The site-specific recombination locus of mycobacteriophage Ms6 determines DNA integration at the tRNAAla gene of Mycobacterium spp.
The site-specific recombination locus of mycobacteriophage Ms6 determines DNA integration at the tRNAAla gene of Mycobacterium spp.
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DOI：
10.1099/00221287-144-12-3397
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发表时间：
1998-12-01
期刊：
影响因子：
2.8
通讯作者：
Moniz-Pereira, J
中科院分区：
文献类型：
--
作者：
Freitas-Vieira, A;Anes, E;Moniz-Pereira, J
鉴定了染色体整合所需的温和分枝杆菌噬菌体Ms 6的遗传决定簇。含attP的片段的DNA序列分析揭示了编码372个氨基酸残基的蛋白质的ORF，其C-末端类似于噬菌体整合酶家族典型的其他保守C-末端区域。比较attP、attB和细菌-原噬菌体接头attL和attR的序列，显示在整合酶基因的5'端附近有一个26 bp的共同核心序列，在此发生重组。对attB染色体区域的核苷酸序列分析表明，核心位点与tRNA（Ala）基因的3'端重叠。构建了整合能力强的质粒载体，并将其有效地插入到耻垢分枝杆菌、母牛分枝杆菌、牛分枝杆菌BCG和结核分枝杆菌H37 Ra的tRNA（Ala）基因处。已证明Ms 6和D29整合系统可联合用于在多个基因座处插入基因。分枝杆菌噬菌体Ms 6的位点特异性整合系统是分枝杆菌遗传分析的新工具，与L5噬菌体家族的整合系统相关性很低。
Genetic determinants of the temperate mycobacteriophage Ms6 required for chromosomal integration were identified. DNA sequence analysis of an attP-containing fragment revealed an ORF encoding a protein of 372 amino acid residues with a C-terminus similar to other conserved C-terminal regions typical of the phage integrase family. Comparison of the sequences of attP, attB and bacteria-prophage junctions attL and attR showed a 26 bp common core sequence, where recombination takes place, near the 5' end of the integrase gene. Nucleotide sequence analysis of the attB chromosomal region showed that the core site overlaps the 3' end of the tRNA(Ala) gene. An integration-proficient plasmid vector was constructed and efficiently inserted at the tRNA(Ala) gene of Mycobacterium smegmatis, Mycobacterium vaccae, Mycobacterium bovis BCG and Mycobacterium tuberculosis H37Ra. It was demonstrated that Ms6 and D29 integrative systems can be used in conjunction for inserting genes at multiple loci. The site-specific integration system of mycobacteriophage Ms6 is a new tool for mycobacterial genetic analysis and is poorly related to those of the L5 bacteriophage family.