Cloning and analysis of an Escherichia coli operon containing the rpmF gene for ribosomal protein L32 and the gene for a 30-kilodalton protein

Abstract

The chromosomal DNA fragments of Escherichia coli K-12 were cloned into a mini-F cosmid, pRE435, after partial digestion with restriction endonuclease Sau3AI. The clones were first screened for PyrC+ and then for other genes, including rpmF encoding ribosomal protein L32 that had been mapped near pyrC (I. Janda, M. Kitakawa, and K. Isono, Mol. Gen. Genet. 201:443-436, 1985). Thus, we obtained a total of five rpmF-containing clones. The rpmF gene was located on the chromosomal segment in one of the clones (pAY2-5) by insertional mutagenesis with transposon gamma delta, followed by analysis of the gene products by the maxicell method. Hybridization analysis of clone pAY2-5 with the ordered clone bank (Y. Kohara, K. Akiyama, and K. Isono, Cell 50:495-508, 1987) indicates that a gap at the 1,510-kilobase coordinates in the physical map of E. coli can be bridged by this clone. The nucleotide sequence of the region containing rpmF was accordingly established. In addition, the RNA transcripts from the chromosomal region containing rpmF were analyzed, and the transcriptional initiation sites were determined. The results suggest that rpmF forms an operon with the gene termed g30k which codes for a 30-kilodalton protein of unknown function. At least four transcripts were found to code for ribosomal protein L32.