Control of expression of the vaccinia virus thymidine kinase gene.

mRNA extracted from vaccinia virus-infected cells early after infection directs cell-free synthesis of enzymatically active viral thymidine kinase (Hruby and Ball, Virology, in press). We used this assay for a specific vaccinia virus mRNA to study the induction and repression of the viral thymidine kinase gene during infection of thymidine kinase-deficient L-cells. As observed previously by other workers, the synthesis of thymidine kinase occurred immediately after infection but was switched off after 4 h later. We observed similar kinetics of accumulation and shutoff under conditions where viral DNA synthesis and late gene expression were inhibited. Cell-free translation of mRNA from infected cells showed that the concentration of functional message for viral thymidine kinase reached a peak 3 to 4 h after infection and then decreased with a half-life of about 1 h. These kinetics indicated that significant levels of thymidine kinase mRNA persisted in cells which had stopped synthesizing the enzyme. Under conditions where late gene expression was inhibited, high concentrations of functional mRNA could be isolated from cells at late times after infection. On the basis of these results, we conclude that the repression of thymidine kinase expression is mediated at the translational level by one or more early or delayed early viral genes. Repression is accompanied by, but does not depend on, the inactivation or degradation of thymidine kinase mRNA, which is a late gene function.     

Control of cAMP-induced gene expression by divergent signal transduction pathways.

A compilation of literature data and recent experiments led to the following conclusions regarding cyclic adenosine 3':5' monophosphate (cAMP) regulation of gene expression. Several classes of cAMP-induced gene expression can be discriminated by sensitivity to stimulation kinetics. The aggregation-related genes respond only to nanomolar cAMP pulses. The prestalk-related genes respond both to nanomolar pulses and persistent micromolar stimulation. The prespore specific genes respond only to persistent micromolar stimulation. The induction of the aggregation- and prestalk-related genes by nanomolar cAMP pulses may share a common transduction pathway, which does not involve cAMP, while involvement of the inositol 1,4,5-trisphosphate (IP3)/Ca2+ pathway is unlikely. Induction of the expression of prespore and prestalk-related genes by micromolar cAMP stimuli utilizes divergent signal processing mechanisms. cAMP-induced prespore gene expression does not involve cAMP and probably also not cyclic guanosine 3'.5' monophosphate (cGMP) as intracellular intermediate. Involvement of cAMP-induced phospholipase C (PLC) activation in this pathway is suggested by the observation that IP3 and 1,2-diacylglycerol (DAG) can induce prespore gene expression, albeit in a somewhat indirect manner and by the observation that Li+ and Ca2+ antagonists inhibit prespore gene expression. Cyclic AMP induction of prestalk-related gene expression is inhibited by IP3 and DAG and promoted by Li+, and is relatively insensitive to Ca2+ antagonists, which indicates that PLC activation does not mediate prestalk-related gene expression. Neither prespore nor prestalk-related gene expression utilizes the sustained cAMP-induced pHi increase as intracellular intermediate.     

Control involving metabolism and gene expression

Control of DNA supercoiling by the free-energy of hydrolysis of ATP that involves gene expression is analyzed in terms of three levels of unconnected metabolic pathways. These are synthesis and breakdown of topoisomerase mRNAs, synthesis and breakdown of topoisomerase proteins and supercoiling and relaxation of DNA. The so-called square-matrix method previously developed for the control of metabolic pathways, is extended to deal with this hierarchical control system. It turns out that also in this case, the matrix of control coefficients is equal to the inverse of the so-called elasticity matrix, which contains all relevant elasticity coefficients as well as information about the structure and connectedness of the pathways involved. For a simpler case of a hierarchy of two systems, we demonstrate that the explicit matrix inversion method may be replaced by an implicit method in which the regulatory effects that run through the other level are described by an additional elasticy coefficient which may then be treated as if local.     

Control elements of muscarinic receptor gene expression

Studies describing the structures of the M1, M2 and M4 muscarinic acetylcholine receptors (mAChR) genes and the genetic elements that control their expression are reviewed. In particular, we focus on the role of the neuron-restrictive silencer element/restriction element-1 (NRSE/RE-1) in the regulation of the M4 mAChR gene. The NRSE/RE-1 was first identified as a genetic control element that prevents the expression of the SCG-10 and type II sodium channel (NaII) genes in non-neuronal cells in culture. The NRSE/RE-1 inhibits gene expression by binding the repressor/silencer protein NRSF/REST, which is present in many non-neuronal cell lines and tissues. Our studies show that although the expression of the M4 mAChR gene is inhibited by NRSF/REST, this inhibition is not always complete. Rather, the efficiency of silencing by NRSF/REST is different in different cells. A plausible explanation for this differential silencing is that the NRSF/RE-1 interacts with distinct sets of promoter binding proteins in different types of cells. We hypothesize that modulation of NRSF/REST silencing activity by these proteins contributes to the cell-specific pattern of expression of the M4 mAChR in neuronal and non-neuronal cells. Recent studies that suggest a more complex role for the NRSE/RE-1 in regulating gene expression are also discussed.     

Control of early gene expression in Dictyostelium

We have examined the expression of a cAMP pulse-repressed and two cAMP pulse-induced genes in response to cAMP and caffeine under a number of different physiological conditions, and in several classes of development mutants altered in cAMP-mediated signal transduction pathways. The data presented help characterize the mutants with regard to early gene expression. Analysis of the data indicates that full induction of the pulse-induced or repression of the pulse-repressed genes requires cycles of activation and adaptation of the cAMP receptor but does not require a rise in intracellular cAMP. Comparison of the results obtained between different mutant classes suggests that repression and activation of the two classes of genes can be uncoupled, implying that different intracellular mechanisms control these processes. In addition, we examined the effects of caffeine and show that it can induce pulse-induced mRNA accumulation in the absence of cAMP.     

Control of developmental gene expression by cell-to-cell interactions in Myxococcus xanthus.

The ssbA mutants of Myxococcus xanthus behave as if they are unable to produce a cell-to-cell signal required for normal development. They are unable to form fruiting bodies or spores on developmental medium. They do sporulate, however, if allowed to develop in mixtures with wild-type cells. Fusions of developmentally induced promoters of M. xanthus to the Escherichia coli lacZ gene were used to characterize the effect of the ssbA mutations on developmental gene expression. Each of the five independent fusions tested was found to be dependent upon the ssbA+ allele for full expression. The ssbA mutants were able to express each of these fusions if the mutants were allowed to develop in mixtures with wild-type (Lac-) cells. These results cannot be explained on the basis of genetic exchange. The data are consistent with regulation of gene expression mediated by cell-to-cell interactions.