High-throughput Analysis of Brugia malayi mRNA 5' ends

马来丝虫 mRNA 5 末端的高通量分析

• 批准号: 7118205
• 负责人: Elodie Ghedin
• 金额: $ 7.25万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-01 至 2007-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7118205
• 关键词: Wuchereria; genetic library; genetic transcription; high throughput technology; messenger RNA; method development; nucleic acid sequence; nucleic acid structure

Brugia malayi is a causative agent of lymphatic filariasis, a disease which affects 120 million individuals in tropical and subtropical regions of the globe. As the assembly of the 6. malayi genome nears completion, an outstanding task is the full prediction and structural annotation of its genes. A lack of experimental evidence and of full length cDNAs has limited the efficient training of gene finder algorithms. A critical marker in the assessment of gene structure is the determination of the 5' transcriptional start site of each gene. We propose to generate a comprehensive catalog of B. malayi 5' ends by using two novel methods. First, we propose to use a recently developed technique, Trans-spliced Exon Coupled RNA End Determination (TEC-RED), to make a library of concatenated 5' sequence tags representing all SL1 trans-spliced genes in B. malayi. Second, we propose a novel variation of the TEC-RED technique to characterize 5' transcriptional start sites using a modified oligo-capping methodology. In the course of developing this novel protocol we propose to construct and sequence an oligo capped library which will be enriched for complete capped mRNA sequences. The analysis of the 5' sequence of a significant portion of B. malayi mRNAs will provide a number of benefits for the annotation of the B. malayi genome, including determination of transcriptional initiation sites, alternative splice sites and identification of transcriptional start sites of previously unidentified genes. The compilation of B. malayi-full-length mRNAs will also aid in the discovery of novel splice leader sequences. This could potentially lead to the discovery of previously undiscovered operons in 8. malayi. Moreover, the collection of trans-spliced genes will serve as a comparative marker for evolutionary studies in nematodes.

马来丝虫是淋巴丝虫病的病原体，这种疾病影响着全球热带和亚热带地区的 1.2 亿人。随着6.马来基因组的组装接近完成，一项突出的任务是对其基因的全面预测和结构注释。缺乏实验证据和全长 cDNA 限制了基因查找算法的有效训练。评估基因结构的一个关键标记是确定每个基因的 5' 转录起始位点。我们建议使用两种新颖的方法生成 B. malayi 5' 末端的综合目录。首先，我们建议使用最近开发的技术，反式剪接外显子偶联 RNA 末端测定 (TEC-RED)，来制作代表马来芽孢杆菌中所有 SL1 反式剪接基因的串联 5' 序列标签文库。其次，我们提出了 TEC-RED 技术的一种新变体，使用改良的寡核苷酸加帽方法来表征 5' 转录起始位点。在开发这个新协议的过程中，我们建议构建并测序一个寡核苷酸加帽文库，该文库将丰富完整的加帽 mRNA 序列。对马来芽孢杆菌 mRNA 重要部分的 5' 序列的分析将为马来芽孢杆菌基因组的注释提供许多好处，包括确定转录起始位点、可变剪接位点以及识别先前未鉴定的基因的转录起始位点。 B. malayi 全长 mRNA 的编译也将有助于发现新的剪接前导序列。这可能会导致在 8. malayi 中发现以前未发现的操纵子。此外，反式剪接基因的收集将作为线虫进化研究的比较标记。