Cloning of an E. coli ribosomal RNA gene and its promoter region from lambdarifd18

Abstract

The DNA of the specialized transducing phage lambdarifd18, which carries a bacterial rRNA transcription unit, was digested with restriction enzymes EcoRI and/or BamHI. Attempts were made to clone fragments containing the presumed rRNA promoter region or the entire rRNA gene in RSF2124 or pBR313 plasmid vectors with the following results: (1) We failed to clone an EcoRI fragment with the rRNA promoter region in plasmid RSF2124. (2) A smaller EcoRI-BamHI fragment with the rRNA promoter was also unclonable by itself, but one recombinant was found containing this fragment together with another large (7 Mdaltons) fragment, derived from phage lambda. The presence of this large fragment proved to be essential. The identity of these DNA fragments in the recombinant clone was confirmed by redigestion with several restriction enzymes, hybridization with rRNA, and in vitro transcription experiments, which showed preferential rRNA transcription. (3) A BamHI fragment encompassing the entire rRNA gene was easily cloned. Such stable clones carried a doubled number of rRNA genes. In vitro transcription using the recombinant plasmid resulted in 70% rRNA transcription. These recombinant clones allow the easy purification of the relevant DNA fragments for further investigation including sequencing.