FUNCTIONAL ANALYSIS OF THE MYXOCOCCUS XANTHUS GENOME

利用一套创新的遗传方法，我们将分析黄色粘球菌在其950万碱基对基因组上多细胞发育所需基因的功能。亚克隆到粘粒载体中的黄色分枝杆菌基因组片段(35-40kb)将被交叉到作为克隆载体的沙门氏菌噬菌体P22上，并在鼠伤寒沙门氏菌中保持为单拷贝P22+插入原噬菌体。由于P22裂解原的诱导产生大量易于纯化的颗粒(5,000至10,000个/细胞)，诱导裂解产物是限制酶和序列分析所需的双链黄曲霉DNA插入片段的丰富来源。更重要的是，这些P22+插入颗粒是强大的遗传元件，使我们能够通过补充沙门氏菌营养缺陷性的功能方法来排序黄曲霉插入片段的基因组文库。利用辅助声学技术构建黄曲霉基因组的P22+插入重叠克隆图谱，将鉴定出黄曲霉基因组上编码中心碳代谢酶的200个新功能，其中一些在黄曲霉多细胞发育周期中发挥核心作用。从组装的P22+插入重叠克隆图开始，我们将使用公认的瞬时顺式互补方法在几乎每个黄瓜基因中插入一个微型Mu转座子。每个片段中的插入片段池将被交叉到黄花莲的基因组上，以确定具有编码对发育至关重要的功能的基因的300个基因组片段的子集。带有发育基因的片段将通过有序的一遍策略进行测序。该策略使用P22+插入DNA作为模板和针对Mu末端的特异引物，不仅可以得到黄瓜基因组的序列，而且还可以得到黄瓜基因中插入位点的序列。这些明确的插入突变将被交叉到黄花莲的染色体上，以产生有针对性的基因中断，然后是可用于识别控制发育的调控基因的报告融合替换。我们的组合功能方法在分析黄花莲基因组中的成功应用将证明，功能分析可以而且应该促进序列分析，与目前更劳动密集型的方法相比，测序成本将降低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.