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)

The bean phenylalanine ammonia-lyase gene 2 (PAL2) is expressed in the early stages of vascular development at the inception of xylem differentiation, associated with the synthesis of lignin precursors. This is part of a complex program of developmental expression regulating the synthesis of functionally diverse phenylpropanoid natural products. Analysis of the expression of PAL2 promoter-beta-glucuronidase gene fusions in transgenic tobacco plants showed that functionally redundant cis elements located between nucleotides -289 and -74 relative to the transcription start site were essential for xylem expression, but were not involved in expression in leaf primordia and stem nodes or in establishing tissue specificity in petals. The -135 to -119 region implicated in xylem expression contains a negative element that suppresses the activity of a cryptic cis element for phloem expression located between -480 and -289. The functional properties of each vascular element are conserved in stem, petiole, and root, even though the xylem and phloem are organized in different patterns in these organs. We conclude that the PAL2 promoter has a modular organization and that tissue-specific expression in the vascular system involves a negative combinatorial interaction, modulation of which may provide a flexible mechanism for modification of tissue specificity.
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PMID:cis-element combinations determine phenylalanine ammonia-lyase gene tissue-specific expression patterns. 149 96

New promoter-probe and terminator-probe shuttle vectors for Escherichia coli and corynebacteria were constructed. These vectors, working with the uidA gene of E. coli as reporter gene, are very useful for the cloning and subsequent analysis of transcriptional regulatory signals. The beta-glucuronidase encoding activity of uidA can be easily detected on agar plates containing chromogenic substrates such as 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-gluc). In the terminator-probe vector pUT2, uidA is expressed from a promoter of the Brevibacterium lactofermentum cryptic plasmid pBL1. Multiple cloning sites (MCS) located immediately upstream of uidA allow introduction and selection of terminators or regulatory signals. In the promoter-probe vector pUT3, transcription readthrough from vector promoters is prevented by a terminator of B. lactofermentum isolated using pUT2. We have successfully used pUT2 and pUT3 to isolate several terminators and promoter regions active in B. lactofermentum and shown that the E. coli strong terminator cartridge omega appears less efficient in corynebacteria.
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PMID:Improved vectors for transcriptional signal screening in corynebacteria. 172 68

Recombinase encoded by the R gene of pSR1 of Zygosaccharomyces rouxii mediates reciprocal recombination between two specific recombination sites (RSs) to induce excision or inversion of the DNA segment that is flanked by the RSs. We report here that site-specific recombination mediated by this system takes place effeciently in tobacco cells. To monitor the recombination events in tobacco cells, we have constructed two types of cryptic beta-glucuronidase reporter gene in such a way that recombination such as inversion of the construct or excision of the intervening sequence results in their expression. When these cryptic reporter constructs were transiently introduced together with the R gene by electroporation into protoplasts of tobacco cells, beta-glucuronidase activity was detected. The cryptic reporter genes, when stably resident in the chromosome of tobacco cells, were also activated by the R gene. Structural analyses of the genomic DNA isolated from these tobacco cells showed that the R protein did in fact catalyze precise recombination between two copies of RSs in tobacco cells, with resultant activation of the cryptic reporter genes. This observation provides the basis for development of a DNA technology whereby large regions of DNA can be manipulated in plant chromosomes. Potential uses of this recombination system are discussed.
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PMID:Operation of an efficient site-specific recombination system of Zygosaccharomyces rouxii in tobacco cells. 175 73

Excision of a DNA segment can occur in Arabidopsis thaliana by reciprocal recombination between two specific recombination sites (RSs) when the recombinase gene (R) from Zygosaccharomyces rouxii is expressed in the plant. To monitor recombination events, we generated several lines of transgenic Arabidopsis plants that carried a cryptic beta-glucuronidase (GUS) reporter gene which was designed in such a way that expression of the reporter gene could be induced by R gene-mediated recombination. We also made several transgenic lines with an R gene linked to the 35S promoter of cauliflower mosaic virus. Each transgenic line carrying the cryptic reporter gene was crossed with each line carrying the R gene. Activity of GUS in F1 and F2 progeny was examined histochemically and recombination between two RSs was analyzed by Southern blotting and the polymerase chain reaction. In seedlings and plantlets of F1 progeny and most of the F2 progeny, a variety of patterns of activity of GUS, including sectorial chimerism in leaves, was observed. A small percentage of F2 individuals exhibited GUS activity in the entire plant. This pattern of expression was ascribed to germinal recombination in the F1 generation on the basis of an analysis of DNA structure by Southern blotting. These results indicate that R gene-mediated recombination can be induced in both somatic and germ cells of A. thaliana by cross-pollination of parental transgenic lines.
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PMID:Visualization of site-specific recombination catalyzed by a recombinase from Zygosaccharomyces rouxii in Arabidopsis thaliana. 761 56

T-DNA tagging with a promoterless beta-glucuronidase (GUS) gene generated a transgenic Nicotiana tabacum plant that expressed GUS activity only in developing seed coats. Cloning and deletion analysis of the GUS fusion revealed that the promoter responsible for seed coat specificity was located in the plant DNA proximal to the GUS gene. A 3.3 kb fragment corresponding to the insertion site was isolated from untransformed plants. No long open reading frames were detected in this region. Northern blots and RNase protection assays failed to detect transcripts from this region in untransformed plants. Furthermore, the insertion site was situated within the N. tomentosiformis genome of the allotetraploid species N. tabacum, in a region which is not conserved within the genus Nicotiana. It is concluded that seed coat-specific GUS expression in this transgenic plant resulted from T-DNA insertion next to a cryptic promoter. These results suggest that at least some of the fusions generated to marker genes in promoter trapping studies are not associated with conventional gene promoters. The possibility that similar insertion events play a role in gene evolution is discussed.
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PMID:T-DNA tagging of a seed coat-specific cryptic promoter in tobacco. 798 15

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.
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PMID:Truncated presequences of mitochondrial F1-ATPase beta subunit from Nicotiana plumbaginifolia transport CAT and GUS proteins into mitochondria of transgenic tobacco. 815 82

We have previously sequenced the complete coding region and the promoter region of the beta-glucuronidase gene of a patient with mild mucopolysaccharidosis type VII (MPS VII) and identified a nonsense mutation in the gene inherited from her mother. The mutation inherited from her father was not found. Here, we have extended the sequence analysis of the introns to cover all putative lariat branch points and putative intronic enhancers, although no nucleotide changes have been found in these regions. Careful analysis of mRNA structure by reverse transcription/polymerase chain reaction (RT-PCR) and direct sequencing has revealed the inclusion of a new exon derived from an antisense Alu-repeat in intron 8 and the skipping of exon 9 in a large proportion of the mRNA of our patient. A 2-bp deletion creating a strong 5'-splice site has subsequently been identified in the paternal gene of the patient (IVS8+0.6kbdelTC). With a sensitive RT-PCR assay, we demonstrate that both the inclusion of the Alu-cassette and the skipping of exon 9 are minor events in control samples and that mRNA with both alterations is only found in the IVS8+0.6kbdelTC carrier. The increased proportion of exon 9 skipping seems to be related to the premature termination of translation. This is the third report of a human disease mutation that creates a splice site and activates an antisense Alu-cassette; the question rises as to how these apparently strong cryptic exons are generally excluded from coding sequences.
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PMID:A mutation (IVS8+0.6kbdelTC) creating a new donor splice site activates a cryptic exon in an Alu-element in intron 8 of the human beta-glucuronidase gene. 992 4

This report describes the use of promoter trap lines from the model plant Arabidopsis thaliana to clone regulatory sequences that mediate guard-cell-specific reporter gene expression. Stomatal guard cells represent a highly differentiated cell type within the epidermis of green tissues of higher plants. They control the stomatal aperture in response to different endogenous and environmental signals in order to optimize carbon fixation while minimizing water loss. We screened available promoter trap lines for guard-cell-specific activation of a beta-glucuronidase (uidA) reporter gene in order to obtain marker lines for guard-cell development and to gain access to regulatory pathways leading to gene expression which is restricted to this cell type. From two lines identified we successfully cloned upstream regulatory sequences. For one line, guard-cell-specific promoter activity was confirmed by re-introducing the uidA gene, fused to the newly identified regulatory sequences, into the Arabidopsis nuclear genome. However, DNA sequences downstream of the uidA/T-DNA insertion sites in the original promoter trap lines revealed no obvious coding regions in the corresponding orientation, indicating that we have probably identified 'cryptic' promoters, being active in guard cells.
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PMID:Cloning of regulatory sequences mediating guard-cell-specific gene expression. 1083 41

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.
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PMID:Expression of intron-containing GUS constructs is reduced due to activation of a cryptic 5' splice site. 1140 28

A limited number of constitutive promoters have been used to direct transgene expression in plants and they are often derived from non-plant sources. Here, we describe novel gene-regulatory elements which are associated with a cryptic constitutive promoter from tobacco, tCUP, and modifications that were made to create a strong gene-expression system that is effective across all living cell types from a wide range of plant species, including several important crops ( Arabidopsis, canola, flax, alfalfa, tobacco). The tCUP 5' untranslated region was mutated to eliminate translational interference by upstream ATGs, and the influence of the Kozak consensus sequence on the levels of a beta-glucuronidase (GUS) reporter gene activity was demonstrated. These modifications resulted in expression that was greatly enhanced in all organs. A TATA consensus sequence was added to the core promoter to complement an existing Initiator (Inr) sequence. Although this addition was known to elevate core promoter activity by 3-fold the additive effect on the overall gene-expression system was marginal in all of the transgenic plants tested. Two transcriptional enhancers were identified and the region containing them were oligomerized, yielding a significant increase in marker gene-expression in some but not all plant species. In general, the enhanced tCUP gene-expression system generated levels of GUS activity which exceeded that of the 35S promoter in most plant species and the elevation in activity occurred uniformly among the various plant organs. The potential benefit of cryptic elements for the construction of gene-expression systems for crop species is discussed
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PMID:A constitutive gene expression system derived from the tCUP cryptic promoter elements. 1258 98


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