Characterization of regulatory mutations causing anaerobic derepression of the sodA gene in Escherichia coli K12: cooperation between cis- and trans-acting regulatory loci

The genetic loci leading to anaerobic derepression of a sodA::lacZ protein fusion in a UV-generated mutant strain (UV14) of Escherichia coli were identified. The mutant (UV14) was found to harbour two altered loci: one is in the trans-regulatory gene fnr (fumarate nitrate reduction) where leucine-129 was changed to glutamine (fnr14), and the second (sodA14) is in the promoter region (cis) of the sodA gene apparently affecting the binding of the Fur (ferric uptake regulation) protein. Introduction of an fnr+ gene into UV14 restored anaerobic repression of sodA::lacZ and restored the ability of the cells to reduce nitrate. However, when either the fnr14 or the sodA14 mutation was introduced into an otherwise wild-type background, only slight anaerobic derepression of sodA was observed. When both the cis- and trans-acting mutations (i.e. sodA14 and fnr14) were combined simultaneously in an otherwise wild-type background, the specific activity of sodA::lacZ expression was comparable to that of the original mutant strain (UV14). Furthermore, a genetically confirmed fur fnr double mutant was also similarly derepressed in anaerobic sodA::lacZ expression. The data presented suggest that the cis-mutation in UV14 (sodA14) affects the Fur-binding site in the sodA promoter, while having no effect on Fnr or Arc mediated repression. Also, a second putative Fnr-binding site that straddles the ribosomal binding-site was identified in the sodA gene.     

Effect of aeration and unsaturated fatty acids on expression of the Saccharomyces cerevisiae alcohol acetyltransferase gene

The reduction of acetate ester synthesis by aeration and the addition of unsaturated fatty acids to the medium has been reported to be the result of the reduction in alcohol acetyltransferase (AATase) activity induced by inhibition of this enzyme. However, regulation of the AATase gene ATF1 has not been reported. In this study, ATF1 gene expression was studied by Northern analysis, and the results showed that the ATF1 gene was repressed both by aeration and by unsaturated fatty acids. The results also showed that the reduction of AATase activity is closely related to the degree of repression of ATF1 mRNA, which suggested that the gene repression is the primary means of reducing AATase activity in vivo. Using the Escherichia coli lacZ gene as a reporter gene, it was shown that a 150-bp fragment of the 5' flanking sequence played a major role in the repression by aeration and unsaturated fatty acid addition.     

Inducible gene expression from defective herpes simplex virus vectors using the tetracycline-responsive promoter system

Herpes simplex virus-based amplicon vectors have been used for gene transfer into cultured neurons and the adult CNS. Since constitutive expression of a foreign gene or overexpression of an endogenous gene may have deleterious effects, the ability to control temporal expression would be advantageous. To achieve inducible gene expression, we have incorporated the tetracycline-responsive promoter system into amplicon vectors and showed, both in vitro and in vivo, that expression can be modulated by tetracycline. Using the firefly luciferase as the reporter gene, maximal repression by tetracycline in hippocampal cultures was about 50-fold. Withdrawal of tetracycline derepressed gene expression, reaching maximal levels within 10-12 h. In contrast, addition of tetracycline to cultures without prior tetracycline exposure inhibited gene expression rapidly; luciferase activity was reduced to less than 8% within 24 h. In adult rat hippocampus, vectors expressing luciferase or the Escherichia coli lacZ were repressed by tetracycline 9- and 60-fold, respectively. Maximum gene expression from the vectors occurred 2-3 days post-infection and declined thereafter. Such decline impeded further induction of expression by withdrawing tetracycline. This study demonstrates the feasibility of incorporating a powerful inducible promoter system into HSV vectors. The development of such an inducible viral vector system for gene transfer into the adult CNS might prove to be of experimental and therapeutic value.