Roberts I, Hylemon P B, Holmes W M
Department of Microbiology, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298-0678.
Microbios. 1988;54(219):87-99.
Promoters which function in Gram-positive organisms show, with few exceptions, remarkable conservation of sequences identical with those in Escherichia coli. An E. coli system was tested to select putative promoters of two anaerobes, the Gram-positive Clostridium absonum and the Gram-negative Bacteroides thetaiotaomicron. Random restriction fragments of chromosomal DNA from these organisms were fused to the galactokinase (galK) gene of E. coli within a plasmid vector. Approximately 10% of these fragments functioned as promoters in E. coli, and a broad range of activities was evident. A single 88 base pair (bp) C. absonum DNA fragment yielded, in the E. coli plasmid vector, approximately the same high activity as that provided by the E. coli galK promoter. Sequence analysis of this fragment showed typical -35 and -10 sequences, with about five -10-like sequences closely flanking each other, some overlapping, and this appears to result in multiple start sites for transcription. The transcriptions of E. coli plasmid fragments in vitro with both E. coli RNA polymerase and C. absonum RNA polymerase showed pairs of transcripts corresponding to two start sites. By colony hybridization with the 88 bp fragment, radioactively labelled, as a probe, a 4.2 kilobase segment of C. absonum chromosomal DNA containing the 88 bp fragment was isolated. About 375 bp of this fragment was sequenced. A putative Shine-Dalgarno sequence and ATG start site were detected, followed by an opening reading frame. Using a sequence about 100 bp downstream from the 88 bp sequence, a 17-base oligonucleotide was synthesized to serve as a primer. With C. absonum RNA as a template, a reverse transcriptase primer extension assay located a pair of transcription start sites just downstream from the 88 bp sequence, proving that the 88 bp sequence functions as a promoter in C. absonum.
在革兰氏阳性菌中起作用的启动子，除少数例外，与大肠杆菌中的启动子序列表现出显著的保守性。对大肠杆菌系统进行了测试，以筛选两种厌氧菌（革兰氏阳性的奇异梭菌和革兰氏阴性的多形拟杆菌）的假定启动子。将这些生物体染色体DNA的随机限制性片段与质粒载体中大肠杆菌的半乳糖激酶（galK）基因融合。这些片段中约10%在大肠杆菌中起启动子的作用，并且表现出广泛的活性。一个88碱基对（bp）的奇异梭菌DNA片段在大肠杆菌质粒载体中产生的活性与大肠杆菌galK启动子提供的活性大致相同。对该片段的序列分析显示出典型的-35和-10序列，约有五个-10样序列彼此紧密相邻，有些相互重叠，这似乎导致了多个转录起始位点。用大肠杆菌RNA聚合酶和奇异梭菌RNA聚合酶对体外的大肠杆菌质粒片段进行转录，结果显示对应于两个起始位点的转录本对。通过用放射性标记的88 bp片段作为探针进行菌落杂交，分离出了奇异梭菌染色体DNA中包含该88 bp片段的4.2千碱基片段。对该片段约375 bp进行了测序。检测到一个假定的Shine-Dalgarno序列和ATG起始位点，随后是一个开放阅读框。利用88 bp序列下游约100 bp的一段序列，合成了一个17碱基的寡核苷酸作为引物。以奇异梭菌RNA为模板，通过逆转录酶引物延伸试验在88 bp序列下游定位了一对转录起始位点，证明该88 bp序列在奇异梭菌中起启动子的作用。
