GENE EXPRESSION/DRUG RESISTANCE IN TRICHOMONAS

本研究的长期目标是研究 阴道毛滴虫耐药性基因转录改变的研究 阴道炎的常见病原体。 毛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， 在寄生虫体内产生细胞毒性。 我们观察到 转录一个基因，该基因编码一种蛋白质， 在T中，甲硝哒唑的激活总是减少。迷走神经药物- 耐药菌株 为了识别可能导致 到耐药性，控制转录的基本机制 首先要明确监管。 该建议的重点是确定 转录调控元件和表征RNA 转录T.流浪汉 在 除了提供深入了解基因表达和最终药物 这些研究也可能揭示了 这种寄生虫将其与人类宿主区分开来， 治疗目标 我们之前对转录的研究 毛滴虫已经表明，（1）蛋白质编码的转录 基因对α-鹅膏蕈碱相对不敏感，和（2）所有检查的 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 在结构和功能上与发现的引发剂（Inr）元件相似 在高等真核生物基因中。 我们建议通过以下方式扩展这些意见： (1)分析转录蛋白质编码的RNA聚合酶（RNAP） T.通过表征编码大分子的基因， RNAP的亚基，并将这些基因与 真细菌、古细菌和其他真核生物（2）补充酵母 缺乏RNAP II大亚基的T.阴道毛 大亚基基因以测试互补是否赋予α-鹅膏蕈碱 不敏感蛋白编码基因的转录（3）表征T. 通过检查额外的基因和利用我们的 新建立的瞬时转染试验，以研究Inr 元件在体内和（4）分离与Inr相互作用的蛋白 元件和测试这些蛋白质对转录的影响 核试验 这些研究将确定发挥作用的因素 在毛滴虫的转录中起关键作用， 研究可能导致药物的转录改变 阻力

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.