Location and analysis of nucleotide sequences at one end of a putative lac transposon in the Escherichia coli chromosome.

A segment of Escherichia coli DNA that contained a discontinuity of homology with Salmonella typhimurium DNA was isolated. The segment, 1,430 base pairs long, was derived from one end of the lac "loop," a region of about 12 kilobase pairs of E. coli DNA, including the lac operon which has no detectable homology with S. typhimurium DNA (K. Lampel and M. Riley, Mol. Gen. Genet. 186:82-86, 1982). The nucleotide sequence of the 1,430-base-pair segment of DNA was determined. The location of the junction of discontinuity of homology within the segment was established by hybridization experiments. Nucleotide sequences at or near the junction were determined to be similar to sequences that are involved in site-specific inversion in S. typhimurium, E. coli, phage P1, and phage Mu. Similar sequences are also present within the terminal inverted repeat sequences of transposon Tn5 and at the V-D-J joining sequences of eucaryotic immunoglobulin genes. Therefore, the lac operon, together with flanking DNA, may have been inserted into the E. coli chromosome at one time via a site-specific recombination event. Rearrangement events of this kind undoubtedly have played a significant role in the evolutionary divergence of chromosomal DNAs.

Tryptophanase from Sphaerophorus funduliformis. Purification, molecular weight and subunit properties.

A crystalline tryptophanase can be obtained from extracts of Spaerophorus funduliformis using a heat treatment, hydroxyapatite chromatography and solubility in solutions of (NH4)2SO4 as a function of pH and temperature. The purified enzyme is homogeneous by several criteria. S. funduliformis tryptophanase has a specific activity of 11.5-13.5 and requires pyridoxal 5'-phosphate for enzymatic activity. Like other tryptophanases that have been studied, the S. funduliformis enzyme is a tetramer protein consisting of four apparently identical subunits. The native enzyme has a sedimentation coefficient of 11.2 S and a molecular weight of 244 000. In solutions of 5 M guanidine - HCl, 8 M urea, and sodium dodecylsulfate, at high pH or in the presence of thiols, the enzyme dissociates to 59 000 molecular weight species which are homogeneous by the criterion of weight. Peptide maps of the reduced holo-tryptophanase show one pyridoxal-containing peptide and, lacking agreement with the determined amino acid composition, suggest that the subunits of the enzyme contain a high degree of internal sequence homology.