Different photodynamic action of proflavine and methylene blue on bacteriophage. II. Mutation induction in extracellularly treated Serratiaphage kappa.
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Bacteriophages↗
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Studies on the mutagenic action of formaldehyde in Drosophila. II. The production of mutations in females and the induction of crossing-over.
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Animals↗
A comparative study of the action of ethyl methane sulfonate and HNO2 on the mutation to streptomycin resistance of Escherichia coli I 12.
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Alkylation↗
The mechanism of the mutagenic action of hydroxylamine. XIV. O-methylhydroxylamine-induced direct mutations in vegetative Sd phage.
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Coliphages↗
Quinolone action in Escherichia coli cells carrying gyrA and gyrB mutations.
We have isolated spontaneous mutant strains of Escherichia coli KL16 showing different levels of nalidixic acid (NAL) resistance. From 40 independent mutants, 36 had gyrA and four had gyrB mutations. Most of the gyrA mutations (30/36) conferred high level NAL resistance. In contrast, the only gyrB mutation that conferred a relatively high level of NAL resistance also determined enhanced susceptibility to quinolones with a piperazinyl substituent at C7 position of the quinolone ring (amphoteric quinolones). This gyrB mutation (denoted gyrB1604), jointly with a gyrA mutation (denoted gyrA972) which confers a high level of quinolone resistance, were used to construct strain IC2476, carrying the two gyr mutant alleles. The susceptibility of this strain to amphoteric quinolones (pipemidic acid, norfloxacin and ciprofloxacin) was similar to that of the gyrA972 single mutant. This result indicates that the change in GyrA subunit which determines a high level of quinolone-resistance has the capacity to mask the hypersusceptibility to amphoteric quinolones promoted by the GyrB1604 mutant subunit. This capacity was further confirmed by studying the effects of ciprofloxacin (CFX) on gyrase inhibition in the gyrA972 gyrB1604 strain.
Anti-Infective Agents↗
Action of BTN1, the yeast orthologue of the gene mutated in Batten disease.
Neuronal ceroid-lipofuscinoses (NCL) are autosomal recessive disorders that form the most common group of progressive neurodegenerative diseases in children, with an incidence as high as 1 in 12,500 live births, and with approximately 440,000 carriers in the United States. Disease progression is characterized by a decline in mental abilities, increased severity of untreatable seizures, blindness, loss of motor skills and premature death. The CLN3 gene, which is responsible for Batten disease, has been positionally cloned. The yeast gene, denoted BTN1, encodes a non-essential protein that is 39% identical and 59% similar to human CLN3. Strains lacking Btn1p, btn1-delta, are resistant to D-(-)-threo-2-amino-1-[p-nitrophenyl]-1,3-propanediol (ANP) in a pH-dependent manner. This phenotype was complemented by expression of human CLN3, demonstrating that yeast Btn1p and human CLN3 share the same function. Here, we report that btn1-delta yeast strains have an abnormally acidic vacuolar pH in the early phases of growth. Furthermore, DNA microarray analysis of BTN1 and btn1-delta strains revealed differential expression of two genes, with at least one, HSP30, involved in pH control. Because Btn1p is located in the vacuole, we suggest that Batten disease is caused by a defect in vacuolar (lysosomal) pH control. Our findings draw parallels between fundamental biological processes in yeast and previously observed characteristics of neurodegeneration in humans.
Cell Division↗
Genetic modifiers of streptomycin resistance in Pneumococcus.
Bryan, Betty E. (University of Rochester, Rochester, N. Y.). Genetic modifiers of streptomycin resistance in pneumococcus. J. Bacteriol. 82:461-470. 1961.-Streptomycin-resistant mutants arising spontaneously in pneumococcus vary widely with respect to the maximal concentrations of streptomycin which they resist. A small random sample of these mutations tested for linkage are allelic or closely linked. One single-step mutant resisting a relatively low level of streptomycin undergoes the following changes: (i) replacement (by a secondary mutation) of the original mutation by an allele conferring a high level of resistance; (ii) reversion to complete sensitivity by back mutation at the originally mutated site; (iii) enhancement by an unlinked modifier gene which confers no resistance by itself but raises the level of resistance of the mutant strain approximately 10-fold. This modifier may have some specificity in its action on the different mutations occurring at a gene locus.The possibility is discussed that multi-step resistance may often originate by acquisition of modifiers.
Drug Resistance, Microbial↗
[Effectiveness of using different radioprotectors for the protection of the sex cells of male mice from the genetic action of radiation, 1. Frequency study of dominant lethal mutations in postspermatogonial cells].
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Animals↗
Mutations of L293 in transmembrane two of the mouse 5-hydroxytryptamine3A receptor alter gating and alcohol modulatory actions.
1 The goal of this study was to determine whether mutations of L293 at the 15' position of TM2 in the 5-HT(3A) receptor alter macroscopic current kinetics, and if these changes could account for alterations in alcohol modulation. Receptor function was assessed in Xenopus oocytes under voltage-clamp and in HEK293 cells with whole-cell patch-clamp recording and rapid drug application. 2 Examination of responses of L293C and L293S receptors to agonist alone revealed enhanced activation, deactivation, and desensitization rates relative to the wild-type receptor. The L293G mutation produced marked slowing of deactivation and desensitization rates. Increased potency of 5-HT and increased efficacy of the partial agonist, DA, was also observed in these mutant receptors. 3 Ethanol and trichloroethanol (TCEt) enhancement of receptor function was reduced or eliminated in receptors containing L293 mutations to C, G, or S. The L293I mutant receptor retained ethanol and TCEt sensitivity. Ethanol and TCEt enhanced activation rate in the wild-type, but not the L293G and L293S receptors. No relationship was observed between any physicochemical property of the substituted amino acids and the change in alcohol potentiation of function. 4 The changes in receptor-channel properties in the mutant receptors support the idea that the L293 residue has important roles in channel gating. Our findings indicate that loss of allosteric modulation by alcohols is not related in any simple way to changes in channel kinetic properties brought about by L293 mutants. We did not observe any evidence that L293 is part of an alcohol binding site.
Allosteric Regulation↗
Pharmacokinetics, biochemical mechanism and mutation accumulation: a comprehensive model of chemical carcinogenesis.
Chemical carcinogenesis is a process beginning with carcinogen absorption and ending with development of a malignant tumor. Individual elements of this process have been studied intensively but no comprehensive model has been developed. This report describes a comprehensive model which incorporates carcinogen pharmacokinetics, biochemical mechanism of action, and the resultant mutation of normal cells to malignancy. Model parameters correspond to specific physiological and biochemical structures and processes. The model was encoded in a simulation language and used to examined biochemical and cellular effects of exposure to an initiator and a promoter. With laboratory validation, the model should be useful for interpretation and design of studies on carcinogenic mechanisms and for risk assessment.
Carcinogens↗
Charged-particle mutagenesis. 1. Cytotoxic and mutagenic effects of high-LET charged iron particles on human skin fibroblasts.
Cytotoxic and mutagenic effects of high-LET charged iron (56Fe) particles were measured quantitatively using primary cultures of human skin fibroblasts. Argon and lanthanum particles and gamma rays were used in comparative studies. The span of LETs selected was from 150 keV/microns (330 MeV/u) to 920 keV/microns (600 MeV/u). Mutations were scored at the hypoxanthine guanine phosphoribosyl transferase (HPRT) locus using 6-thio-guanine (6-TG) for selection. Exposure to these high-LET charged particles resulted in exponential survival curves. Mutation induction, however, was fitted by the linear model. The relative biological effectiveness (RBE) for cell killing ranged from 3.7 to 1.3, while that for mutation induction ranged from 5.7 to 0.5. Both the RBE for cell killing and the RBE for mutagenesis decreased with increasing LET over the range of 1.50 to 920 keV/microns. The inactivation cross section (sigma i) and the action cross section for mutation induction (sigma m) ranged from 32.9 to 92.0 microns2 and 1.45 to 5.56 X 10(-3) microns2; the maximum values were obtained by 56Fe with an LET of 200 keV/microns. The mutagenicity (sigma m/sigma i) ranged from 2.05 to 7.99 X 10(-5) with an inverse relationship to LET.
Cell Survival↗
Systematic mutation screening of the pro-opiomelanocortin gene: identification of several genetic variants including three different insertions, one nonsense and two missense point mutations in probands of different weight extremes.
Pro-opiomelanocortin (POMC) is the precursor of melanocortins (adrenocorticotropin: ACTH, beta-endorphin, beta-lipotropin: beta-LPH, corticotropin like intermediate peptide, alpha-, beta- and gamma-melanocyte-stimulating hormone: alpha-, beta- and gamma-MSH) some of which act in the brain to reduce food intake and are potential mediators of leptin action. Recently, three different mutations in the POMC gene (POMC) were identified in two unrelated children that lead to early-onset extreme obesity, adrenal insufficiency, and red hair pigmentation. In the present study we systematically screened the coding region of POMC in 96 extremely obese children and adolescents, 60 healthy underweight individuals and 46 patients with anorexia nervosa (AN) and identified several variants. a) A 9 and an 18 base pair insertion (9bp and 18bp: AGC AGC GGC and AGC AGC GGC AGC AGC GGC, respectively, between codon 73 and 74; 1,2). These in-frame variants lead to the insertion of three or six amino acids (Ser-Ser-Gly; Ser-Ser-Gly-Ser-Ser-Gly) carboxy-terminal to gamma-MSH. Frequencies of the 9bp insertion allele varied between 3 and 5% among the different study groups (Pearson's chi2 P>0.5). b) Both an out-of-frame 6 bp insertion (within codon 176: GGG CCC) leading to the insertion of two amino acids (Arg-Ala) and a premature stop-codon (G-7316-T: Glu-180-Stop) within the gamma-LPH sequence were maternally inherited in an obese female proband. This proband inherited another missense mutation from her father (Glu-188-Gly). c) A missense mutation (G-7016-A; Asp-80-Asn) was observed in a single patient with AN who also harboured the 9bp insertion on a paternally derived haplotype. d) The allelic co-occurence of two silent mutations (C-6982-T and C-7285-T) was detected in two obese subjects. e) Two further silent mutations (C-3832-T; C-7111-G) were detected in an underweight and an obese subject, respectively. We conclude that the POMC gene harbors several different polymorphisms and mutations, none of which can readily be associated with the phenotypes under study.
Adolescent↗
Mutational analysis of the substrate binding/catalytic domains of human M form and P form phenol sulfotransferases.
Human monoamine (M) form and simple phenol (P) form phenol sulfotransferases (PSTs) are greater than 93% identical in their primary sequences and yet display distinct substrate specificities and other enzymatic properties. Through the generation and characterization of a series of chimeric PSTs, we have previously demonstrated two highly variable regions within their sequences to be responsible for determining their substrate phenotypes (Sakakibara, Y., Takami, Y., Nakayama, T., Suiko, M., and Liu, M.-C. (1998) J. Biol. Chem. 273, 6242-6247). By employing the site-directed mutagenesis technique, the present study aims to identify and quantitatively evaluate the specific amino acid residues critical to the substrate binding and catalysis in these two enzymes. Twelve mutated M-PSTs and seven mutated P-PSTs were generated, expressed, and purified. Enzymatic characterization showed that, of the twelve mutated M-PSTs, mutations at residues Asp-86, Glu-89, and Glu-146 resulted in a dramatic decrease in V(max)/K(m) with dopamine as substrate, being greater than 450 times for the D86A/E89I/E146A mutated M-PST. With p-nitrophenol as substrate, the V(max)/K(m) determined for the D86A/E89I/E146A-mutated M-PST increased more than 25 times and approached that determined for the wild-type P-PST. These results indicated that the concerted action of the three mutated residues (D86A, E89I, and E146A) is sufficient for the conversion of the substrate phenotype of M-PST to that of P-PST. Among the mutated P-PSTs, the I89E- and A146E-mutated P-PSTs displayed considerable deviations in V(max)/K(m) with dopamine or p-nitrophenol as substrate. No corresponding changes, however, were detected with the opposite compound as substrate. These results indicated that, in contrast to M-PST, mutations at Ala-86, Ile-89, and Ala-146 to the corresponding residues in M-PST are not sufficient for rendering the change of P-PST substrate phenotype to that of M-PST. For both M-PSTs and P-PSTs, mutations at Lys-48 or His-108 led to the loss of sulfotransferase activities, indicating their importance in the catalytic mechanism.
Amino Acid Sequence↗
Variants of Escherichia coli giving the appearance of mixed growths in urine.
Urines from patients with symptoms of urinary tract infection yielded mixed growths of different colony types of Escherichia coli. The different colony types were found to be variants of single infecting strains caused by mutation or by phase action. It is suggested that care should be exercised in the interpretation of apparently mixed growths from urine.
Bacteriophages↗
Anti-Müllerian hormone in disorders of sex determination and differentiation.
Masculinisation of internal and external genitalia during foetal development depends on the existence of two discrete testicular hormones: Leydig cell-secreted testosterone drives the differentiation of the Wolffian ducts, the urogenital sinus and the external genitalia, whereas Sertoli cell-produced anti-Müllerian hormone (AMH) provokes the regression of Müllerian ducts. The absence of AMH action in early foetal life results in the formation of the Fallopian tubes, the uterus and the upper third of the vagina. In 46,XY foetuses, lack of AMH may result from testicular dysgenesis affecting both Leydig and Sertoli cell populations: in this case persistence of Müllerian remnants is associated with ambiguous or female external genitalia. Alternatively, defective AMH action may result from mutations of the genes encoding for AMH or its receptor: in this condition known as Persistent Müllerian Duct Syndrome, testosterone production is normal and external genitalia are normally virilised. Finally, AMH may be normally secreted in intersex patients with defects restricted to androgen synthesis or action, resulting in patients with female or ambiguous external genitalia with no Müllerian derivatives.
Anti-Mullerian Hormone↗
Determinants of promoter-specific activity by glucocorticoid receptor.
Glucocorticoid receptor (GR) is expressed at essentially equal levels in almost all tissues and cell types. Remarkably, glucocorticoids themselves regulate transcription in vivo in both a promoter- and tissue-specific manner. Thus, specific systems must be in place to regulate receptor action within certain cells and at certain promoters. To address two specific aspects of these systems, we have analyzed promoter-specific activity of GR using two different, well studied promoters (termed simple and composite promoters) from which GR activates transcription. The simple promoter depends only on the receptor for glucocorticoid-responsive transcriptional activation, while GR activity at the composite promoter depends on additional transcription factors. We have compared the action of several GR ligands at these promoters and demonstrate fundamental differences in the activities of these ligands on receptor activity. Furthermore, these compounds induce unique conformational changes in receptor, resulting in promoter-specific receptor function. We have identified critical amino acid residues within GR which, when mutated, genetically distinguish the action of GR at these promoters. Taken together, the data indicate that the presence of only the receptor and the ligand is not sufficient to allow activation of transcription. An additional system of regulation influences receptor action in both a tissue- and promoter-selective fashion, suggesting that multiple, regulated surfaces of the receptor respond to the cellular environment and determine the spectrum of GR activities. These functional surfaces may be induced or regulated by ligand binding, by the DNA sequence to which receptor is bound, or by the nonreceptor factors resident at the promoter or in the tissue.
Animals↗
Kinds and locations of mutations induced in the hypoxanthine-guanine phosphoribosyltransferase gene of human T-lymphocytes by 1-nitrosopyrene, including those caused by V(D)J recombinase.
The detection of an increase in the frequency of mutants in the hypoxanthine-guanine phosphoribosyltransferase (HPRT) gene of circulating T-cells has been proposed as a method to evaluate the biological effects of human exposure to environmental mutagens. We exposed adult human T-cells in vitro to 1-nitrosopyrene (1-NOP), a partially reduced metabolite of 1-nitropyrene, a ubiquitous environmental carcinogen. In populations of T-cells from two unrelated donors, a dose of 1-NOP that reduced survival to 40% of the untreated cells increased the HPRT mutant frequency 6 to 7 times over the background frequency of 5 x 10(-6). The coding region of 35 independent mutants was amplified by polymerase chain reaction and sequenced. Single base substitutions were found in 63% of the mutants (22 of 35). These were distributed randomly throughout the gene. Most of the substitutions (82%) involved G-C base pairs, mainly G.C-->A.T transitions and G.C-->T.A transversions. Fifteen mutants were lacking one or more exons; 9 of the 15 were lacking exons 2 and 3. Examination showed that at least four of the latter had resulted from V(D)J recombinase acting illegitimately to recombine sites located in introns 1 and 3 of the HPRT gene. T-cells from a second unrelated donor were exposed to 1-NOP and 38 additional independent mutants were analyzed. The results indicated that such mutations occurred at a frequency of 2.4 x 10(-6) compared to a background frequency of less than 0.3 x 10(-6). This recombinase, which plays an important role in leukemogenesis, is normally present in developing, but not mature, B- and T-cells such as those used here as target cells for 1-NOP. The present study is the first report showing that exposure to an environmental carcinogen can cause mutations induced by the action of this enzyme.
Amino Acid Sequence↗
Mutations of the phage lambda nutL region that prevent the action of Nun, a site-specific transcription termination factor.
Phage HK022 encodes a protein, Nun, that promotes transcription termination within the pL and pR operons of its relative, phage lambda. The lambda sequences required for termination had previously been shown to overlap the nut sites, which are essential for transcription antitermination during normal lambda growth. To further specify the Nun target and to determine its relation to the nut sites, we constructed deletion and base substitution mutations of the lambda nutL region and measured Nun-dependent reduction of the expression of a downstream reporter gene. The shortest construct that retained full Nun responsiveness was a 42-bp segment that included both boxA and boxB, sequences that have been implicated in lambda antitermination. Deletion of boxA reduced Nun termination, and deletion of both sequences eliminated Nun termination. Base substitutions in boxA and the proximal portion of boxB impaired Nun termination, while base substitutions between boxA and boxB, in the distal portion of boxB, and immediately downstream from boxB had no appreciable effect. The termination defect of all of the base substitution mutations was relieved by increasing the level of Nun protein; in contrast, the deletions and a multiple-base substitution did not regain full Nun responsiveness at elevated Nun concentrations. We also asked if these mutant nut regions retained their ability to interact with N, the lambda-encoded antitermination protein. A qualitative assay showed that mutations within boxA or boxB reduced interaction, while mutations outside boxA and boxB did not. These data show that (i) the recognition sites for N and Nun overlap to a very considerable extent but are probably not identical and (ii) a high concentration of Nun promotes its interaction with mutant nut sites, a behavior also reported to be characteristic of N.
Bacteriophage lambda↗