• Journal of Sericultural and Entomological Science
• /
• v.41 no.2
• /
• pp.102-107
• /
• 1999

To analysis a promoter strength of Atographa californica nucler polyhedrosis virus (AcNPV) IE1 gene, an immediate viral gene, ${\beta}$-glactosidase gene as a reporter gene was introduced under the control of the IE1 promoter. The restriction fragment containing IE1 promoter and ${\beta}$-galctosidase gene from pAcIE1-gal were inserter into pBacPAK9 to yield transfer vector pAcNPV-IE1-gal. The pAcNPV-IE1-gal was cotransfected with AcNPV genomic DNA BacPAK6 into Sf9 cells to produce recombinant baculovirus AcNPV-IE1-gal. In addition, recombinant bacvulovirus AcNPV-gal, which express ${\beta}$-galac-tosidase under the control of the polyhedrin promoter, was constrer, was constructed to compared with AcNPV-IE1-gal. The recombinant viruses were respectively infected into Sf9 cells and characterized by the virus titer and expression of ${\beta}$-galactoxidase in Sf9 cells. The promoter strength of IE1 and polyhedrin promoters was determined by the amount of ${\beta}$-galactosidase secreted into medium by viral infection. The titer of AcNPV-IE1-Gal determined by plaque assays in Sf9 cells was similar to that of AcNPV-gal. However, expression level of ${\beta}$-galactosidase by AcNPV-IE1-gal was significantly lower than that by AcNPV-gal. In conclusion, promoter strength of IE1 was approximately 25-fold lower than that of polyhedrin.

• The Journal of Korean Society of Virology
• /
• v.28 no.3
• /
• pp.267-274
• /
• 1998

Human cytomegalovirus (HCMV) has the ability to activate the expression of many viral and cellular genes. Among various viral proteins, the immediate early proteins (IE1-72kDa, IE2-86kDa) have been known to be potent transactivators. The product of c-jun proto-oncogene is important in cell activation and differentiation. Here, we tried to find out if the IE could activate the c-jun promoter and also tried to identify the responsible sequence elements in the c-jun activation by IE1-72kDa. We found HCMV IE expression transactivated the c-jun promoter in human embryonal lung fibroblasts (HEL). The activation fold by IE1-72kDa, IE2-86kDa and IE2-55kDa was 23, 35, and 5, respectively. When the expression of each IE was combined, it showed synergism. Expression of (IE1-72kDa + IE2-86kDa) and (IE1-72kDa + IE2-86kDa + IE2-55kDa) resulted in 131 and 162 fold increase, respectively. The c-jun promoter region between -117 and -59 contains binding sites for the transcription factors Spl, CAAT, AP-l like (ATF/CREB), and MEF2. Transient expression assays were performed using various reporter plasmids containing the c-jun promoter-regulatory region linked to the luciferase gene and a plasmid expressing HCMV IE1 gene. Deletional and point mutational analysis showed that the sequence between -225 to -160 and the CTF binding site were involved in the up-regulation of c-jun promoter.

• International Journal of Industrial Entomology and Biomaterials
• /
• v.15 no.2
• /
• pp.131-135
• /
• 2007

Open reading frame 4 of Bombyx mori nucleopolyhedrovirus (BmNPV), designated as Bm4, is a gene whose function is completely unknown. With the recently developed BmNPV bacmid and a modified pFastBac1 whose polyhedrin promoter was replaced with ie1 promoter, a recombinant bacmid expressing Bm4-EGFP fusion protein under the control of ie1 promoter in BmN cells was successfully constructed. The result not only showed that the polyhedrin promoter can be replaced efficiently with other promoters to direct the expression of foreign gene in BmN cells by using Bac-to-Bac/BmNPV baculovirus expression system but also laid the foundation for rescue experiment of Bm4 deletion mutant due to the ability of ie1 promoter to direct gene expression throughout the infection cycle.

• BMB Reports
• /
• v.35 no.6
• /
• pp.553-561
• /
• 2002

A gene that encodes a protein homologue to baculoviral IE-1 was identified and sequenced in the genome of the Choristoneura fumiferana granulovirus (ChfuGV). The gene has an 1278 nucleotide (nt) open-reading frame (ORF) that encodes 426 amino acids with an estimated molecular weight of 50.33 kDa. At the nucleotide level, several cis-acting regulatory elements were detected within the promoter region of the ie-1 gene of ChfuGV along with other studied granuloviruses (GVs). Two putative CCAAT elements were detected within the noncoding leader region of this gene; one was located on the opposite strand at -92 and the other at -420 nt from the putative start triplet. Two baculoviral late promoter motifs (TAAG) were also detected within the promoter region of the ie-1 gene of ChfuGV. A single polyadenylation signal, AATAAA, was located 18nt downstream of the putative translational stop codon of ie-1 from ChfuGV. At the protein level, the amino acid sequence data that was derived from the nucleotide sequence in ChfuGV IE-1 was compared to those of the Cydia pomonella granulovirus (CpGV), Xestia c-nigrum granulovirus (XcGV) and Plutella xylostella granulovirus (PxGV). The C-terminal regions of the granuloviral IE-1 sequences appeared to be more conserved when compared to the N-terminal regions. A domain, similar to the basic helix-loop-helix like (bHLH-like) domain in NPVs, was detected at the C-terminal region of IE-1 from ChfuGV (residues 387 to 414). A phylogenetic tree for baculoviral IE-1 was constructed using a maximum parsimony analysis. A phylogenetic estimation demonstrates that ChfuGV IE-1 is most closely related to that of CpGV.

• Journal of Microbiology and Biotechnology
• /
• v.24 no.12
• /
• pp.1719-1727
• /
• 2014

In the present study, whispovirus immediate early 1 promoter (ie-1) was used to initiate surface expression of the hemagglutinin (HA) protein of Egyptian H5N1 avian influenza virus (AIV) by using the baculovirus expression vector system. The HA gene and whispovirus ie-1 promoter sequence were synthesized as a fused expression cassette (ie1-HA) and successfully cloned into the pFastBac-1 transfer vector. The recombinant vector was transformed into DH10Bac competent cells, and the recombinant bacmid was generated via site-specific transposition. The recombinant bacmid was used for transfection of Spodoptera frugiperda (Sf-9) insect cells to construct the recombinant baculovirus and to induce expression of the HA protein of H5N1 AIV. The recombinant glycoprotein expressed in Sf-9 cells showed hemadsorption activity. Hemagglutination activity was also detected in both extra- and intracellular recombinant HAs. Both the HA and hemadsorption activities were inhibited by reference polyclonal anti-H5 sera. Significant expression of the recombinant protein was observed on the surface of infected insect cells by using immunofluorescence. SDS-PAGE analysis of the expressed protein revealed the presence of a visually distinguishable band of ~63 kDa in size, which was absent in the non-infected cell control. Western blot analysis confirmed that the distinct 63 kDa band corresponded to the recombinant HA glycoprotein of H5N1 AIV. This study reports the successful expression of the HA protein of H5N1 AIV. The expressed protein was displayed on the plasma membrane of infected insect cells under the control of whispovirus ie-1 promoter by using the baculovirus expression vector system.

• Korean Journal of Microbiology
• /
• v.31 no.1
• /
• pp.72-78
• /
• 1993

Since the 'promoter/enhancer region of the major immediate early (IE) ~ene of human cytomegalovirus (HCMV) contains the cyclic AMP (cAMP) response element (CRE) consensus sequence, it was reasonable to hypothesize that cAMP might affect HCMV replication. Cyclic AMP modulating drugs such as 8-bromoadenosine 3',5'-cyclic monophosphate (BrA), and papaverine were used to affect the intracellular levels of cAMP, and the effects of the drugs on HCMV replication were studied. While papaverine effectively inhibited HCMV multiplication and DNA synthesis, BrA exerted little effect on the production of infectious HCMV yields. The synthesis of DNA in HCMV-infected cells appeared to be stimulated by BrA In order to understand the effect of cAMP on the expression of HCMV major IE gene, plasmid (pCMVIE/CAT) containing a reporter gene driven by HCMV IE promoter was transfected into either permissive human embryo lung (HEL) cells or nonpermissive cells. PL,Javerine, which has been reported to block the HCMV-induced increase in cAMP, reduced the expression of pCMVIE/CA T in permissive HEL cells. Treatment of transfected cells with BrA increased the expression of HCMV major IE promoter not only in HEL cells, but also in nonpermissive HeLa and Vero cells. Therefore, it seems that the expression of HCMV major IE gene is regulated by cAMP.

• International Journal of Industrial Entomology and Biomaterials
• /
• v.3 no.1
• /
• pp.43-49
• /
• 2001

We have cloned and characterized an immediate early-1 gene, iel, which is activated immediately upon entrance of the viral genome into the cell nucleus, from Bombyx mori nuclear polyhedrosis virus (BmNPV) K1 strain. This gene encodes a protein 584 amino acids with a predicted molecular weight of 67 kDa. The promoter and coding regions of BmNPV-K1 ie1 showed high homology with Autographa californica nuclear polyhedrosis virus and BmNPV T3 strain. The BmNPV-K1 ie1 was different from amino acid sequence at 4 positions in BmNPV T3. The location of ie1 gene in the BmNPV-K1 genome was confirmed by Southern blot analysis and its expression patterns at the transcriptional level in the infected cells were confirmed by Nerthern hybridization analysis.

• Journal of Sericultural and Entomological Science
• /
• v.40 no.2
• /
• pp.111-116
• /
• 1998

To construct transformed Bm5 cells, Autographa californica nuclear polyhedrosis virus (AcNPV)IE1 gene, an immediate early viral gene was firstly used in this study. AcNPV IE1 gene, which shares on 95.3% uncleotide sequence homology with Bombyx mori nuclear polyhedrosis virus (BmNPV) IE1 gene, was isolated and cloned into pBluescript. Neomycin gene from pKO-neo was inserted under the control of the IE1 promoter to yield pAcIE1-neo. The plasmid pAcIE1-neo was transfected into Bm5 or Sf9 cells, and neomycin-resistant cells were selected in TC100 medium containing 10% fetal bovine serum (FBS) and 1 mg/$m\ell$ G418 for two weeks. Individual clones were picked and each was amplified for further characterization. The genomic DNA from neomycin-resistnt cells was isolated and characterized by PCR using AcNPV IE1 gene-specific primers and by Southern blot analysis using neomycin gene probe. We concluded that AcNPV IE1 gene was functional in B. moridrived Bm5 cells as well as Spodaptera frugiperda-derived Sf9 cells to produce stably-transformed insect cells.

• Korean Journal of Poultry Science
• /
• v.50 no.4
• /
• pp.261-266
• /
• 2023

The skeletal muscles of livestock play a crucial role as protein sources for humans, and the consumption of poultry meat is steadily increasing worldwide. Numerous genes, including myogenic regulatory factors, are involved in myogenesis, and precise regulation of them is essential. In this study, genes specifically expressed in muscles were selected, and their promoters were cloned and analyzed. The analysis of gene expression in various tissues of animals revealed that many genes exhibited specific expression patterns in skeletal muscles, with TNNT3, TNNC2, and MYF6 genes showing similar patterns in poultry. The promoter regions of three genes were amplified by polymerase chain reaction to sizes of 1.2 kb, 1.03 kb, and 1.43 kb, respectively. These fragments were then inserted at the front of the enhanced green fluorescent protein gene in vectors. It was confirmed that the sequences of three promoters closely matched the chicken genome sequences. Upon introducing vectors with each promoter into QM7 quail muscle cells, all three promoters successfully induced the expression of the green fluorescent protein. The brightness of the green fluorescence in each promoter was approximately seven times dimmer compared to the control, CMV-IE promoter. It is predicted that more than 230 transcription factors can bind to each promoter, especially various transcription factors expressed in muscles, including myogenic regulatory factors such as MYF5, MYOD, and MYOG. These promoters can be valuable for studying gene expression in poultry muscle cells, and further research is needed to precisely investigate the regulatory region of gene expression in promoters.

• The Journal of Korean Society of Virology
• /
• v.29 no.2
• /
• pp.137-144
• /
• 1999

Transformation efficiency, virus multiplication and foreign gene expression were characterized in the insect cells transformed with Autographa californica nuclear polyhedrosis virus (AcNPV) immediate early 1 gene (IE1). Transformation efficiency of insect cells by AcNPV IE1 gene vector horboring foreign gene was approximately 8-fold higher in the Sf9 cells transformed previously with AcNPV IE1 gene than in the normal Sf9 cells. Virus multiplication and foreign gene expression of recombinant baculovirus in the Sf9 cells transformed with AcNPV IE1 gene were similar to those of the normal Sf9 cells. These results suggest that transformed cells displaying foreign gene product by using AcNPV IE1 gene promoter will be useful for the diverse applications of insect cells.