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Recent evidence bearing on cellular senescence has come from cell fusion and clonal lifespan studies. To date, no model has been developed that is consistent with the recent findings. The purpose of this paper is to present a mechanism for cell senescence that is qualitatively consistent with the experimental evidence.
The functional locus for alpha-amylase (Amy) in Drosophila miranda is in the evolutionarily new X2 chromosome. X2 evolved from an autosome in response to an ancestral autosome-Y translocation that gave rise to the "neo-Y" chromosome of this species. Y-linked Amy, if still present in the ancestrally translocated element, is unexpressed. Dosage compensation for amylase activity was examined in larvae of the S 204 strain. Since dietary glucose is known to repress Amy expression in Drosophila melanogaster, dosage compensation of amylase activity in male larvae of D. miranda was tested by rearing larvae of both sexes on yeast diets with or without a glucose supplement. The WT 10 strain of Drosophila persimilis, a sibling species in which Amy is autosomally linked, was used as a reference for tests of amylase activity differences between the sexes. On the diet with glucose, Amy expression was repressed in both WT 10 and S 204 larvae and male larvae of S 204 displayed dosage compensation for amylase activity. On the nonrepressing diet consisting of yeast alone, S 204 continued to display dosage compensation.
To investigate the importance of the 3'-untranslated region (UTR) of the mouse cholesterol 7alpha-hydroxylase (cyp7) mRNA in post-transcriptional regulation of expression of the cyp7 gene, chimaeric genes encoding mRNA containing the structural sequence of chloramphenicol acetyltransferase (CAT) linked to either the 3'-UTR of the mouse cyp7 mRNA or the SV40 early gene mRNA were constructed. The human cytomegalovirus (CMV) promoter was used to drive the expression of all the chimaeric genes. Thus the transgenes had identical sequences in the promoter, the regions encoding the 5'-UTR and translated sequence but differed in the region encoding the 3'-UTR of their respective mRNA species. The transgene containing the entire cyp7 3'-UTR (designated CMV.CAT.CYP7) gave rise to CAT activity in transfected hepatoma cells that was one-quarter of that obtained in cells transfected with the transgene containing the SV40 3'-UTR (designated CMV.CAT.SV40). The 3'-UTR of the cyp7 mRNA contains sequences resembling AU-rich elements (AREs). Deleting eight of nine putative AREs from the CYP7 3'-UTR sequence increased the CAT activity to a level greater than that observed for CMV.CAT. SV40, whereas deletion of the intron region had no effect. These results show that the AREs of the 3'-UTR of the cyp7 mRNA decrease transgene expression. Bile acids are known to repress the expression of the cyp7 gene. To test whether the 3'-UTR of the cyp7 mRNA has a role in this process, the expression of the chimaeric genes was evaluated in hepatoma cells competent for bile acid uptake. Conjugated bile acids, but not unconjugated bile acids, further decreased the expression of the CMV.CAT.CYP7 transgene. The same bile acids had no effect on the expression of the CMV.CAT.SV40 transgene. Deletion of the intron from the cyp7 sequence did not alter the CAT activity compared with the parental plasmid, and also did not alter the sensitivity of the transgene to the conjugated bile acids. Deletion of the AREs from the cyp7 3'-UTR, which increased the expression of the transgene, did not abolish the sensitivity of the transgene to repression by conjugated bile acids. Thus the 3'-UTR of the mouse cyp7 mRNA also contains elements that facilitate the further repression of transgene expression in the presence of conjugated bile acids. The results indicate that the 3'-UTR of the mouse cyp7 mRNA contains information specifying regulation at the post-transcriptional level.
Escherichia coli K-12 possesses two active transport systems for arginine, two for ornithine, and two for lysine. In each case there is a low- and a high-affinity transport system. They have been characterized kinetically and by response to competitive inhibition by arginine, lysine, ornithine and other structurally related amino acids. Competitors inhibit the high-affinity systems of the three amino acids, whereas the low-affinity systems are not inhibited. On the basis of kinetic evidence and competition studies, it is concluded that there is a common high-affinity transport system for arginine, ornithine, and lysine, and three low-affinity specific ones. Repression studies have shown that arginine and ornithine repress each other's specific transport systems in addition to the repression of their own specific systems, whereas lysine represses its own specific transport system. The common transport system was found to be repressible only by lysine. A mutant was studied in which the uptake of arginine, ornithine, and lysine is reduced. The mutation was found to affect both the common and the specific transport systems.
In vivo synthesis of the mitochondrial elongation factors T and G in the yeast Saccharomyces fragilis can be repressed. Enzymatic activity assays and immunochemical titration methods reveal that cells grown in the presence of 8% glucose or in the absence of oxygen contain relatively lower amounts of mitochondrial elongation factors than cells grown in the presence of lactate. In contrast, in vivo production of the cytoplasmic elongation factors 1 and 2 does not respond to such a change of extracellular conditions. The rate of growth does not affect the level of the mitochondrial elongation factors. Production of both enzymes is almost constant during logarithmic growth, but decreases when the stationary phase is reached. Chloramphenicol, an inhibitor of mitochondrial protein synthesis, does not block but, rather, seems to enhance the in vivo synthesis of mitochondrial T or G.
A potato (Solanum tuberosum) cDNA encoding an isoform of disproportionating enzyme (stDPE2) was identified in a functional screen in Escherichia coli. The stDPE2 protein was demonstrated to be present in chloroplasts and to accumulate at times of active starch degradation in potato leaves and tubers. Transgenic potato plants were made in which its presence was almost completely eliminated. It could be demonstrated that starch degradation was repressed in leaves of the transgenic plants but that cold-induced sweetening was not affected in tubers stored at 4 degrees C. No evidence could be found for an effect of repression of stDPE2 on starch synthesis. The malto-oligosaccharide content of leaves from the transgenic plants was assessed. It was found that the amounts of malto-oligosaccharides increased in all plants during the dark period and that the transgenic lines accumulated up to 10-fold more than the control. Separation of these malto-oligosaccharides by high-performance anion-exchange chromatography with pulsed-amperometric detection showed that the only one that accumulated in the transgenic plants in comparison with the control was maltose. stDPE2 was purified to apparent homogeneity from potato tuber extracts and could be demonstrated to transfer glucose from maltose to oyster glycogen.
Maf1 is an essential and specific mediator of transcriptional repression in the RNA polymerase (pol) III system. Maf1-dependent repression occurs in response to a wide range of conditions, suggesting that the protein itself is targeted by the major nutritional and stress-signaling pathways. We show that Maf1 is a substrate for cAMP-dependent PKA in vitro and is differentially phosphorylated on PKA sites in vivo under normal versus repressing conditions. PKA activity negatively regulates Maf1 function because strains with unregulated high PKA activity block repression of pol III transcription in vivo, and strains lacking all PKA activity are hyperrepressible. Nuclear accumulation of Maf1 is required for transcriptional repression and is regulated by two nuclear localization sequences in the protein. An analysis of PKA phosphosite mutants shows that the localization of Maf1 is affected via the N-terminal nuclear localization sequence. In particular, mutations that prevent phosphorylation at PKA consensus sites promote nuclear accumulation of Maf1 without inducing repression. These results indicate that negative regulation of Maf1 by PKA is achieved by inhibiting its nuclear import and suggest that a PKA-independent activation step is required for nuclear Maf1 to function in the repression of pol III transcription. Finally, we report a previously undescribed phenotype for Maf1 in tRNA gene-mediated silencing of nearby RNA pol II transcription.
The carAB operon of Escherichia coli K-12, which encodes the two subunits of carbamoyl-phosphate synthetase (glutamine hydrolyzing) [carbon-dioxide: L-glutamine amido-ligase (ADP-forming, carbamate-phosphorylating); EC 6.3.5.5], is cumulatively repressed by arginine and the pyrimidines. We describe the structure of the control region of carAB and the sequence of the carA gene. Nuclease S1 mapping experiments show that two adjacent tandem promoters within the carAB control region serve as initiation sites. The upstream promoter P1 is controlled by pyrimidines; the downstream promoter P2 is regulated by arginine. Attenuation control does not appear to be involved in the expression of carAB. A possible mechanism by which control at these promoters concurs to produce a cumulative pattern of repression is discussed. The translational start of carA is atypical; it consists of a UUG or AUU codon.
Three sequences are required for complete repression of the aroF promoter by the TyrR repressor protein. Two of these operator sites lie adjacent to each other and overlap the -35 region of the aroF promoter while the third lies about 70 base pairs upstream of the promoter. An aroF-cat (chloramphenicol acetyltransferase) gene fusion has been used to assay the effect of DNA insertions that alter the distance between the two promoter-proximal and the third, distal, operator sites on the repression of the aroF promoter in vivo. The distal site contributes to the repression of the promoter up to a distance of about 400 base pairs and its effect is not dependent on its separation from the first and second sites by an integral number of turns of the DNA helix.
Mutants of Salmonella have been isolated which luck either end product inhibition or repression of leucine biosynthesis. In a minimal saltsglucose medium, growth is not impaired by the lack of either control mechanism alone. The loss of both control mechanisms, however, leads to a 43 percent reduction in cell yield and a 1.5-fold reduction in the growth rate.
If bacteria are incapable of forming and incorporating proteins into the cytoplasmic membranes in all phases of the cell cycle, then not all cells from an asynchronous culture should be capable of growth when switched to a new carbon and energy source whose metabolism requires new membrane function. The transfer of an inducible culture to low lactose provides such a situation since the cells cannot grow unless galactoside permease can function to concentrate the lactose internally. From such experiments, it was concluded that the Y gene product of the lac operon is synthesized, incorporated, and can start functioning in active transport, at any time throughout the bulk of the cell cycle. Not only were the lags before growth re-ensued much shorter than would be expected if the membrane transport capability could only be developed in a small portion of the cycle, but brief pulses of a gratuitous inducer shortened the lags much further. Three types of Escherichia coli ML 30 culture were studied: cells that had exhausted the limiting glucose; cells taken directly from glucose-limited chemostats; and a washed suspension of highly catabolite repressed cells from cultures grown in high levels of glucose and gluconate. The growth studies reported here were performed on-line with a minicomputer. They represent at least an order of magnitude increase in accuracy in estimating growth parameters over previous instrumentation.
Repression of the lac promoter may be achieved in two different ways: either by interference with the action of RNA polymerase or by interference with CAP activation. We investigated cooperative repression of the Escherichia coli lac operon by systematic conversion of its three natural operators (O1, O2 and O3) on the chromosome. We find that cooperative repression by tetrameric Lac repressor increases with both quality and proximity of the interacting operators. A short distance of 92 bp allows effective repression by two very weak operators (O3, O3). The cooperativity of lac operators is discussed in terms of a local increase of repressor concentration. This increase in concentration depends on flexible DNA which allows loop formation.
In Saccharomyces cerevisiae harvested from early exponential growth on glucose-containing media, the specifc activities of proteinases A and B, carboxypeptidase Y, and the inhibitors IA, IB, IC of these three proteinases, respectively, are found to be 10-30% of the specific activities observed in media without glucose, containing acetate as a carbon source; the activities of two aminopeptidases in glucose-grown cells were 30-50% of those in acetate-grown cells. In contrast to fructose-biphosphatase, phosoenolpyruvate carboxykinase, and cytoplasmic malate dehydrogenase, which are inactivated after the addition of glucose to derepressed cells, the proteinases and inhibitors are not inactivated after glucose addition, but appear to be repressed. Growth of the yeast on poor nitrogen sources or starvation for nitrogen results in 2-3 fold increases in the levels of most proteinases and peptidases, but this effect is not observed with glucose as the carbon source.
The transcriptional activator LAC9, a GAL4 homolog of Kluyveromyces lactis which mediates lactose and galactose-dependent activation of genes involved in the utilization of these sugars can also confer glucose repression to those genes. Here we report on the isolation and characterization of LAC9-2, an allele which encodes a glucose-sensitive activator in contrast to the one previously cloned. A single amino acid exchange of leu-104 to tryptophan is responsible for the glucose-insensitive phenotype. The mutation is located within the Zn-finger-like DNA binding domain which is highly conserved between LAC9 and GAL4. Glucose repression is also eliminated by duplication of the LAC9-2 allele. The data indicate that LAC9 is a limiting factor for beta-galactosidase gene expression under all growth conditions and that glucose reduces the activity of the activator.
The symbiotic, nitrogen-fixing bacterium Rhizobium sp. 32H1 is a specialized ammonium producer during symbiosis. However, during free-living growth, Rhizobium 32H1 assimilates ammonium very poorly. Two pathways of ammonium assimilation exist in enteric bacteria. One is mediated by glutamate dehydrogenase, and the other is mediated by glutamine synthetase-glutamate synthase. The former pathway is altogether inoperative in Rhizobium 32H1; the latter pathway operates at a slow rate and is under strict negative control by ammonium itself. Rhizobium 32H1 glutamine synthetase activity is modulated by both repression-derepression and reversible adenylylation. For a biochemical process lacking an alternative pathway, such a regulatory pattern exacerbates the very process. This suggests that Rhizobium 32H1 restricts its own ammonium assimilation to maximize the contribution of fixed nitrogen to the host plant during symbiosis.
A negative control mutant of the nitrogen assimilation control protein, NAC, has been isolated. Mutants with the leucine at position 111 changed to a nonhydrophobic residue activate transcription from hut and ure promoters, but fail to repress gdhA expression. This failure does not result from failure to bind to either of the two sites required for gdhA repression, but the binding at those sites is altered in the mutant. It appears that the NAC negative control mutants fail to form the complex structures (probably tetramers) formed by wild-type NAC at the gdhA promoter.
When strain C3 of Klebsiella pneumoniae is grown on a minimal medium with excess glucose, isocitrate dehydrogenase, malate dehydrogenase, and succinate dehydrogenase specific activities increase in the last period of the exponential growth phase and in the beginning of the stationary phase. Glucose exhaustion does not alter the development of malate dehydrogenase and succinate dehydrogenase, but specific activities are higher than those obtained with excess glucose. In contrast, glucose exhaustion can be correlated with a decrease of isocitrate dehydrogenase specific activity in the stationary phase. Induction of strain C3 isocitrate dehydrogenase by glucose in complex medium and repression by cAMP in mineral medium were observed. Glucose induction and the NADP/NADPH ratio are suggested as regulatory mechanisms controlling isocitrate dehydrogenase synthesis in the Enterobacteriaceae, but the former appears to be restricted to some Klebsiella strains.
A stable HepG2 cell line harboring a human cholesterol 7 alpha-hydroxylase (CYP7A) minigene/luciferase reporter gene construct was selected for studying transcriptional regulation of CYP7A gene promoter. Insulin and phorbol 12-myristate-13-acetate (PMA) strongly repressed the promoter activity as measured with luciferase activity expressed in the cells. The promoter activity of the 5' progressive deletion/luciferase reporter gene constructs was studied in a transient transfection assay in HepG2 cells. PMA represses the promoter activity and the response elements were localized in the -184/-151 and -134/-81 regions. Insulin also represses the promoter activity and response element was mapped in the -298/-81 region. Surprisingly, glucocorticoid receptor (GR) strongly inhibited promoter activity in the presence of dexamethasone, and response elements were localized in the -298/-151 and the -150/+24 regions. Thyroid hormone receptor also repressed promoter activity and response elements were localized in the -150/+24 and upstream regions. Cotransfection of CYP7A chimeric constructs with an expression vector carrying liver-enriched transcription factor HNF3 alpha stimulated the reporter gene activity, but cotransfection with GR plasmid interfered with the HNF3 alpha-stimulated activity possibly through competition for binding to overlapping GR/HNF3 binding sites. Thus, human cholesterol 7 alpha-hydroxylase gene promoter is strongly repressed by insulin, PMA, and steroid/thyroid hormones and results in the low level of cholesterol 7 alpha-hydroxylase expression in the human liver.