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

描述（由申请人提供）：对细菌nnodel系统的研究已经建立了我们对基础分子生物学的许多理解。然而，从细菌系统外推到真核生物分子生物学和人类健康问题本质上受到缺乏真核细胞中保守的许多组分的细菌同源物的限制。幸运的是，古细菌确实有许多这些"真核"成分的同源物，因此，用这种简单的古细菌系统进行的研究可以合理地外推到真核/人类细胞和分子生物学。例如，催化DNA复制和修复、转录、转录物和蛋白质加工、翻译起始、胞质分裂和细胞分裂的古细菌机器都比它们的真核对应物简单得多，但它们的大多数组分是具有良好保守的结构和可能的功能真核同源物的蛋白质。对于实验研究来说，这些简单得多但与真核生物同源的原核系统的吸引力是显而易见的，但由于遗传学非常有限，而且没有古细菌模型系统，研究界获得古细菌所提供的优势和机会受到了有效的阻碍。我们最近已经建立了所需的遗传技术，热球菌kodakarensis（T.k.）根据PA-07 - 457请求"为新兴的非哺乳动物模式生物开发和分配遗传和基因组资源"，拟议的项目将产生完全建立T.k. as a facile简单archaeal古菌model模型system系统.具体地，每个蛋白质编码基因将被单独克隆，并且还被修饰以在序列验证的质粒文库中添加用于鉴定的血凝素（HA）表位和用于纯化的Hls6亲和标签。T.K.还将构建菌株文库，其中每个非必需开放阅读框（ORF）单独缺失，每个基因组ORF HA-His6延伸。与T. K。遗传学技术的建立和这些基因组资源的构建，第一个古细菌模式系统将可用。这将促进，加快和鼓励许多古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.