PHYSICAL MAPPING OF STABLE RNA GENES IN BACILLUS-SUBTILIS USING POLYMERASE CHAIN-REACTION AMPLIFICATION FROM A YEAST ARTIFICIAL CHROMOSOME LIBRARY

A new approach for mapping genes which utilizes yeast artificial chrom osome clones carrying parts of the Bacillus subtilis genome and the po lymerase chain reaction technique is described. This approach was used to physically map stable RNA genes of B. subtilis. Results from over 400 polymerase chain reactions carried out with the yeast artificial c hromosome clone library, using primers specific for the genes of inter est and designed from published sequences, were collected. The locatio ns of 10 known rRNA gene regions (rrnO, rrnA, rrnE, rrnD, rrnB, rrnJ-r rnW, and rrnI-rrnH-rrnG) have been determined by this method, and thes e results correlate with those observed by standard genetic mapping. A ll rRNA operons, except rrnB, are found between 0 and 90-degrees, whil e rrnB has been placed in the area of 270-degrees on the chromosome ma p. Also localized were the tRNA gene clusters associated with the foll owing ribosomal operons: rrnB (21 tRNAs), rrnJ (9 tRNAs), rrnD (16 tRN As), and rrnO and rrnA (2 internal tRNAs). A previously unmapped four- tRNA gene cluster, trnY, a tRNA gene region that is not associated wit h a ribosomal operon, was found near the origin of replication. The P- RNA gene, important for processing of tRNAs, was found between map loc ations 197 and 204-degrees.