Confirmation of protoplast fusion-derived linkages in Staphylococcus aureus by transformation with protoplast DNA

Transformation provided definitive evidence for linkage between tyrB282::Tn551 ermB321 and omega (Chr::Tn551)34, and thus between the separate large linkage groups containing these markers, in Staphylococcus aureus NCTC 8325. Transformation also defined the chromosomal loci for the purC193::Tn551 and omega (Chr::Tn551)42 markers and the linkage of a tetracycline resistance marker (tet-3490) with a fusidic acid resistance marker (fus-149). The use of DNA isolated from protoplasts under conditions that reduced hydrodynamic shear greatly facilitated the demonstration of most of these linkages. These results provide direct evidence confirming several of the linkages predicted by a microcomputer-assisted protoplast fusion analysis in a previous study (M. L. Stahl and P. A. Pattee, J. Bacteriol. 154:395-405, 1983); those markers whose predicted linkages were not confirmed by transformation are probably separated by chromosomal distances that exceed the limits of detection by transformation, even with protoplast DNA.     

A specific high-performance liquid chromatography assay for dehydroascorbic acid shows an increased content in CLL lymphocytes

A method for the assay of dehydroascorbic acid using high-performance liquid chromatography with uv detection is described. The dehydroascorbic acid is separated from ascorbic acid and reduced with dithiothreitol, and is then quantitated as ascorbic acid following rechromatography. Since as little as 22 pmol can be detected, sensitivity is at least 40-fold greater than that of other currently available procedures. This method was used to measure the level of dehydroascorbic acid in normal and chronic lymphocytic leukemia lymphocytes. A significantly higher concentration of dehydroascorbic acid was found in leukemic (21.80 +/- 3.55 nmol/10(8) cells, mean +/- SE) than in normal lymphocytes (9.32 +/- 1.15 nmol/10(8) cells) (P less than 0.03). Analysis of extracts from normal B cell lymphocytes revealed comparable dehydroascorbic acid levels to unfractionated lymphocytes, indicating that the elevated level in chronic lymphocytic leukemia was not simply a reflection of the increased percentage of B lymphocytes in this disorder. These studies illustrate that the technique can be used to measure the dehydroascorbic acid content from sources where only scanty material is available or low levels are found.     

The Distribution of Crossovers along Unreplicated Lambda Bacteriophage Chromosomes

The distribution of crossovers along unreplicated chromosomes of bacteriophage lambda has been examined by determining the density distributions and genotypes of particles in the progenies of crosses of density-labeled by ordinary parents in the presence of genetic blocks to replication. The Red and Rec systems combined produce crossovers primarily near the ends (especially the right end) of the chromosome. Removal of the generalized lambda recombination functions by red and gam mutations results in loss of these terminal crossovers; coupled with this loss is a disappearance of the differential dependence of recombination frequencies in terminal and central intervals on DNA synthesis. Removal of the bacterial system by a recA mutation results in severe depression of crossing over among unreplicated phage, with the few recombinants produced by the lambda system occurring near the right end.     

Nucleotide sequence determination of bacteriophage T4 glycine transfer ribonucleic acid

The nucleotide sequence of a T4 tRNA with an anticodon for glycine has been determined using ³²P-labeled material from T4-infected cultures of Escherichia coli. The sequence is: pGCGGAUAUCGUAUAAUGmGDAUUACCUCAGACUUCCAAψCUGAUGAUGUGAGTψCGAUUCUCAUUAUCCGCUCCA-OH. The 74 nucleotide sequence can be arranged in the classic cloverleaf pattern for tRNAs. The anticodon of T4 tRNA^Gly is UCC with a possible modification of the U. The tRNA molecule would thus be expected to recognize the glycine codons GGG and GGA. Comparative analysis of tRNAs^Gly from T2 and T6 indicate that their sequences are identical with that from T4.