Characterization of Promoter Activities of Four Different Japanese Flounder Promoters in Transgenic Zebrafish

An important consideration in transgenic research is the choice of promoter for regulating the expression of a foreign gene. In this study several tissue-specific and inducible promoters derived from Japanese flounder Paralichthys olivaceus were identified, and their promoter activity was examined in transgenic zebrafish. The 5′ flanking regions of the Japanese flounder complement component C3, gelatinase B, keratin, and tumor necrosis factor (TNF) genes were linked to green fluorescence protein (GFP) as a reporter gene. The promoter regulatory constructs were introduced into fertilized zebrafish eggs. As a result we obtained several stable transgenic zebrafish that displayed green fluorescence in different tissues. Complement component C3 promoter regulated GFP expression in liver, and gelatinase B promoter regulated it in the pectoral fin and gills. Keratin promoter regulated GFP expression in skin and liver. TNF gene promoter regulated GFP expression in the pharynx and heart. TNF promoter had lipoplysaccharide-inducible activity, such that when transgenic embryos were immersed lipopolysaccharide, GFP expression increased in the epithelial tissues. These 4 promoters regulated the expression of GFP in different patterns in transgenic zebrafish.     

Characterization of an ethanol-inducible promoter system in Catharanthus roseus hairy roots

Efforts to engineer Catharanthus roseus hairy roots to produce commercially significant amounts of valuable compounds, such as the terpenoid indole alkaloids vinblastine and vincristine, require the development of tools to study the effects of overexpressing key metabolic and regulatory genes. The use of inducible promoters allows researchers to control the timing and level of expression of genes of interest. In addition, use of inducible promoters allows researchers to use a single transgenic line as both the control and experimental line, minimizing the problems associated with clonal variation. We have previously characterized the use of a glucocorticoid-inducible promoter system to study the effects of gene overexpression within the terpenoid indole alkaloid pathway on metabolite production. Here the feasibility of using an ethanol-inducible promoter within C. roseus hairy roots is reported. This ethanol-inducible promoter is highly sensitive to ethanol concentration with a concentration of 0.005% ethanol causing a 6-fold increase in CAT reporter activity after 24 h of induction. The ethanol-inducible CAT activity increased 24-fold over a 72-h induction period with 0.5% ethanol.     

A novel robust heat-inducible promoter for heterologous gene expression in Tetrahymena thermophila

An increasing amount of data has revealed the importance of inducible promoters in ciliate research and in ciliate-related industries. However, knowledge about these promoters and related genes is relatively sparse. Here we report a novel inducible promoter from a Tetrahymena cytoplasmic Hsp70 gene member, HSP70-2. The reported promoter was able to induce the endogenous gene up to ～9000-fold after a short heat shock treatment and this remarkable feature has been retained when a relatively short region of the promoter was introduced into a reporter construct followed by transformation. During the recovery period following a short heat shock, both the mRNA and protein levels of the reporter gene were maintained high up to two hours. A constant heat shock treatment to the transformed cells led to a stabilization of the reporter mRNA up to at least six hours and the reporter protein continued to accumulate up to around three hours. The promoter strength appears to be similar to that of the cadmium-induced metallothionein gene (MTT1) promoter. Therefore, the HSP70-2 promoter represents an attractive alternative for the over-expression of proteins in Tetrahymena, and the promoter-reporter gene construct used in this study is an ideal tool to help in understanding the regulation mechanisms of heat shock genes in ciliates.     

利用GFP/RFP双荧光指示载体鉴定特异性启动子功能

在基因表达定位或启动子调控模式的研究中, 多以gusA作为报告基因。但由于部分组织中高内源GUS背景活性或转化手段的限制, 使判断基因表达定位或调控时存在很大误差。为了解决上述问题, 本实验将报道基因绿色荧光蛋白(GFP)和红色荧光蛋白(RFP)融合构建双荧光标记瞬时表达载体pBI221-RFP/GFP。该载体以CaMV35S启动子驱动GFP确定转化效率, 通过鉴定阳性个体的红色荧光活性分析目的基因或启动子的表达模式。并通过番茄E8和西瓜AGPL1果实特异启动子验证了该载体在启动子调控模式研究中的应用可行性。结果表明pBI221-RFP/GFP是一个可以在基因和启动子功能验证中应用的高效瞬时表达载体。     

A rapid reporter system using GFP as a reporter protein for identification and screening of synthetic stationary-phase promoters in Escherichia coli

To develop a rapid reporter system for the screening of stationary-phase promoters in Escherichia coli, the expression pattern of the green fluorescent protein (GFP) during bacterial cultivation was compared with that of the commonly used β-galactosidase. Using GFP with enhanced fluorescence, the expression pattern of both reporter systems GFP and β-galactosidase were similar and showed a typical induction of gene activity of the reporter genes, i.e. increase of expression at the transition from exponential to stationary phase. The expression was affected by the culture medium, i.e. in contrast to the complex medium (LB medium), the stationary-phase specific induction was only observed in synthetic medium (M9) when amino acids were added, whereas there was generally no induction in MOPS medium. To develop a rapid screening method on agar plates for stationary-phase promoters, a photographic approach was used, continued with computational image treatment. A screening method is presented which enables an on-line monitoring of gene activity.     

Beta-lactamase reporter system for selecting high-producing yeast clones

In modern production of protein biopharmaceuticals, a good screening and selection method of high-producing clones can dramatically influence the whole production process and lead to lower production costs. We have created a rapid, simple, and inexpensive method for selecting high-producing clones in the yeast Pichia pastoris that is based on the beta-lactamase reporter system. By integrating the reporter gene and the gene of interest into the same genome locus, it was possible to use beta-lactamase activity as a measure of the expression level of the protein of interest. A novel expression vector with two independent expression cassettes was designed and tested using green fluorescent protein (GFP) as a model. The first cassette contained the GFP gene under the control of a strong, inducible AOX1 promoter, while the second cassette consisted of the beta-lactamase reporter gene under the control of a weak constitutive YPT1 promotor. High-producing GFP clones were selected directly on the plates based on the color change after hydrolysis of the beta-lactamase substrate added to the medium. beta-lactamase activity was found to positively correlate with GFP fluorescence. The reporter system described is widely applicable-it can be easily applied to other, also pharmaceutically relevant proteins and to other yeast expression systems, such as Saccharomyces cerevisiae and Hansenula polymorpha.     

Both the <Emphasis Type="Italic">AOX1</Emphasis> promoter and the <Emphasis Type="Italic">FLD1</Emphasis> promoter work together in a <Emphasis Type="Italic">Pichia pastoris</Emphasis> expression vector

We constructed a Pichia pastoris expression vector with two strongly inducible promoters (an alcohol oxidase 1 promoter and a formaldehyde dehydrogenase 1 promoter) based on pPIC9 k. To test the function of these promoters, the vector was used to co-express two genes that encode for green fluorescent protein (GFP) and a portion of a gelatin gene (an intra- and extracellular protein). The gelatin gene was placed under the control of PAOX1, while the GFP was under the control of PFLD1. The two proteins were simultaneously expressed upon induction with 0.5% (v/v) methanol. The two promoters functioned effectively and their coexistence on one vector did not affect their efficiency in protein expression. Thus, it was possible to simultaneously induce the expression of at least two proteins from one vector, using two different promoters.     

白血病细胞中不同启动子驱动外源基因表达能力差异分析

为了分析不同启动子在白血病细胞中驱动外源基因表达能力的差异,文章选择了4种含不同启动子EF1α、PGK、Ubiquitin和CMV驱动的GFP报告基因的慢病毒载体,用以感染4种不同的白血病细胞株——NB4、HL60、Kasumi和THP1,利用荧光显微镜、流式细胞仪和荧光定量PCR的方法检测其GFP表达效率,发现EF1α启动子驱动GFP表达的能力最强,CMV最弱,PGK和Ubiquitin则介于两者之间。该结果提示在白血病细胞中研究基因功能时,应根据不同的研究需求选择最为合适的启动子。     

Identification of the cadmium-inducible Hansenula polymorpha SEO1 gene promoter by transcriptome analysis and its application to whole-cell heavy-metal detection systems

The genomewide gene expression profiling of the methylotrophic yeast Hansenula polymorpha exposed to cadmium (Cd) allowed us to identify novel genes responsive to Cd treatment. To select genes whose promoters can be useful for construction of a cellular Cd biosensor, we further analyzed a set of H. polymorpha genes that exhibited >6-fold induction upon treatment with 300 muM Cd for 2 h. The putative promoters, about 1,000-bp upstream fragments, of these genes were fused with the yeast-enhanced green fluorescence protein (GFP) gene. The resultant reporter cassettes were introduced into H. polymorpha to evaluate promoter strength and specificity. The promoter derived from the H. polymorpha SEO1 gene (HpSEO1) was shown to drive most strongly the expression of GFP upon Cd treatment among the tested promoters. The Cd-inducible activity was retained in the 500-bp deletion fragment of the HpSEO1 promoter but was abolished in the further truncated 250-bp fragment. The 500-bp HpSEO1 promoter directed specific expression of GFP upon exposure to Cd in a dose-dependent manner, with Cd detection ranging from 1 to 900 muM. Comparative analysis of the Saccharomyces cerevisiae SEO1 (ScSEO1) promoter revealed that the ScSEO1 promoter has a broader specificity for heavy metals and is responsive to arsenic and mercury in addition to Cd. Our data demonstrate the potential use of the HpSEO1 promoter as a bioelement in whole-cell biosensors to monitor heavy metal contamination, particularly Cd.     

Adenovirus-mediated gene transfer in primary murine adipocytes

The transfer of genes into primary murine adipocytes using an adenovirus system has been developed. A recombinant adenovirus was constructed (expressing green fluorescent protein [GFP] under the control of the strong cytomegalovirus [CMV] promoter and a luciferase reporter gene under the control of the weak adipocyte promoter keratinocyte lipid-binding protein [KLBP/FABP5]) and incubated with primary adipocytes from C57BL/6J mice. Analysis of infected cells by confocal microscopy detected GFP expression in both the cytoplasm and nucleus of adipocytes with a 64% efficiency of infection. To demonstrate the applicability of this method in the study of gene regulation, adenovirus-infected adipocytes exhibited significant levels of luciferase activity even from a weak promoter. TPA treatment of infected adipocytes increased luciferase activity, consistent with previous studies indicating that the KLBP/FABP5 gene is up-regulated by phorbol esters.These results provide an efficient, convenient, and sensitive method to transiently infect primary murine adipocytes, facilitating protein expression or permitting analysis of reporter gene activity from both viral and endogenous promoters.     

Hypochlorous acid activates the heat shock and soxRS systems of Escherichia coli.

A series of plasmids, containing fusions of different stress promoters to lux reporter genes, was used in an attempt to monitor the defense circuits activated upon exposure of Escherichia coli to sublethal doses of free chlorine. A significant level of activation was exhibited by promoters of three heat shock genes (grpE, dnaK, and lon), in an rpoH-dependent manner. The promoter of micF, a gene under the control of the soxRS regulon, was also strongly induced, but not in a soxR mutant. This induction was not affected by sodA and sodB mutations, implying that it did not involve oxygen radical activity. Free-chlorine activation of both heat shock and soxRS regulons required an exposure of less then I s in duration. The oxyR or the SOS regulons were apparently not induced by free chlorine (as judged by lack of activation of katG and recA, respectively), and neither was the universal stress (uspA) protein.     

Green fluorescent protein is superior to blue fluorescent protein as a quantitative reporter of promoter activity in <Emphasis Type="Italic">E. coli</Emphasis>

Fluorescent proteins related to and derived from green fluorescent protein (GFP) are widely used as tools for investigating a wide range of biological processes. In particular, GFP and its relatives have been used extensively as qualitative reporters of gene expression in many different organisms, but relatively few studies have investigated fluorescent proteins as quantitative reporters of gene expression. GFP has some limitations as a reporter gene, including possible toxicity when expressed at high levels. Therefore, it would be useful if other fluorescent proteins could be identified for use as quantitative reporters. Toward this end, we investigated BFP as a quantitative reporter of promoter activity in E. coli and directly compared it with GFPuv using a set of well-characterized synthetic constitutive promoters. The fluorescence produced in E. coli strains expressing GFPuv or BFP grown on solid medium was quantified using a CCD camera and fluorimetry. GFPuv consistently gave more reliable and statistically significant results than did BFP in all assays. Correspondingly, we found that the signal-to-noise ratio for GFPuv fluorescence is substantially higher than for BFP. We conclude that, under the conditions assessed in this study, GFPuv is superior to BFP as a quantitative reporter of promoter activity in E. coli. J. Bayes, M. Calvey, L. Reineke, A. Colagiavanni, and M. Tscheiner made equivalent contributions to this work.     

Dual promoter expression system with insulator ensures a stringent tissue-specific regulation of two reporter genes in the transgenic fish

The precise control of spatiotemporal expression of target genes is crucial when establishing transgenic animals, and the introduction of genes for fluorescent marker proteins is inevitable for accelerating research at molecular levels. To assist this, we constructed a novel dual promoter expression vector for two independent reporter genes, green fluorescent protein (GFP) and red fluorescent protein (mCherry). Their expression is designed under the control of two distinct tissue-specific promoters, e.g. zebrafish cardiac muscle-specific promoter (cmlc2) and medaka skeletal muscle-specific promoter (myl2) derived from the myosin light chain 2 genes, and they are placed in a head-to-head orientation. After microinjecting the dual promoter expression vector into fertilized eggs of medaka, the developing fish embryos and the resulting transgenic fish lines showed strong GFP signal in the whole body (skeletal muscle) and mCherry signal in the heart (cardiac muscle). However, weak GFP signal was observed in the heart, indicating a leakiness of the skeletal muscle promoter. To improve the stringency of dual promoter expression, we inserted two chicken-derived insulators, e.g. tandem copies of the core sequence (250 bp) of cHS4 (5′-hypersensitive site-4 chicken beta-globin insulator), in the boundary of two promoters. The dual promoter expression vector with insulator now ensured the stringent tissue-specific expression in the transgenic fish lines. Thus, our dual promoter expression system with insulator is compatible to the conventional IRES and fused reporter gene systems and will be an alternative method to produce the transgenic fishes.     

Green fluorescent protein as a quantitative reporter of relative promoter activity in E. coli

Green fluorescent protein (GFP) has become a valuable tool for the detection of gene expression in prokaryotes and eukaryotes. To evaluate its potential for quantitation of relative promoter activity in E. coli, we have compared GFP with the commonly used reporter gene lacZ, encoding beta-galactosidase. We cloned a series of previously characterized synthetic E. coli promoters into GFP and beta-galactosidase reporter vectors. Qualitative and quantitative assessments of these constructs show that (a) both reporters display similar sensitivities in cells grown on solid or liquid media and (b) GFP is especially well suited for quantitation of promoter activity in cells grown on agar. Thus, GFP provides a simple, rapid and sensitive tool for measuring relative promoter activity in intact E. coli cells.     

Development of Lactococcus lactis encoding fluorescent proteins,GFP, mCherry and iRFP regulated by the nisin-controlled gene expression system

Fluorescent proteins are useful reporter molecules for a variety of biological systems. We present an alternative strategy for cloning reporter genes that are regulated by the nisin-controlled gene expression (NICE) system. Lactoccocus lactis was genetically engineered to express green fluorescent protein (GFP), mCherry or near-infrared fluorescent protein (iRFP). The reporter gene sequences were optimized to be expressed by L. lactis using inducible promoter pNis within the pNZ8048 vector. Expression of constructions that carry mCherry or GFP was observed by fluorescence microscopy 2 h after induction with nisin. Expression of iRFP was evaluated at 700 nm using an infrared scanner; cultures induced for 6 h showed greater iRFP expression than non-induced cultures or those expressing GFP. We demonstrated that L. lactis can express efficiently GFP, mCherry and iRFP fluorescent proteins using an inducible expression system. These strains will be useful for live cell imaging studies in vitro or for imaging studies in vivo in the case of iRFP.     

Analysis of the activity of promoters from two photosynthesis-related genes psaF and petH of spinach in a monocot plant, rice

The subunit III of photosystem I and ferredoxin-NADP(+)-oxidoreductase are encoded by nuclear genes, namely psaF and petH. The activity of their promoters from spinach has been evaluated in transgenic tobacco earlier. Evaluation of the activity of these Dicotyledoneae-specific promoters has been carried out in a monocot system (i.e. rice) by transient gene expression system, based on electroporation-mediated gene delivery into protoplasts from leaves and roots. It has been found that various promoter deletions show higher activity in leaf protoplasts and elements for quantitative response are widely distributed. Transgenic rice has also been produced with a petH promoter and gus reporter gene construct. Although petH promoter is a weak promoter in comparison to the 35S promoter, it expresses well in green tissues and could be useful for plant genetic engineering.