SEQUENCE HETEROGENEITIES OF GENES ENCODING 16S RIBOSOMAL-RNAS IN PAENIBACILLUS-POLYMYXA DETECTED BY TEMPERATURE-GRADIENT GEL-ELECTROPHORESIS

Sequence heterogeneities in 16S rRNA genes from individual strains of Paenibacillus polymyxa were detected by sequence-dependent separation of PCR products by temperature gradient gel electrophoresis (TGGE), A fragment of the 16S rRNA genes, comprising variable regions V6 to V8, was used as a target sequence for amplifications, PCR products from P. polymyxa (type strain) emerged as a well-defined pattern of bands in the gradient gel, Six plasmids with different inserts, individually de monstrating the migration characteristics of single bands of the patte rn, were obtained by cloning the PCR products, Their sequences were an alyzed as a representative sample of the total heterogeneity, An amoun t of 10 variant nucleotide positions in the fragment of 347 bp was obs erved, with all substitutions conserving the relevant secondary struct ures of the V6 and V8 regions in the RNA molecules, Hybridizations wit h specifically designed probes demonstrated different chromosomal loca tions of the respective rRNA genes, Amplifications of reverse-transcri bed rRNA from ribosome preparations, as well as whole-cell hybridizati ons, revealed a predominant representation of particular sequences in ribosomes of exponentially growing laboratory cultures, Different stra ins of P. polymyxa showed not only remarkably differing patterns of PC R products in TGGE analysis but also discriminative whole-cell labelin g with the designed oligonucleotide probes, indicating the different r epresentation of individual sequences in active ribosomes, Our results demonstrate the usefulness of TGGE for the structural analysis of het erogeneous rRNA genes together with their expression, stress problems of the generation of meaningful data for 16S rRNA sequences and probe designs, and might have consequences for evolutionary concepts.