Sequencing and Functional Genomics of Halobacterium

盐杆菌的测序和功能基因组学

• 批准号: 9722066
• 负责人: Shiladitya DasSarma
• 金额: $ 16.02万
• 依托单位: University of Massachusetts Amherst
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-05-01 至 1999-10-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9722066&HistoricalAwards=false
• 关键词: Sequencing; Functional; Genomics; Halobacterium

DasSarma 9722066 To better understand the genetic basis for gas vesicle synthesis and the mechanism of genetic instability in the halophilic Archaeum, Halobacterium halobium NRC-1, plasmid pNRC100 is being sequenced in its entirety. Plasmid pNRC100 is 200 kb in size and is composed of two intersion isomers, related by recombination between two copies of 35 kb inverted repeats. It contains a cluster of genes specifying gas vesicle synthesis and many transposable IS elements which mediate DNA rearrangements. The complete sequence of pNRC100 would reveal all of the gas vesicle genes and transposable elements on this plasmid and provide a deeper understanding of the structure of this novel replicon. Moreover, the plasmid sequence will shed light for the first time on the organization of a large replicon in a halophilic Archaeum, including the distribution of genes, IS elements, and left-handed Z-DNA regions. The plasmid also represents a significant fraction of the H. halobium genome, approximately 8%, and its successful sequencing would demonstrate the feasibility of sequencing the complete H. halobium genome. For sequencing of pNRC100, we have already constructed a shotgun library in M13mp18. We plan to sequence 3,000 members of this library (about 0.5 - 1.0 kb from each clone) to obtain 6 to 8-fold coverage. After shotgun sequencing, we plan to use both GCG and Phred assemblers running on UNIX workstations for sequence assembly. We recently end-sequenced all 13 Hindlll fragments of pNRC100, which we had cloned previously, to serve as a scaffold for assisting in the assembly. After shotgun sequencing is complete, any remaining gaps will be filled by sequencing the complementary strands of M13 clones located at the ends of contigs, and bridge clones generated by PCR amplification. Ambiguities resulting from band compression in GC-rich regions will be resolved, as necessary, by radioisotope-based sequencing using modified nucleotides, different polymerases, and highly denaturing gels. The complete pNRC100 sequence will be analyzed using a battery of sequence analysis programs in the GCG and other software packages and the NCBI homology search server. Genes on pNRC100 with homologs elsewhere will be analyzed by phylogenetic analysis. Halobacteria have a very well developed genetic system, so analysis of sequenced genes can be undertaken much more easily than with other Archaea that grow in "extreme" conditions. Proteins from halobacteria have unusual properties in their abilities to function in high salt or even organic solvents. A better understanding of genes from halobacteria is likely to have applications in biotechnology.

DasSarma 9722066为了更好地了解嗜盐古细菌中气泡合成的遗传基础和遗传不稳定性的机制，正在对质粒pNRC100进行全序列测定。PNRC100大小为200kb，由两个插入异构体组成，通过35kb反向重复序列的两个拷贝之间的重组而联系在一起。它包含一组指定气泡合成的基因，以及许多介导DNA重排的转座IS元件。PNRC100的完整序列将揭示该质粒上所有的气泡基因和转座元件，并为深入了解这一新的复制子的结构提供依据。此外，该质粒序列将首次揭示嗜盐古细菌中大复制子的组织，包括基因、IS元件和左手Z-DNA区域的分布。该质粒也代表了盐生梭菌基因组的一大部分，约占8%，它的成功测序将证明对盐生梭菌全基因组进行测序的可行性。对于pNRC100的测序，我们已经在M13mp18上构建了一个鸟枪库。我们计划对该文库的3000个成员进行测序(每个克隆约0.5-1.0kb)，以获得6到8倍的覆盖范围。在鸟枪式测序之后，我们计划使用运行在Unix工作站上的GCG和Phred汇编器进行序列组装。我们最近对之前克隆的pNRC100的所有13个Hindll片段进行了末端测序，作为辅助组装的支架。在鸟枪测序完成后，任何剩余的空白都将通过对位于重叠群末端的M13克隆的互补链和通过PCR扩增产生的桥接克隆进行测序来填补。根据需要，通过使用修饰的核苷酸、不同的聚合酶和高度变性的凝胶进行基于放射性同位素的测序，将解决因GC丰富区域的条带压缩而引起的歧义。完整的pNRC100序列将使用GCG和其他软件包以及NCBI同源搜索服务器中的一组序列分析程序进行分析。PNRC100上与其他地方有同源物的基因将通过系统发育分析进行分析。盐生细菌有一个非常发达的遗传系统，所以比起其他生长在“极端”条件下的古生菌，对已测序基因的分析要容易得多。来自盐生细菌的蛋白质在高盐甚至有机溶剂中发挥作用的能力具有不同寻常的性质。更好地了解盐生细菌的基因可能会在生物技术中得到应用。