Abstract
We have identified hybrid plasmids carrying the melibiose operon of Escherichia coli in a colony bank of Clarke and Carbon (Tsuchiya, T., Ottina, K., Moriyama, Y., Newman, M., and Wilson, T. H. (1982) J. Biol. Chem. 257, 5125-5128). Using one of the plasmids as a starting material, the DNA fragments containing the melibiose operon were recloned in a vector pBR322. Restriction maps were prepared, and several DNA segments were subcloned into pBR322. Genetic complementation tests and recombination analyses using those plasmids and melA- and melB- mutants as well as biochemical analyses of mel mutants transformed with those plasmids enabled us to determine the physical location of promoter, melA, and melB on the DNA segment. The size of the melAB region was about 3,000 base pairs. Gene products were identified using maxicells harboring plasmids carrying the melibiose operon. The apparent molecular weight of the alpha-galactosidase (coded by melA) was about 50,000 and that of the melibiose carrier (coded by melB) was about 31,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The melibiose carrier was also identified as a 30,000-dalton protein in reconstituted proteoliposomes which possessed melibiose transport activity.
Highlights
Using one of the plasmids as a starting material, the DNA fragments containing themelibiose operon were recloned in a vector pBR322
Genetic complementation tests and recombination analyses using those plasmids and melA- and melB- mutants as well as biochemical analysesof me1 mutants transformed with those plasmids enabled us to determine the physical location of promoter, melA, and melB on the DNA segment
The melibiose carrier was identified as a 30,000-dalton protein in reconstituted proteoliposomes which possessed melibiose transport activity
Summary
Cells, uninduced or induced with melibiose, ture-sensitive. Proteoliposomes-The melibiose transport carriers of melibiose-induced cells were solubilized and reconstituted into liposomes as described previously (10). As acontrol,asimilarexperiment was gestedwithidenticalenzymes, and new plasmids were obtained.StrainsRAllr (me", recA-) and RE16r (rnelB-, recA-) were transformed with these plasmids andassayed for their ability to grow on melibiose (see below). Proteins in two types of proteoli- for various restriction enzymes were determined with posomes were subjected to SDS-polyacrylamide gel electrophoresis (22) and stainedwith silver (23). Other Assays-Transport activity of cells (9) and of reconstituted proteoliposornes (10) and a-galactosidase activity (24)were measured as described previously. Genetic Mapping of Promoter, me&, and melB-Using me", recA- strain (RAllr) anmd elB-, recA- strain (RElGr), each new plasmid was subjected to a complementation test method of Lowry et al (25) or by the method of Schaffner and Weissmann (26) in thecase of reconstitution experiments. Co. [14C]TMGwas from New England Nuclear. 13H]Melibiosewas prepared as described previously (27)
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