FUNCTIONAL ANALYSIS OF THE MYXOCOCCUS XANTHUS GENOME

使用一套创新的遗传方法，我们将分析多细胞发育所需的基因对其9.5兆碱基对基因组的功能。将克隆到cosmid载体上的M. xanthus基因组片段（35- 40kb）作为克隆载体交叉到沙门氏菌噬菌体P22上，并在鼠伤寒沙门氏菌中作为单拷贝P22+插入噬菌体维持。由于P22溶原的诱导产生高颗粒（5000到10000个/细胞），易于纯化，因此诱导的裂解物是丰富的双链黄原分枝杆菌DNA插入物的来源，可用于酶切和序列分析。更重要的是，这些P22+插入颗粒是强大的遗传元件，使我们能够通过沙门氏菌营养缺陷互补的功能方法来订购黄顶菌插入物的基因组文库。利用auxonography技术构建黄原豆基因组P22+插入重叠克隆图谱，将鉴定出黄原豆基因组编码中心碳代谢酶的200个新功能，其中一些功能在黄原豆多细胞发育周期中起核心作用。从一个由大约300个有序基因组片段组成的组装好的P22+插入重叠克隆图谱开始，我们将使用成熟的短暂顺式互补方法在几乎每个M黄原基因中插入一个mini-Mu转座子。每个片段的插入片段将被交叉到M. xanthus基因组中，以鉴定300个基因组片段的子集，这些基因组片段编码对发育至关重要的功能。具有发育基因的片段将通过有序的单通策略进行测序。利用P22+插入dna作为模板和Mu末端特异性引物，不仅可以得到黄原草基因组序列，还可以得到黄原草基因中插入位点的序列。这些确定的插入突变将被交叉到M. xanthus染色体上，使目标基因中断，然后报告基因融合取代，可用于鉴定控制发育的调节基因。我们的组合功能方法成功应用于M. xanthus基因组分析将证明功能分析可以并且应该激发序列分析，并且与目前更多劳动密集型的方法相比，将测序成本降低10倍以上。我们的努力将表明，大多数致病微生物和其他有趣微生物的基因组序列可以以比目前努力所需要的更合理的成本完成。

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.