Cloning of chromosomal genes in Streptococcus pneumoniae.
Cloning of chromosomal genes in Streptococcus pneumoniae.
复制标题
肺炎链球菌染色体基因的克隆。
DOI：
10.1073/pnas.78.11.7028
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发表时间：
1981
影响因子：
11.1
通讯作者：
Lacks,SA
中科院分区：
文献类型：
--
作者：
Stassi,DL;Lopez,P;Espinosa,M;Lacks,SA
开发了肺炎链球菌分子克隆系统。使用赋予四环素抗性的多拷贝质粒pMV158（5.4kb）和pLS1（4.3kb）作为载体，在该物种的宿主细胞中克隆肺炎链球菌的染色体基因。含有编码麦芽糖淀粉酶的 malM 基因的 3.3-kilobase 限制性片段被克隆到与该片段缺乏染色体同源性的缺失突变体中。重组质粒 pLS70 可以将超过 50% 的受体群体转化为麦芽糖利用。淀粉麦芽糖酶占含有 pLS70 的细胞蛋白质的 10%。具有缺失的衍生物 pLS69 似乎通过产生更少的酶而获得了选择优势。在同源染色体基因存在的情况下，克隆了含有磺胺抗性的 sul-d 基因的 10 kb 限制性片段。重组质粒的从头建立与内源质粒的转化一样频繁。尽管在肺炎链球菌转化过程中摄取DNA时会进行加工，但仍可引入重组质粒。考虑并比较了肺炎链球菌和枯草芽孢杆菌细胞中重组 DNA 的重建模型。
A system for molecular cloning in Streptococcus pneumoniae was developed. The multicopy plasmids pMV158 (5.4 kilobases) and pLS1 (4.3 kilobases), which confer tetracycline resistance, were used as vectors to clone chromosomal genes of S. pneumoniae in host cells of this species. A 3.3-kilobase restriction fragment containing the malM gene, which codes for amylomaltase, was cloned in a deletion mutant lacking chromosomal homology with the fragment. The recombinant plasmid pLS70, could transform over 50% of a recipient population to maltose utilization. Amylomaltase constituted up to 10% of the protein of cells containing pLS70. A derivative with a deletion, pLS69, appeared to gain a selective advantage by producing less enzyme. A 10-kilobase restriction fragment containing the sul-d gene for sulfonamide resistance was cloned in the presence of the homologous chromosomal gene. De novo establishment of a recombinant plasmid was just as frequent as transformation in an endogenous plasmid. Despite the processing of DNA during uptake in the transformation of S. pneumoniae, recombinant plasmids can be introduced. Models for the reconstruction of recombinant DNA in cells of S. pneumoniae and Bacillus subtilis are considered and compared.