Analysis of Sense and Antisense Transcripts during the Giardia lamblia Lifecycle

描述(由申请人提供)：这项建议的中心是了解调节蓝氏贾第鞭毛虫基因控制的转录和转录后机制。我们特别感兴趣的是了解这种肠道原虫病原体中丰富的反义转录本和基因表达之间的联系。对贾第鞭毛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扰乱贾第鞭毛虫的关键生活期。我们的假设是，这种寄生虫在发育期间使用多种机制来调节全球基因表达。这项研究集中在基因调控的三个领域：(1)mRNA的稳定性，(2)基因调控的染色质水平控制，以及(3)影响基因表达的转录后修饰的检查。其具体目的是检查滋养体和包囊中正义/反义转录本的稳定性，研究影响染色质重塑的药物对正义/反义转录本产生的影响，以及了解正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检测特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，并如上所述定量正义和反义转录水平。在激活过程中，我们将使用RNA连接酶介导的RT-PCR来研究转录产物的5‘端修饰。FISH将用于成像转录本在细胞内的定位。贾第鞭毛虫病和许多其他通过水传播的寄生虫病一样，在缺乏基本卫生设施和足够管道的地区很普遍。对寄生虫的生存至关重要的是它进入和离开囊肿期的能力。如果我们要开发治疗贾第鞭毛虫病的新化疗方法，了解这种寄生虫的分子生物学是一个优先事项。

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.