Hargreaves J A, Keon J P
Department of Cell Biology, University of Bristol, UK.
FEMS Microbiol Lett. 1996 Jun 1;139(2-3):203-7. doi: 10.1111/j.1574-6968.1996.tb08203.x.
A gene (ERG11) encoding cytochrome P450 sterol 14 alpha-demethylase (P450(14DM)) was isolated from the maize pathogen, Ustilago maydis, by amplifying part of the coding region of the gene using PCR and by employing the amplified DNA fragment as a hybridization probe to recover the complete gene from an U. maydis lambda EMBL3 genomic library. The deduced amino acid sequence of the U. maydis gene showed homology to P450(14DM)s from other organisms and contained specific motifs which were hallmarks of P450s. Expression of the gene in an U. maydis mutant (A20) deficient in P450(14DM) led to only a partial restoration of P450(14DM) activity. Accumulation of ergosta-7,22-dienol and ergost-7-enol in A20 transformants containing the ERG11 gene implied that an additional mutation affecting sterol delta 5,6-desaturase activity accompanied the P450(14DM) lesion.
通过使用聚合酶链反应（PCR）扩增基因的部分编码区，并将扩增的DNA片段用作杂交探针，从玉米病原体玉米黑粉菌（Ustilago maydis）中分离出编码细胞色素P450固醇14α-脱甲基酶（P450(14DM)）的基因（ERG11），以从玉米黑粉菌λEMBL3基因组文库中回收完整基因。玉米黑粉菌基因推导的氨基酸序列与来自其他生物体的P450(14DM)具有同源性，并包含作为P450特征的特定基序。该基因在缺乏P450(14DM)的玉米黑粉菌突变体（A20）中的表达仅导致P450(14DM)活性的部分恢复。含有ERG11基因的A20转化体中麦角甾-7,22-二烯醇和麦角甾-7-烯醇的积累表明，影响甾醇δ5,6-去饱和酶活性的另一个突变与P450(14DM)损伤同时发生。
