Histidine Operon Research Articles

Visualization of Type IV-A1 CRISPR-mediated repression of gene expression and plasmid replication.

Type IV CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) effector complexes are often encoded on plasmids and are proposed to prevent the replication of competing plasmids. The Type IV-A1 CRISPR-Cas system of Pseudomonas oleovorans additionally harbors a CRISPR RNA (crRNA) that tightly regulates the transcript levels of a chromosomal target and represents a natural CRISPR interference (CRISPRi) tool. This study investigates CRISPRi effects of this system using synthetic crRNAs against genome and plasmid sequences. Targeting of reporter genes revealed extended interference in P. oleovorans and Escherichia coli cells producing recombinant CRISPR ribonucleoprotein (crRNP) complexes. RNA sequencing (RNA-seq) analyses of Type IV-A1 CRISPRi-induced transcriptome alterations demonstrated highly effective long-range downregulation of histidine operon expression, whereas CRISPRi effects of dCas9 remained limited to the vicinity of its binding site. Single-molecule microscopy uncovered the localization dynamics of crRNP complexes. The tracks of fluorescently labeled crRNPs co-localized with regions of increased plasmid replication, supporting efficient plasmid targeting. These results identify mechanistic principles that facilitate the application of Type IV-A1 CRISPRi for the regulation of gene expression and plasmid replication.

Library Selection with a Randomized Repertoire of (βα)8-Barrel Enzymes Results in Unexpected Induction of Gene Expression.

The potential of the frequently encountered (βα)8-barrel fold to acquire new functions was tested by an approach combining random mutagenesis and selection in vivo. For this purpose, the genes encoding 52 different phosphate-binding (βα)8-barrel proteins were subjected to error-prone PCR and cloned into an expression plasmid. The resulting mixed repertoire was used to transform different auxotrophic Escherichia coli strains, each lacking an enzyme with a phosphate-containing substrate. After plating of the different transformants on minimal medium, growth was observed only for two strains, lacking either the gene for the serine phosphatase SerB or the phosphoserine aminotransferase SerC. The same mutants of the E. coli genes nanE (encoding a putative N-acetylmannosamine-6-phosphate 2-epimerase) and pdxJ (encoding the pyridoxine 5'-phosphate synthase) were responsible for rescuing both ΔserB and ΔserC. Unexpectedly, the complementing NanE and PdxJ variants did not catalyze the SerB or SerC reactions in vitro. Instead, RT-qPCR, RNAseq, and transcriptome analysis showed that they rescue the deletions by enlisting the help of endogenous E. coli enzymes HisB and HisC through exclusive up-regulation of histidine operon transcription. While the promiscuous SerB activity of HisB is well-established, our data indicate that HisC is promiscuous for the SerC reaction, as well. The successful rescue of ΔserB and ΔserC through point mutations and recruitment of additional amino acids in NanE and PdxJ provides another example for the adaptability of the (βα)8-barrel fold.

The Mutagenic Study of Benzimidazole Analogues: Structure-Activity Relationship (SAR)

Event Abstract Back to Event The mutagenic study of benzimidazole analogues: structure-activity relationship (SAR) Nurul Hafizan Azahar1, Siti Soleha Abdullah1, Rozaini Abdullah2, Norizan Ahmat3 and Hasiah Ab Hamid1* 1 Department of Biomedical Sciences, Faculty of Medicine and Health sciences, University of Putra Malaysia, Malaysia 2 Department of Environmental and Occupational Health, Putra Malaysia University, Malaysia 3 School of Chemistry and Environment, University Technology Mara, Malaysia Background Benzimidazole analogues have diverse range of biological activities including anti-ulcer, anti-hypertensive, anti-viral, anti-fungal, anti-inflammatory and anti-cancer. Previous studies revealed some of the analogues can induce mutation despite their biological activities. This study aims to screen for potential mutagenic activities of novel benzimidazole analogues using Ames test and study their structure-activity relationship (SAR). Methods Ames test was carried out on two strains of Salmonella typhimurium; TA98 and TA100 that carry different mutations in their various genes in histidine operon, which protocols complied with OECD 471 Guideline for Testing of Chemicals. The preliminary tests including growth curve and genetic analysis were carried out prior to Ames test in order to determine the optimum incubation hour and genotypes of the bacterial strains respectively. The bacterial growth curve was carried out by incubating the bacterial suspension for 52 hours. The optical density of the bacteria was recorded for every two hours at 660nm wavelength and the graph of optical density versus time was plotted. The genotypes of the bacterial strains used were determined by genetic analysis which includes tests on dependency on histidine and/or biotin, presence of rfa mutation, urvB deletion as well as pKM101 plasmid. All data were analysed using Two-Way ANOVA, followed by post-hoc Tukey test. The SAR was analysed based on the results of Ames test. Results The optimum incubation hour for both strains was 14 hours. Both bacterial strains show dependency on histidine with presence of rfa mutation, urvB deletion and pKM101 plasmid. Besides, all analogues tested; NN-1-5, NN-1-7, NN-1-9 and NN-1-18 show no mutagenic activity. Conclusion There was no significant difference in the mutagenic activity among all analogues despite their difference in the number and position of hydroxyl groups. Keywords: benzimidazole, Ames test, Salmonella typhimurium, Structure-activity relationship (SAR), MUTAGENIC Conference: International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18) “Seizing Opportunities and Addressing Challenges of Precision Medicine”, Putrajaya, Malaysia, 3 Dec - 5 Feb, 2019. Presentation Type: Poster Presentation Topic: Cancer Citation: Azahar N, Abdullah S, Abdullah R, Ahmat N and Ab Hamid H (2019). The mutagenic study of benzimidazole analogues: structure-activity relationship (SAR). Front. Pharmacol. Conference Abstract: International Conference on Drug Discovery and Translational Medicine 2018 (ICDDTM '18) “Seizing Opportunities and Addressing Challenges of Precision Medicine”. doi: 10.3389/conf.fphar.2018.63.00012 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 30 Sep 2018; Published Online: 17 Jan 2019. * Correspondence: Dr. Hasiah Ab Hamid, Department of Biomedical Sciences, Faculty of Medicine and Health sciences, University of Putra Malaysia, Kuala Lumpur, Malaysia, hasiah@upm.edu.my Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Nurul Hafizan Azahar Siti Soleha Abdullah Rozaini Abdullah Norizan Ahmat Hasiah Ab Hamid Google Nurul Hafizan Azahar Siti Soleha Abdullah Rozaini Abdullah Norizan Ahmat Hasiah Ab Hamid Google Scholar Nurul Hafizan Azahar Siti Soleha Abdullah Rozaini Abdullah Norizan Ahmat Hasiah Ab Hamid PubMed Nurul Hafizan Azahar Siti Soleha Abdullah Rozaini Abdullah Norizan Ahmat Hasiah Ab Hamid Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Surveying Mutagenic and Anti-mutagenic Effects of Citrus Limon

Background and objective: Cancer is one of the main causes of mortality in the world and countless mutagenic chemical compound kill millions of cancer patients every year. Scientists are searching for natural foodstuff to prevent cancer. The present paper examines anti-mutagenic and anti-carcinogenic effects of citrus lemon.Methodology: Human astrocytoma cancerous cell line was cultured in DMEM (Gibco) with FBS10% (fetal bovine serum), L-glutamine, penicillin, and streptomycin at 37ºC for two days. Then the samples were treated with citrus lemon juice and biotic potential of cells was examined through the MTT method. Anti-mutagenic and anti-carcinogenic effects of citrus lemon were studied through back mutation assessment standard method (Ames test). The test was done using Salmonella typhimurium strain (TA100) with the mutation in histidine operon that needs external histidine source. The strain creates the reverse colony in presence of carcinogenic agents (sodium azide). Findings: Based on the MTT method, human astrocytoma cancerous cell line demonstrated significant cellular death compared with the control groups (p<0.01). Ames test showed that the juice prevented back mutation and rate of inhibition, based on the anti-mutagenic effect, of half-ripen and ripen citrus lemon was 71.7% and 34.3% respectively. In addition, the anti-carcinogenic effect of half-ripen and ripen citrus lemon were 83.3% and 50% respectively. Conclusion: Anti-mutagenic and anti-carcinogenic effects of citrus lemon were examined for the first time. The results showed that in this regard half-ripen citrus lemon was more effective than ripening citrus lemon.

Probing the Mechanism by which Proteins Designed De Novo Function in Living Cells

One goal of synthetic biology is the design of a collection of novel proteins to create an artificial proteome. The Hecht lab made initial progress toward this goal by producing a library of stable novel proteins that are not similar to any naturally occurring proteins, but nonetheless function in vivo. The E. coli strain ΔserB, which cannot grow on minimal media due to the chromosomal deletion of phosphoserine phosphatase (which is essential for the biosynthesis of serine), was transformed with a library of de novo designed proteins and screened for growth. Four novel proteins, named SynSerB1, 2, 3, and 4, were found to rescue this strain on minimal media. Since the pure SynSerB proteins do not perform the reaction that wild-type SerB performs in the cell, we suspect that the SynSerB proteins may function by altering the expression of an endogenous protein. Assaying the transcriptome of cells expressing SynSerB3 revealed an overexpression of the histidine operon, including HisB, a phosphatase that can ut...

Comparative proteomic analysis of Salmonella typhimurium LT2 and its hisG gene inactivated mutant

A hisG gene, encoding ATP phosphoribosyl phosphothansferase, insertion mutation was found to effected the production of four different proteins in Salmonella Typhimurium LT2 as a results of comparative analysis of two-dimensional analysis of hisG mutant (Δhis87) and the conrol strain (LT2). MALDI-TOF analysis of these protein spots which were expresed in S. Typhimurium LT2 but not its mutant Δhis87, showed that these proteins were; GMP synthase, arginine-tRNA ligase (Arginyl-tRNA synthetase), SopE and SifA. All of these proteins are virulence-associated proteins in Salmonella. Thus we have concluded that the proper function of histidine operon is very crucial for virulence of S. Typhimurium.

Histidine biosynthesis, its regulation and biotechnological application in Corynebacterium glutamicum

l-Histidine biosynthesis is an ancient metabolic pathway present in bacteria, archaea, lower eukaryotes, and plants. For decades l-histidine biosynthesis has been studied mainly in Escherichia coli and Salmonella typhimurium, revealing fundamental regulatory processes in bacteria. Furthermore, in the last 15 years this pathway has been also investigated intensively in the industrial amino acid-producing bacterium Corynebacterium glutamicum, revealing similarities to E. coli and S. typhimurium, as well as differences. This review summarizes the current knowledge of l-histidine biosynthesis in C. glutamicum. The genes involved and corresponding enzymes are described, in particular focusing on the imidazoleglycerol-phosphate synthase (HisFH) and the histidinol-phosphate phosphatase (HisN). The transcriptional organization of his genes in C. glutamicum is also reported, including the four histidine operons and their promoters. Knowledge of transcriptional regulation during stringent response and by histidine itself is summarized and a translational regulation mechanism is discussed, as well as clues about a histidine transport system. Finally, we discuss the potential of using this knowledge to create or improve C. glutamicum strains for the industrial l-histidine production.

Assessment of the antimutagenic effect of Doash tea extract fractions

Origanum majorana L (Doash) is one of the traditional remedy that is used as a tea and to treat ailments, in the Kingdom of Saudi Arabia. The present study has attempted to evaluate the inhibitory action of Doash fractions on the bioactivation of selected food mutagens and direct-acting mutagens. Four Sallmonella bacterial strains (TA98, TA97, TA100 and TA1530) were used in the present study. These strains contain different mutations in the histidine operon, allowing the bacteria to detect different types of mutation. The two strains (TA98 and TA97) are capable of detecting frameshift mutations, while TA100 and TA1530 are able to detect base-pair substitutions. The liver homogenate and other subcellular fractions were prepared. Identification of Doash fractions was conducted using the high-performance liquid chromatography/mass spectrometry system. The results of the present study demonstrated that the Doash tea fraction components have the ability to reduce the in vitro mutagencity of several promutagens, which were employed in this study. Fraction No. 5 (with the highest content of solid) was the most potent inhibitor of the mutagenicity of all promutagens employed in this study. The antimutagenic effect of Doash tea extract, and its various fractions, was pronounced, indicating that the metabolism of cytochrome P450 1A2 isozyme is likely to be inhibited.

Evaluation of mutagenic activity in the Nakdong river and advanced drinking water treatment processes

The previously proved method of Ames test was followed to measure the mutagenic activity. We used Salmonella typhimurium which has special gene mutation site with a single compound characteristic and histidine operon, and measured at the four (Gumi, Gangjung, Gachang, Hyunpung) branch points of Nakdong river for one year. We used Standard Methods for general water analysis with the items as water temperature, pH, DO, BOD, COD, KMnO4, T-N, T-P, NH3-N, TOC, and AOX. Hyunpung branch, where the Nakdong and the Gumho river seems to be mixed enough, showed higher mutagenic activity than the other 3 branches. Every point of Nakdong river showed the TA98 mutagenic activity higher than 2, while TA100 was lower than 2. Most of the mutagenic activity was detected from methanol extracts. These results indicate that Nakdong river water quality is getting deteriorated, and that Nakdong river is influencing the mutagenic activity by Frame-shift type mutation and its polar substances. Correlations (R) between mutagenic activity and concentrations of water analysis items as TOC, AOX, BOD, COD, T-N, and T-P, resulted as 0.768, 0.787, 0.743, 0.688, 0.757, 0.628, respectively, and mutagenic activities of TA98 and TA100, after the treatment of ozone and activated carbon, decreased by chlorine pre-treatment. Finally, if the injected concentration of ozone was higher than 1.0 mL, TA98 and TA100 showed lower mutagenic activities than 2.

Regulation of Carbon and Nitrogen Utilization by CbrAB and NtrBC Two-Component Systems in Pseudomonas aeruginosa

The global effect of the CbrAB and NtrBC two-component systems on the control of carbon and nitrogen utilization in Pseudomonas aeruginosa was characterized by phenotype microarray analyses with single and double mutants and the isogenic parent strain. The tested compounds were clustered based on the growth phenotypes of these strains, and the results clearly demonstrated the pivotal roles of CbrAB and NtrBC in carbon and nitrogen utilization, respectively. Growth of the cbrAB deletion mutant on arginine, histidine, and polyamines used as the sole carbon source was abolished, while growth on the tricarboxylic acid (TCA) cycle intermediates was sustained. In this study, suppressors of the cbr mutant were selected from minimal medium containing l-arginine as the sole carbon and nitrogen source. These mutants fell into two groups according to the ability to utilize histidine. The genomic library of a histidine-positive suppressor mutant was constructed, and the corresponding suppressor gene was identified by complementation as an ntrB allele. Similar results were obtained from four additional suppressor mutants, and point mutations of these ntrB alleles resulting in the following changes in residues were identified, with implications for reduced phosphatase activities: L126W, D227A, P228L, and S229I. The Ntr systems of these ntrB mutants became constitutively active, as revealed by the activity profiles of glutamate dehydrogenase, glutamate synthase, and glutamine synthetase. As a result, these mutants not only regain the substrate-specific induction on catabolic arginine and histidine operons but are also expressed to higher levels than the wild type. While the DeltacbrAB ntrB(Con) mutant restored growth on many N-containing compounds used as the carbon sources, its capability to grow on TCA cycle intermediates and glucose was compromised when ammonium served as the sole nitrogen source, mostly due to an extreme imbalance of carbon and nitrogen regulatory systems. In summary, this study supports the notion that CbrAB and NtrBC form a network to control the C/N balance in P. aeruginosa. Possible molecular mechanisms of these two regulatory elements in the control of arginine and histidine operons used as the model systems are discussed.

Histidine Auxotrophy in Commensal and Disease-Causing Nontypeable Haemophilus influenzae

Histidine biosynthesis is one of the best studied metabolic pathways in bacteria. Although this pathway is thought to be highly conserved within and between bacterial species, a previous study identified a genetic region within the histidine operon (his) of nontypeable strains of Haemophilus influenzae (NTHI) that was more prevalent among otitis media strains than among throat commensal NTHI strains. In the present study, we further characterized this region and showed that genes in the complete his operon (hisG, -D, -C, -NB, -H, -A, -F, and -IE) are >99% conserved among four fully sequenced NTHI strains, are present in the same location in these four genomes, and are situated in the same gene order. Using PCR and dot blot hybridization, we determined that the his operon was significantly more prevalent in otitis media NTHI strains (106/121; 87.7%) than in throat strains (74/137; 54%) (prevalence ratio, 1.62; P<0.0001), suggesting a possible role in middle ear survival and/or acute otitis media. NTHI strains lacking the his operon showed attenuated growth in histidine-restricted media, confirming them as his-negative auxotrophs. Our results suggest that the ability to make histidine is an important factor in bacterial growth and survival in the middle ear, where nutrients such as histidine may be found in limited amounts. Those isolates lacking the histidine pathway were still able to survive well in the throat, which suggests that histidine is readily available in the throat environment.

A novel phenotype based on esterase electrophoretic polymorphism for the differentiation of Lactococcus lactis ssp. lactis and cremoris

To evaluate the esterase phenotype in Lactococcus lactis strains isolated from traditional Tunisian dairy products. A collection of 55 L. lactis strains isolated from traditional fermented milk products and three reference strains were identified at species and subspecies level using molecular methods targeted to the 16S rRNA gene and to the histidine operon. The genotypic data obtained allowed the identification of the strains as L. lactis ssp. lactis and L. lactis ssp. cremoris with the prevalence of the ssp. lactis. The phenotypic identification based on arginine hydrolysis, the growth at 40 degrees C and in presence of 4% NaCl showed several discrepancy with the identification data based on genotypic analysis. Additional experiments carried out evaluating the esterase electrophoretic patterns revealed four classes of esterases identified on the basis of their electrophoretic mobility and specific activity on alpha- and beta-naphthyl ester of acetate and propionate. Esterase profiles discriminated the strains in two main groups corresponding to the subspecies cremoris and lactis according to a DNA-based identification. The evaluation of esterase activity represents a novel phenotype for the taxonomic discrimination of the L. lactis ssp. lactis and cremoris. Besides the DNA-based techniques that allow the rapid and accurate species/subspecies identification, the electrophoretic esterase profiles of L. lactis strains represents: (i) a new phenotypic tool to understand the physiology and the ecology of this species; and (ii) a new test for the potential selection of flavour producing strains.

A "gain of function" mutation in a protein mediates production of novel modified nucleosides.

The mutation sufY204 mediates suppression of a +1 frameshift mutation in the histidine operon of Salmonella enterica serovar Typhimurium and synthesis of two novel modified nucleosides in tRNA. The sufY204 mutation, which results in an amino-acid substitution in a protein, is, surprisingly, dominant over its wild-type allele and thus it is a "gain of function" mutation. One of the new nucleosides is 5-methylaminomethyl-2-thiouridine (mnm(5)s(2)U34) modified by addition of a C(10)H(17) side chain of unknown structure. Increased amounts of both nucleosides in tRNA are correlated to gene dosage of the sufY204 allele, to an increased efficiency of frameshift suppression, and to a decreased amount of the wobble nucleoside mnm(5)s(2)U34 in tRNA. Purified tRNA(Gln)(cmnm(5)s(2)UUG) in the mutant strain contains a modified nucleoside similar to the novel nucleosides and the level of aminoacylation of tRNA(Gln)(cmnm(5)s(2)UUG) was reduced to 26% compared to that found in the wild type (86%). The results are discussed in relation to the mechanism of reading frame maintenance and the evolution of modified nucleosides in tRNA.

The Origin and Evolution of Operons: The Piecewise Building of the Proteobacterial Histidine Operon

The structure and organization of 470 histidine biosynthetic genes from 47 different proteobacteria were combined with phylogenetic inference to investigate the mechanisms responsible for assembly of the his pathway and the origin of his operons. Data obtained in this work showed that a wide variety of different organization strategies of his gene arrays exist and that some his genes or entire his operons are likely to have been horizontally transferred between bacteria of the same or different proteobacterial branches. We propose a "piecewise" model for the origin and evolution of proteobacterial his operons, according to which the initially scattered his genes of the ancestor of proteobacteria coded for monofunctional enzymes (except possibly for hisD) and underwent a stepwise compacting process that reached its culmination in some gamma-proteobacteria. The initial step of operon buildup was the formation of the his "core," a cluster consisting of four genes (hisBHAF) whose products interconnect histidine biosynthesis to both de novo synthesis of purine metabolism and that occurred in the common ancestor of the alpha/beta/gamma branches, possibly after its separation from the epsilon one. The following step was the formation of three mini-operons (hisGDC, hisBHAF, hisIE) transcribed from independent promoters, that very likely occurred in the ancestor of the beta/gamma-branch, after its separation from the alpha one. Then the three mini-operons joined together to give a compact operon. In most gamma-proteobacteria the two fusions involving the gene pairs hisN-B and hisI-E occurred. Finally the gamma-proteobacterial his operon was horizontally transferred to other proteobacteria, such as Campylobacter jejuni. The biological significance of clustering of his genes is also discussed.

Analysis of mutagenic activity of airborne particulate matter, standard reference materials and reference compounds using base pair-specific Salmonella typhimurium tester strains

The mutagenicity profiles of organic extracts of airborne dust samples from Mannheim, Germany, and two standard reference materials (SRM) as well as eight compounds with different chemical properties were investigated using tester strains Salmonella typhimurium TA700 x (Ames II Assay). Each strain of this series carries a unique missense mutation in the histidine operon and is reverted by only one specific base substitution out of six possible changes. Mutation patterns of eight compounds with different modes of genotoxic action reveal significant differences. Samples of airborne particulate matter (APM) from an industrialized town in Germany (Mannheim) were collected for five consecutive days once a month for 1 year using an automatic high-volume air sampler. Samples taken from Monday to Friday were Soxhlet-extracted and prepared according to standard methods. Although the threshold limit for the least active strains is not triggered by all samples, it can be concluded that mutation patterns of the samples do not vary between different seasons. Standard reference materials (SRMs) were prepared and tested using the same methods. SRMs and APM samples from Mannheim reveal similar mutagenicity profiles in TA700 x strains. The comparison of the mutagenicity profiles of air dust extracts from Mannheim and the SRMs, respectively, with reference compounds investigated so far shows some similarities although the patterns do not fit perfectly. Mutagenicity profiles of TA700 x-activity of nitro-aromatic compounds published so far are similar to those of APM collected in Mannheim, Germany, as well as to standard reference materials 1648 and 1649.

Activation of bis-electrophiles to mutagenic conjugates by human O6-alkylguanine-DNA alkyltransferase.

O(6)-Alkylguanine DNA-alkyl transferase (AGT) has been shown to conjugate both 1,2-dibromoethane and dibromomethane, yielding AGT inactivation, DNA-AGT cross-linking, and enhanced mutagenicity. A variety of related chemicals were examined to determine if similar phenomena occur. Among the compounds examined in these systems (histidine operon reversion in Escherichia coli and Salmonella typhimurium tester strains), a strong halide order was generally observed, with increasing activities in the order I > Br >> Cl. At least one Br atom appeared to be required for human AGT-dependent mutations, and compounds with only Cl did not inhibit AGT and were not activated to genotoxins. Of a series of haloforms tested (CHX(3), X = Br or Cl), all were without effect. Among a series of alpha,omega-disubstituted dihaloalkanes (Br or I), the inactivation of AGT increased with methylene chain length (at least up to n = 5) but the most mutagenic activity (AGT-dependent) was seen with n = 1-3. The effects with n = 1 or 2 were expected from previous results; the mutagenic effect with n = 3 and the reduction with n > 3 may represent a balance between AGT reaction, stability, and reactivity, in the absence of anchimeric assistance. A strong AGT-dependent mutation was observed for 1,3-butadiene diepoxide. We conclude that numerous bis-electrophiles show AGT-dependent activation to mutagenic conjugates. Haloforms and dichloroalkanes are therefore not an issue, but bromohaloalkanes and 1,3-butadiene diepoxide are potential problems. These observations are of relevance in considering toxicity and risks of some chemicals used in industrial applications.

Direct Stimulation of the λpaQ Promoter by the Transcription Effector Guanosine-3′,5′-(bis)pyrophosphate in a Defined in Vitro System

The bacterial response to nutritional deprivation, called the stringent response, results in the introduction of the specific nucleotide guanosine-3',5'-(bis) pyrophosphate (ppGpp). This nucleotide interacts with RNA polymerase and alters its action so that transcription from certain promoters is inhibited, whereas transcription from others seems to be activated. The exact mechanism of transcriptional stimulation by ppGpp in vivo remains unknown. A passive control model has been proposed according to which transcription inhibition during the stringent response at several very active promoters, like those for rRNA and tRNA genes, makes more free RNA polymerase (RNAP) molecules available for transcription at promoters with weak binding affinities for RNAP, thus leading to their passive activation. Among promoters whose transcription is activated by ppGpp in vivo is the histidine operon promoter (hisGp). However, in vitro it is only possible to demonstrate this effect in a coupled transcription-translation system. Here we demonstrate, using another in vivo ppGpp-stimulated promoter, the phage lambdapaQ promoter, that activation by ppGpp in a defined in vitro system is direct. A systematic study of ppGpp effects on the stimulation of paQ revealed that, as in the case of promoters inhibited by this nucleotide, ppGpp decreases the half-life of paQ open complexes. Our results also indicate that the equilibrium binding affinity of RNA polymerase to paQ seems not to be affected in the presence of ppGpp. Our data indicate that the mechanism underlying ppGpp stimulation of paQ is due to an increased rate of productive open complex formation.

The ames test

Terms to be familiar with before you start to solve the test: Polycyclic aromatic hydrocarbons, microsomal fraction, histidine operon, mutation, induction, repression, translation, Ca2+ channel, Ca2+ ATPase, steroid receptor, cytochrome P450, cytochrome c, direct and indirect carcinogen, procarcinogen, mutagenic compounds, tumor initiation, tumor promoter, complete and incomplete carcinogens, apoptosis.

The Ames Salmonella/microsome mutagenicity assay

The Ames Salmonella/microsome mutagenicity assay ( Salmonella test; Ames test) is a short-term bacterial reverse mutation assay specifically designed to detect a wide range of chemical substances that can produce genetic damage that leads to gene mutations. The test employs several histidine dependent Salmonella strains each carrying different mutations in various genes in the histidine operon. These mutations act as hot spots for mutagens that cause DNA damage via different mechanisms. When the Salmonella tester strains are grown on a minimal media agar plate containing a trace of histidine, only those bacteria that revert to histidine independence ( his +) are able to form colonies. The number of spontaneously induced revertant colonies per plate is relatively constant. However, when a mutagen is added to the plate, the number of revertant colonies per plate is increased, usually in a dose-related manner. The Ames test is used world-wide as an initial screen to determine the mutagenic potential of new chemicals and drugs. The test is also used for submission of data to regulatory agencies for registration or acceptance of many chemicals, including drugs and biocides. International guidelines have been developed for use by corporations and testing laboratories to ensure uniformity of testing procedures. This review provides historical aspects of how the Ames was developed and detailed procedures for performing the test, including the design and interpretation of results.

Histidine operon deattenuation in dnaA mutants of Salmonella typhimurium correlates with a decrease in the gene dosage ratio between tRNA(His) and histidine biosynthetic loci.

Expression of the histidine operon of Salmonella typhimurium is increased in dnaA(Ts) mutants at 37 degrees C. This effect requires an intact his attenuator and can be suppressed by increasing the gene copy number of the hisR locus, which encodes the tRNA(His). We present data which suggest that the his deattenuation defect in dnaA(Ts) mutants results from the loss of a gene dosage gradient between the hisR locus, close to oriC, and the his operon, far from oriC. Some of the conclusions drawn here may apply to other operons as well.