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 obtained transgenic maize plants by using high-velocity microprojectiles to transfer genes into embryongenic cells. Two selectable genes were used to confer resistance to either chlorsulfuron or phosphinothricin, and genes encoding either E. coli beta-glucuronidase or firefly luciferase were used as markers to provide convenient assays for transformation. When regenerated without selection, only two of the eight transformed embryogenic calli obtained produced transgenic maize plants. With selection, transgenic plants were obtained from three of the other eight calli. One of the two initial lines produced 15 fertile transgenic plants. The progeny of these plants contained and expressed the foreign genes. Luciferase expression could be visualized, in the presence of added luciferin, by overlaying leaf sections with color film.
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PMID:Inheritance and expression of chimeric genes in the progeny of transgenic maize plants. 136 94

We have investigated whether reporter genes influence cytoplasmic regulation of gene expression in tobacco and Chinese hamster ovary (CHO) cells. Two genes, uidA encoding beta-glucuronidase (GUS) from Escherichia coli and Luc, encoding firefly luciferase (LUC), were used to analyze the ability of a cap, polyadenylated tail, and the 5'- and 3'-untranslated regions (UTR) from tobacco mosaic virus (TMV) to regulate expression. The regulation associated with the 5' cap structure and the TMV 5'-UTR, both of which enhance translational efficiency, was reporter gene-independent. The poly(A) tail and the TMV 3'-UTR, which is functionally equivalent to a poly(A) tail, increase translational efficiency as well as mRNA stability. The regulation associated with these 3' ends was highly reporter gene-dependent; their effect on GUS expression was almost an order of magnitude greater than that on LUC expression. In tobacco, the tenfold reporter gene effect on poly(A) tail or TMV 3'-UTR function could not be explained by a differential impact on mRNA stability; GUS and LUC mRNA half-life increased only twofold when either the poly(A) tail or TMV 3'-UTR was present. In CHO cells, however, GUS mRNA was stabilized to a greater extent by a poly(A) tail or the TMV 3'-UTR than was LUC mRNA.
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PMID:Post-transcriptional regulation in higher eukaryotes: the role of the reporter gene in controlling expression. 188 10

Maize transposable elements, when inserted in or near genes, alter expression by several transcriptional and post-transcriptional mechanisms. Three independent, unstable insertions of the transposable element Mutator (Mu) into the first intron of the Alcohol dehydrogenase-1 (Adh1) gene have been shown to decrease expression [Strommer et al. (1982). Nature 300, 542-544]. We have developed an approach to elucidate the underlying molecular mechanisms responsible for the mutant phenotypes. Mu1 elements were inserted into Adh1-S intron 1 in vitro to create plasmid facsimiles of the mutant alleles. The Mu1 element was also inserted at novel positions within intron 1 to create new mutations. The Mu1/intron constructions were placed between the Adh1-S promoter/exon 1 segment and a reporter gene (firefly luciferase or beta-glucuronidase), and these chimeric gene constructs were tested in transient assays in maize protoplasts. When compared with the appropriate control, the Mu1 insertions decreased reporter gene expression to levels approximating the alcohol dehydrogenase enzyme activities observed for the Adh1-S mutants in vivo. The Mu1 insertions also showed a polarity effect with luciferase expression increasing as the insertions were placed nearer the 3' splice junction. In addition, Mu1 insertions within a different intron, actin intron 3, also significantly reduced luciferase expression, indicating that Mu1 insertions within introns are likely to diminish expression in many genes. The presence of the Mu1 sequences was correlated with decreased levels of steady-state luciferase transcript. Deletion analysis of the Mu1 element and RNase mapping indicate that the transposable element contains RNA processing signals in its central region that are largely responsible for the decrease in expression.
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PMID:Insertion of Mu1 elements in the first intron of the Adh1-S gene of maize results in novel RNA processing events. 196 75

The inclusion of the alcohol dehydrogenase 1-S(Adh 1-S) intron 1 in the transcription unit of maize gene constructs has been shown to increase gene expression in cultured maize cells. We have extended these studies with Adh1-S intron 1 using the firefly luciferase, Escherichia coli beta-glucuronidase and chloramphenicol acetyltransferase reporter genes adjoined to different plant promoters and find enhancement of transient gene expression in all cases but one. We also show that the enhancement phenomenon can be mediated by the third intron of the maize actin gene. In all cases tested, the inclusion of an intron results in increased levels of steady-state RNA. The degree of enhancement depends on the exon sequences flanking the intron; flanking exons also influence the efficiency of intron splicing. Unexpectedly, unspliced RNAs accumulate during the transient assay.
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PMID:Intron enhancement of gene expression and the splicing efficiency of introns in maize cells. 200 94

We have isolated and determined DNA sequence for the 5'-flanking regions of three Arabidopsis thaliana polyubiquitin genes, UBQ3, UBQ10, and UBQ11. Comparison to cDNA sequences revealed the presence of an intron in the 5'-untranslated region at the same position immediately upstream of the initiator methionine codon in each of the three genes. An intron at this position is also present in two sunflower and two maize polyubiquitin genes. An intron is also found in the 5'-untranslated regions of several animal polyubiquitin genes, although the exact intron position is not conserved among them, and none are in the same position as those in the higher plant polyubiquitin genes. Chimeric genes containing the 5'-flanking regions of UBQ3, UBQ10, and UBQ11 in front of the coding regions for the reporter enzyme Escherichia coli beta-glucuronidase (GUS) were constructed. When introduced transiently into Arabidopsis leaves via microprojectile bombardment, all resulted in readily detectable levels of GUS activity that were quantitatively similar. The introns of UBQ3 and UBQ10 in the corresponding promoter fragments were removed by replacement with flanking cDNA sequences and chimeric genes constructed. These constructs resulted in 2.5- to 3-fold lower levels of marker enzyme activity after transient introduction into Arabidopsis leaves. The UBQ10 promoter without the 5' intron placed upstream of firefly luciferase (LUX) resulted in an average of 3-fold lower LUX activity than from an equivalent construct with the UBQ10 intron. A UBQ3 promoter cassette was constructed for the constitutive expression of open reading frames in dicot plants and it produced readily detectable levels of GUS activity in transient assays.
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PMID:The intron of Arabidopsis thaliana polyubiquitin genes is conserved in location and is a quantitative determinant of chimeric gene expression. 838 9

The 897 bp 5' flanking region of the rice storage protein glutelin gene (GluA-3) was analyzed to define cis-regulatory elements responsible for the seed specific expression by progressive 5' deletions and gain-of-function experiments using a truncated CaMV 35S promoter (-90/+9). These constructs were transcriptionally fused either to the firefly luciferase (LUX) or beta-glucuronidase (GUS) reporter gene and introduced into tobacco. It was shown that a 121 bp sequence between -437 and -317 from the transcriptional start site is important for the quantitative regulation of the GluA-3 gene expression. This region contained at least two functionally different cis-regulatory elements. One acts as a general positive element and the other is involved in a specific negative regulation in non-seed tissues.
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PMID:cis-regulatory elements responsible for quantitative regulation of the rice seed storage protein glutelin GluA-3 gene. 872 Sep 27

The ubiquitin pathway targets proteins for degradation through the post-translational covalent attachment of the 76 amino acid protein ubiquitin to epsilon-amino lysyl groups on substrate proteins. Two instability determinants recognized by the ubiquitin pathway in Saccharomyces cerevisiae have been identified. One is described by the N-end rule and requires specific destabilizing residues at the substrate protein N-termini along with a proximal lysyl residue for ubiquitin conjugation. The second is a linear uncleavable N-terminal ubiquitin moiety. The ability of these two determinants to function in higher plants was investigated in tobacco protoplast transient transfection assays using DNA encoding variants of well characterized reporter enzymes as substrates: firefly luciferase that is localized to peroxisomes (pxLUC), a cytosolic version of LUC (cLUC), and Escherichia coli beta-glucuronidase (GUS). cLUC with phenylalanine encoded at its mature N-terminus was 10-fold less abundant than cLUC with methionine at its mature N-terminus. GUS with phenylalanine encoded at its mature N-terminus was 3-fold less abundant than GUS with methionine at its mature N-terminus. The presence of a uncleavable N-terminal ubiquitin fusion resulted in 50-fold lower protein accumulation of cLUC, but had no effect on GUS. Both instability determinants had a much larger effect on cLUC than on pxLUC, suggesting that these degradation signals are either unrecognized or poorly recognized in the peroxisomes.
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PMID:Engineering in vivo instability of firefly luciferase and Escherichia coli beta-glucuronidase in higher plants using recognition elements from the ubiquitin pathway. 961 5

: We report the genetic transformation of two marine diatoms by microparticle bombardment. The pennate diatom Phaeodactylum tricornutum was transformed with the bacterial gene Sh ble from Streptoalloteichus hindustanus, which confers resistance to the antibiotics phleomycin and zeocin. Transformants contained between 1 and 10 copies of the exogenous DNA integrated into the genome by illegitimate recombination at apparently random locations. Transformation efficiencies were around 10(-6), and individual cell lines could be maintained at -80 degrees C following cryopreservation. Also, P. tricornutum could be transformed simultaneously with two different plasmids, one containing the Sh ble gene and another containing the firefly luciferase gene (LUC) under control of a promoter derived from a fucoxanthin, chlorophyll a/c-binding protein gene (FCP). In these cotransformants, LUC activity was light inducible. The transient transformation of the centric diatom Thalassiosira weissflogii with the bacterial beta-glucuronidase (GUS) gene has also been achieved using similar transformation technology. The availability of gene transfer protocols for marine diatoms, together with a range of functional reporter genes and regulated expression systems, will permit molecular dissection of their biology and allow an assessment of the biotechnological potential of these organisms.
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PMID:Transformation of Nonselectable Reporter Genes in Marine Diatoms. 1038 98

In Plasmodium parasites the fusion of gametes to form a fertilized zygote and morphogenesis into the motile ookinete are critical developmental stages in the parasite's complex life cycle. In analogous developmental stages of metazoan organisms 3' gene flanking regions are critical in the regulation of gene expression. To determine whether these mechanisms are conserved in the protozoan parasite we studied the 3' gene flanking elements necessary for the expression of Pgs28, the major surface protein of mature zygotes and ookinetes of the chicken malaria Plasmodium gallinaceum. The DNA sequence of the pgs28 3' gene flanking region contains 7 eukaryotic polyadenylation consensus signals (AATAAA/ATTAAA). An unusual 82% T-rich region is located 55 nucleotides upstream of the fifth polyadenylation signal (ATTAAA). The pgs28 mRNA terminates approximately 20 nucleotides from the polyadenylation signal in a poly (A) tail. To determine whether the T-rich region and polyadenylation signals were necessary for Pgs28 protein expression, sexual stage parasites were transfected with plasmids containing deletions of these elements utilizing firefly luciferase (LUC) and beta-glucuronidase (GUS) as markers of transient gene transfection. The parasites were allowed to develop in vitro to the ookinete stage and assayed for enzymatic activity. Cells transfected with plasmids containing deletions of the T-rich region or fifth eukaryotic polyadenylation consensus signal expressed 89 and 92%, less enzymatic activity respectively than those transfected with the full length pgs28 3' gene flanking region. The U-rich element and fifth eukaryotic polyadenylation consensus sequence within the pgs28 3' UTR are therefore necessary for Pgs28 protein expression.
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PMID:3' UTR elements enhance expression of Pgs28, an ookinete protein of Plasmodium gallinaceum. 1061 99

We describe new tools for functional analysis of the tomato genome based on insertional mutagenesis with the maize Ac/Ds transposable elements in the background of the miniature cultivar Micro-Tom. 2932 F3 families, in which Ds elements transposed and were stabilized, were screened for phenotypic mutations. Out of 10 families that had a clear mutant phenotype, only one mutant was Ds-tagged. In addition, we developed promoter trapping using the firefly luciferase reporter gene and enhancer trapping, using beta-glucuronidase (GUS). We show that luciferase can be used as a non-invasive reporter to identify, isolate and regenerate somatic sectors, to study the time course of mutant expression, and to identify inducible genes. Out of 108 families screened for luciferase activity 55% showed expression in the flower, 11% in the fruit and 4% in seedlings, suggesting a high rate of Ds insertion into genes. Preferential insertion into genes was supported by the analysis of Ds flanking sequences: 28 out of 50 sequenced Ds insertion sites were similar to known genes or to ESTs. In summary, the 2932 lines described here contain 2-3 Ds inserts per line, representing a collection of approximately 7500 Ds insertions. This collection has potential for use in high-throughput functional analysis of genes and promoter isolation in tomato.
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PMID:Technical advance: a high throughput system for transposon tagging and promoter trapping in tomato. 1084 44


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