• 主题
高级搜索  >
历史搜索 清空历史
首页> 美国卫生研究院文献>Eukaryotic Cell >C-5(6) Sterol Desaturase from Tetrahymena thermophila: Gene Identification and Knockout Sequence Analysis and Comparison to Other C-5(6) Sterol Desaturases
【2h】

C-5(6) Sterol Desaturase from Tetrahymena thermophila: Gene Identification and Knockout Sequence Analysis and Comparison to Other C-5(6) Sterol Desaturases

机译：嗜热四膜虫的C-5（6）甾醇去饱和酶：基因鉴定和敲除序列分析和与其他C-5（6）甾醇去饱和酶的比较

代理获取
本网站仅为用户提供外文OA文献查询和代理获取服务，本网站没有原文。下单后我们将采用程序或人工为您竭诚获取高质量的原文，但由于OA文献来源多样且变更频繁，仍可能出现获取不到、文献不完整或与标题不符等情况，如果获取不到我们将提供退款服务。请知悉。
页面导航
  • 摘要
  • 著录项
  • 相似文献
  • 相关主题

摘要

The gene coding for a C-5(6) sterol desaturase in Tetrahymena thermophila, DES5A, has been identified by the knockout of the TTHERM_01194720 sequence. Macronucleus transformation was achieved by biolistic bombardment and gene replacement through phenotypic assortment, using paromomycin as the selective agent. A knockout cell line (KO270) showed a phenotype consistent with that of the DES5A deletion mutant. KO270 converted only 6% of the added sterol into the C-5 unsaturated derivative, while the wild type accumulated 10-fold larger amounts under similar conditions. The decreased desaturation activity is specific for the C-5(6) position of lathosterol and cholestanol; other desaturations, namely C-7(8) and C-22(23), were not affected. Analysis by reverse transcription-PCR reveals that DES5A is transcribed both in the presence and absence of cholestanol in wild-type cells, whereas the transcribed gene was not detected in KO270. The growth of KO270 was undistinguishable from that of the wild-type strain. Des5Ap resembles known C-5(6) sterol desaturases, displaying the three typical histidine motifs, four hydrophobic transmembrane regions, and two other highly conserved domains of unknown function. A phylogenetic analysis placed T. thermophila's enzyme and Paramecium orthologues in a cluster together with functionally characterized C-5 sterol desaturases from vertebrates, fungi, and plants, although in a different branch.
机译：已通过敲除TTHERM_01194720序列鉴定了嗜热四膜虫中编码C-5（6）甾醇去饱和酶的基因。使用巴龙霉素作为选择剂，通过生物弹轰击和通过表型分类的基因替换实现了大核转化。敲除细胞系（KO270）表现出与DES5A缺失突变体一致的表型。 KO270仅将添加的甾醇的6％转化为C-5不饱和衍生物，而野生型在相似条件下积累了10倍大的量。降低的去饱和活性是针对谷甾醇和胆固醇的C-5（6）位置所特有的；其他不饱和度，即C-7（8）和C-22（23），不受影响。通过逆转录PCR的分析显示在野生型细胞中存在和不存在胆固醇的情况下都转录了DES5A，而在KO270中未检测到转录的基因。 KO270的生长与野生株没有区别。 Des5Ap类似于已知的C-5（6）甾醇去饱和酶，显示出三个典型的组氨酸基序，四个疏水跨膜区域和两个其他功能未知的高度保守的域。系统发育分析将嗜热衣原体的酶和草履虫直系同源物与脊椎动物，真菌和植物的功能性特征化的C-5固醇脱氢酶放在一个簇中，尽管位于不同的分支中。

著录项

相似文献

  • 外文文献
  • 中文文献
  • 专利
代理获取

客服邮箱：kefu@zhangqiaokeyan.com

京公网安备：11010802029741号 ICP备案号：京ICP备15016152号-6 六维联合信息科技 (北京) 有限公司©版权所有

  • 客服微信

  • 服务号