INSERTIONAL MUTAGENESIS BY RANDOM CLONING OF ANTIBIOTIC-RESISTANCE GENES INTO THE GENOME OF THE CYANOBACTERIUM SYNECHOCYSTIS STRAIN PCC-6803
INSERTIONAL MUTAGENESIS BY RANDOM CLONING OF ANTIBIOTIC-RESISTANCE GENES INTO THE GENOME OF THE CYANOBACTERIUM SYNECHOCYSTIS STRAIN PCC-6803
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DOI：
10.1128/jb.171.6.3449-3457.1989
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发表时间：
1989-06-01
影响因子：
3.2
通讯作者：
THURIAUX, P
中科院分区：
文献类型：
--
作者：
LABARRE, J;CHAUVAT, F;THURIAUX, P
将HaeII基因片段随机连接到宿主基因组的HaeII DNA片段上，转化兼性异养蓝藻（synhocystis sp.） PCC 6803菌株。用连接到AvaII宿主DNA片段的AvaII Kmr标记进行了类似的转化。将抗性标记整合到宿主基因组中导致稳定的Kmr和Cmr转化子频率高。对单个转化子的物理分析表明，这一结果是由于同源重组导致Cmr（或Kmr）基因插入宿主基因组的两个HaeII（或AvaII）位点之间，并在这些位点之间精确删除宿主DNA。相比之下，与宿主DNA同源的环状DNA分子的整合交叉在这种蓝藻中非常罕见。菌株PCC 6803在标准生长条件下每个细胞大约有12个基因组拷贝，这使得化学或紫外线诱变诱导的隐性突变的检测变得复杂。通过插入转化对宿主DNA的随机破坏为蓝藻提供了一种方便的转座子诱变替代方法，并有助于克服传统诱变产生隐性突变所遇到的困难。
The facultative heterotrophic cyanobacterium Synehocystis sp. strain PCC 6803 was transformed by HaeII Cmr fragments ligated at random to HaeII DNA fragments of the host genome. A similar transformation was done with an AvaII Kmr marker ligated to AvaII host DNA fragments. Integration of the resistance markers into the host genome led to a high frequency of stable Kmr and Cmr transformants. Physical analysis of individual transformants indicated that this result was due to homologous recombination by conversionlike events leading to insertion of the Cmr (or Kmr) gene between two HaeII (or AvaII) sites of the host genome, with precise deletion of the host DNA between these sites. In contrast, integrative crossover of circular DNA molecules with homology to the host DNA is very rare in this cyanobacterium. Strain PCC 6803 was shown to have about 12 genomic copies per cell in standard growth conditions, which complicates the detection of recessive mutations induced by chemical or UV mutagenesis. Random disruption of the host DNA by insertional transformation provides a convenient alternative to transposon mutagenesis in cyanobacteria and may help to overcome the difficulties encountered in generating recessive mutants by classical mutagenesis.