CLONING, MAPPING AND MOLECULAR ANALYSIS OF THE PYRG (OROTIDINE-5'-PHOSPHATE DECARBOXYLASE) GENE OF ASPERGILLUS-NIDULANS
CLONING, MAPPING AND MOLECULAR ANALYSIS OF THE PYRG (OROTIDINE-5'-PHOSPHATE DECARBOXYLASE) GENE OF ASPERGILLUS-NIDULANS
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DOI：
10.1016/0378-1119(87)90201-0
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发表时间：
1987-01-01
期刊：
影响因子：
3.5
通讯作者：
MAY, GS
中科院分区：
文献类型：
--
作者：
OAKLEY, BR;RINEHART, JE;MAY, GS
我们已经修改了Ballance等人[Biochem.Biophys.通信资源112（1983）284-289]，以提高构巢曲霉中的转化率。用改进的方法，我们已经能够补充pyrG 89，一个在乳清酸核苷-5”-磷酸脱羧酶基因的突变。通过用pBR 329中的野生型（wt）序列文库转化nidulans。我们已经从一个这样的细菌中回收了一个携带A. nidulans序列，其有效地互补pyrG 89。在三个实验中，该质粒平均产生1985个稳定转化体/μ g转化DNA。我们已经通过基因和Southern杂交分析了其中的十个。在5个转化体中，单个拷贝的转化质粒已整合在pyrG基因座上，在100个拷贝中，几个拷贝的pJR 15已整合在该基因座上，在100个拷贝中，几个拷贝的质粒已整合到其它位点上，在3个转化体中，wt等位基因显然取代了突变等位基因，而没有整合pBR 329序列。序列和S1核酸酶保护分析表明，pJR 15含有一个基因，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酸核苷-5“-磷酸脱羧酶具有很强的同源性。我们得出结论，该基因是野生型pyrG等位基因。最后，我们将该基因的5“-和3”-非编码序列以及内含子剪接序列与其他基因进行了比较。已经将pyrG基因座定位到连锁群I上fpaB和GalD基因座之间的区域。
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 plamsid (pJR15) that carries an A. nidulans sequence that complements pyrG89 efficiently. In three experiments, this plamsid gave an average of 1985 stable transformants/.mu.g 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.