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PubMed1987

Human T-lymphoblastoid cell lines with high and low abilities to produce interferon-gamma constitutively and their susceptibilities to interferon.

A human T-lymphoblastoid cell line, TCL-Fuj, produces large amounts of interferon (IFN)-gamma constitutively. A variant cell line, 2M, was derived from it. Both cell lines express similar surface antigen markers, but differ in surface morphology. Compared with the parent TCL-Fuj cell line, 2M produced less IFN-gamma constitutively but more in response to IFN inducers. The IFNs produced constitutively and on stimulation with inducers were analyzed by SDS-polyacrylamide gel electrophoresis. In TCL-Fuj cells, the constitutive and induced IFNs consisted of the same molecular species (22K and 39K). In 2M cells, smaller IFNs were produced constitutively (18K and 32K) and induction resulted in a marked increase of 22K molecules. These two cell lines also differed in sensitivity to the antiviral activity of IFN. Other T-lymphoblastoid cell lines, HPB-ALL and TCL-Fuj 4 cells, which did not produce IFN-gamma were permissive for vesicular stomatitis virus (VSV) replication; its growth was markedly suppressed by IFN-gamma and -alpha. TCL-Fuj cells were also permissive for VSV, but were not susceptible to the antiviral effect of the IFNs. In contrast, in 2M cells the multiplication of VSV was restricted; the viral yield was further reduced by the IFNs and increased by treatment with anti-human IFN-gamma serum. Several clonal cell lines derived from TCL-Fuj and 2M cells had characteristics similar to the respective parent cell lines. The growth of both cell lines was not affected by IFN-gamma or by -alpha. The separation of antiviral and anti-proliferative susceptibilities was peculiar to 2M cells unlike other cell lines.

Cell Line↗
PubMed2004

Metalloendopeptidase EC3.4.24.15 is constitutively released from the exofacial leaflet of lipid rafts in GT1-7 cells.

Metalloendopeptidase EC3.4.24.15 (EP24.15) is a physiologically important neuropeptide-degrading enzyme involved in the metabolism of multiple neuropeptides. The mechanism of release of EP24.15 from neuronal cells is multimodal, being both constitutive and stimulatable. Previous studies have characterized stimulated EP24.15 secretion, yet little is understood concerning constitutive release of the peptidase. Utilizing the mouse hypothalamic neuronal GT1-7 cell line, we demonstrate that EP24.15 exists within lipid rafts in the plasma membrane, and that the enzyme is localized to the exofacial leaflet of lipid rafts. Further, we have found that biotinylated EP24.15 on the extracellular surface is released into the cell media in a fashion similar to constitutive release. In addition, classical and non-classical secretion pathway inhibitors were employed to understand the release of EP24.15 into surrounding cell media. The non-classical secretion inhibitor glyburide, a blocker of ATP-sensitive K+ channels, decreased the amount of constitutively released EP24.15 in cell media of GT1-7 cells. With these data, we conclude that EP24.15 association with lipid rafts on the extracellular surface precedes constitutive release of the peptidase into the extracellular milieu for its action on neuropeptides.

Biotinylation↗
PubMed1998

Altered expression of constitutive type and inducible type heat shock proteins in response of D-galactosamine-sensitized mice to lipopolysaccharide as an experimental endotoxic shock model.

The expression of heat shock proteins (HSPs) as stress-induced proteins was studied in mice injected with D-galactosamine (D-GalN) and lipopolysaccharide (LPS) as an experimental endotoxic shock model. The expression of constitutive type heat shock protein 70 (HSC70) was significantly reduced in livers of mice injected with D-galactosamine and lipopolysaccharide, while its expression was unaffected in livers of mice injected with D-galactosamine or lipopolysaccharide alone. The expression of other constitutive type heat shock proteins, namely HSP60, HSP32 and HSP25 was also reduced in mice injected with D-galactosamine and lipopolysaccharide. On the other hand, inducible type HSP70 was detected in livers from mice injected with D-galactosamine and lipopolysaccharide, but not in livers from mice injected with D-galactosamine or lipopolysaccharide alone. Simultaneous injection of anti-tumor necrosis factor (TNF)-alpha antibody prevented the liver from reduced expression of constitutive type HSC70, and lead to marked expression of inducible type HSP70 in the liver. Reduced expression of constitutive type HSC70 was also found when D-galactosamine and recombinant TNF-alpha was injected. Therefore, TNF-alpha was suggested to play a critical role on altered expression of constitutive HSC70 and inducible type HSP70 in response of D-galactosamine-sensitized mice to lipopolysaccharide.

Animals↗
PubMed1997

Epidermal thickness, skin pigmentation and constitutive photosensitivity.

The important factors for UV sensitivity in humans are considered to be the skin pigmentation and the epidermal thickness. In this study on 73 Caucasians (age 20-85 years), we investigated in UV unexposed buttock skin the relationship between the UV sensitivity and constitutive skin pigmentation and thickness of the stratum corneum and the cellular part of the epidermis, in 34 normal people and in 39 skin cancer patients (20) patients with cutaneous malignant melanoma and 19 patients with basal cell carcinoma of the skin). Skin pigmentation was measured by skin reflectance spectroscopy, and UV sensitivity by phototest with a solar simulator. Thicknesses of the stratum corneum and the cellular part of the epidermis were determined by light microscopic evaluation of skin biopsies from the phototest areas. We found that epidermal thickness was independent of skin type and was not correlated to constitutive skin pigmentation. Thickness of the stratum corneum was statistically not different in normal persons and in skin cancer patients (P = 0.41) and was independent of gender (P = 0.61) and age (P = 0.56), while thickness of the cellular epidermis decreased with age (P < 0.01). Stratum corneum thickness was found to be of minor importance for the constitutive UV sensitivity (accounting for on average 11% of the total photoprotection), which was mainly determined by the constitutive skin pigmentation (goodness-of-fit for correlation r = 0.83). A theoretical model for the relationship of UV dose to induction of clinical erythema grade and skin pigmentation and thickness of the stratum corneum was developed. Objective measurements of skin pigmentation in UV unexposed skin by skin reflectance spectroscopy in Caucasians, normal people and people with cutaneous malignant melanoma and basal cell carcinoma of the skin predicts the constitutive UV sensitivity with a high degree of precision.

Adult↗
PubMed2004

Inhibitory regulation of constitutive transient receptor potential-like cation channels in rabbit ear artery myocytes.

In the present study we have investigated an inhibitory pathway regulating a constitutively active Ca(2+)-permeable non-selective cation conductance (I(cat)) in rabbit ear artery smooth muscle cells. Constitutive single channel activity of I(cat) was recorded in cell-attached and inside-out patches with similar unitary conductance values. In inside-out patches with relatively high constitutive activity the G-protein activator GTPgammaS inhibited channel activity which was reversed by the protein kinase C (PKC) inhibitor chelerythrine indicating a G-protein pathway inhibits channel activity via PKC. Spontaneous channel activity was also suppressed by the G-protein inhibitor GDPbetaS suggesting a G-protein is also involved in initiation of constitutive channel activity. Bath application of antibodies to G(alphaq)/G(alpha11) enhanced channel activity whereas anti-G(alpha1-3)/G(alphao) antibodies decreased basal channel activity which suggests that G(alphaq)/G(alpha11) and G(alphaiota)/G(alphao) proteins initiate, respectively, the inhibitory and excitatory cascades. The phospholipase C (PLC) inhibitor U73122 increased spontaneous activity which implies a role for PLC in the inhibitory pathway. Bath application of the diacylycerol (DAG) analogue 1-oeoyl-2-acetyl-sn-glycerol (OAG) decreased the probability of channel opening (NP(o)) and this was reversed by chelerythrine. Application of the PKC activator phorbol 12, 13-dibutyrate (PDBu) and chelerythrine, respectively, decreased and increased NP(o). These data indicate that spontaneously active cation channels are inhibited by a tonic inhibitory pathway involving G(alphaq)/G(alpha11)-mediated stimulation of PLC to generate DAG which activates PKC to inhibit channel opening. There were some patches with relatively low NP(o) and it was evident that the inhibitory pathway was particularly marked in these cases. Moreover in the latter patches GTPgammaS and OAG caused marked increases in NP(o). Together with inhibitory effects of GDPbetaS and anti-G(alpha1-3)/G(alphao) antibodies the results suggest that there is constitutive G(alphai)/G(alphao) protein activity leading to channel opening via a DAG-mediated but PKC-independent mechanism. Finally, with whole-cell recording it is shown that noradrenaline increases I(cat) and the noradrenaline-evoked response is markedly potentiated by PKC inhibition. This latter observation shows that PKC also limits agonist-evoked I(cat) in these arterial myocytes.

Animals↗
PubMed1998

A multiaxial constitutive law for mammalian left ventricular myocardium in steady-state barium contracture or tetanus.

The constitutive law of the material comprising any structure is essential for mechanical analysis since this law enables calculation of the stresses from the deformations and vice versa. To date, there is no constitutive law for actively contracting myocardial tissue. Using 2,3-butanedione monoxime to protect the myocardium from mechanical trauma, we subjected thin midwall slices of rabbit myocardium to multiaxial stretching first in the passive state and then during steady-state barium contracture or during tetani in ryanodine-loaded tissue. Assuming transverse isotropy in both the passive and active conditions, we used our previously described methods (Humphrey et al., 1990a) to obtain both passive and active constitutive laws. The major results of this study are: (1) This is the first multiaxial constitutive law for actively contracting mammalian myocardium. (2) The functional forms of the constitutive law for barium contracture and ryanodine-induced tetani are the same but differ from those in the passive state. Hence, one cannot simply substitute differing values for the coefficients of the passive law to describe the active tissue properties. (3) There are significant stresses developed in the cross-fiber direction (more than 40 percent of those in the fiber direction) that cannot be attributed to either deformation effects or nonparallel muscle fibers. These results provide the foundation for future mechanical analyses of the heart.

Animals↗
PubMed2006

Constitutive and 3-methylcholanthrene-induced rat ALDH3A1 expression is mediated by multiple xenobiotic response elements.

The rat class 3 aldehyde dehydrogenase gene (ALDH3A1) is expressed constitutively or by xenobiotic induction depending on the tissue in which it occurs. Although the mechanism that mediates inducible expression has been well characterized, relatively little is known about constitutive regulatory mechanisms. Previous ALDH3A1 promoter analyses have indicated that primary regulatory regions within the ALDH3A1 5' flanking region exert similar effects on both constitutive and inducible ALDH3A1 expression. However, promoter gene analyses that served as the basis of early work were limited by the lack of sufficient 5' flanking region sequence. To gain a more complete picture of how the 5' flanking region regulates both modes of expression, we have subcloned an 8.0-kilobase (kb) fragment from the 5' flanking region of the ALDH3A1 gene and subjected it to reporter gene analyses. We found a region located between 4.8 and 7.8 kb upstream of the noncoding first exon that drives strong ALDH3A1 reporter activity. This region contains xenobiotic response element consensus sequences that mediate constitutive and inducible ALDH3A1 reporter gene expression. Using the new generation of ALDH3A1 reporter constructs, we were unable to confirm the presence of a negative regulatory region that was apparent in previous studies using a shorter fragment of the 5' flanking region. We also demonstrate that 3-methylcholanthrene induces ALDH3A1 expression above high constitutive background in corneal epithelial cells.

Aldehyde Dehydrogenase↗
PubMed2006

Point mutations in either subunit of the GABAB receptor confer constitutive activity to the heterodimer.

The GABA receptor (GABABR) is a class C G protein-coupled receptor (GPCR) that functions as an obligate heterodimer, composed of two heptahelical subunits, GABABR subunit 1 (R1) and GABABR subunit 2 (R2). In this study, we generated and pharmacologically characterized constitutively active GABABR mutants as novel tools to explore the molecular mechanisms underlying receptor function. A single amino acid substitution, T290K, in the R1 agonist binding domain results in ligand-independent signaling when this mutant subunit is coexpressed with wild-type R2. Introduction of a Y690V mutation in the putative G protein-coupling domain of R2 is sufficient to confer moderate constitutive activity when this subunit is expressed alone. Activity of the Y690V mutant can be markedly enhanced with coexpression of wild-type R1. Coexpression of both mutant subunits (R1-T290K and R2-Y690K) leads to a further increase in basal signaling. Potencies of the full agonists R-(+)-beta-(aminomethyl)-4-chlorobenzenepropanoic acid hydrochloride (baclofen) and GABA are increased at the constitutively active versus the corresponding wild-type receptors. The mutant GABABR variants provided a sensitive probe enabling detection of inverse or partial agonist activity of molecules previously considered neutral antagonists. Our studies using constitutively active isoforms provide independent support for a model of GABABR function that takes into account 1) ligand binding by R1, 2) signal transduction by R2, and 3) modulation of R2-induced function by R1. Furthermore, we demonstrate that certain hallmark features of constitutive activity as originally established with class A GPCRs (e.g., enhanced agonist potency and affinity), are more generally applicable, as suggested by our finding with a class C heterodimeric receptor.

Amino Acid Substitution↗
PubMed1999

Constitutively active alpha-1b adrenergic receptor mutants display different phosphorylation and internalization features.

We compared the phosphorylation and internalization properties of constitutively active alpha-1b adrenergic receptor (AR) mutants carrying mutations in two distant receptor domains, i.e., at A293 in the distal part of the third intracellular loop and at D142 of the DRY motif lying at the end of the third transmembrane domain. For the A293E and A293I mutants the levels of agonist-independent phosphorylation were 150% and 50% higher than those of the wild-type alpha-1b AR, respectively. On the other hand, for the constitutively active D142A and D142T mutants, the basal levels of phosphorylation were similar to those of the wild-type alpha-1b AR and did not appear to be further stimulated by epinephrine. Overexpression of the guanyl nucleotide binding regulatory protein-coupled receptor kinase GRK2 further increases the basal phosphorylation of the A293E mutant, but not that of D142A mutant. Both the wild-type alpha-1b AR and the A293E mutant could undergo beta-arrestin-mediated internalization. The epinephrine-induced internalization of the constitutively active A293E mutant was significantly higher than that of the wild-type alpha-1b AR. In contrast, the D142A mutant was impaired in its ability to interact with beta-arrestin and to undergo agonist-induced internalization. Interestingly, a double mutant A293E/D142A retained very high constitutive activity and regulatory properties of both the A293E and D142A receptors. These findings demonstrate that two constitutively activating mutations occurring in distant receptor domains of the alpha-1b AR have divergent effects on the regulatory properties of the receptor.

Adrenergic Agonists↗
PubMed2003

The interaction of a constitutively active arrestin with the arrestin-insensitive 5-HT(2A) receptor induces agonist-independent internalization.

5-HT(2A) serotonin receptors are unusual among G-protein coupled receptors in that they can be internalized and desensitized, in some cell types, in an arrestin-independent manner. The molecular basis of the arrestin-insensitivity of 5-HT(2A) receptors is unknown but is probably caused, in part, by the apparent lack of agonist-induced 5-HT(2A) receptor phosphorylation. Because the arrestin-insensitivity of 5-HT(2A) receptors is cell-type selective, we used a "constitutively active" arrestin mutant that can interact with agonist-activated but nonphosphorylated receptors. We show here that this "constitutively active" arrestin mutant (Arr2-R169E) can force 5-HT(2A) receptors to be regulated by arrestins. Cotransfection of 5-HT(2A) receptors with Arr2-R169E induced agonist-independent 5-HT(2A) receptor internalization, and a constitutive translocation of the Arr2-R169E mutant to the plasma membrane, whereas wild-type Arrestin-2 had no effect. Additionally, Arr2-R169E, unlike wild-type arrestin-2, induced a significant decrease in efficacy of agonist-induced phosphoinositide hydrolysis with an unexpected increase in agonist potency. Radioligand binding assays demonstrated that the fraction of receptors in the high-affinity agonist binding-state increased with expression of Arr2-R169E, indicating that Arr2-R169E stabilizes the agonist-high affinity state of the 5-HT(2A) receptor (R*). Intriguingly, the agonist-independent interaction of Arr2-R169E with 5-HT(2A) receptors was inhibited by inverse agonist treatment and is thus probably caused by the high level of 5-HT(2A) receptor constitutive activity. This is the first demonstration that a constitutively active arrestin mutant can both induce agonist-independent internalization and stabilize the agonist-high affinity state of an arrestin-insensitive G protein coupled receptor.

Amino Acid Substitution↗
PubMed1995

Colonization of Transgenic Tobacco Constitutively Expressing Pathogenesis-Related Proteins by the Vesicular-Arbuscular Mycorrhizal Fungus Glomus mosseae.

We studied the effect of constitutive expression of pathogenesis-related proteins (PRs) in tobacco plants on vesicular-arbuscular mycorrhiza. Tobacco lines genetically transformed to express various PRs constitutively under the control of the cauliflower mosaic virus 35S promoter of tobacco were examined. Immunoblot analysis and activity measurements demonstrated high levels of expression of the PRs in the root systems of the plants. Constitutive expression of the following acidic isoforms of tobacco PRs did not affect the time course or the final level of colonization by the vesicular-arbuscular mycorrhizal fungus Glomus mosseae: PR-1a, PR-3 (=PR-Q), PR-Q(prm1), PR-4, and PR-5. Similarly, constitutive expression of an acidic cucumber chitinase, of a basic tobacco chitinase with and without its vacuolar targeting peptide, of a basic (beta)-1,3-glucanase, and of combinations of PR-Q and PR-Q(prm1) or basic chitinase and basic (beta)-1,3-glucanase did not affect colonization by the mycorrhizal fungus. A delay of colonization by G. mosseae was observed in tobacco plants constitutively expressing the acidic isoform of tobacco PR-2, a protein with (beta)-1,3-glucanase activity.

Journal Article↗
PubMed1975

Isolation and characterization of D-serine deaminase constitutive mutants by utilization of D-serine as sole carbon or nitrogen source.

Mutants constitutive for D-serine deaminase (Dsdase) synthesis were isolated by utilizing D-serine as sole nitrogen or carbon source in the chemostat. This method generated only regulatory constitutive (dsdC) mutants. The altered dsdC gene product in these strains is apparently able to bind D-serine more efficiently than the wild-type dsdC+ gene product--a selective advantage. Constitutive synthesis of Dsdase in all of these dsdC mutants is extremely sensitive to catabolite repression, and catabolite repression is reversed by the addition of D-serine. Of the 15 mutants generated by this method, none are suppressible by supD, supE, or supF. Mutations to a low level of constitutivity (maximal specific activity of 9) occur much more frequently than mutations to a high level (maximal specific activity of 79). High level constitutive synthesis of Dsdase results from the synthesis of an altered dsdC gene product--not from loss of ability to form the dsdC product. Dsdase synthesis is not regulated by the nitrogen supply in the medium, as nitrogen starvation does not result in the derepression of Dsdase synthesis.

Chromosome Mapping↗
PubMed1976

Constitutive synthesis of enzymes of the protocatechuate pathway and of the beta-ketoadipate uptake system in mutant strains of Pseudomonas putida.

Mutant Pseudomonas putida strains that produce constitutive levels of the beta-ketoadipate uptake system are selected by the sequential transfer of cultures between mineral growth media supplemented with the noninducing growth substrate succinate and growth media containing beta-ketoadipate as the sole carbon and energy source. The mutant strains also produce constitutively three catabolic enzymes that give rise to beta-ketoadipate from the metabolic precursor beta-carboxy-cis, cis-muconate, and thus a single regulatory gene appears to govern the expression of the enzymes as well as the uptake system. The three enzymes that convert beta-carboxy-cis, cis-muconate to beta-ketoadipate are induced to higher levels when the orgainisms are grown with p-hydroxybenzoate (a compound that is catabolized via beta-ketoadipate); the beta-ketoadipate uptake system is partially repressed when the cells are grwon at the expense of p-hydroxybenzoate. The transferase that acts upon beta-ketoadipate remains inducible in the constitutive mutant strains. Thus a minimum of three biosynthetic controls must be exerted over the expression of the five genes. Since the regulatory mutation does not alter the expression of the gene for the transferase, the physiological target of the selection procedure appears to be mutant strains that produce the uptake system constitutively. Levels of the uptake system are higher in uninduced constitutive mutant cultures than in induced cultures of the wild type. Hence procedures analogous to the one we employed may be of general use in obtaining mutant strains that produce high levels of uptake systems.

Adipates↗
PubMed1986

Naturally occurring Staphylococcus epidermidis plasmid expressing constitutive macrolide-lincosamide-streptogramin B resistance contains a deleted attenuator.

A naturally occurring constitutive macrolide-lincosamide-streptogramin B (MLS) resistance plasmid, pNE131, from Staphylococcus epidermidis was chosen to study the molecular basis of constitutive expression. Restriction and functional maps of pNE131 are presented along with the nucleotide sequence of ermM, the gene which mediates constitutive MLS resistance. Sharing 98% sequence homology within the 870-base-pair Sau3A-TaqI fragment, ermM appears to be almost identical to ermC, the inducible MLS resistance determinant from S. aureus (pE194). The two genes share nearly identical sequences, except in the 5' promoter region of ermM. Constitutive expression of ermM is due to the deletion of 107 base pairs relative to ermC; the deletion removes critical sequences for attenuation, resulting in constitutive methylase expression.

Amino Acid Sequence↗
PubMed1987

Regulation of two nickel-requiring (inducible and constitutive) hydrogenases and their coupling to nitrogenase in Methylosinus trichosporium OB3b.

Two uptake hydrogenases were found in the obligate methanotroph Methylosinus trichosporium OB3b; one was constitutive, and a second was induced by H2. Both hydrogenases could be assayed by measuring methylene blue reduction anaerobically or by coupling their activity to nitrogenase acetylene reduction activity in vivo in an O2-dependent reaction. The H2 concentration for half-maximal activity of the inducible and constitutive hydrogenases in both assays was 0.01 and 0.5 bar (1 and 50 kPa), respectively, making it easy to distinguish these enzymes from one another both in vivo and in vitro. Hydrogen uptake was shown to be coupled to ATP synthesis in methane-starved cells. Methane, methanol, formate, succinate, and glucose all repressed the H2-mediated synthesis of the inducible hydrogenase. Furthermore, this enzyme was only expressed in N-starved cultures and was repressed by NH4+ and NO3-; synthesis of the constitutive hydrogenase was not affected by excess N in the growth medium. In nickel-free, EDTA-containing medium, the activities of these two enzymes were negligible; however, both enzyme activities appeared rapidly following the addition of nickel to the culture. Chloramphenicol, when added along with nickel, had no effect on the rapid appearance of either the constitutive or inducible activity, indicating that nickel is not required for synthesis of the hydrogenase apoproteins. These observations all suggest that these hydrogenases are nickel-containing enzymes. Finally, both hydrogenases were soluble and could be fractionated by 20% ammonium sulfate; the constitutive enzyme remained in the supernatant solution, while the inducible enzyme was precipitated under these conditions.

Adenosine Triphosphate↗
PubMed1997

Constitutive expression of fibronectin binding in Streptococcus pyogenes as a result of anaerobic activation of rofA.

Protein F is a fibronectin-binding surface protein of Streptococcus pyogenes (group A streptococcus) that mediates adherence to host cells. A gene product encoded by rofA activates transcription of the gene that encodes protein F (prtF) and was identified in a strain of S. pyogenes that expressed high levels of protein F under all conditions tested. Insertional inactivation of rofA in this strain results in a phenotype similar to that of other strains where high-level transcription of prtF occurs only in response to increased oxygen tension. In this study, we have compared the regulation of prtF and rofA in O2-regulated and constitutive strains in order to gain further insight into the function of rofA. Comparison of the prtF and rofA transcripts by S1 nuclease and primer extension assays indicated that the same promoters for each transcript are used in both O2-regulated and constitutive strains. However, analyses of rofA-lacZ reporter alleles revealed that a key difference between strains involves regulation of rofA itself. In O2-regulated strains, expression of rofA was elevated following culture under conditions of reduced O2 tension. However, a much more robust activation of rofA expression was observed when constitutive strains were grown under similar conditions. Exchange of reporter and rofA alleles between strains demonstrated that host genetic background, and not the sequence of the respective rofA allele or regulatory region, dictates the expression phenotype. Activation of rofA required RofA, and RofA was shown to bind specifically to DNA containing the promoters for rofA and prtF. Finally, overexpression of either allele of rofA caused constitutive expression of prtF regardless of host background. These data suggest a model where anaerobic expression of prtF in constitutive hosts is controlled at the level of transcription of rofA and implicate additional factors in this regulatory pathway.

Adhesins, Bacterial↗
PubMed1997

Escherichia coli cells expressing a mutant glyV (glycine tRNA) gene have a UVM-constitutive phenotype: implications for mechanisms underlying the mutA or mutC mutator effect.

Transfection of M13 single-stranded viral DNA bearing a 3,N4-ethenocytosine lesion into Escherichia coli cells pretreated with UV results in a significant elevation of mutagenesis at the lesion site compared to that observed in untreated cells. This response, termed UVM, for UV modulation of mutagenesis, is induced by a variety of DNA-damaging agents and is distinct from known cellular responses to DNA damage, including the SOS response. This report describes our observation, as a part of our investigation of the UVM phenomenon, that E. coli cells bearing a mutA or mutC allele display a UVM-constitutive phenotype. These mutator alleles were recently mapped (M. M. Slupska, C. Baikalov, R. Lloyd, and J. H. Miller, Proc. Natl. Acad. Sci. USA 93:4380-4385, 1996) to the glyV (mutA) and glyW (mutC) tRNA genes. Each mutant allele was shown to arise by an identical mutation in the anticodon sequence such that the mutant tRNAs could, in principle, mistranslate aspartate codons in mRNA as glycine at a low level. Because a UVM-constitutive phenotype resulting from a mutation in a tRNA gene was unexpected, we undertook a series of experiments designed to test whether the phenotype was indeed mediated by the expression of mutant glycine tRNAs. We placed either a wild-type or a mutant glyV gene under the control of a heterologous inducible promoter on a plasmid vector. E. coli cells expressing the mutant glyV gene displayed all three of the following phenotypes: (i) missense suppression of a test allele, (ii) a mutator phenotype measured by mutation to rifampin resistance, and (iii) a UVM-constitutive phenotype. These phenotypes were not associated with cells expressing the wild-type glyV gene or with cells in which the mutant allele was present but was not transcriptionally induced. These observations provide strong support for the idea that expression of mutant tRNA can confer a mutator phenotype, including the UVM-constitutive phenotype observed in mutA and mutC cells. However, our data imply that low-level mistranslation of the epsilon subunit of polymerase III probably does not account for the observed UVM-constitutive phenotype. Our results also indicate that mutA deltarecA double mutants display a normal UVM phenotype, suggesting that the mutA effect is recA dependent. The observations reported here raise a number of intriguing questions and raise the possibility that the UVM response is mediated through transient alteration of the replication environment.

Anticodon↗
PubMed2004

Isolation of SOS constitutive mutants of Escherichia coli.

The bacterial SOS regulon is strongly induced in response to DNA damage from exogenous agents such as UV radiation and nalidixic acid. However, certain mutants with defects in DNA replication, recombination, or repair exhibit a partially constitutive SOS response. These mutants presumably suffer frequent replication fork failure, or perhaps they have difficulty rescuing forks that failed due to endogenous sources of DNA damage. In an effort to understand more clearly the endogenous sources of DNA damage and the nature of replication fork failure and rescue, we undertook a systematic screen for Escherichia coli mutants that constitutively express the SOS regulon. We identified mutant strains with transposon insertions in 42 genes that caused increased expression from a dinD1::lacZ reporter construct. Most of these also displayed significant increases in basal levels of RecA protein, confirming an effect on the SOS system. As expected, this collection includes genes, such as lexA, dam, rep, xerCD, recG, and polA, which have previously been shown to cause an SOS constitutive phenotype when inactivated. The collection also includes 28 genes or open reading frames that were not previously identified as SOS constitutive, including dcd, ftsE, ftsX, purF, tdcE, and tynA. Further study of these SOS constitutive mutants should be useful in understanding the multiple causes of endogenous DNA damage. This study also provides a quantitative comparison of the extent of SOS expression caused by inactivation of many different genes in a common genetic background.

DNA Damage↗