Expression of an enhancer-binding protein in insect cells transfected with the Autographa californica nuclear polyhedrosis virus IE1 gene.
The baculovirus Autographa californica nuclear polyhedrosis virus contains an element known as homologous region 5 (hr5) which is an enhancer of delayed-early viral gene expression. To begin to identify proteins that interact with hr5, DNA-protein interactions were analyzed by using extracts from Spodoptera frugiperda cells and a fragment of DNA containing the left half of the hr5 enhancer. This 252-bp DNA fragment contains two copies of a 30-bp direct repeat (DR30) and two copies of a 24-bp imperfect palindrome contained within a 60-bp direct repeat (DR60). Extracts prepared from normal S. frugiperda cells and cells transfected with pUC8 lacked enhancer-binding proteins. However, when gel shift assays were performed with extracts from cells transfected with a plasmid containing the viral trans-activator IE1 gene, two DNA-protein complexes were formed. Both DNA-protein complexes were specifically inhibited by competition with a 60-bp oligonucleotide corresponding to DR60 but not by competition with a different oligonucleotide corresponding to DR30. Formation of the two complexes did not appear to involve cooperative interactions between binding proteins. When DR60 was used as a probe, a single complex was formed. To measure the enhancer activity of DR60, a reporter plasmid was constructed that contained DR60 cloned upstream of the reporter chloramphenicol acetyltransferase gene under the control of the delayed-early 39K promoter. Transient expression analysis indicated that the oligonucleotide increased expression of this gene 300-fold over the level obtained in the absence of any enhancer sequences.
Guarino
L A
LA
Department of Entomology, Texas A & M University, College Station 77843-2475.
Dong
W
W
D000818:Animals; D016367:Baculoviridae / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000502:physiology*; D004742:Enhancer Elements, Genetic; D017874:Immediate-Early Proteins; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009036:Moths; D011401:Promoter Regions, Genetic; D015534:Trans-Activators / Q000235:genetics* / Q000502:physiology; D014162:Transfection; D014764:Viral Proteins / Q000235:genetics*
C069183:Ac-IE-1 protein, Autographa californica nucleopolyhedrovirus; D004268:DNA-Binding Proteins; D017874:Immediate-Early Proteins; D015534:Trans-Activators; D014764:Viral Proteins