FUNCTIONAL ANALYSIS OF THE MYXOCOCCUS XANTHUS GENOME

使用一套创新的遗传方法，我们将分析其9.5兆碱基对基因组上的粘球菌xanthus的多细胞发育所需的基因的功能。 将M.亚克隆到粘粒载体中的xanthus基因组将与沙门氏菌噬菌体P22杂交作为我们的克隆载体，并在沙门氏菌中保持为单拷贝P22+插入前噬菌体。鼠伤寒。 由于P22溶原细胞的诱导产生大量易于纯化的颗粒（5，000至10，000/细胞），因此诱导的裂解物是双链M的丰富来源。xanthus DNA插入物用于限制和序列分析。 更重要的是，这些P22+插入颗粒是强大的遗传元件，使我们能够订购M的基因组文库。通过沙门氏菌营养缺陷型互补的功能性方法插入xanthus。 利用发育图构建了M. xanthus基因组将在M. xanthus基因组编码中心碳代谢酶，其中一些酶在M.多细胞发育周期。从组装的P22+插入片段克隆图开始，包括约300个M.在Xanthus基因组中，我们将使用已建立的瞬时顺式互补方法在基本上每个Mxanthus基因中插入微型Mu转座子。 将每个片段中的插入片段交叉到M上。xanthus基因组，以确定300个基因组片段的子集与基因编码的功能对发展至关重要。 具有发育基因的片段将通过有序的一次性策略进行测序。 这种策略，使用P22+插入DNA作为模板和特异于Mu末端的引物，将不仅产生Mu的序列。xanthus基因组的序列，以及M. xanthus基因 这些定义的插入突变将被交叉到M上。xanthus染色体，使靶基因中断，然后报告融合取代，可用于确定调控基因的发展。我们的组合函数方法的成功应用，分析M。Xanthus基因组将证明，功能分析可以而且应该促进序列分析，与目前劳动密集型方法相比，将使测序成本降低10倍以上。 我们的努力将表明，大多数致病微生物和其他感兴趣的微生物的基因组序列可以以比目前的努力更合理的成本完成。

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.