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)

To assess the suitability of transgenic peas as a host for protein production from the perspective of ease of recovery, a strain containing recombinant beta-glucuronidase with poly(histidine) tail (GUSH6) was evaluated for solubility of the target protein in relation to native components (proteins, carbohydrates, and phenolics). Recovery of the recombinant GUSH6 from aqueous extracts by immobilized metal affinity chromatography with coupled Co(2+) yielded a nearly pure product with IDA (enrichment factor (EF) = 260) or NTA (EF = 200) resin. Single-step recoveries were also possible by isoelectric precipitation (EF = 4), polyelectrolyte precipitation (EF = 1.5), and anion-exchange chromatography (EF = 3.1), but enrichment factors were low.
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PMID:Recombinant protein purification from pea. 1500 47

Cytokinins are plant hormones that may play essential and crucial roles in various aspects of plant growth and development. Although the functional significance of exogenous cytokinins as to the proliferation and differentiation of cells has been well documented, the biological roles of endogenous cytokinins have remained largely unknown. The recent discovery of the Arabidopsis Histidine Kinase 4 (AHK4)/CRE1/WOL cytokinin receptor in Arabidopsis thaliana strongly suggested that the cellular response to cytokinins involves a two-component signal transduction system. However, the lack of an apparent phenotype in the mutant, presumably because of genetic redundancy, prevented us from determining the in planta roles of the cytokinin receptor. To gain insight into the molecular functions of the three AHK genes AHK2, AHK3, and AHK4 in this study, we identified mutational alleles of the AHK2 and AHK3 genes, both of which encode sensor histidine kinases closely related to AHK4, and constructed a set of multiple ahk mutants. Application of exogenous cytokinins to the resultant strains revealed that both AHK2 and AHK3 function as positive regulators for cytokinin signaling similar to AHK4. The ahk2 ahk4 and ahk3 ahk4 double mutants and the ahk single mutants grew normally, whereas the ahk2 ahk3 double mutants exhibited a semidwarf phenotype as to shoots, such as a reduced leaf size and a reduced influorescence stem length. The growth and development of the ahk2 ahk3 ahk4 triple mutant were markedly inhibited in various tissues and organs, including the roots and leaves in the vegetative growth phase and the influorescence meristem in the reproductive phase. We showed that the inhibition of growth is associated with reduced meristematic activity of cells. Expression analysis involving AHK:beta-glucuronidase fusion genes suggested that the AHK genes are expressed ubiquitously in various tissues during postembryonic growth and development. Our results thus strongly suggest that the primary functions of AHK genes, and those of endogenous cytokinins, are triggering of the cell division and maintenance of the meristematic competence of cells to prevent subsequent differentiation until a sufficient number of cells has accumulated during organogenesis.
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PMID:Histidine kinase homologs that act as cytokinin receptors possess overlapping functions in the regulation of shoot and root growth in Arabidopsis. 1515 80

Two-component signaling systems, involving His kinases, His-containing phosphotransfer proteins, and response regulators, have been implicated in plant responses to hormones and environmental factors. Genomic analysis of Arabidopsis supports the existence of 22 response regulators (ARRs) that can be divided into at least two distinct groups designated type-A and type-B. Phylogenetic analysis indicates that the type-B family is composed of one major and two minor subfamilies. The expression of the type-B ARRs was examined by using both reverse transcription-PCR and beta-glucuronidase fusion constructs. The major subfamily of type-B ARRs showed particularly high expression in regions where cytokinins play a significant role, including cells in the apical meristem region and in young leaves that would be undergoing cell division. Multiple members within this same subfamily of type-B ARRs were expressed near the root tip with highest expression in the root elongation zone. beta-Glucuronidase-fusions to full-length ARR2, ARR12, and ARR19 were nuclear localized, consistent with a role in transcriptional regulation. These data suggest that differing expression levels of the type-B ARRs may play a role in modulating the cellular responses to cytokinin.
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PMID:Type-B response regulators display overlapping expression patterns in Arabidopsis. 1517 62

ZPT2-related proteins that have two canonical Cys-2/His-2-type zinc-finger motifs in their molecules are members of a family of plant transcription factors. To characterize the role of this type of protein, we analyzed the function of Arabidopsis L. Heynh. genes encoding four different ZPT2-related proteins (AZF1, AZF2, AZF3, and STZ). Gel-shift analysis showed that the AZFs and STZ bind to A(G/C)T repeats within an EP2 sequence, known as a target sequence of some petunia (Petunia hybrida) ZPT2 proteins. Transient expression analysis using synthetic green fluorescent protein fusion genes indicated that the AZFs and STZ are preferentially localized to the nucleus. These four ZPT2-related proteins were shown to act as transcriptional repressors that down-regulate the transactivation activity of other transcription factors. RNA gel-blot analysis showed that expression of AZF2 and STZ was strongly induced by dehydration, high-salt and cold stresses, and abscisic acid treatment. Histochemical analysis of beta-glucuronidase activities driven by the AZF2 or STZ promoters revealed that both genes are induced in leaves rather than roots of rosette plants by the stresses. Transgenic Arabidopsis overexpressing STZ showed growth retardation and tolerance to drought stress. These results suggest that AZF2 and STZ function as transcriptional repressors to increase stress tolerance following growth retardation.
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PMID:Arabidopsis Cys2/His2-type zinc-finger proteins function as transcription repressors under drought, cold, and high-salinity stress conditions. 1533 55

Our previous results demonstrated that the DNAbeta satellite (Y10beta) associated with Tomato yellow leaf curl China virus Y10 isolate (TYLCCNV-Y10) is essential for induction of leaf curl symptoms in plants and that transgenic expression of its betaC1 gene in Nicotiana plants induces virus-like symptoms. In the present study, in vitro DNA binding activity of the betaC1 proteins of Y10beta and DNAbeta (Y35beta) found in the Tobacco curly shoot virus Y35 isolate (TbCSV-Y35) were studied following their expression as six-His fusion proteins in Escherichia coli. Electrophoretic mobility shift assays and UV cross-linking experiments revealed that betaC1 proteins could bind both single-stranded and double-stranded DNA without size or sequence specificity. Suppression of green fluorescent protein (GFP) transgene silencing was observed with the new leaves of GFP-expressing Nicotiana benthamiana plants coinoculated by TYLCCNV-Y10 plus Y10beta or by TbCSV-Y35 plus Y35beta. In a patch agroinfiltration assay, the transiently expressed betaC1 gene of Y10beta or Y35beta was able to suppress host RNA silencing activities and permitted the accumulation of high levels of GFP mRNA in the infiltrated leaf patches of GFP transgenic N. benthamiana plants. The betaC1 protein of Y10beta accumulated primarily in the nuclei of plant and insect cells when fused with beta-glucuronidase or GFP and immunogold labeling showed that the betaC1 protein is present in the nuclei of infected N. benthamiana plants. A mutant version of Y10beta carrying the mutations within the putative nuclear localization sequence of the Y10 betaC1 protein failed to induce disease symptoms, suppress RNA silencing, or accumulate in the nucleus, suggesting that nuclear localization of the betaC1 protein is a key requirement for symptom induction and silencing suppression.
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PMID:A Begomovirus DNAbeta-encoded protein binds DNA, functions as a suppressor of RNA silencing, and targets the cell nucleus. 1605 68

In flowering plants, sperm cells develop in the pollen cytoplasm and are transported through floral tissues to an ovule by a pollen tube, a highly polarized cellular extension. After targeting an ovule, the pollen tube bursts, releasing two sperm that fertilize an egg and a central cell. Here, we identified the gene encoding Arabidopsis HAP2, demonstrating that it is allelic to GCS1. HAP2 is expressed only in the haploid sperm and is required for efficient pollen tube guidance to ovules. We identified an insertion (hap2-1) that disrupts the C-terminal portion of the protein and tags mutant pollen grains with the beta-glucuronidase reporter. By monitoring reporter expression, we showed that hap2-1 does not diminish pollen tube length in vitro or in the pistil, but it reduces ovule targeting by twofold. In addition, we show that the hap2 sperm that are delivered to ovules fail to initiate fertilization. HAP2 is predicted to encode a protein with an N-terminal secretion signal, a single transmembrane domain and a C-terminal histidine-rich domain. These results point to a dual role for HAP2, functioning in both pollen tube guidance and in fertilization. Moreover, our findings suggest that sperm, long considered to be passive cargo, are involved in directing the pollen tube to its target.
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PMID:Arabidopsis HAP2 (GCS1) is a sperm-specific gene required for pollen tube guidance and fertilization. 1707 65

In response to wounding and pathogens, jasmonate (JA) serves as a signal molecule for both induction and repression of gene expression. To examine defense-regulated gene repression in Arabidopsis (Arabidopsis thaliana), we have identified a nonclassical arabinogalactan protein (AGP) gene, AGP31, and show that its mRNA decreased to about 30% of its original level within 8 h in response to methyl JA (MeJA) treatment of whole 7-d-old seedlings. Wounding and abscisic acid treatment had similar effects. MeJA suppression primarily depends on the action of the JA-signaling protein, COI1, as shown by much lower MeJA suppression in coi1-1 mutant plants. The main mechanism of mRNA suppression by MeJA is repression of transcription, as shown by nuclear run-on experiments. The AGP31 protein shares features with several known and putative nonclassical AGPs from other species: a putative signal peptide, a histidine-rich region near the N terminus followed by a repetitive proline-rich domain, and a cysteine-rich C-terminal PAC (for proline-rich protein and AGP, containing cysteine) domain. Positive Yariv reagent interaction demonstrated that the protein is an AGP. Monosaccharide analysis of purified AGP31 indicated it is a galactose-rich AGP. Expression of an AGP31-enhanced green fluorescent protein fusion protein in transgenic cells revealed that the AGP31 protein was localized to the cell wall. AGP31 promoter-beta-glucuronidase reporter gene analysis showed expression in the vascular bundle throughout the plant, except in the flower. In the flower, beta-glucuronidase staining occurred throughout the pistil, except in the stigma. The strong preferential expression in vascular tissues suggests that AGP31 may be involved in vascular tissue function during both the defense response and development.
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PMID:A nonclassical arabinogalactan protein gene highly expressed in vascular tissues, AGP31, is transcriptionally repressed by methyl jasmonic acid in Arabidopsis. 1788 91

The expression of a fusion protein formed between the avian infectious bronchitis virus M protein and the bacterial enzyme beta-glucuronidase (GUS) in plants promotes the formation of new organization of the endoplasmic reticulum in tobacco plants. This unusual organization of the membranes, never present in nontransformed plants, has been explained by the oligomerization of the GUS domains of the IBVM-GUS fusion proteins. These specific organized membranes could have broad implications for biotechnology since their formation could be used as a mechanism for retaining and accumulating resident proteins in specific and discrete membrane compartments. In this study, we have shown that the unusual organization of native membranes due to overexpression of the IBVM-GUS fusion gene in tobacco transgenic plants and calli is present at higher levels in plant cell suspensions than in plant tissues. In these cell suspensions, IBVM-GUS protein was continuously synthesized and accumulated throughout the cell culture. An enrichment of the chimeric IBVM-GUS protein corresponding to a five-fold increase in the microsomal fractions was achieved and the GUS enzyme did not show any modification on enzyme kinetics. However, the GUS activity could be differentially distributed in the fractions eluted at different pH suggesting differences in the surface topography of histidine residues for this recombinant GUS.
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PMID:Targeted protein accumulation promoted by autoassembly and its recovery from plant cells. 1826 77

The signal transduction of the phytohormone cytokinin is mediated by a multistep histidine-to-aspartate phosphorelay system. One component of this system are B-type response regulators, transcription factors mediating at least part of the response to cytokinin. In planta functional analysis of this family is hampered by the high level of functional redundancy of its 11 members. We generated a dominant repressor version of the Arabidopsis (Arabidopsis thaliana) response regulator ARR1 (ARR1-SRDX) using chimeric repressor silencing technology in order to study the extent of the contribution of B-type response regulators to cytokinin activities. In a protoplast test system, ARR1-SRDX suppressed ARR6:beta-glucuronidase reporter gene activation by different B-type ARRs. 35S:ARR1-SRDX transgenic Arabidopsis plants showed phenotypic changes reminiscent of plants with a reduced cytokinin status, such as a strongly reduced leaf size, an enhanced root system, and larger seeds. Several bioassays showed that 35S:ARR1-SRDX plants have an increased resistance toward cytokinin. The rapid induction of a large part of the cytokinin response genes was dampened. The transcript levels of more than 500 genes were more than 2.5-fold reduced in 35S:ARR1-SRDX transgenic seedlings, suggesting a broad function of B-type ARRs. Collectively, the suppression of pleiotropic cytokinin activities by a dominant repressor version of a B-type ARR indicates that this protein family is involved in mediating most, if not all, of the cytokinin activities in Arabidopsis. In addition, a role for B-type ARRs in mediating cross talk with other pathways is supported by the resistance of 35S:ARR1-SRDX seeds to phytochrome B-mediated inhibition of germination by far-red light. This study demonstrates the usefulness of chimeric repressor silencing technology to overcome redundancy in transcription factor families for functional studies.
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PMID:The transcriptional repressor ARR1-SRDX suppresses pleiotropic cytokinin activities in Arabidopsis. 1850 77

The aim of this work is to increase the efficiency of the biodegradation of polychlorinated biphenyls (PCBs) by the introduction of bacterial genes into the plant genome. For this purpose, we selected the bphC gene encoding 2,3-dihydroxybiphenyl-1,2-dioxygenase from Pseudomonas testosteroni B-356 to be cloned into tobacco plants. The dihydroxybiphenyldioxygenase enzyme is the third enzyme in the biphenyl degradation pathway, and its unique function is the cleavage of biphenyl. Three different constructs were designed and prepared in E. coli: the bphC gene being fused with the beta-glucuronidase (GUS) gene, with the luciferase (LUC) gene, and with histidine tail in three separate plant cloning vectors. The GUS and LUC genes were chosen because they can be used as markers for the easy detection of transgenic plants, while histidine tail better enables the isolation of protein expressed in plant tissue. The prepared vectors were then introduced into cells of Agrobacterium tumefaciens. The transient expression of the prepared genes was first studied in cells of Nicotiana tabacum. Once this ability had been established, model tobacco plants were transformed by agrobacterial infection with the bphC/GUS, bphC/LUC, and bphC/His genes. The transformed regenerants were selected on media using a selective antibiotic, and the presence of transgenes and mRNA was determined by PCR and RT-PCR. The expression of the fused proteins BphC/GUS and BphC/LUC was confirmed histochemically by analysis of the expression of their detection markers. Western blot analysis was performed to detect the presence of the BphC/His protein immunochemically using a mouse anti-His antibody. Growth and viability of transgenic plants in the presence of PCBs was compared with control plants.
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PMID:Cloning the bacterial bphC gene into Nicotiana tabacum to improve the efficiency of PCB phytoremediation. 1868 52


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