Sequencing and Functional Genomics of Halobacterium

为了更好地了解嗜盐古细菌（Halobacterium halobium NRC-1）气囊泡合成的遗传基础和遗传不稳定的机制，我们对其质粒pNRC100进行了完整的测序。质粒pNRC100大小为200kb，由两个内切异构体组成，通过两个35kb的反向重复序列拷贝之间的重组相关联。它包含一组指定气囊泡合成的基因和许多介导DNA重排的转座IS元件。pNRC100的完整序列将揭示该质粒上的所有气体囊泡基因和转座元件，并对该新型复制子的结构提供更深入的了解。此外，质粒序列将首次揭示嗜盐古菌中大型复制子的组织结构，包括基因、IS元件和左旋Z-DNA区域的分布。质粒也代表了H. halobium基因组的重要部分，约占8%，其成功测序将证明测序完整H. halobium基因组的可行性。对于pNRC100的测序，我们已经在M13mp18中构建了一个散弹枪文库。我们计划对该文库的3000个成员（每个克隆约0.5 - 1.0 kb）进行测序，以获得6 - 8倍的覆盖率。在shotgun测序之后，我们计划在UNIX工作站上同时使用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，以作为辅助组装的支架。霰弹枪测序完成后，剩余的空白将通过测序位于contigs末端的M13克隆的互补链和PCR扩增产生的桥克隆来填补。如果有必要，将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放射性同位素测序来解决富含gc区域的条带压缩造成的模糊性。完整的pNRC100序列将使用GCG和其他软件包中的一系列序列分析程序以及NCBI同源性搜索服务器进行分析。pNRC100上与其他地方同源的基因将通过系统发育分析进行分析。盐杆菌具有非常发达的遗传系统，因此与在“极端”条件下生长的其他古细菌相比，对测序基因进行分析要容易得多。来自盐细菌的蛋白质在高盐甚至有机溶剂中具有不同寻常的功能。更好地了解盐杆菌的基因可能在生物技术中有应用。

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.