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首页> 外文期刊>Molecular and Cellular Biology >Direct and indirect gene replacements in Aspergillus nidulans.
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Direct and indirect gene replacements in Aspergillus nidulans.

机译：Nidulans中的直接和间接基因替代。

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We performed three sets of experiments to determine whether cloned DNA fragments can be substituted for homologous regions of the Aspergillus nidulans genome by DNA-mediated transformation. A linear DNA fragment containing a heteromorphic trpC+ allele was used to transform a trpC- strain to trpC+. Blot analysis of DNA from the transformants showed that the heteromorphic allele had replaced the trpC- allele in a minority of the strains. An A. nidulans trpC+ gene was inserted into the argB+ gene, and a linear DNA fragment containing the resultant null argB allele was used to transform a trpC- argB+ strain to trpC+. Approximately 30% of the transformants were simultaneously argB-. The null argB allele had replaced the wild-type allele in a majority of these strains. The A. nidulans SpoC1 C1-C gene was modified by removal of an internal restriction fragment and introduced into a trpC- strain by transformation with a circular plasmid. A transformant containing a tandem duplication of the C1-C region separated by plasmid DNA was self-fertilized, and trpC- progeny were selected. All of these had lost the introduced plasmid DNA sequences, whereas about half had retained the modified C1-C gene and lost the wild-type copy. Thus, it is possible with A. nidulans to replace chromosomal DNA sequences with DNA fragments that have been cloned and modified in vitro by using either one- or two-step procedures similar to those developed for Saccharomyces cerevisiae.
机译：我们进行了三组实验，以确定克隆的DNA片段是否可以通过DNA介导的转化来替代构巢曲霉基因组的同源区域。包含异质性trpC +等位基因的线性DNA片段用于将trpC-菌株转化为trpC +。对来自转化体的DNA的印迹分析表明，异质等位基因已取代了少数菌株中的trpC-等位基因。将构巢曲霉（A.nidulans）的trpC +基因插入到argB +基因中，并使用包含所得的无效argB等位基因的线性DNA片段将trpC-argB +菌株转化为trpC +。大约30％的转化体同时是argB-。在大多数菌株中，无效的argB等位基因已取代了野生型等位基因。通过去除内部限制片段来修饰构巢曲霉SpoC1 C1-C基因，并通过用环状质粒转化将其引入到trpC菌株中。将含有通过质粒DNA分离的C1-C区域的串联重复序列的转化子进行自我受精，然后选择trpC-后代。所有这些都丢失了引入的质粒DNA序列，而大约一半保留了修饰的C1-C基因并丢失了野生型拷贝。因此，用构巢曲霉可以用与酿酒酵母相似的一步或两步方法，用体外克隆和修饰的DNA片段代替染色体DNA序列。

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