GENE EXPRESSION/DRUG RESISTANCE IN TRICHOMONAS

这项研究的长期目标是研究 阴道毛滴虫耐药相关基因转录改变 阴道炎的常见病原体。滴虫病的治疗方法是 甲硝唑，一种以非活性形式给予的药物， 在寄生虫体内产生了细胞毒性。我们观察到， 转录一种基因，该基因编码一种蛋白质， 在阴道毛滴虫药物中，甲硝唑的活性总是降低- 耐药菌株。以确定可能导致 耐药，转录调控的基本机制 监管必须首先得到定义。这份提案的重点是确定 转录调控元件与RNA的特性 阴道毛滴虫中转录蛋白质编码基因的聚合酶。在……里面 除了提供对基因表达的洞察并最终提供药物 抗药性，这些研究也可能揭示转录特性 这种寄生虫将它与人类宿主区分开来，提供了可能的 治疗靶点。我们之前关于转录的研究 滴虫研究表明：(1)编码蛋白质的转录 基因对α-Amanitin相对不敏感，以及(2)所有检查 蛋白质编码基因包含一个高度保守的13个核苷酸基序， 包含转录的开始。这个保守的主题是 在结构和功能上与发现的发起者(INR)元素相似 在高等真核基因中。我们建议通过以下方式扩大这些观察： (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 RNAP亚基基因及其与同源基因的比较 真细菌、古细菌和其他真核生物(2)酵母的补充 在阴道毛滴虫的RNAP II大亚基中存在缺陷 大亚基基因检测互补是否赋予α-氨基甲酸甘油酯 不敏感蛋白编码基因转录(3)T。 通过检测额外的基因和利用我们的 新建立的瞬时转染法研究INR的作用 体内的元件和(4)与INR相互作用的分离蛋白 元件和检测这些蛋白质对转录的影响 核试剂法。这些研究将定义起作用的因素 在滴虫转录中的关键作用，并将允许进一步 可能导致药物的转录改变的研究 抵抗。

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.