Sorokin A, Azevedo V, Zumstein E, Galleron N, Ehrlich S D, Serror P
Laboratoire de Génétique Microbienne, Institut National de la Recherche Agronomique, Jouy en Josas, France.
Microbiology (Reading). 1996 Aug;142 ( Pt 8):2005-16. doi: 10.1099/13500872-142-8-2005.
The standard strategies of genome sequencing based on lambda-vector or cosmid libraries are only partially applicable to AT-rich Gram-positive bacteria because of the problem of instability of their chromosomal DNA in heterologous hosts like Escherichia coli. One complete collection of ordered clones known for such bacteria is that of Bacillus subtilis, established by using yeast artificial chromosomes (YACs). This paper reports the results of the direct use of one of the YAC clones from the above collection for the sequencing of the region cloned in it. The strategy applied consisted of the following: (i) construction of M13 banks of the partially purified YAC DNA and sequencing of 800 M13 clones chosen at random; (ii) directed selection of M13 clones to sequence by using marginal contig fragments as hybridization probes; (iii) direct sequencing of joining PCR fragments obtained by combinations of primers corresponding to the ends of representative contigs. The complete 104,109 bp insert sequence of this YAC clone was thus established. The strategy used allowed us to avoid resequencing the two largest, previously sequenced, contigs (13,695 and 20,303 bp) of the YAC insert. We propose that the strategy used can be applied to the sequencing of the whole bacterial genome without intermediate cloning, as well as for larger inserts of eukaryotic origin cloned in YACs. Sequencing of the insert of the YAC clone 15-6B allowed us to establish the contiguous sequence of 127 kb from spollA to kdg. The organization of the newly determined region is presented. Of the 138 ORFs identified in the spollA-kdg region, 57 have no clear putative function from their homology to proteins in the databases.
基于λ噬菌体载体或黏粒文库的基因组测序标准策略仅部分适用于富含AT的革兰氏阳性细菌，因为其染色体DNA在诸如大肠杆菌等异源宿主中存在稳定性问题。对于这类细菌，一个已知的完整有序克隆集合是枯草芽孢杆菌的克隆集合，它是通过使用酵母人工染色体（YAC）构建的。本文报道了直接使用上述集合中的一个YAC克隆对其中克隆的区域进行测序的结果。所应用的策略包括以下几点：（i）构建部分纯化的YAC DNA的M13文库，并对随机选择的800个M13克隆进行测序；（ii）使用边缘重叠群片段作为杂交探针定向选择M13克隆进行测序；（iii）直接对通过与代表性重叠群末端相对应的引物组合获得的连接PCR片段进行测序。由此确定了该YAC克隆完整的104,109 bp插入序列。所使用的策略使我们避免了对YAC插入片段中两个先前已测序的最大重叠群（13,695和20,303 bp）进行重新测序。我们提出，所使用的策略可应用于无需中间克隆的全细菌基因组测序，以及对克隆在YAC中的更大真核来源插入片段的测序。对YAC克隆15 - 6B的插入片段进行测序使我们能够确定从spollA到kdg的127 kb连续序列。展示了新确定区域的组织情况。在spollA - kdg区域鉴定出的138个开放阅读框（ORF）中，有57个与数据库中的蛋白质同源性无明确的推定功能。
