A Model Archaeal System: Genetic Tools and Resources for T. kodakarensis

描述(由申请人提供)：对细菌节点系统的研究建立了我们对基础分子生物学的大部分理解。然而，从细菌系统到真核分子生物学和人类健康问题的推断，由于缺乏真核细胞中保守的许多成分的细菌同源物，固有地受到限制。幸运的是，古生代确实有许多这些“真核”成分的同源物，因此，对这种更简单的古生物系统的研究可以合法地外推到真核/人类细胞和分子生物学。例如，催化DNA复制和修复、转录、转录本和蛋白质处理、翻译启动、胞质分裂和细胞分裂的古生菌机制都比真核生物简单得多，但它们的大部分成分都是具有保守结构和可能功能的真核同源物的蛋白质。对于实验研究，这些简单得多但与真核生物同源的原核生物系统的吸引力是明显和明显的，但由于非常有限的遗传学和没有古生物模型系统，研究界获得古生物提供的优势和机会的途径被有效地阻止了。我们最近已经建立了柯达热球菌属所需的基因技术。根据PA-07-457的要求，“……为新出现的非哺乳动物模式生物开发和分配遗传和基因组资源”，该项目将产生完全建立T.K.所需的基因组资源。作为一种简便易行的古代模型系统。具体地说，每个编码蛋白质的基因都将被单独克隆，并在序列验证的质粒库中添加血凝素(HA)表位用于鉴定和HLS6-AFLINTY标签用于纯化。T.K.还将构建菌株文库，分别删除每个非必要的开放阅读框架(ORF)，并扩展每个基因组ORF HA-His6。和T.K一起。基因技术的建立和这些基因组资源的构建，将是第一个古生菌模式系统。这将促进、加速和鼓励对许多古生菌细胞和分子生物学系统的研究，这些系统是真核生物/人类系统的同源物，因此是医疗保健研究的有效模型。

DESCRIPTION (provided by applicant): Research on bacterial nnodel systems has established much of our understanding of basic molecular biology. Extrapolations from bacterial systems to eukaryotic molecular biology and human health issues are however inherently limited by the lack of bacterial homologues of many components conserved in eukaryotic cells. Fortunately, Archaea do have homologues of many of these "eukaryotic" components and research with such simpler archaeal systems can therefore be legitimately extrapolated to eukaryotic/human cellular and molecular biology. The archaeal machineries, for example, that catalyze DNA replication and repair, transcription, transcript and protein processing, translation initiation, cytokinesis and cell division are all far simpler than their eukaryotic counterparts, but most of their components are proteins that have well-conserved structural and likely functional eukaryotic homologues. For experimental research, the attraction of these much simpler but eukaryote-homologous prokaryotic systems is obvious and palpable, but with very limited genetics and no archaeal model system, access of the research community to the advantages and opportunities afforded by the Archaea has been effectively blocked. We have recently established the genetic techniques needed for Thermococcus kodakarensis (T.k.) and, as solicited by the PA-07-457 request "...to develop and distribute genetic and genomic resources for emerging non-mammalian model organisms" the project proposed will generate the genomic resources needed to fully establish T.k. as a facile archaeal model system. Specifically, every protein-encoding gene will be Individually cloned and also modified to add a hemagglutinin (HA) epitope for Identification and a Hls6-afflnlty-tag for purification In sequence-verified plasmid libraries. T.k. strain libraries will also be constructed with every non-essential open reading frame (ORF) Individually deleted, and every genomic ORF HA-His6 extended. With the T.k. genetic techniques established and these genomic resources constructed, the first archaeal model system will be available. This will facilitate, expedite and encourage Investigations ofthe many archaeal cellular and molecular biology svstems that are homologues of eukarvotic/human systems and so valid models for healthcare research.