Villafane R, Bechhofer D H, Narayanan C S, Dubnau D
Department of Microbiology, Public Health Research Institute, City of New York, Inc., New York 10016.
J Bacteriol. 1987 Oct;169(10):4822-9. doi: 10.1128/jb.169.10.4822-4829.1987.
pE194, a 3.7-kilobase plasmid, confers resistance to macrolide, lincosamide, and streptogramin B antibiotics. The previously identified cop and repF genes of pE194 have been further localized by molecular cloning and mutational analysis together with DNA sequencing. The CfoIB fragment of pE194 is capable of autonomous replication and contains both genes. Most of this region has been resequenced, and two errors reported in a previous study have been corrected. The corrected sequence indicates that the replication region contains a single large open reading frame, which we propose encodes the repF product. Northern blot (RNA blot) analysis of this region detected six transcripts, all transcribed in the same direction as one another and opposite to repF. A 350-base transcript is synthesized from the region containing cop. No in vivo transcript for the repF gene was detected, but a protein was observed in an in vitro transcription-translation system which appears to be its product. An ochre mutation was inserted in the putative repF open reading frame, and a nonsense fragment was detected in the in vitro system. When carried passively on a pUB110 replicon, this mutant product appears capable of inhibiting pE194 replicons in trans. The pE194 origin of replication has been localized to within 200 bases.
pE194是一种3.7千碱基的质粒，赋予对大环内酯类、林可酰胺类和链阳菌素B抗生素的抗性。通过分子克隆、突变分析以及DNA测序，对先前鉴定出的pE194的cop和repF基因进行了进一步定位。pE194的CfoIB片段能够自主复制，并且包含这两个基因。该区域的大部分已重新测序，纠正了先前研究中报告的两个错误。校正后的序列表明，复制区域包含一个单一的大开放阅读框，我们认为它编码repF产物。对该区域的Northern印迹（RNA印迹）分析检测到六个转录本，它们均以相同的方向转录，且与repF的转录方向相反。一个350碱基的转录本由包含cop的区域合成。未检测到repF基因的体内转录本，但在体外转录-翻译系统中观察到一种蛋白质，似乎是其产物。在假定的repF开放阅读框中插入了一个赭石突变，并在体外系统中检测到一个无义片段。当被动携带在pUB110复制子上时，这种突变产物似乎能够反式抑制pE194复制子。pE194的复制起点已定位在200个碱基范围内。
