RESUMO
The occurrence of DNA looping is ubiquitous. This process plays a well-documented role in the regulation of prokaryotic gene expression, such as in regulation of the Escherichia coli lactose (lac) operon. Here we present two complementary methods for high-resolution in vivo detection of DNA/protein binding within the bacterial nucleoid by using either chromatin immunoprecipitation combined with phage λ exonuclease digestion (ChIP-exo) or chromatin endogenous cleavage (ChEC), coupled with ligation-mediated polymerase chain reaction (LM-PCR) and Southern blot analysis. As an example, we apply these in vivo protein-mapping methods to E. coli to show direct binding of architectural proteins in the Lac repressor-mediated DNA repression loop.
Assuntos
Imunoprecipitação da Cromatina , DNA Bacteriano , Escherichia coli , DNA Bacteriano/metabolismo , DNA Bacteriano/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Imunoprecipitação da Cromatina/métodos , Ligação Proteica , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Óperon Lac , Reação em Cadeia da Polimerase/métodos , Southern Blotting , Bacteriófago lambda/genética , Bacteriófago lambda/metabolismoRESUMO
Systems like the prototypical lac operon can reliably hold repression of transcription upon DNA replication across cell cycles with just 10 repressor molecules per cell and behave as if they were at equilibrium. The origin of this phenomenology is still an unresolved question. Here, we develop a general theory to analyze strong perturbations in quasi-equilibrium systems and use it to quantify the effects of DNA replication in gene regulation. We find a scaling law linking actual with predicted equilibrium transcription via a single kinetic parameter. We show that even the lac operon functions beyond the physical limits of naive regulation through compensatory mechanisms that suppress non-equilibrium effects. Synthetic systems without adjuvant activators, such as the cAMP receptor protein (CRP), lack this reliability. Our results provide a rationale for the function of CRP, beyond just being a tunable activator, as a mitigator of cell cycle perturbations.
Assuntos
Ciclo Celular , Replicação do DNA , Ciclo Celular/genética , Óperon Lac , Regulação da Expressão Gênica , Proteína Receptora de AMP Cíclico/metabolismo , Proteína Receptora de AMP Cíclico/genética , Transcrição Gênica , Escherichia coli/genética , Escherichia coli/metabolismo , CinéticaRESUMO
Optogenetics' advancement has made light induction attractive for controlling biological processes due to its advantages of fine-tunability, reversibility, and low toxicity. The lactose operon induction system, commonly used in Escherichia coli, relies on the binding of lactose or isopropyl ß-d-1-thiogalactopyranoside (IPTG) to the lactose repressor protein LacI, playing a pivotal role in controlling the lactose operon. Here, we harnessed the light-responsive light-oxygen-voltage 2 (LOV2) domain from Avena sativa phototropin 1 as a tool for light control and engineered LacI into two light-responsive variants, OptoLacIL and OptoLacID. These variants exhibit direct responsiveness to light and darkness, respectively, eliminating the need for IPTG. Building upon OptoLacI, we constructed two light-controlled E. coli gene expression systems, OptoE.coliLight system and OptoE.coliDark system. These systems enable bifunctional gene expression regulation in E. coli through light manipulation and show superior controllability compared to IPTG-induced systems. We applied the OptoE.coliDark system to protein production and metabolic flux control. Protein production levels are comparable to those induced by IPTG. Notably, the titers of dark-induced production of 1,3-propanediol (1,3-PDO) and ergothioneine exceeded 110% and 60% of those induced by IPTG, respectively. The development of OptoLacI will contribute to the advancement of the field of optogenetic protein engineering, holding substantial potential applications across various fields.
Assuntos
Escherichia coli , Isopropiltiogalactosídeo , Óperon Lac , Repressores Lac , Luz , Optogenética , Isopropiltiogalactosídeo/farmacologia , Repressores Lac/metabolismo , Repressores Lac/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/efeitos da radiação , Optogenética/métodos , Regulação Bacteriana da Expressão Gênica/efeitos da radiação , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Engenharia de Proteínas/métodos , Avena/genética , Avena/metabolismo , Avena/efeitos da radiaçãoRESUMO
HMG (high mobility group) proteins are a diverse family of nonhistone chromosomal proteins that interact with DNA and a wide range of transcriptional regulators to regulate the structural architecture of DNA. HMGXB4 (also known as HMG2L1) is an HMG protein family member that contains a single HMG box domain. Our previous studies have demonstrated that HMGXB4 suppresses smooth muscle differentiation and exacerbates endotoxemia by promoting a systemic inflammatory response in mice. However, the expression of Hmgxb4 in vivo has not fully examined. Herein, we generated a mouse model that harbors a gene trap in the form of a lacZ gene insertion into the Hmgxb4 gene. This mouse enables the visualization of endogenous HMGXB4 expression in different tissues via staining for the ß-galactosidase activity of LacZ which is under the control of the endogenous Hmgxb4 gene promoter. We found that HMGXB4 is widely expressed in mouse tissues and is a nuclear protein. Furthermore, the Hmgxb4 gene trap mice exhibit normal cardiac function and blood pressure. Measurement of ß-galactosidase activity in the Hmgxb4 gene trap mice demonstrated that the arterial injury significantly induces Hmgxb4 expression. In summary, the Hmgxb4 gene trap reporter mouse described here provides a valuable tool to examine the expression level of endogenous Hmgxb4 in both physiological and pathological settings in vivo.
Assuntos
Proteínas de Grupo de Alta Mobilidade , Camundongos Endogâmicos C57BL , Animais , Masculino , Camundongos , beta-Galactosidase/metabolismo , beta-Galactosidase/genética , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Óperon Lac/genética , Camundongos Transgênicos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Proteins that are kinetically stable are thought to be less prone to both aggregation and proteolysis. We demonstrate that the classical lac system of Escherichia coli can be leveraged as a model system to study this relation. ß-galactosidase (LacZ) plays a critical role in lactose metabolism and is an extremely stable protein that can persist in growing cells for multiple generations after expression has stopped. By attaching degradation tags to the LacZ protein, we find that LacZ can be transiently degraded during lac operon expression but once expression has stopped functional LacZ is protected from degradation. We reversibly destabilize its tetrameric assembly using α-complementation, and show that unassembled LacZ monomers and dimers can either be degraded or lead to formation of aggregates within cells, while the tetrameric state protects against proteolysis and aggregation. We show that the presence of aggregates is associated with cell death, and that these proteotoxic stress phenotypes can be alleviated by attaching an ssrA tag to LacZ monomers which leads to their degradation. We unify our findings using a biophysical model that enables the interplay of protein assembly, degradation, and aggregation to be studied quantitatively in vivo. This work may yield approaches to reversing and preventing protein-misfolding disease states, while elucidating the functions of proteolytic stability in constant and fluctuating environments.
Assuntos
Escherichia coli , Óperon Lac , Proteólise , beta-Galactosidase , beta-Galactosidase/metabolismo , Escherichia coli/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Agregados Proteicos , Estabilidade EnzimáticaRESUMO
Pasteurella multocida is a zoonotic conditional pathogen that infects multiple livestock species, causing substantial economic losses in the animal husbandry industry. An efficient markerless method for gene manipulation may facilitate the investigations of P. multocida gene function and pathogenesis of P. multocida. Herein, a temperature-sensitive shuttle vector was constructed using lacZ as a selection marker, and markerless glgB, opa, and hyaE mutants of P. multocida were subsequently constructed through blue-white colony screening. The screening efficiency of markerless deletion strains was improved by the lacZ system, and the method could be used for multiple gene deletions. However, the fur mutant was unavailable via this method. Therefore, we constructed a pheSm screening system based on mutated phenylalanine tRNA synthetase as a counterselection marker to achieve fur deletion mutant. The transformed strain was sensitive to 20 mM p-chloro-phenylalanine, demonstrating the feasibility of pheSm as a counter-selective marker. The pheSm system was used for markerless deletions of glgB, opa, and hyaE as well as fur that could not be screened by the lacZ system. A comparison of screening efficiencies of the system showed that the pheSm counterselection system was more efficient than the lacZ system and broadly applicable for mutant screening. The methods developed herein may provide valuable tools for genetic manipulation of P. multocida.IMPORTANCEPasteurella multocida is a highly contagious zoonotic pathogen. An understanding of its underlying pathogenic mechanisms is of considerable importance and requires efficient species-specific genetic tools. Herein, we propose a screening system for P. multocida mutants using lacZ or pheSm screening markers. We evaluated the efficiencies of both systems, which were used to achieve markerless deletion of multiple genes. The results of this study support the use of lacZ or pheSm as counterselection markers to improve counterselection efficiency in P. multocida. This study provides an effective genetic tool for investigations of the virulence gene functions and pathogenic mechanisms of P. multocida.
Assuntos
Pasteurella multocida , Animais , Pasteurella multocida/genética , Óperon Lac , Vetores Genéticos , FenilalaninaRESUMO
Bis(2-ethylhexyl) phthalate, generally known as DEHP is a synthetic compound mainly used as a plasticizer to make polyvinyl chloride products flexible and soft. The present work aimed to study the toxicity of Bis(2-ethylhexyl) phthalate on the third instar larvae of transgenic Drosophila melanogaster(hsp70-lacZ) Bg9. The hsp70 gene is associated with the ß-galactosidase in our present transgenic strain therefore, the more activity of ß-galactosidase will indirectly correspond to hsp70 expression. The third instar larvae were allowed to feed on the diet for 24 h having 0.001, 0.005, 0.01, and 0.02 M of Bis(2-ethylhexyl) phthalate at the final concentration. After the exposure of 24hrs, the larvae were subjected to ONPG assay, X-gal staining, trypan blue exclusion test, oxidative stress markers assays, and comet assay. A dose-dependent increase in hsp70 expression, tissue damage, Glutathione-S-transferase (GST) activity, lipid peroxidation, monoamine oxidase, caspase-9 & 3, protein carbonyl content (PCC), DNA damage and decrease in the glutathione (GSH) content, delta-aminolevulinic acid dehydrogenase (áº-ALD-D) and acetylcholinesterase activity were observed in the larvae exposed to 0.005, 0.01, 0.02 M of Bis-(2-ethylhexyl) phthalate. The dose of 0.001 M of Bis(2-ethylhexyl) phthalate did not showed any toxic effects and hence can be considered as No Observed Adverse Effect Level (NOAEL) for Bis(2-ethylhexyl) phthalate. The study supports the use of Drosophila for the evaluation of possible toxic effects associated with synthetic compounds.
Assuntos
Dietilexilftalato , Drosophila melanogaster , Ácidos Ftálicos , Animais , Carbonilação Proteica , Larva , Óperon Lac , Acetilcolinesterase/metabolismo , Animais Geneticamente Modificados/metabolismo , Drosophila , Glutationa/metabolismo , beta-Galactosidase/metabolismo , Dietilexilftalato/metabolismoRESUMO
Laccase genes produce laccase enzymes that play a crucial role in the production of lignin and oxidation reactions within plants. Lignin is a complex polymer that provides structure and toughness to the cell walls of numerous fruit plants. The LAC genes that encode laccase enzymes play vital roles in plant physiology, including the synthesis of pigments like PA that contribute to the colors of fruits, and in defending against pathogens and environmental stresses. They are crucial for fruit development, ripening, structural maintenance in plants, and adaptation to various environmental factors. As such, these genes and enzymes are essential for plant growth and development, as well as for various biotechnological applications in environmental remediation and industrial processes. This review article emphasizes the significance of genes encoding laccase enzymes during fruit growth, specifically pertaining to the strengthening of the endocarp through lignification. This process is crucial for ensuring fruit defense and optimizing seed scattering. The information gathered in this article will aid breeders in producing future fruit-bearing plants that are resistant to disease, cost-effective, and nutrient-rich.
Assuntos
Frutas , Lignina , Lignina/metabolismo , Lacase/metabolismo , Óperon Lac , Sementes/metabolismo , Regulação da Expressão Gênica de PlantasRESUMO
High-resolution NMR spectroscopy enabled us to characterize allosteric transitions between various functional states of the dimeric Escherichia coli Lac repressor. In the absence of ligands, the dimer exists in a dynamic equilibrium between DNA-bound and inducer-bound conformations. Binding of either effector shifts this equilibrium toward either bound state. Analysis of the ternary complex between repressor, operator DNA, and inducer shows how adding the inducer results in allosteric changes that disrupt the interdomain contacts between the inducer binding and DNA binding domains and how this in turn leads to destabilization of the hinge helices and release of the Lac repressor from the operator. Based on our data, the allosteric mechanism of the induction process is in full agreement with the well-known Monod-Wyman-Changeux model.
Assuntos
Proteínas de Escherichia coli , Repressores Lac/genética , Repressores Lac/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulação Alostérica/genética , Escherichia coli/metabolismo , DNA/metabolismo , Estrutura Secundária de Proteína , Óperon Lac/genéticaRESUMO
BACKGROUND: Hydroxyapatite (HAp) presents similarities with the human bone structure and presents properties such as biodegradability, biocompatibility, and osteoconductivity, which favors its use in prostheses implants and enables its use as a vehicle for the delivery of photosensitizers (PS) from systems of release (DDS) for photodynamic therapy applications Methods: In this work was to synthesized hydroxyapatite microspheres (meHAp), encapsulated with chloroaluminium phthalocyanine (ClAlPc), for DDS. meHAp was synthesized using vaterite as a template. The drug was encapsulated by mixing meHAp and a 50.0 mg.mL-1 ClAlPc solution. Photochemical, photophysical, and photobiological studies characterized the system. RESULTS: The images from the SEM analysis showed the spherical form of the particles. All spectroscopic results showed excellent photophysical parameters of the drug studied when served in the meHAp system. The incorporation efficiency was 57.8 %. The trypan blue exclusion test results showed a significant reduction (p < 0.05) in cell viability for the groups treated with PDT at all concentrations above 250 µg.mL-1. In 9 L/lacZ gliosarcoma cells, PDT mediated at concentrations from 250 to 62.5 µg.mL-1 reduced cell viability by more than 98 %. In the cell internalization study, it was possible to observe the internalization of phthalocyanines at 37 °C, with the accumulation of PS in the cytoplasm and inside the nucleus in the two tested concentrations. CONCLUSIONS: From all the results presented throughout the article, the meHAp system shows promise for use as a modified release system (DSD) in photodynamic therapy.
Assuntos
Gliossarcoma , Fotoquimioterapia , Humanos , Fármacos Fotossensibilizantes , Fotoquimioterapia/métodos , Durapatita , Óperon Lac , Microesferas , Sistemas de Liberação de MedicamentosRESUMO
Mutagenicity testing is an essential component of health safety assessment. Duplex Sequencing (DS), an emerging high-accuracy DNA sequencing technology, may provide substantial advantages over conventional mutagenicity assays. DS could be used to eliminate reliance on standalone reporter assays and provide mechanistic information alongside mutation frequency (MF) data. However, the performance of DS must be thoroughly assessed before it can be routinely implemented for standard testing. We used DS to study spontaneous and procarbazine (PRC)-induced mutations in the bone marrow (BM) of MutaMouse males across a panel of 20 diverse genomic targets. Mice were exposed to 0, 6.25, 12.5, or 25 mg/kg-bw/day for 28 days by oral gavage and BM sampled 42 days post-exposure. Results were compared with those obtained using the conventional lacZ viral plaque assay on the same samples. DS detected significant increases in mutation frequencies and changes to mutation spectra at all PRC doses. Low intra-group variability within DS samples allowed for detection of increases at lower doses than the lacZ assay. While the lacZ assay initially yielded a higher fold-change in mutant frequency than DS, inclusion of clonal mutations in DS mutation frequencies reduced this discrepancy. Power analyses suggested that three animals per dose group and 500 million duplex base pairs per sample is sufficient to detect a 1.5-fold increase in mutations with > 80% power. Overall, we demonstrate several advantages of DS over classical mutagenicity assays and provide data to support efforts to identify optimal study designs for the application of DS as a regulatory test.
Assuntos
Medula Óssea , Taxa de Mutação , Masculino , Camundongos , Animais , Procarbazina/toxicidade , Mutagênicos/toxicidade , Mutação , Testes de Mutagenicidade/métodos , Camundongos Transgênicos , Óperon LacRESUMO
Agents that suppress the toxic effect of arecoline (a chemical present in the Areca nut fruit) have become a need of the hour owing to its several harmful effects on human beings. Although some drug molecules have been developed for this purpose, yet, simple, easy to prepare, and economical molecules with remarkable potency are still a challenge to design. The present work thus becomes important as it involves the synthesis of a new charge transfer complex (CTC) material, which has, for the first time, been screened to investigate its effect on the toxic effects of arecoline. The newly designed material (CL), which is generated from the reaction between 2,4,6-trinitrophenol (TNP) and pyrazole (PYZ), has been crystallized by a slow evaporation method and characterized by employing spectral studies including single crystal X-ray crystallography. Spectrophotometry studies with the inclusion of the Benesi-Hildebrand equation reveal 1 : 1 stoichiometry and physical parameters of CL. Assays were used for determining the protective effect of CL against arecoline. CL was found to (dose-dependently) decrease ß-galactosidase activity, damage in tissue and DNA damage caused by arecoline (80 µM) in the third-instar larvae of the transgenic Drosophila melanogaster (hsp70-lacZ)Bg9. The possible mechanism of this effect was explored through fluorescence and UV-vis spectroscopy. The possibility of suppression of arecoline action on the muscarinic acetylcholine receptor 1-G11 protein complex (found in the cell membrane) in the presence of CL was studied theoretically by molecular docking. Density functional theory (DFT) also theoretically supported various aspects of the designed material concerning the energy profile of the orbitals (HOMO-LUMO) as well as the energy minimized structure. Furthermore, time dependent (TD) DFT corroborated the electronic properties of the designed material.
Assuntos
Arecolina , Drosophila melanogaster , Animais , Animais Geneticamente Modificados , Arecolina/toxicidade , Drosophila melanogaster/metabolismo , Proteínas de Choque Térmico HSP70/metabolismo , Óperon Lac , Larva , Simulação de Acoplamento Molecular , Tomografia Computadorizada por Raios XRESUMO
Reverse genetics (RG) systems have been instrumental for determining the molecular aspects of viral replication, pathogenesis, and for the development of therapeutics. Here, we demonstrate that genes encoding the influenza surface antigens hemagglutinin and neuraminidase have varying stability when cloned into a common RG plasmid and transformed into Escherichia coli. Using GFP as a reporter, we demonstrate that E. coli expresses the target genes in the RG plasmid at low levels. Incorporating lac operators or a transcriptional terminator into the plasmid reduced expression and stabilized the viral genes to varying degrees. Sandwiching the viral gene between two lac operators provided the largest contribution to stability and we confirmed the stabilization is Lac repressor-dependent and crucial for subsequent plasmid propagations in E. coli. Viruses rescued from the lac operator-stabilized plasmid displayed similar kinetics and titers to the original plasmid in two different viral backbones. Together, these results indicate that silencing transcription from the plasmid in E. coli helps to maintain the correct influenza gene sequence and that the lac operator addition does not impair virus production. It is envisaged that sandwiching DNA segments between lac operators can be used for reducing DNA segment instability in any plasmid that is propagated in E. coli which express the Lac repressor.
Assuntos
Escherichia coli , Influenza Humana , Humanos , Escherichia coli/genética , Escherichia coli/metabolismo , Repressores Lac/genética , Genética Reversa , Plasmídeos/genética , Óperon LacRESUMO
Undergraduate student engagement in research increases retention and degree completion, especially for students who are underrepresented in science. Several approaches have been adopted to increase research opportunities including curriculum based undergraduate research opportunities (CUREs), in which research is embedded into course content. Here we report on efforts to tackle a different challenge: providing research opportunities to students engaged in remote learning or who are learning at satellite campuses or community colleges with limited research infrastructure. In our project we engaged students learning remotely or at regional centers to map gene expression in the mouse brain. In this project we mapped expression of the Down syndrome cell adhesion molecule like 1 (Dscaml1) gene in mouse brain using a LacZ expression reporter line. Identifying where Dscaml1 is expressed in the brain is an important next step in determining if its roles in development and function in the retina are conserved in the rest of the brain. Students working remotely reconstruct brain montages and annotated Dscaml1 expression in the brain of mice carrying one or two copies of the gene trap. We built on these findings by further characterizing Dscaml1 expression in inhibitory neurons of the visual pathway. These results build on and extend previous findings and demonstrate the utility of including distance learners in an active research group for both the student learners and the research team. We conclude with best practices we have developed based on this and other distance learner focused projects.
Assuntos
Neurônios , Estudantes , Animais , Camundongos , Humanos , Óperon Lac , Neurônios/metabolismo , Retina , Encéfalo , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismoRESUMO
Glioma-associated oncogene homolog 1 (Gli1) marks a subpopulation of endogenous mesenchymal stem cells (MSCs) characterized by perivascular location. Here, we present an optimized immunofluorescence staining protocol to identify resident Gli1+ MSCs in fixed/frozen bone sections from LacZ transgenic mice. This protocol describes the preparation of fixed/frozen tissue sections and the use of LacZ immunofluorescent staining for the in vivo characterization of endogenous MSCs, regarding their specific identity and specialized niches, and is applicable to LacZ-expressing cells of diverse organs. For complete details on the use and execution of this protocol, please refer to Chen et al. (2020).
Assuntos
Células-Tronco Mesenquimais , Camundongos , Animais , Camundongos Transgênicos , Óperon Lac , Proteína GLI1 em Dedos de Zinco , Coloração e Rotulagem , ImunofluorescênciaRESUMO
Lac operon is the standard regulator used to control the orthogonality of T7RNA polymerase (T7RNAP) and T7 promoter inEscherichia coli BL21(DE3) strain for protein expression. However,E. coliNissle 1917 (EcN), the unique probiotic strain, has seldom been precisely adapted to the T7 system. Herein, we applied bioinformatics analysis on Lac operon from different strains, and it was observed that a weak promoter for LacI repressor existed in EcN. Furthermore, X-gal assay revealed a strong expression of lacZ in EcN. We demonstrated that Lac operon significantly affected the protein expression in the two T7-derived EcN, in which T7RNAP was integrated at lambda (ET7L) and HK022 (ET7H), respectively. Different combinations of replication origin, chaperonin GroELS, inducer, and medium were explored to fine-tune the best strain with tyrosine ammonia-lyase (TAL) for p-coumaric acid (pCA) production, which is one of the essential bioactive compounds for human health. Finally, the highest pCA conversion of 78.8% was achieved using RRtL (plasmid form) under the optimum condition, and a 51.5% conversion was obtained with L::Rt strain which has integrated T7-RtTAL at HK022 of ET7L in the simulated gut environment. The appropriate reprogramming of T7RNAP expedites EcN as an effective and promising cell factory for live bacterial therapeutics in the future.
Assuntos
RNA Polimerases Dirigidas por DNA , Escherichia coli , Humanos , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Óperon Lac , Óperon/genéticaRESUMO
DNA looping has emerged as a central paradigm of transcriptional regulation, as it is shared across many living systems. One core property of DNA looping-based regulation is its ability to greatly enhance repression or activation of genes with only a few copies of transcriptional regulators. However, this property based on a small number of proteins raises the question of the robustness of such a mechanism with respect to the large intracellular perturbations taking place during growth and division of the cell. Here we address the issue of sensitivity to variations of intracellular parameters of gene regulation by DNA looping. We use the lac system as a prototype to experimentally identify the key features of the robustness of DNA looping in growing Escherichia coli cells. Surprisingly, we observe time intervals of tight repression spanning across division events, which can sometimes exceed 10 generations. Remarkably, the distribution of such long time intervals exhibits memoryless statistics that is mostly insensitive to repressor concentration, cell division events, and the number of distinct loops accessible to the system. By contrast, gene regulation becomes highly sensitive to these perturbations when DNA looping is absent. Using stochastic simulations, we propose that the observed robustness to division emerges from the competition between fast, multiple rebinding events of repressors and slow initiation rate of the RNA polymerase. We argue that fast rebinding events are a direct consequence of DNA looping that ensures robust gene repression across a range of intracellular perturbations.
Assuntos
Divisão Celular , DNA Bacteriano , Óperon Lac , Divisão Celular/genética , DNA Bacteriano/química , Escherichia coli/crescimento & desenvolvimento , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Repressores Lac/genética , Repressores Lac/metabolismo , Conformação de Ácido Nucleico , Análise de Célula ÚnicaRESUMO
Here, we describe an in vivo approach to visualize CD11c+ cells in atherosclerosis. In particular, we use a protocol for X-Gal staining of immune cells within atherosclerotic plaques, which can be used as an alternative to analyze plaque composition and cell-specific molecules in atherogenesis. LacZ knockin mice have to be bred to mice carrying the CD11ccre recombinase-both brought onto an ApoE-/- background-to be able to visualize this cell type of interest in the plaques by X-Gal staining. With this approach, different immune cells in atherogenesis can be examined. For complete details on the use and execution of this protocol, please refer to Sauter et al. (2021).
Assuntos
Aterosclerose , Placa Aterosclerótica , Animais , Aterosclerose/genética , Antígeno CD11c/genética , Óperon Lac/genética , Camundongos , Camundongos Knockout , Placa Aterosclerótica/genéticaRESUMO
Lactose utilization is one of the general biochemical characteristics of Escherichia coli, and the lac operon is responsible for this phenotype, which can be detected on lactose-containing media, such as MacConkey agar, after 24 h of incubation. However, some Shiga toxin-producing E. coli (STEC) O121:H19 strains exhibit an unusual phenotype called delayed lactose utilization (DLU), in which lactose utilization can be detected after 48 h of cultivation but not after only 24 h of cultivation. Insertion of an insertion sequence (IS), IS600, into the lacZ gene appears to be responsible for the DLU phenotype, and exposure to lactose has been reported to be necessary to observe this phenotype, but the mechanism underlying these phenomena remains to be elucidated. Here, we performed detailed analyses of the lactose utilization abilities of a set of O121:H19 strains and their mutants and found that IS-excision enhancer (IEE)-mediated excision of IS600 reactivates the lacZ gene and that the selective proliferation of IS-cured subclones in lactose-supplemented culture medium is responsible for the expression of the DLU phenotype. In addition, we analyzed the patterns of IS insertion into the lacZ and iee genes in the global O121:H19 population and revealed that while there are O121:H19 strains or lineage/sublineages that contain the IS insertion into iee or intact lacZ and thus do not show the DLU phenotype, most currently circulating O121:H19 strains contain IS600-inserted lacZ and intact iee and thus exhibit this phenotype. IMPORTANCE Insertion sequences (ISs) can modulate gene expression by gene inactivation or activation. While phenotypic changes due to IS insertion/transposition are frequently observed, gene reactivation by precise or simple IS excision rarely occurs. In this study, we show that IS600 is excised from the lacZ gene by IS-excision enhancer (IEE) during the cultivation of Shiga toxin-producing Escherichia coli (STEC) O121:H19 strains that show an unusual phenotype called delayed lactose utilization (DLU). This excision rescued their lactose utilization defect, and the subsequent selective proliferation of IS-cured subclones in lactose-containing medium resulted in the expression of the DLU phenotype. As we also show that most currently circulating O121:H19 strains exhibit this phenotype, this study not only provides information helpful for the isolation and identification of O121:H19 STEC but also offers novel insights into the roles of IS and IEE in the generation of phenotypic variation in bacterial populations.
Assuntos
Proteínas de Escherichia coli , Lactose , Escherichia coli Shiga Toxigênica , Elementos de DNA Transponíveis , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Óperon Lac , Lactose/metabolismo , Fenótipo , Toxina Shiga/genética , Escherichia coli Shiga Toxigênica/genéticaRESUMO
Here, we describe a free, web-based simulation of the lac operon, "LacOp," that is designed to enhance the learning of prokaryotic gene regulation and pathways in advanced high school and undergraduate genetics courses. This new electronic resource was created by a team of students in an advanced undergraduate course and is hosted online (http://flask-env.rnwhymamqf.us-west-2.elasticbeanstalk.com/lacop). LacOp has a simple web interface compatible with a range of devices, including smartphones. To determine whether the LacOp simulation enhances student learning from traditional instruction, we introduced the lac operon to undergraduate genetics students through a traditional classroom experience followed by use of the LacOp simulation. Students worked on their own using the included tutorial to create and test the effect of various genotypes on E. coli lactose metabolism and regulation. Upon completion of the tutorial, students showed measurable gains in conceptual understanding of the lac operon. These students also reported a generally favorable opinion of the LacOP simulation as a use of their instructional time.