Entire Replication Research Articles

Herpesvirus saimiri open reading frame 50 (Rta) protein reactivates the lytic replication cycle in a persistently infected A549 cell line.

Herpesviruses occur in two distinct forms of infection, lytic replication and latent persistence. In this study, we investigated the molecular mechanisms that govern the latent-lytic switch in the prototype gamma-2 herpesvirus, herpesvirus saimiri (HVS). We utilized a persistently HVS-infected A549 cell line, in which HVS DNA is stably maintained as nonintegrated circular episomes, to assess the role of the open reading frame 50 (ORF 50) (Rta) proteins in the latent-lytic switch. Northern blot analysis and virus recovery assays determined that the ORF 50a gene product, when expressed under the control of a constitutively active promoter, was sufficient to reactivate the entire lytic replication cycle, producing infectious virus particles. Furthermore, although the ORF 50 proteins of HVS strains A11 and C488 are structurally divergent, they were both capable of inducing the lytic replication cycle in this model of HVS latency.

Intermediates in the assembly pathway of the double-stranded RNA virus phi6.

The double-stranded RNA bacteriophage phi6 contains a nucleocapsid enclosed by a lipid envelope. The nucleocapsid has an outer layer of protein P8 and a core consisting of the four proteins P1, P2, P4 and P7. These four proteins form the polyhedral structure which acts as the RNA packaging and polymerase complex. Simultaneous expression of these four proteins in Escherichia coli gives rise to procapsids that can carry out the entire RNA replication cycle. Icosahedral image reconstruction from cryo-electron micrographs was used to determine the three-dimensional structures of the virion-isolated nucleocapsid and core, and of several procapsid-related particles expressed and assembled in E. coli. The nucleocapsid has a T = 13 surface lattice, composed primarily of P8. The core is a rounded structure with turrets projecting from the 5-fold vertices, while the procapsid is smaller than the core and more dodecahedral. The differences between the core and the procapsid suggest that maturation involves extensive structural rearrangements producing expansion. These rearrangements are co-ordinated with the packaging and RNA polymerization reactions that result in virus assembly. This structural characterization of the phi6 assembly intermediates reveals the ordered progression of obligate stages leading to virion assembly along with striking similarities to the corresponding Reoviridae structures.

Polynucleotide phosphorylase of Escherichia coli is required for the establishment of bacteriophage P4 immunity.

Bacteriophage P4's superinfection immunity mechanism is unique among those of other known bacteriophages in several respects: (i) the P4 immunity factor is not a protein but a short, stable RNA (CI RNA); (ii) in the prophage the expression of the replication operon is prevented by premature transcription termination rather than by repression of transcription initiation; (iii) transcription termination is controlled via RNA-RNA interactions between the CI RNA and two complementary target sequences on the nascent transcript; and (iv) the CI RNA is produced by processing of the same transcript it controls. It was thought that several host-encoded factors may participate in the molecular events required for P4 immunity expression, i.e., RNA processing, RNA-RNA interactions, and transcription termination. To identify such factors we searched for Escherichia coli mutations that affect P4 lysogenization. One such mutation, bfl-1, severely reduced P4's lysogenization frequency and delayed both the disappearance of the long transcripts that cover the entire replication operon and the appearance of the CI RNA. By physical mapping and genetic analysis we show that bfl-1 is allelic to pnp, which codes for polynucleotide phosphorylase, a 3'-to-5' exonucleolytic enzyme. A previously isolated pnp null mutant (pnp-7) exhibited a phenotype similar to that of bfl-1. These results indicate that the polynucleotide phosphorylase of E. coli is involved with the maturation pathway of bacteriophage P4's RNA immunity factor.

Aldehyde-induced protein-DNA crosslinks disrupt specific stages of SV40 DNA replication.

Aldehydes with specific protein-DNA crosslinking ability disrupted simian virus 40 (SV40) DNA replication to cause replication fork failure by the 40S intermediate pathway, in which replicating viral genomes become inactivated and torsionally stressed. In contrast, aldehydes without detectable protein-DNA crosslinking ability had no effect on SV40 DNA replication during the 10 min exposure times employed. This indicates that protein-DNA crosslinks block either DNA polymerase or the entire replication complex. Replication failure by the 40S pathway is known to initiate recombinational events in the damaged SV40 replicons. Similar events in cellular replicons may play a role in the clastogenic effects of formaldehyde. In addition, formaldehyde and acrolein caused accumulation of catenated (topologically linked) SV40 daughter chromosomes--a signature of topoisomerase II inhibition.

Exon-intron-like pattern of the first propagating molecule?

A biogeochemical scenario is proposed, according to which prebiotic self-replication of RNA-like molecules evolved gradually from domain propagation to entire strand replication. The hypothetical replicating entities that formed an evolutionary continuity in time, the prebioectons, were characterized by self-structuring, and started their evolution from strands possessing one or more templatable domains. The inherent self-structuring implies a very early coevolution of the replicating molecules with prebiotic peptides, in line with the cassette model of Cedergren and Grosjean (1987). The proposed approach can be partially tested in the laboratory.

Detection of a 10 kb DNA replication intermediate in human melanoma cells.

DNA replication in human melanoma cells is investigated by lysing the cells in dilute alkali. This lysis condition results in the release from parental DNA of the single-stranded DNA fragments located in active replicating units. The size of the released DNA should theoretically range from that of Okazaki-fragments up to that of the entire replication unit. However, the results showed that the released DNA replication intermediates which are detected range in the size between Okazaki-fragments up to 10 kb DNA fragments. The 10 kb DNA fragments show a discrete appearance in agarose gel electrophoresis. Moreover the kinetic results indicate that the ligation of the 10 kb DNA fragments to form high molecular weight DNA is a late step. A prerequisite for the release of this DNA fragment as a discrete population is that there are gaps in the continuity of the newly synthesized DNA spaced roughly 10 kb away from each other.

PED100, a conjugative F plasmid derivative without insertion sequences.

The largest HindIII fragment of F includes the entire replication and transfer regions, and its circularisation with ligase gave the conjugative plasmid pED100. This plasmid, which contains none of the F insertion sequences, was essentially unable to mobilise the E. coli chromosome or to give integrative suppression of a dnaA strain.

Action of inhibitors of macromolecule formation on duplication of B. subtilis phage 2C-DNA

The action exerted by inhibitors of DNA, RNA and protein synthesis on the replication of B. subtilis phage 2C and on the duplication of its DNA was investigated. Virus yield was reduced by all the antibiotics tested, the most active drugs being actinomycin, rifampicin and the mixture of the two virginiamycin components VM and VS. The effect of these inhibitors on virion formation was irreversible, whereas that of chloramphenicol, nalidixic acid, mitomycin and of VM and VS separately was fully reversible. The phage genome, containing hydroxymethyluracil instead of thymine, was labeled by adding [ 3H]uracil to infected cells. Hence, this precursor was used to monitor the action of inhibitors on viral DNA replication. Actinomycin prevented completely viral DNA formation at any moment of the viral cycle, whereas the effect of rifampicin was restricted to the eclipse phase. Duplicational and translational inhibitors (mitomycin, nalidixic acid, chloramphenicol, VM and VS), were partly inhibitory during the entire replication cycle, the extent of inhibition of DNA synthesis being inversely related to the time elapsed from infection. The implication of these findings on the pattern of macromolecule formation during the viral cycle is discussed.

Discontinuous duplication of both strands of virus 2C DNA.

When Bacillus subtilis infected with virus 2C was labeled with short pulses of tritiated uracil (a specific precursor of the hydroxymethyluracil replacing thymine in 2C chromosome) and the cell lysate was fractionated in alkaline sucrose gradients, most of the radioactivity was found within a lowmolecular-weight peak of DNA (“Okazaki pieces”) of about 2×106 daltons. After longer pulses, segments of large size and full strands were the main labeled products. A shift from low to high-molecular-weight nascent DNA took place slowly during the eclipse phase, and rapidly during the maturation phase. This suggested a lagging of the ligase behind the polymerase at early stages. During the entire replication cycle, pulse-labeled DNA was present within the replication fork in its native form with no involvement of proteins. Furthermore, the amount of lowmolecular-weight pulse-labeled DNA hybridizing with the L strand was higher than that annealing with the H strand. To explain this finding it is suggested that the polymerization or the ligation of small segments is faster for one strand than for the other. Finally, the DNA fraction which is resistant to single-strand specific nuclease was about 10% when alkali-denatured Okazaki pieces were neutralized at 4C and was increased about five-fold upon selfannealing. It is concluded that both strands of 2C DNA duplicate in a discontinuous fashion through the formation of Okazaki pieces, which are held by hydrogen bonds initially and subsequently by phosphodiester linkages. It is proposed also that ligase action is faster for the L strand than for the H strand, and that this phenomenon might be related to both the polarity of chain growth and the asymmetry of transcription of 2C chromosome.

A simplified technique for in vivo analysis of sister-chromatid exchanges using 5-bromodeoxyuridine tablets.

A small tablet of 5-bromodeoxyuridine (BrdU), implanted subcutaneously in a mouse, provides sustained release of base analog sufficient to effect substitution of DNA throughout an entire replication period. As illustrated by studies of mouse bone-marrow and spleen cells in the presence or absence of cyclophosphamide, this depot method of BrdU administration greatly simplifies in vivo analysis of sister-chromatid-exchange formation.

Specificity of genetic elements controlling regulation of early functions in temperate bacteriophages

We have investigated the specificity of genetic elements from several related temperate phages. Hybrid phages were constructed, all of which have the head and tail genes of coliphage λ, but which carry various combinations of positive regulator (“ N”) genes, promoters, transcription termination sites, repressor genes, operators, and DNA replication genes from coliphage λ, coliphage 21, and the Salmonella phage P22. The structures of these hybrid phages were inferred from the method of their construction and from their genetic properties. These inferences were then checked, where feasible, by analysis of DNA hetero-duplexes in the electron microscope. Genetic experiments using these hybrid phages suggest: 1. (1) That the DNA replication genes of phages λ and P22 cannot complement each other; however, the entire DNA replication regions of these phages can be substituted for one another. 2. (2) That the product of gene 24 of P22 (which encodes a positive regulator analogous to the N gene product of λ) is less specific for its target sites (i.e. the sites of recognition and/or action) than the regulators specified by the N genes of phages λ and 21; i.e. the P22 N product can substitute, to some degree, for the λ and 21 N products in growth and complementation tests whereas the λ and 21 N products are apparently specific for their own target sites. 3. (3) That the efficiency of N function may depend, under certain circumstances, not only on the nature of N regulation sites at or near the phage promoter but also on the nature of the distal termination signals at which the N product functions.

Induction of mitotic gene conversion with nitrous acid, 1-methyl-3-nitro-1-nitrosoguanidine and other alkylating agents in Saccharomyces cerevisiae

1. Mitotic gene conversion was induced at three different hetero-allelic loci of the yeast Saccharomyces cerevisiae with nitrous acid, 1-methyl-3-nitro-1-nitrosoguanidine, N-nitroso-N-methylurethane, 1-nitroso-imidazolidone-2 and N-nitroso-N-methylcaproamide. 2. All agents were already able to induce mitotic gene conversion at very high frequencies (1×10-3 and more) at doses of low toxicity. 3. Induced mitotic gene conversion occurs in normal mitotic cells and does not result from a meiosis-like condition. 4. Mitotic gene conversion can be accomplished in non-dividing cells provided a certain degree of metabolic activity is possible. An entire DNA replication cycle, however, is not required. 5. Chemically induced mitotic gene conversion occurs as a process with localized effects. There is, however, a certain coincidence between conversional events and recombination in the near vicinity. Moreover, chemically induced gene conversion too is a non-reciprocal process. 6. Mitotic gene conversion is a biologically relevant process in diploids as it generates specific genetic alterations at much higher frequencies than do mutation or mitotic recombination especially in the case of heterozygosis due to multiple allelism. 7. It is pointed out that mitotic gene conversion possibly is common in all diploid organism. Moreover, one well analysed example of genetically determined tumorous growth in intraspecies hybrids in the genus Nicotiana is most readily explained by mitotic gene conversion. Further, mitotic gene conversion of spontaneous or induced origin is discussed as an important genetic process leading to cancer or to other diseases in man.