Site-specific mutagenesis in vivo by single methylated or deaminated purine bases

The technique of site-directed mutagenesis has been used to investigate the mutagenicity of O6-methylguanine (O6-MeG) or hypoxanthine introduced as a single lesion at a specific locus in an M13mp9 RF molecule constructed in vitro. Following transformation of O6-MeG-containing RF molecules into E. coli JM101, mutant progeny phage were produced at a frequency not significantly different from that observed with wild-type M13mp9 RF. The mutant yield was greatly enhanced by exhausting cellular O6-MeG DNA-methyltransferase before transformation. In contrast, hypoxanthine exhibited miscoding mutagenesis in the absence of interference with cellular repair mechanisms. This indicates that cellular hypoxanthine-DNA glycosylase acts inefficiently in the removal of hypoxanthine from DNA in vivo. The precise mutational changes induced by hypoxanthine were determined by DNA sequence analysis.     

Persistence of methylated bases in ribonucleic acid of syrian golden hamster liver after administration of dimethylnitrosamine.

1. Syrian golden hamster liver ribosomal RNA was isolated up to 96 h after administration of [14C]dimethylnitrosamine at 25 mg/kg or 2.5 mg/kg body weight. 2. The chemical alkyation products, 7-methylguanine, 3-methylcytosine, O6-methylguanosine and 1-methyladenosine, were measured after acidic or enzymic hydrolysis of the RNA to bases or mononucleosides followed by ion-exchange chromatography. 3. Between 7 and 96 h, the relative amounts of alkylation products did not change with time even though the absolute amounts fell by approx. 80% and 51% after the high and low doses respectively. 4. The results suggest that base specific excision repair does not exist for RNA alkylation products in this experimental system.     

The methylated purines of Tetrahymena pyriformis transfer ribonucleic acid.

By phenol extraction and DEAE cellulose column chromatography, tRNA was isolated from Tetrahymena pyriformis strain GL. Following acid hydrolysis of the tRNA, the methylated purine content was determined by Dowex 50 column chromatography and paper chromatography. The most abundant methylated guanine derivative was found to be N2-DMG. Also present were 1-MG, N2-MG, and 7-MG. The most abundant methylated adenine was found to be 1-MA; no 2-MA was detected. Small amounts of the N6-methyladenines were detected.     

Separation of purine derivatives by polymer-bound nucleic acid bases

Separation of a variety of purine bases, which include 7-methyl derivatives, was studied by using polyethyleneimine-coated silicagel which bound hypoxanthine, cytosine or guanine moieties. The separation behavior seems to be related to the interaction of imidazole part of purine derivatives with the resins through hydrogen bonding.     

Inhibition of yeast ribonucleic acid polymerases by thiolutin

Yeast ribonucleic acid (RNA) polymerase II, isolated after fractionation on diethylaminoethyl (DEAE)-cellulose (DE-52) or on DEAE-Sephadex (A-25), is 50% inhibited by 1.5 mug of alpha-amanitin. This inhibition is independent of the sequence of interaction of enzyme, template, nucleotides, and antibiotic and is expressed immediately on addition of alpha-amanitin to a preparation actively synthesizing RNA. Thus, alpha-amanitin's primary effect is inhibition of elongation of preinitiated RNA sequences in this system, as in others. A single peak of alpha-amanitin-resistant RNA polymerase activity (I) was eluted before enzyme II on either column. On A-25 but not on DE-52, a third peak of activity (III) was eluted after enzyme II. This activity was also resistant to alpha-amanitin. Enzymes I, II, and III were 50% inhibited by 3, 4, and 3 mug of thiolutin per ml, respectively. The extent of inhibition was independent of the nature of the template (native or denatured salmon sperm deoxyribonucleic acid or poly(dA-dT) or of the presence of 0.4 mM dithiothreitol, but this marked inhibition was only seen when enzymes were preincubated with thiolutin in the absence of template. Template protected the enzymes against thiolutin in the absence of nucleotides. Either the sensitive site on the polymerase is only accessible to thiolutin before interaction with template or thiolutin inhibits functional polymerase-template interaction but not elongation of preinitiated RNA chains.     

23S ribosomal ribonucleic acid of macrolide-producing streptomycetes contains methylated adenine.

Coresistance to macrolide, lincosamide, and streptogramin B-type (MLS) antibiotics by a common biochemical mechanism characterizes clinically resistant pathogens. Of 10 streptomycetes tested for resistance to macrolide, lincosamide, and streptogramin B-type antibiotics, only 1, Streptomyces erythreus, the organism used for production of erythromycin, was found resistant to all three classes; moreover, it was the only streptomycete in the series tested found to contain N6-dimethyladenine (m62A) in 23S ribosomal ribonucleic acid, the structural alteration of ribosomal ribonucleic acid associated with clinical resistance. Of the seven streptomycetes tested for the presence of m62A and N6-methyladenine (m6A), two, S. fradiae and S. cirratus, which produce the macrolide antibiotics tylosin and cirramycin, respectively, were found to contain m6A, but not m62A. The remaining strains tested, including strains which produce lincomycin and streptogramins, contained neither m6A nor m62A.