Miao V P, Rountree M R, Selker E U
Institute of Molecular Biology, University of Oregon, Eugene 97403, USA.
Genetics. 1995 Apr;139(4):1533-44. doi: 10.1093/genetics/139.4.1533.
In a variety of organisms, DNA-mediated transformation experiments commonly produce transformants with multiple copies of the transforming DNA, including both selected and unselected molecules. Such "cotransformants" are much more common than expected from the individual transformation frequencies, suggesting that subpopulations of cells, or nuclei, are particularly competent for transformation. We found that Neurospora crassa transformants selected for gene replacement at the am gene had not efficiently incorporated additional DNA, suggesting that nuclei that undergo transformation by homologous recombination are not highly competent at integration of DNA by illegitimate recombination. Spheroplasts were treated with DNA fragments homologous to am and with an Escherichia coli hph plasmid. Transformants were initially selected for hph (hygromycinR), allowed to conidiate to generate homokaryons and then selected for either Am- (gene replacements) or hph. Surprisingly, most am replacement strains were hygromycinS (124/140) and carried no extraneous DNA (116/140). Most transformants selected for hph also had ectopic copies of am DNA and/or multiple copies of hph sequences (32/35), generally at multiple sites, confirming that efficient cotransformation could occur. To test the implication that cotransformation involving gene replacement and ectopic integration is rare, we compared the yields of am replacement strains with or without prior selection for hph. The initial selection did not appreciably help (or hinder) recovery of strains with replacements.
在多种生物体中，DNA介导的转化实验通常会产生带有多个转化DNA拷贝的转化体，包括已选择和未选择的分子。这种“共转化体”比根据单个转化频率预期的要常见得多，这表明细胞或细胞核的亚群对转化特别敏感。我们发现，在am基因处选择用于基因替换的粗糙脉孢菌转化体并没有有效地整合额外的DNA，这表明通过同源重组进行转化的细胞核在通过非同源重组整合DNA方面并不高效。原生质球用与am同源的DNA片段和大肠杆菌hph质粒处理。转化体最初选择hph（潮霉素抗性），使其分生以产生同核体，然后选择Am-（基因替换）或hph。令人惊讶的是，大多数am替换菌株是潮霉素敏感型（124/140）且不携带额外的DNA（116/140）。大多数选择hph的转化体也有am DNA的异位拷贝和/或hph序列的多个拷贝（32/35），通常在多个位点，证实了高效共转化可能发生。为了测试涉及基因替换和异位整合的共转化很少见这一观点，我们比较了预先选择hph和未预先选择hph的情况下am替换菌株的产量。最初的选择对替换菌株的回收没有明显帮助（或阻碍）。
