Genetic and structural characterization of endA. A membrane-bound nuclease required for transformation of Streptococcus pneumoniae.
Genetic and structural characterization of endA. A membrane-bound nuclease required for transformation of Streptococcus pneumoniae.
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DOI：
10.1016/s0022-2836(05)80259-1
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发表时间：
1990-06
影响因子：
5.6
通讯作者：
Antonio Puyet;B. Greenberg;S. Lacks
中科院分区：
文献类型：
--
作者：
Antonio Puyet;B. Greenberg;S. Lacks
将编码肺炎链球菌膜核酸酶的endA基因克隆到链球菌载体中，该基因是遗传转化中DNA摄取所必需的。这是通过基因的插入诱变、突变等位基因的克隆和通过质粒建立的染色体促进取代野生型等位基因来实现的。携带endA+基因的质粒补充了染色体中含有endA −的细胞，以恢复DNA酶活性和转化能力。DNA序列测定表明，该基因编码一个分子量为30 kDa的蛋白EndA，其氨基端具有典型的膜转运信号序列，体外合成结果表明，该基因的翻译产物不经进一步加工即具有酶活性。与在细胞膜中发现的EndA比较表明，该酶保留了其信号序列，该序列显然将亲水性蛋白质锚定在膜上。从末端A附近的核苷酸序列以及各种插入和缺失的影响来看，似乎末端A是包含至少两个其他基因的操纵子中的最后一个基因。这些上游基因和下游基因都不是细胞活力或可转化性所必需的。
TheendAgene encoding the membrane nuclease ofStreptococcus pneumoniae, which is necessary for DNA uptake in genetic transformation, was cloned in a streptococcal vector. This was accomplished by insertional mutagenesis of the gene, cloning of the mutant allele, and substitution of the wild-type allele by chromosomal facilitation of plasmid establishment. Plasmids carrying theendA+gene complemented cells withendA−in the chromosome to restore DNAase activity and transformability. Determination of its DNA sequence showed the gene to encode a 30 kDa protein, EndA, with a typical signal sequence for membrane transport at its amino end.In vitrosynthesis of EndA showed the initial translation product to be enzymatically active without further processing. Comparison with EndA found in cell membranes indicated that the enzyme retained its signal sequence, which apparently anchored the otherwise hydrophilic protein to the membrane. From the nucleotide sequence in the vicinity ofendAand the effect of various insertions and deletions, it appears thatendAis the last gene in an operon containing at least two other genes. Neither of these upstream genes, nor the downstream gene, are essential for either cell viability or transformability.