Orbach M J, Schneider W P, Yanofsky C
Department of Biological Sciences, Stanford University, California 94305-5020.
Mol Cell Biol. 1988 May;8(5):2211-3. doi: 10.1128/mcb.8.5.2211-2213.1988.
An arg-2 mutant of Neurospora crassa was transformed to prototrophy with a pBR322-N. crassa genomic DNA library. Repeated attempts to recover the integrated transforming DNA or segments thereof by digestion, ligation, and transformation of Escherichia coli, with selection for the plasmid marker ampicillin resistance, were unsuccessful. Analyses of a N. crassa transformant demonstrated that the introduced DNA was heavily methylated at cytosine residues. This methylation was shown to be responsible for our inability to recover transformants in standard strains of E. coli; transformants were readily obtained in a strain which is deficient in the two methylcytosine restriction systems. Restriction of methylated DNA in E. coli may explain the general failure to recover vector or transforming sequences from N. crassa transformants.
用pBR322-粗糙脉孢菌基因组DNA文库将粗糙脉孢菌的一个arg-2突变体转化为原养型。通过对大肠杆菌进行消化、连接和转化，并选择质粒标记氨苄青霉素抗性，多次尝试回收整合的转化DNA或其片段均未成功。对一个粗糙脉孢菌转化体的分析表明，导入的DNA在胞嘧啶残基处高度甲基化。这种甲基化被证明是我们无法在大肠杆菌标准菌株中回收转化体的原因；在缺乏两种甲基胞嘧啶限制系统的菌株中很容易获得转化体。大肠杆菌中甲基化DNA的限制可能解释了从粗糙脉孢菌转化体中普遍无法回收载体或转化序列的原因。
