In vivo inactivation of the Streptococcus mutans recA gene mediated by PCR amplification and cloning of a recA DNA fragment.
In vivo inactivation of the Streptococcus mutans recA gene mediated by PCR amplification and cloning of a recA DNA fragment.
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PCR 扩增和recA DNA 片段克隆介导的体内变形链球菌recA 基因失活。
DOI：
10.1016/0378-1119(92)90626-z
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发表时间：
1992
期刊：
影响因子：
3.5
通讯作者：
Faustoferri,RC
中科院分区：
文献类型：
--
作者：
QuiveyJr,RG;Faustoferri,RC
RecA蛋白在致病性口腔链球菌中的失活将有助于这些菌株中潜在毒力因子的遗传分析。通过比较不同细菌的RecA核苷酸（nt）序列，发现RecA氨基酸（aa）序列的两个高度保守区域可以作为合成简并寡脱氧核苷酸引物的基础，用于从链球菌中扩增RecA同源物。因此，使用引物混合物通过PCR扩增变形链球菌染色体的693-bp片段。将扩增的片段克隆，并通过与大肠杆菌recA基因探针杂交和nt序列测定来确认其身份。该片段与S. mutansGS-5与E.大肠杆菌和枯草芽孢杆菌RecA酶。帖前在体外通过插入失活对mutans recA片段进行诱变，并使用等位基因交换使其返回染色体。分离得到的S.突变株比野生型菌株对紫外线照射更敏感。此外，将线性标记物并入染色体的能力在假定的S中被消除。变形杆菌recA菌株，从而表明RecA在这些微生物中的功能失活。
The inactivation of the RecA protein in pathogenic oral streptococci would facilitate genetic analysis of potential virulence factors in these strains. Comparison ofrecAnucleotide (nt) sequences from a number of bacteria has suggested that two regions of highly conserved RecA amino acid (aa) sequence could be used as a basis for synthesizing degenerate oligodeoxyribonucleotide primers with which to amplifyrecAhomologues from the streptococci. Accordingly, primer mixtures were used to amplify a 693-bp fragment of theStreptococcus mutanschromosome by PCR. The amplified fragment was cloned and its identity confirmed via hybridization to anEscherichia coli recAgene probe and by nt sequence determination. TherecAhomologue fragment fromS. mutansGS-5 was 63% and 75% homologous to the deduced aa sequences of theE. coliandBacillus subtilisRecA enzymes, respectively. TheS. mutans recAfragment was mutagenized in vitro via insertional inactivation and returned to the chromosome using allelic exchange. The resulting strains ofS. mutanswere shown to be substantially more sensitive to UV irradiation than the wild-type strain. Further, the ability to incorporate linear markers into the chromosome was abolished in putativeS. mutans recAstrains, thus indicating the functional inactivation of RecA in these microorganisms.