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      Evaluation of the impact of ul54 gene-deletion on the global transcription and DNA replication of pseudorabies virus

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          Abstract

          <p class="first" id="P1">Pseudorabies virus (PRV) is an animal alpha-herpesvirus with a wide host range. PRV has 67 protein-coding genes and several non-coding RNA molecules, which can be classified into three temporal groups, immediate early, early and late classes. The <i>ul54</i> gene of PRV and its homolog <i>icp27</i> of herpes simplex virus have a multitude of functions, including the regulation of viral DNA synthesis and the control of the gene expression. Therefore, abrogation of PRV <i>ul54</i> function was expected to exert a significant effect on the global transcriptome and on DNA replication. Real-time PCR and real-time RT-PCR platforms were used to investigate these presumed effects. Our analyses revealed a drastic impact of the <i>ul54</i> mutation on the genome-wide expression of PRV genes, especially on the transcription of the true late genes. A more than two hour delay was observed in the onset of DNA replication, and the amount of synthesized DNA molecules was significantly decreased in comparison to the wild-type virus. Furthermore, in this work, we were able to successfully demonstrate the utility of long-read SMRT sequencing for genotyping of mutant viruses. </p>

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          Is Open Access

          Characterization of DNA methyltransferase specificities using single-molecule, real-time DNA sequencing

          DNA methylation is the most common form of DNA modification in prokaryotic and eukaryotic genomes. We have applied the method of single-molecule, real-time (SMRT®) DNA sequencing that is capable of direct detection of modified bases at single-nucleotide resolution to characterize the specificity of several bacterial DNA methyltransferases (MTases). In addition to previously described SMRT sequencing of N6-methyladenine and 5-methylcytosine, we show that N4-methylcytosine also has a specific kinetic signature and is therefore identifiable using this approach. We demonstrate for all three prokaryotic methylation types that SMRT sequencing confirms the identity and position of the methylated base in cases where the MTase specificity was previously established by other methods. We then applied the method to determine the sequence context and methylated base identity for three MTases with unknown specificities. In addition, we also find evidence of unanticipated MTase promiscuity with some enzymes apparently also modifying sequences that are related, but not identical, to the cognate site.
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            Full-Length Isoform Sequencing Reveals Novel Transcripts and Substantial Transcriptional Overlaps in a Herpesvirus

            Whole transcriptome studies have become essential for understanding the complexity of genetic regulation. However, the conventionally applied short-read sequencing platforms cannot be used to reliably distinguish between many transcript isoforms. The Pacific Biosciences (PacBio) RS II platform is capable of reading long nucleic acid stretches in a single sequencing run. The pseudorabies virus (PRV) is an excellent system to study herpesvirus gene expression and potential interactions between the transcriptional units. In this work, non-amplified and amplified isoform sequencing protocols were used to characterize the poly(A+) fraction of the lytic transcriptome of PRV, with the aim of a complete transcriptional annotation of the viral genes. The analyses revealed a previously unrecognized complexity of the PRV transcriptome including the discovery of novel protein-coding and non-coding genes, novel mono- and polycistronic transcription units, as well as extensive transcriptional overlaps between neighboring and distal genes. This study identified non-coding transcripts overlapping all three replication origins of the PRV, which might play a role in the control of DNA synthesis. We additionally established the relative expression levels of gene products. Our investigations revealed that the whole PRV genome is utilized for transcription, including both DNA strands in all coding and intergenic regions. The genome-wide occurrence of transcript overlaps suggests a crosstalk between genes through a network formed by interacting transcriptional machineries with a potential function in the control of gene expression.
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              A versatile class of positive-selection vectors based on the nonviability of palindrome-containing plasmids that allows cloning into long polylinkers

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                Author and article information

                Journal
                Archives of Virology
                Arch Virol
                Springer Science and Business Media LLC
                0304-8608
                1432-8798
                September 2017
                June 2 2017
                September 2017
                : 162
                : 9
                : 2679-2694
                Article
                10.1007/s00705-017-3420-3
                5927779
                28577213
                370a9f0a-d019-4ee6-96f0-7faff469535e
                © 2017

                http://www.springer.com/tdm

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