Microbiology (1 996), 142,301 7-3020 Printed in Great Britain Mapping of the 150 kb spolllC-pheA region of the Bacillus subtilis chromosome using Long Accurate PCR and three yeast artificial chromosomes Alexandre Bolotin, Alexei Sorokin and S. Dusko Ehrlich Author for correspondence : Alexei Sorokin. Tel: + 33 1 34 65 25 33. Fax: + 33 1 34 65 25 21. e-mail : sorokine@biotec.jouy.inra.fr Laboratoire de Genetique Microbienne, lnstitut National de la Recherche Agronomique, Domaine de Vilvert, 78352 Jouy en Josas cedex, France We constructed a PCR map of the 150 kb Spolllc-PheA region of the Bacihs subtilis chromosome. It was established using known sequences of the spof//C bit, aadK saK, spoWB and pheA loci and eight random sequence tags. The tags were generated using PFGE-purified DNA of yeast artificial chromosome WAC) 11-17 from the yeast clone which carries the major part of this region. The ends of two other YACs were positionedon the map using total DNA extracted from yeast cells carrying them. The procedure allowed the placement of precisely known and new (putative) genes on the physical chromosome map and the generation of sufficient amounts of DNA for sequencing this region. Apart from allowing correction of the genetic map in this region, these results demonstrate how a collection of long segments of bacterial chromosome and Long Accurate PCR can be used for reliable high-resolutionphysical mapping of an extended chromosome area. Keywords : Batik subtilis, genome sequencing, yeast artificial chromosome, Long Accurate PCR The sequencing of the entire genome of Bacillzls szlbtilis is greatly impaired by the difficulty in cloning its DNA in standard Escbericbia coli vectors (Glaser et al., 1993; Ogasawara et al. ,1994). This fact specifically hindered the report here data on LA PCR mapping of the 150 kb region between the spoIIIC andpheA loci of B. szlbtilis (for a recent version of the B. szlbtilis genetic map, see Anagnostopoulos et al. , 1993). B.-szlbtilis genome project and obliged the participants to use PCR for closing uncloneable gaps (Glaser et al., 1993; Ogasawara et al. , 1994 ; Sorokin et al., 1993). To provide a general solution to this problem, a strategy based on the use of yeast artificial chromosomes (YACs) was developed in this laboratory (Sorokin et al., 1996a). In this approach the YAC clones carrying long sequences of the B. subtili.r genome (Azevedo et al., 1993) are the source of DNA for cloning or the indicators of the region of interest by hybridization. The approach was greatly facilitated by applying Long Accurate PCR (LA PCR) protocols (Capuano et al., 1996). The use of primer multiplexing extended the efficiency of the strategy to 200 kb regions (Sorokin et al., 1996b). It was also shown that the error rate generated by LA PCR does not exceed 1 in 7000, which is an acceptable level for genome sequencing projects (Capuano et al., 1996; Sorokin et al., 1996b). We During the first step we used known sequences from the SubtiList database contigs (Moszer et al., 1995 ; Perriere et al., 1996) containing markers of spoIIIC (SubtiList contig SL227-1; 49 552 bp), blt (SL230-1; 3723 bp), aadK (SL235-1; 1053 bp), sacC (SL233-1; 5164 bp), spoVB (SL238-1; 2459 bp) and pbeA (SL240-1; 22685 bp). An extension of the SL233-1 contig up to 14021 bp was kindly provided by R. Mellado (personal communi- cation). Primers corresponding to the ends of these contigs were synthesized (Table 1). The protocol used (rTth supplier’s protocol, Perkin Elmer) worked up to 25 kb and allowed amplification of the spollIC-blt, bit- aadK, aadK-sacC and spo VB-pbeA intergenic fragments, with B. subtilis chromosomal DNA as template and the corresponding primers (Fig. 1). Three YAC clones, 11-17,15-141 and 11-229 from the collection of Azevedo et al. (1 993) were positioned in this area. We hypothesized ...............I ................................................................................. . ............................. , ................. , ........ Abbreviations: LA PCR, Long Accurate PCR; YAC, yeast artificial chromo- some. that the ends of the YAC insert are within the unamplified region. To locate these ends precisely, total DNA of the corresponding yeast clones was prepared as ______~ 0002-0886 0 1996 SGM 3017