MITOTIC GENE CONVERSION, RECIPROCAL RECOMBINATION AND GENE REPLACEMENT AT THE BENA, BETA-TUBULIN, LOCUS OF ASPERGILLUS-NIDULANS
MITOTIC GENE CONVERSION, RECIPROCAL RECOMBINATION AND GENE REPLACEMENT AT THE BENA, BETA-TUBULIN, LOCUS OF ASPERGILLUS-NIDULANS
复制标题
DOI：
10.1007/bf00339600
复制
发表时间：
1988-08-01
期刊：
影响因子：
--
通讯作者：
OAKLEY, BR
中科院分区：
文献类型：
--
作者：
DUNNE, PW;OAKLEY, BR
我们已经开发了一个程序，用于确定有丝分裂重组的中断复制的转化质粒序列的benA，β-微管蛋白，构巢曲霉基因座的整合所创建的速率。用质粒AIpGM 4转化携带苯菌灵抗性benA等位基因的菌株，所述质粒AIpGM 4携带粗糙脉孢菌的野生型benA等位基因和pyr 4（乳清酸核苷-5“-磷酸脱羧酶）基因，产生了具有两侧为两个benA等位基因（一个野生型和一个苯菌灵抗性）的质粒序列的中断复制。此类转化体在高水平苯菌灵存在下不会生长。有丝分裂重组导致野生型benA等位基因的丢失或野生型向突变型等位基因的转化，导致细胞核仅携带苯菌灵抗性等位基因。可以在具有高苯菌灵的培养基上选择含有这样的核的分生孢子，从而允许容易地定量有丝分裂重组。我们发现产生苯菌灵抗性分生孢子的重组率为4.6 × 10 - 6。10-4.导致缺乏质粒序列的苯菌灵抗性分生孢子的相互重组以2.0 × 104的速率发生。10-4并且导致苯菌灵抗性分生孢子的基因转化以2.6 × 10 - 6的速率发生。10-4.我们选择了导致5-氟乳清酸上pyr 4序列丢失的相互重组，并使用这种选择对突变型benA等位基因与野生型等位基因进行两步基因置换。
We have developed a procedure for determining the rates of mitotic recombination of an interrupted duplication created by integration of transforming plasmid sequences at the benA, beta-tubulin, locus of Aspergillus nidulans. Transformation of a strain carrying a benomyl-resistant benA allele with plasmid AIpGM4, which carries the wild-type benA allele and the pyr4 (orotidine-5''-phosphate decarboxylase) gene of Neurospora crassa, creates an interrupted duplication with plasmid sequences flanked by two benA alleles, one wild type and one benomyl resistant. Such transformants will not grow in the presence of high levels of benomyl. Mitotic recombination causes the loss of the wild-type benA allele or conversion of the wild-type to the mutant allele resulting in nuclei carrying only the benomyl-resistant allele. Conidia containing such nuclei can be selected on media with high benomyl allowing easy quantitation of mitotic recombination. We found that the rate of recombination giving rise to benomyl-resistant conidia was 4.6 .times. 10-4. Reciprocal recombination leading to benomyl-resistant conidia lacking plasmid sequences occurred at a rate of 2.0 .times. 10-4 and gene conversion leading to benomyl-resistant conidia occurred at a rate of 2.6 .times. 10-4. We selected for reciprocal recombination leading to loss of pyr4 sequences on 5-fluoro-orotic acid and used this selection for two-step gene replacement of a mutant benA allele with the wild-type allele.