Bacterial expression system with tightly regulated gene expression and plasmid copy number

A new Escherichia coli host/vector system has been engineered to allow tight and uniform modulation of gene expression and gamma origin (ori) plasmid copy number. Regulation of gamma ori plasmid copy number is achieved through arabinose-inducible expression of the necessary Rep protein, pi, whose gene was integrated into the chromosome of the host strain under control of the P(BAD) promoter. gamma ori replication can be uniformly modulated over 100-fold by changing the concentration of l-arabinose in the growth medium. This strain avoids the problem of all-or-nothing induction of P(BAD) because it is deficient in both arabinose uptake and degradation genes. Arabinose enters the cell by a mutant LacY transporter, LacYA177C, which is expressed from the host chromosome. Although this strain could be compatible with any gamma ori plasmid, we describe the utility of a gamma ori expression vector that allows especially tight regulation of gene expression. With this host/vector system, it is possible to independently modulate gene expression and gene dosage, facilitating the cloning and overproduction of toxic gene products. We describe the successful use of this system for cloning a highly potent toxin, Colicin E3, in the absence of its cognate immunity protein. This system could be useful for cloning genes encoding other potent toxins, screening libraries for potential toxins, and maintaining any gamma ori vector at precise copy levels in a cell.     

New methods for tightly regulated gene expression and highly efficient chromosomal integration of cloned genes for Methanosarcina species

A highly efficient method for chromosomal integration of cloned DNA into Methanosarcina spp. was developed utilizing the site-specific recombination system from the Streptomyces phage φC31. Host strains expressing the φC31 integrase gene and carrying an appropriate recombination site can be transformed with non-replicating plasmids carrying the complementary recombination site at efficiencies similar to those obtained with self-replicating vectors. We have also constructed a series of hybrid promoters that combine the highly expressed M. barkeri PmcrB promoter with binding sites for the tetracycline-responsive, bacterial TetR protein. These promoters are tightly regulated by the presence or absence of tetracycline in strains that express the tetR gene. The hybrid promoters can be used in genetic experiments to test gene essentiality by placing a gene of interest under their control. Thus, growth of strains with tetR-regulated essential genes becomes tetracycline-dependent. A series of plasmid vectors that utilize the site-specific recombination system for construction of reporter gene fusions and for tetracycline regulated expression of cloned genes are reported. These vectors were used to test the efficiency of translation at a variety of start codons. Fusions using an ATG start site were the most active, whereas those using GTG and TTG were approximately one half or one fourth as active, respectively. The CTG fusion was 95% less active than the ATG fusion.     

Inducible,tightly regulated and non-leaky neuronal gene expression in mice

The Tetracycline (Tet)-controlled inducible system is the most widely used reversible system for transgene expression in mice with over 500 lines created to date. Although this system has been optimized over the years, it still has limitations such as residual transgene expression when turned off, referred to as leakiness. Here, we present a series of new Tet-OFF transgenic mice based on the second generation tetracycline-responsive transactivator system. The tTA-Advanced (tTA2^S) is expressed under control of the neuron-specific Thy1.2 promoter (Thy-OFF), to regulate expression in the mouse brain. In addition, we generated a lacZ reporter line, utilizing the P _tight Tet-responsive promoter (P _tight–lacZ), to test our system. Two Thy-OFF transgenic lines displaying two distinct patterns of expression were selected. Oral doxycycline treatment of Thy-OFF/P _tight–lacZ mice demonstrated tight transgene regulation with no leak expression. These new Thy-OFF mice are valuable for studies in a broad range of neurodegenerative diseases such as Alzheimer’s disease and related forms of dementia, where control of transgene expression is critical to understanding mechanisms underlying the disease. Furthermore, P _tight–lacZ reporter mice may be widely applicable.     

An expression vector containing a rhamnose-inducible promoter provides tightly regulated gene expression in Burkholderia cenocepacia

Infection of the respiratory tract caused by Burkholderia cepacia complex poses a serious risk for cystic fibrosis (CF) patients due to the high morbidity and mortality associated with the chronic infection and the lack of efficacious antimicrobial treatments. A detailed understanding of the pathogenicity of B. cepacia complex infections is hampered in part by the limited availability of genetic tools and the inherent resistance of these isolates to the most common antibiotics used for genetic selection. In this study, we report the construction of an expression vector which uses the rhamnose-regulated P(rhaB) promoter of Escherichia coli. The functionality of the vector was assessed by expressing the enhanced green fluorescent protein (eGFP) gene (e-gfp) and determining the levels of fluorescence emission. These experiments demonstrated that P(rhaB) is responsive to low concentrations of rhamnose and it can be effectively repressed with 0.2% glucose. We also demonstrate that the tight regulation of gene expression by P(rhaB) promoter allows us to extend the capabilities of this vector to the identification of essential genes.     

Development of a tightly regulated and highly inducible ecdysone receptor gene switch for plants through the use of retinoid X receptor chimeras

Chemical inducible gene regulation systems provide essential tools for the precise regulation of transgene expression in plants and animals. Recent development of a two-hybrid ecdysone receptor (EcR) gene regulation system has solved some of the drawbacks that were associated with the monopartate gene switch. To further improve the versatility of the two-hybrid EcR gene switch for wide spread use in plants, chimeras between Homo sapiens retinoid X receptor (HsRXR) and insect, Locusta migratoria RXR (LmRXR) were tested in tobacco protoplasts as partners with Choristoneura fumiferana EcR (CfEcR) in inducing expression of the luciferase reporter gene. The RXR chimera 9 (CH9) along with CfEcR, in a two-hybrid format gave the best results in terms of low-background expression levels in the absence of ligand and high-induced expression levels of the reporter gene in the presence of nanomolar concentrations of the methoxyfenozide ligand. The performance of CH9 was further tested in corn and soybean protoplasts and the data obtained was compared with the other EcR switches that contained the wild-type LmRXR or HsRXR as EcR partners. In both transient expression studies and stable transformation experiments, the fold induction values obtained with the CH9 switch were several times higher than the values obtained with the other EcR switches containing LmRXR or HsRXR. The new CfEcR two-hybrid gene switch that uses the RXR CH9 as a partner in inducing reporter gene expression provides an efficient, ligand-sensitive and tightly regulated gene switch for plants.     

A tightly regulated and reversibly inducible siRNA expression system for conditional RNAi-mediated gene silencing in mammalian cells

BACKGROUND: RNA interference (RNAi) is a powerful and widely used gene silencing strategy for studying gene function in mammalian cells. Transient or constitutive expression of either small interfering RNA (siRNA) or short hairpin RNA (shRNA) results in temporal or persistent inhibition of gene expression, respectively. A tightly regulated and reversibly inducible RNAi-mediated gene silencing approach could conditionally control gene expression in a temporal or spatial manner that provides an extremely useful tool for studying gene function involved in cell growth, survival and development. MATERIAL AND METHODS: In this study, we have developed a lactose analog isopropyl thiogalactose (IPTG)-responsive lac repressor-operator-controlled RNA polymerase III (Pol III)-dependent human RNase P RNA (H1) promoter-driven inducible siRNA expression system. To demonstrate its tight regulation, efficient induction and reversible inhibition, we have used this system to conditionally control the expression of firefly luciferase and human tumor suppressor protein p53 in both transient transfection cells and established stable clones. RESULTS: The results showed that this inducible siRNA expression system could efficiently induce conditional inhibition of these two genes in a dose- and time-dependent manner by administration of the inducing agent IPTG as well as being fully reverted after withdrawal of IPTG. In particular, this system could conditionally inhibit the expression of both the genes in not only established stable clones but also transient transfection cells, which should greatly increase its usefulness and convenience. CONCLUSIONS: The results presented in this study clearly indicate that this inducible siRNA expression system could efficiently, conditionally and reversibly inhibit gene expression with only very low or undetectable background silencing effects under non-inducing condition. Thus, this inducible siRNA expression system provides an ideal genetic switcher allowing the inducible and reversible control of specific gene activity in mammalian cells.     

Discovering tightly regulated and differentially expressed gene sets in whole genome expression data

MOTIVATION: Recently, a new type of expression data is being collected which aims to measure the effect of genetic variation on gene expression in pathways. In these datasets, expression profiles are constructed for multiple strains of the same model organism under the same condition. The goal of analyses of these data is to find differences in regulatory patterns due to genetic variation between strains, often without a phenotype of interest in mind. We present a new method based on notions of tight regulation and differential expression to look for sets of genes which appear to be significantly affected by genetic variation. RESULTS: When we use categorical phenotype information, as in the Alzheimer's and diabetes datasets, our method finds many of the same gene sets as gene set enrichment analysis. In addition, our notion of correlated gene sets allows us to focus our efforts on biological processes subjected to tight regulation. In murine hematopoietic stem cells, we are able to discover significant gene sets independent of a phenotype of interest. Some of these gene sets are associated with several blood-related phenotypes. AVAILABILITY: The programs are available by request from the authors.     

Toxic protein expression in Escherichia coli using a rhamnose-based tightly regulated and tunable promoter system

The refinement of tightly regulated prokaryotic expression systems that permit functional expression of toxic recombinant proteins is a continually evolving process. Unfortunately, the current best promoter options are either tightly repressed and produce little protein, or produce substantial protein but lack the necessary repression to avoid mutations stimulated by leaky expression in the absence of inducer. In this report, we present three novel prokaryotic expression constructs that are tightly regulated by L-rhamnose and D-glucose. These expression vectors utilize the Escherichia coli rhaT promoter and corresponding regulatory genes to provide titratable, high-level protein yield without compromising clone integrity. Together, these components may enable the stable cloning and functional expression of otherwise toxic proteins.     

The estrogen receptor binds tightly to its responsive element as a ligand-induced homodimer

Extracts containing wild-type or mutant human estrogen receptor (ER) have been used to study the binding of ER to its responsive element (ERE). Estradiol (E2) or the antiestrogen hydroxytamoxifen is required for ER binding as assayed by gel retardation. The DNA binding domain (DBD) encompasses the highly conserved region C. Both intact ER-E2 complexes and ER mutants truncated for the hormone binding domain (HBD) bind as dimers to an ERE. However, an HBD-truncated ER binds less tightly to an ERE than an intact ER-E2 complex. The DBD and HBD contain a constitutive and a stronger ER-induced dimerization function, respectively. Thus, in addition to inducing the activation function associated with the HBD, estrogen plays a crucial role in the formation of stable ER dimers that bind tightly to ERE.     

Development of a tightly regulatable copper-mediated gene switch system in dermatophytes

Targeted gene deletion is now available for molecular genetic research of dermatophytes, and the physiological roles of several genes have been elucidated. However, this method cannot be applied to essential genes, which can be potential drug targets. To overcome this limitation, we have developed a conditional gene knockdown system using a copper-responsive promoter. The promoter sequence of the copper transporter gene CTR4 (P(CTR4)) and that of the copper efflux pump gene CRP1 (P(CRP1)) derived from Trichophyton rubrum were examined for their response to copper in Arthroderma vanbreuseghemii. P(CTR4) was demonstrated to repress expression of a reporter gene in the presence of copper, while the activity of P(CRP1) was induced by addition of copper. Importantly, P(CTR4) regulated the gene expression more tightly. Furthermore, when P(CTR4) was applied to regulate the expression of the endogenous genes ERG1 and TRP5, their conditional mutants exhibited decreased growth activity under the repressive conditions. These results suggest that the P(CTR4)-based gene regulation system represents a powerful tool for identification and characterization of a broad range of genes, including essential genes, in dermatophytes.     

逆转录病毒表达系统及其在外源蛋白高效表达中的应用

逆转录病毒表达系统是一种新的重组蛋白高效表达系统,它由逆转录病毒载体,包膜蛋白载体和包装细胞系构成,在基因治疗和生物制药领域都有很大的应用潜力。逆转录病毒和宿主细胞基因组的重组倾向于发生在转录活性区;口炎疱疹病毒-G蛋白 (vesicular stomatitis virus G, VSV-G)能有效扩大逆转录病毒感染宿主细胞的范围、提高逆转录病毒的感染效率;用高滴度的重组病毒感染细胞,经过简单的筛选即可获得高表达细胞株。本文对逆转录病毒表达系统的组成、感染的特点和机制及其应用前景作了概述。     

Stable expression of pertussis toxin in Bordetella bronchiseptica under the control of a tightly regulated promoter.

Pertussis toxin (PT) is an essential component of accellular vaccines against whooping cough. However, the industrial production of PT from Bordetella pertussis is impaired by slow growth and poor yields. To overcome these problems, we have constructed a minitransposon containing the tox operon under the control of a tightly regulated promoter responsive to an aromatic inducer. The expression cassettes have been integrated into the chromosome of Bordetella bronchiseptica 5376 and ATCC 10580 bvg. Five recombinant clones containing the tox operon under the control of the Psal promoter, which is activated by the product of nahR, were further characterized. The recombinant clones expressed PT after only 3 h of induction with sodium salicylate at levels similar to those of B. pertussis grown for 24 h. The stability of the engineered phenotype was 100% after 72 h of growth without selective pressure. The growth pattern was not modified either under noninducing conditions or in the presence of the inducer at low concentrations, suggesting that strain performance would not be affected in bioreactors when uncoupled from gene expression. Recombinant PT, which was localized mainly in the periplasm, was purified by affinity chromatography. The recombinant protein was immunologically indistinguishable from wild-type PT and retained its biological activity as determined by the CHO cell-clustering test. These recombinant clones appear to be useful tools for the cost-effective production of PT under conditions of improved biosafety, as demonstrated by the inducible expression of PT uncoupled from the bacterial biomass in a nonvirulent and fast-growing B. bronchiseptica background.     

Two tobacco AP1-like gene promoters drive highly specific,tightly regulated and unique expression patterns during floral transition,initiation and development

The genetic engineering of agronomic traits requires an array of highly specific and tightly regulated promoters that drive expression in floral tissues. In this study, we isolated and characterized two tobacco APETALA1-like (AP1-like) promoters (termed NtAP1La and NtAP1Lb1) in transgenic plants using the GUS reporter system, along with tissue-specific ablation analyses. Our results demonstrated that the two promoters are active in floral inflorescences but not in vegetative apical meristems or other vegetative tissues, as reflected by strong GUS staining and DT-A-mediated ablation of apical shoot tips during reproductive but not vegetative growth. We also showed that the NtAP1Lb1 promoter was more active than NtAP1La in inflorescences, as the former yielded higher frequencies and greater phenotypic evidence of tissue ablation compared to the latter. We further revealed that both promoters were uniformly expressed in the meristems of stage 1 and 2 floral buds, but were differentially expressed in floral organs later during development. While NtAP1La was found to be active in stage 4–5 carpels, later becoming confined to ovary tissue from stage 9 onwards, NtAP1Lb1 activity was apparent in all floral organs from stages 3 to 7, becoming completely absent in all floral organs from stage 11 onward. Therefore, it seems that the two tobacco promoters have acquired similar but distinct inflorescence-, floral meristem- and floral organ-specific and development-dependent regulatory features without any leaky activity in vegetative tissues. These features are novel and have rarely been observed in other flower-specific promoters characterized to date. The potential application of these promoters for engineering sterility, increasing biomass production and modifying flower architecture, as well as their putative use in flower-specific transgene excision, will be discussed.     

It's time to flower: the genetic control of flowering time

In plants, successful sexual reproduction and the ensuing development of seeds and fruits depend on flowering at the right time. This involves coordinating flowering with the appropriate season and with the developmental history of the plant. Genetic and molecular analysis in the small cruciform weed, Arabidopsis, has revealed distinct but linked pathways that are responsible for detecting the major seasonal cues of day length and cold temperature, as well as other local environmental and internal signals. The balance of signals from these pathways is integrated by a common set of genes to determine when flowering occurs. Excitingly, it has been discovered that many of these same genes regulate flowering in other plants, such as rice. This review focuses on recent advances in how three of the signalling pathways (the day-length, vernalisation and autonomous pathways) function to control flowering.