Molecular studies of Chlorella virus DNA and protein modifications

Chlorella virus SC-1A encodes at least seven genes related to DNA restriction-modification, including four sequence-specific \rm N\sp6-adenine methyltransferases (M.CviSI-SIV), one cytosine methyltransferase (M.CviSV), one endonuclease (R.CviSIII), and one methyltransferase pseudogene. Genes encoding the three methyltransferases, M.CviSI, M.CviSII, and M.CviSIII, were cloned, sequenced, and mapped. The three methyltransferase genes were scattered throughout SC-1A genome. The predicted amino acid sequences of M.CviSI (TGC\sp{\rm m}A) and M.CviSIII (TCG\sp{\rm m}A) were compared to their isomethylomers; some important sequence motifs are conserved among Chlorella isomethylomers. The sequences of M.CviSII (C\sp{\rm m}ATG) and its promoter region were almost identical to those of its isomethylomer M.CviAII (C\sp{\rm m}ATG) from virus PBCV-1. Sequencing of a SC-1A genomic fragment revealed the presence of a gene with strong homology with eukaryotic serine/threonine specific protein kinases. The homologous gene from virus PBCV-1 was also cloned and sequenced. The predicted amino acid sequence of the protein kinases from PBCV-1 and SC-1A has 94% identity. Both Chlorella protein kinases contain all the highly conserved motifs present in all eukaryotic serine/threonine specific protein kinases. The Chlorella virus protein kinase was overexpressed in E.coli. Protein kinase activity was restored from the denatured inclusion body by in vitro folding. Both SC-1A and PBCV-1 package protein kinase activity(ies) into their virion. Chlorella virus PBCV-1 contains three glycoproteins, the major capsid protein Vp54 and two minor proteins Vp280 and Vp260. The oligosaccharide(s) are apparently attached via an O-linkage. Vp54, along with two other viral proteins (Vp51 and Vp27.5), are labeled with myristic acid via an amide linkage. The carboxyl-terminal 33 kDa cyanogen bromide cleavage fragment of Vp54 contains both myristylation and glycosylation sites. The putative gene encoding PBCV-1 and its serotype EPA-1 glycoprotein 2 was cloned and sequenced; Vp260 contains 15 similar repeats of 61 to 65 amino acids. The EPA-1 protein lacked four of the amino acid repeats and contains a duplication of one of the repeat sequences

Molecular studies of Chlorella virus DNA and protein modifications

Chlorella virus SC-1A encodes at least seven genes related to DNA restriction-modification, including four sequence-specific \rm N\sp6-adenine methyltransferases (M.CviSI-SIV), one cytosine methyltransferase (M.CviSV), one endonuclease (R.CviSIII), and one methyltransferase pseudogene. Genes encoding the three methyltransferases, M.CviSI, M.CviSII, and M.CviSIII, were cloned, sequenced, and mapped. The three methyltransferase genes were scattered throughout SC-1A genome. The predicted amino acid sequences of M.CviSI (TGC\sp{\rm m}A) and M.CviSIII (TCG\sp{\rm m}A) were compared to their isomethylomers; some important sequence motifs are conserved among Chlorella isomethylomers. The sequences of M.CviSII (C\sp{\rm m}ATG) and its promoter region were almost identical to those of its isomethylomer M.CviAII (C\sp{\rm m}ATG) from virus PBCV-1. Sequencing of a SC-1A genomic fragment revealed the presence of a gene with strong homology with eukaryotic serine/threonine specific protein kinases. The homologous gene from virus PBCV-1 was also cloned and sequenced. The predicted amino acid sequence of the protein kinases from PBCV-1 and SC-1A has 94% identity. Both Chlorella protein kinases contain all the highly conserved motifs present in all eukaryotic serine/threonine specific protein kinases. The Chlorella virus protein kinase was overexpressed in E.coli. Protein kinase activity was restored from the denatured inclusion body by in vitro folding. Both SC-1A and PBCV-1 package protein kinase activity(ies) into their virion. Chlorella virus PBCV-1 contains three glycoproteins, the major capsid protein Vp54 and two minor proteins Vp280 and Vp260. The oligosaccharide(s) are apparently attached via an O-linkage. Vp54, along with two other viral proteins (Vp51 and Vp27.5), are labeled with myristic acid via an amide linkage. The carboxyl-terminal 33 kDa cyanogen bromide cleavage fragment of Vp54 contains both myristylation and glycosylation sites. The putative gene encoding PBCV-1 and its serotype EPA-1 glycoprotein 2 was cloned and sequenced; Vp260 contains 15 similar repeats of 61 to 65 amino acids. The EPA-1 protein lacked four of the amino acid repeats and contains a duplication of one of the repeat sequences

Targeted Integration of T-DNA into the Tobacco Genome at Double-Stranded Breaks: New Insights on the Mechanism of T-DNA Integration

Agrobacterium tumefaciens T-DNA normally integrates into random sites in the plant genome. We have investigated targeting of T-DNA by nonhomologous end joining process to a specific double-stranded break created in the plant genome by I-CeuI endonuclease. Sequencing of genomic DNA/T-DNA junctions in targeted events revealed that genomic DNA at the cleavage sites was usually intact or nearly so, whereas donor T-DNA ends were often resected, sometimes extensively, as is found in random T-DNA inserts. Short filler DNAs were also present in several junctions. When an I-CeuI site was placed in the donor T-DNA, it was often cleaved by I-CeuI endonuclease, leading to precisely truncated targeted T-DNA inserts. Their structure requires that T-DNA cutting occurred before or during integration, indicating that T-DNA is at least partially double stranded before integration is complete. This method of targeting full-length T-DNA with considerable fidelity to a chosen break point in the plant genome may have experimental and practical applications. Our findings suggest that insertion at break points by nonhomologous end joining is one normal mode of entry for T-DNA into the plant genome

Acetosyringone treatment duration affects large T-DNA molecule transfer to rice callus

Abstract Background Large T-DNA fragment transfer has long been a problem for Agrobacterium-mediated transformation. Although vector systems, such as the BIBAC series, were successfully developed for the purpose, low transformation efficiencies were consistently observed. Results To gain insights of this problem in monocot transformation, we investigated the T-strand accumulation of various size of T-DNA in two kinds of binary vectors (one copy vs. multi-copy) upon acetosyringone (AS) induction and explored ways to improve the efficiency of the large T-DNA fragment transfer in Agrobacterium-mediated rice transformation. By performing immuno-precipitation of VirD2-T-strands and quantitative real-time PCR assays, we monitored the accumulation of the T-strands in Agrobacterium tumeficiens after AS induction. We further demonstrated that extension of AS induction time highly significantly improved large-size T-DNA transfer to rice cells. Conclusions Our data provide valuable information of the T-strand dynamics and its impact on large T-DNA transfer in monocots, and likely dicots as well