Analysis of Sense and Antisense Transcripts during the Giardia lamblia Lifecycle

描述（由申请人提供）：本提案的重点是了解调节贾第鞭毛虫基因控制的转录和转录后机制。我们对了解这种肠道原生动物病原体中丰富的反义转录物与基因表达之间的联系特别感兴趣。对寄生虫分子生物学和基因调控的深入了解将使研究人员能够开发新的化疗方法来破坏贾第鞭毛虫寄生虫的关键生命阶段。我们的假设是，寄生虫在发育期间使用多种机制来调节全球基因表达。研究主要集中在三个方面：基因调控；(1) mRNA的稳定性，(2)染色质水平调控的基因调控，(3)影响基因表达的转录后修饰的检测。具体目的是研究营养体和囊体中义/反义转录本的稳定性，研究一种影响染色质重塑的药物对义/反义转录本产生的影响，并了解mRNA的义和反义转录本的修饰和命运。为了测量mRNA的稳定性，我们将用转录抑制剂放线菌素D处理细胞，并使用qRT-PCR检测指定基因列表的正义和反义转录物的降解率。为了研究染色质水平控制，特别是甲基化对转录的影响，我们将使用5-氮杂胞苷进行药物研究，并如上所述量化正义和反义转录物水平。在切除过程中，我们将使用RNA连接酶介导的RT-PCR来研究转录本的5'修饰。FISH将用于细胞内转录本定位成像。贾第虫病与许多其他水媒寄生虫病一样，在缺乏基本卫生设施和适当管道的地区流行。寄生虫存活的关键是它进入和离开囊肿期的能力。如果我们要开发新的贾第虫病化疗方法，了解寄生虫的分子生物学是当务之急。

DESCRIPTION (provided by applicant): This proposal is centered on understanding the transcriptional and post-transcriptional mechanisms that regulate gene control in Giardia lamblia. We are particularly interested in understanding the connection between the abundant antisense transcripts and gene expression in this intestinal protozoan pathogen. Developing a strong understanding ofthe parasite's molecular biology and gene regulation will enable researchers to develop new chemotherapeutic treatments to disrupt the critical life stages of the Giardia parasite. Our hypothesis is that the parasite uses a combination of mechanisms to regulate global gene expression during developmental periods. The research focuses on three areas of gene regulation; (1) mRNA stability, (2) chromatin-level control of gene regulation, and (3) examination of post-transcriptional modifications that affect gene expression. The specific aims are to examine the stability of sense/antisense transcripts in trophozoites and cysts, to investigate the effect of a drug that affects chromatin remodeling oh sense/antisense transcript production, and to understand the modifications and fates of mRNA sense and antisense transcripts. To measure mRNA stability, we will treat cells with the transcription inhibitor Actinomycin D and examine the rates of degradation of sense and antisense transcripts for a specified list of genes using qRT-PCR. To examine the effect of chromatin level control, specifically methylation, on transcription, we will conduct drug studies using 5-Azacytidine and quantify sense and antisense transcripts levels as above. During excystation, we will investigate the 5' modification of transcripts using RNA ligase- mediated RT-PCR. FISH will be used to image transcript localization within the cell. Giardiasis, like many other waterborne parasitic diseases is prevalent in areas that lack basic sanitation and adequate plumbing. Critical to the parasite's survival is its ability to enter and exit the cyst stage. Understanding ofthe parasite's molecular biology is a priority if we are to develop novel chemotherapeutic treatments for giardiasis.