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首页> 外文期刊>Molecular and Cellular Biology >Cloning of the PYR3 gene of Ustilago maydis and its use in DNA transformation.
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Cloning of the PYR3 gene of Ustilago maydis and its use in DNA transformation.

机译：玉米Us的PYR3基因的克隆及其在DNA转化中的应用。

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The Ustilago maydis PYR3 gene encoding dihydroorotase activity was cloned by direct complementation of Escherichia coli pyrC mutations. PYR3 transformants of E. coli pyrC mutants expressed homologous transcripts of a variety of sizes and regained dihydroorotase activity. PYR3 also complemented Saccharomyces cerevisiae ura4 mutations, and again multiple transcripts were expressed in transformants, and enzyme activity was regained. A 1.25-kilobase poly(rA)+ PYR3 transcript was detected in U. maydis itself. Linear DNA carrying the PYR3 gene transformed a U. maydis pyr3-1 pyrimidine auxotroph to prototrophy. Hybridization analysis revealed that three different types of transformants could be generated, depending on the structure of the transforming DNA used. The first type involved exchange of chromosomal mutant gene sequences with the cloned wild-type plasmid sequences. A second type had integrated linear transforming DNA at the chromosomal PYR3 locus, probably via a single crossover event. The third type had integrated transforming DNA sequences at multiple sites in the U. maydis genome. In the last two types, tandemly reiterated copies of the transforming DNA were found to have been integrated. All three types had lost the sensitivity of the parental pyr3-1 mutant to UV irradiation. They had also regained dihydroorotase activity, although its level did not correlate with the PYR3 gene copy number.
机译：通过直接互补的大肠杆菌pyrC突变克隆了编码双氢乳清酶活性的乌斯地亚哥maydis PYR3基因。大肠杆菌pyrC突变体的PYR3转化体表达了各种大小的同源转录本，并恢复了二氢乳清酶活性。 PYR3还补充了酿酒酵母ura4突变，并且在转化体中再次表达了多个转录本，并恢复了酶活性。在maydis本身中检测到1.25千碱基的poly（rA）+ PYR3转录本。带有PYR3基因的线性DNA将美狄氏酵母pyr3-1嘧啶营养缺陷型转化为原养型。杂交分析表明，取决于所用转化DNA的结构，可以产生三种不同类型的转化体。第一类涉及染色体突变基因序列与克隆的野生型质粒序列的交换。第二种可能是通过一次交叉事件在染色体PYR3位点整合了线性转化DNA。第三类在梅迪斯氏菌基因组的多个位点具有整合的转化DNA序列。在最后两种类型中，发现重组DNA的串联重复拷贝已整合。所有这三种类型都失去了亲本pyr3-1突变体对UV辐射的敏感性。他们也恢复了二氢乳清酶活性，尽管其水平与PYR3基因拷贝数无关。

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