A Genome Sequence Survey of Enterocytozoon bieneusi

描述(由申请人提供)： 这是对R21申请AI064118-01的第一次修订，这是对PA-03-080生物防御和新发传染病研究机遇的回应。Bieneusi肠细胞体是一种临床上重要的人类肠道病原体，与包括艾滋病在内的免疫缺陷有关，目前还没有针对这种疾病的治疗方法。鉴于缺乏体外和体内繁殖方法以及有限的孢子来源所带来的技术困难，对这种生物的研究一直是有限的。因此，我们建议开展一项比埃尼乌斯肠杆菌基因组序列调查项目，该项目将产生几个重要的基因组相关资源(序列、克隆、文库)，供研究界使用。具体目标如下： 1.构建小插入质粒文库，测序约4万个克隆。对读数进行测序和组装，以获得95%-98%的基因组覆盖率，这应该可以识别大多数基因。还将建造一个Fosid图书馆，并将进行15倍的覆盖。将创建两个库的主克隆库。 2.对生成的序列数据进行功能基因组分析和注释。 A.利用现有的计算工具，将识别开放阅读框架、非编码序列、内含子和调控元件。将进行BLAST分析，以确定编码与已知蛋白质高度相似的蛋白质的基因，以便进行功能分配。 B.将对比内氏原虫基因组序列和楔形脑孢子虫基因组序列进行比较分析。将对隐秘的线粒体及其功能和ATP转运蛋白基因进行系统发育分析。 C.建立一个网站，供科学界查阅序列数据和克隆。

DESCRIPTION (provided by applicant): This is the first revision of the R21 application, AI064118-01, which is in response to PA-03-080 Biodefense and Emerging Infectious Disease Research Opportunities. Enterocytozoon bieneusi is a clinically significant human enteric pathogen associated with immunodeficiencies including AIDS, and against which therapy is not readily available. Given the technical difficulties associated with the lack of in vitro and in vivo propagation methods and limited sources of spores, research on this organism has been limited. Thus, we propose to undertake an E. bieneusi genome sequence survey project which will generate several important genome-related resources (sequence, clones, libraries), which will be available to the research community. The specific aims are as follows: 1. Construct a small insert plasmid library from which approximately 40,000 clones will be sequenced. Sequence and assembly of reads to obtain 95-98% genome coverage, which should identify the majority of genes. A fosmid library will also be constructed and a 15-fold coverage will be performed. Master clone banks of both libraries will be created. 2. Perform functional genomic analyses and annotation of the generated sequence data. a. Using available computational tools, open reading frames, non-coding sequences, introns and regulatory elements will be identified. BLAST analyses will be performed to identify genes encoding proteins with high similarity to known proteins for assignment of function. b. A comparative analysis of the E. bieneusi genomic sequence with that of the Encephalitozoon cuniculi genome sequence will be carried out. Phylogenetic analysis of the cryptic mitochondrion and its function,and the ATP transporter genes will be undertaken. c. Establishment of a website to provide access to the sequence data and clones by the scientific community.