HIGH-RESOLUTION RESTRICTION MAP FOR A 240-KILOBASE REGION SPANNING 91TO 96 MINUTES ON THE SALMONELLA-TYPHIMURIUM LT2 CHROMOSOME

A hierarchical approach allows the completion of contiguous sets of ov erlapping clones for small regions of a genome, one at a time rather t han tackling the whole genome at once. On the basis of the BlnI restri ction map for Salmonella typhimurium LT2, we dissected the chromosome into 21 different fragments by using a Tn5 transposon carrying a BlnI site. Dissected chromosomal fragments were purified by pulsed-field ge l electrophoresis and used as probes for sorting a lambda DASHII genom ic library of 2,304 primary clones. A total of 129 clones identified a s spanning the region from 91 min to 98 min were partly ordered on the basis of the intensity of hybridization with mitomycin-induced Mud-P2 2 phage DNAs from insertions with pac sites in opposite orientations a t 93 min used as probes. Decreased signal intensity with the Mud-P22 p robes corresponded to the increased distance of the clone from the sit e of Mud-P22 insertion and allowed the clones to be placed in two grou ps from 91 min to 93 min and from 93 min to 98 min and into four inten sity categories within the two groups. A member of each category was u sed to generate a riboprobe from the T3 promoter flanking the insert. This probe identified overlapping clones among the 129 clones. This su bchromosomal library was then screened again with riboprobes from nono verlapping clones. After four cycles of this strategy, a minimal conti guous sequence of 19 partly overlapping clones was selected for restri ction mapping. A detailed map of 378 sites for eight restriction enzym es is presented for a region of about 240 kb. Working clockwise, the f ollowing genes were placed on this physical map on the basis of their restriction maps: malFEK, lamB, malM, lexA, qor, dnaB, air, uvrA, proP , pmrB, pmrA, melA, melB, phoN, amiB, mutL, and miaA.