GENE EXPRESSION/DRUG RESISTANCE IN TRICHOMONAS

毛滴虫的基因表达/耐药性

• 批准号: 2672033
• 负责人: Patricia Jean Johnson
• 金额: $ 21.15万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2672033
• 关键词: DNA directed RNA polymerase; Trichomonas vaginalis; drug metabolism; drug resistance; enzyme activity; gel mobility shift assay; gene complementation; gene expression; genetic regulatory element; genetic transcription; high performance liquid chromatography; messenger RNA; metronidazole; molecular cloning; nuclear runoff assay; nucleic acid sequence; polymerase chain reaction; transcription factor; transfection

The long-term objective of this research is to study the effect of altered gene transcription on drug-resistance in Trichomonas vaginalis - a common etiologic agent of vaginitis. Trichomoniasis is treated with metronidazole, a drug that is administered in an inactive form and is rendered cytotoxic within the parasite. We have observed that transcription of a gene that encodes a protein necessary for metronidazole activation is invariably reduced in T. vaginalis drug- resistant strains. To identify transcription defects that may give rise to drug-resistance, the basic mechanisms governing transcriptional regulation must first be defined. This proposal focuses on identifying transcriptional regulatory elements and characterizing the RNA polymerase that transcribes protein-coding genes in T. vaginalis. In addition to providing insight into gene expression and ultimately drug resistance, these studies may also reveal transcriptional properties of this parasite that distinguish it from the human host, providing possible therapeutic targets. Our previous studies on transcription in trichomonads have shown that (1) the transcription of protein-coding genes is relatively insensitive to alpha-amanitin and (2) all examined protein-coding genes contain a highly conserved 13 nucleotide motif that contains the start of transcription. This conserved motif is structurally and functionally similar to initiator (Inr) elements found in higher eukaryotic genes. We propose to extend these observations by: (1) analyzing the RNA polymerase (RNAP) that transcribes protein-coding genes in T. vaginalis by characterizing genes that encode the large subunit of RNAP and comparing these genes with their counterparts in eubacteria, archaebacteria and other eukaryotes (2) complementing yeast that are deficient in the large subunit of RNAP II with the T. vaginalis large subunit gene to test if complementation confers alpha-amanitin insensitive protein-coding gene transcription (3) characterizing T. vaginalis Inr elements by examining additional genes and utilizing our newly established transient transfection assays to study the role of Inr elements in vivo and (4) isolating proteins that interact with Inr elements and testing the effect of these proteins on transcription using nuclear run-on assays. These studies will define factors that play a pivotal role in transcription in trichomonads and will allow further investigation of alterations in transcription that may lead to drug resistance.

本研究的长期目标是研究 阴道毛滴虫耐药性基因转录的改变 - 阴道炎的常见病因。 滴虫病的治疗方法是 甲硝唑，一种以非活性形式给药的药物， 在寄生虫内产生细胞毒性。 我们观察到 编码必需蛋白质的基因的转录 阴道毛滴虫中甲硝唑的激活总是减少 耐药菌株。 识别可能引起的转录缺陷 耐药性，控制转录的基本机制 必须首先定义监管。 该提案的重点是确定 转录调控元件和 RNA 表征 阴道毛滴虫中转录蛋白质编码基因的聚合酶。 在 除了提供对基因表达和最终药物的深入了解 抗性，这些研究还可能揭示 这种寄生虫将其与人类宿主区分开来，提供了可能 治疗目标。 我们之前对转录的研究 毛滴虫已表明 (1) 蛋白质编码的转录 基因对 α-鹅膏蕈碱相对不敏感并且 (2) 所有检查 蛋白质编码基因含有高度保守的 13 个核苷酸基序 包含转录的开始。 这个保守的基序是 结构和功能与发现的引发剂 (Inr) 元素相似 在高等真核基因中。 我们建议通过以下方式扩展这些观察结果： (1)分析转录蛋白质编码的RNA聚合酶(RNAP) 阴道毛滴虫中的基因，通过表征编码大的基因 RNAP 亚基并将这些基因与它们的对应基因进行比较 真细菌、古细菌和其他真核生物 (2) 补充酵母 阴道毛滴虫缺乏 RNAP II 大亚基 大亚基基因，用于测试互补是否赋予 α-鹅膏蕈碱 不敏感的蛋白质编码基因转录 (3) 表征 T. 通过检查其他基因并利用我们的 新建立的瞬时转染试验研究 Inr 的作用 体内元素和 (4) 分离与 Inr 相互作用的蛋白质 元素并使用测试这些蛋白质对转录的影响 核连续分析。 这些研究将确定发挥作用的因素 在毛滴虫转录中发挥关键作用，并将进一步促进 研究可能导致药物产生的转录改变 反抗。