Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.3.1.28 (chloramphenicol acetyltransferase)
 5,100 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The 39k promoter of the baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) is transactivated by the viral protein IE1 and further stimulated by viral enhancer elements and the viral coactivator IE2. To identify additional viral proteins that regulate expression from the 39k promoter, we performed cotransfection experiments with clones of viral DNA and a plasmid containing the chloramphenicol acetyltransferase (CAT) gene under the control of the 39k promoter. Our results indicated that cotransfection of a 39cat construct with plasmids containing IE1 and the EcoRI-S fragment of viral DNA stimulated CAT activity 2.5-fold as compared to cells transfected in the absence of EcoRI-S. EcoRI-S encodes P35, a protein that suppresses apoptosis in AcNPV-infected cells. Primer extension analysis showed that the increase in CAT activity was due to a corresponding increase in mRNA, indicating that P35 may increase transcription from the 39k promoter. To determine whether P35 could activate the 39k promoter in the absence of IE1, the p35 open reading frame was cloned under the control of the ie1 promoter. Cotransfections of 39cat and IE-P35 in the presence and absence of pIE1 indicated that activation of 39k by P35 required the IE1 protein. Cotransfection of plasmids encoding P35 and IE1 did not lead to an increase in the expression of IE1. This suggests that the mechanism of P35 is different than that of IE2 which activates 39k indirectly by increasing the expression of IE1. Cotransfection experiments indicated that IE2 and P35 act in a synergistic fashion.
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PMID:Expression of the 39k promoter of Autographa californica nuclear polyhedrosis virus is increased by the apoptotic suppressor P35. 809 69

Viral expression systems offer the ability to generate high levels of a particular protein within a relatively short period of time. In particular, alphavirus constructs based on Sindbis virus (SV) and Semliki Forest virus (SFV) are promising vehicles as they are cytoplasmic vectors with the potential for high expression levels. Two such alphavirus vectors were utilized during the current study to infect two commercially relevant cell lines, baby hamster kidney (BHK) and Chinese hamster ovary (CHO); the first was a fully competent SV derivative carrying the gene for chloramphenicol acetyltransferase (dsSV-CAT), while the second was a replication deficient SFV construct containing the human interleukin-12 (IL-12) p35 and p40 genes (SFV-IL-12). Since infection with these vectors induced apoptosis in both cell lines, the present effort was dedicated to determining the ability of anti-apoptosis genes to limit the cell death associated with these virus constructs. Infection with the dsSV-CAT vector resulted in the rapid death of BHK and CHO cells within 4 days, a phenomenon which was considerably delayed by stably overexpressing bcl-2 or bcl-x(L). In fact, cellular lifespans were doubled in both BHK-bcl2 and CHO-bclx(L) cells relative to the parental cell lines. Furthermore, the presence of these gene products provided increases of up to 2-fold in recombinant CAT production. Overexpression of bcl-2 and bcl-x(L) also altered the response of these cells upon infection with SFV-IL-12. While the parental cell lines were completely nonviable within 1 week, the BHK-bcl2, BHK-bclx(L), and CHO-bclx(L) cells each recovered from the infection, resuming exponential growth and regaining viabilities of over 90% by 9 days post-infection. Total IL-12 productivities were nearly doubled by Bcl-2 and Bcl-x(L) in the CHO cells, although this effect was apparently cell-line specific, as the native BHK cells were able to secrete more IL-12 than either of its transfected derivatives. Regardless, the presence of the anti-apoptosis genes allowed the production of IL-12 to be maintained, albeit at low levels, from each of the cell lines for the duration of the culture process. Therefore, overexpression of bcl-2 family members can have a significant impact on culture viabilities and recombinant protein production during alphavirus infections of mammalian cells.
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PMID:Part I. Bcl-2 and Bcl-x(L) limit apoptosis upon infection with alphavirus vectors. 1064 29