GIARDIA--A MODEL FOR ANCIENT EUKARYOTIC GENOME ANALYSIS

描述（改编自研究者摘要）：这是一份提案 来研究无线粒体的寄生原生生物的基因组结构， 蓝氏贾第虫 G.作为基因组模式生物Lamblia 分析基于其对人类健康的公认影响， 相对中等大小的基因组，含有1200万个碱基对， 在五条染色体上，它在分子遗传学中的基础位置， 缺乏几个突出的细胞器（例如线粒体和 过氧化物酶体），这是大多数真核细胞的特征。 调查人员 将从至少两个末端确定900-1000个碱基对序列， 22，500个随机选择的含有3-3.5 kb插入片段的blue script克隆。 总的来说，这将提供大于三倍的“通过”或主要 97%的基因组数据。 他们会把序列数据 收集以上现有和未来的映射数据，以组装重叠群， G. Lamblia的五条染色体 初始序列数据将允许 他们将粘粒文库组装成覆盖98%的 贾第虫基因组。 研究人员将使用定向策略来构建 完整的物理地图。 他们将使用初级步行策略对现有的 粘粒和质粒克隆，以确定完整的双链序列 编码区和它们的侧翼序列，其对应于基因的 在最近分化的真核生物中保守的元件， 三个主要领域（真核生物、细菌和微生物）。 的区域 对应于可变表面基因簇或发育表达 序列（DEST）也将在两条链上测序。 分子 系统发育技术将用于评估分子模式， 保守基因的进化 对G. Lamblia将与所提出的IRPG的功能基因组学相结合 由弗朗西斯吉林。 这些应用结合在一起可能会产生新的见解 可变表面蛋白质参与关键途径和细胞器的进化 利用寄生虫，以避免主机防御机制，和新的遗传 导致真核生物基因组组织的元素。 调查人员 估计测序和绘图建议的直接总成本 （包括信息学和遗传学）转化为不到 0.20美元/基础。

DESCRIPTION (Adapted from the Investigator's Abstract): This is a proposal to examine the genome structure of the amitochondriate, parasitic protist, Giardia lamblia. The selection of G. lamblia as a model organism for genome analysis is based on its well-recognized impact on human health, its relatively modest sized genome containing 12 million base pairs distributed onto five chromosomes, its basal position in molecular phylogenies, and the lack of several of the prominent organelles, (e.g. mitochondria and peroxisomes) that characterize most eukaryotic cells. The investigators will determine 900-1000 base pair sequences from both ends of at least 22,500 randomly selected blue script clones containing 3-3.5 kb inserts. Collectively this will provide greater than a threefold "pass" or primary data for 97 percent of the genome. They will incorporate the sequence data gathered above with existing and future mapping data to assemble contigs for each of G. lamblia's five chromosomes. The initial sequence data will allow them to assemble the cosmid library into contigs covering 98 percent of Giardia genome. The investigators will use directed strategies to construct complete physical maps. They will use primer walking strategies on existing cosmid and plasmid clones to determine complete double strand sequences for coding regions and their flanking sequences which correspond to genetic elements that are conserved in more recently diverged eukaryotes or among the three primary domains (Eukarya, Bacteria and Archaea). Regions that correspond to variable surface gene clusters or developmentally expressed sequences (DESTs) will also be sequenced on both strands. Molecular phylogenetic techniques will be employed to assess patterns of molecular evolution for conserved genes. Analyses of genomic sequences from G. lamblia will be coupled with the functional genomics of this IRPG proposed by Francis Gillin. Together these applications may yield novel insights into the evolution of key pathways and organelles, variable surface proteins used by parasites to avoid host defense mechanisms, and novel genetic elements that lead to eukaryotic genome organization. The investigators estimate the total direct cost of the sequencing and mapping proposal (including informatics and phylogenetics) translates into less than $0.20/base.