We have isolated and partially characterized 30 large polyhedral, dsDNA containing (greater than 300 kbp), plaque forming viruses which infect a unicellular, eukaryotic, Chlorella-like green alga. The plaque assay, the ability to synchronously infect the host, the short life cycle, and the ability of the viruses to undergo homologous recombination make them excellent model systems for studying gene regulation and expression in a photosynthetic eukaryote. These are the first plant-virus systems amenable to standard bacteriophage technology. At least some of these viruses, whose genomes contain various levels of methylated bases (0.1 to 47% 5mC and 0 to 37% 6mA), encode for DNA modification and restriction systems. The virus infected algae are a new source of type II DNA restriction endonucleases and the first source from a nonprokaryotic system. Our objectives are to: (i) clone and characterize some of the methyltransferase and restriction endonuclease genes. Sequencing these genes will provide information not only on the promoters and other regulatory elements of early virus genes but also allow us to compare them to genes for bacterial enzymes which recognize identical base sequences. (ii) Characterize viruses containing mutations in their modification and restriction systems; comparing the mutant viruses with their parents may reveal the biological function of these systems. (iii) Determine the structure of virus DNA replicative intermediates and the intracellular site of virus DNA replication and DNA methylation. (iv) Examine progeny viruses form Chlorella simultaneously infected with two viruses possessing different modification and restriction systems to determine if only one or both viruses replicate in the same cell. (v) Isolate and characterize virus DNA and RNA polymerases from different virus-host combinations and compare the ability of the enzymes to function with different methylated DNA templates.
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