Isolation and expression of cloned hook protein gene from Caulobacter crescentus.
Isolation and expression of cloned hook protein gene from Caulobacter crescentus.
复制标题
新月柄杆菌钩蛋白基因克隆的分离和表达。
DOI：
10.1073/pnas.79.16.4863
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发表时间：
1982
影响因子：
11.1
通讯作者：
Newton,A
中科院分区：
文献类型：
--
作者：
Ohta,N;Chen,LS;Newton,A
先前对新月柄杆菌的遗传分析表明，主要鞭毛亚基钩蛋白、鞭毛蛋白A和鞭毛蛋白B的周期性合成以某种方式与染色体复制偶联。为了在分子水平上研究鞭毛基因表达的调节，我们分离了编码72，000-道尔顿钩蛋白的基因。特异性125 I标记的抗钩蛋白IgG用于筛选4，500个克隆的杂合cDNA L47.1库，并将克隆的基因产物的肽图与纯化的钩蛋白进行比较。来自阳性λ克隆和质粒亚克隆的DNA的限制性分析表明，钩蛋白的结构基因包含在2.3-内切酶（kb）BamHI片段上。通过证明具有融合到大肠杆菌脂蛋白基因-乳糖基因启动子-pIN-II操纵子区的2.3-kb片段的菌株中的钩蛋白基因的诱导来确定转录方向。初步的基因组分析表明，hook基因在C.新月形染色体这些结果表明，在细胞周期中的钩蛋白基因的周期性表达不涉及一个主要的或持久的重排的2.3 kb的编码序列在细胞周期。
Previous genetic analysis of Caulobacter crescentus showed that the periodic synthesis of hook protein, flagellin A, and flagellin B, the major flagellar subunits, is coupled in some way to chromosome replication. To examine the regulation of flagellar gene expression at the molecular level, we isolated the gene that codes for the 72,000-dalton hook protein. A specific 125I-labeled anti-hook protein IgG was used to screen a hybrid lambdaL47.1 bank of 4,500 clones and to compare peptide maps of the cloned gene product with purified hook protein. Restriction analysis of DNA from the positive lambda clones and plasmid subclones showed that the structural gene for the hook protein is contained on a 2.3-kilobase (kb) BamHI fragment. The direction of transcription was established by demonstrating the inducibility of hook protein gene in strains with the 2.3-kb fragment fused to the Escherichia coli lipoprotein gene-lactose gene promoter-operator region of pIN-II. Preliminary genomic analysis showed that the hook gene occupies a single location on the C. crescentus chromosome. These results suggest that the periodic expression of the hook protein gene in the cell cycle does not involve a major or persistent rearrangement of the 2.3-kb coding sequence during the cell cycle.