Itoh Y, Scott B
Department of Plant Pathology, Tottori University, Japan.
Curr Genet. 1994 Nov-Dec;26(5-6):468-76. doi: 10.1007/BF00309936.
Mutations in a spore pigmentation locus (brs; brown spore) in Penicillium paxilli were isolated at a relatively-high frequency (0.17%) following integrative transformation of the hygromycin-resistance plasmid pAN7-1. A molecular analysis of four independently-isolated Brs- mutants showed that all contained pAN7-1 integrated at a single-site that was unique for each mutant. A previously-described Brs- mutant, YI-34 (Itoh et al. 1994), was a two-site integration. Three of the mutants had multiple copies of pAN7-1 arranged in head-to-tail tandem arrays. A 9.6-kb BamHI junction fragment was cloned from one of these, YI-33, by plasmid rescue and used to isolate two overlapping lambda clones, lambda WB33-1 and lambda WB33-2, that span about 30 kb in the region of the wild-type locus. When genomic digests of the five Brs- mutants were probed with these lambda clones all of them were found to contain an extensive deletion through a common region of the P. paxilli genome. Subsequent attempts to generate one-step gene replacements within a 4.5-kb EcoRI fragment at the wild-type locus resulted in the isolation of Brs- mutants at a frequency of 1.6%, but all mutants with this phenotype were also found to contain an extensive genomic deletion. Therefore, a common outcome of both heterologous and homologous plasmid integration at this locus is deletion formation.
在对潮霉素抗性质粒pAN7 - 1进行整合转化后，以相对较高的频率（0.17%）分离到了滑锈伞青霉孢子色素沉着位点（brs；棕色孢子）的突变体。对四个独立分离的Brs⁻突变体进行分子分析表明，所有突变体都含有整合在单个位点的pAN7 - 1，且每个突变体的整合位点都是独特的。先前描述的一个Brs⁻突变体YI - 34（Itoh等人，1994年）是双位点整合。其中三个突变体有多个pAN7 - 1拷贝以头对尾串联阵列排列。通过质粒拯救从其中一个突变体YI - 33中克隆了一个9.6 kb的BamHI连接片段，并用于分离两个重叠的λ克隆，λWB33 - 1和λWB33 - 2，它们跨越野生型位点区域约30 kb。当用这些λ克隆对五个Brs⁻突变体的基因组消化产物进行探针杂交时，发现它们都通过滑锈伞青霉基因组的一个共同区域发生了广泛缺失。随后尝试在野生型位点的一个4.5 kb EcoRI片段内进行一步基因替换，结果以1.6%的频率分离到了Brs⁻突变体，但所有具有这种表型的突变体也都被发现含有广泛的基因组缺失。因此，在这个位点进行异源和同源质粒整合的一个共同结果是形成缺失。
