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PubMed1969

Mapping of deletions and substitutions in heteroduplex DNA molecules of bacteriophage lambda by electron microscopy.

Electron microscopy of heteroduplex DNA molecules, composed of one strand of Escherichia coli phage lambda(+) DNA annealed to the complementary DNA strand of a lambda deletion or substitution mutant, permits visualization, as well as precise measurements and mapping, of the unpaired single-stranded regions of nonhomology in the otherwise double-stranded molecules. In the lambdab2 mutant, the central segment (13 percent) of the lambda(+) DNA molecule is shown to be deleted. In the hybrid phages lambda(i434) and lambda(i21) a segment of the right arm of the lambda(+) genome (5.5 or 7.6 to 9 percent) is replaced by the corresponding immunity regions of phage 434 (3.3 percent or phage 21 (4 percent) DNA. The b5 region in the lambdab5 mutant appears to be identical to the i(21) segment. From these data it is possible to estimate the size and posiion of those lambda genes which are replaced by the i(434) and i(21) segments. The method permits preparing complete physical maps of viral genomes with a precision heretofore unattainable.

Centrifugation, Density Gradient↗
PubMed1979

Cloning of herpes simplex type 1 DNA fragments in a bacteriophage lambda vector.

DNA isolated from defective and nondefective virions of herpes simplex type 1 (HSV-1) (strain Patton) was digested with restriction endonucleases, and the resulting DNA fragments were inserted in the EK2 coliphage vector lambdagtWES . lambdaB. The recombinant DNA was encapsidated in vitro under P4 maximum containment conditions. These lambda-HSV1 hybrids were purified and amplified, and the DNA was isolated in the P4 facility. DNA, free of viable phage and bacteria, was removed from P4 conditions and analyzed. Represented among the hybrids studied to date are DNA fragments from about 50 percent of the normal HSV-1 genome. The hybrids derived from defective HSV-1 DNA fragments demonstrate the existence of many similar but not identical classes of defective genomes.

Chromosome Mapping↗
PubMed1971

Inactivation of Escherichia coli, F' episomes at transfer, and bacteriophage lambda by psoralen plus 360-nm light: significance of deoxyribonucleic acid cross-links.

We have investigated some biological consequences of light-induced psoralen-deoxyribonucleic acid (DNA) adducts and find that for several Escherichia coli functions (killing of strain AB2480 recA13 uvrA6, inactivation of phage lambda plaque-forming ability in wild type and uvrA6 hosts, loss of ability to transmit intact Flac(+) episomes), a light exposure sufficient for production of a single cross-link per DNA molecule correlates well with the biological consequence. Although one cross-link per genome is apparently lethal to recA13 uvr(-) strains, mutants carrying the recA13 or uvrA6 markers survive light exposures producing 6.7 and 16 cross-links per genome, respectively, and wild-type cells recover from 65 psoralen cross-links. Evidently, the excision and recombinational repair systems complement one another in reconstructing an intact genome from cellular DNA containing psoralen photoproducts. The above bacterial and phage strains, in which DNA repair processes are minimized, are also extremely sensitive to pyrimidine dimer-forming 254-nm UV light (without psoralen), and were expected to respond similarly to formation of psoralen-pyrimidine base monoadducts in their DNA. Since the biological inactivation by psoralen correlates well with cross-link formation, we suggest that the sensitizing action of this drug primarily derives from its ability to form DNA cross-links.

Cell Survival↗
PubMed1972

Degradation of bacteriophage lambda deoxyribonucleic acid after restriction by Escherichia coli K-12.

Wild-type bacteria which restrict the deoxyribonucleic acid (DNA) of infecting phage when the phage do not carry the proper host modification rapidly degrade that restricted DNA to acid-soluble products. The purified restriction enzyme acts as an endonuclease in vitro to cleave restrictable DNA and does not further degrade the DNA fragments produced. We have examined mutants of Escherichia coli K-12 which lack various nucleases in order to determine which nucleases are involved in the rapid acid solubilization in vivo of unmodified lambda DNA following restriction. Bacteria which are wild type, recA(-), or polA1(-) degrade about 50% of the unmodified phage DNA within 10 min of infection, with little subsequent degradation. Mutants which are recB(-) or recC(-) degrade unmodified DNA very slowly, solubilizing about 15% of the DNA by 10 min after infection. Two classes of phenotypic revertants of recB(-)/C(-) mutants were also tested. Bacteria which are sbcA(-) restrict poorly and do not degrade much of the restricted DNA. Bacteria which are sbcB(-) restrict normally. This mutation does not appear to affect degradation of restricted phage DNA in recB(-)/C(-) mutants, but such degradation is decreased in recB(+)/C(+) bacteria. The presence of a functional lambda exonuclease gene is not required for degradation after restriction.

Adenosine Triphosphate↗
PubMed1973

Isolation of a mutant of Escherichia coli defective in cytosine-specific deoxyribonucleic acid methylase activity and in partial protection of bacteriophage lambda against restriction by cells containing the N-3 drug-resistance factor.

A mutant (designated mec(-)) of Escherichia coli F(+) 100 endo I(-)su(+) r(K) (-)m(K) (+) has been isolated which is defective in cytosine-specific deoxyribonucleic acid (DNA) methylase activity. The DNA of this mutant, as well as the DNA of phages lambda and fd propagated in it, is virtually devoid of 5-methyl-cytosine (MeC); in contrast, the mutation has no significant effect on the level of N(6)-methyladenine in DNA. Phage lambda grown on the mec(-) mutant is more strongly restricted by N-3-containing cells than is lambda grown on the mec(+) parent. These results suggest that methylation of certain cytosine residues by the E. coli K-12 enzyme partially protects lambda DNA from either the N-3 restriction nuclease or against secondary degradation subsequent to N-3-specific degradation. Analysis of the MeC level in viral and cellular DNA obtained from mec(+), mec(+) (m(N3) (+)), and mec(-) (m(N3) (+)) strains has led to the conclusion that the R-factor controlled DNA-cytosine methylase may be capable of methylating a sequence(s) which is a substrate for the K-12 enzyme.

Adenine↗