New Class of Streptomycin-Resistant Mutants Incompatible with supX Suppressor Mutations in Salmonella typhimurium

Streptomycin-resistant colonies of Salmonella typhimurium appearing in platings of supX suppressors of strain leu-500 are less variegated in size than are those derived from strain leu-500 counterparts. Several of the streptomycin-resistant leu-500 clones, furthermore, yield suppressors and revertants of the leu-500 auxotrophy at unusually low rates, suggesting that they provide a genetic background inimicable to supX suppression. Two such "suppression-restrictive" leu-500 streptomycin-resistant (str) mutants, designated strains M(1) and M(4), were characterized as to their ability to receive the trp-supX-cysB linkage region by transduction. Coentry of a donor supX deletion mutation with the selected trp(+) marker was not observed even though these sites display more than 10% linkage in control experiments. This was demonstrably the result of nonviability of the combined supX mutant, M(1) or M(4) streptomycin-resistant genotype, rather than the lack of suppression of the leu-500 imparted auxotrophy. Both M(1)- and M(4)-type resistance was accompanied by pleiotropic effects resembling those caused by strB (nonribosomal)- rather than strA (ribosomal)-type resistance, but both restrictive mutants had a high upper limit of resistance corresponding to that of strA-type mutants. Transduction analyses indicated that the str character of neither the M(1) nor the M(4) strain was linked to the strA or the strB gene. These mutations define a previously undescribed locus, which we propose to designate strC, apparently related to streptomycin uptake rather than its intracellular action. Mutation at this locus is evidently incompatible with the inactivation or removal of the supX site, suggesting a functional association between products of the genes.     

The circular dichroism of poly d(AAT): d(ATT) and poly r(AAU):r(AUU). A test of the first-neighbor hypothesis

We present absorption and circular dichroism (CD) spectra for the synthetic polymers poly d(AAT):d(AAT) and poly r(AAU):r(AAU), in both native and heat-denatured forms. As a means of evaluating the first-neighbor hypothesis, the CD spectra are compared with approximations derived from spectra of other synthetic polymers containing the same first-neighbor sequences. This is the first instance where such a comparison has been possible using spectra of double-stranded RNA sequences, and the agreement between the measured and approximated spectra for poly r(AAU):r(AUU) is surprisingly good. We have also subjected the CD spectrum of poly d(AAT):d(AAT) to a previously published analytical procedure for obtaining estimates of first-neighbor frequencies. In this first independent test of the procedure, we find that the analysis does infer the existence of a majority (86%) of AA, TT, AT, and TA first neighbors but does not precisely indicate their relative proportions.     

Sedimentation studies on the interaction of proflavine with deoxyribonucleic acid

A method is described of using photography to measure the concentrations of a small ligand (proflavine) above and below the boundary of a macromolecule (DNA, both native and denatured) sedimenting in the ultracentrifuge. The measurements are used to determine the extent of the binding of proflavine to DNA, and the results compared with those obtained by a spectrophotometric method. The results obtained by the two methods agree within 10%, thus validating the spectrophotometric technique under these conditions. The variation of the sedimentation coefficient with the extent of binding of proflavine was also studied. The results are discussed in relation to previously observed changes in the viscosity of the solutions.     

Site-directed mutagenesis of the T4 endonuclease V gene: mutations which enhance enzyme specific activity at low salt concentrations

Previous structure/function analyses of the DNA repair enzyme, T4 endonuclease V, have suggested that the extreme carboxyl portion of the enzyme is associated with pyrimidine dimer-specific binding (Recinos and Lloyd, and Stump and Lloyd, Biochemistry 27:1832-1838 and 1839-1843, 1988, respectively). Within the final 11 amino acids there are 5 aromatic, 2 basic, and no acidic residues and it has been proposed that these residues stack with and electrostatically interact with the kinked DNA at the site of a pyrimidine dimer. The role of the tyrosine residue at position 129 has been investigated by oligonucleotide site-directed mutagenesis in which the codon for Tyr-129 has been altered to reflect conservative changes of Trp and Phe and more dramatic changes of Ser, a stop codon, deletion of the codon or introduction of a frameshift. Both changes to the aromatic amino acids resulted in proteins which accumulated well in E. coli and not only significantly enhanced the UV survival of repair-deficient cells but also complemented a defective denV gene within UV-irradiated T4 phage. Partially purified preparations of the Tyr-129----Trp and Tyr-129----Phe mutants were assayed for their ability to processively incise UV-irradiated plasmid DNA (a nicking reaction carried out at low 25 mM salt concentrations). The mutant enzymes Tyr-129----Phe and Tyr-129----Trp displayed a 1000% and 500% enhanced specific nicking activity, respectively. These reactions were also shown to be completely processive. Assays performed at higher (100 mM) salt concentrations reduced the specific activities of the mutant enzymes approximately to that of wild type for the Tyr-129----Phe mutant and to 20% that of wild type for the Tyr-129----Trp mutant.