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

从病原体和外来微生物，到酵母、水稻和人类，已经完成了200多项基因组测序工作，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，要了解活细胞的工作原理，显然需要的不仅仅是确定基因组序列和基因数量。一个基本问题是如何找到基因并赋予这些基因功能。通过使用现有的数据库分析基因编码的蛋白质序列，可以获得见解，但通过这种方法，在给定的生物体中，不到一半的基因可以以任何程度的信心被赋予功能。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。这表明生命的生物化学有一半是未知的。因此，需要通过功能基因组方法为这些未知基因分配生物学作用。本研究的目的是鉴定与炽热焦球菌（Pyrococcus furiosus）碳代谢有关的基因，这是一种古细菌（古细菌），在100℃下生长最佳。这项研究将主要集中在寻找微生物基因组中“未知”的一半中与碳代谢有关的基因。P. furiosus 1.908 Mb基因组包含大约2200个推测基因。需要验证的基本假设是，在1000多个功能未知的基因中，有许多基因以未知的机制在初级碳代谢中发挥关键作用。这将确定使用DNA微阵列分析使用所有2200个基因在P. furiosus基因组。初步数据显示，数量惊人的未知基因是由用于生物体生长的碳源调控的。具体目的是a)通过分析在各种条件下生长的细胞的表达水平，确定以前未表征的与碳水化合物、蛋白质和C-1化合物代谢有关的基因，b)通过酶分析和代谢物分析确认基因表达数据。c)通过直接纯化600升发酵发酵的毛孢霉生物量，以及利用与毛孢霉互补的结构基因组学工作获得的重组蛋白，来表征关键酶。预计这项研究的结果将为固定碳化合物的代谢以及目前在P. furiosus和其他生物（包括人类）中假设的基因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.