Biomedical subjects

A Quiñones

Publications and source records attributed to A Quiñones.

At least 19 recordsLinked to original sources

PubMed2001

Identification of genotoxic stress in human cells by fluorescent monitoring of p53 expression.

The tumor suppressor protein p53 is induced upon DNA damage essentially by post-translational regulatory mechanisms, which lead to a substantial increase of p53 levels. To exploit this essential property of p53, we developed a novel reporter system for monitoring accumulation and subcellular translocation of p53 protein, which is able to function as a simple test for detecting mutagenic and genotoxic stress in human cells. For this purpose, we constructed a plasmid with a specific translational TP53::EGFP gene fusion and selected stable transfected clones in the human cell line HEK293, in which p53 is functionally stabilized due to the expression of the transgenic adenoviral E1A oncoproteins. HEK293-TP53::EGFP clones may be used as a living cell system for monitoring not only of the induction of p53 protein in the cell, but also of its subcellular localization. Using this human reporter cell system, we examined levels of p53 by fluorescence microscopy and by FACS analysis following treatment with several classes of genotoxic and carcinogenic compounds. All tested DNA damaging agents caused a significant increase of intracellular p53-EGFP levels in a concentration-dependent manner. On the other hand, non-genotoxic carcinogens and stress conditions that cannot damage DNA were not able to induce p53-EGFP accumulation. The induction effect caused by genotoxic stress was found to be dependent on the endogenous p53 status, because it was not observed in p53-deficient cell lines. This corroborates the notion that p53 may be used as an universal sensor for genotoxic stress and demonstrates the usefulness of HEK293-p53-EGFP cells as a reporter system for identification of mutagens and genotoxic carcinogens in human cells by means of visualizing and monitoring intracellular p53 levels and localization.

4-Nitroquinoline-1-oxide↗

PubMed1997

Treatment with DNA-damaging agents increases expression of polA'-'lacZ gene fusions in Escherichia coli K-12.

The polA gene of Escherichia coli encodes the DNA polymerase I that is involved in DNA replication and repair. In contrast to the extensive body of data on the structure and function of polymerase I, there is little information available concerning the mechanisms that govern polA expression. Here, we studied the expression of the polA gene using translational fusions to lacZ. We found that treatment with the DNA-damaging agents 4-nitroquinoline-N-oxide (4-NQO), UV light mitomycin C (MC) and methyl methanesulfonate (MMS) leads to enhanced expression of polA'-'lacZ fusions. The increase in expression of polA reflects stimulation of transcription from a single promoter, as determined by S1 nuclease analyses. This was not observed in mutants that are blocked in induction of the SOS regulon. However, mutants with defective excision repair were more susceptible to polA stimulation. These results support the hypothesis that increased polA expression may be important for the ability to repair bulky DNA adducts that interfere with replication.

4-Nitroquinoline-1-oxide↗

PubMed1997

Discoordinate gene expression of gyrA and gyrB in response to DNA gyrase inhibition in Escherichia coli.

The intracellular level of DNA supercoiling is regulated in Escherichia coli by a homeostatic control mechanism that includes DNA gyrase and topoisomerase I gene expression. Despite several biochemical and genetical evidence that supports the existence of a homeostatic regulation mechanism, there are only few studies focusing gyrA and gyrB gene expression in connection to the mechanism involved in the regulation of DNA supercoiling in vivo. To study DNA gyrase gene expression and to be able to isolate mutants with altered expression of DNA gyrase, we constructed a new chromosomal reporter system based on two translational fusions of gyrA and gyrB to lacZ Using this stable monitor system in a robust wild type, we simultaneously studied the influence of several inhibitors of DNA gyrase (quinolones and coumarins) on gyrA and gyrB gene expression as well as on the intracellular level of DNA supercoiling. Surprisingly, we found a delayed and differential response of gyrA and gyrB gene expression following inhibition of DNA gyrase by quinolones or coumarins. Whereas both groups of drugs were able to increase the expression of gyrA, the gyrB gene expression was only induced by the coumarins. Although the action of the quinolones was able to alter DNA supercoiling, we never observed any induction of gyrB from the chromosome. These results revealed that the gene expressio of gyrA appears to be more sensitive to alterations in DNA supercoiling than the gyrB gene expression and suggest that probably additional regulatory mechanisms on the post-translational level might be involved in the regulation of DNA supercoiling and DNA gyrase gene expression.

Aminocoumarins↗

PubMed1997

DnaA protein stimulates polA gene expression in Escherichia coli.

The polA gene of Escherichia coli encodes DNA polymerase I that is involved in DNA replication and repair. Despite the wide knowledge about structure and function of DNA polymerase I, there is little insight into the regulatory mechanisms involved in polA expression. DnaA is the initiator protein for DNA replication in E. coli. There are two putative DnaA-binding sites within the extended promoter region of polA. In this work we studied the influence of altered levels of DnaA protein on polA expression. We found that DnaA overproduction increases polA expression in stationary-phase cultures. The stimulation effect was independent of rpoS, which encodes the sigma factor for stationary-phase-inducible genes. However, it was modulated by ppGpp. Comparative S1 analyses revealed that the induction was based on transcriptional stimulation. Footprinting experiments demonstrated that DnaA binds only to the proximal DnaA box near the polA promoter. These results suggest an additional role for DnaA as transcriptional activator of polA at least under certain physiological conditions.

Bacterial Proteins↗

PubMed1997

The ssb-113 allele suppresses the dnaQ49 mutator and alters DNA supercoiling in Escherichia coli.

Mutations in the dnaQ gene, which encodes the proofreading epsilon-subunit of the DNA polymerase III holoenzyme, lead to a mutator phenotype caused by enhanced error rates during DNA replication. In this paper, we studied the influence of ssb mutations on the dnaQ49 mutator, because of the involvement of SSB protein in DNA replication. We found that the ssb-113 mutation suppresses the mutator phenotype of dnaQ49. The suppression effect resulted from an enhanced expression of the dnaQ49 allele as determined by experiments with gene fusions. S1 nuclease analysis revealed that the increased dnaQ expression is based on transcriptional activation of the dnaQP2 promoter. This seems to be the consequence of an increased DNA supercoiling in the ssb-113 mutant, which also influenced further functions that are sensitive to alterations in DNA supercoiling. These results support the hypothesis that the expression of the epsilon-subunit of DNA polymerase III may additionally be modulated by DNA supercoiling, and suggest a possible role for DNA topology in mutagenesis.

Alleles↗

PubMed1996

The antitumor agent cisplatin inhibits DNA gyrase and preferentially induces gyrB gene expression in Escherichia coli.

Cisplatin is a widely used anticancer agent that exerts its biological activity principally by damaging DNA. Although detailed knowledge exists concerning mechanisms that lead to cisplatin adducts in DNA, there are few insights into the processes that result in its antitumor action. To explore some of the cellular responses elicited by cisplatin treatment, we studied its influence on DNA supercoiling and DNA gyrase gene expression in E. coli. We found that cisplatin inhibits DNA gyrase in a concentration-dependent manner leading to a transient alteration of DNA supercoiling and to an induction of gyrase gene expression. The induction effect was asymmetrical, affecting gyrB stronger than gyrA. Furthermore, we studied the influence of cisplatin on the supercoiling activity of purified DNA gyrase in vitro and found that cisplatin was an efficient inhibitor of DNA gyrase in the standard assay. However, cisplatin was an excellent inhibitor when added to DNA gyrase before it could interact with its substrate. In this assay GyrB was also more affected by cisplatin than GyrA. This strongly suggests that cisplatin inhibits DNA gyrase primarily by direct interaction with the enzyme. The data from this work present evidence that further cellular responses following cisplatin treatment include DNA gyrase inhibition, altered DNA supercolling and enhanced DNA gyrase gene expression. This suggests an important role of DNA topology in the induction of defense mechanisms against the action of cisplatin in addition to the processes related to DNA damage and repair.

Antibodies↗

PubMed1995

Expression of the dnaB gene of Escherichia coli is inducible by replication-blocking DNA damage in a recA-independent manner.

The replicative DNA helicase encoded by the dnaB gene is essential for chromosomal DNA replication in Escherichia coli. The DnaB protein is a component of the phi X-type primosome which is regarded as a model system for lagging strand synthesis of the chromosome. Using translational lacZ fusions at the plasmid and chromosomal levels, we studied the influence of DNA-damaging agents on dnaB gene expression. We found that DNA damage caused by mitomycin C, methyl methanesulphonate, 4-nitro-quinoline N-oxide, and UV irradiation led to a moderate, but significant induction of dnaB gene expression. Comparative S1 analysis of transcripts in untreated and induced cells demonstrated that the induction is due to increased transcription from the dnaB promoter. In contrast to other DNA damage-inducible replication genes, such as dnaA, dnaN, dnaQ, and polA, expression of which is not inducible in recA and lexA mutants, the induction of dnaB was also observed in a recA1 mutant. These results show that the induction of dnaB gene expression by replication-blocking DNA damage is due to a mechanism other than the indirectly SOS-dependent induction of the other DNA replication genes. Moreover, the data suggest that replication proteins are involved in recovery from replication-blocking DNA damage in two different ways--on the one hand at the level of initiation and on the other hand at the level of elongation.

Bacterial Proteins↗

PubMed1991

Expression of the dnaA gene of Escherichia coli is inducible by DNA damage.

The DnaA protein is the key DNA initiation protein in Escherichia coli. Using transcriptional and translational fusions, comparative S1 nuclease mapping and immunoblot analysis, the regulation of dnaA in relation to inducible responses to DNA damage was studied. We found that DNA damage caused by mitomycin C (MC) and methyl methanesulfonate (MMS) led to a significant induction of the dnaA gene. These results strongly suggest that in response to DNA damage which inhibits DNA replication, an increased initiation capacity is induced at oriC and that, in addition to the known auto-repression, a new regulatory mechanism may be involved in the control of dnaA gene expression. Furthermore, this mechanism might be indirectly related to the SOS regulon, because lexA and recA mutants, which block the induction of the SOS response, prevent dnaA induction by MMS and MC.

Bacterial Proteins↗

PubMed1991

DNA lesions that block DNA replication are responsible for the dnaA induction caused by DNA damage.

The initiation protein DnaA of Escherichia coli regulates its own expression autogenously by binding to a 9 bp consensus sequence, the dnaA box, between the promoters dnaAP1 and dnaAP2. In this study, we analysed dnaA regulation in relation to DNA damage and found dnaA expression to be inducible by DNA lesions that inhibit DNA replication. On the other hand, coding DNA lesions were not able to induce dnaA expression. These results suggest that an additional regulatory mechanism is involved in dnaA gene expression and that DnaA protein may play a role in cellular responses to DNA damage. Furthermore, they strongly suggest that in response to DNA replication inhibition by DNA damage, and enhanced (re)initiation capacity is induced by oriC.

Bacterial Proteins↗

PubMed1991

New perspectives for perfusion imaging in echocardiography.

The rapid injection into the coronary circulation of solutions containing microbubbles produces an ultrasonic contrast effect in the myocardium. The time-intensity curves generated by sequential videodensitometric analysis of contrast intensity, which is corrected for myocardial background intensity, resembles the curves used in indicator dilution techniques. Studies done in vitro have demonstrated a direct relation between contrast intensity and amount of microbubbles. In animal studies, measurements derived from these curves correlated with changes in myocardial blood flow assessed by microspheres but with varying controversial results. Differences between investigators in technique of injection and size of microbubbles injected are among the list of factors that will alter the time-intensity curve and explain some of the differences in results between investigators. Several factors limit the application of myocardial contrast echocardiography (MCE) to the quantitation of myocardial blood flow. Nevertheless, the results from multiple investigations suggest that the technique is sensitive to changes in perfusion and may be applicable to the evaluation of regional coronary reserve and assessment of the results of revascularization procedures. Peak intensity and area under the time-intensity curve have provided consistent results between investigators; therefore, these measurements have been used to assess regional coronary reserve in experimental studies and patients with coronary artery disease. Although the results are encouraging, they cannot distinguish well between normal and mild impairments in coronary reserve and are subject to larger reproducibility errors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Regulation of the dnaQ gene of Escherichia coli in mutants expressing the SOS regulon constitutively.

Current models for the mechanism of SOS mutagenesis in E. coli propose the involvement of a new or modified DNA polymerase III holoenzyme in error-prone replicative bypass of bulky DNA lesions assuming an inhibited or excluded 3'----5' proofreading exonuclease function of DNA polymerase. By promotor fusion to galK, gene fusion to lacZ and Sl analysis the in vivo regulation of dnaQ coding the proofreading subunit of DNA polymerase III holoenzyme was analyzed under conditions of induced or constitutive SOS expression. The results presented here clearly show that, at least on the level of gene expression no regulatory event seems to contribute to the assumed decrease of proofreading activity during SOS mediated error-prone bypassing of bulky lesions. On the contrary, an increase in dnaQ gene expression was observed following treatment with some SOS inducing agents which produces bulky DNA lesions.

DNA Polymerase III↗

PubMed1989

Induction of dnaN and dnaQ gene expression in Escherichia coli by alkylation damage to DNA.

The dnaN and dnaQ genes encode the beta-subunit and the epsilon-subunit of the DNA polymerase III holoenzyme. By transcriptional fusions to the galK gene, translational fusions to lacZ and comparative S1 mapping analysis, we investigated the in-vivo regulation of dnaN and dnaQ. We found that DNA damage caused by the alkylating agent methyl methanesulphonate (MMS) leads to a significant induction in dnaN and dnaQ gene expression suggesting a requirement of increased amounts of at least some DNA polymerase III holoenzyme subunits for recovery from DNA damage caused by MMS. These results are first evidences that subunits of the DNA polymerase III holoenzyme are DNA damage inducible. This MMS induction of dnaN and dnaQ gene expression is unrelated to the adaptive response. It was not observed in lexA and recA mutants which abolish the induction of the SOS response.

Cloning, Molecular↗

PubMed1989

Expression of the dnaN and dnaQ genes of Escherichia coli is inducible by mitomycin C.

The dnaN and dnaQ genes encode the beta subunit and the epsilon subunit of the DNA polymerase III holoenzyme. Using translational fusions to lacZ we found that DNA damage caused by mitomycin C induces expression of the dnaA and dnaQ genes. This induction was not observed in lexA and recA mutants which block the induction of the SOS response, suggesting a relationship between the mechanism(s) of genetic control of DNA polymerase III holoenzyme and the SOS regulatory network. Nevertheless, there is evidence that the mitomycin C induction of dnaN and dnaQ is not a simple lexA-regulated process, because nalidixic acid (an excellent SOS inducer) does not increase dnaN and dnaQ gene expression, and the time course of induction is abnormally slow.

DNA Damage↗

PubMed1988

Discoordinate gene expression in the dnaA-dnaN operon of Escherichia coli.

The dnaN gene of Escherichia coli encodes the beta-subunit of the DNA polymerase III holoenzyme. Previous work has established that dnaN lies immediately downstream of dnaA and that both genes may be cotranscribed from the dnaA promoters; no promoter for dnaN has been described. We investigated the in vivo regulation of transcription of the dnaN gene by transcriptional fusions to the galK gene, translational fusion to the lacZ gene and S1 mapping analysis. We found that there are at least three dnaN promoters residing entirely in the reading frame of the preceding dnaA gene, and that transcription from these promoters can occur independently of dnaA transcription which, however, extends at least up to dnaN. Furthermore, we found evidence for the inducibility of the dnaN promoters in a dam background under conditions of simultaneously reduced dnaA transcription. These results are consistent with the hypothesis that although dnaA and dnaN are organized in an operon considerable discoordinate transcription can occur, thus uncoupling dnaN and dnaA regulation, when needed.

Bacterial Proteins↗

PubMed1988

Expression of the Escherichia coli dnaQ (mutD) gene is inducible.

By promoter fusion to the galK gene and comparative S1 analysis we investigated the in vivo regulation of transcription of the dnaQ gene which encodes the epsilon-subunit of the DNA polymerase III holoenzyme carrying the 3'----5' exonucleolytic proofreading function. Induction of a mutagenic stress situation by treatment with the base analogue 2-aminopurine (2-AP) leads to an increase in dnaQ transcription. S1 mapping analysis of the two dnaQ transcripts revealed a differential promoter activation for this 2-AP induced increase in dnaQ transcription. In addition, a similar galK promoter fusion with the dnaN gene coding for the beta-subunit of the DNA polymerase III holoenzyme revealed that dnaN transcription is also 2-AP inducible as judged by galactokinase activity. This is the first evidence for the inducibility of dnaQ gene expression (and possibly of other genes of the DNA polymerase II holoenzyme) and is discussed in relation to DNA repair mechanisms.

2-Aminopurine↗

PubMed1987

Differential suppressor effects of the ssb-1 and ssb-113 alleles on uvrD mutator of Escherichia coli in DNA repair and mutagenesis.

We have constructed double mutants carrying either ssb-1 or ssb-113 alleles, which encode temperature-sensitive single strand DNA binding proteins (SSB), and the uvrD::Tn5 allele causing deficiency in DNA helicase II, and have examined sensitivity to ultraviolet light (UV), recombination and spontaneous as well as UV-induced mutagenesis. We have found in a recA+ background that (i) none of the ssb uvrD double mutants was more sensitive to UV than either single mutant; (ii) the ssb-1 allele partially suppressed the strong UV sensitivity of uvrD::Tn5 mutants; (iii) in the recA730 background with constitutive SOS expression, the ssb-1 and ssb-113 alleles suppressed the strong UV-sensitivity caused by the uvrD::Tn5 mutation; (iv) in ssb-113 mutants, the level of recombination was reduced only 10-fold but 100-fold in ssb-1 mutants, showing that there was no correlation between the DNA repair deficiency and the recombination deficiency; (v) the hyper-recombination phenotype of the uvrD::Tn5 mutant was suppressed by the addition of either the ssb-1 or the ssb-113 allele; (vi) no addition of the spontaneous mutator effects promoted by the uvrD::Tn5 and the ssb-113 alleles was observed. These results suggest a possible functional interaction between SSB and Helicase II in DNA repair and mutagenesis.

Adenosine Triphosphatases↗

PubMed1987

Reduced transcription of the rnh gene in Escherichia coli mutants expressing the SOS regulon constitutively.

We have analysed the transcription levels for the convergently overlapping Escherichia coli genes for the DNA polymerase III proofreading function (dnaQ) and ribonuclease H (rnh). The two tandem dnaQ promoters are about three times more active than the single rnh promoter as shown by analysing the level of in vivo transcription using dnaQ-galK and rnh-galK fusions. In E. coli mutants constitutively expressing the pleiotropic SOS response, which includes activities that enhance DNA repair, recombination and mutagenesis, a strong reduction in rnh transcription was observed. The lexA51 recA441 double mutant which fully expresses the SOS response shows the strongest reduction in rnh transcription and the highest increase in dnaQ transcription. Nuclease S1 mapping supported the finding that a constitutive expression of SOS function leads to a strong reduction in rnh transcription.

DNA Polymerase III↗

PubMed1986

Mutational specificity of a proof-reading defective Escherichia coli dnaQ49 mutator.

The dnaQ (mutD) gene product which encodes the epsilon-subunit of the DNA polymerase III holoenzyme has a central role in controlling the fidelity of DNA replication because both mutD5 and dnaQ49 mutations severely decrease the 3'-5' exonucleolytic editing capacity. It is shown in this paper that more than 95% of all dnaQ49-induced base pair substitutions are transversions of the types G:C-T:A and A:T-T:A. Not only is this unusual mutational specificity precisely that observed recently for a number of potent carcinogens such as benzo(a) pyrene diolepoxide (BPDE) and aflatoxin B1 (AFB1), which are dependent on the SOS system to mutagenize bacteria, but it is also seen for the constitutively expressed SOS mutator activity in E. coli tif-1 strains as well as for the SOS mutator activity mediated gap filling of apurinic sites. Because the G:C-T:A and A:T-T:A transversions can either result from the insertion of an adenine across from apurinic sites or arise due to the incorporation of syn-adenine opposite a purine base, we postulate that the DNA polymerase III holoenzyme also has a reduced discrimination ability in a dnaQ49 background. The introduction of a lexA (Ind-) allele, which prevents the expression of SOS functions, led to a significant reduction in the dnaQ49-caused mutator effect. Both, the mutational specificity observed and the partial lexA+ dependence of the mutator effect provoke a reanalysis of the hypothesis that the DNA polymerase III holoenzyme can be converted into the postulated but until now unidentified SOS polymerase.

Anticodon↗