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Missing contact footprinting with formic acid as a modifying reagent was used to examine specific IE-1 binding contacts to double-stranded oligonucleotides that contained either a consensus hr repeat sequence or a sequence from the pe38 promoter, which is down regulated by IE-1. The hr repeat sequences contain two consensus IE-1 binding motifs (IBMs) flanking a central EcoRI site that are oriented...
The genome of theLymantria disparmultinucleocapsid nucleopolyhedrovirus (LdMNPV) was sequenced and analyzed. It is composed of 161,046 bases with a G + C content of 57.5% and contains 163 putative open reading frames (ORFs) of ≥150 nucleotides. Homologs were found to 95 of the 155 genes predicted for theAutographa californicaMNPV (AcMNPV) genome. More than 9% of the LdMNPV genome was occupied by 16...
The late expression factor-5 gene (lef-5) ofAutographa californicamultinucleocapsid polyhedrovirus (AcMNPV) is required for late gene expression. In this paper, we demonstrate that LEF-5 interacts with itself in the yeast two-hybrid system and in glutathione–S-transferase affinity assays. Deletion analysis suggested that the C-terminal 71 amino acids (aa) were not required for interaction. However,...
A region of theLymantria disparmultinucleocapsid nuclear polyhedrosis virus (LdMNPV) genome containing the homolog of the baculovirusie-1 gene was identified using a series of overlapping cosmids and individual plasmids in a transient transcriptional expression assay. Sequence analysis of the active region identified two ORFs, one of which is 32% identical to AcMNPV ORF141 (ie-0) and contains a putative...
Homologous regions (hrs), which are present at eight dispersed locations on theAutographa californicamultinucleocapsid nuclear polyhedrosis virus genome, are composed of repeated imperfect palindromes within directly repeated sequences.Hrsact as transcriptional enhancers of RNA polymerase II-mediated transcription and as origins of DNA replication when incorporated into plasmids and tested in transient...
The virus-induced RNA polymerase fromAutographa californicanuclear polyhedrosis virus-infectedSpodoptera frugiperdacells was separated from the three host nuclear RNA polymerases by DEAE–Sephadex chromatography and then purified through two more steps: heparin–agarose chromatography and glycerol gradient ultracentrifugation. Fractions from each of these purification steps have been assayedin vitrofor...
A transient DNA replication assay was used to identify genes located within m.u. 90.5-7.0 of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) genome that influenced replication of a reporter plasmid containing an OpMNPV origin of replication, when cotransfected into uninfected Lymantria dispar cells. The viral transactivator ie-1 and a 2.4-kb subclone were found to be...
Using a transient complementation assay for DNA replication of an origin-containing reporter plasmid, a 2.4-kb region (m.u. 48.3-50.1) of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) genome was identified that contains a gene essential for OpMNPV DNA replication. This region was sequenced and open reading frames were identified. Replication assays using subclones...
Hr1a consists of two 30-bp imperfect palindrome sequences separated by 58 bp and each palindrome contains a naturally occurring EcoRI site at its core. Plasmid subclones of the hr1a-containing AcMNPV Hin dIII-N fragment were examined for their ability to replicate in virus-infected (Spodoptera frugiperda) Sf9 cells, and to stimulate transcription when linked in cis with a 39K gene promoter-β-glucuronidase...
A transient replication assay for the identification of baculovirus genes that are essential for replication of an origin-containing reporter plasmid was established for the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV). Using a replication origin located on the OpMNPV Hind III-N fragment, we identified a subset of cosmids and plasmids from an OpMNPV cosmid library that...
A 7.5-kb region (96.8-2.5 m.u), called Op5, of the Orgyia pseudotsugata multinucleocapsid nuclear polyhedrosis virus (OpMNPV) genome that contains an origin of DNA replication was characterized. This region replicates several times more efficiently and is unrelated to the previously identified putative origin of replication located on the viral HindIII-N fragment. In contrast to HindIII-N, the origin...
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