MAPPING NON-SELECTABLE GENES OF ESCHERICHIA-COLI BY USING TRANSPOSON TN10 - LOCATION OF A GENE AFFECTING PYRUVATE OXIDASE
MAPPING NON-SELECTABLE GENES OF ESCHERICHIA-COLI BY USING TRANSPOSON TN10 - LOCATION OF A GENE AFFECTING PYRUVATE OXIDASE
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DOI：
10.1128/jb.151.3.1279-1289.1982
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发表时间：
1982-01-01
影响因子：
3.2
通讯作者：
CRONAN, JE
中科院分区：
文献类型：
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作者：
CHANG, YY;CRONAN, JE
利用Tabor等人的方法，通过筛选[1-14C]丙酮酸产生14CO2的方法，筛选出丙酮酸氧化酶缺陷型大肠杆菌K-12。(J.细菌素)128：485-486,1976)。其中一个病变(指定为PoxA)将丙酮酸氧化酶活性降低到正常水平的10%至15%，但生长良好。为了定位这种不可选择的突变，我们分离了将转座子Tn10插入到靠近poxA位点的染色体中的菌株，并定位了转座子。通过以下步骤分离这些插入片段：(I)通过转座从lambda：：Tn10载体中制备Tn10插入到两个不同HFR菌株染色体中的池；(Ii)这些携带Tn10的菌株随后与PoxA受体菌株交配，并选择四环素抗性(TetR)重组子；(Iii)筛选TetR重组子以从[1-14C]丙酮酸中产生14CO2。与转导相比，这种方法在poxA基因附近插入Tn10的丰度超过40倍。计算表明，其他基因应该也会有类似的浓缩。这是由于与转导杂交相比，共轭杂交中选择标记和未选择标记之间有很强的连锁关系的图谱间隔要大得多。使用HFR接合转移可以通过获得数百个而不是数千个菌落来分离与非选择基因紧密连锁的转座子插入。利用与poxA基因座共转导大于98%的Tn10插入片段，我们将poxA基因定位在大肠杆菌的遗传图谱上。PoxA基因定位于94min，靠近PSD基因。基因顺时针顺序为AMPA、poxA、PSD、PURA。PoxA突变是隐性的，似乎是一个调控基因。
Mutants of Escherichia coli K-12 deficient in pyruvate oxidase were isolated by screening for the production of 14CO2 from [1-14C]pyruvate by the method of Tabor et al. (J. Bacteriol. 128:485-486, 1976). One of these lesions (designated poxA) decreased the pyruvate oxidase activity to 10 to 15% of the normal level but grew well. To map this nonselectable mutation, we isolated strains having transposon Tn10 inserted into the chromosome close to the poxA locus and mapped the transposon. These insertions were isolated by the following procedure: (i) pools of Tn10 insertions into the chromosomes of two different Hfr strains were prepared by transposition from a lambda::Tn10 vector; (ii) these Tn10-carrying strains were then mated with a poxA recipient strain, and tetracycline-resistant (Tetr) recombinants were selected; (iii) the Tetr recombinants were then screened for 14CO2 production from [1-14C]pyruvate. This method was shown to give a greater than 40-fold enrichment of insertions of Tn10 near the poxA gene as compared with transduction. Calculations indicate that a similar enrichment should be expected for other genes. The enrichment is due to the much greater map interval over which strong linkage between selected and unselected markers is found in conjugational crosses as compared with transductional crosses. The use of Hfr conjugative transfer allows isolation of transposon insertions closely linked to a nonselectable gene by scoring hundreds rather than thousands of colonies. Using a Tn10 insertion greater than 98% cotransduced with the poxA locus, we mapped the poxA gene on the E. coli genetic map. The poxA locus is located at 94 min, close to the psd locus. The clockwise gene order is ampA, poxA, psd, purA. The poxA mutation is recessive and appears to be a regulatory gene.