MUTATIONS IN THE ESCHERICHIA-COLI DNAG GENE SUGGEST COUPLING BETWEEN DNA-REPLICATION AND CHROMOSOME PARTITIONING
MUTATIONS IN THE ESCHERICHIA-COLI DNAG GENE SUGGEST COUPLING BETWEEN DNA-REPLICATION AND CHROMOSOME PARTITIONING
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DOI：
10.1128/jb.173.3.1268-1278.1991
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发表时间：
1991-02-01
影响因子：
3.2
通讯作者：
LUPSKI, JR
中科院分区：
文献类型：
--
作者：
GROMPE, M;VERSALOVIC, J;LUPSKI, JR
11个条件性致死dnaG（Ts）突变的化学切割之间形成的异源双链聚合酶链反应扩增的DNA从野生型和突变dnaG基因。 这需要末端标记异源双链的一条DNA链，在错配位点用羟胺或四氧化锇（OsO 4）化学修饰链，并用哌啶切割它们。 对切割产物进行测序，其大小对应于相对于标记引物的突变位置。 然后通过DNA序列分析确定确切的碱基对变化。 dnaG 3、dnaG 308和dnaG 399突变在dnaG中间附近的135个核苷酸内相互映射。 dnaG的“parB”等位基因距离dnaG的3'端36 bp，距离dnaG 2903下游9 bp;两者似乎都导致异常染色体分配和弥漫性类核染色。 dnaG 2903等位基因的抑制子（sdgA 5）在终止子T1内定位于dnaG基因的5'端。 携带dnaG 2903且携带或不携带sdgA 5抑制子的等基因菌株通过相差显微镜和荧光显微镜的组合进行分析，所述荧光显微镜用4 '，6-二脒基-2-苯基吲哚染色DNA并使分配染色体可视化。 突变dnaG等位基因的过表达纠正了与正常表达的dnaG 2903相关的异常弥散性类核染色表型。 dnaG基因内的突变似乎聚集成两个区域，这可能代表引发酶蛋白内不同的功能结构域。
Eleven conditional lethal dnaG(Ts) mutations were located by chemical cleavage of heteroduplexes formed between polymerase chain reaction-amplified DNAs from wild-type and mutant dnaG genes. This entailed end labeling one DNA strand of the heteroduplex, chemically modifying the strands with hydroxylamine or osmium tetroxide (OsO4) at the site of mismatch, and cleaving them with piperidine. The cleavage products were electrophoresed, and the size corresponded to the position of the mutation with respect to the labeled primer. Exact base pair changes were then determined by DNA sequence analysis. The dnaG3, dnaG308, and dnaG399 mutations map within 135 nucleotides of one another near the middle of dnaG. The "parB" allele of dnaG is 36 bp from the 3' end of dnaG and 9 bp downstream of dnaG2903; both appear to result in abnormal chromosome partitioning and diffuse nucleoid staining. A suppressor of the dnaG2903 allele (sdgA5) maps within the terminator T1 just 5' to the dnaG gene. Isogenic strains that carried dnaG2903 and did or did not carry the sdgA5 suppressor was analyzed by a combination of phase-contrast and fluorescence microscopy with 4', 6-diamidino-2-phenylindole to stain DNA and visualize the partitioning chromosome. Overexpression of the mutant dnaG allele corrected the abnormal diffuse-nucleoid-staining phenotype associated with normally expressed dnaG2903. The mutations within the dnaG gene appear to cluster into two regions which may represent distinct functional domains within the primase protein.