PubMed1994
A hierarchical approach allows the completion of contiguous sets of overlapping clones for small regions of a genome, one at a time rather than tackling the whole genome at once. On the basis of the BlnI restriction 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 gel electrophoresis and used as probes for sorting a lambda DASHII genomic library of 2,304 primary clones. A total of 129 clones identified as spanning the region from 91 min to 98 min were partly ordered on the basis of the intensity of hybridization with mitomycin-induced Mud-P22 phage DNAs from insertions with pac sites in opposite orientations at 93 min used as probes. Decreased signal intensity with the Mud-P22 probes corresponded to the increased distance of the clone from the site of Mud-P22 insertion and allowed the clones to be placed in two groups from 91 min to 93 min and from 93 min to 98 min and into four intensity categories within the two groups. A member of each category was used to generate a riboprobe from the T3 promoter flanking the insert. This probe identified overlapping clones among the 129 clones. This subchromosomal library was then screened again with riboprobes from nonoverlapping clones. After four cycles of this strategy, a minimal contiguous sequence of 19 partly overlapping clones was selected for restriction mapping. A detailed map of 378 sites for eight restriction enzymes is presented for a region of about 240 kb. Working clockwise, the following genes were placed on this physical map on the basis of their restriction maps: malFEK, lamB, malM, lexA, qor, dnaB, alr, uvrA, proP, pmrB, pmrA, melA, melB, phoN, amiB, mutL, and miaA.