Preponderance of Fis-binding sites in the R6K gamma origin and the curious effect of the penicillin resistance marker on replication of this origin in the absence of Fis.
Preponderance of Fis-binding sites in the R6K gamma origin and the curious effect of the penicillin resistance marker on replication of this origin in the absence of Fis.
复制标题
R6K gamma 起点中 Fis 结合位点的优势以及青霉素抗性标记在 Fis 不存在的情况下对该起点复制的奇怪影响。
DOI：
10.1128/jb.178.16.4965-4974.1996
复制
发表时间：
1996
影响因子：
3.2
通讯作者：
Filutowicz,M
中科院分区：
文献类型：
--
作者：
Wu,F;Wu,J;Ehley,J;Filutowicz,M
该蛋白与质粒R6K γ起源的10个位点结合。在质粒编码的pi启动蛋白和三种宿主编码蛋白（dna、整合宿主因子和RNA聚合酶）的γ起源中，fish结合位点重叠了所有先前鉴定的结合位点。然而，Fis对R6K复制的要求取决于复制的pi蛋白变体的使用，奇怪的是，质粒上的抗生素抗性标记。在fish缺陷细胞中，复制pi变异体不能驱动携带编码青霉素抗性（Penr）的bla基因的R6K γ源质粒的复制，但可以驱动携带编码氯霉素抗性（Cmr）的氯霉素乙酰转移酶基因的同源质粒的复制。相比之下，在缺乏Fis蛋白的情况下，野生型pi驱动的R6K复制不受抗生素抗性标记的影响。单独来说，这些因素（复制pi、Fis缺乏或药物标记）都不能阻止R6K的复制。这种复制缺陷不是由培养基中的青霉素或失控复制引起的，也不受bla基因相对于起源的方向的影响。当部分bla编码片段被删除但bla启动子完好无损时，复制仍然受到抑制。然而，通过在γ起源两侧插入转录终止子，复制得以恢复，这表明在没有Fis的情况下，bla基因的过量转录可能会使pi复制突变驱动的复制失活。这项研究表明，载体序列，如药物标记，在复制研究中可能不是无关紧要的，正如通常认为的那样。
Fis protein is shown here to bind to 10 sites in the gamma origin of plasmid R6K. The Fis-binding sites overlap all the previously identified binding sites in the gamma origin for the plasmid-encoded pi initiator protein and three host-encoded proteins, DnaA, integration host factor, and RNA polymerase. However, the requirement of Fis for R6K replication depends on the use of copy-up pi-protein variants and, oddly, the antibiotic resistance marker on the plasmid. In Fis-deficient cells, copy-up pi variants cannot drive replication of R6K gamma-origin plasmids carrying the bla gene encoding resistance to penicillin (Penr) but can drive replication of plasmids with the same origin but carrying the chloramphenicol acetyltransferase gene encoding chloramphenicol resistance (Cmr). In contrast, R6K replication driven by wild-type pi is unaffected by the antibiotic resistance marker in the absence of Fis protein. Individually, none of these elements (copy-up pi, Fis deficiency, or drug markers) prevents R6K replication. The replication defect is not caused by penicillin in the medium or runaway replication and is unaffected by the orientation of the bla gene relative to the origin. Replication remains inhibited when part of the bla coding segment is deleted but the bla promoter is left intact. However, replication is restored by insertion of transcriptional terminators on either side of the gamma origin, suggesting that excess transcription from the bla gene may inactivate replication driven by pi copy-up mutants in the absence of Fis. This study suggests that vector sequences such as drug markers may not be inconsequential in replication studies, as is generally assumed.