lacZ Research Articles

Discovering Prokaryotic Gene Regulation by Building and Investigating a Computational Model of the lac Operon

Discovering Prokaryotic Gene Regulation by Building and Investigating a Computational Model of the lac Operon

Stability and Hopf bifurcation analysis for a Lac operon model with nonlinear degradation rate and time delay.

In this paper, we construct a discrete time delay Lac operon model with nonlinear degradation rate for mRNA, resulting from the interaction among several identical mRNA pieces. By taking a discrete time delay as bifurcation parameter, we investigate the nonlinear dynamical behaviour arising from the model, using mathematical tools such as stability and bifurcation theory. Firstly, we discuss the existence and uniqueness of the equilibrium for this system and investigate the effect of discrete delay on its dynamical behaviour. Absence or limited delay causes the system to have a stable equilibrium, which changes into a Hopf point producing oscillations if time delay is increased. These sustained oscillation are shown to be present only if the nonlinear degradation rate for mRNA satisfies specific conditions. The direction of the Hopf bifurcation giving rise to such oscillations is also determined, via the use of the so-called multiple time scales technique. Finally, numerical simulations are shown to validate and expand the theoretical analysis. Overall, our findings suggest that the degree of nonlinearity of the model can be used as a control parameter for the stabilisation of the system.

High-yield Production of Granulocyte-macrophage Colony-stimulating Factor in E. coli BL21 (DE3) By an Auto-induction Strategy.

A novel strategy to increase protein expression yield is unintended induction of expression in complex media, called auto-induction. This method can be used to express proteins under control of the lac promoter without any need to monitor bacterial growth pattern, and addition of specific expression inducers such as Isopropyl β-D-1-thiogalactopyranoside (IPTG) at proper time. In the present study, a codon optimized gene encoding granulocyte-macrophage colony stimulating factor (GM-CSF) was cloned and over-expressed in Escherichia coli BL21 (DE3) using both conventional inducer-based and auto-induction approaches in a shake flask scale and the yield of GM-CSF expression and biomass production was identified. Results showed higher biomass production and expression yield for GM-CSF in case of auto-induction comparing with IPTG-induction. The auto-induction approach was also performed in a fed batch fermentation process in a 2-L bioreactor scale. The feeding strategy yielded an amount of 300 mg/L GM-CSF within 20 h of induction. However, most of the over-expressed GM-CSF was produced as inclusion bodies and following purification and refolding, a final yield of 90 mg/L was achieved. These results suggest that auto-induction approach can be effectively applied in fed-batch fermentation for the large scale production of GM-CSF; however, further optimization of purification process is obligatory to increase the purification yield.

Origin of Lactose Fermentation in <i>Kluyveromyces lactis</i> by Interspecies Transfer of a Neofunctionalized Gene Cluster During Domestication

Humans have used yeasts to make cheese and kefir for millennia, but the ability to ferment the milk sugar lactose is found in only a few yeast species, of which the foremost is Kluyveromyces lactis. Two genes, LAC12 (lactose permease) and LAC4 (lactase), are sufficient for lactose uptake and hydrolysis to glucose and galactose. Here, we show that these genes have a complex evolutionary history in the genus Kluyveromyces that is likely the result of human activity during domestication. We show that their ancestral role was in a combined lactose and cellobiose assimilation system, which used Lac12 to import both of these sugars into the cell, and then hydrolyzed lactose using Lac4, and cellobiose using a cellobiase, Cel2. A second cellobiose transporter Cel1 was also present ancestrally. In the K. lactis lineage, however, the ancestral LAC12 and LAC4 were lost and a separate upheaval in the sister species K. marxianus resulted in loss of CEL1 and quadruplication of LAC12. Among its four LAC12 genes, one became neofunctionalized as an efficient lactose transporter capable of supporting fermentation, specifically in dairy strains of K. marxianus where it formed a LAC4-LAC12-CEL2 gene cluster, while another remained a cellobiose transporter. Then, the ability to ferment lactose was acquired by K. lactis var. lactis by introgression of a 15 kb subtelomeric region containing LAC12 and LAC4 from a dairy strain of K. marxianus. The genomic history of the LAC genes shows that strong selective pressures were imposed on yeasts by early dairy farmers.

Stability and Hopf bifurcation analysis of lac Operon model with distributed delay and nonlinear degradation rate.

We propose a simple model of lac operon that describes the expression of B-galactosidase from lac Z gene in Escherichia coli, through the interaction among several identical mRNA. Our goal is to explore the complex dynamics (i.e. the oscillation phenomenon) of this architecture mediated by this interaction. This model was theoretically and numerically investigated using distributed time delay. We considered the average delay as a bifurcation parameter and the nonlinear degradation rate as a control parameter. Sufficient conditions for local stability were gained by using the Routh-Hurwitz criterion in the case of a weak delay kernel. Then we proved that Hopf bifurcation happened and the direction of the periodic solution was determined using multiple time scale technique. Our results suggest that the interaction among several identical mRNA plays the main role in gene regulation.

Клонирование кДНК S100B в E. coli под контролем индуцибельного промотора, и ее гетерологичная экспрессия

В работе разработана стратегия клонирования и экспрессии кДНК S100B с использованием эффективной и многофункциональной онлайн программы Genome compiler на основе векторной системы pBT7-N-His, имеющей промотор РНК-полимеразы фага Т7, участок клонирования и экспрессии с ключевыми элементами контроля и регуляции процессов транскрипции и трансляции. Экспрессия белка S100B подтверждена процедурой SDS-ПААГЭ по молекулярной массе выявленных на электрофорезе полос с динамикой накопления относительно контроля при увеличении времени инкубации после индукции с IPTG. Полученная гетерологичная экспрессия белка S100B с сохранением основных физико-химических свойств и биологической активностью открывает хорошие перспективы его применения в качестве информативного маркера состояний организме в норме и при патологии, и в качестве ценного объекта направленной терапии. To investigate the structure and functions of the S100B protein in cells and tissues, the heterologous expression of S100B cDNA was studied on E. coli. A strategy for cloning and expression of S100B cDNA was developed using an efficient and multifunctional online program, Genome Compiler, based on a pBT7-N-His vector system, with a which had a phage T7 RNA polymerase promoter and a cloning and expression site with key elements for control and regulation of transcription and translation. The E. coli strain, BL-21 (DE3) containing the DE3 prophage with the T7 RNA polymerase gene under the control of the lac promoter was used for the expression. The expression of S100B protein was confirmed by the SDS-PAGE procedure, and preliminary studies were performed to optimize the expression procedure. The obtained heterologous expression of the protein with preserved basic physicochemical properties and biological activity opens good prospects for using it as an informative marker of the body state in normal and pathological conditions and as a valuable target of therapy.

Function Perturbation Impact on Feedback Stabilization of Boolean Control Networks.

Function perturbation analysis of Boolean networks is an important topic in the study of gene regulation due to gene mutation or immeasurable variables. This brief studies the function perturbation impact on feedback stabilization of Boolean control networks (BCNs) by using the algebraic state space representation approach. First, the state feedback stabilization control design of BCNs is recalled and the function perturbation problem is formulated. Second, given a state feedback stabilizer, it is robust to the considered function perturbation if one of the following three cases happens: 1) the block where the function perturbation occurs is different from the block which is affected by the state feedback stabilizer (Case 1); 2) when Case 1 does not happen, the perturbed column converges to the equilibrium faster than the original column (Case 2); 3) when Cases 1 and 2 do not happen, the perturbed column does not belong to the reachable set of the original column (Case 3). Third, when the perturbed column belongs to the reachable set of the original column, a constructive procedure is proposed to modify the given state feedback stabilizer to be robust to the function perturbation. Finally, the obtained new results are applied to the function perturbation analysis of lactose operon in Escherichia coli. The main novelty of this brief is to develop a new theoretical framework for the robustness of feedback controllers of BCNs with respect to function perturbation, which is not solved in the existing literature.

A Single-Plasmid Genome Editing System for Metabolic Engineering of Lactobacillus casei.

Genome engineering of Lactobacillus casei, an important industrial microorganism for dairy fermented product, currently relies on inefficient and time-consuming double crossover events. In this study, we developed an easy-to-use genome engineering strategy for metabolic engineering of L. casei for acetoin production. Plasmid pMSP456-Cre, that contains prophage recombinase operon LCABL_13040-50-60 driven by the nisin-controlled inducible expression (NICE) system and the site-specific recombinase gene cre under the control of the promoter of the lactose operon from L. casei, was constructed. Using this plasmid, integration of a hicD3 gene linear donor cassette (up-lox66-cat-lox71-down) was catalyzed by the LCABL_13040-50-60 recombinase and the cat gene was excised by the Cre/lox system with an efficiency of 60%. To demonstrate this system for sequential and iterative knocking out genes in L. casei, another three genes (pflB, ldh and pdhC) related to acetoin production were deleted with the efficiencies of 60, 40, and 60%, respectively. The yielding quadruple mutant could produce a ∼18-fold higher amount of acetoin than the wild-type and converted 59.8% of glucose to acetoin in aerobic. Therefore, these results proved this simple genome engineering strategy have potential in metabolic engineering of L. casei for production of high value-added metabolites.

Cloning and Heterologous Expression of Plantaricin ZJ5, a Novel Bacteriocin from Lactobacillus plantarum ZJ5, in Escherichia coli

A novel bacteriocin, plantaricin ZJ5 (PZJ5) was yielded from Lactobacillus plantarum ZJ5, cloned, and produced in Escherichia coli BL21 (DE3) pLys. The PZJ5 structural gene was fused with a Trx tag, and cloned into the pET32a plasmid under the control of the inducible lac operon. Induction was performed with isopropyl-β-D-thiogalactopyranoside (IPTG), with subsequent overexpression of the fusion protein, followed by purification to homogeneity via His affinity chromatography. Recombinant E. coli produced greater quantities of PZJ5 than L. plantarum ZJ5, and PZJ5 in E. coli was expressed in the form of soluble material. Biologically active PZJ5 was recovered by cleaving the purified fusion protein using enterokinase. The released PZJ5 demonstrated antibacterial activity against Micrococcus luteus. In this study, an inexpensive biological method using a Trx fusion system was presented, and for the first time, bacteriocin PZJ5 was expressed and purified in E. coli.

Pharmacological inhibition of pigmentation inCryptococcus

Melanin formation is a promising target for antifungal development. We screened a collection of 727 compounds that were previously approved for clinical use in humans for inhibition of pigmentation in Cryptococcus gattii, a lethal fungal pathogen that causes damage to both immunocompetent and immunocompromised hosts. The pyrimidine analogues flucytosine (5-fluorocytosine [5-FC]), 5-fluorouracil (5-FU) and carmofur were identified as efficient inhibitors of pigmentation in the C. gattii model. Since melanin synthesis is enzymatically catalyzed by laccase in Cryptococcus, we investigated whether inhibition of pigmentation by the pyrimidine analogues was laccase-mediated. Enzyme activity and expression of LAC genes were not involved in the effects of the pyrimidine analogues, suggesting alternative cellular targets for inhibition of pigmentation. To address this hypothesis, we screened a collection of approximately 8000 mutants of C. gattii that were produced by insertional mutation after incubation with Agrobacterium tumefaciens and identified a gene product required for the anti-pigmentation activity of 5-FC as a beta-DNA polymerase. Reduced expression of this gene affected capsule formation and urease activity, suggesting essential roles in the cryptococcal physiology. These results demonstrate a previously unknown antifungal activity of 5-FC and reveal a promising target for the development of novel antifungals.

Transcriptional analysis of the laccase-like gene from Burkholderia cepacia BNS and expression in Escherichia coli.

Bacterial laccases have received considerable attention because of several advantages associated with the higher environmental stability of these enzymes compared with fungal laccases. In this study, a laccase-like gene from Burkholderia cepacia BNS was successfully cloned. This gene was found to encode a mature protein of 279 amino acids that exhibited laccase activity in dimer form. The mature protein was found to contain approximately 4mol of copper per monomer, and the metal ion-binding sites were predicted. BC_lacL gene transcription levels were analyzed by qRT-PCR to study expression patterns in the presence of different putative inducers (copper ions, guaiacol, veratryl alcohol, vanillin, coniferaldehyde, p-coumaric acid, sinapic acid, and ferulic acid). Copper ions had a positive effect on both transcription levels and intracellular laccase activity. Interestingly, upon induction with sinapic acid, BC_lacL gene transcription was lower than in the presence of copper ions, but laccase activity was highest under these conditions. The BC_lacL protein expressed in Escherichia coli exhibited a specific activity of 7.81U/mg with 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) as the substrate and 12.3U/mg with 2,6-dimethoxyphenol (2,6-DMP) as the substrate after purification through Ni-affinity chromatography. The optimal activity and kinetic parameters of the recombinant BC_lacL protein were observed (kcat/Km = 3.96s-1μM-1) at a pH of 4.0 at 55°C for ABTS oxidization and (kcat/Km = 11.6s-1μM-1) at a pH of 10.0 at 75°C for 2,6-DMP oxidization. The protein exhibited high stability in an alkaline environment, with a half-life of more than 12h. The same results were obtained via decolorization of eight dyes. Hence, this laccase-like enzyme may have potential industrial applications.

Valuation of agro-industrial wastes as substrates for heterologous production of \u03b1-galactosidase

BackgroundThe recycling of agro-industrial wastes is at present limited by the availability of efficient and low-cost enzyme cocktails. The use of these materials as culture media to produce the enzymes can contribute to the profitability of the recycling process and to the circular economy. The aim of this work is the construction of a recombinant yeast strain efficient to grow in mixed whey (residue of cheese making) and beet molasses (residue of sugar manufacture) as culture medium, and to produce heterologous α-galactosidase, an enzyme with varied industrial applications and wide market.ResultsThe gene MEL1, encoding the α-galactosidase of Saccharomyces cerevisiae, was integrated (four copies) in the LAC4 locus of the Kluyveromyces lactis industrial strain GG799. The constructed recombinant strain produces high levels of extracellular α-galactosidase under the control of the LAC4 promoter, inducible by lactose and galactose, and the native MEL1 secretion signal peptide. K. lactis produces natively beta-galactosidase and invertase thus metabolizing the sugars of whey and molasses. A culture medium based on whey and molasses was statistically optimized, and then the cultures scaled-up at laboratory level, thus obtaining 19 U/mL of heterologous α-galactosidase with a productivity of 0.158 U/L h, which is the highest value reported hitherto from a cheap waste-based medium.ConclusionsA K. lactis recombinant strain was constructed and a sustainable culture medium, based on a mixture of cheese whey and beet molasses, was optimized for high productivity of S. cerevisiae α-galactosidase, thus contributing to the circular economy by producing a heterologous enzyme from two agro-industrial wastes.

Transcriptomic Insights Into the Growth Phase- and Sugar-Associated Changes in the Exopolysaccharide Production of a High EPS-Producing Streptococcus thermophilus ASCC 1275.

In a previous study, incorporation of high exopolysaccharide (EPS) producing dairy starter bacterium Streptococcus thermophilus ASCC 1275 was found to improve functionality of low fat mozzarella cheese and yogurt. This bacterium in its eps gene cluster has a unique pair of chain length determining genes, epsC- epsD, when compared to other sequenced S. thermophilus strains. Hence, the aim of this study was to understand the regulatory mechanism of EPS production in this bacterium using transcriptomic analysis to provide opportunities to improve the yield of EPS. As sugars are considered as one of the major determinants of EPS production, after preliminary screening, we selected three sugars, glucose, sucrose and lactose to identify the EPS producing mechanism of this bacterium in M17 medium. Complete RNA-seq analysis was performed using Illumina HiSeq 2000 sequencing system on S. thermophilus 1275 grown in three different sugars at two-time points, 5 h (log phase) and 10 h (stationary phase) to recognize the genes involved in sugar uptake, UDP-sugar formation, EPS assembly and export of EPS outside the bacterial cell. S. thermophilus 1275 was found to produce high amount of EPS (∼430 mg/L) in sucrose (1%) supplemented M17 medium when compared to other two sugars. Differential gene expression analysis revealed the involvement of phosphoenolpyruvate phosphotransferase system (PEP-PTS) for glucose and sucrose uptake, and lacS gene for lactose uptake. The pathways for the formation of UDP-glucose and UDP-galactose were highly upregulated in all the three sugars. In the presence of sucrose, eps1C1D2C2D were found to be highly expressed which refers to high EPS production. Protein homology study suggested the presence of Wzx/Wzy-dependent EPS synthesis and transport pathway in this bacterium. KEGG pathway and COG functional enrichment analysis were also performed to support the result. This is the first report providing the transcriptomic insights into the EPS production mechanism of a common dairy bacterium, S. thermophilus.

GlaR (YugA)-a novel RpiR-family transcription activator of the Leloir pathway of galactose utilization in Lactococcus lactis IL1403.

Bacteria can utilize diverse sugars as carbon and energy source, but the regulatory mechanisms directing the choice of the preferred substrate are often poorly understood. Here, we analyzed the role of the YugA protein (now designated GlaR—Galactose–lactose operon Regulatory protein) of the RpiR family as a transcriptional activator of galactose (gal genes) and lactose (lac genes) utilization genes in Lactococcus lactis IL1403. In this bacterium, gal genes forming the Leloir operon are combined with lac genes in a single so‐called gal–lac operon. The first gene of this operon is the lacS gene encoding galactose permease. The glaR gene encoding GlaR lies directly upstream of the gal–lac gene cluster and is transcribed in the same direction. This genetic layout and the presence of glaR homologues in the closest neighborhood to the Leloir or gal–lac operons are highly conserved only among Lactococcus species. Deletion of glaR disabled galactose utilization and abrogated or decreased expression of the gal–lac genes. The GlaR‐dependent regulation of the gal–lac operon depends on its specific binding to a DNA region upstream of the lacS gene activating lacS expression and increasing the expression of the operon genes localized downstream. Notably, expression of lacS‐downstream genes, namely galMKTE, thgA and lacZ, is partially independent of the GlaR‐driven activation likely due to the presence of additional promoters. The glaR transcription itself is not subject to catabolite control protein A (CcpA) carbon catabolite repression (CRR) and is induced by galactose. Up to date, no similar mechanism has been reported in other lactic acid bacteria species. These results reveal a novel regulatory protein and shed new light on the regulation of carbohydrate catabolism in L. lactis IL1403, and by similarity, probably also in other lactococci.

Influence of Ba Substitutions on Microstructure and Magnetic Properties of La–Ca–Co System Permanent Ferrite

La0.6Ca0.4−xBaxO⋅n/2(Fe1−yCoy)2O3(x = 0, 0.05, 0.1, 0.15 and 0.2) permanent ferrite materials were prepared by ceramic technique. The topography and the structure were characterized by scanning electron microscopy (SEM) and x-ray diffraction (XRD), respectively. The magnetic properties were measured using vibrating sample magnetometer (VSM) and digital magnetometer. Single M-phase and flake-like grains were obtained for samples without Ba substitution(x = 0), and the magnetic properties of the pre-sintering La–Ca–Co ferrite were as follows: Br = 440 mT, Hcb = 296 kA/m, Hcj = 426 kA/m, (BH)max = 36.6 kJ/m3 and Hk/Hcj = 0.83. When Ca was substituted by Ba, pre-sintered powder presents single M-phase structure and close three-dimensional size of grains, resulting in an improved rectangle ratio of sintered magnet. The rectangle ratio of sintered magnet reaches a maximum of 0.93 when x = 0.1. By optimizing process parameters, M-type La–Ca–Ba–Co sintered magnet was prepared with Br = 445 mT, Hcb = 337 kA/m, Hcj = 420 kA/m, (BH)max = 38.6 kJ/m3 and Hk/Hcj = 0.95.

Production of microhomologous-mediated site-specific integrated LacS gene cow using TALENs

Gene editing tools (Zinc-Finger Nucleases, ZFN; Transcription Activator-Like Effector Nucleases, TALEN; and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas)9, CRISPR-Cas9) provide us with a powerful means of performing genetic engineering procedures. A combinational approach that utilizes both somatic cell nuclear transfer (SCNT) and somatic cell gene editing facilitates the generation of genetically engineered animals. However, the associated research has utilized markers and/or selected genes, which constitute a potential threat to biosafety. Microhomologous-mediated end-joining (MMEJ) has showed the utilization of micro-homologous arms (5–25 bp) can mediate exogenous gene insertion. Dairy milk is a major source of nutrition worldwide. However, most people are not capable of optimally utilizing the nutrition in milk because of lactose intolerance. Sulfolobus solfataricus β-glycosidase (LacS) is a lactase derived from the extreme thermophilic archaeon Sulfolobus solfataricus. Our finally aim was to site-specific integrated LacS gene into cow's genome through TALEN-mediated MMEJ and produce low-lactose cow. Firstly, we constructed TALENs vectors which target to the cow's β-casein locus and LacS gene expression vector which contain TALEN reorganization sequence and micro-homologous arms. Then we co-transfected these vectors into fetal derived skin fibroblasts and cultured as monoclone. Positive cell clones were screened using 3′ junction PCR amplification and sequencing analysis. The positive cells were used as donors for SCNT and embryo transfer (ET). Lastly, we detected the genotype through PCR of blood genomic DNA. This resulted in a LacS knock-in rate of 0.8% in TALEN-treated cattle fetal fibroblasts. The blastocyst rate of SCNT embryo was 27%. The 3 months pregnancy rate was 20%. Finally, we obtained 1 newborn cow (5%) and verified its genotype. We obtained 1 site-specific marker-free LacS transgenic cow. It provides a basis to solve lactose intolerance by gene engineering breeding. This study also provides us with a new strategy to facilitate gene knock-ins in livestock using techniques that exhibit improved biosafety and intuitive methodologies.

Versatile use of Azospirillum brasilense strains tagged with egfp and mCherry genes for the visualization of biofilms associated with wheat roots

This study reports the introduction of egfp or mCherry markers to the Sp245, Sp7, and M40 wild-type strains of Azospirillum brasilense and the hhkB (encoding for a putative hybrid histidine kinase) minus mutant an isogenic strain of A. brasilense Sp245 to monitor colonization of wheat (Triticum aestivum). Two plasmids were constructed: (1) the pJMS-2 suicide plasmid derived from pSUP202 and harboring the mCherry gene expressed under the constitutive kanamycin resistance promoter to create a cis tag and (2) the broad-range plasmid pMP2449-5 that carries the mCherry gene under the lac promoter, which is derived from the plasmid pMP2444; to create the in trans tag. The stability of the plasmids encoding egfp and mCherry were confirmed in vitro for seven days of bacterial growth, and then, the A. brasilense strains harboring the plasmids were studied under nonselective conditions for adherence to seeds and, at seven or 14 days post-inoculation, for wheat root colonization. The utility of the labeled strains was proven by observation, using fluorescence microscopy and confocal laser scanning microscopy (CLSM) in wheat plants inoculated with the labeled strains and compared with the CFU g–1 for seed and wheat root. The method was suitable for observation of the in situ formation of mini-colonies, enabled visualization of bacterial colonization sites on large root fragments, and showed adherence to germinated seeds and root colonization of all strains by cell counts and direct microscopic examination. Thus, we are able to quantify the structures of the biofilms formed by each strain.

Establishment of a low-dosage-IPTG inducible expression system construction method in Escherichia coli.

The lac operon is a delicate inducible gene expression element in bacteria. To efficiently induce gene expression, a sufficient dosage of an inducer, usually that of 500-1000 µM isopropyl β-D-1-thiogalactopyranoside (IPTG), is required to keep repressor LacI from its binding sites, which is a heavy cost burden in low-value-added products. So we propose a strategy to reduce the required dosage of IPTG by restricting LacI expression. To test this strategy, we employed a reconstructed IPTG inducible expression system based on lac operon, Promoter(lacO)-target gene-PtacL-lacI, where a modified promoter, Ptac, with a random synthetic library (PtacL) to instead of PlacI to optimize LacI expression in Escherichia coli. Finally, the PtacL mutant, PtacL4, which could maintain the same repression effect as the original PlacI while reducing the required dosage of IPTG from 500 to 20 µM, was selected. This method is simple and efficient and can be of a good reference point for attempts to reduce inducer concentration in the IPTG or similar inducible expression systems.

PO-002 Angiotensin II-induced hypertension increases the mutation frequency in the rat kidney

IntroductionEpidemiological studies revealed an increased risk for kidney cancer in hypertensive patients. In many of these patients, the blood pressure regulating renin angiotensin aldosterone system (RAAS) is activated. A stimulated RAAS can lead to oxidative stress and DNA damage, as we have shown both in vitro and in animal models of hypertension. Here, we used a rat model to quantify mutations generated by 20 weeks of angiotensin II-infusion.Material and methodsBigBlue+/- rats, which carry a transgenic lacI gene for mutation analysis, were treated with 0.4 mg angiotensin II/kg/day with the help of osmotic minipumps. Urinary samples were collected in week 15 by placing the rats into metabolic cages. After 20 weeks of treatment the kidneys were isolated and analysed for oxidative stress markers, DNA damage and mutation frequency.Results and discussionsAngiotensin II-treated animals showed significantly increased levels of blood pressure. Urinary and blood parameters of oxidative stress to proteins and nucleobases were increased. Lipoxygenation products were reduced in urine compared to the control animals, pointing to impaired renal excretion of these substances. Oxidative stress and the surrogate marker for DNA double-strand breaks, γ-H2AX, was significantly increased in the renal cortex. Further, a significant elevation of the mutation frequency in kidney DNA was detected. Sequencing results revealed the presence of G->T transversions in the mutated reporter gene of the angiotensin II-treated animals while no such transversions were found in the mutated reporter gene from the control animals. G->T transversions are mutations arising typically from the unrepaired oxidised base modification 8-oxodG.ConclusionThis study shows evidence that the oxidative stress and DNA damage previously observed in vitro and in animal models of hypertension after angiotensin II treatment is indeed associated with the formation of mutations in rat kidneys, providing further evidence for a cancer initiating potential of increased angiotensin II concentrations.

Determining the Transcription Rates Yielding Steady-State Production of mRNA in the Lac Genetic Switch of Escherichia coli.

To elucidate the regulatory dynamics of the gene expression activation and inactivation, an in silico biochemical model of the lac circuit in Escherichia coli was used to evaluate the transcription rates that yield the steady-state mRNA production in active and inactive states of the lac circuit. This result can be used in synthetic biology applications to understand the limits of the genetic synthesis. Since most genetic networks involve many interconnected components with positive and negative feedback control, intuitive understanding of their dynamics is often difficult to obtain. Although the kinetic model of the lac circuit considered involves only a single positive feedback, the developed computational framework can be used to evaluate supported ranges of other reaction rates in genetic circuits with more complex regulatory networks. More specifically, the inducible lac gene switch in E. coli is regulated by unbinding and binding of the inducer-repressor complexes to or from the DNA operator to switch the gene expression on and off. The dependency of mRNA production at steady state on different transcription rates and the repressor complexes has been studied by computer simulations in the Lattice Microbe software. Provided that the lac circuit is in active state, the transcription rate is independent of the inducer-repressor complexes present in the cell. In inactive state, the transcription rate is dependent on the specific inducer-repressor complex bound to the operator that inactivates the gene expression. We found that the repressor complex with the largest affinity to the operator yields the smallest range of the feasible transcription rates to yield the steady state while the lac circuit is in inactive state. In contrast, the steady state in active state can be obtained for any value of the transcription rate.