The baculovirus promoter OpIE2 sequence has inhibitory effect on the activity of the cytomegalovirus (CMV) promoter in HeLa and HEK-293 T cells.

Understanding how promoters work in non-host cells is complex. Nonetheless, understanding this process is crucial while performing gene expression modulation studies. This study began with the process of constructing a shuttle vector with CMV and OpIE2 promoters in a tandem arrangement to achieve gene expression in both mammalian and insect cells, respectively. In this system, inhibitory regions in the 5' end of the OpIE2 insect viral promoter were found to be blocking the activity of the CMV promoter in mammalian cells. Initially, the OpIE2 promoter was cloned downstream of the CMV promoter and upstream of the EGFP reporter gene. After introducing the constructed shuttle vector to insect and mammalian cells, a significant drop in the CMV promoter activity in mammalian cells was observed. To enhance the CMV promoter activity, several modifications were made to the shuttle vector including site-directed mutagenesis to remove all ATG codons from the downstream promoter (OpIE2), separating the two promoters to eliminate the effect of transcription interference between them, and finally, identifying some inhibitory regions in the OpIE2 promoter sequence. When these inhibitory regions were removed, high expression levels in insect and mammalian cells were maintained. In conclusion, a shuttle vector was constructed that works efficiently in both mammalian and insect cell lines in the absence of baculovirus infection or gene expression. Moreover, the shuttle vector can be used as a platform to further study the reason for this inhibition, which may give new insights about transcription and promoters' mode of action in both insect and mammalian hosts.

Effect of Temporal Expression of Integral Membrane Proteins by Baculovirus Expression Vector System.

Integral membrane proteins (IMPs) are popular target for drugs, but their resolved structures have been overlooked when compared with cytosolic proteins. The main reason is that IMPs usually need intensive post-translational modifications and they are bound to membranes, which increase the complexity of purifying or crystalizing them. Although different expression systems are used to express IMPs, baculovirus is considered one of the most successful expression systems for those proteins. Despite that, there are always unknown discrepancies in the level of IMPs expression in the baculovirus expression system. Retrospective studies have shown that expression of an immunoglobulin (anti-Chymase mouse monoclonal IgG1) driven by vp39 promoter was more efficient compared to its expression under polyhedrin (polh) promoter; however, this conclusion was not tested on different IMPs to generalize such a conclusion. In this study, the expression of eight different IMPs has been compared under vp39 and polh promoters of Autographa californica nucleopolyhedrovirus. Although different IMPs have shown different patterns of expression, the expression driven by vp39 promoter was found to be generally more efficient than the polh promoter.

Phenol-oxidizing laccases from the termite gut.

cDNAs encoding two gut laccase isoforms (RfLacA and RfLacB) were sequenced from the termite Reticulitermes flavipes. Phylogenetic analyses comparing translated R. flavipes laccases to 67 others from prokaryotes and eukaryotes indicate that the R. flavipes laccases are evolutionarily unique. Alignments with crystallography-verified laccases confirmed that peptide motifs involved in metal binding are 100% conserved in both isoforms. Laccase transcripts and phenoloxidase activity were most abundant in symbiont-free salivary gland and foregut tissue, verifying that the genes and activities are host-derived. Using a baculovirus-insect expression system, the two isoforms were functionally expressed with histidine tags and purified to near homogeneity. ICP-MS (inductively coupled plasma - mass spectrometry) analysis of RfLacA identified bound metals consisting mainly of copper (∼4 copper molecules per laccase protein molecule and ∼3 per histidine tag) with lesser amounts of calcium, manganese and zinc. Both recombinant enzyme preparations showed strong activity towards the lignin monomer sinapinic acid and four other phenolic substrates. By contrast, both isoforms displayed much lower or no activity against four melanin precursors, suggesting that neither isoform is involved in integument formation. Modification of lignin alkali by the recombinant RfLacA preparation was also observed. These findings provide evidence that R. flavipes gut laccases are evolutionarily distinct, host-derived, produced in the salivary gland, secreted into the foregut, bind copper, and play a role in lignocellulose digestion. These findings contribute to a better understanding of termite digestion and gut physiology, and will assist future translational studies that examine the contributions of individual termite enzymes in lignocellulose digestion.

Transcriptional analysis of a major capsid protein gene from Spodoptera exigua ascovirus 5a.

The major capsid protein (mcp) gene of Spodoptera exigua ascovirus 5a (SeAV-5a) was confirmed by aphidicolin viral DNA replication inhibition analysis to be a late gene. The 5' and 3' ends of mcp gene transcripts have been mapped. Primer extension analyses indicated that transcription of the mcp gene initiates from a cytosine 25 nucleotides (nt) upstream of the translation start codon. Two independent approaches by 3' rapid amplification of cDNA ends (3' RACE) and oligo (dT) cellulose binding assay suggested that SeAV-5a mcp mRNA is polyadenylated. Analyses by 3' RACE also revealed that mcp transcripts terminate at a U, either at 26 or 38 nt downstream of the translation stop codon. The putative 5' transcription control region of the SeAV-5a mcp gene shares similarities with other ascoviruses and Chilo iridescent virus (CIV), containing a conserved TATA-box-like motif (TAATTAAA) and an ATTTGATCTT motif upstream of it. The 3' downstream regions of the mcp gene of all the ascoviruses examined and CIV can form a stem-loop structure, and the ends of the mcp gene transcripts of SeAV-5a are within the predicted stem-loop region. This suggests that the stem-loop structure of the mcp gene might be involved in transcription termination.