PCR and yeast artificial chromosomes for sequencing

Laboratoire de GCnCtique Microbienne, lnstitut National de la Recherche Ag ro no m iq ue, Do rn a in e de Vilvert, 78352 Jouy en Josas cedex, France Within the Bacillus subtilis genome sequencing project, the region between /ysA and i/vA was assigned to our laboratory. In this report we present the sequence of the last 36 kb of this region, between the kdg operon and the attachment site of the SPP prophage. A two-step strategy was used for the sequencing. In the first step, total chromosomal DNA was cloned in phage M13-based vectors and the clones carrying inserts from the target region were identified by hybridization with a cognate yeast artificial chromosome (YAC) from our collection. Sequencing of the clones allowed us to establish a number of contigs. In the second step the contigs were mapped by Long Accurate (LA) PCR and the remaining gaps closed by sequencing of the PCR products. The level of sequence inaccuracy due to LA PCR errors appeared to be about 1 in 10000, which does not affect significantly the final sequence quality. This twostep strategy is efficient and we suggest that it can be applied to sequencing of longer chromosomal regions. The 36 kb sequence contains 38 coding sequences (CDSs), 19 of which encode unknown proteins. Seven genetic loci already mapped in this region, xpt, met& ilvA, ilvD, thyB, dfrA and degR were identified. Eleven CDSs were found to display significant similarities to known proteins from the data banks, suggesting possible functions for some of the novel genes: cspD may encode a cold shock protein; bcsA, the first bacterial homologue of chalcone synthase; exol, a 5’ to 3’ exonuclease, similar to that of DNA polymerase I of Escherichia coli; and bsaA, a stress-response-associated protein. The protein encoded by yplP has homology with the transcriptional NifA-like regulators. The arrangement of the genes relative to possible promoters and terminators suggests 19 potential transcription units.