Molecular cloning of equine herpesvirus type 1 DNA: analysis of standard and defective viral genomes and viral sequences in oncogenically transformed cells.
Abstract: Genomic DNA sequences of equine herpesvirus type 1 (EHV-1) have been cloned as BamHI and EcoRI restriction fragments into the plasmid pBR322 and propagated in Escherichia coli. With the exception of two EcoRI restriction fragments that reside in the S region of the viral genome, all of the cloned fragments demonstrated the same electrophoretic mobilities, restriction cleavage sites, and blot-hybridization patterns as did the parent fragments produced by BamHI or EcoRI digestion of virion DNA. The EcoRI J fragment and the BamHI E fragment of the L-region terminus were cloned after the addition of appropriate linker oligonucleotides. Fragments originating from each of the two isomeric forms of EHV-1 DNA were contained in this library of clones. Supramolar DNA fragments present only in the DNA of defective interfering (DI) particles of EHV-1 were generated from Bgl II digestion of DNA preparations enriched for EHV-1 DI particles and were cloned as Bgl II and EcoRI fragments into the plasmid vector. The cloned viral sequences represented in this defective genome mapped to the S region of EHV-1 DNA. Blot-hybridization analysis of EHV-1 transformed and tumor cell DNAs with the cloned BamHI B fragment confirmed that subgenomic viral sequences are present and indicated that those sequences map to the viral genome between 0.32 and 0.43 map unit.
Publication Date: 1981-11-01 PubMed ID: 6273883PubMed Central: PMC349114DOI: 10.1073/pnas.78.11.6684Google Scholar: Lookup
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- Journal Article
- Research Support
- Non-U.S. Gov't
- Research Support
- U.S. Gov't
- Non-P.H.S.
- Research Support
- U.S. Gov't
- P.H.S.
Summary
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This research investigates the DNA structure of equine herpesvirus type 1 (EHV-1). The DNA of the virus was inserted into a plasmid and then copied or cloned in E.coli to better understand their genomic composition. This study also looked at defective interfering particles of the virus and found conclusions about how these might interact with regular virus particles.
Cloning of EHV-1 DNA
- The DNA of equine herpesvirus type 1 (EHV-1) was cloned using special enzymes called BamHI and EcoRI which allow specific sections of the DNA to be cut out and inserted into a plasmid in Escherichia coli (a type of bacteria).
- In the plasmid, the DNA sequence can generate copies of itself which allows further analysis.
- Most of the cloned fragments showed the same traits as the original EHV-1 DNA fragments, the exceptions were two fragments that are part of the ‘S region’ of the viral genome.
Linker Oligonucleotides and Defective Interfering Particles
- Two parts of the virus’s DNA, the EcoRI J fragment and the BamHI E fragment, were specifically cloned after adding linker oligonucleotides, short synthetic DNA sequences that help in the cloning process.
- Defective interfering (DI) particles of EHV-1 which are incomplete virus particles that can interfere with the replication of proper virus particles, were also studied. DNA was prepared from a population of DI particles and cloned into the plasmid.
- The cloned DI sequences matched with the S region of the EHV-1 DNA, indicating where these defective particles fit into the picture of the entire EHV-1 genome.
Confirmation of Viral Sequences in Transformed and Tumor Cells
- The presence of specific virus sequences was confirmed within the DNA of cells that had been transformed by EHV-1. This could indicate that the virus has altered the DNA of these cells to facilitate its own reproduction.
- The sequences present were found to be between the 0.32 and 0.43 map unit of the viral genome, hence this region may play a key role in the virus’s oncogenic (cancer-causing) activity.
Cite This Article
APA
Robinson RA, Tucker PW, Dauenhauer SA, O'Callaghan DJ.
(1981).
Molecular cloning of equine herpesvirus type 1 DNA: analysis of standard and defective viral genomes and viral sequences in oncogenically transformed cells.
Proc Natl Acad Sci U S A, 78(11), 6684-6688.
https://doi.org/10.1073/pnas.78.11.6684 Publication
Researcher Affiliations
MeSH Terms
- Animals
- Cell Line
- Cell Transformation, Neoplastic
- Cell Transformation, Viral
- Cloning, Molecular
- Cricetinae
- DNA Restriction Enzymes
- DNA, Recombinant / metabolism
- DNA, Viral / genetics
- Embryo, Mammalian
- Escherichia coli / genetics
- Herpesviridae / genetics
- Nucleic Acid Hybridization
- Plasmids
Grant Funding
- AI-02032 S-507-RR05386 / NIAID NIH HHS
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Citations
This article has been cited 12 times.- Chen M, Harty RN, Zhao Y, Holden VR, O'Callaghan DJ. Expression of an equine herpesvirus 1 ICP22/ICP27 hybrid protein encoded by defective interfering particles associated with persistent infection.. J Virol 1996 Jan;70(1):313-20.
- Harty RN, Caughman GB, Holden VR, O'Callaghan DJ. Characterization of the myristylated polypeptide encoded by the UL1 gene that is conserved in the genome of defective interfering particles of equine herpesvirus 1.. J Virol 1993 Jul;67(7):4122-32.
- Harty RN, Holden VR, O'Callaghan DJ. Transcriptional and translational analyses of the UL2 gene of equine herpesvirus 1: a homolog of UL55 of herpes simplex virus type 1 that is maintained in the genome of defective interfering particles.. J Virol 1993 Apr;67(4):2255-65.
- Miller G, Rabson M, Heston L. Epstein-Barr virus with heterogeneous DNA disrupts latency.. J Virol 1984 Apr;50(1):174-82.
- Baumann RP, Dauenhauer SA, Caughman GB, Staczek J, O'Callaghan DJ. Structure and genetic complexity of the genomes of herpesvirus defective-interfering particles associated with oncogenic transformation and persistent infection.. J Virol 1984 Apr;50(1):13-21.
- Ruyechan WT, Dauenhauer SA, O'Callaghan DJ. Electron microscopic study of equine herpesvirus type 1 DNA.. J Virol 1982 Apr;42(1):297-300.
- Baumann RP, Sullivan DC, Staczek J, O'Callaghan DJ. Genetic relatedness and colinearity of genomes of equine herpesvirus types 1 and 3.. J Virol 1986 Mar;57(3):816-25.
- Harty RN, Colle CF, Grundy FJ, O'Callaghan DJ. Mapping the termini and intron of the spliced immediate-early transcript of equine herpesvirus 1.. J Virol 1989 Dec;63(12):5101-10.
- Baumann RP, Yalamanchili VR, O'Callaghan DJ. Functional mapping and DNA sequence of an equine herpesvirus 1 origin of replication.. J Virol 1989 Mar;63(3):1275-83.
- Harty RN, O'Callaghan DJ. An early gene maps within and is 3' coterminal with the immediate-early gene of equine herpesvirus 1.. J Virol 1991 Jul;65(7):3829-38.
- Holden VR, Yalamanchili RR, Harty RN, O'Callaghan DJ. ICP22 homolog of equine herpesvirus 1: expression from early and late promoters.. J Virol 1992 Feb;66(2):664-73.
- Zhao Y, Holden VR, Harty RN, O'Callaghan DJ. Identification and transcriptional analyses of the UL3 and UL4 genes of equine herpesvirus 1, homologs of the ICP27 and glycoprotein K genes of herpes simplex virus.. J Virol 1992 Sep;66(9):5363-72.
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