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PubMed1969

A mutant of Escherichia coli permitting replication of two F factors.

Control mechanisms of replication of bacterial genetic elements are poorly understood at present. We have studied one such mechanism involving replication of the F factor in Escherichia coli. The F factor can replicate either autonomously in F(+) or F' strains, or as an integral part of the chromosome in Hfr strains. We have shown that presence of either an integrated F factor or a free F factor prevents replication of a second free F factor. Two integrated F factors can replicate in the same cell. The present experiments show that when a F'lac element was transferred by mating into an Hfr strain, it had to become integrated into the chromosome in order to persist. With a recombination-deficient (recA) Hfr strain as recipient, the frequency of F'lac integration was greatly reduced. This permitted us to isolate a mutant Hfr strain in which the F'lac element was able to replicate autonomously. The mutation has most likely occurred in the integrated F factor itself. Availability of this new recA mutant Hfr strain facilitates genetic analysis of the F factor, since in recA(+) Hfr strains frequent integration of a free F factor into the chromosome obscures recognition of complementation and recombination between two free F factors.

Chromosomes, Bacterial↗
PubMed1969

Defective phage and chromosome mobilization in Pseudomonas putida.

The transfer of transducing phage DNA in association with the mandelate genetic region of Pseudomonas putida strain PRS1 (termed pfdm) has been achieved by growing together mandelate-positive PpG2 cells harboring pfdm as an extrachromosomal element and mandelate-deleted PpG1 strains. This transfer is analogous to sexual conjugation in the enterobacteria. The transfer of pfdm elements is always associated with chromosome mobilization and some rare recombinants acquire genetic donor ability. We have therefore concluded that the pfdm elements are responsible for initiation of chromosome mobilization in a manner not yet fully understood.

Bacteriophages↗
PubMed1971

Strand-specific supercoiled DNA-protein relaxation complexes: comparison of the complexes of bacterial plasmids ColE1 and ColE2.

Certain bacterial plasmids can be isolated as unique complexes of supercoiled circular DNA and protein. These complexes are distinguished by the conversion of the supercoiled DNA to the relaxed or open-circular DNA form upon treatment with ionic detergents, proteases, or alkali. This report demonstrates that the open-circular DNA resulting from the pronase-induced relaxation of the complexes of colicinogenic factors E(1) (ColE(1)) and E(2) (ColE(2)) possesses a strand-specific break. In each case this break is found in the heavy strand of the DNA as defined by CsCl centrifugation in the presence of poly(U,G). In addition, the ColE(1) and ColE(2) complexes exhibit certain properties that are plasmid specific. Heat treatment, and to a lesser extent pronase treatment, inactivates the ColE(2) complex, making it insensitive to agents that formerly were capable of inducing relaxation (conversion of the DNA to the open-circular form). In contrast, the ColE(1) complex is not inactivated by these treatments. The potential role of these strand-specific relaxation complexes in DNA replication is discussed.

Bacterial Proteins↗
PubMed1971

Bacterial mutants defective in plasmid formation: requirement for the lon + allele.

Bacterial mutants defective in plasmid formation were selected for their inability to be transduced to chloramphenicol resistance by bacteriophage PlCM. The mutants isolated are indistinguishable from a lon mutant strain. Both the lon strain and the mutants isolated here show very poor lysogenization by P1 and very low transduction to Gal(+) by the plasmid formation of the lambdagal(8)-Nam7am53cI(857). The lon(+) gene function of the host bacteria is indispensable for plasmid formation, even though P1 and lambda(+) can grow normally on the lon strains and lambda(+) can be integrated normally in lon as well as in the lon(+) bacteria. Phage mutants that can persist as plasmids in lon strains were isolated from P1CM. The function of the lon(+) gene is discussed with regard to plasmid formation.

Chloramphenicol↗
PubMed1972

Endonucleolytic activity from Micrococcus luteus that acts on -ray-induced damage in plasmid DNA of Escherichia coli minicells.

A partially purified extract from Micrococcus luteus contains endonucleolytic activity toward ultraviolet (UV)-irradiated deoxyribonucleic acid (DNA). We found that the same extract also acts on superhelical, covalently-closed circular lambdadv DNA isolated from gamma-irradiated minicells of E. coli. The introduction of nicks in isolated covalently-closed circular DNA by an endonuclease in the extract results in relaxed circles, and these two circular DNA species are easily distinguishable by their sedimentation properties in alkaline sucrose. The frequency with which the endonuclease-susceptible lesions are produced in superhelical DNA is only marginally enhanced when (60)Co gamma-rays are administered to an aerobic rather than an anoxic minicell suspension. The ratio of endonuclease-sensitive defects to single-strand scissions, induced by gamma-irradiation in air, is about 1:3. The nuclease-sensitive lesions disappear from gamma-irradiated minicells during incubation after radiation presumably as a consequence of excision repair. Since the addition of UV-irradiated calf-thymus DNA depresses the ability of the M. luteus extract to attack not only UV-damaged E. coli DNA (a known substrate for the so-called UV-specific endonuclease that catalyzes the initial single-strand incision adjacent to the structural defect) but, also lambdadv DNA injured by gamma-rays, we conclude that physicochemical alterations induced by both types of radiation are recognized by one and the same endonuclease.

Animals↗
PubMed1972

Hybridization of variants of chloramphenicol acetyltransferase specified by fi + and fi - R factors.

An fi(-) type of R factor for transferable chloramphenicol resistance carries the structural gene for a novel species of chloramphenicol acetyltransferase (EC 2.3.1.99). The enzyme associated with the fi(-) plasmid is distinct from that described for fi(+) R factors. The fi(+)- and fi(-)-related variants of chloramphenicol acetyltransferase were hybridized in vivo and in vitro. Both techniques yielded only a single symmetrical heteromeric species rather than the three (A(3)B(1), A(2)B(2), and A(1)B(3)) hybrids predicted from the tetrameric (identical monomers) structure of chloramphenicol acetyltransferase.

Acetyltransferases↗
PubMed1972

DNA nucleotide sequence restricted by the RI endonuclease.

The sequence of DNA base pairs adjacent to the phosphodiester bonds cleaved by the RI restriction endonuclease in unmodified DNA from coliphage lambda has been determined. The 5'-terminal nucleotide labeled with (32)P and oligonucleotides up to the heptamer were analyzed from a pancreatic DNase digest. The following sequence of nucleotides adjacent to the RI break made in lambda DNA was deduced from these data and from the 3'-dinucleotide sequence and nearest-neighbor analysis obtained from repair synthesis with the DNA polymerase of Rous sarcoma virus [Formula: see text] The RI endonuclease cleavage of the phosphodiester bonds (indicated by arrows) generates 5'-phosphoryls and short cohesive termini of four nucleotides, (p)A(p)A(p)T(p)T. The most striking feature of the sequence is its symmetry.

Adenine Nucleotides↗
PubMed1972

Genetic and physicochemical characterization of Escherichia coli strains carrying fused F' elements derived from KLF1 and F57.

Although the two F' elements, F57 (carrying his(+)) and KLF1 (carrying leu(+)), cannot normally coexist in the same cell, crosses between RecA(-) strains of Escherichia coli carrying these two elements gave rise, at a low frequency, to progeny carrying both the his(+) and leu(+) markers in an extrachromosomal state. Genetic studies showed that the his(+) and leu(+) markers were now linked, and centrifugation studies of F' DNA isolated from these strains confirmed the presence of a new species of DNA molecule of altered size. It is proposed that the his(+) and leu(+) genes in these strains are covalently linked on a single "fused" F' element.

Acridines↗