Temperature-Sensitive Mutants for the Replication of Plasmids in ESCHERICHIA COLI II. Properties of Host and Plasmid Mutations

Host mutations in Escherichia coli K12 selected for the temperature-sensitive replication of the bacterial plasmid colicinogenic factor E(1) (ColE(1)) exhibit a pleiotropic effect with respect to the effect of the mutation on other extra-chromosomal elements. The mutations also vary with respect to the time of incubation of the cells at 43 degrees C required for complete cessation of ColE(1) DNA synthesis. While the synthesis of the bacterial chromosome appears unaffected, supercoiled ColE(1) DNA replication stops immediately in some mutants and gradually decreases during several generations of cell growth before stopping in others. Mutations isolated in the ColE(1) plasmid resulted in only a gradual cessation of ColE(1) DNA synthesis over several generations of cell growth at 43 degrees C. Conjugal transfer of the ColE(1) and ColV factors occurs normally in the host mutants when the transfer is carried out at the permissive temperature; however, the presence of a group I mutation in the donor cell prohibited conjugal transfer of either plasmid DNA at 43 degrees C to a normal recipient cell. Similarly, the presence of this mutation in the recipient prevented the establishment of ColE(1) or ColV in the mutant recipient cell upon conjugation with a normal donor at 43 degrees C. Various host ColE(1) replication mutants carrying either ColE(1) or ColE(2) were also defective in the mitomycin C-induced production of colicin E(1) or colicin E(2) at 43 degrees C. The majority of the host mutations examined exhibited a temperature sensitivity to growth in deoxycholate in addition to the inhibition of plasmid DNA replication, suggesting a membrane alteration in these mutants when grown at the restrictive temperature.     

Temperature-Sensitive Mutants for the Replication of Plasmids in ESCHERICHIA COLI I. Isolation and Specificity of Host and Plasmid Mutations

Temperature-sensitive mutants of Escherichia coli defective in the replication of the plasmid colicinogenic factor E1 (ColE(1)) were isolated following mutagenesis of E. coli K12 strain carrying the ColE(1) factor. Following the mutagenic treatment an enrichment procedure utilizing the replacement of thymine with bromouracil in the ColE(1) DNA duplicated at the restrictive temperature was used. The mutants isolated following this enrichment step were the result of a mutation event either in the host chromosome or in the ColE(1) plasmid. The host mutants fell into three phenotypic classes based on the effect each mutation had on the maintenance of a variety of other extrachromosomal DNA elements. Phenotypic class I mutations affected all E. coli plasmids, both the I and F sex factor types as well as the ColE(1) factor. Phenotypic class II mutations affected the maintenance of the ColE(1) and the F sex factor type plasmids and not the I type, while phenotypic class III mutations affected only ColE(1) replication. None of these mutations was found to have a significant effect on the replication of the E. coli chromosome. The plasmid-linked mutations fell into two phenotypic classes on the basis of the ability of the Flac episome to complement the mutation in the ColE(1) plasmid.     

A Dominant Constitutive phoR Mutation in ESCHERICHIA COLI

A dominant constitutive mutation of the phoR locus controlling alkaline phosphatase synthesis in Escherichia coli is described. Its phenotype can be explained by the production of a poisonous subunit of the phoR gene product. The phoR gene product is inferred to consist of at least 3 or 4 subunits.     

Functional Interchangeability of DNA Replication Genes in SALMONELLA TYPHIMURIUM and ESCHERICHIA COLI Demonstrated by a General Complementation Procedure

Twenty-four genes from Salmonella typhimurium that affect DNA replication were isolated from a lambda-Salmonella genomic library by lysogenic complementation of temperature-sensitive mutants of Salmonella or E. coli, using a new plaque complementation assay. The complementing lambda clones, which make red plaques in this assay, and noncomplementing mutant derivatives, which make uncolored plaques, were used to further characterize the temperature-sensitive Salmonella mutants and to establish the functional similarity of E. coli and Salmonella DNA replication genes. For 17 of 18 E. coli mutants representing distinct loci, a Salmonella gene that complemented the mutant was found. This result indicates that single Salmonella replication proteins are able to function in otherwise all E. coli replication complexes and suggests that the detailed properties of Salmonella and E. coli replication proteins are very similar. The other seven Salmonella genes that were cloned were unrelated functionally to any E. coli genes examined. --As an aid to the derivation of chromosomal mutations affecting some of the cloned genes, a general method was developed for placing a transposon in the Salmonella chromosome in a segment corresponding to cloned DNA. Chromosomal mutations were derived in Salmonella affecting a gene (dnaA) that was cloned by complementation of an E. coli mutant by using the transposon-encoded drug resistance as a selectable marker in local mutagenesis.     

The Infidelity of Conjugal DNA Transfer in ESCHERICHIA COLI

The accuracy of replication and transfer of a lacI gene on an F' plasmid was measured. Following conjugal transfer of the F', a small but reproducible increase (1.8-fold) in the frequency of lacI- mutations was detected. Among these, however, the frequency of nonsense mutations was 15-fold higher than in the absence of transfer. This corresponds to a 300-fold increase in the rate of base substitutions per round of replication compared with normal vegetative DNA replication. The amber mutational spectra revealed that, following conjugal transfer, mutation frequencies were increased markedly at all sites detected. In addition, an increase in G:C leads to A:T transitions was noted and was due almost entirely to an enhanced proportion of mutants recovered at the spontaneous hotspots (amber sites 6, 15 and 34). recA-dependent processes were not responsible for the increase in mutation, since similar results were observed with various recA- donor and recipient combinations. These results demonstrate that the fidelity of conjugal DNA replication is considerably lower than that of vegetative DNA replication.     

大肠杆菌L型生物学特性及致病性的研究

我们对人工诱导的大肠杆菌L型从生物学特性、致病性以及耐药性R质粒传递等方面进行了初步的研究。结果表明:L型菌从菌体形态、菌落特征、生化反应等方面与原型菌有较大差别;原型大肠杆菌和相应的L型菌接合试验均阳性,但后者接合频率较前者明显低;将L型菌经膀胱和尾静脉感染小鼠,引起实验鼠脏器的间质性炎症,通过病理学和细菌学检查证明,L型菌可直接引起脏器感染,并非只有返祖后才能致病。     

A Temperature-Sensitive Mutation Affecting Synthesis of Outer Arm Dyneins in Tetrahymena thermophila

ABSTRACT. We have characterized a novel, temperature-sensitive mutation affecting motility in Tetrahymena thermophila . Mutants grew and divided normally at the restrictive temperature (38°C), but became nonmotile. Scanning electron microscopic analysis indicated that nonmotile mutants contained the normal number of cilia and that the cilia were of normal length. Transmission electron microscopic analysis indicated that axonemes isolated from nonmotile mutants lacked outer dynein arms, so the mutation was named oad I ( outer arm defficient ). Motile mutants shifted to 38° C under conditions that prevent cell growth and division (starvation) remained motile suggesting that once assembled into axonemes at the permissive temperature (28° C) the outer arm dyneins remain functional at 38° C. Starved, deciliated mutants regenerated a full complement of functional cilia at 38° C, indicating that the mechanism that incorporates the outer arm dynein into developing axonemes is not affected by the oad I mutation. Starved, nonmotile mutants regained motility when shifted back to 28° C, but not when incubated with cycloheximide. We interpret these results to rule out the hypothesis that the oad I mutation affects the site on the microtubules to which the outer arm dyneins bind. Axonemes isolated from mutants grown for one generation at 38° C had a mean of 6.0 outer arm dyneins, and axonemes isolated from mutants grown for two generations at 38° C had a mean of 3.2 outer arm dyneins. Taken together, these results indicate that the oad I mutation affects the synthesis of outer arm dyneins in Tetrahymena .     

Partial Characterization of a Temperature-Sensitive Mutation Affecting Acetyl Coenzyme A Carboxylase in Escherichia coli K-12

A temperature-sensitive mutation in Escherichia coli K-12 was shown to affect acetyl coenzyme A carboxylase and to map at min 63. This site is designated fabE.     

Replication Process of the Parvovirus H-1 III. Factors Affecting H-1 RF DNA Synthesis

Replication of the single-stranded DNA parvovirus H-1 involves the synthesis of a double-stranded DNA replicative form (RF). In this study, the metabolism of RF DNA was examined in parasynchronous hamster embryo cells. The initiation of RF DNA replication was found to occur late in S phase, as was the synthesis of the DNA upon which subsequent viral hemagglutinin synthesis is dependent. Evidence is presented which indicates that initiation of RF replication requires proteins synthesized in late S phase, but that concomittant protein synthesis is not required for the continuation of RF replication. The data also suggest a requirement for viral protein(s) for progeny strand synthesis. Incorporation of 5-bromo-2'-deoxyuridine (BUdR) into viral DNA resulted in an "all-or-none" inhibition of viral hemagglutinin and viral antigen synthesis. BUdR inactivation of viral protein function was used to explore the time of synthesis of viral DNA serving as template for viral RNA synthesis and the effect of viral protein on RF replication and progeny strand synthesis. Results of this study suggest that parental RF DNA is synthesized shortly after infection, and that viral mRNA is transcribed from only a few copies of the viral genome in each cell. They also support the conclusion that viral protein is inhibitory to RF DNA replication. Density labeling of RF DNA with BUdR, allowing separation of viral strand DNA (V) from viral complementary strand (C), provided additional data in support of the above findings.     

Function of DNA Polymerase III in DNA Replication

RECENTLY an in vitro system for DNA replication has been described. This system could be divided into two fractions (A and B) both of which are necessary for proper DNA replication¹. Fraction A, the “soluble” fraction, contains those proteins which do not tightly bind to membranes or native DNA. Fraction B, the “insoluble” fraction, consists of DNA and membranous structures and proteins which are bound to either of them. It was shown that the soluble fraction contains at least one component which is needed at about in vivo concentration¹. Studies of one such component are described in the following.