During productive infections of cells with the gammaherpesvirus, herpesvirus saimiri (HVS), a polyadenylated RNA of 2.2-2.4 kb accumulates to form a large fraction of virus-specified RNA. This transcript is from the virus thymidylate synthase (TS) gene and its synthesis, like that of late mRNAs encoding the virus structural proteins, is sensitive to an inhibitor of virus DNA synthesis (phosphonoacetic acid, PAA). Transcription which is insensitive to PAA occurs from many parts of the HVS genome, including the EcoRI-D, EcoRI-E, EcoRI-I, and HindIII-G fragments. A 1.6-kb RNA from EcoRI-I/E and a 1.3-kb RNA from HindIII-G accumulate in HVS-infected cells incubated in the continuous presence of cycloheximide, and thus represent immediate-early (IE) class transcripts. The 1.3-kb message from HindIII-G is the predominant stable RNA under these conditions; accumulation of the 1.6-kb transcript from EcoRI-I/E (which encodes the previously characterized 52-kDa IE phosphoprotein) is markedly more dependent on the multiplicity of infection. The sequence of a 2.5-kbp region of the HindIII-G fragment has been determined and a single major open reading frame is present within the boundaries of the 1.3-kb IE RNA. Comparison of the amino acid sequence of the encoded protein (IE-G) with current databases of protein sequences failed to demonstrate significant similarities with herpesvirus proteins, but did detect a significant similarity with a region of the protein specified by an open reading frame in the LTR of mouse mammary tumor virus. The function of the IE gene in HindIII-G and the basis for the distinctive multiplicity dependence of IE transcription from the 52-kDa gene remain to be established.
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http://dx.doi.org/10.1016/0042-6822(90)90288-3 | DOI Listing |
Currently, the most common approach for manufacturing GMP-grade adeno-associated virus (AAV) vectors involves transiently transfecting mammalian cells with three plasmids that carry the essential components for production. The requirement for all three plasmids to be transfected into a single cell and the necessity for high quantities of input plasmid DNA, limits AAV production efficiency, introduces variability between production batches, and increases time and labor costs. Here, we developed an all-in-one, single-plasmid AAV production system, called AAVone.
View Article and Find Full Text PDFNat Commun
January 2025
State Key Laboratory of Microbial Technology, Institute of Microbial Technology, Helmholtz International Lab for Anti-infectives, Shandong University-Helmholtz Institute of Biotechnology, Shandong University, Qingdao, Shandong, China.
Modular polyketide synthases (mPKSs) are multidomain enzymes in bacteria that synthesize a variety of pharmaceutically important compounds. mPKS genes are usually longer than 10 kb and organized in operons. To understand the transcriptional and translational characteristics of these large genes, here we split the 13-kb busA gene, encoding a 456-kDa three-module PKS for butenyl-spinosyn biosynthesis, into three smaller separately translated genes encoding one PKS module in an operon.
View Article and Find Full Text PDFJ Gen Virol
December 2023
School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, QLD, Australia.
Flavivirids are small, enveloped, positive-sense RNA viruses from the family with genomes of ~9-13 kb. Metatranscriptomic analyses of metazoan organisms have revealed a diversity of flavivirus-like or flavivirid viral sequences in fish and marine invertebrate groups. However, no flavivirus-like virus has been identified in amphibians.
View Article and Find Full Text PDFBMC Biotechnol
August 2023
State Key Laboratory of Pig Genetic Improvement and Production Technology, Jiangxi Agricultural University, Nanchang, 330045, China.
Background: Porcine fetal fibroblasts (PFFs) are important donor cells for generating genetically modified pigs, but the transfection efficiencies of PFFs are often unsatisfactory especially when large-size vectors are to be delivered. In this study, we aimed to optimize the transfection conditions for delivery of a large-size vector in PFFs using Lonza 4D-Nucleofector™ vessels and strips.
Methods: We firstly delivered a 13 kb Cas9-EGFP and a 3.
Theranostics
June 2023
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, Chengdu 610041, P. R. China.
CRISPR-Cas13a is an efficient tool for robust RNA knockdown with lower off-target effect, which may be a potentially powerful and safe tool for cancer gene therapy. However, therapeutic effect of current cancer gene therapy that targeting monogene was compromised by the multi-mutational signal pathway alterations of tumorigenesis. Here, hierarchically tumor-activated nanoCRISPR-Cas13a (CHAIN) is fabricated for multi-pathway-mediated tumor suppression by efficient microRNA disruption .
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