Mapping of the vaccinia virus thymidine kinase gene by marker rescue and by cell-free translation of selected mRNA

Weir, Jerry P.; Bajszár, G.; Moss, Bernard

doi:10.1073/pnas.79.4.1210

Cited by 137 publications

(116 citation statements)

References 34 publications

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“…This observation contrasts the previously reported requirement of carrier DNA for optimal marker rescue in another poxvirus, vaccinia (27,28). The difference is likely not due to differential uptake into the cells, because CaPO4 precipitates enter the cytoplasm of cells with great efficiency, even in the absence of DNA (29).…”

Section: Discussioncontrasting

confidence: 56%

Sequence-nonspecific replication of transfected plasmid DNA in poxvirus-infected cells.

DeLange¹,

McFadden²

1986

Proc. Natl. Acad. Sci. U.S.A.

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A system in which transfected plasmid DNA replicates in the cytoplasm of poxvirus-infected cells is described. A variety of recombinant plasmids was introduced into poxvirus-infected cells by transfection, and replication of input plasmid DNA was monitored by (i) digestion with restriction enzymes that discriminate between input methylated plasmid DNA and unmethylated DNA produced by replication in mammalian cells; (it) amplification of intracellular plasmid DNA; and (Wi) density shift analysis in the presence of BrdUrd. Replication of plasmid DNA was observed in the cytoplasm of cells infected with the tumorigenic leporipoxviruses Shope fibroma virus (SFV) and myxoma, and less extensively with the orthopoxvirus vaccinia, but not in uninfected cells. Unexpectedly, all input plasmids tested, including pBR322, pUC13, polyoma, PM2, 4X174 replicative form (RF), and M13 RF, replicated with equal efficiency in SFV-infected cells, indicating that no specific replication origin sequence is required. The transfected plasmid DNA was replicated concomitantly with the infecting poxviral DNA and by 24 hr post-transfection, it resided predominantly in high molecular weight Dpn I-resistant head-to-tail tandem repeats. The failure to detect unreplicated Dpn I-sensitive plasmid concatemers early in replication together with the absence of significant levels of integrated plasmid sequences in the poxviral genome suggest that replication of the transfected plasmid DNA is not the consequence of nonhomologous recombination of concatemeric plasmid DNA into the poxvirus genome, but rather of an autonomous process that is dependent on trans-acting replication factors produced during virus infection, and that does not require a specific origin sequence on the substrate plasmid DNA.Poxviruses replicate their double-stranded linear genomes in factories or "micro-nuclei" within the cytoplasm of infected cells (1-3). The observation that these viruses are capable of replication in enucleated cells suggests that poxvirus DNA synthesis is a relatively autonomous process and may not require host functions (4-6). Electron microscopic observation of the replicating vaccinia genome (7-9) and in vivo labeling experiments (10-12) suggest that DNA replication starts near the genomic termini, but definitive evidence that a specific poxviral origin sequence exists is lacking. To further localize and examine the role of viral sequences during replication, we have utilized the transfection of exogenous plasmids into virus-infected cells, an approach that has previously been used with success to identify and characterize various functional viral DNA domains, including replication origins (13)(14)(15)(16)(17)(18).In this communication, we report on the fate of transfected plasmids with and without viral DNA inserts after their introduction into cells infected with either vaccinia virus or the tumorigenic poxvirus, Shope fibroma virus (SFV). Al-though preliminary experiments using standard calcium phosphate (CaPO4) precipitation protocols had indic...

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Section: Discussioncontrasting

confidence: 56%

Sequence-nonspecific replication of transfected plasmid DNA in poxvirus-infected cells.

DeLange¹,

McFadden²

1986

Proc. Natl. Acad. Sci. U.S.A.

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“…At the other extreme of viral complexity, Sam and Dumbell (1981) and Weir, Bajszar and Moss (1982) have reported marker rescue of mutants of vaccinia virus with genomie fragments of vaccinia viral DNA. Weir and colleagues showed that methotrexate-resistant TK+ (thymidine kinase) vaccinia virus was formed in cells coinfeeted with TK~ virus and wild-type (i.e.…”

Section: Construction Of Hybrid Virusesmentioning

confidence: 99%

Vaccines for the Future?

1982

Aust J Exp Biol Med

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“…All of the HindlII fragments, except HindIII-J, were alternatively introduced with tk or aprt in order to determine if different vectors could influence the biological properties of the transformants. Since the HindIII-J fragment contains the vaccinia virus tk gene (Hruby & Ball, 1982;Weir et al, 1982), this fragment was introduced into recipient cells using G418 selection because this marker is not involved in nucleic acid metabolism.…”

Section: (Accepted 21 March 1984)mentioning

confidence: 99%

Expression of Cloned Vaccinia Virus DNA Sequences Introduced into Animal Cells

Boni

Esteban

Pellicer

1984

Journal of General Virology

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SUMMARYIndividual cloned HindlII fragments of vaccinia virus DNA were introduced into cells by DNA-mediated gene transfer. The presence of individual fragments in the different transformants was confirmed by Southern blot hybridization analysis. Several of the transformants were found to express viral sequences at various levels. The sizes of the transcripts containing vaccinia virus sequences were highly heterogeneous, with no discrete species of RNA. Positive clones contained vaccinia virus sequences in both the poly(A) ÷ and poly(A)-RNA fractions, although the prevalence of these sequences was variable in the two fractions. The S1 nuclease map of the 5' end of the transcripts from transformants containing the HindlII-J fragment revealed a unique 5' end, similar to RNA from virus-infected cells• In contrast, analysis of the 3' end of RNAs from these transformants showed a high degree of heterogeneity, which might explain the heterogeneity found in Northern blot patterns. In this report, it is shown for the first time that in cells transformed with vaccinia virus DNA there is proper initiation for, at least, the viral thymidine kinase gene.

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Mapping of the vaccinia virus thymidine kinase gene by marker rescue and by cell-free translation of selected mRNA

Cited by 137 publications

References 34 publications

Sequence-nonspecific replication of transfected plasmid DNA in poxvirus-infected cells.

Sequence-nonspecific replication of transfected plasmid DNA in poxvirus-infected cells.

Vaccines for the Future?

Expression of Cloned Vaccinia Virus DNA Sequences Introduced into Animal Cells

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