DNA replication of wheat dwarf geminivirus vectors: effects of origin structure and size.
Wheat dwarf virus (WDV) is a representative member of subgroup I of the Geminiviridae, a unique plant DNA virus family. Since geminivirus DNA replication occurs in the host cell nucleus exclusively via double-stranded DNA intermediates, a considerable interest has arisen to use them as expression vectors. We have used particle bombardment to introduce WDV vectors into cultured wheat cells and to analyze the fate of input DNA and the accumulation of newly replicated DNA. Under our conditions, we have found that input DNA, which can be detected immediately after DNA delivery, is rapidly degraded. Newly replicated viral DNA appears approximately 1 day after DNA delivery and reaches a maximum at Days 2-4. Afterward, the total amount of viral DNA is maintained for several days. We have observed a progressive decrease in the relative amount of supercoiled DNA and, concomitantly, an increase in plasmid forms migrating as open circular and nicked DNA. GUS expression from the virion-sense WDV promoter is also maximal 2-3 days after DNA delivery and then it declines to negligible levels 8 days afterward. These results support the conclusion that, under these conditions, reporter gene expression depends on the accumulation of newly replicated, supercoiled plasmid DNA and not on input plasmid DNA. We have also analyzed the effects of WDV origin structure and plasmid size on the accumulation of newly replicated plasmid DNA. Our results lead us to conclude that the replication efficiency of WDV-derived plasmids depends largely on plasmid size. Interestingly, sequences downstream of the initiation site, which in WDV confer an intrinsic curvature to the large intergenic region, seem to have a small effect on the efficiency of plasmid accumulation.
Suárez-López
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Centro de Biología Molecular Severo Ochoa, CSIC, Universidad Autónoma,Cantoblanco, Madrid, Spain.
Gutiérrez
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D002478:Cells, Cultured; D004261:DNA Replication; D004278:DNA, Superhelical / Q000096:biosynthesis; D004279:DNA, Viral / Q000096:biosynthesis; D017791:Geminiviridae / Q000235:genetics / Q000502:physiology*; D017930:Genes, Reporter; D005822:Genetic Vectors; D005966:Glucuronidase / Q000096:biosynthesis; D007700:Kinetics; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis; D014162:Transfection / Q000379:methods; D014908:Triticum / Q000821:virology*; D014771:Virion / Q000235:genetics / Q000502:physiology; D014779:Virus Replication
D004278:DNA, Superhelical; D004279:DNA, Viral; D011994:Recombinant Proteins; D005966:Glucuronidase