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PubMed1972

Expression of ribosomal protein genes as analyzed by bacteriophage Mu-induced mutations.

The organization of ribosomal protein genes and the gene (fus) for a protein chain elongation factor, EF G, in Escherichia coli were studied with a merodiploid strain that has an episome with genetic markers, ery(r), spc(r), str(r), and fus(r), and a chromosome with markers ery(s), spc(s), str(s), and fus(s). The ery locus determines a 50S ribosomal protein and the spc and str loci determine 30S ribosomal proteins. The phenotype of the diploid strain is sensitive to all of the four antibiotics, erythromycin (Ery), spectinomycin (Spc), streptomycin (Str), and fusidic acid (Fus). Analysis of antibiotic-resistant mutants induced by bacteriophage Mu in the diploid strain indicates that these four genes, and probably many other ribosomal protein genes linked to them, are transcribed as a single unit, and the direction of the transcription is in the order of ery, spc, str, and fus.

Bacterial Proteins↗

PubMed1972

Characterization by electron microscopy of fused F-prime factors in Escherichia coli.

During previous attempts to find mutants in which incompatibility between two F-prime (F') factors of E. coli is abolished, we found that two F' factors harbored in the same cell occasionally recombined to form a single genetic unit. Thus, a fused F'trp(+)arg(+) factor was obtained from a mating of a strain carrying recA F'trp(+) with a strain carrying recA F'arg(+). In the present paper physical fusion of the two F' factors, inferred from genetic studies, is confirmed by electron microscopy. F DNA was extracted, after detergent lysis, from four fused F' strains: the parental F'trp(+) and F'arg(+) strains and three F(+) strains. This supercoiled DNA was separated on ethidium bromide-cesium chloride gradients and converted to the open circular from before electron microscopy. The contour lengths in mum of the various DNA preparations were: F'trp(+), 59.4; F'arg(+), 33.2; the four F'trp(+)arg(+) factors, 38.5, 39.6, 63.2, and 73.5: the three F factors, 31.1, 31.1, and 31.0. Thus, different fused F' factors formed from the same parental F' factors vary in length, but are shorter than the sum of the parental F' factors.

Centrifugation, Density Gradient↗

PubMed1972

The stabilization of episomal integration by genetic inversion: a general hypothesis.

In F' cells of Escherichia coli K12, recombination in the region of genetic duplication between chromosome and F-genote produces a rapid alteration between the integrated and detached states of the episome. Nevertheless, F' strains can give rise to Hfr cells in which the F-genote has undergone stable integration. We have found that one such Hfr has retained the genetic duplication as well as the recombination proficiency of the parental F' strain; to account for the failure of recombination to detach the episome in this Hfr, we have postulated the inversion of one of the duplicated regions. A general model is presented, showing that an inversion must prevent episome detachment by any combination of genetic crossovers.

Chromosome Aberrations↗

PubMed1972

Nonchromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA.

Transformation of E. coli cells treated with CaCl(2) to multiple antibiotic resistance by purified R-factor DNA is reported. Drug resistance is expressed in a small fraction of the recipient bacterial population almost immediately after uptake of DNA, but full genetic expression of resistance requires subsequent incubation in drugfree medium before antibiotic challenge. Transformed bacteria acquire a closed circular, transferable DNA species having the resistance, fertility, and sedimentation characteristics of the parent R factor. Covalently-closed, catenated, and open (nicked) circular forms of R-factor DNA are all effective in transformation, but denaturation and sonication abolish the transforming ability of R-factor DNA in this system.

Anti-Bacterial Agents↗

PubMed1972

Isolation of supercoiled colicinogenic factor E 1 DNA sensitive to ribonuclease and alkali.

The synthesis of the covalently-closed, circular DNA form of colicinogenic factor E(1) (ColE(1)) continues in Escherichia coli cells after the addition of chloramphenicol. A large portion of the purified supercoiled ColE(1) DNA molecules made in the presence of chloramphenicol are converted to the open circular DNA form after treatment with alkali (pH 13), RNase A, or RNase H. These treatments do not significantly affect the covalently-closed form of ColE(1) DNA isolated from normally growing E. coli cells. The open circular product resulting from treatment of supercoiled ColE(1) DNA with RNase A possesses a single break in one strand of the circular duplex. The site sensitive to RNase A occurs with equal probability in either of the complementary strands. Both synthesis of ColE(1) DNA and the formation of supercoiled ColE(1) DNA sensitive to RNase A or alkali are prevented by the inhibitor of RNA synthesis, rifampicin. These results indicate that covalently-closed ColE(1) DNA containing one or more ribonucleotides accumulates during ColE(1) replication in the presence of chloramphenicol. It is proposed that this incorporated RNA served as a primer during the initiation of synthesis of ColE(1) DNA and that its removal from the circular DNA is inhibited in cells incubated in the presence of chloramphenicol.

Carbon Isotopes↗

PubMed1972

Relationship of Flac replication and chromosome replication.

The time of replication of a bacterial plasmid, Flac, during the division cycle of Escherichia coli has been estimated in exponentially growing cultures and at various times after a shift from minimal medium to a richer medium (a shift-up). There is a variation in the cell age at which the capacity to synthesize beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) doubles (assumed to be a measure of the time at which the Flac plasmid replicates) when this capacity is measured at various times during the shift-up, and with increasing steady-state exponential growth rate. Cells growing at slow and moderate growth rates exhibit Flac replication in the middle of the division cycle. With increasing time after a shift-up or with increasing growth rate the plasmid replicates at earlier times, eventually at cell division, and finally in the older cells. This variation in the cell age at which the plasmid replicates is similar to the variation in cell age at which chromosome initiation occurs during a shift-up, although plasmid replication occurs slightly before initiation of chromosome replication.

Amino Acids↗

PubMed1973

A transmissible plasmid controlling camphor oxidation in Pseudomonas putida.

Earlier papers demonstrated an extensive genetic exchange among fluorescent Pseudomonads; this one documents for genes specifying enzymes of peripheral dissimilation an extrachromosomal array, segregation, and frequent interstrain transfer. An hypothesis is presented of a general mechanism for the formation and maintenance of metabolic diversity. The example used, the path of oxidative cleavage of the carbocyclic rings of the bicyclic monoterpene D- and L-camphor, terminates in acetate release and isobutyrate chain debranching. By transduction, two gene linkage groups are shown for the reactions before and after isobutyrate. The group for reactions before isobutyrate is plasmid borne, contransferable by conjugation, mitomycin curable, and shows a higher segregation rate from cells that are multiplasmid rather than carrying a single plasmid. The genes that code for isobutyrate and essential anaplerotic and amphibolic metabolism are chromosomal. By conjugation plasmid-borne genes are transferred at a higher frequency than are chromosomal, and are transferred in homologous crosses more frequently than between heterologous species. Most isobutyrate-positive fluorescent pseudomonad strains will accept and express the camphor plasmid.

Camphor↗

PubMed1973

Genetic regulation of octane dissimilation plasmid in Pseudomonas.

The enzymes responsible for the oxidation of n-octane to octanoic acid or beyond in Pseudomonas oleovorans are octane inducible and are coded by genes borne on a transmissible extrachromosomal element. The octane to octanoate enzymes induced by octane are repressed by octanol. The chromosome also carries genes coding octanol oxidation enzymes that, in contrast, are induced by octanol, not by octane. The octane plasmid has been transferred from P. oleovorans to several other fluorescent Pseudomonas species. In exconjugants, the presence of both octane and camphor plasmids enhances their segregation rate.

Alcohol Oxidoreductases↗

PubMed1973

Recircularization and autonomous replication of a sheared R-factor DNA segment in Escherichia coli transformants.

Controlled shearing of R-factor DNA leads to formation of fragments carrying an antibiotic resistance gene present on, but not expressed by, the intact R-factor. Transformation of CaCl(2)-treated E. coli by such fragments yields an autonomously replicating tetracycline-resistance plasmid (Tc6-5) that contains only a small fraction of the genome of the parent R-factor, and lacks both its fertility functions and its other drug-resistance determinants. Although the Tc6-5 plasmid is not self-transmissible, it can interact and/or recombine with conjugally-proficient plasmids that promote its transfer to other bacteria.

Bromides↗

PubMed1973

Genetic fusion of incompatible plasmids in Pseudomonas.

The genes specifying enzymes responsible for the degradation of camphor and octane occur on transmissible plasmids in Pseudomonas putida strain PpG1 and P. oleovorans. Since the presence of the plasmids is vital for the oxidative metabolism of camphor or octane (by the cells) in the absence of other carbon sources, such naturally occurring, energy-generating plasmids have been designated as degradative plasmids. The two degradative plasmids, CAM and OCT, are incompatible with each other and cannot coexist in the same cell. By the use of UV irradiation and suitable selection techniques, it has been possible to fuse these two plasmids so they become part of the same replicon and coexist. Such a technique might be useful in introducing several degradative pathways in the same cell.

Alkanes↗

PubMed1974

Interaction between mendelian and non-mendelian genes. Regulation of the transmission of non-mendelian genes by a mendelian gene in Chlamydomonas reinhardtii.

The non-Mendelian genetic element of a given mating type of Chlamydomonas reinhardtii was first doubly marked by succesive mutageneses and then transferred into a number of offspring cells of the opposite mating type. In subsequent crosses of these offspring clones, the transmission pattern of the original non-Mendelian markers was analyzed. The results indicate that the mating-type gene of a given cell regulates the transmission and perhaps the recombination of the non-Mendelian genes regardless of whether the non-Mendelian markers were isolated originally in the same cell or obtained from another cell of the opposite mating type through genetic transmission. The regulation of the transmission of non-Mendelian genes in C. reinhardtii is thus apparently different from the autonomous control of the transmission of non-Mendelian mitochondrial genes in yeast by a genetic factor located on the mitochondrial genome.

Chlamydomonas↗

PubMed1974

Relaxation complexes of plasmids ColE1 and ColE2: unique site of the nick in the open circular DNA of the relaxed complexes.

The product of the induced relaxation of supercoiled DNA-protein relaxation complexes of colicinogenic factors E1 (ColE1) and E2 (ColE2) is an open circular DNA molecule with a strand-specific nick. Cleavage of the open circular DNA of each relaxed complex with the EcoRI restriction endonuclease demonstrates that the single-strand break is at a unique position. The site of the single-strand break in the relaxed ColE1 complex is approximately the same distance from the EcoRI cleavage site as the origin of ColE1 DNA replication.

Binding Sites↗

PubMed1974

Utilization of two distinct modes of replication by a hybrid plasmid constructed in vitro from separate replicons.

A hybrid plasmid, pSC134, that codes for two distinct sets of replication functions has been constructed in vitro by ligation of EcoRI endonuclease-cleaved pSC101 and Col E1 plasmid replicons, and has been introduced into Escherichia coli by transformation. The replication properties of the pSC134 plasmid in DNA polymerase I-defective mutants or in the presence of chloramphenicol indicate that this hybrid plasmid can utilize the functionally distinct modes of replication specified by both of its parent replicons.

Carbon Radioisotopes↗

PubMed1974

Mitochondrial genetics in Bakers' yeast: a molecular mechanism for recombinational polarity and suppressiveness.

Recombinational polarity and suppressiveness are two well-known but puzzling cytoplasmic genetic phenomena in bakers' yeast, Saccharomyces cerevisiae. Little progress has been made in characterizing the underlying molecular mechanisms of these phenomena. In this paper we describe a molecular model for recombinational polarity that is compatible with the available genetic evidence. The model stresses the role of small deletions and excision/repair processes in otherwise canonical recombinational events. According to the model, both phenomena require recombination and may share mechanistic elements.

Crosses, Genetic↗

PubMed1974

Termination point of replication of colicin E1 plasmid DNA in cell extracts.

Closed-circular monomeric molecules were one of the major products of replication of colicin E1 plasmid DNA in cell extracts. However, when the plasmid DNA synthesized in the reaction mixture was labeled for 3 min after 27 min of incubation, most of the label was found in open-circular molecules. The open-circular molecules were converted to closed-circular molecules upon further incubation. The newly replicated open-circular molecules had a nick or small gap in their newly synthesized strand. The interruption was located at approximately 20% of the molecular length from the single site of cleavage by restriction endonuclease EcoR1 and at or very close to the origin of replication. The addition of 10 mM nicotinamide mononucleotide instead of nicotinamide adenine dinucleotide to extracts did not significantly affect the kinetics of the colicin E1 plasmid DNA synthesis. However, in the presence of nicotinamide mononucleotide the formation of completely replicated closed-circular molecules was suppressed and, instead, open-circular molecules accumulated with an interruption in the newly synthesized strand at the termination point of replication, which was located at or very close to the origin of replication.

Cell-Free System↗

PubMed1974

Replication of colicin E1 plasmid DNA in cell extracts.

Cell extracts were prepared from Escherichia coli carrying colicin E1 plasmid. The DNA in extracts was almost exclusively closed-circular DNA of the plasmid. Labeled deoxyribonucleotides were incorporated into DNA in extracts. DNA of colicin E1 plasmid was the sole DNA product, and was composed of completely replicated molecules and a class of replicative intermediates. The intermediates carried an average of approximately two pieces of DNA fragments that had a sedimentation coefficient of approximately 6 S and were not covalently attached to the parental DNA strands. The replication was initiated on closed-circular molecules and the complete molecules were synthesized semiconservatively. The DNA synthesis depended on the four ribonucleoside triphosphates and was sensitive to rifampicin. A round of DNA replication, once initiated, was completed in the presence of rifampicin, indicating that RNA synthesis is involved in the initiation of replication of the plasmid DNA. Most of the replicated molecules were isolated in super-coiled structures. These results indicate that this soluble system is capable of carrying out a complete round of replication of colicin E1 plasmid DNA.

Cell-Free System↗

PubMed1974

Genome construction between bacterial species in vitro: replication and expression of Staphylococcus plasmid genes in Escherichia coli.

Genes carried by EcoRI endonuclease-generated fragments of Staphylococcus plasmid DNA have been covalently joined to the E. coli antibiotic-resistance plasmid pSC101, and the resulting hybrid molecules have been introduced into E. coli by transformation. The newly constructed plasmids replicate as biologically functional units in E. coli, and express genetic information carried by both of the parent DNA molecules. In addition, electron microscope heteroduplex analysis of the recombinant plasmids indicate that they contain DNA sequences derived from E. coli and Staphylococcus aureus. Recombinant molecules can transform other E. coli cells for penicillin-resistance markers originally carried by the staphylococcal plasmid, and can be transferred among E. coli strains by conjugally proficient transfer plasmids.

Centrifugation, Density Gradient↗

PubMed1974

Replication of colicin E1 plasmid DNA in cell extracts. II. Selective synthesis of early replicative intermediates.

The major products of colicin E1 plasmid DNA synthesis in cell extracts are completely replicated molecules and a class of molecules containing newly synthesized small DNA fragments. The addition of 10% glycerol and/or 2 mM spermidine to extracts blocks synthesis of completely replicated molecules while enhancing synthesis of molecules containing newly synthesized DNA fragments. The latter molecules, which contain on the average two DNA fragments of approximately 6 S, are early replicative intermediates for synthesis of completely replicated molecules. Synthesis of the intermediates is sensitive to rifampicin and depends on RNA synthesis. RNA components are linked to the 6S DNA molecules.

Centrifugation, Density Gradient↗