Cloning of the recA gene from a free-living leptospire and distribution of RecA-like protein among spirochetes.
Cloning of the recA gene from a free-living leptospire and distribution of RecA-like protein among spirochetes.
复制标题
从自由生活的钩端螺旋体中克隆 recA 基因以及 RecA 样蛋白在螺旋体中的分布。
DOI：
10.1128/aem.57.1.183-189.1991
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发表时间：
1991
影响因子：
4.4
通讯作者：
Gherardini,FC
中科院分区：
文献类型：
--
作者：
Stamm,LV;Parrish,EA;Gherardini,FC
利用大肠埃希菌recA基因突变，从cosmid基因组DNA文库中分离到携带双歧钩端螺旋体血清型recA基因的重组质粒。克隆的血清型patoc recA基因有效地恢复了对紫外线辐射和甲磺酸甲酯的抗性。重组能力也得到了恢复，这是通过从重复突变的lacZ基因中形成Lac+重组来衡量的。此外，克隆的recA基因增加了大肠杆菌recA突变体溶原中自发和丝裂霉素c诱导的lambda噬菌体的产生。克隆的recA基因产物在大细胞中被鉴定为多肽，Mr为43,000。制备的抗大肠杆菌RecA蛋白的抗体与血清型patoc RecA蛋白发生交叉反应，表明结构守恒。南方杂交数据显示，血清型patoc的recA基因与疑问乳杆菌、illini瘦体菌和大肠杆菌的recA基因存在分化。除疑问螺旋体血清型的RecA蛋白外，钩端螺旋体血清型和伊利尼螺旋体血清型的RecA蛋白在钠二酸处理细胞后合成水平升高。可检测到的RecA水平与先前的研究相关，表明双歧乳杆菌和伊利尼乳杆菌的自由活细胞比寄生的疑问乳杆菌血清型对dna损伤剂的抵抗力强得多。在毒力强的梅毒螺旋体和伯氏疏螺旋体细胞中未检测到RecA蛋白。
A recombinant plasmid carrying the recA gene of Leptospira biflexa serovar patoc was isolated from a cosmid library of genomic DNA by complementation of an Escherichia coli recA mutation. The cloned serovar patoc recA gene efficiently restored resistance to UV radiation and methyl methanesulfonate. Recombination proficiency was also restored, as measured by the formation of Lac+ recombinants from duplicated mutant lacZ genes. Additionally, the cloned recA gene increased the spontaneous and mitomycin C-induced production of lambda phage in lysogens of an E. coli recA mutant. The product of the cloned recA gene was identified in maxicells as a polypeptide with an Mr of 43,000. Antibodies prepared against the E. coli RecA protein cross-reacted with the serovar patoc RecA protein, indicating structural conservation. Southern hybridization data showed that the serovar patoc recA gene has diverged from the recA gene of L. interrogans, Leptonema illini, and E. coli. With the exception of the RecA protein of L. interrogans serovar hardjo, the RecA protein of the Leptospira serovars and L. illini were synthesized at elevated levels following treatment of cells with nalidixic acid. The level of detectable RecA correlated with previous studies demonstrating that free-living cells of L. biflexa serovars and L. illini were considerably more resistant to DNA-damaging agents than were those of parasitic L. interrogans serovars. RecA protein was not detected in cells of virulent Treponema pallidum or Borrelia burgdorferi.