Chang L F, Cunningham T S, Gatzek P R, Chen W J, Kohlhaw G B
Genetics. 1984 Sep;108(1):91-106. doi: 10.1093/genetics/108.1.91.
By complementation of an alpha-isopropylmalate synthase-negative mutant of Saccharomyces cerevisiae (leu4 leu5), a plasmid was isolated that carried a structural gene for alpha-isopropylmalate synthase. Restriction mapping and subcloning showed that sequences sufficient for complementation of the leu4 leu5 strain were located within a 2.2-kilobase SalI-PvuII segment. Southern transfer hybridization indicated that the cloned DNA was derived intact from the yeast genome. The cloned gene was identified as LEU4 by integrative transformation that caused gene disruption at the LEU4 locus. When this transformation was performed with a LEU4fbr LEU5 strain, the resulting transformants had lost the 5',5',5'-trifluoro-D,L-leucine resistance of the recipient strain but were still Leu+. When it was performed with a LEU4 leu5 recipient, the resulting transformants were Leu-. The alpha-isopropylmalate synthase of a transformant that carried the LEU4 gene on a multicopy plasmid (in a leu5 background) was characterized biochemically. The transformant contained about 20 times as much alpha-isopropylmalate synthase as wild type. The enzyme was sensitive to inhibition by leucine and coenzyme A, was inactivated by antibody generated against alpha-isopropylmalate synthase purified from wild type and was largely confined to the mitochondria. The subunit molecular weight was 65,000-67,000. Limited proteolysis generated two fragments with molecular weights of about 45,000 and 23,000. Northern transfer hybridization showed that the transformant produced large amounts of LEU4-specific RNA with a length of about 2.1 kilonucleotides. The properties of the plasmid-encoded enzyme resemble those of a previously characterized alpha-isopropylmalate synthase that is predominant in wild-type cells. The existence in yeast of a second alpha-isopropylmalate synthase activity that depends on the presence of an intact LEU5 gene is discussed.
通过对酿酒酵母α-异丙基苹果酸合酶阴性突变体（leu4 leu5）进行互补，分离出了一个携带α-异丙基苹果酸合酶结构基因的质粒。限制性酶切图谱分析和亚克隆表明，足以互补leu4 leu5菌株的序列位于一个2.2千碱基的SalI - PvuII片段内。Southern印迹杂交表明，克隆的DNA完整地来源于酵母基因组。通过导致LEU4位点基因破坏的整合转化，将克隆基因鉴定为LEU4。当用LEU4fbr LEU5菌株进行这种转化时，所得转化体失去了受体菌株对5',5',5'-三氟-D,L-亮氨酸的抗性，但仍然是亮氨酸营养缺陷型（Leu+）。当用LEU4 leu5受体进行转化时，所得转化体是亮氨酸营养缺陷型（Leu-）。对在多拷贝质粒上携带LEU4基因的转化体（在leu5背景下）的α-异丙基苹果酸合酶进行了生化特性分析。该转化体所含的α-异丙基苹果酸合酶约为野生型的20倍。该酶对亮氨酸和辅酶A的抑制敏感，被针对从野生型纯化的α-异丙基苹果酸合酶产生的抗体所灭活，并且主要定位于线粒体。亚基分子量为65,000 - 67,000。有限的蛋白酶解产生了两个分子量约为45,000和23,000的片段。Northern印迹杂交表明，该转化体产生了大量长度约为2.1千核苷酸的LEU4特异性RNA。质粒编码酶的特性类似于先前在野生型细胞中占主导地位的已表征的α-异丙基苹果酸合酶的特性。讨论了酵母中第二种依赖完整LEU5基因存在的α-异丙基苹果酸合酶活性的存在情况。
