Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.2.1.31 (beta-glucuronidase)
7,680 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We showed previously that a gene, designated AX92, which is expressed at an early stage of cortex differentiation in the root apex of oilseed rape seedlings, is also expressed in embryos. To compare AX92 gene regulation during embryo-genesis and postembryonic growth, we constructed a chimeric gene consisting of AX92 5' and 3' untranslated and flanking regions fused with a beta-glucuronidase protein coding region. We showed that the chimeric gene is active in both developing cortex cells in the root apical meristems of transgenic oilseed rape seedlings and in cortex cells at the root end of embryonic axes. To determine whether the AX92 gene is regulated by a common mechanism in embryos and seedlings, we analyzed the expression of modified chimeric genes. We showed that the AX92 chimeric gene is regulated combinatorially and that DNA sequences located 3' of the protein coding region are necessary for its activation in the root cortex of both embryos and seedlings. Our results suggest that common regulatory sequences are required to activate the gene in the embryonic and postembryonic root cortex.
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PMID:Downstream DNA sequences are required to activate a gene expressed in the root cortex of embryos and seedlings. 147 53

Deletion analysis has previously shown that the major gibberellic acid (GA)- and abscisic acid (ABA)-responsive elements in the promoter of a high-pI alpha-amylase gene of barley are located downstream of -174 (Jacobsen and Close, 1991). We have used transient expression assays in barley aleurone protoplasts to identify sequences between -174 and +53 that confer GA and ABA responsiveness on expression of a beta-glucuronidase reporter gene. Using alpha-amylase promoter fragments and synthetic oligonucleotides fused to minimal promoters, we have shown that the hormone-responsive region is located between -174 and -108. A single copy of this region fused to a minimal alpha-amylase promoter (-41) conferred both GA- and ABA-responsive expression on the reporter gene comparable to the positive control, Am(-174)IGN. Multiple copies of this region were able to activate even greater levels of expression. Site-directed mutagenesis was used to determine the functional importance of the conserved motifs (-169pyrimidine box, -143TAACAAA box, and -124TATCCAC box) and nonconserved intervening sequences within the region between -174 and -108. Our results showed that both the TAACAAA and TATCCAC boxes play an important role in GA-regulated expression. We propose that the TAACAAA box is a gibberellin response element, that the TATCCAC box acts cooperatively with the TAACAAA box to give a high level of GA-regulated expression, and that together these motifs form important components of a gibberellin response complex in high-pI alpha-amylase genes. The TAACAAA box also appears to be the site of action of ABA.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Gibberellin-responsive elements in the promoter of a barley high-pI alpha-amylase gene. 147 56

Inhibition of flower pigmentation in transgenic petunia plants was previously accomplished by expressing an antisense chalcone synthase (chs) gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter. This chimeric gene was not effective in inhibiting pigmentation in anthers, presumably because the viral CaMV 35S promoter was insufficiently expressed in cell types of this organ in which the pigments are produced. Insertion of the anther box, a homologous sequence found in other genes expressed in anthers, resulted in a modified expression pattern driven by this promoter, as monitored by the beta-glucuronidase (gus) gene. In addition to the basic CaMV 35S expression pattern in anthers, GUS activity was observed in tapetum cells when the modified promoter was fused to the gus gene. This promoter construct was subsequently used to drive an antisense chs gene in transgenic petunia, which led to the inhibition of pigment synthesis in anthers of five of 35 transformants. Transgenic plants with white anthers were male sterile due to an arrest in male gametophyte development. This finding indicated that flavonoids play an essential role in male gametophyte development.
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PMID:Antisense inhibition of flavonoid biosynthesis in petunia anthers results in male sterility. 149 95

The pool of tubulin protein in tissues of Arabidopsis is provided by the expression of multiple alpha-tubulin (TUA) and beta-tubulin genes. Whereas most tubulin genes are expressed in many tissues, previous evidence suggested that the TUA1 gene might be expressed primarily in pollen. We now report a detailed analysis of TUA1 expression during Arabidopsis development. In RNA from tissues of dissected flowers, TUA1 transcripts were detected only in stamens and mature pollen. Chimeric genes containing TUA1 5' flanking DNA fused to the beta-glucuronidase (GUS) coding region were used to create transgenic Arabidopsis plants. Plants containing a chimeric gene with 533 bp of 5' flanking sequence were analyzed by histochemical assay to localize GUS expression within the plant. The blue product of GUS enzyme activity accumulated very rapidly in postmitotic pollen grains. Much lower levels of GUS activity were detected in anthers with uninucleate pollen grains, in flower receptacles, and in a few vegetative tissues. Analysis of 5' deletions of the TUA1 promoter suggested that 97 bp of 5' flanking DNA is sufficient to drive GUS expression in pollen and young anthers, whereas at least 380 bp is required to detect GUS expression in the receptacle. Examination of the TUA1 promoter sequence revealed several motifs that are repeated within the TUA1 promoter and are similar to sequences in other pollen-specific promoters.
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PMID:Preferential expression of an alpha-tubulin gene of Arabidopsis in pollen. 149 10

The NIa proteinase encoded by tobacco etch potyvirus catalyzes six processing events, three of which occur by an autoproteolytic mechanism. Autoproteolysis is necessary to cleave the boundaries of both NIa and the 6-kDa protein, which is located adjacent to the N terminus of NIa in the viral polyprotein. As a consequence, NIa may exist in a free form or in a transient polyprotein form containing the 6-kDa protein. While the majority of NIa molecules localize to the nuclei of infected cells, a fraction of the NIa pool is attached covalently to the 5' terminus of genomic RNA in the cytoplasm. To determine whether the presence of the 6-kDa protein affects the nuclear transport properties of NIa, we have generated transgenic plants that express genes encoding a reporter enzyme, beta-glucuronidase (GUS), fused to NIa or NIa-containing polyproteins. The NIa/GUS fusion protein was detected by histochemical analysis in the nucleus. Similarly, an NIa/GUS fusion protein that arose by autoproteolysis of a 6-kDa/NIa/GUS polyprotein was found in the nucleus. In contrast, fusion protein consisting of 6-kDa/NIa/GUS, which failed to undergo proteolysis because of the presence of a Cys-to-Ala substitution in the proteolytic domain of NIa, was detected in the cytoplasm. The inhibition of NIa-mediated nuclear transport was not due to the Cys-to-Ala substitution, since this alteration had no effect on translocation in the absence of the 6-kDa protein. These results indicate that the 6-kDa protein impedes nuclear localization of NIa and suggest that subcellular transport of NIa may be regulated by autoproteolysis.
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PMID:Regulation of nuclear transport of a plant potyvirus protein by autoproteolysis. 150 Dec 98

Stable transformation of Norway spruce tissue has been obtained following bombardment of mature somatic embryos with pRT99gus, a plasmid that contains neo coding for NPTII, and gusA, coding for beta-glucuronidase, both fused to the CaMV 35S promoter. At least 8 lines have been stably transformed (over 15 months in culture) following bombardment and selection on kanamycin. Polymerase chain reaction analyses showed a high frequency of cotransformation of the gusA and neo genes. The frequency of coexpression of the selected and unselected markers was 100%. DNA/DNA hybridization of one transformed line provided conclusive evidence of stable integration and showed copy numbers of over 10 plasmid sequences per genome. None of the transformed lines has remained embryogenic.
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PMID:Genetic transformation of Norway spruce (Picea abies (L.) Karst) using somatic embryo explants by microprojectile bombardment. 151 Nov 38

The tobacco etch potyvirus (TEV) polyprotein is processed by three virus-encoded proteinases, termed Nla, HC-Pro, and the 35-kDa proteinase. The 35-kDa proteinase is derived from the amino-terminal region of the polyprotein. Analysis of polyproteins containing beta-glucuronidase fused to the expected carboxy terminus of the 35-kDa proteinase confirmed the previously identified Tyr304-Ser305 dipeptide as the cleavage site between the 35-kDa proteinase and HC-Pro. The 35-kDa proteinase of TEV was unable to catalyze proteolysis when synthetic substrate polyproteins were supplied in a bimolecular or trans reaction, suggesting that processing occurs by an autolytic mechanism. The results of a mutational analysis within the 35-kDa proteolytic domain indicated that His214, Asp223, Ser256, and Asp288 were required for optimal autoproteolytic activity. Replacement of Ser256 with either Thr or Cys resulted in low but detectable proteinase activity, as did substitution of Asp223 and Asp288 with Glu. These results are consistent with the hypothesis that the 35-kDa proteinase resembles cellular serine-type proteinases, with Ser256 functioning as the nucleophilic residue within the active site. Cleavage mediated by the 35-kDa proteinase has been shown previously to occur after polyprotein synthesis in wheat germ extracts and transgenic plants, but not in rabbit reticulocyte lysate. We were able to demonstrate that processing in vitro may require a heat-labile factor present in wheat germ extracts.
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PMID:Mutational analysis of the tobacco etch potyviral 35-kDa proteinase: identification of essential residues and requirements for autoproteolysis. 152 35

Many late embryogenesis abundant (Lea) protein genes in plants are regulated by abscisic acid (ABA). The RNA level of a carrot gene, DC8, increases in response to ABA in developing seeds. However, DC8 cannot be induced by ABA in adult tissues. We used chimeric genes made of various DC8 promoter fragments fused to beta-glucuronidase (GUS) to analyze the transcriptional regulation of DC8. DC8:GUS expression was measured in electroporated carrot protoplasts and in stably transformed carrots. The region of the DC8 promoter from -170 to -51 contained ABA-responsive sequences that required a 5' upstream region for high levels of expression in embryogenic callus protoplasts. 505 bp of the DC8 promoter conferred GUS expression in stably transformed somatic and zygotic embryos. DC8:GUS was expressed only in tissues formed in the seed. This includes cells in the embryo, the endosperm and the germinating seedlings. Gel retardation and competition experiments were performed to analyze the embryo nuclear protein-DNA binding activities in vitro. No binding activity was detected on the putative ABA-responsive region; however the 5' upstream regions located between -505 and -301 interacted with embryo nuclear factors. An additional site of DNA-protein interaction was located between positions -32 and +178. The nuclear proteins that bind these sequences were found in the embryo nuclei only, not in the nuclei from leaves or roots.
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PMID:Transcriptional regulation of a seed-specific carrot gene, DC8. 153 2

The double-stranded DNA copy corresponding to the 5'-nontranslated alpha beta-leader of potato virus X (PVX) genomic RNA (positions -3 to-85 according to AUG initiator) was chemically synthesized and fused to the transcription plasmids containing three different reporter genes: neomycinphosphotransferase type II (NPT II) gene, Bacillus thuringiensis coleopteran-specific toxic protein gene and beta-glucuronidase (GUS) gene. Expression of the reporter genes in vitro and in plant protoplasts (in the case of GUS gene) reveals that the alpha beta-leader of PVX RNA acts as a translation enhancer despite the presence of the upstream vector-derived sequence and irrespective of the length of the spacer sequence preceding the reporter genes.
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PMID:Translation enhancing properties of the 5'-leader of potato virus X genomic RNA. 153 1

Thionins cause the irreversible inactivation of beta-glucuronidase (GUS) in vitro in a dose- and time-dependent manner. The enzyme is also sensitive to externally added thionins when expressed in the cytoplasmic compartment of tobacco protoplasts transformed with the Gus gene under the 35S promoter of the cauliflower mosaic virus. In protoplasts transformed with the Gus gene fused to a signal peptide, where GUS is translocated into the lumen of the endoplasmic reticulum, the activity is significantly increased both by externally-added and by transiently-expressed thionin, suggesting that it interferes with GUS secretion.
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PMID:Effects of thionins on beta-glucuronidase in vitro and in plant protoplasts. 153 4


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