Göran Magnusson

Replication of polyoma DNA in isolated nuclei. I. Characterization of the system from mouse fibroblast 3T6 cells.

Abstract Nuclei isolated from polyoma virus-infected mouse fibroblast 3T6 cells incorporated radioactive [3H]dTTP into the replicative intermediate of polyoma DNA. The replicative intermediate was characterized by centrifugation through neutral and alkaline sucrose gradients, isopycnic banding in a propidiumiodide/cesium chloride gradient, by chromatography on benzoylated naphthoylated DEAE-cellulose and DNA-DNA hybridization. A small but significant amount of radioactivity was incorporated into mature polyoma DNA. The incorporation of radioactivity into polyoma DNA required the presence of dATP, dCTP and dGTP and was stimulated by ATP, Mg2+ and monovalent cations. A substantial part of the radioactivity was incorporated into short chains during the first five minutes of incubation while after 30 minutes most of the incorporation had occurred into longer chains which, in an alkaline sucrose gradient, sedimented close to 16 s, the sedimentation coefficient of unit-length linear polyoma DNA. The time-course of the reaction was studied at 25, 30 and 35 °C. The incorporation was non-linear at all temperatures but reached a similar plateau value in each case. It is proposed that the nuclei in vitro retained the capacity to elongate replicative intermediate molecules present at the time of their isolation but only had a very limited ability to initiate the replication of new polyoma DNA molecules.

Non-contiguous segments of the polyoma genome required in cis for DNA replication

The boundaries of the origin of polyoma DNA replication have been analyzed using a set of deletion mutants. The majority of these had small deletions, 5 to 30 basepairs in size, which together removed most of the non-translated sequences of the genome. The phenotype of the mutants was characterized by analysis of infectivity, transforming ability and DNA synthesis. All mutants with reduced or abolished infectivity had corresponding defects of viral DNA synthesis. The effect of the deletion was cis-acting, since the replication of the mutants was not stimulated by the presence of wild-type DNA. Deletions causing a reduction of DNA synthesis were found at two sites. The first at the 32 base-pair inverted repeat sequence and the neighbouring A . T tract previously implicated in the initiation of DNA synthesis, and the second close to the late genes. The two sites were separated by at least 60 base-pairs of non-essential DNA. Only one mutant with a deletion at the second site was unable to express early gene functions. The mutants were constructed by linearization, shortening and recircularization of polyoma DNA inserted into the plasmid pBR322. The mutagenesis was directed at restriction endonuclease BglI or PvuII cleavage sites. The BglI-directed mutagenesis was focussed to polyoma DNA by using as a vector a derivative of pBR322 resistant to cleavage by BglI.

Ethidium-bromide-inhibited restriction endonucleases cleave one strand of circular DNA

We analyzed the effect of ethidium bromide (EtBr) on the cleavage of closed circular pBR322 DNA molecules by six restriction enzymes which make staggered or flush cuts (EcoRI, HindIII, BglI, PstI, HincII, PvuII). EtBr concentrations and reaction temperatures were determined at which DNA molecules with single-strand breaks were the major reaction product of digestion by all the enzymes. However, the amounts of intermediates which could be isolated differed for various enzymes. The results extend previous studies, showing that sequential cleavage of the DNA strands probably is a general property of restriction endonucleases.

Replication of polyoma DNA in isolated nuclei: II. Evidence for semi-conservative replication

Abstract Nuclei from polyoma-infected mouse fibroblast 3T6 cells catalysed the incorporation of [ 3 H]dTTP into the replicative intermediate of polyoma DNA. The nature of this process was studied by the introduction of a density label into the viral DNA. For this purpose dBrUTP replaced dTTP in the in vitro system and the product formed in the presence of [ 14 C]dATP was analysed by equilibrium centrifugation in neutral CsCl or alkaline Cs 2 SO 4 gradients. The results suggest that the density label was incorporated into newly synthesized progeny strands and that the incorporation process represented semi-conservative replication. In a second type of experiment the replicative intermediate was first pulselabelled in vivo with [ 3 H]thymidine. The isolated nuclei were then incubated in vitro with dBrUTP and [ 14 C]dATP. From an analysis of the distribution of the two isotopes in buoyant density gradients we propose that in vitro synthesis represented elongation of essentially all replicative intermediate molecules present in the nuclei at the time of isolation. From the observed density shifts we could calculate that the initial rate of the in vitro elongation process corresponded to a replication time of about 20 minutes and thus may amount to about 20% of the reported in vivo rate.

Synthesis of polyoma DNA by isolated nuclei

Abstract Nuclei from polyoma or mock infected baby mouse kidney, 3T3 or 3T6 cells were incubated under conditions suitable for DNA synthesis. The labeled DNA thus formed by the infected nuclei was by several criteria indistinguishable from the replicative intermediate formed in intact cells as first described by Bourgeaux et al. (1).

Cellular Mobile Genetic Elements in the Regulatory Region of the Pneumotropic Mouse Polyomavirus Genome: Structure and Function in Viral Gene Expression and DNA Replication

ABSTRACT DNA from the murine pneumotropic virus was extracted from virus in lung tissue of infected mice, and the regulatory region of the genome was amplified by PCR. The regulatory region of individual plasmid cloned DNA molecules appeared to have heterogeneous enhancer segments, whereas the protein-coding part of the genome had a uniform length. Nucleotide sequence analysis revealed that the majority of the DNA molecules had a structure differing from the standard type. A 220-bp insertion at nucleotide position 142 with a concomitant deletion of nucleotides 143 to 148 was prominent. There were two variants of the 220-bp insertion, differing at two nucleotide positions at one of the termini. Other DNA molecules had complete or partial deletions of these structures and surrounding sequences in the viral enhancer. However, the end of the insertion at nucleotide 142 was frequently preserved. The viral early and late promoter activity of the variant regulatory regions was tested in a luciferase reporter assay by using transfected NIH 3T3 cells. In relation to the standard-type DNA, all variants, including a G272T mutant, had much stronger late promoters. In contrast, the early promoter activity was influenced in a positive or negative direction by individual mutations. Also, the activity of the viral origin of DNA replication was affected by the sequence variation of the regulatory region, although the effects were smaller than for the late promoter. Analysis by Southern blotting and quantification using dot blots showed that approximately 103 copies of material related to the 220-bp insert in murine pneumotropic virus DNA was present in mouse and human DNA but not in Escherichia coli DNA. Moreover, analysis by PCR indicated that there were multiple copies in the mouse genome of sequences that were identical or closely related to the 220-bp viral DNA segment. These data together with the nucleotide sequence analysis suggest that the 220-bp insertion is related to a transposable element of a novel type.

Structure at restriction endonuclease MboI cleavage sites protected by actinomycin D or distamycin A

Restriction endonuclease MboI cleavage of DNA was inhibited by actinomycin D and distamycin A. The two inhibitors protected different subsets of the 8 cleavage sites in polyoma DNA. The cleavage reactions were analyzed both in the presence of minimal inhibitory concentrations of the compounds and at higher concentrations, allowing cleavage at only 1 site/DNA molecule. The experiments showed that cleavage sites most efficiently protected by actinomycin D had putative inhibitor binding sites at a distance of 1–2 base pairs from the MboI recognition sequence. Distamycin A, in contrast, apparently has to bind immediately adjacent to the MboI recognition sequence to protect from cleavage.

Effect on polyomavirus T-antigen function of mutations in a conserved leucine-rich segment of the DnaJ domain.

ABSTRACT The N-terminal part of the mouse polyomavirus T antigens contains a highly conserved segment (-LLELLKL-), including amino acid residues 13 to 19. The sequence motif is predicted to form alpha helix I in the DnaJ domain of the T antigens. Four mutants with conservative substitutions of amino acid residues 13 and 14 were constructed. Of the four substitutions, L13M, L13I, L13V, and L14V, only L13V resulted in a phenotypic change. In transfected mouse cells, L13V large T antigen showed a more than 100-fold-reduced viral DNA synthesis. The viral replication could not be rescued by cotransfection of the cells with DNA expressing small t antigen or a large T antigen truncated at the C terminus that would compensate for a defect in host cell stimulation. In contrast to the effect on DNA replication, the L13V substitution in large T antigen did not prevent complex formation with Hsc70 and the Rb protein. Also, the activity of the protein in transactivation of transcription from the adenovirus E2 promoter was unimpaired, showing that the transcription factor E2F was released from pRb. The L13V substitution also caused a defect in small t antigen. However, this phenotypic change was due to protein instability. In contrast, middle T antigen with the L13V substitution remained stable and functional in cellular transformation. Together, the data show that the effect of the L13V substitution did not abrogate the Hsc70 interaction of the DnaJ domain. However, it is possible that the substitution of amino acid residue 13 affected specific DnaJ functions of large T antigen.

Replication of polyoma DNA accumulation of early replicative intermediates during hydroxyurea inhibition

Abstract Inhibition of polyoma DNA synthesis by hydroxyurea appears to affect the initiation of new rounds of DNA synthesis to a lesser extent than chain elongation. Viral DNA synthesized in hydroxyurea-treated 3T6 cultures showed an accumulation of early replicative intermediates, in which the progeny strands were shorter than 20% of the genome length. No such accumulation was found for polyoma DNA replicating in control cells, where the replicative intermediates were evenly distributed at all stages of replication.

Viable polyoma virus variant with two origins of DNA replication

Abstract A viable evolutionary variant of polyoma virus, ev 1001, produced small clear plaques in assays on mouse 3T3 cells. The kinetics of virus formation during a single infection cycle showed that progeny virus was produced more rapidly than in cells infected by wild-type virus. However, an early cytopathic effect on the infected cells appeared to interrupt the process, since the yield of virus was low. Infection of semipermissive rat cells by ev 1001 virus resulted in unusually high transformation frequencies, particularly at low multiplicities of infection. The biological properties of ev 1001 can all be explained by the effect of a duplication of a 250-base pair segment of the polyoma genome in a region located between the 5′-ends of the early and late genes. The evolutionary variant has two origins of DNA replication, as judged by the high rate of DNA synthesis and strong interference with the formation of wild-type virus in coinfection experiments. The variant ev 1001 also induced mouse cells to synthesize cellular DNA more efficiently than wild-type virus. This effect might result from an increased expression of the viral early genes.