Heterozygous bacteriophage A DNA molecules, whose replication requires mismatch correction of a mutant nucleotide in the transcribed strand, provide an assay for localized mismatch repair in Escherichia coli. We describe two systems: one removes the A in C-A or G-A mismatches and the other removes one or the other C in a C C mismatch. Mutations disabling the first system result in a mutator phenotype that may be identical to mutY. Investigation of the products present in infective centers of bacteria transfected with artificially constructed heteroduplex DNA molecules of bacteriophage A has provided evidence for the mode of action of two mismatch repair systems in E. coli (5, 6). One of these, the adenine methylationdirected mismatch repair system, has been shown to play an important role in replication fidelity by repairing errors in a newly replicated DNA strand, distinguishing that strand by the absence of methylation at GATC sequences. This repair process directs the replacement of long tracts of the undermethylated strand (several kilobases) when a base pair mismatch is present (7). Some of the functions repaired for this mismatch repair system are encoded by mutL, mutS, mutH, and mutU (uvrD) (8). A localized repair activity became evident when the methyl-directed mismatch repair system was disabled as a result of mutations in mutH or mutU (5). This localized repair results in separation of very closely linked markers, requires the functions mutL and mutS, is independent of adenine methylation, efficiently corrects the T of G-T mismatches resulting from a C to T transition mutation in the inner C in CC(A/T)G sites (6), and is similar to the very short patch repair described by Lieb (9, 10).Serendipitous evidence of additional localized mismatch repair (11) suggested a strategy that allows the investigation of repair capacities, one mismatch at a time. DNA is isolated from phage harboring conditional (amber) mutations in functions essential for DNA replication, the strands are separated, and complementary strands from each of two different mutants are hybridized. The heteroduplex molecules are packaged in vitro and the assembled phage can be plated under permissive conditions, to determine the phage yield, or on hosts that do not suppress the amber mutations. In the latter case, plaque formation requires repair of the mutant nucleotide on the transcribed strand, without co-correction of the neighboring wild-type nucleotide that is, in turn, mismatched with a mutant nucleotide on the untranscribed strand. Thus, plaque formation reflects repair of a particular mismatch. This strategy has revealed the presence of two mismatch repair capabilities present in E. coli. One of these functions removes the A of C-A and G-A mismatches and the other corrects a C in a C C mismatch. When the A removal function is disabled, the bacteria display a mutator phenotype.

Earlier investigations of pneumococcal transformation revealed a function (hex+) responsible for severely reducing the transformation yield of certain markers. A mutational alteration (hex-) responsible for the loss of this function has been transferred into a hex+ strain to permit a comparison of the hex+ and hex-phenoitypes in an isogenic background.The loss of the hex+ function results both in a loss of the capacity to eliminate the low efficiency markers in transformation and a substantial increase in the spontaneous mutation rate. These properties of the hex-strain could result from the loss of a capacity to eliminate certain classes of mismatched base pairs that occur as intermediates in both transformation and mutagenesis.Genetic transformation of pneumococcus proceeds by the insertion of a single-strand fragment of donor DNA into the DNA of the recipient bacterium. The product, a heteroduplex structure, is also heterozygous when a mutational difference distinguishing the donor and recipient strains occurs within the heteroduplex region (1-4). For several markers the yield of transformants begins to approach the number of donor genomes in the DNA taken up by the cells (5, 6). These markers are referred to as high efficiency (HE) markers.For other markers the yield of viable transformants, when compared to the yield of HE transformants, is substantially reduced. A striking class of such markers, that include point mutations, show a 10-to 20-fold reduction in relative yield of transformants and are referred to as low efficiency (LE) markers (7,8).Pneumococcal strains which do not discriminate between high and low efficiency markers have been identified among existing stock cultures as well as after exhaustive mutagenesis (10, 11; Fox, M. S., unpublished results). In order to examine the phenotype of these mutants, the mutation responsible for the loss of capacity to discriminate [referred to as hex-by Lacks (10)1 was introduced, by transformation, into our standard discriminating strain (hex+). The integration efficiencies of both HE and LE markers are the same in two of these hex-strains and are the same as the efficiency observed for HE markers in our otherwise isogenic hex+ strain.The hex+ function appears to be responsible for the selective loss of transformants harboring certain base-pair mismatches. Since spontaneously occurring point mutations would be expected to exist transiently as heterozygotes, harboring basepair mismatches, it was thought that the presence of the hex4 function might result in the loss of some of these mutants by the same mechanism that is responsible for the loss of the LE transformants.The spontaneous mutation rates at several loci are indeed substantially lower in the discriminating strain than in the nondiscriminating strain. Examination, using the discriminating hex+ recipient, of the integration efficiencies of markers arising as spontaneous mutants in the hex+ strain and the hex-strain substantiate the proposal that the reduced mutation rate observed in the hex+ s...

The appearance of transformed foci after x-irradiation of the C3H 10T1/2 line of murine cells requires extensive proliferation followed by prolonged incubation under conditions of confluence. When the progeny of irradiated cells are resuspended and plated to determine the number of potential transformed foci, the absolute yield is constant over a wide range of dilutions and is similar to that observed in cultures that have not been resuspended. In addition, for cells exposed to a given x-ray dose, the number of transformed foci per dish is independent of the number of irradiated cells. These observations suggest that few, if any, of the transformed clones occur as a direct consequence of the x-ray exposure and challenge the hypothesis that transformed foci are the clonal products of occasional cells that have experienced an x-ray-induced mutational change. Rather, it appears that at least two steps are involved. We suggest that exposure to x-rays result in a change, for example, the induction or expression of some cell function, in many or all of the cells and that this change is transmitted to the progeny of the surviving cells; a consequence of this change is an enhanced probability of the occurrence of a second step, transformation, when these cells are maintained under conditions of confluence.

No abstract

In the transformation of Diplococcus pneumonia, fragments of the transforming deoxyribonucleate (DNA) are inserted into the genome of recipient transformable bacteria. This entire process requires little or no net DNA synthesis., 2 Immediately following its fixation, the transforming DNA can be reisolated from the transformed bacterial population and can be demonstrated to be without biological activity. The newly introduced DNA recovers its activity with a half time of about 3 min and thereafter replicates in synchrony with the bulk DNA of the recipient bacteria." Lacks4 has examined the inactive transforming DNA extracted from transformed bacteria. Some of the material was degraded and the remainder was denatured. He concluded that the denatured DNA was singlestranded and proposed an integration mechanism on this basis.We will present evidence demonstrating that the transforming DNA extracted, prior to its replication, from transformed bacteria is a hybrid, which is apparently formed with the DNA of the recipient bacteria and which extends over a region of about one or two million daltons. Furthermore, the newly introduced DNA appears to be covalently linked to the DNA of the recipient bacteria.Experimental.-Transforming DNA carrying the density labels deuterium and nitrogen-15 was isolated from a streptomycin-resistant, p-nitrobenzoic acid-sensitive strain of pneumococcus,5 RF6S. The bacteria were allowed many generations of growth in a heavy-isotope-labeled medium containing p32 in the form of phosphate. The nutrients in the medium were in the form of sugar and amino acid extracts from algae that had been grown in a deuterium, nitrogen-15-substituted medium. The algal extracts were generously provided by H. Crespi.The bacteria were centrifuged, washed two times in 0.15 M NaCl, 0.01 M versene, pH 7.5, and then lysed with a mixture of sodium dodecyl sulphate and deoxycholate at a final concentration of 0.05% and 0.025%, respectively. The lysate was shaken with chloroform and isoamyl alcohol2 and either (a) treated with boiled ribonuclease, alcohol precipitated, redissolved in M/40 phosphate buffered saline, pH 7.5, and filtered through an HA Millipore filter before using, or (b) DNA reisolated from a preparative equilibrium density gradient centrifugation in CsCl.Equilibrium density gradient centrifugation was performed by adding DNA samples to a concentrated solution of CsCl (Trona) to give a final density of 1.70, a final volume of 1.5 ml, and a DNA concentration of less than 10 gg/ml. Samples were centrifuged at 36,000 rpm and 210C for 42 hr. The centrifuge tubes were punctured with a needle, and 26-32 fractions of two drops each were collected. The drops were diluted with 5 vol of M/40 phosphate buffered saline, pH 7.5, and aliquots were scanned for radioactivity and assayed for biological activity. Both the donor marker, streptomycin resistance, and the recipient marker, p-nitrobenzoic acid resistance, were assayed on the test strain RF6 which is sensitive to both drugs. Under these conditions, native "hea...