promoter. Results obtained from transient expression studies showed that the low-temperature regulation of BN115 expression involves a possible enhancer region between bp -1107 and -802 and a second positive regulatory region located between bp -302 and -274. Deletion analyses and results from replacement with a truncated cauliflower mosaic virus 35S promoter suggest that the minimal size required for any maintenance of low-temperature GUS expression is a -300-bp fragment. Within this fragment are two 8-bp elements with the sequence TGGCCGAC, which are identical to those present in the positive regulatory region of the promoter of the homologous Arabidopsis cor15a gene and to a 5-bp core sequence in the low-temperature- and dehydration-responsive elements identified in the promoter regions of several cold-responsive Arabidopsis thaliana genes.