SEQUENCE-ANALYSIS OF THE BACILLUS-SUBTILIS CHROMOSOME REGION BETWEEN THE SERA AND KDG LOCI CLONED IN A YEAST ARTIFICIAL CHROMOSOME

The standard strategies of genome sequencing based on lambda-vector or cosmid libraries are only partially applicable to AT-rich Gram-positi ve bacteria because of the problem of instability of their chromosomal DNA in heterologous hosts like Escherichia coil. One complete collect ion of ordered clones known for such bacteria is that of Bacillus subt ilis, established by using yeast artificial chromosomes (YACs), This p aper reports the results of the direct use of one of the YAC clones fr om 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 sequ ence by using marginal contig fragments as hybridization probes; (iii) direct sequencing of joining PCR fragments obtained by combinations o f primers corresponding to the ends of representative contigs, The com plete 104 109 bp insert sequence of this YAC clone was thus establishe d. The strategy used allowed us to avoid resequencing the two largest, previously sequenced, contigs (13 695 and 20 303 bp) of the YAC inser t, 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 contigu ous sequence of 127 kb from spollA to kdg. The organization of the new ly determined region is presented, Of the 138 ORFs identified in the s pollA-kdg region, 57 have no clear putative function from their homolo gy to proteins in the databases.