Replication of vaccinia DNA in mouse L cells. IV. Protein synthesis and viral DNA replication

The requirement for protein synthesis during vaccinia DNA replication in mouse L cells was investigated. Within the first 30 min after reversal of a hydroxyurea (HU) block, viral DNA replication was not affected in cells treated with cycloheximide (100 μg/ml) to inhibit protein synthesis. During this period the intermediates in DNA replication detected, the rate of chain elongation, and the accumulation of crosslinked viral DNA molecules were all identical to those observed in vaccinia-infected cells not treated with cycloheximide. Thereafter, DNA replication, as measured by incorporation of [³H]thymidine, was inhibited in cycloheximide-treated infected cells (>90%, 2 hr post-HU reversal). Inhibition of viral DNA synthesis was further demonstrated by the sparse appearance and failure of cytoplasmic viral factories to increase in size after HU reversal, when protein synthesis was inhibitied. Density labeling of replicating viral DNA molecules with bromodeoxyuridine and analysis of equilibrium density centrifugation in CsCl showed that hybrid moelcules (hl, ? = 1.77 g/ml) accumulated in cycloheximide-treated cells. The hybrid molecules were not converted to “heavy” viral DNA (hh, ? = 1.825 g/ml), as was observed to occur during viral DNA replication in cells continuously synthesizing protein. The results of these experiments showed that after an initial round of viral DNA replication was completed, new protein synthesis was required to initiate additional rounds of viral DNA replication. The dissociation of viral DNA molecules, synthesized after HU reversal, from cytoplasmic DNA complexes was inhibited by cycloheximide but not rifampin. Continuous protein synthesis, apparently to permit expression of a “late” viral function, not related to viral assembly is required for release of the newly replicated viral genomes from complexes.     

Direct double-stranded DNA sequencing with baculovirus genomes.

Double-stranded DNA sequencing with the modified T7 DNA polymerase (Sequenase) was performed directly with nuclear polyhedrosis virus DNA genomes. The conditions were optimized to allow for a rapid and unambiguous sequence analysis of nuclear polyhedrosis virus genomes.     

Replication of vaccinia DNA in mouse L cells I. In vivo DNA synthesis

Replication of vaccinia DNA was analyzed in lysates of intact, vaccinia-infected L cells prepared under conditions which preserved the structure of replicating and mature viral DNA molecules. The techniques employed permitted the separation of viral and host DNA, as confirmed by DNA-DNA hybridization. Sedimentation analysis in alkaline sucrose gradients showed that, during the period of maximum vaccinia DNA replication in L cells [2–3 hr postinfection (h.p.i.)], 10–12 S viral DNA fragments were preferentially labeled by short pulses (0.5–10 min) of [³H]thymidine. About 20–30% of the pulse-labeled DNA was hydrolyzed by nuclease S,. These results support the view that viral DNA replication was discontinuous and may involve single-stranded DNA intermediates. Pulse-chase experiments showed that the 10–12 S fragments elongated into 30–50 S “intermediate-sized” DNA species and finally into 70–72 S (full length) viral DNA in about 30 min, which would require the incorporation of 6500 nucleotides/min. The conversion of mature viral DNA (70–72 S) into mature, cross-linked DNA (which sedimented at 92–94 S and 102–106 S in alkaline sucrose gradients) occurred late in infection (5–6 h.p.i.), when virion assembly had begun. Replicating viral DNA molecules were pulse-labeled with [³H]thymidine and chased with bromodeoxyuridine (BrdU); the labeled DNA species were analyzed by equilibrium density centrifugation in CsCl. Hybrid (HL) molecules (? = 1.77 g/cm³) were detected, demonstrating that viral DNA replication was semiconservative. Analysis of replicating viral DNA molecules in ethidium bromide-CsCl gradients at equilibrium failed to show the presence of circular or superhelical duplexes. This result and the fact that no viral DNA molecules of greater than unit length were labeled during long or short pulses suggest that viral DNA replication is symmetrical.     

Replication of vaccinia DNA in mouse L cells. II. In vitro DNA synthesis in cytoplasmic extracts.

Cytoplasmic extracts prepared from vaccinia virus-infected L cells catalyzed the incorporation of labeled deoxynucleotide triphosphates into DNA which hybridized with vaccinia virus DNA. The incorporation of [³H]thymidine 5′ triphosphate ([³H]TTP) into DNA was shown to be dependent on the presence of all four deoxynucleoside triphosphates and incorporation was stimulated twofold by the addition of ATP, NAD, and ribonucleoside triphosphates. The incorporation of [³H]TTP in vitro was linear for 10 min and continued at a reduced rate for 30 min at 30°. The viral DNA synthesized in vitro was analyzed by sedimentation in alkaline-sucrose gradients and by isopycnic centrifugation in CsCl gradients. Alkaline-sucrose sedimentation analysis showed that replication of in vitro labeled DNA was discontinuous. Small fragments (～10 S) were synthesized in vitro in 10–30 sec which appeared to elongate so that after 30 min of synthesis the in vitro synthesized molecules cosedimented with in vivo labeled viral DNA species of 10–70 S. No molecules of greater length than mature viral single-stranded DNA (Type 1, 70–72 S) were observed when cell extracts prepared 3 hr postinfection were employed. Replication of viral DNA in vitro was symmetrical. No evidence for circular or superhelical DNA duplex molecules was obtained when in vitro synthesized DNA was analyzed by equilibrium density centrifugation in CsCl containing ethidium bromide.     

Cooperative sequence modules determine replication initiation sites at the human beta-globin locus

The human beta globin locus contains two adjacent replicators, each capable of initiating DNA replication when transferred from its native locus to ectopic sites. Here, we report a detailed analysis of the sequence requirements for replication initiation from these replicators. In both replicators, initiation required a combination of an asymmetric purine:pyrimidine sequence and several AT-rich stretches. Modules from the two replicators could combine to initiate replication. AT-rich sequences were essential for replicator activity: a low frequency of initiation was observed in DNA fragments that included a short stretch of AT-rich sequences, whereas inclusion of additional AT-rich stretches increased initiation efficiency. By contrast, replication initiated at a low level without the asymmetric purine:pyrimidine modules but they were required in synergy to achieve efficient initiation. These data support a combinatorial model for replicator activity and suggest that the initiation of DNA replication requires interaction between at least two distinct sequence modules.     

Requirement of adenovirus type 12 gene 401 function for initiation of virus DNA synthesis.

The highly oncogenic human adenovirus type 12 temperature sensitive mutant. H12ts401, is unable to maintain the growth characteristics of transformed cells at the non-permissive temperature. In lytic infection, the 401 gene function is required to produce virus DNA. In the present study, virus DNA synthesized in ts401-infected human cells after temperature shift-up was characterized. No apparent suppression of DNA chain elongation or ligation occurs at the non-permissive temperature, but, as shown by density labelling, new initiation of virus DNA replication is inhibited under this condition. The results indicate that the 401 gene function is involved in the initiation of virus DNA synthesis in the lytic cycle.     

Replication intermediates of herpes simplex virus DNA.

The DNA synthesized in herpes simplex virus (HSV)-infected nuclei in vivo was analyzed by chromatography on benzoylated naphthoylated DEAE-cellulose columns. Viral-DNA molecules that elute with caffeine contain single-stranded DNA sequences sensitive to a micrococcal endonuclease that cleaves single-stranded DNA only. These viral-DNA molecules behave kinetically as precursors to mature virion DNA, and their transition to the mature double-stranded form occurs within a period of 10 to 20 min. Electron microscopy revealed viral-DNA molecules at different stages of replication. The mechanism of HSV-DNA replication is discussed.     

Replication of herpesvirus DNA. II. Sedimentation characteristics of newly synthesized DNA.

The sedimentation behavior of viral DNA synthesized during short labeling periods at various times after infection was investigated. These experiments indicated that viral DNA synthesis may be divided into the following two phases: (1) During the early phase, newly synthesized DNA is associated with structures which sediment with S values up to approximately twice that of mature viral DNA; (2) during the later phase, newly synthesized DNA is associated with structures that sediment much more rapidly. Both at early and later times after infection, approximately 20% of the newly synthesized DNA sediments in sucrose gradients more slowly than does mature viral DNA. Furthermore, after isopycnic centrifugation in CsCl, most of the newly synthesized viral DNA sediments in sucrose gradients more slowly than does mature viral DNA. The smaller than unit-size, newly synthesized viral DNA molecules are breakage products resulting from the fragility of newly synthesized DNA. These molecules fragment, not only because of their fragility in the regions of the replicative forks but also because of the presence of fragile sites at other positions along the newly synthesized DNA molecule. Experiments dealing with the transfer of parental DNA to progeny virions show that most parental viral DNA strands that are transferred to progeny virions retain their integrity. Breakage and reunion of parental viral DNA strands with progeny DNA is a relatively rare event.     

Characterization of DNA synthesis and chloroplast DNA replication initiation in a Petunia hybrida chloroplast lysate system

Summary An in vitro chloroplast DNA synthesizing lysate system prepared from purified chloroplasts of Petunia hybrida leaves has been developed. Both co-isolated endogeneous chloroplast (cp)DNA and externally added DNA can be used as DNA templates in the system. The system contains a -like DNA polymerase as determined by using DNA polymerase-specific inhibitors and synthetic templates. The molecular weight of this enzyme is about 85 kd. Part of the DNA synthesizing activity is repair synthesis. When a chimaeric plasmid containing a fragment with a potential cpDNA replication origin is used as a template (pPCY62), specific initiation of DNA synthesis is observed on this fragment which strongly suggests that the in vitro chloroplast lysate system is also capable of replication initiation.     

Analysis of mutations at the fragile X locus using the DNA probe Ox1.9.

In this study, 40 families segregating for fragile X [fra (X)] syndrome were examined for the presence of a mutation within the FMR-1 gene. Using the DNA probe Ox1.9, both carriers and affected individuals were found to contain an insertion/amplification-type of mutation with somatic instability. Variability in the size of the mutation, which ranged from less than 0.2 kb to approximately 13 kb, was observed both between individuals (even from the same family) and within individuals, who showed a smear rather than a discrete band(s) on Southern blot analysis. Transmission of the mutation by males resulted in little change of its size, while transmission by females usually resulted in an increase in size. Correlations were observed between the size of inserted/amplified DNA and the level of chromosome fragility and the presence or absence of mental impairment. Overall, a mutation was detected in 66 of 67 (99%) clinically affected males, in 12 of 13 (92%) transmitting males and in 95 of 112 (85%) carrier females. Equivocal results were obtained in 12 (11%) of the carrier females. No mutation was detected in 58 females and 33 males predicted to be normal by linkage, or in one female and 36 normal control males. These results strongly suggest that the mutation detected by Ox1.9 is closely associated with the cytogenetic and clinical expression of fra (X) syndrome. Additionally, the use of this probe along with other probe/enzyme combinations should provide a sensitive clinical assay for the detection of carriers of fra (X) syndrome.     

Evolutionary variants of simian virus 40: cloned substituted variants containing multiple initiation sites for DNA replication.

Two cloned evolutionary variants of simian virus 40 (SV40) containing substitutions of cellular DNA have been characterized by restriction endonuclease analysis, electron microscopic heteroduplex mapping, and DNA-DNA hybridization. Each variant genome is made up of a small, tandemly repeated segment of DNA consisting of cellular DNA and that portion of the SV40 genome containing the initiation signal for viral DNA replication. Cellular DNA sequences are different in the two variants, indicating that recombination between cell DNA and SV40-DNA can occur at more than one site. However, one end of the SV40 segment (0.68 SV40 map-units) is the same in each variant. The data suggest that substituted variants arise by integration of SV40-DNA into cellular DNA followed by excision of a small substituted genome which is subsequently amplified to a size compatible with encapsidation; the presence of multiple initiation signals in each molecule results in selective replication.     

In situ hybridisation with digoxigenin-labelled DNA probes for detection of viral genomes.

The applicability of a recently developed non-radioactive DNA labelling and detection method, which uses the digoxigenin (DIG) enzyme linked immunosorbent assay (ELISA) system, for the detection of viral infections in pathology specimens by in situ hybridisation, was examined. Its efficacy was compared with that of biotin and radioisotope labelling methods. Three cases of progressive multifocal leucoencephalopathy, two of verruca vulgaris, and seven cases of laryngeal papilloma were studied. The sensitivity of the DIG labelled probe was almost the same as that of a 35S-labelled probe in the dot-blot hybridisation test. Using in situ hybridisation with 35S-labelled and DIG labelled probes, the levels of the hybridised signals detected were similar. The biotin labelled probe was less sensitive, particularly in the cases of laryngeal papilloma. The DIG labelling and detection method was highly sensitive and applicable to the detection of viral infection by ISH, and is preferable to a radiolabelled probe, especially when in situ hybridisation is done in the pathology laboratory.