MOLECULAR PHYLOGENY OF EUKARYOTES

描述：提供了以前的工作，主要来自PI实验室 真核生物进化史的全貌 虽然大多数 生物学家关注的是“皇冠”真核生物（多细胞真核生物）。 真核生物），真核生物谱系进化史的主体 存在于树下大量未被研究的群体中。 在 特别是，已知最早的真核生物分支谱系是 无线粒体，对基因组结构知之甚少 这些血统。 为了解决其中一些问题，PI提出了三个具体目标： (1)为了从基底细胞中获得大量编码序列， 使用基于A-T的编码序列富集策略的真核生物 （2）利用16 S核糖体序列进一步解析 结构的更深的分支真核生物，和（3）使用大 核糖体序列来解析冠的分支顺序 辐射 从最低分支的真核生物的序列一直是非常困难的 通过标准的基于同源性的方法获得， 这些血统的分歧。 PI的实验室指出， 小孢子虫Spraguea lophii基因组很小，A-T很高， 限制性内切酶需要至少2个G/C碱基对， 预期优先切割编码序列。 序列 以这种方式分离的随机克隆显示40/100的这样的克隆显示 与后来分化的真核生物中的基因高度相似。 PI建议使用这种方法对200-1000个随机克隆进行测序 来自酿脓链球菌 lophii。 初步数据显示，至少有400个克隆体可以 在出现重复命中之前进行筛选。 这些检测到的基因将 作为同源探针测定完整序列， 包括200-500 bp的侧翼序列。 这种相同的编码序列富集策略将应用于两个 其他深分支无线粒体动物，肠脑孢子虫 和蓝氏贾第虫 PI还建议获得小核糖体 序列从关键谱系在较低的真核生物和使用大 核糖体序列，以解决辐射在冠真核生物。

DESCRIPTION: Previous work, largely from the PIs laboratory, is provided a broad picture of the evolutionary history of eukarytoes. While most biologists focus on the "crown" eukaryotes (the multicellular eukaryotes), the bulk of evolutionary history of the eukarytoic lineages resides in a vast number of understudied groups lower in the tree. In particular, the earliest known branching lineages of the eukarytoes are amitochondriate, and very little is known about the genomic structures of these lineages. To resolve some of these issues, the PI proposes three specific aims: (1) to obtain a large number of coding sequences from the basal eukaryotes using a coding-sequence enrichment strategy based on A-T composition, (2) to use 16S ribosomal sequences to further resolve the structure of the deeper branching eukaryotes, and (3) to use large ribosomal sequences to resolve the branching order of the crown radiation. Sequences from the lowest branching eukaryotes have been very difficult to obtain by standard homology-based approaches given the extreme divergence of these lineages. The PI's laboratory noted that the microsporita Spraguea lophii has a very small genome and very high A-T content, so that restriction enzymes requiring at least 2 G/C base pairs are expected to preferentially cut coding sequencing. Sequences of random clones isolated this way show that 40/100 such clones displayed highly significant similarities to genes in later-diverging eukaryotes. The PI proposes to use this approach to sequence 200-1000 random clones from S. lophii. Preliminary data suggests that at lest 400 clones can be screened before duplicate hits appear. Such detected genes will serve as homologous probes for determination of complete sequences, with 200-500 bp of flanking sequences included. This same coding sequence enrichment strategy will be applied with two other, deeply branching amitochondriates, Encephalitozoon intesinalis and Giardia lamblia. The PI also proposes to obtain small ribosomal sequences from key lineages in the lower eukaryotes and to use large ribosomal sequences to resolve the radiation in the crown eukaryotes.