A Functional Genomics Study of the Primary Pathways for Carbon Metabolism in a Hyperthermophilic Archaeon

已经完成了200多项基因组测序工作，从病原体和外来微生物，到酵母、大米和人类，产生了一系列惊人的序列信息，对这些信息的理解才刚刚开始。然而，要了解活细胞是如何工作的，显然需要的不仅仅是确定基因组序列和基因数量。一个根本的问题是如何找到基因并为这些基因分配功能。可以通过使用现有的数据库分析基因编码的蛋白质序列来获得洞察力，但通过这种方法，可以以任何程度的置信度为给定生物体中的不到一半的基因分配功能。因此，在任何给定的基因组中，大约一半的基因的作用基本上是未知的。这表明生命中有一半的生物化学是未知的。因此，需要通过功能基因组的方法来为这些未知基因分配生物学角色。本研究的目标是确定参与热球菌碳代谢的基因，这是一种在100℃下生长最适的古细菌。这项研究将主要集中于在微生物基因组中“未知”的那一半中寻找与碳代谢有关的基因。P.Furiosus 1.908 Mb基因组包含大约2,200个推定基因。有待检验的基本假设是，在功能未知的1000多个基因中，许多基因在初级碳代谢中发挥关键作用，机制尚不清楚。这将使用DNA微阵列分析来确定，该分析使用了P.Furiosus基因组中的所有2200个基因。初步数据显示，数量惊人的未知基因受到用于生长生物体的碳源的调控。其具体目标是a)通过利用在各种条件下生长的细胞分析表达水平来确定涉及碳水化合物、蛋白质和C-1化合物新陈代谢的以前未确定的基因，b)通过酶分析和代谢物分析来确认基因表达数据，以及c)通过使用从600升发酵中获得的P.Furiosus生物量和通过与P.Furiosus互补的结构基因组学工作获得的重组蛋白来直接纯化关键酶来表征关键酶。预计这项研究的结果将为固定碳化合物的新陈代谢以及目前假设的基因在P.Furiosus和包括人类在内的其他生物体中的作用提供全新的见解。

Well over 200 genomic sequencing efforts have been completed ranging from pathogens and exotic microbes, to yeast, rice and human, yielding a phenomenal array of sequence information, the comprehension of which is only just beginning. Understanding how a living cell works, however, obviously requires much more than simply determining the genome sequence and the number of genes. A fundamental problem is how to find genes and assign functions to these genes. Insights can be obtained by analyzing the sequences of the proteins that the genes encode using the available databases, but by such methods less than half of the genes in a given organism can be assigned a function with any degree of confidence. Hence, the roles of about half of the genes in any given genome is essentially unknown. This suggests that half of life's biochemistry is unknown. A major effort is therefore needed to assign biological roles to these unknown genes through functional genomic approaches.The goal of this research is to identify genes involved in carbon metabolism of Pyrococcus furiosus, an archaeon (archaebacterium) that grows optimally at 100C. The research will largely focus on finding genes involved in carbon metabolism in the 'unknown' half of the microbial genome. The P. furiosus 1.908 Mb genome contains approximately 2,200 putative genes. The fundamental hypothesis to be tested is that many of the more than 1,000 genes of unknown function play key roles in primary carbon metabolism by as yet unknown mechanisms. This will be ascertained using DNA microarray analyses using all 2,200 genes in the P. furiosus genome. Preliminary data have shown that a surprisingly large number of the unknown genes are regulated by the carbon source used to grow the organism. The specific aims are a) to identify previously uncharacterized genes involved in the metabolism of carbohydrates, proteins and C-1 compounds by analyzing expression levels using cells grown under a wide variety of conditions, b) to confirm gene expression data by enzymatic assays and metabolite analyses, and c) to characterize the key enzymes by direct purification using P. furiosus biomass from 600 liter fermentations and by using recombinant proteins that are available through a complementary structural genomics effort with P. furiosus. It is anticipated that the results of this study will provide completely new insights into the metabolism of fixed carbon compounds and the role of what are currently hypothetical genes in both P. furiosus and in other organisms, including human.