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PubMed1980

Quantitation of cis versus trans regulation of mouse beta-glucuronidase. Action of alleles of the Gus-r locus determining androgen inducibility.

Genetic studies have shown that the induction of beta-glucuronidase in mouse kidney in response to androgens is under the control of the Gus-r gene, closely linked to the beta-glucuronidase structural gene, Gus-s, on mouse Chromosome 5. Despite the fact that a single structural gene codes for beta-glucuronidase, enzyme molecules show considerable charge heterogeneity, presumably as a result of post-translational events. Because the enzyme is a tetramer, this heterogeneity has complicated examination of the cis versus trans action of the Gus-r gene. In order to resolve the problem, we have developed a quantitative method for determining the amount of beta-glucuronidase derived from each structural allele in mice heterozygous for beta-glucuronidase electrophoretic mobility. The method involves separation and quantitation of genetically variant cyanogen bromide peptides from the enzyme. Using this technique, we show that Gus-r acts cis, suggesting that it directly affects the template activity of the glucuronidase structural gene.

Alleles↗

PubMed1994

The expression of a chimeric Phaseolus vulgaris nodulin 30-GUS gene is restricted to the rhizobially infected cells in transgenic Lotus corniculatus nodules.

In Phaseolus vulgaris there is a nodulin family, Npv30, of ca. 30 kDa, as detected in an in vitro translation assay [2]. We isolated a gene (npv30-1) for one of the members of this family. The nucleotide sequence of the promoter of npv30-1 contains nodule-specific motifs common to other late nodulin genes. The promoter was fused to the GUS reporter gene; this chimeric fusion was introduced into Lotus corniculatus via Agrobacterium rhizogenes transformation. GUS activity was only detected in the infected cells of the nodules of transgenic plants. By contrast, the expression of a 35S-GUS construct was restricted to the uninfected cells and the vascular tissue.

Base Sequence↗

PubMed1995

Novel GUS expression patterns following transposition of an enhancer trap Ds element in Arabidopsis.

Enhancer trap derivatives of the maize Dissociation (Ds) transposon were introduced into Arabidopsis thaliana. The enhancer trap Ds was so designed that upon transposition to sites containing regulatory sequences in adjacent genomic DNA, transcription of a Ds-borne beta-glucuronidase (GUS) gene would be activated. Sixty percent of all transposition events were associated with GUS expression patterns including one linked to a mutant phenotype. Patterns of GUS expression were found in various organs and were stably inheritable in the F4 and F5 progenies. These results demonstrate the potential value of the technique as a means for detection of developmentally regulated genes and analysis of their function. The enhancer trap construct used in our experiments, as well as the seeds of primary transformants are publicly available.

Arabidopsis↗

PubMed1993

The Gus-e locus regulates estrogen repression of androgen-induced beta-glucuronidase expression in mouse kidney.

Both enzyme activity and mRNA concentration of beta-glucuronidase were measured in kidneys of mice treated with testosterone and the synthetic estrogen, diethylstilbestrol. Six congenic strains, all having a C57BL6/J genetic background but each having a different haplotype of the beta-glucuronidase gene complex, were compared. In each strain the induction caused by androgen was partially repressed by estrogen. The extent of this antagonism varied among the six haplotypes and was not coordinate with the extent of induction by androgen alone. Antagonism appears to be regulated by at least two alleles of a new locus, Gus-e, within the beta-glucuronidase gene complex. Repression by estrogen, like induction by androgen, appears to take place primarily at the transcriptional level. Kinetic studies revealed that estrogen causes the androgen response curve to plateau earlier and at a lower level. This suggests that estrogen increases the rate of gene deactivation rather than decreasing the rate of gene activation. Isoelectric focusing of beta-glucuronidase from Gus-ea and Gus-eb mice and their F1 progeny revealed that the genes are regulated in cis. Together, these findings support a model in which both sex hormones exert their effects on separate DNA response elements located in close proximity to the gene or within the gene itself.

Androgens↗

PubMed2006

The GUS reporter-aided analysis of the promoter activities of a rice metallothionein gene reveals different regulatory regions responsible for tissue-specific and inducible expression in transgenic Arabidopsis.

To gain a better understanding of the regulatory mechanism of plant metallothionein (MT) genes, a chimeric expression unit consisting of the beta-glucuronidase (gusA) reporter gene under the control of a 1,324 bp fragment of the rice MT (ricMT) promoter was introduced into Arabidopsis via Agrobacterium tumefaciens. The strongest histochemical staining for GUS activity was observed in the cotyledons and hypocotyls of the transgenic seedlings and in the stigma, filaments and anthers of young and mature flowers, and especially in the wounded tissues of transgenic plants. In contrast, a relatively low level of reporter gene expression was seen in the young roots of transgenic seedlings and no GUS activity was detected in the stems, seeds and leaves, but GUS activity was observed in cotyledons and the first two true leaves. Promoter analysis of 5' deletions further identified several important regions responsible for organ-specific expression including roots, flowers and wound induction, light and ABA, Cu and Zn responses. These findings demonstrate that a 1,324 bp fragment of the rice MT promoter performs a complicated transcriptional regulation with clearly functional regions in a model plant, and provide an important insight into the transcriptional regulation mechanisms that operate the temporal- and spatial-specific expression and stress responses of the rice MT gene. These results suggest that the ricMT promoter and its functional regions are potentially useful in genetic engineering of plants to express the desired genes whose products are preferentially needed in roots, flowers and wound induction.

Arabidopsis↗

PubMed1995

The effect of Ca-formaldehyde fixation on the activity of the reporter enzyme beta-glucuronidase (GUS) in transgenic tobacco.

The inhibition of beta-glucuronidase (GUS) activity by Ca-formaldehyde fixation was investigated in leaves of transgenic tobacco (Nicotiana tabacum L.) expressing the beta-glucuronidase uidA reporter gene from Escherichia coli. One-hour fixation of a free-floating leaf disc in a cold fixative decreased the activity of GUS by 60% as compared with the untreated control disc kept in a moist chamber. The inhibition was identical in the periphery and centre of the disc. Fixation including initial vacuum infiltration of the fixative led to almost complete inactivation of the enzyme. The incubation of discs in H2O, either with or without vacuum infiltration, did not lower GUS activity.

Calcium↗

PubMed1993

A wheat histone H3 promoter confers cell division-dependent and -independent expression of the gus A gene in transgenic rice plants.

To investigate developmental regulation of wheat histone H3 gene expression, the H3 promoter, which has its upstream sequence to -1711 (relative to the cap site as +1), was fused to the coding region of the gus A gene (-1711H3/GUS) and introduced into a monocot plant, rice. Detailed histochemical analysis revealed two distinct types of GUS expression in transgenic rice plants; one is cell division-dependent found in the apical meristem of shoots and roots and in young leaves, and another is cell division-independent detected in flower tissues including the anther wall and the pistil. In this study, replication-dependent expression occurring in non-dividing cells which undergo endoreduplication could not be discriminated from strict replication-independent expression. The observed expression pattern in different parts of roots suggested that the level of the H3/GUS gene expression is well correlated with activity of cell division in roots. To identify 5' sequences of the H3 promoter necessary for an accurate regulation of the GUS expression, two constructs containing truncated promoters, -908H3/GUS and -185H3/GUS, were analyzed in transiently expressed protoplasts, stably transformed calli and transgenic plants. The results indicated that the region from -909 to -1711 contains the positive cis-acting element(s) and that the proximal promoter region (up to -185) containing the conserved hexamer, octamer and nonamer motifs is sufficient to direct both cell division-dependent and -independent expression. The use of the meristem of roots regenerated from transformed calli for the analysis of cell division-dependent expression of plant genes is discussed.

Base Sequence↗

PubMed1997

Expression of ACC oxidase promoter-GUS fusions in tomato and Nicotiana plumbaginifolia regulated by developmental and environmental stimuli.

The enzyme ACC oxidase, catalysing the last step in the biosynthesis of the plant hormone ethylene, is encoded by a small multigene family in tomato, comprising three members, LEACO1, LEACO2 and LEACO3. LEACO1 is the major gene expressed during ripening, leaf senescence, and wounding (Barry et al., 1996). To investigate the transcriptional regulation of ACC oxidase gene expression, chimeric fusions between the beta-glucuronidase reporter gene and 97 bp of 5' UTR plus 124, 396 and 1825 bp, respectively, of 5' untranscribed LEACO1 sequence were constructed and introduced into Lycopersicon esculentum (Mill cv. Ailsa Craig) and Nicotiana plumbaginifolia. Analysis of transgenic tomatoes indicated that the region containing nucleotides -124 to +97 of the LEACO1 gene is sufficient to confer a marked increase in GUS activity during fruit ripening, albeit at very low levels. Fusion of 396 and 1825 bp of LEACO1 upstream sequence resulted in strong and specific induction of GUS expression in situations known to be accompanied by enhanced ethylene production. Reporter gene expression was similar to that of the endogenous LEACO1 gene, with major increases especially during fruit ripening, senescence and abscission of leaves and, to a lesser extent, of flowers. Analysis of transgenic N. plumbaginifolia plants confirmed the pattern of LEACO1 promoter activity detected in tomato leaves and flowers. Reporter gene expression was also induced following wounding, treatment with ethylene, and pathogen infection. Histochemical analysis illustrated localized GUS activity in the pericarp of ripening fruit, abscission zones of senescent petioles and unfertilized flowers, and at wound sites. These results demonstrate that ACC oxidase is regulated at the transcriptional level in a wide range of cell types at different developmental stages and in response to several external stimuli.

Amino Acid Oxidoreductases↗

PubMed2003

Fluorescence microscopy: a powerful technique to detect low GUS activity in vascular tissues.

We have previously shown that the Eucalyptus gunnii EgCAD2 promoter was preferentially expressed in vascular tissues in different transgenic plants (poplar, tobacco, Arabidopsis and grapevine). In order to delineate the cis elements governing this vascular expression pattern, promoter deletion analysis was performed allowing us to identify the proximal region [-340/-124] as essential for vascular cambium/xylem-specific expression. In plants transformed with the smallest promoter region [-124/+117], the GUS activity was difficult to detect using conventional bright field microscopy. To overcome this problem, we used fluorescence microscopy, enabling us to show that the [-124/+117] region contained cis-elements driving activity in phloem fibres but not in secondary xylem. The technical improvement of the histochemical detection of GUS activity using fluorescence microscopy enables accurate investigation of low GUS activity in phenol-rich tissues.

Eucalyptus↗

PubMed1995

Hairpin ribozyme-mediated cleavage of the full-length beta-glucuronidase (GUS) mRNA.

We designed three hairpin ribozymes to cleave Escherichia coli beta-glucuronidase (GUS) mRNA and tested those activities in vitro. One of the ribozymes was designed to form 9 base pairs in total with the target GUS mRNA, and the other two ribozymes had longer substrate binding sites. All ribozymes cleaved the model substrate (100 bases long) at the predicted target site. Two ribozymes containing longer substrate binding sites cleaved the substrate much more efficiently than the other ribozyme containing shorter substrate binding site. Also, the ribozymes with long substrate binding sites had high activity against the full-length GUS mRNA (1.9 kilobases) and maintained the activity even at a low temperature, 26 degrees C, a general growth condition of plant cells. Effects of the substrate binding site length of the ribozyme on cleavage activity are discussed.

Base Composition↗

PubMed2005

Histochemical and fluorometric assays for uidA (GUS) gene detection.

Transgenic plant production has been intimately connected to the beta-glucuronidase (uidA or GUS) gene used as a reporter marker gene. The enzyme stability and the high sensitivity and amenability of the GUS assay to qualitative (histochemical assay) and to quantitative (fluorometric or spectrophotometric assay) detection are some of the reasons that explain the extensive use of uidA gene in plant genetic transformation. Methods for uidA gene detection have been thoroughly described in the literature. The aim of this chapter is to describe the basic protocols needed for GUS detection in a plant genetic transformation laboratory.

Citrus↗

PubMed2005

[Study on positive control for GM papaya (55-1) detection method by GUS (beta-glucuronidase) assay].

A suitable positive control was investigated for histochemical assay (GUS-examining method) to detect genetically modified (GM) papaya (55-1), currently undergoing a safety assessment in Japan. Six different kinds of test papers were soaked with beta-glucuronidase solution and examined for GUS activity. The test papers made of nylon and glass fiber turned blue, and were stable for fifteen months at -20 degrees C. They are concluded to be useful as positive controls in the GUS-examining method for inspection of GM papaya (55-1).

Carica↗

PubMed2004

Shoot regeneration from GUS-transformed tomato (Lycopersicon esculentum) hairy root.

To study the influence of genetic background on the transformation and regeneration of cultivated tomato plants, hairy root lines of tomato (Lycopersicon esculentum) were obtained by inoculating the hypocotyl explants of three tomato cultivars with the Agrobacterium rhizogenes strain DCAR-2, which harbors the pBI-121 binary vector. The Ri-T-DNA transformation into the plant DNA was confirmed by both of mikimopine and GUS assay analyses. The regeneration efficiency from hairy root explants was assessed. The data indicated that white embryonic calli were formed within two weeks in the presence of 2 mgl(-1) 2, 4-D plus 0.25 mgl(-1) kinetin. Adventitious shoots emerged from the embryonic callus in the presence of 1 mgl(-1) GA3 along with 0.5 mgl(-1) NAA. The regeneration frequency was higher in the cultivar UC-97, followed by Momotaro and then Edkawi. Molecular confirmation of the integration of the GUS gene into the hairy root-derived plants genomes was done via PCR using GUS-specific primers and also using Southern blotting analysis. Our data shows that regeneration is possible from hairy roots of the cultivated tomato and this system could be used to produce transgenic tomato plants expressing the genes present in Agrobacterium rhizogenes binary vectors.

Adenine↗

PubMed2005

[Transient expression of GUS gene controlled by different regulator sequences in rice and Populus tomrutosa].

GUS gene controlled by several kinds of regulator sequences (promoters, TMVOmega enhancer and fragments of 18S rRNA gene from Arabidopsis thaliane) were introduced into the calli of rice and Populus tomrutosa respectively by gene gun. The transient expression of GUS gene in the calli of rice and P. tomrutosa was investigated. The results showed: (1) in the calli of rice, Ubil promoter was the most effective one among the several regulator sequences and CaMV35S with fragments of 18S rRNA gene is the least effective one; (2) in the calli of P. tomrutosa, CaMV35S promoter with 18S rDNA fragments was the most effective one among the several regulator sequences; and (3) tandem CaMV35S-Ubil promoter with TMVOmega sequence could not enhance significantly the expression of GUS gene in the calli of rice and P. tomrutosa.

Biolistics↗

PubMed1997

Promoter sequences of two homologous pectin esterase genes from Chinese cabbage (Brassica campestris L. ssp. pekinensis) and pollen-specific expression of the GUS gene driven by a promoter in tobacco plants.

The promoter regions of two genomic clones, GBAN215-6 and GBAN215-12 from Chinese cabbage (Brassica campestris L. ssp. pekinensis), were sequenced. The nucleotide sequences of their promoter regions were compared with that of the Bp19 pollen-specific gene of Brassca napus. High nucleotide sequence homologies were observed among these three genes in the region between 210 bp upstream and the putative transcription start site. A sequence motif TGTGGTG, which is similar to that of the PB core motif (TGTGGTT) of two tomato pollen-specific genes, LAT52 and LAT56, was present in these two cloned genes. To determine regulatory sequences responsible for the anther-specific expression of the gene BAN215-6, two recombinant plasmids, pBPE3 (-274- + 109) and pBPE4 (-816- + 109) containing different lengths of the promoter fused with the GUS gene, were constructed and introduced into tobacco plants by Agrobacterium-mediated transformation. The result showed that the 383 bp (-274- + 109) of the BAN215-6 promoter region was sufficient for the anther-specific expression of the GUS gene. The GUS expression in a tobacco plants transformed with these constructs was first detected in uninucleate microspores and persisted at in vitro germinated pollen tubes. The expression level was increased during anther development, reaching the highest level in mature pollens.

Brassica↗

PubMed1994

Truncated presequences of mitochondrial F1-ATPase beta subunit from Nicotiana plumbaginifolia transport CAT and GUS proteins into mitochondria of transgenic tobacco.

The mitochondrial F1-ATPase beta subunit (ATPase-beta) of Nicotiana plumbaginifolia is nucleus-encoded as a precursor containing an NH2-terminal extension. By sequencing the mature N. tabacum ATPase-beta, we determined the length of the presequence, viz. 54 residues. To define the essential regions of this presequence, we produced a series of 3' deletions in the sequence coding for the 90 NH2-terminal residues of ATPase-beta. The truncated sequences were fused with the chloramphenicol acetyl transferase (cat) and beta-glucuronidase (gus) genes and introduced into tobacco plants. From the observed distribution of CAT and GUS activity in the plant cells, we conclude that the first 23 amino-acid residues of ATPase-beta remain capable of specifically targeting reporter proteins into mitochondria. Immunodetection in transgenic plants and in vitro import experiments with various CAT fusion proteins show that the precursors are processed at the expected cleavage site but also at a cryptic site located in the linker region between the presequence and the first methionine of native CAT.

Amino Acid Sequence↗

PubMed1992

Site-specific oligodeoxynucleotide binding to maize Adh1 gene promoter represses Adh1-GUS gene expression in vivo.

There is a 36 bp tract of extreme homopurine/homopyrimidine (PuPy) asymmetry in the maize Adh1 gene promoter (from -44 to -79) that is S1-hypersensitive in plasmids under supercoil tension. Oligodeoxynucleotides corresponding to the PuPy tract were designed to examine the secondary structure of the region and address the possible role of the tract in gene regulation. On the basis of oligodeoxynucleotide band-shift and DNase I footprinting analyses, it was concluded that the homopyrimidine oligodeoxynucleotide can form a triple helix with the duplex PuPy tract in vitro. Transient assays in protoplasts, suspension cells, and seedling roots show that the homopyrimidine oligodeoxynucleotide is also capable of repressing Adh1-GUS gene expression during co-transformation, presumably by the formation of a triple helix with the PuPy tract in vivo. The complementary homopurine oligodeoxynucleotide would not form a triple helix in vitro, nor would it repress Adh1-GUS in vivo. We propose that triple helix formation is a potential regulatory phenomenon in vivo, and that an intraregion triple helix could occur within the Adh1 promoter via the formation of H-DNA.

Alcohol Dehydrogenase↗

PubMed1994

Jasmonate-inducible expression of a potato cathepsin D inhibitor-GUS gene fusion in tobacco cells.

A potato gene encoding cathepsin D inhibitor (CDI) is expressed constitutively in tubers and flower buds and it is inducible in leaves upon wounding of the tissue or by treatment with methyl jasmonate (MJA). A fusion gene (CDI:GUS) in which the 2.4 kb long promoter of the CDI gene was translationaly fused with the coding sequence for beta-glucuronidase (GUS) showed MJA-inducible expression in transformed tobacco cells in suspension. The maximum level of induction by MJA was obtained in the absence of auxin and repression of MJA-inducible expression of the fusion gene by auxin was released by aphidicolin, the results suggesting that MJA-inducible expression is repressed during active cell division. JA and MJA showed similar activities in inducing the expression of the fusion gene, while other JA-related compounds such as cucurbic acid, tuberonic acid and dihydrojasmonic acid neither induced expression of the fusion gene nor inhibited the MJA-inducible expression of the fusion gene. Methyl dihydrojasmonate specifically stimulated the MJA-inducible expression of the fusion gene. The MJA-inducible expression of the fusion gene was observed even with a 100 bp long promoter of the CDI gene albeit with significantly decreased level of expression compared to the 2.4 kb long promoter. The 100 bp long CDI promoter did not contain a G-box or hexamer motif that had been implicated in the MJA-responsive expression of several other plant genes. Further mutagenesis of the 100 bp long promoter by deletion or oligonucleotide insertion suggested that although a sequence between -100 and -82 is required for the MJA-responsive expression, the presence of this sequence alone does not confer the MJA-responsive expression.

2,4-Dichlorophenoxyacetic Acid↗