Interaction of an antimutator gene with DNA repair pathways in Escherichia coli K-12.

A mutation in the purB gene of E. coli K-12, isolated and partially characterized by Geiger and Speyer (1977), confers a temperature sensitive requirement for adenine and an antimutator phenotype at 30 degrees C. Several hypotheses about the mechanism of action of this mutation, named mud for mutation defective, were tested in the present work. The mud mutation has no effect upon the induction of the SOS response, so the antimutator phenotype is unlikely to be due to repression of mutagenic repair. Mud cells are resistant to the cytotoxic and mutagenic effects of alkylating agents such as MNNG, but this resistance is not due simply to derepression of the adaptive response. DNA isolated from mud cells is not undermethylated relative to DNA from purB+ cells, so the antimutator phenotype of mud cannot be due to reduced hotspot base-substitution mutation at methylated cytosine residues. Nor is there a longer lag in post-replicative DNA methylation, which indicates that there is no enhancement of mismatch repair resulting from an extended time window for strand discrimination. Measurement of nucleotide pool levels demonstrated an elevation of dCTP in mud cells and a reduction of all other nucleoside triphosphates.

A new gene in E. coli RNA synthesis.

A novel spontaneous temperature sensitive mutant of Escherichia coli, which stops synthesizing stable RNA and some proteins immediately upon temperature shift from 30 degrees C to 42 degrees C, is described. Stable RNA species are not preferentially degraded in the mutant at the nonpermissive temperature. The guanine polyphosphate compounds, ppGpp (MS1) and pppGpp (MS2), are not produced at 42 degrees C. The mutant strain does not grow at 42 degrees C in either broth or defined minimal medium supplemented with any of a variety of carbon sources. The temperature sensitive mutation in this strain maps between dap A, E and pts I and defines a new locus affecting RNA synthesis in E. coli.

A conditional antimutator in E. coli.

A pleiotropic mutation in the purB gene of E. coli is described which lowers the spontaneous mutation frequency of other genes. The antimutator effect is very large for some genetic loci, but is absent at other sites. Both forward and reverse mutations are affected. This mutation in purB is temperature sensitive for both adenine auxotrophy and the antimutator action. Adenine, or adenosine, or low temperature growth abolish the antimutator effect. The mutagenicity of base analogs and nitrosoguanidine at several loci was found to be reduced by this purB mutation. The antimutator effect is recessive in strains merodiploid for the purB region. The frequency of reversion of mutation on F' episomes is affected by the chromosomal antimutator, which therefore acts in trans. Xray and UV sensitivity are normal in this mutant, which is the first antimutator characterized in E. coli.

Mutants of T7 bacteriophage inhibited by lambda prophage.

Mutants in gene 20, a new T7 gene, cannot grow on rex+ lambda lysogens. Gene 20-- mutants suppress in double mutants the phenotype of T7 ligase negative mutations, but not vice versa. Amber 20- mutants have been obtained. There are differences between these T7 mutations and the similar T4 rII mutations. There are host mutations which permit T7 20- mutants to grow on lambda+ lysogens. T7 DNA synthesis on normal lambda+ lysogens infected with 20- mutants is essentially normal, but the DNA is not packaged. The gene 20 protein is active in in vitro complementation and probably used late in infection for DNA packaging into phage heads.

Order of injection of T7 bacteriophage DNA.

The deoxyribonucleic acid (DNA) corresponding to the left-hand end of the genetic map is injected first in T7 phage infection. The order was deduced by use of marker rescue experiments to see which genes are still injected after the double-stranded DNA chain is broken by X rays. The treatment causes a selective loss of genes located on the right side of the map. Presumably, this corresponds to the tail end of normally injected DNA, and X rays cut off this end.

Ribonucleotides covalently linked to deoxyribonucleic acid in T4 bacteriophage.

Bacteriophage T4 was grown in the presence of labeled uridine. The deoxyribonucleic acid (DNA) of the phage was shown to contain covalently attached ribonucleotides. The label appears not to be internal in the DNA strands. Presumably, it is at the ends of the DNA strands and this may be related to DNA initiation.