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)

A flower-predominant cDNA for a gene, termed OsChia 1;175, was isolated from a cDNA library of rice pistils. Northern blot and RT-PCR analyses revealed that the OsChia 1;175 gene is highly expressed in floral organs (pistils, stamens and lodicules at the heading stage) but not or at an extremely low level in vegetative organs. OsChia 1;175 encodes a protein that consists of 340 amino acid residues, and the putative mature protein shows 52% to 63% amino acid identity to class I chitinases of rice or other plants. The phylogenetic tree shows that the OsChia 1;175 protein is a new type of plant class I chitinase in rice. The expression of OsChia 1;175 in vegetative organs is not induced by several chemicals, UV, and wounding. The soluble putative mature OsChia 1;175 protein expressed in Escherichia coli exhibited chitinase activity in the assay with colloidal chitin as a substrate. Genomic Southern analysis revealed that the OsChia 1;175 gene was organized as a low-copy gene family. The rice genomic library was screened and a genome clone corresponding to OsChia 1;175 was isolated. The transcription start sites of the OsChia 1;175 gene were mapped by primer extension analysis. The 1.2 kb putative promoter region of the OsChia 1;175 gene was fused to the GUS (beta-glucuronidase) gene, and this chimeric gene was introduced to rice by Agrobacterium-mediated transformation. The flower-predominant gene expression was identified also in the transgenic rice plants. The high promoter activity was detected in the stigmas, styles, stamens and lodicules in transgenic plants. The possible functions of OsChia 1;175 are discussed.
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PMID:Flower-predominant expression of a gene encoding a novel class I chitinase in rice (Oryza sativa L.). 1089 May 35

The carrot (Daucus carota L.) EP3 chitinase was shown to be essential for somatic embryo formation in a carrot mutant cell line. We identified the Arabidopsis thaliana (L.) Heynh. ortholog of the carrot EP3-3 chitinase gene, designated as AtEP3/AtchitIV and analyzed its expression in Arabidopsis by means of reverse transcription-polymerase chain reaction and promoter::beta-glucuronidase and luciferase fusions. As in carrot, the gene is expressed during somatic embryogenesis in "nursing" cells surrounding the embryos but not in embryos themselves. In plants, gene expression is found in mature pollen and growing pollen tubes until they enter the receptive synergid, but not in endosperm and integuments as in carrot. Post-embryonically, expression is found in hydathodes, stipules, root epidermis and emerging root hairs, indicating that the Arabidopsis chitinase may have a function that is not restricted to embryogenesis.
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PMID:Expression pattern of the Arabidopsis thaliana AtEP3/AtchitIV endochitinase gene. 1152 12

Nuclear matrix attachment regions (MARs) are thought to influence the expression of flanking genes. In this study, we investigated the activation of genes by tobacco MARs that had previously been identified in the 5' region of the basic class I chitinase gene, CHNS0. In transgenic tobacco cells, a construct consisted of the 35S promoter of cauliflower mosaic virus (CaMV) fused to a beta-glucuronidase gene (uidA) with 5' MAR elements was expressed at a 10-fold higher level than a similar construct without MAR sequences. However, expression of a similar construct with 3' MARs and of a construct with a truncated (-46) 35S minimal promoter and uidA with 5' MARs was not similarly enhanced, suggesting that MARs might act by increasing the activity of downstream enhancers. Deletion analysis of the MAR sequences revealed that the function of the MARs that increased the expression of the transgene was redundant. Moreover, assays of the transient expression of transgenes suggested that MAR elements might be involved in the structure and organization of chromatin. To examine the influence of MARs on chromatin structure, we investigated the effects of micrococcal nuclease (MNase) on the DNA in the reporter gene around the MARs. Analysis of the time-course of digestion of nuclei with MNase revealed that the 35S promoter region with 5' MARs was much more sensitive to MNase than the same region without MARs, suggesting that MARs might mediate the opening of chromatin in the region of a downstream promoter, with consequent enhancement of transcription.
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PMID:Matrix attachment regions enhance transcription of a downstream transgene and the accessibility of its promoter region to micrococcal nuclease. 1267 55

Chitinases are ubiquitous proteins that occur in all plants in multiple isoforms. We have isolated the ChtC2 gene encoding an unusual, basic (class I) chitinase from potato ( Solanum tuberosum L.). In contrast to other chitinase genes, ChtC2 is not activated by infection, but rather constitutively expressed in leaves and stems where it is restricted to epidermal cells. Sequence analysis revealed a number of potential regulatory elements in the promoter, but most striking was the presence of a 319-bp direct repeat located between -333 and -968 upstream of the transcription start site. For a functional analysis, a 1,322-bp promoter fragment and two 5' deletions of 782 bp and 162 bp in length were translationally fused to the beta-glucuronidase (GUS) reporter gene and used for transient expression studies by particle bombardment. All promoter constructs conferred expression of GUS activity in different epidermal cell types of potato leaves. Expression in parenchyma cells of the leaf mesophyll was not detectable with any of the ChtC2 gene promoter constructs, in contrast to the pattern observed with the 35S promoter from cauliflower mosaic virus. The epidermis-specific expression of the reporter gene was confirmed using transgenic potato plants containing the fusion of the entire ChtC2 promoter with the GUS reporter. Histochemical analysis indicated that the promoter was only active in epidermal cells of leaves.
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PMID:The promoter of the potato chitinase C gene directs expression to epidermal cells. 1273 75

Plant vacuoles are multi-functional, developmentally varied and can occupy up to 90% of plant cells. The N-terminal propeptide (NTPP) of sweet potato sporamin and the C-terminal propeptide (CTPP) of tobacco chitinase have been developed as models to target some heterologous proteins to vacuoles but so far tested on only a few plant species, vacuole types and "payload" proteins. Most studies have focused on lytic and protein-storage vacuoles, which may differ substantially from the sugar-storage vacuoles in crops like sugarcane. Our results extend the evidence that NTPP of sporamin can direct heterologous proteins to vacuoles in diverse plant species and indicate that sugarcane sucrose-storage vacuoles (like the lytic vacuoles in other plant species) are hostile to heterologous proteins. A low level of cytosolic NTPP-GFP (green fluorescent protein) was detectable in most cell types in sugarcane and Arabidopsis, but only Arabidopsis mature leaf mesophyll cells accumulated NTPP-GFP to detectable levels in vacuoles. Unexpectedly, efficient developmental mis-trafficking of NTPP-GFP to chloroplasts was found in young leaf mesophyll cells of both species. Vacuolar targeting by tobacco chitinase CTPP was inefficient in sugarcane, leaving substantial cytoplasmic activity of rat lysosomal beta-glucuronidase (GUS) [ER (endoplasmic reticulum)-RGUS-CTPP]. Sporamin NTPP is a promising targeting signal for studies of vacuolar function and for metabolic engineering. Such applications must take account of the efficient developmental mis-targeting by the signal and the instability of most introduced proteins, even in "storage vacuoles".
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PMID:Efficient developmental mis-targeting by the sporamin NTPP vacuolar signal to plastids in young leaves of sugarcane and Arabidopsis. 1537 94

The activity of constitutive promoters was compared in transgenic alfalfa plants using two marker genes. Three promoters, the 35S promoter from cauliflower mosaic virus (CaMV), the cassava vein mosaic virus (CsVMV) promoter, and the sugarcane bacilliform badnavirus (ScBV) promoter were each fused to the beta-glucuronidase (gusA) gene. The highest GUS enzyme activity was obtained using the CsVMV promoter and all alfalfa cells assayed by in situ staining had high levels of enzyme activity. The 35S promoter was expressed in leaves, roots, and stems at moderate levels, but the promoter was not active in stem pith cells, root cortical cells, or in the symbiotic zones of nodules. The ScBV promoter was active primarily in vascular tissues throughout the plant. In leaves, GUS activity driven by the CsVMV promoter was approximately 24-fold greater than the activity from the 35S promoter and 38-fold greater than the activity from the ScBV promoter. Five promoters, the double 35S promoter, figwort mosaic virus (FMV) promoter, CsVMV promoter, ScBV promoter, and alfalfa small subunit Rubisco (RbcS) promoter were used to control expression of a cDNA from Trichoderma atroviride encoding an endochitinase (ech42). Highest chitinase activity in leaves, roots, and root nodules was obtained in plants containing the CsVMV:ech42 transgene. Plants expressing the endochitinase were challenged with Phoma medicaginis var. medicaginis, the causal agent of spring black stem and leaf spot of alfalfa. Although endochitinase activity in leaves of transgenic plants was 50- to 2650-fold greater than activity in control plants, none of the transgenic plants showed a consistent increase in disease resistance compared to controls. The high constitutive levels of both GUS and endochitinase activity obtained demonstrate that the CsVMV promoter is useful for high-level transgene expression in alfalfa.
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PMID:A comparison of constitutive promoters for expression of transgenes in alfalfa (Medicago sativa). 1551 94

Chitinase gene expression has been shown to be transcriptionally regulated by a number of inducers, including ethylene, elicitors, and pathogen attack. To investigate the mechanism(s) responsible for induction of chitinase gene expression in response to various stimuli, we have developed a transient gene expression system in bean (Phaseolus vulgaris) protoplasts that is responsive to ethylene and elicitor treatment. This system was used to study the expression of a chimeric gene composed of the 5' flanking sequences of a bean endochitinase gene fused to the reporter gene beta-glucuronidase linked to a 3' fragment from nopaline synthase. Addition of 1-aminocyclopropane-1-carboxylic acid, the direct precursor of ethylene, or elicitors such as chitin oligosaccharides or cell wall fragments derived from Colletotrichum lagenarium, to transformed protoplasts resulted in a rapid and marked increase in the expression of the chimeric gene. The kinetics and dose response for these treatments were similar to those observed for the native gene in vivo. Analyses of 5' deletion mutants in the protoplast system indicated that DNA sequences located between -305 and -236 are important for both ethylene and elicitor induction of the reporter gene.
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PMID:Regulation of a chitinase gene promoter by ethylene and elicitors in bean protoplasts. 1666 5

A putative class I basic chitinase gene, assigned as psiBCH, was cloned from a tomato breeding line NC 24E. The gene contains a coding region with two introns. The predicted psiBCH open reading frame (ORF) is 971 bp and exhibits 81-88% identity at the nucleotide level with known class I basic chitinase genes from the Solanaceae family. However, the presence of a stop codon caused by a frameshift in the ORF of psiBCH makes it unusual among the other class I plant basic chitinases. This stop codon might be involved in the lower accumulation of fully spliced psiBCH RNA caused by nonsense-mediated decay (NMD), which is an RNA surveillance system universally found in eukaryotes. Sequence analysis of the 1883-bp 5'-flanking region of the psiBCH gene revealed the presence of potential wound-response promoter elements. To study the transcriptional regulation of the psiBCH gene, its 5'-flanking region containing the putative promoter was fused to the gus reporter gene and introduced into the tobacco genome via Agrobacterium tumefaciens-mediated transformation. Transgenic plants were functionally assayed for beta-glucuronidase activity. The psiBCH promoter drives the reporter gene expression in response to wounding stimuli. psiBCH promoter-GUS analysis indicates that wound-response of the tobacco transgene was rapid and localized in the wounded area following mechanical wounding. Therefore, our results suggest that the psiBCH promoter can provide targeted expression of genes, such as protease inhibitors in response to pest attack.
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PMID:A frameshift in the coding region of a novel tomato class I basic chitinase gene makes it a pseudogene with a functional wound-responsive promoter. 1671 38

Co-transformation of Oryza sativa L. var. Pusa Basmati1 was done using an Agrobacterium tumefaciens strain harbouring a single-copy cointegrate vector and a multi-copy binary vector in the same cell. The T-DNA of the cointegrate vector pGV2260::pSSJ1 carried the hygromycin phosphotransferase (hph) and beta-glucuronidase (gus) genes. The binary vector pCam-chi11, without a plant selectable marker gene, harboured the rice chitinase (chi11) gene under maize ubiquitin promoter. Co-transformation of the gene of interest (chi11) with the selectable marker gene (hph) occurred in 4 out of 20 T(0) plants (20%). Segregation of hph from chi11 was accomplished in two (CoT6 and CoT23) of the four co-transformed plants in the T(1) generation. The selectable marker-free (SMF) lines CoT6 and CoT23 harboured single copies of chi11. Homozygous SMF T(2) plants were established in the lines CoT6 and CoT23. Northern and Western blot analysis of the homozygous SMF lines showed high level of transgene expression. In comparison to untransformed controls, chitinase specific activity was 66- and 22-fold higher in the homozygous SMF T(2) plants of lines CoT6 and CoT23, respectively. The lines CoT6 and CoT23 exhibited 38 and 40% reduction in sheath blight disease, respectively.
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PMID:Generation of selectable marker-free sheath blight resistant transgenic rice plants by efficient co-transformation of a cointegrate vector T-DNA and a binary vector T-DNA in one Agrobacterium tumefaciens strain. 1866 52

Novozym 234 has been traditionally used to prepare protoplasts for genetic transformation of fungi. Since it is no longer on the market, a new enzyme cocktail was defined to protoplast Aspergillus niger. The cocktail consists of lysing enzymes from Trichoderma harzianum, chitinase from Streptomyces griseus and beta-glucuronidase from Helix pomatia.
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PMID:An enzyme cocktail for efficient protoplast formation in Aspergillus niger. 1904 7


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