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首页> 美国政府科技报告 >Overcoming a Defect in Generalized Recombination in the Marine Fish Pathogen'Vibrio anguillarum' 775: Construction of a recA Mutant by Marker Exchange
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Overcoming a Defect in Generalized Recombination in the Marine Fish Pathogen'Vibrio anguillarum' 775: Construction of a recA Mutant by Marker Exchange

机译：克服海洋鱼类病原体'鳗弧菌'775中广义重组的缺陷：通过标记交换构建reca突变体

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A defect in generalized recombination has prevented the use of marker exchangefor the construction of specific chromosomal mutations in the marine fish pathogen Vibrio anguillarum 775. Through the use of large segments of homologous DNA, we were successful in overcoming this defect and used marker exchange to construct a recA mutant of V. anguillarum H775-3. A recombinant cosmid carrying the recA gene of V. anguillarum 775 in the center of a 25-kb cloned DNA insert was isolated by complementation of methyl methanesulfonate (MMS) sensitivity in Escherichia coli HB101. The recA gene was inactivated by inserting a kanamycin resistance gene into recA, and the mutant gene was subsequently introduced into V. anguillarum H775-3 by conjugal mobilization. Isolation of recombinants between cosmid-borne recA::kan sequences and chromosomal DNA was facilitated by the introduction of an incompatible plasmid, and Southern hybridization was used to verify the presence of recA::kan in the chromosomal DNA of the recA mutant. V. anguillarum carring recA::kan was considerable more sensitive to UV radiation and to MMS than was its parent, and near wild-type levels of resistance to MMS and UV light were restored by introduction of cloned recA genes from both E coli and V. anguillarum.

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