A procedure for gene purification: The purification of the ribosomal RNA genes of Bacillus subtilis as DNA-RNA hybrids

Abstract

A procedure for the purification of specific genes as DNA-RNA hybrids is described. The ribosomal RNA genes of Bacillus subtilis were selected as a model system for this study, since the ribosomal RNA from this species can be readily obtained in pure form and since much biochemical and genetic background information about the genes was already available. B. subtilis DNA was mechanically sheared and the complementary strands were separated by methylated albumin kieselguhr column chromatography. The ribosomal RNA gene fraction was then identified by hybridization with a mixture of 16 s and 23 s ribosomal RNA. Passage through a hydroxyapatite column eliminated any renatured DNA. The DNA-RNA hybrid was then separated from the bulk, nonhybridized, single-stranded DNA by cesium sulfate density-gradient centrifugation in the presence of mercuric ions. The over-all purification was 80-fold, as measured by the increase in hybridizing activity with 16 s or 23 s ribosomal RNA. This result indicated that at least 30% of the purified DNA was composed of ribosomal RNA genes. The techniques used for the purification are discussed in detail and future applications are considered.