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PubMed1990

Maize sucrose synthase-1 promoter directs phloem cell-specific expression of Gus gene in transgenic tobacco plants.

Tobacco plants were transformed by using a chimeric gene construction, in which a corn sucrose synthase-1 gene (Sh) promoter was used to direct expression of the beta-glucuronidase (Gus) reporter gene. Expression of Sh-Gus activity in these plants was found to be cell type specfifc. GUS activity was detected only in the phloem cells but not in any other cell types of vegetative tissues. In addition, Sh-Gus expression was found to be anaerobically inducible in tobacco roots. Sh-Gus was also expressed at high levels in the endosperm tissue of maturing tobacco seeds. We thus demonstrated that the corn Sh promoter can direct cell-type-specific and inducible expression in a heterologous dicotyledonous plant.

Journal Article↗

PubMed1995

beta-Glucuronidase (GUS) transposons for ecological and genetic studies of rhizobia and other gram-negative bacteria.

A series of transposons are described which contain the gusA gene, encoding beta-glucuronidase (GUS), expressed from a variety of promoters, both regulated and constitutive. The regulated promoters include the tac promoter which can be induced by IPTG, and nifH promoters which are symbiotically activated in legume nodules. One transposon contains gusA with a strong Shine-Dalgarno translation initiation context, but no promoter, and thus acts as a promoter-probe transposon. In addition, a gus operon deletion strain of Escherichia coli, and a transposon designed for use in chromosomal mapping using PFGE, are described. The GUS transposons are constructed in a mini-Tn5 system which can be transferred to Gram-negative bacteria by conjugation, and will form stable genomic insertions. Due to the absence of GUS activity in plants and many bacteria of economic importance, these transposons constitute powerful new tools for studying the ecology and population biology of bacteria in the environment and in association with plants, as well as for studies of the fundamental molecular basis of such interactions. The variety of assays available for GUS enable both quantitative assays and spatial localization of marked bacteria to be carried out.

Base Sequence↗

PubMed1988

Genomic organization and sequence of the Gus-s alpha allele of the murine beta-glucuronidase gene.

The Gus-s alpha allele of the mouse beta-glucuronidase gene exhibits a high degree of inducibility by androgens due to its linkage with the Gus-r alpha regulatory locus. We isolated Gus-s alpha on a 28-kilobase pair fragment of mouse chromosome 5 and found that it contains 12 exons and 11 intervening sequences spanning 14 kilobase pairs of this genomic segment. The mRNA cap site was identified by ribonuclease protection and primer extension analyses which revealed an unusually short 5' noncoding sequence of 12 nucleotides. Proximal regulatory sequences in the 5'-flanking DNA and the complete sequence of the Gus-s alpha mRNA transcript were also determined. Comparison of the amino acid sequence determined from the Gus-s alpha nucleotide sequence with that of human beta-glucuronidase indicated that the two human mRNA species differ due to alternate splicing of an exon homologous to exon 6 of the mouse gene.

Alleles↗

PubMed2002

[Molecular evolution of beta-glucuronidase in vitro: obtaining thermotolerant GUS gene].

The Escherichia coli beta-glucuronidase gene (gus) has been developed as a reporter gene for plants, and has been widely used for over a decade. Both chromogenic and fluorogenic GUS substrates have been synthesized, allowing rapid nonradioactive assays. The use of the Escherichia coli enzyme beta-glucuronidase (GUS) as a reporter in gene expression studies is limited by some plants and plant-associated bacteria express endogenous glucuronidase activities. The use of the enzyme as a reporter in transgenic plants is limited by high false positive. Laboratory evolution methods were used to enhance the thermostability and activity of the beta-glucuronidase. Using plasmid pBI121 as template, a 1.8 kb specific product was amplified and cloned into the vector pBluescript SK. The result of nucleotide sequence analysis was the same as reported. In vitro recombination (DNA shuffling), which involves DNase I digestion, primerless PCR, and primer PCR was used to generate mutant libraries. The mutant GUS3-3 gene was isolated after three rounds of mutation, DNA shuffling, and screening. The GUS3-3 enzyme can resistant high temperature up to 80 degrees C for 30 min. The nucleotide sequence analysis showed 99.2% homology between the GUS-ck gene from pBI121 and GUS3-3 gene. The deduced amino acid sequence demonstrated that 11 amino acid was changed. The Tm value of GUS3-3 is 80 degrees C and increased by 25 degrees C above GUS-ck (55 degrees C). The researches indicated the feasibility of the molecular evolution of beta-glucuronidase in vitro to improve enzymatic thermostability.

Amino Acid Sequence↗

PubMed2001

Molecular characterisation of Arabidopsis lines tagged with a promoterless gus gene.

Promoter trapping has been performed through integration of a T-DNA containing a promoterless beta-glucuronidase (gus) reporter gene into the genome of Arabidopsis thaliana. A collection of T-DNA-tagged Arabidopsis lines has been produced with the vector pdeltagusBin19. Part of this collection was screened for lines with specific gus expression patterns. Here we report on the identification of two lines with gus expression in anthers and seeds. Both lines harbour complex T-DNA inserts. In one line, the integration of the T-DNA causes a male sterility phenotype and gus expression is developmentally regulated in anthers and flower bases. In the other line expression of gus is seen in the anther and seed endosperm.

Arabidopsis↗

PubMed1994

Chemiluminescent reporter gene assays: sensitive detection of the GUS and SEAP gene products.

Chemiluminescent assays are described for the secreted alkaline phosphatase (SEAP) and beta-glucuronidase (GUS) reporter gene products. These assays provide simple, sensitive, non-isotopic alternatives to existing detection methods and are performed in microplate or tube luminometers or in a scintillation counter. The SEAP reporter gene product is secreted from mammalian cells and is thus easily detected in a sample of culture medium. Sensitive detection of secreted placental alkaline phosphatase is performed with CSPD chemiluminescent alkaline phosphatase substrate, and approximately 3 fg of enzyme can be detected. GUS has become the major reporter gene used for the analysis of plant gene expression. Sensitive chemiluminescent detection of GUS activity can be performed with an assay system we have developed using Glucuron, a beta-glucuronidase substrate. This chemiluminescent assay detects 60 fg of GUS and is linear over six orders of magnitude of enzyme concentration. CSPD and Glucuron substrates have been incorporated into two new chemiluminescent reporter gene assay kits for SEAP and GUS.

Alkaline Phosphatase↗

PubMed1990

Localized transient expression of GUS in leaf discs following cocultivation with Agrobacterium.

A chimaeric gene has been constructed that expresses beta-D-glucuronidase (GUS) in transformed plant tissues, but not in bacterial cells. This gene has proved extremely useful for monitoring transformation during the period immediately following gene transfer from Agrobacterium tumefaciens. GUS expression was detectable 2 days after inoculation, peaked at 3-4 days and then declined; if selection was imposed expression increased again after 10-14 days. The extent of transient expression after 4 days correlated well with stable integration as measured by kanamycin resistance, hormone independence, and gall formation. Histochemical staining of inoculated leaf discs confirmed the transient peak of GUS expression 3-4 days after inoculation. The most surprising result was that the blue staining was concentrated in localized zones on the circumference of the disc; within these zones, essentially all the cells appeared to be expressing GUS. We suggest that the frequency of gene transfer from Agrobacterium is extremely high within localized regions of leaf explants, but that the frequency of stable integration is several orders of magnitude lower.

Base Sequence↗

PubMed1992

Seed-specific repression of GUS activity in tobacco plants by antisense RNA.

beta-Conglycinin, the 7S storage protein of soybean, is expressed only in seeds and is regulated predominantly by gene transcription [5]. We applied an antisense strategy to modify expression of a beta-glucuronidase (uidA or gusA) gene in seeds using a promoter from a beta-conglycinin gene. Transgenic tobacco plants harboring the gusA gene under the control of the CaMV 35S promoter were retransformed with a gene construct comprising the beta-conglycinin promoter fused to the gusA gene in the antisense orientation. Double transformants were regenerated and transformation was confirmed by Southern blot hybridization. Seed-specific repression of GUS activity was observed in lines containing high copy numbers of the antisense gusA transgene. Suppression of GUS activity was correlated with the amounts of (-) sense gusA transcript detected and concomitantly with a decrease in gusA transcript levels. Furthermore, the amount of suppression of GUS activity was greatest during mid to late stages of seed development, when expression of the alpha' promoter is high. These results indicate that suppression of GUS activity is due to expression of the antisense gene.

Antigens, Plant↗

PubMed1992

Potato granule-bound starch synthase promoter-controlled GUS expression: regulation of expression after transient and stable transformation.

Chimaeric genes of promoter sequences from the potato gene encoding granule-bound starch synthase (GBSS) and the beta-glucoronidase (GUS) reporter gene were used to study GBSS expression and regulation. Analysis of stable transformants revealed that a GBSS promoter sequence of 0.4 kb was sufficient to result in tissue-dependent GUS expression: levels in stably transformed microtubers exceeded levels in corresponding leaves by orders of magnitude. GBSS-GUS constructs could be transiently expressed in leaf protoplasts from wild-type and amylose-free potato lines, etuberosum Solanum brevidens, Nicotiana tabacum and Arabidopsis thaliana. Transient expression levels in potato leaf protoplasts were clearly lower than in corresponding suspension cell protoplasts. This lower expression in leaf protoplasts could not be elevated by increasing DNA concentrations during transfection. Light incubation of electroporated suspension cell protoplasts reduced transient GBSS-GUS expression, whereas incubation of transfected protoplasts in media with different sucrose concentrations did not affect transient expression levels. However, electroporated protoplasts, isolated from suspensions, which had been grown on media with increasing amounts of sucrose showed a sucrose concentration-dependent transient expression profile. This indicates that studying GBSS regulation by transient expression experiments needs pre-treatment of the protoplast source. Sequence data of the GBSS promoter were compared to those of two other potato alleles.

Alleles↗

PubMed1991

Transgene expression variability (position effect) of CAT and GUS reporter genes driven by linked divergent T-DNA promoters.

Forty-five individually transformed clonal tobacco callus lines were simultaneously assayed for both chloramphenicol acetyltransferase (CAT) and beta-glucuronidase (GUS) activity resulting from expression of introduced reporter genes driven by the adjacent and divergent mannopine (mas) promoters. Excluding lines in which one or both of the enzyme activities was essentially zero, the activities of the reporter genes varied by as much as a factor of 136 (CAT) and 175 (GUS) between individual transformants. Superimposed upon the high degree of inter-clonal expression variability was an intra-clonal variability of 3-4-fold. The observed degree of intra-clonal reporter gene activity may be more extreme because of the regulatory characteristics of the mannopine promoters, but must still be addressed when considering the limitations of reporter gene-based analysis of transgene function and structure. There was no consistent correlation between the expression levels of the introduced CAT and GUS genes since the ratio of GUS to CAT activities (nmol min-1 mg-1) within individual lines varied from 0.05 to 49. Even divergent transcription from two directly adjacent promoter regions (both contained within a 479 bp TR-DNA fragment) is insufficient to guarantee concurrent expression of two linked transgenes. Our quantitative data were compared to published data of transgene expression variability to examine the overall distribution of expression levels in individual transformants. The resulting frequency distribution indicates that most transformants express introduced transgenes at relatively low levels, suggesting that a potentially large number of Agrobacterium-mediated transformation events may result in silent transgenes.

Chloramphenicol O-Acetyltransferase↗

PubMed1991

Expression of a chimaeric granule-bound starch synthase-GUS gene in transgenic potato plants.

Granule-bound starch synthase is the key enzyme in amylose synthesis. The regulation of this gene was investigated using a chimaeric gene consisting of a 0.8 kb 5' upstream sequence of the granule-bound starch synthase gene from potato and the beta-glucuronidase gene which was introduced into potato using an Agrobacterium tumefaciens binary vector system. The chimaeric gene was highly expressed in stolons and tubers, whereas the expression in leaves, stems or roots from greenhouse-grown plants was relatively low. However, leaves from in vitro grown plantlets exhibited an elevated GUS expression. The expression of the chimaeric gene was inducible in leaves by growth on relatively high concentrations of sucrose, fructose and glucose and was about 30- to 50-fold higher than in leaves from greenhouse-grown plants. The granule-bound starch synthase gene is expressed organ-specifically since stolons and tubers showed GUS activities 125- to 3350-fold higher than in leaves. The activities in these two organs are 3- to 25-fold higher than the expression of the CaMV-GUS gene. Histochemical analysis of different tissues showed that only certain regions of leaves and roots express high GUS activities. Stolons and tubers show high expression.

Agrobacterium tumefaciens↗

PubMed2006

Development of RNR3- and RAD54-GUS reporters for testing genotoxicity in Saccharomyces cerevisiae.

S. cerevisiae RNR3 and RAD54 gene transcription becomes strongly activated upon DNA damage. This property was used to construct yeast strains in which DNA damage can be monitored by a very sensitive fluorogenic assay in a convenient 96-well microtiter plate format. These strains carried stably integrated fusions of RNR3 or RAD54 promoters to the E. coli beta-glucuronidase GUS gene. GUS activity was measured by fluorogenic detection, a method that greatly increases the precision and sensitivity of the assay. Detection levels were similar to those of real-time quantitative PCR methods and close to the limits of biological response. The two reporters differed in terms of fold-induction, activation kinetics, sensitivity and specificity upon exposure to a variety of genotoxic compounds. While RNR3-GUS showed the fastest response, RAD54-GUS showed the highest sensitivity: similar to previous reported sensitivities for bacterial and eukaryotic genotoxic detection systems. These reporter strains may complement current genotoxicity tests, but they also have the advantages of higher flexibility, requirement for shorter incubation times, and the capability of being fully automated. In addition, the intrinsic features of the system facilitate its easy improvement by genetic manipulating the yeast strain or by introducing mammalian metabolizing enzymes.

DNA Damage↗

PubMed2003

Sugar acts as a regulatory signal on the wound-inducible expression of SbHRGP3::GUS in transgenic plants.

SbHRGP3 encodes an HRGP whose expression is correlated with the cessation of root elongation in soybean. The wound-inducible expression of SbHRGP3 interestingly requires sucrose although wounding alone induces the expression of many HRGP genes. To examine whether sugar serves as a specific signal on the wound-inducible expression or whether sugar is required to provide ATP, we examined SbHRGP3:: GUS expression in transgenic tobacco plants. Various oligosaccharides including non-metabolizable sugar induced SbHRGP3:: GUS expression in transgenic plants. The inhibitors of photosynthesis and of cellular respiration did not affect the wound-inducible expression of SbHRGP3:: GUS. However, the induction was significantly affected by PCMBS, an inhibitor of active apoplastic phloem loading of sucrose, suggesting that SbHRGP3:: GUS expression in phloem tissues requires translocated sucrose. We therefore propose that sugar acts as a specific regulatory signal on the wound-inducible expression of SbHRGP3, rather than acting as a simple provider of ATP.

Carbohydrates↗

PubMed2001

Expression of intron-containing GUS constructs is reduced due to activation of a cryptic 5' splice site.

An intron-containing beta-glucuronidase (GUS) gene has been used widely in promoter analyses and as a plant transformation marker. Maximal plant gene expression requires accurate and efficient removal of the intron from the expressed pre-mRNA transcripts by splicing. Detailed analysis of splicing of potato ST-LS1 and pea legumin introns from GUS constructs revealed the activation of a cryptic 5' splice site in the GUS coding sequence 4 nt upstream from the authentic intron 5' splice site. About 40% of transcripts utilised the cryptic 5' splice site in tobacco protoplasts, reducing the translational potential of expressed pre-mRNA. The same cryptic splicing event was evident in transgenic tobacco leaves but at reduced levels. Mutations that removed the cryptic 5' splice site are associated with a two-fold enhancement in GUS activity in tobacco protoplasts, highlighting the need for careful examination of introns and their sites of insertion into gene constructs to minimise variability in gene activity and maximise gene expression.

Glucuronidase↗

PubMed1995

Vectors with the gus reporter gene for identifying and quantitating promoter regions in Saccharomyces cerevisiae.

The Escherichia coli beta-glucuronidase-encoding gene gusA is useful as a reporter gene in a variety of organisms. In this report, we describe the development of two related vectors, pGUS1 and pGUS2, which can be used to identify and quantitate activities of the promoter regions from yeast genes. Both vectors contain several unique restriction sites upstream from gus and the yeast CYC1 transcription terminator downstream from gus. In addition, pGUS2 carries the yeast ADH1 transcriptional terminator sequence upstream from gus, in order to block read-through transcription originating in vector sequences. Both vectors were tested after cloning the well-characterized GAL1,10 promoter region from yeast. These GAL1,10-containing plasmids demonstrated appropriate regulation of the reporter in response to carbon sources. The pGUS1 and pGUS2 vectors provide a simple, reliable and extremely sensitive reporter-gene system that allows quantitative measurement of promoter activity of yeast DNA sequences. Furthermore, the presence of a terminator sequence upstream from gus in pGUS2 should facilitate analysis and quantitation of expression from weak promoters.

Alcohol Dehydrogenase↗

PubMed2003

Molecular detection and beta-glucuronidase expression of gus-marked Bacillus subtilis L-form bacteria in developing Chinese cabbage seedlings.

AIM: To detect L-form bacteria in developing Chinese cabbage seedlings. METHODS AND RESULTS: Stable Bacillus subtilis L-forms were genetically modified to express the gus gene (encoding beta-glucuronidase). Germinated seeds of Chinese cabbage were soaked in mannitol based suspensions of the L-form bacteria or with mannitol alone and after washing were grown in aseptic conditions on plant growth medium. Histochemical staining of beta-glucuronidase activity (X-gluc) and Polymerase Chain Reaction (PCR) detection of the gus gene were achieved in the L-form associated seedlings. beta-Glucuronidase was localized in discrete spots, mainly in the roots with staining, and was also observed in the cotyledons and base of stems. Correlation was observed between PCR detection of the gus gene and histochemical staining with detection in similar tissues. Stable L-form bacteria were non-culturable after their association with plant material. CONCLUSIONS: The gus reporter gene system with its associated histological staining for enzyme activity was used successfully for detecting B. subtilis L-form bacteria in plant material. SIGNIFICANCE AND IMPACT OF THE STUDY: These molecular marked L-forms should provide a specific and sensitive technique for detecting L-form bacteria in planta and offer a method for further understanding the L-form/plant association.

Bacillus subtilis↗

PubMed1991

Upstream sequences of rice proliferating cell nuclear antigen (PCNA) gene mediate expression of PCNA-GUS chimeric gene in meristems of transgenic tobacco plants.

The transgenic tobacco plants have been generated that express the E. coli beta-glucuronidase (GUS) gene under control of the promoter from the rice proliferating cell nuclear antigen (PCNA, DNA polymerase auxiliary protein) gene. GUS expression detected in situ by staining with the chromogenic substrate, 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-Gluc), was restricted to meristems in the organs of the transgenic tobacco plants. This expression responded to the phytohormones which promote callus formation. Furthermore, in situ thymidine uptake showed that the GUS expression pattern corresponded well to the active sites of DNA synthesis. Deletion analysis of the 5' upstream sequence confined the GUS expression pattern to a fragment extending 263 bp upstream of the transcription start site of the rice PCNA gene. Thus, we have identified this fragment as a main regulatory element of the rice PCNA gene promoter.

Blotting, Northern↗

PubMed2001

Extracellular calmodulin stimulates light-independent RbcS-GUS expression in suspension-cultured cells of transgenic tobacco.

The RbcS genes encode the small subunits of rubisco; the expression of these genes is controlled in a light-dependent and independent manner. It has been reported that intracellular calmodulin (CaM) is involved in light-dependent RbcS expression. In this report, the role of extracellular CaM in regulating expression of RbcS in darkness was examined. The time course of expression of RbcS-GUS and that of the secretion of CaM in the suspended transgenic tobacco cells in darkness were very similar. Both showed initial increase followed by decline with maximum CaM secretion preceding maximum GUS expression by 24 h. The concentration of CaM in the culture medium is regulated light independently. Purified CaM alone added to the media enhanced RbcS-GUS expression in darkness. The addition of membrane-impermeable CaM inhibitors, such as anti-CaM antiserum or W7-agarose, repressed the expression of RbcS-GUS in darkness, but this inhibitory effect was completely reversed by adding exogenous purified CaM. These results provide the first evidence that extracellular CaM is involved in the regulation of light-independent RbcS gene expression.

Calmodulin↗