Oakley B R, Rinehart J E, Mitchell B L, Oakley C E, Carmona C, Gray G L, May G S
Department of Molecular Genetics, Ohio State University, Columbus 43210.
Gene. 1987;61(3):385-99. doi: 10.1016/0378-1119(87)90201-0.
We have modified the transformation procedures of Ballance et al. [Biochem. Biophys. Res. Commun. 112 (1983) 284-289] to give increased rates of transformation in Aspergillus nidulans. With the modified procedures we have been able to complement pyrG89, a mutation in the orotidine-5'-phosphate decarboxylase gene of A. nidulans, by transformation with a library of wild-type (wt) sequences in pBR329. We have recovered, by marker rescue from one such transformant, a plasmid (pJR15) that carries an A. nidulans sequence that complements pyrG89 efficiently. In three experiments, this plasmid gave an average of 1985 stable transformants/micrograms of transforming DNA. We have analyzed ten of these genetically and by Southern hybridization. In five transformants a single copy of the transforming plasmid had integrated at the pyrG locus, in one transformant several copies of pJR15 had integrated at this locus, in one transformant several copies of the plasmid had integrated into other sites, and in three transformants, the wt allele had apparently replaced the mutant allele with no integration of pBR329 sequences. Sequence and S1 nuclease protection analysis revealed that pJR15 contains a gene that predicts an amino acid sequence with regions of strong homology to the orotidine-5'-phosphate decarboxylases of Neurospora crassa and Saccharomyces cerevisiae. We conclude that this gene is the wt pyrG allele. Finally, we have compared the 5'- and 3'-noncoding sequences and intron splice sequences to other genes of A. nidulans and have mapped the pyrG locus to a region between the fpaB and galD loci on linkage group I.
我们对巴伦斯等人[《生物化学与生物物理学研究通讯》112(1983)284 - 289]的转化程序进行了改进，以提高构巢曲霉的转化效率。采用改进后的程序，我们能够通过用pBR329中的野生型（wt）序列文库进行转化，来互补构巢曲霉乳清苷 - 5'-磷酸脱羧酶基因中的pyrG89突变。我们从一个这样的转化体中通过标记拯救回收了一个质粒（pJR15），该质粒携带一个能有效互补pyrG89的构巢曲霉序列。在三个实验中，该质粒每微克转化DNA平均产生1985个稳定转化体。我们对其中十个进行了遗传学分析和Southern杂交分析。在五个转化体中，转化质粒的单拷贝整合到了pyrG位点；在一个转化体中，多个pJR15拷贝整合到了该位点；在一个转化体中，多个质粒拷贝整合到了其他位点；在三个转化体中，野生型等位基因显然取代了突变等位基因，且没有pBR329序列的整合。序列和S1核酸酶保护分析表明，pJR15包含一个基因，该基因预测的氨基酸序列与粗糙脉孢菌和酿酒酵母的乳清苷 - 5'-磷酸脱羧酶有高度同源区域。我们得出结论，这个基因就是野生型pyrG等位基因。最后，我们将5'和3'非编码序列以及内含子剪接序列与构巢曲霉的其他基因进行了比较，并将pyrG位点定位到了连锁群I上fpaB和galD位点之间的区域。
