Adaptation of Escherichia coli to respiratory conditions: regulation of gene expression.
Explore the source record for details and available documents.
Aerobiosis↗
SEARCH · Search PubMed
Results for “conditional gene expression”
Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
The conditional inhibition of gene expression in cultured Drosophila cells by antisense RNA.
Genes producing antisense RNA are becoming important tools for the selective inhibition of gene expression. Experiments in different biological systems, targeting different mRNAs have yielded diverse results with respect to the success of the technique and its mechanism of action. We have examined the potential of three antisense genes, whose transcription is driven by a Drosophila metallothionein promoter, to inhibit the expression of alcohol dehydrogenase (ADH) or a microtubule associated protein (205K MAP) in cultured Drosophila cells. Expression of ADH was significantly reduced upon induction of the anti-ADH genes. The ADH mRNA does not appear to be destabilized by the presence of antisense RNA but rather exists at similar levels in hybrid form. Hybrids are detected with both spliced and unspliced ADH RNA. In contrast to these results, antisense genes producing antisense RNA in great excess to 205K MAP mRNA, which is itself far less abundant than the ADH mRNA, failed to show any inhibition of 205K MAP expression.
Alcohol Dehydrogenase↗
Effect of culture conditions on PLP and MAG gene expression in rat glioma C6 cells.
The effects of culture conditions on the expression of myelin-specific genes, i.e. proteolipid protein (PLP) and myelin-associated glycoprotein (MAG) in rat glioma C6 cells was studied. Early passage (40-46) cells had higher steady-state level of PLP- and MAG-specific mRNA than late (100) passage cells when grown in defined (serum-free) medium. The PLP gene expression was increased whereas the MAG gene expression was reduced in the presence of 10% fetal calf serum in either passage. The level of both PLP- and MAG-specific messages was also directly related to the cell density indicating cell contact-induced stimulation of the gene expression. Furthermore, the cells apparently secrete factors into the medium, which upregulate the gene expression in autocrine fashion. The results also indicate a dissimilarity of regulatory mechanisms involved in the expression of the PLP and MAG genes.
Animals↗
A yeast protein with homology to the beta-subunit of G proteins is involved in control of heme-regulated and catabolite-repressed genes.
The product of the Saccharomyces cerevisiae AER2 gene is responsible for maintaining repression of at least two distinct regulatory pathways: heme activation/repression and catabolite repression. Mutations in the gene caused an eightfold increase in the expression of the heme-activated CYC1 gene in the absence of heme, a substantial increase in the expression of the heme-repressed ANB1 gene in the presence of heme, and a 13-fold increase in the expression of the catabolite-repressed GAL1 gene in the presence of glucose. Lesser or no increases in the expression of these genes were observed under derepressed or activation conditions. The aer2 mutations also caused a large increase in CYC7 gene expression under all conditions; this gene is subject to heme activation/repression, as well as catabolite repression. The AER2 gene was cloned and the sequence determined. The large open reading frame contiguous with the transcript from the complementing region encoded a 713-amino acid polypeptide chain with extensive homology to the beta-subunit of G proteins. The sequence revealed that AER2 is the TUP1 gene. A deletion mutation was constructed and the null phenotype was the same as the original mutants. The aer2 null mutant was shown to have increased aerobic and anaerobic levels of RNA encoding the ROX1 repressor, normally expressed only aerobically and responsible for the aerobic repression of ANB1 expression. The increase in both ROX1 and ANB1 RNAs aerobically in this mutant suggests that the repressor is nonfunctional in the mutant.
Amino Acid Sequence↗
The msf gene causes condition-specific shifts in global gene expression in Haemophilus influenzae.
UNLABELLED: Haemophilus influenzae is a diverse human-restricted bacterium that normally colonizes the healthy nasopharynx but also causes common infections. Comparisons of clinical isolate genomes previously identified a gene, msf, that contained Sel1-like repeats that were associated with clinical disease. Mutant analysis had further found that msf improved survival in macrophages and increased systemic infection in an animal model. However, the role of msf in other conditions and its molecular function remain unknown. To identify protein-protein interactions with Msf, a yeast two-hybrid screen against an H. influenzae prey library was conducted, which found potential interactions with lipoprotein exporter protein LolD and an autotransporter adhesin Hap. To identify effects of msf on gene expression, we compared wild-type and mutant strains grown in multiple culture conditions by RNA-seq. The results indicate that msf modulates global gene expression in a condition-dependent manner, exerting an especially strong influence in starved surface-attached biofilm cells. The few consistent changes in mutants' planktonic exponential and stationary phases included decreased expression of two paralogous autotransporter adhesins. By contrast, mutant cells in starved surface-attached biofilms had dramatic changes in expression, including upregulation of protein translation and downregulation of alternative carbon metabolism. However, assays of 24 hour biofilm phenotypes found only subtle gene expression changes. Together, the results point to a speculative model of Msf functioning as an envelope-associated chaperone whose presence affects the relative expression of proteins at the outer membrane. IMPORTANCE: Comparing genomes from different clinical isolates of the same pathogenic bacterial species has identified genes associated with virulence, but many of these are understudied or have no known function. The msf gene was previously implicated as a virulence factor in Haemophilus influenzae, a common cause of mucosal diseases including middle-ear and chronic lung infections. This study finds that the msf gene causes condition-specific changes in gene expression, with especially dramatic changes in starved surface-attached biofilm cells. Along with identification of putative protein-protein interaction partners, the results provide new clues as to the molecular and cellular function of Msf, potentially as an envelope-associated chaperone involved in membrane protein trafficking. Understanding how virulence-associated genes like msf modulate bacterial responses to the environment may help explain why some bacterial strains remain harmless colonizers while others become pathogens.
Haemophilus influenzae↗
Ablation of Drosophila photoreceptor cells by conditional expression of a toxin gene.
We have used toxin-mediated ablation to study some aspects of visual system development in Drosophila melanogaster. To devise a method that permits the conditional expression of a cellular toxin, we introduced an amber mutation into the diphtheria toxin-A-chain gene. In transgenic animals, this toxin gene can be activated by providing the gene for an amber suppressor tRNA. By coupling this toxin gene to the photoreceptor cell-specific promoter of the chaoptic gene, photoreceptor cells could be specifically ablated during development. Photoreceptor cell-specific markers normally activated during pupal development failed to appear after midpupation. Photoreceptor cells were absent from the retinas of adult flies at eclosion. We have assessed the consequences of photoreceptor cell ablation for eye and optic lobe development. We suggest that the larval photoreceptor nerve is not essential, in the late larval stages, for retinula photoreceptor cell axons to achieve their proper projection pattern in the brain. Moreover, while retinula photoreceptor innervation is initially required for the development of normal optic ganglia, the ablation of these cells in midpupation has no discernible effect. This approach to cell-specific ablation should be generally applicable to the study of cellular functions in development and behavior.
Animals↗
[Role of enzymatic DNA methylation in cell differentiation and carcinogenesis].
The enzymatic methylation of specific cytosine residues in DNA plays a part in controlling gene expression. Low methylation levels may be a necessary condition for gene expression. The chemical carcinogens exert their effect on the enzymatic methylation of mammalian DNA and can cause hypomethylation. Demethylated sites do not become remethylated in the subsequent cell cycles. The consequence of DNA hypomethylation may be both stimulation of cell differentiation and initiation of carcinogenesis.
Animals↗
Thioredoxin reductase-dependent inhibition of MCB cell cycle box activity in Saccharomyces cerevisiae.
Mlu1 cell cycle box (MCB) elements are found near the start site of yeast genes expressed at G1/S. Basal promoters dependent on the elements for upstream activating sequence activity are inactive in Deltaswi6 yeast. Yeast were screened for mutations that activated MCB reporter genes in the absence of Swi6. The mutations identified a single complementation group. Functional cloning revealed the mutations were alleles of the TRR1 gene encoding thioredoxin reductase. Although deletion of TRR1 activated MCB reporter genes, high copy expression did not suppress reporter gene activity. The trr1 mutations strongly (20-fold) stimulated MCB- and SCB (Swi4/Swi6 cell cycle box)-containing reporter genes, but also weakly (3-fold) stimulated reporter genes that lacked these elements. The trr1 mutations did not affect the level or periodicity of three endogenous MCB gene mRNAs (TMP1, RNR1, and SWI4). Deletion of thioredoxin genes TRX1 and TRX2 recapitulated the stimulatory effect of trr1 mutations on MCB reporter gene activity. Conditions expected to oxidize thioredoxin (exposure to H2O2) induced MCB gene expression, whereas conditions expected to conserve thioredoxin (exposure to hydroxyurea) inhibited MCB gene expression. The results suggest that thioredoxin oxidation contributes to MCB element activation and suggest a link between thioredoxin-oxidizing processes such as ribonucleotide reduction and cell cycle-specific gene transcription.
Cell Cycle↗
Synthesis of early vaccinia-virus-specific enzymes under conditions of immediate early gene expression.
Cycloheximide reversal experiments in chick embryo fibroblasts and mouse L-929 cells indicate that the poxvirus-induced enzymes DNA polymerase and 'alkaline' DNase are immediate early gene products of the virus. In contrast to the vaccinia-WR-coded enzyme under conditions of immediate early gene expression the cowpox-virus-induced DNA polymerase is made only in very small amounts. The studies are consistent with the notion that all poxvirus-specific early proteins may be immediate early viral gene products.
Animals↗
Intron-specific stimulation of anaerobic gene expression and splicing efficiency in maize cells.
Most of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) genes characterized in plants and algae to date have one intron very close to the 5' end of the gene. To study the functional relevance of some of these introns for gene expression we have analysed the influence of three 5' introns on transient gene expression of the anaerobically inducible maize GapC4 promoter in maize cells. Under aerobic conditions, reporter gene expression is increased in the presence of the first introns of the GapC4 and GapC1 genes, and the first intron of the nuclear encoded chloroplast-specific GapA1 gene. In contrast, the GapC4 intron increases anaerobic gene expression above the level obtained for the intronless construct, while anaerobic expression of constructs harboring the GapA1 and GapC1 introns was similar to the anaerobic expression level of the intronless construct. Splicing analysis revealed that the GapC4 intron is processed more efficiently under anaerobic conditions, while no change in splicing efficiency is observed for the GapC1 and the GapA1 introns when subjected to anaerobic conditions. These results suggest that an increase in splicing efficiency contributes to the anaerobic induction of the maize GapC4 gene.
Base Sequence↗
Construction and characterization of herpes simplex virus type 1 mutants with conditional defects in immediate early gene expression.
The herpes simplex virus type 1 (HSV-1) mutant in 1814 contains an insertion mutation in the coding sequence for the virion transactivator protein VP16 and is thus impaired for the activation of immediate early (IE) gene expression. This virus was modified further by introducing the Moloney murine leukemia virus LTR promoter in place of the upstream sequences controlling expression of the IE regulatory protein ICPO, to yield mutant in 1820. In almost all cell types tested, in 1820 initiated infection less efficiently than in 1814, behaving as if lacking both VP16 and ICPO functions, but in BHK cells in 1820 was less impaired than in 1814. A rescuant of in 1820 at the VP16 locus, in 1825, also exhibited a host range phenotype, initiating replication as efficiently as wild-type HSV-1 in BHK cells but inefficiently in other cell types. In 1825 was unable to complement an ICPO null mutant in restricted cells, demonstrating that the promoter exchange prevented the expression of ICPO protein in functionally significant amounts. The novel host range properties of in 1820 provided a basis for the construction of additional viruses conditionally impaired for IE gene expression and assessment of their value as prototype vectors. Production of an HSV-1 mutant multiply defective in the expression of IE gene products was achieved by introduction of the temperature-sensitive mutation of HSV-1 tsK, which inactivates the IE transcription activator ICP4 at nonpermissive temperatures, into in 1820 to produce in 1820K. This mutant could be propagated effectively in BHK cells at 31 degrees but was effectively devoid of the major regulators ICPO, ICP4, and VP16 in other cells types at 38.5 degrees. Cultures could withstand infection with 5 PFU of in 1820K per cell without detectable cytopathology and could be reseeded to form colonies at approximately 90% efficiency. A derivative of in 1820K containing the Escherichia coli lacZ gene controlled by the human cytomegalovirus (HCMV) major IE promoter expressed low but detectable levels of beta-galactosidase in almost all cells after infection of cultures at 5 PFU per cell and incubation at 38.5 degrees. Cultures infected with 5 PFU per cell of an in 1820K derivative expressing neomycin phosphotransferase (npt) controlled by the HCMV IE promoter were resistant to killing by the antibiotic G418 for up to 3 days, and cell survival correlated with the retention of functional levels of npt. Mutants based on in 1820K can thus express foreign gene products in virtually all cells in a culture under conditions in which cytotoxicity is eliminated, demonstrating that progressive reduction of IE gene expression is an important step in the design of HSV-1-derived vectors.
Animals↗
Conditional expression of RPA190, the gene encoding the largest subunit of yeast RNA polymerase I: effects of decreased rRNA synthesis on ribosomal protein synthesis.
The synthesis of ribosomal proteins (r proteins) under the conditions of greatly reduced RNA synthesis were studied by using a strain of the yeast Saccharomyces cerevisiae in which the production of the largest subunit (RPA190) of RNA polymerase I was controlled by the galactose promoter. Although growth on galactose medium was normal, the strain was unable to sustain growth when shifted to glucose medium. This growth defect was shown to be due to a preferential decrease in RNA synthesis caused by deprivation of RNA polymerase I. Under these conditions, the accumulation of r proteins decreased to match the rRNA synthesis rate. When proteins were pulse-labeled for short periods, no or only a weak decrease was observed in the differential synthesis rate of several r proteins (L5, L39, L29 and/or L28, L27 and/or S21) relative to those of control cells synthesizing RPA190 from the normal promoter. Degradation of these r proteins synthesized in excess was observed during subsequent chase periods. Analysis of the amounts of mRNAs for L3 and L29 and their locations in polysomes also suggested that the synthesis of these proteins relative to other cellular proteins were comparable to those observed in control cells. However, Northern analysis of several r-protein mRNAs revealed that the unspliced precursor mRNA for r-protein L32 accumulated when rRNA synthesis rates were decreased. This result supports the feedback regulation model in which excess L32 protein inhibits the splicing of its own precursor mRNA, as proposed by previous workers (M. D. Dabeva, M. A. Post-Beittenmiller, and J. R. Warner, Proc. Natl. Acad. Sci. USA 83:5854-5857, 1986).
Base Sequence↗
Non-mitogenic T cell activation signals are sufficient for induction of human immunodeficiency virus transcription.
The expression of human immunodeficiency virus type 1 (HIV) is enhanced after T cell activation due to the interaction of cell-encoded nuclear factors with binding sites in the viral long terminal repeats (LTR). We studied the minimal signal transduction requirements for induction of HIV transcription during T cell activation. Monoclonal antibodies (mAb) against the T cell receptor/CD3 complex induced interleukin (IL) 2 production as well as HIV-LTR-directed gene expression in Jurkat T cells. Addition of cyclosporin A or buffering of intracellular Ca2+ changes did not abolish this LTR-directed gene expression but did block IL 2 production. In contrast, interference with protein kinase C (PKC) activation did inhibit both IL 2 production and LTR-driven gene expression. Under all conditions HIV-LTR-directed gene expression correlated with gene expression induced by the NF-kB binding enhancer, but not by the NF-AT or OCT-1 binding sites. In accordance with observations by Verweij, Geerts and Aarden on the CD28 co-stimulatory activation of IL2 transcription via an NF-kB-like activity, stimulation of the CD2, CD28 and CD44 accessory molecules was tested to mimick physiological activation signals independent of T cell receptor triggering. mAb directed against CD2 and CD44 only marginally induced the LTR. Next, non-mitogenic stimulation by mAb against CD28 clearly induced the HIV-LTR- and NF-kB- but not NF-AT- and OCT-1-driven chloramphenicol acetyltransferase CAT expression, showing a direct effect on gene expression via this receptor. Taken together, this report shows that non-mitogenic T cell activation signals are sufficient to induce HIV transcription. The finding that these signals may be delivered by receptors that are not dependent on antigen-specific activation may have important implications for our understanding of HIV pathogenesis.
Antigens, CD↗
Mutagenesis of Bordetella pertussis with transposon Tn5tac1: conditional expression of virulence-associated genes.
The Tn5tac1 transposon contains a strong outward-facing promoter, Ptac, a lacI repressor gene, and a selectable Kanr gene. Transcription from Ptac is repressed by the lacI protein unless an inducer (isopropyl-beta-D-thiogalactopyranoside [IPTG]) is present. Thus, Tn5tac1 generates insertion mutations in Escherichia coli with conditional phenotypes because it is polar on distal gene expression when IPTG is absent and directs transcription of these genes when the inducer is present. To test the usefulness of Tn5tac1 in Bordetella pertussis, a nonenteric gram-negative bacterial pathogen, we chose the bifunctional adenylate cyclase-hemolysin determinant as an easily scored marker to monitor insertional mutagenesis. Tn5tac1 delivered to B. pertussis on conjugal suicide plasmids resulted in Kanr exconjugants at a frequency of 10(-3) per donor cell, and nonhemolytic (Hly-) mutants were found among the Kanr colonies at a frequency of about 1%. Of eight independent Kanr Hly- mutants, two were conditional and exhibited an Hly+ phenotype only in the presence of IPTG. Using a new quantitative assay for adenylate cyclase based on high-pressure liquid chromatography, we found that enzymatic activity in these two strains was specifically induced at least 500-fold in a dose-dependent fashion over the range of 0 to 125 microM IPTG. These data show that Ptac serves as a promoter, lacI is expressed and is functional, and IPTG can induce Ptac transcription in B. pertussis. Adenylate cyclase expression in whole cells, culture supernatants, and cell extracts from these strains depended upon IPTG, suggesting that the insertions do not merely alter secretion of adenylate cyclase-hemolysin. Other virulence determinants under control of the vir locus are expressed normally, implying that these Tn5tac1 insertions specifically regulate adenylate cyclase-hemolysin expression. We conclude that Tn5tac1 insertion mutations permit sensitive, exogenous control over the expression of genes of interest, providing a useful tool for studying virulence and other important traits of diverse bacterial species.
Adenylyl Cyclases↗
Basic conditions for the drug selection and transient gene expression in the cultured cell line of Bombyx mori.
We established basic conditions for transient gene expression and selection of antibiotics in the cultured cell line of silkworm, Bombyx mori, by use of the promoter of the heat shock protein (hsp70) gene of Drosophila melanogaster. The control promoter (hsp70) promoted the expression of chloramphenicol acetyltransferase (CAT) gene ligated at the downstream, dependent on the orientation of the promoter in the silkworm cell. The cell line is able to be supplied for the promoter assay of the silkworm genes. The concentration for the drug selection was determined as 0.75 mg/ml on neomycin analog, G418 (geneticin).
Animals↗
Phage Trojan horses: a conditional expression system for lethal genes.
The EcoRI restriction enzyme (ENase) cleaves DNA molecules within the sequence GAATTC. Cells expressing this lethal activity normally make a second enzyme, the M.EcoRI methyltransferase (MTase), which protects their chromosomal DNA by modifying the EcoRI recognition sites. To isolate mutants of the EcoRI ENase, its gene was cloned into a filamentous phage vector (M13mp18) under control of the lac promoter. Normally, filamentous phages (M13, f1 and their derivatives) form turbid plaques by impairing the growth of their host cell without killing it. In contrast, phages expressing the EcoRI ENase kill the host cell, but survive long enough to produce plaques which are very clear. Expression of the M.EcoRI MTase rescues the host and restores turbid plaque formation. EcoRI ENase mutants were isolated by screening for mutants that make turbid, instead of clear, plaques on an M- host. This conditional expression system may be useful for cloning and mutating genes for other toxic proteins.
Base Sequence↗
Cascade regulation of terminal adipocyte differentiation by three members of the C/EBP family of leucine zipper proteins.
Terminal differentiation of cultured 3T3-L1 fibroblasts to the adipogenic phenotype is potently stimulated by dexamethasone (DEX) and methylisobutylxanthine (MIX). Previous studies have shown that these hormones induce the expression of genes encoding two members of the CCAAT/enhancer binding protein (C/EBP) family of transcription factors. In the absence of new protein synthesis DEX activates the gene encoding C/EBP delta. Likewise, MIX is a direct inducer of C/EBP beta gene expression. Optimal conditions for differentiation entail a 2-day period wherein confluent fibroblasts are exposed to DEX and MIX, followed by removal of the hormones and subsequent culture in the presence of insulin and fetal bovine serum. During the early phase of differentiation, high levels of C/EBP delta and C/EBP beta accumulate. These transcription factors diminish during the terminal phase of differentiation and come to be replaced by a third member of the C/EBP family, C/EBP alpha. Conclusive evidence has already shown that C/EBP alpha regulates terminal adipocyte differentiation, turning on the battery of fat-specific genes required for the synthesis, uptake, and storage of long chain fatty acids. Here we provide evidence that C/EBP delta and C/EBP beta play early catalytic roles in the differentiation pathway, relaying the effects of the hormonal stimulants DEX and MIX in a cascade-like fashion, leading to the activation of the gene encoding C/EBP alpha. Conditions facilitating the precocious expression of either C/EBP delta or C/EBP beta were observed to accelerate adipogenesis and, in the case of C/EBP beta, relieve dependence on the early hormonal stimulants. Likewise, conditions that prevented the expression of functional C/EBP beta effectively blocked terminal differentiation. Finally, we have discovered that ectopic expression of C/EBP beta in multipotential NIH-3T3 cells results in their conversion into committed adipoblasts capable, upon hormonal stimulation, of synchronous and uniform differentiation into fat-laden adipocytes.
1-Methyl-3-isobutylxanthine↗
Acetaldehyde-induced stimulation of collagen synthesis and gene expression is dependent on conditions of cell culture: studies with rat lipocytes and fibroblasts.
Acetaldehyde has been proposed as a mediator of fibrogenesis in alcoholic liver disease, based in part on its ability to stimulate collagen synthesis by hepatic lipocytes in late primary or passaged culture. In this study, we examined the effect of acetaldehyde on rat lipocytes and fibroblasts at various stages of culture, in an effort to determine whether culture-related events influence responsiveness to this compound. Lipocytes from normal rat liver were studied in primary culture at 3 and 7 days after plating; fibroblasts were studied in subculture, at subconfluent and confluent densities. Both cell types were incubated with 100 microM acetaldehyde for 24 hr followed by measurement of collagen synthesis and type I collagen gene expression. Acetaldehyde had no effect on lipocytes at either 3 or 7 days in primary culture. The inability of acetaldehyde to stimulate collagen synthesis in primary culture was not attributable to toxicity, because cell morphology and total protein synthesis were identical in both treated and untreated cultures. Fibroblasts exhibited a variable response to acetaldehyde that was dependent on cell density: subconfluent cells contained similar amounts of type I collagen mRNA in both the presence and absence of acetaldehyde, whereas confluent cells exhibited a 2- to 3-fold increase in collagen mRNA levels upon acetaldehyde exposure. To determine whether quiescent lipocytes would respond to acetaldehyde in a culture system that mimics the hepatic environment in vivo, lipocytes were plated in coculture with hepatocytes on a basement membrane gel and incubated with 20 mM ethanol for 72 hr. Direct communication between these two cell types did not provoke lipocyte activation, even in the setting of ethanol oxidation.(ABSTRACT TRUNCATED AT 250 WORDS)
Acetaldehyde↗