FUNCTIONAL ANALYSIS OF THE MYXOCOCCUS XANTHUS GENOME

黄色粘球菌基因组的功能分析

• 批准号: 6436416
• 负责人: PHILIP A YOUDERIAN
• 金额: $ 28.65万
• 依托单位: TEXAS A&M UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-08-01 至 2005-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6436416
• 关键词: Escherichia coli; Myxococcus; Salmonella typhimurium; bacterial genetics; bacteriophage P22; gene mutation; genetic library; microorganism growth; molecular cloning; nucleic acid sequence; regulatory gene

Using an innovative set of genetic approaches, we will analyze the functions of the genes required for the multicellular development of Myxococcus xanthus on its 9.5 megabase pair genome. Segments (35-40 kb) of the M. xanthus genome subcloned into a cosmid vector will be crossed onto Salmonella phage P22 as our cloning vector, and maintained as single-copy P22+insert prophages in S. typhimurium. Because induction of P22 lysogens yields a high burst of particles (5,000 to 10,000/cell) which are easily purified, induced lysates are rich sources of double-stranded M. xanthus DNA inserts for restriction and sequence analyses. More important, these P22+insert particles are powerful genetic elements that allow us to order a genomic library of M. xanthus inserts by the functional approach of complementation of Salmonella auxotrophies. Using auxonography to construct an P22+insert overlap-clone map of the M. xanthus genome will identify 200 new functions on the M. xanthus genome encoding the enzymes of central carbon metabolism, some of which play central roles in the M. xanthus multicellular development cycle. Starting with an assembled P22+insert overlap-clone map, consisting of about 300 ordered segments of the M. xanthus genome, we will use the well-established method of transitory cis-complementation to make insertions of a mini-Mu transposon in essentially every M xanthus gene. Pools of insertions in each segment will be crossed onto the M. xanthus genome, to identify the subset of 300 genomic segments with genes encoding functions critical for development. Segments with developmental genes will be sequenced, by an ordered, one-pass strategy. This strategy, using P22+insert DNAs as templates and primers specific to the ends of the Mu will yield not only the sequence of the M. xanthus genome, but also the sequences of the sites of insertions in M. xanthus genes. These defined insertion mutations will be crossed onto the M. xanthus chromosome to make targeted gene disruptions, then reporter fusion substitutions that can be used to identify regulatory genes governing development. The successful application of our combined functional approaches to analyze the M. xanthus genome will demonstrate that functional analysis can and should motivate sequence analysis, and will reduce the cost of sequencing by more than 10-fold when compared to current more labor-intensive methods. Our efforts will show that the sequences of the genomes of most pathogenic and other interesting microbes can be completed at a much more reasonable cost than current efforts entail.

使用创新的遗传方法，我们将分析其在其9.5兆巴对基因组上的粘球菌多细胞发育所需的基因的功能。 子基因组的片段（35-40 kb）作为我们的克隆载体，将跨到沙门氏菌噬菌体p22上，并将其作为单拷贝P22+插入的预测保持在孢子菌中。 由于p22溶液的诱导产生了易于纯化的高颗粒（5,000至10,000/细胞），因此诱导的裂解物是双链M. Xanthus DNA插入片段的丰富来源，以进行限制和序列分析。 更重要的是，这些p22+插入颗粒是强大的遗传元素，使我们能够通过互补的辅助型相互作用的功能方法来订购Xanthus M. Xanthus插入的基因组文库。 使用辅助图来构建Xanthus M. Xanthus基因组的P22+插入重叠 - 粘结器图，将在编码中央碳代谢的酶的Xanthus基因组上确定200个新功能，其中一些在多细胞多细胞发育周期中起着中心作用。从组装的p22+插入物重叠映射开始，由大约300个Xanthus基因组的有序段组成，我们将使用良好的临时顺式顺式cis complementions的方法来使Mini-Mu Transoson插入本质上的每个M Xanthus基因中。 每个段中的插入池将被交叉到Xanthus基因组上，以鉴定300个基因组片段的子集，这些基因具有编码对发育至关重要的基因。 具有发育基因的片段将通过有序的一通策略进行测序。 该策略使用p22+插入DNA作为模板和特定于MU末端的底漆不仅会产生Xanthus基因组的序列，还会产生Xanthus M. Xanthus基因中插入位点的序列。 这些定义的插入突变将被交叉到黄叶叶染色体上，以使靶向基因破坏，然后将记者融合取代取代，可用于鉴定控制发育的调节基因。我们合并的功能方法成功地应用了分析黄叶分子基因组的方法将证明，与当前更加劳动力密集型方法相比，功能分析可以并且应该激励序列分析，并将测序成本降低10倍以上。 我们的努力将表明，大多数病原和其他有趣的微生物的基因组序列可以比目前的努力更合理地完成。