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At least 613 records · Page 34Linked to original sources
PubMed1998

Induction of cyclooxygenase-2 by activated Ha-ras oncogene in Rat-1 fibroblasts and the role of mitogen-activated protein kinase pathway.

Elevated cyclooxygenase-2 (COX-2) expression and activity have been observed in several different transformed cell types that express mutated ras genes. To investigate the mechanism of increased COX-2 expression following Ras-mediated transformation, Rat-1:iRas cell line was transfected with an Ha-RasVal-12 cDNA expression vector that is under the transcriptional control of the lac operon and is inducible with isopropyl-1-thio-beta-D-galactopyranoside (IPTG). IPTG treatment caused parallel increases in the levels of Ha-Ras and COX-2 proteins in Rat-1:iRas cells. The increased expression of COX-2 was accompanied by increased prostaglandin E2 production. Selective inhibition of COX-2 activity suppressed the production of prostaglandin E2 by >90% but did not alter the progress of the morphological transformation. The level of COX-2 mRNA was up-regulated by activated Ha-Ras. Induction of Ras increased the transcription of COX-2 by 44.3 +/- 10.1% and increased the half-life of COX-2 mRNA by approximately 3.5-fold. A specific mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) inhibitor (PD 98059) caused a delay in both the activation of ERK1/2 and the induction of COX-2 in IPTG-induced Rat-1:iRas cells. Inhibition of ERK activity by PD 98059 also suppressed the induction of COX-2 by epidermal growth factor in intestinal epithelial cells and significantly reduced the expression of COX-2 in Ha-Ras-transformed rat intestinal epithelial cells. ERK activity appears to be required for induction of COX-2 by Ras.

Animals↗
PubMed1987

DNA bending induced by the catabolite activator protein allows ring formation of a 144 bp DNA.

The effect of the catabolite activator protein, CAP, on the ligation of a 144 bp DNA was examined. This DNA has EcoRI ends and contains the lac operon CAP site and promoter-operator region. At low DNA concentrations (nM) and 37 degrees C the presence of CAP and cAMP enables T4 ligase to convert the linear duplex to a covalently closed ring. Nuclease digestion and sedimentation equilibrium studies show that the ring is a monomer circle. Ring formation does not occur in the absence of either CAP or cAMP. The kinetics of ring closure, and the bimolecular joining of two fragments were measured. The presence of CAP decreased the rate of bimolecular joining of the EcoRI ends of linear DNAs. Thus the measured rates of ring closure are likely to be a lower limit for this process. Closure reactions carried out with ethidium bromide indicate that CAP induced bending rather than twisting is responsible for ring formation. The all or none nature of the closure reaction suggests that persistence length DNAs may be useful in a simple assay for protein induced DNA bending.

Cyclic AMP↗
PubMed1988

Turn of promotor DNA by cAMP receptor protein characterized by bead model simulation of rotational diffusion.

The rotation diffusion of DNA double helices and their complexes with the cAMP receptor protein (CRP) has been simulated by bead models, in order to derive information on their structure in solution by comparison with results obtained from dichroism decay measurements. Straight DNA double helices are simulated by linear, rigid strings of overlapping beads. The radius of the beads and the length of the string are increased simultaneously by the same increments from initial outer dimensions derived from crystallographic data to final values, which are fitted to experimental rotation time constants observed for short DNA fragments (less than 100 bp). The final values reflect the solvated structure with the same 'solvation layer' added in all three dimensions. The protein is simulated by overlapping beads, which are assembled to a structure very similar to that found by x-ray crystallography. Complexes of the protein with DNA are formed with the centres of palindromic DNA sites at the centre of the two helix-turn-helix-motifs of the protein with some overlap of the two components. Simulation of the experimental data obtained for CRP complexes with specific DNA in the presence of cAMP requires strong bending of the double helices. According to our simulation the DNA is almost completely wrapped around the protein both in the complexes with a 62 bp fragment containing the standard CRP site and with a 80 bp fragment containing the second binding site of the lac operon. Simulations of the data obtained for a 203 bp fragment with both binding sites suggest that the two bound CRP proteins are in contact with each other and that the DNA is wrapped around the two protein dimers. A stereochemical model is suggested with a tetrahedral arrangement of the four protein subunits, which provides the advantage that two binding sites of the protein formed by two subunits each are located favorable for tight contacts to two binding sites on bent DNA, provided that the DNA sites are separated by an integer number of helix turns. In summary, the simulations demonstrate strong bending, which can be reflected by an arc radius in the range around 50 A. According to these data the overall bending angle of our longest DNA fragment is approximately 180 degrees, and thus the protruding ends are sufficiently close to each other such that RNA polymerase, for example, could contact both helical segments.

DNA↗
PubMed2001

RADACK, a stochastic simulation of hydroxyl radical attack to DNA.

RADACK was conceived to simulate the radiation-induced attack to different DNA forms and complexes. It allows to separately calculate the probability of attack to each reactive atom of the sugar and of the base and takes into account the sequence-dependent structure of DNA as known from crystallographic or NMR studies or resulting from molecular modelling. The calculations are aimed to assess sequence-, structure- and ligand-dependent modulation of damages of sugar and bases, leading to single strand breaks (frank strand breaks, FSB) and alkali-labile base modifications (alkali-revealed breaks, ARB), respectively. The modelling procedure and the results of simulations for some representative structures (B, Z and quadruplex forms) are here described and discussed. The calculated relative probabilities of OH* radical attack to all reaction sites are compared to experimental FSB and ARB values. By a fitting procedure, the relative efficiencies of conversion of the C4' and C5'-centred radicals into FSB, epsilon (C4'): epsilon (C5'), and the relative efficiencies of base radicals- to- ARB conversion, epsilon(T) : epsilon(A) : epsilon(C) : epsilon(G), are then deduced for each DNA form. The ability of the model to account for the distribution of damages in DNA-ligand complexes is proven by its successful application to two DNA-protein systems : the lac repressor-lac operator complex and the nuclcosome core.

Bacterial Proteins↗
PubMed2001

Radiosensitivity of DNA in a specific protein-DNA complex: the lac repressor-lac operator complex.

PURPOSE: To calculate the probability of radiation-induced frank strand breakage (FSB) at each nucleotide in the Escherichia coli lac repressor-lac operator system using a simulation procedure. To compare calculated and experimental results. To asses the contribution of DNA conformational changes and of the masking by the protein to DNA protection by the repressor. MATERIALS AND METHODS: Two structures of the complex were extracted from the PDB databank: crystallography- and NMR-based structures. Calculations were made of the accessibility of the atoms mainly involved in strand breakage (H4' and H5') to O&Hdot; and of the FSB probabilities, along: (1) DNA in the complex; (2) DNA in the complex depleted of the repressor; and (3) a linear DNA having the same sequence. An 80bp fragment bearing the operator was irradiated alone or in presence of the repressor. The relative probabilities of FSB at each nucleotide were determined using sequencing gel electrophoresis. RESULTS: Calculations predict modulation of the accessibility of H4' and H5' atoms and of the probabilities of FSB along the DNA fragments of complexes. This is due to the protein-induced conformational change and to masking by bound protein. The best agreement with the experimental FSB was observed for calculations that use the crystallography-based structure. CONCLUSIONS: For specific DNA-protein complexes, our calculations can predict the protein radiolytic footprints on DNA. They show the significant contribution of the protein-induced DNA conformational change to DNA protection.

Bacterial Proteins↗
PubMed2001

Introduction of an RNA stability element at the 5'-end of an antisense RNA cassette increases the inhibition of target RNA translation.

This communication describes improvement strategies used on a previously described two-unit antisense RNA cassette system. This cassette system encodes RNA with noncontiguous regions of complementarity to a bacterial target RNA, lacI mRNA. One of the units of complementarity was contained within an RNA stem-loop resembling that of the very efficient, naturally occurring antisense RNA CopA. As relatively low inhibitory activity was obtained previously, we tested variants in which several stem-loops were combined within one RNA, each of them directed against a different stretch of target RNA. One to four stem-loop RNAs were tested and found to be relatively ineffective, likely because of low metabolic stability. To increase the intracellular stability of these and other antisense RNAs, a stabilizer element (stem-loop derived from gene 32 mRNA of phage T4) was inserted at their 5'-ends. The results indicate that addition of this element indeed increased antisense RNA efficiency in vivo. As expected, this effect was primarily due to a longer antisense RNA half-life, as shown by RNA abundance (Northern analysis) and decay rates (rifampicin runout experiments). In summary, the results reported indicate that rational design of antisense RNA is feasible, but that the degree of inhibition (approximately 75% maximum inhibition) accomplished here could still be improved.

Bacterial Proteins↗
PubMed1993

Mutational specificity of 2-nitro-3,4-dimethylimidazo[4,5-f]quinoline in the lacI gene of Escherichia coli.

2-Nitro-3,4-dimethylimidazo[4,5-f]quinoline (NO2-MeIQ) is the nitro derivative of 2-amino-3,4-dimethylimidazo[4,5-f]quinoline (MeIQ), one of the food pyrolysis product mutagens and carcinogens. The mutational specificity of NO2-MeIQ was determined using the lacI system in an Escherichia coli strain, EE125, harbouring the plasmid pKM101. NO2-MeIQ acts as a direct-acting mutagen towards this strain, and induces a broad range of mutational alterations. At higher doses; G:C-->T:A transversions dominate the mutational spectrum. Complex mutations were also observed, and the involvement of the SOS system in the processing of the mutations induced by NO2-MeIQ is suggested.

Bacterial Proteins↗
PubMed2001

Chromosomal DNA demethylation specified by protein binding.

In the present study, we utilize the well-characterized Escherichia coli lac repressor/operator system to demonstrate that protein binding can lead to demethylation at the binding sites in the chromosome. Similar to the findings using the episome, we found that the presence of LacI in the cells can lead to demethylation of methylated lacO in the chromosome and the LacI inhibitor, isopropyl-beta-D-thiogalactopyranoside (IPTG), can prevent demethylation of the methylated lacO. The lacO sites become progressively more demethylated over time with the presence of LacI, supporting the role of protein occupancy in demethylation targeting. These results validate our earlier conclusions using a stable episomal system, and establish for the first time that protein binding can specify sites of demethylation in the chromosome.

Bacterial Proteins↗
PubMed2002

Plasticity in protein-DNA recognition: lac repressor interacts with its natural operator 01 through alternative conformations of its DNA-binding domain.

The lac repressor-operator system is a model system for understanding protein-DNA interactions and allosteric mechanisms in gene regulation. Despite the wealth of biochemical data provided by extensive mutations of both repressor and operator, the specific recognition mechanism of the natural lac operators by lac repressor has remained elusive. Here we present the first high-resolution structure of a dimer of the DNA-binding domain of lac repressor bound to its natural operator 01. The global positioning of the dimer on the operator is dramatically asymmetric, which results in a different pattern of specific contacts between the two sites. Specific recognition is accomplished by a combination of elongation and twist by 48 degrees of the right lac subunit relative to the left one, significant rearrangement of many side chains as well as sequence-dependent deformability of the DNA. The set of recognition mechanisms involved in the lac repressor-operator system is unique among other protein-DNA complexes and presents a nice example of the adaptability that both proteins and DNA exhibit in the context of their mutual interaction.

Bacterial Proteins↗
PubMed1991

Spontaneous mutation in the Escherichia coli lacI gene.

To gain more detailed insight into the nature and mechanisms of spontaneous mutations, we undertook a DNA sequence analysis of a large collection of spontaneous mutations in the N-terminal region of the Escherichia coli lacI gene. This region of circa 210 base pairs is the target for dominant lacI mutations (i-d) and is suitable for studies of mutational specificity since it contains a relatively high density of detectable mutable sites. Among 414 independent i-d mutants, 70.8% were base substitutions, 17.2% deletions, 7.7% additions and 4.3% single-base frameshifts. The base substitutions were both transitions (60%) and transversions (40%), the largest single group being G.C----A.T (47% of base substitutions). All four transversions were observed. Among the 71 deletions, a hotspot (37 mutants) was present: an 87-bp deletion presumably directed by an 8-bp repeated sequence at its endpoints. The remaining 34 deletions were distributed among 29 different mutations, either flanked (13/34) or not flanked (21/34) by repeated sequences. The 32 additions comprised 29 different events, with only two containing a direct repeat at the endpoints. The single-base frameshifts were the loss of a single base from either repeated (67%) or nonrepeated (33%) bases. A comparison with the spectrum obtained previously in strains defective in DNA mismatch correction (mutH, mutL, mutS strains) yielded information about the apparent efficiency of mismatch repair. The overall effect was 260-fold but varied substantially among different classes of mutations. An interesting asymmetry was uncovered for the two types of transitions, A.T----G.C and G.C----A.T being reduced by mismatch repair 1340- and 190-fold, respectively. Explanations for this asymmetry and its possible implications for the origins of spontaneous mutations are discussed.

Bacterial Proteins↗
PubMed1993

The mutational specificity of two Escherichia coli dnaE antimutator alleles as determined from lacI mutation spectra.

In a companion study we have described the isolation of a series of mutants of Escherichia coli that replicate their DNA with increased fidelity. These mutants carry a mutation in the dnaE gene, encoding the alpha (polymerase) subunit of DNA polymerase III holoenzyme, which is responsible for the faithful replication of the bacterial chromosome. The mutants were detected as suppressors of the high mutability of a mutL strain (defective in postreplicative mismatch correction), in which mutations may be considered to arise predominantly from errors of DNA replication. To investigate the specificity of these antimutator effects, we have analyzed spectra of forward mutations in the N-terminal part of the lacI gene (i-d mutations) for two of the mutL dnaE derivatives (dnaE911 and dnaE915), as well as the control mutL strain. DNA sequencing of over 600 mutants revealed that in the mutL background both antimutator alleles reduce specifically transition mutations (A.T-->G.C and G.C-->A.T). However, the two alleles behave differently in this respect. dnaE911 reduces A.T-->G.C more strongly than it does G.C-->A.T, whereas the reverse is true for dnaE915. Second, dnaE911 does not appear to affect either transversion or frameshift mutations, whereas dnaE915 displays a distinct mutator effect for both. This mutator effect of dnaE915 for frameshift mutations was confirmed by the frequency of reversion of the trpE9777 frameshift mutation. The discovery that dnaE antimutator alleles possess distinct specificities supports the notion that DNA polymerases discriminate against errors along multiple pathways and that these pathways can be influenced independently.

Alleles↗
PubMed1997

A genetic strategy to demonstrate the occurrence of spontaneous mutations in nondividing cells within colonies of Escherichia coli.

A genetic strategy was designed to examine the occurrence of mutations in stationary-phase populations. In this strategy, a parental population of cells is able to survive under both permissive and restrictive conditions whereas mutants at a particular target locus exhibit a conditional-lethal phenotype. Thus, by growing the population to stationary phase under restrictive conditions and then shifting it to permissive conditions, mutations that had arisen in stationary phase can be studied without confounding effects caused by the occurrence of similar mutations during growth of the population. In two different applications of this strategy, we have studied the reversion to Lac+ in stationary phase of several Lac- mutations in Escherichia coli. Our results indicate that a variety of spontaneous point mutations and deletions, particularly those that are sensitive to the mechanisms of replication slippage (for their generation) and methyl-directed mismatch repair (for their correction), can arise in nondividing populations of cells within a colony. The frequency of their occurrence was also elevated in mutS strains, which are defective in such mismatch repair. These data have relevance to the ongoing debate on adaptive or directed mutations in bacteria.

Bacterial Proteins↗
PubMed1972

Mutator gene studies in Escherichia coli: the mutS gene.

We report here on a study of a mutator gene (mutS) that causes transition mutations in Escherichia coli. We have used the trpA system to show that A:T-->G:C and G:C-->A:T transitions occur. Not all A:T pairs are equally susceptible to mutS action however, since the A:T pair at the trpA223 site reverts at a frequency similar to, if not identical with, the frequency in a mut(+) background. Presumably this is a consequence of neighboring bases, because other A:T pairs are reverted by mutS in the same gene; and an A:T pair in the lac operon is reverted at two widely separated points on the chromosome, and in two orientations relative to the trp sense strand. In addition, we have shown that the mutS1 allele is recessive to wild type, and trans active.

Alleles↗
PubMed1996

Aflatoxin B1 induced lacI mutation in liver and kidney of transgenic mice C57BL/6N: effect of phorone.

Transgenic C57BL/6N mice containing a lambda shuttle vector carrying a lacI target and an alpha-lacZ reporter gene have been used to study the modulating effect of phorone, a glutathione-depleting agent, on the mutagenic activity of aflatoxin B1 (AFB1) in vivo. Animals were treated with AFB1 (8 mg/kg) for four consecutive days and the animals sacrificed 21 days after the last treatment. Treatment with AFB1 alone did not result in a significant increase in mutation frequency in the liver and kidney. When the animals were treated with phorone 4 h prior to treatment with AFB1 a significant increase in mutation frequency was observed in the liver (4-fold) and kidney (1.5-fold). Phorone treatment did not increase the AFB1-induced mutation frequency in the lung and intestine. DNA sequence analyses of 30 independent clones isolated from the liver of AFB1-treated animals showed that G:C --> T:A transversion (60%) was the predominant mutational event. Mutations within the lacI gene could not be detected in seven of 30 mutants. The mutations were randomly distributed throughout the coding sequences of the lacI gene and no hotspots for the mutations were observed. However, codons 86 and 928 appeared to be major sites for mutation. The study shows that the transgenic mouse in vivo mutagenesis model can be used to study the influence of effect-modifying compounds on the mutagenic activity of known carcinogens.

Aflatoxin B1↗
PubMed1998

Spontaneous and ENU-induced mutation spectra at the cII locus in Big Blue Rat2 embryonic fibroblasts.

Big Blue Rat2 embryonic fibroblasts carry the lambda-Liz shuttle vector which is also present in the Big Blue mouse and rat. Mutations in the Big Blue systems have most often been measured at the lacI locus. However, a method for positive selection of mutations at the lambda cII locus was recently described. This assay appears to have many advantages over the use of lacI as a mutational target, but it has yet to be well characterized in mammalian mutagenesis studies. The objective of these studies was to determine the spontaneous and ethylnitrosourea (ENU)-induced mutant frequencies (MFs) and mutational spectra at cII using Big Blue Rat2 embryonic fibroblasts. The average spontaneous MF was 13 +/- 1.4 x 10(-5). The average induced MF was 60 +/- 10 x 10(-5) 10 days following a 30 min treatment with 0.1 mg/ml ENU. Eighty four independent spontaneous mutants were sequenced: 23 (27.4%) were frameshift mutations and 61 (72.6%) were base substitutions. Two spontaneous frameshift hotspots were detected, both in mononucleotide runs. G:C-->A:T transitions were the most common type of base substitution in cII; of these 71% occurred at CpG sites. The ENU-induced mutational spectrum at cII (44 mutants) consisted of 42 base substitutions (95.5%) and two -1 frameshift mutations (4.5%). Compared with the spontaneous spectrum, the ENU-induced spectrum had significantly fewer frameshift mutations (4.5 versus 27%) and base substitutions occurred predominantly at A:T base pairs (71 versus 34%). Overall, the spontaneous cII mutational spectrum reported here differs slightly from spontaneous spectra reported at the Big Blue lacI locus, but the mutational spectra and base substitution MFs following treatment with ENU were comparable at both loci. These data support the continued use of cII as a selectable marker in mutagenesis studies involving cells or tissues that carry a lambda transgene.

Amino Acid Sequence↗
PubMed1998

Role of DNA repair by (A)BC excinuclease and Ogt alkyltransferase in the final distribution of LacI-d mutations induced by N-butyl-N-nitrosourea in Escherichia coli.

In the absence of nucleotide excision repair, the additional deficiency of the DNA alkyltransferase (ATase) encoded by the constitutive ogt gene of Escherichia coli caused a marked increase in mutation induction by N-butyl-N-nitrosourea (BNU). Irrespective of the presence or absence of the Ogt ATase, little mutagenic response was detected in Uvr+ bacteria in the concentration range 0-8 mM BNU, indicating that most premutagenic DNA lesions induced at these concentrations are efficiently recognized and repaired by the nucleotide excision repair system. Increased susceptibility to mutagenesis by BNU was detected in Uvr- Ogt+ bacteria, but the Uvr- Ogt- double mutant exhibited much higher sensitivity. These data suggest that the Ogt ATase can replace to a great extent the repair capacity of the (A)BC excinuclease. Forward mutations induced by 6 mM BNU within the initial part of the lacI gene of E.coli were recovered from Uvr+ Ogt-, Uvr- Ogt+ and Uvr- Ogt- bacteria. A total of 454 independent mutations were characterized by DNA sequence analysis. The BNU-induced spectra were dominated by G:C-->A:T transitions, consistent with the major role of the O6-alkylguanine miscoding lesion in mutagenesis by alkylating agents. Specific sites for G:C-->A:T transitions were recovered more or less frequently in one genetic background versus the others, giving statistically significant differences among the spectra (P < 10(-6)). We examined the influence of DNA repair by (A)BC excinuclease and Ogt ATase on the 5'-flanking base associated with the BNU-induced G:C-->A:T transitions; preferences different from those previously reported for other alkylnitrosoureas were detected. We discuss how these differences might be caused by BNU producing branched chain derivatives, in addition to the expected linear chain adducts, and by possible preferences with respect to both the initial distribution of O6-butylguanine lesions and their repairability.

Bacterial Proteins↗
PubMed1999

Hydrogen peroxide and coffee induce G:C-->T:A transversions in the lacI gene of catalase-defective Escherichia coli.

The mutagenicity of hydrogen peroxide (H2O2) was compared with that of coffee, a complex mixture which generates H2O2. An Escherichia coli strain defective in catalase activity (katG katE double mutant) and carrying a single copy mucAB (pRW144) plasmid was constructed to enhance the mutagenic response to oxidants. The ability of the mucAB genes to influence the type, frequency and distribution of H2O2-induced mutations was also investigated in isogenic bacteria lacking pRW144. Induced mutational spectra were characterized and compared with that of spontaneous mutagenesis. A total of 444 independent forward mutations affecting the first 210 bp of the lacI gene were identified by DNA sequence analysis. The spontaneous mutation spectrum showed no bias (P = 0.52) for substitutions at G:C base pairs. In contrast, in the H2O2-induced spectrum substitutions occurred preferentially at G:C base pairs (P < 0.0001) with a preponderance of G:C-->T:A transversions (43.4% of H2O2-induced mutants versus 17.3% of spontaneous mutants). These data support the view that 7,8-dihydro-8-oxoguanine is the main premutagenic lesion induced by H2O2 and that catalase-defective bacteria have elevated levels of 8-oxoguanine in chromosome DNA after H2O2 exposure. Coffee produced a similar distribution of mutational events as H2O2 (P > 0.05), suggesting that this compound may be the main cause of the coffee-induced mutagenesis. The presence of plasmid pRW144 did not affect the frequency of H2O2-induced G:C-->T:A transversions, but caused an increase in A:T-->T:A transversions and a decrease in -1 base frameshifts. Although the frequencies of G:C-->T:A transversions were similar in all three induced spectra (H2O2 and coffee +/- pRW144), differences were observed in location of mutations throughout the target gene.

Acatalasia↗
PubMed1984

Targeted mutagenesis in vitro: lac repressor mutations generated using AMV reverse transcriptase and dBrUTP.

We have cloned the gene for the lac operon repressor (lacI) of Escherichia coli into the M13 related phage f1. Mutagenesis of the lacI gene was performed in vitro by filling dsDNA molecules gapped over the lacI gene with Avian Myeloblastosis Virus (AMV) reverse transcriptase. LacI mutants are found at a frequency of 1 in 10(4) using a genetic screen in vivo. For two-thirds of the 60 mutants, lesions were identified within the first 400 bases of lacI, by dideoxy sequencing. An unexpectedly wide range of different lesions were observed, including transitions, transversions, and deletions (of which the most common were the removal of single base pairs). The replacement of dTTP by dBrUTP in the filling reaction resulted in a doubling of deletions in the sample population as well as the anticipated T to C and C to T transitions. Although the lacI gene has been extensively studied in vivo, the power of this technique for mutagenesis in vitro is demonstrated by the generation of three previously undescribed lacI mutations.

Amino Acid Sequence↗