Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.15 (pectinase)
 2,440 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The Erwinia chrysanthemi kdgR gene encodes a repressor that negatively regulates the expression of genes involved in pectinolysis and in pectinase secretion. The cloned kdgR gene was overexpressed in Escherichia coli by using a phage T7 system. Overproduced repressor was purified to homogeneity by two chromatographic steps. Gel retardation and DNase I protection experiments demonstrated the specific binding of the KdgR protein to the operators of pectinase genes (pelA, pelB, pelC, pelE), to the operator of genes involved in pectin catabolism (kdgT, ogl, kduI-kdgF) and to that of the outT gene involved in pectinase secretion. These interactions involved one (pelA, pelB, kduI-kdgF, outT) or several operator sites (pelC, pelE, ogl, kdgT) that generally overlap the promoter. Despite the presence of potential KdgR binding sites (KdgR-box) in the regulatory regions of four genes involved in pectin catabolism (kdgC, kduD, pem, kdgA) and in a pectinase secretion gene outC, no DNA-repressor complex could be observed by in vitro experiments. By using a missing contact experiment on the coding strand of ogl and pelE regulatory regions, a new KdgR-binding consensus was proposed. This new consensus, constituted by two half motifs (AATGAAAACT)N(NTCGATTTCTA), is well conserved in the operators which interact in vitro with the KdgR repressor. In contrast, this repressor-recognized motif is degenerated in the other operators that cannot interact in vitro with the repressor. These results suggest the existence of different regulation mechanisms mediated by the KdgR protein for the two classes of operators.
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PMID:Specific interactions of Erwinia chrysanthemi KdgR repressor with different operators of genes involved in pectinolysis. 810 32

The tomato fruit consists of a thick, fleshy pericarp composed predominantly of highly vacuolated parenchymatous cells, which surrounds the seeds. During ripening, the activation of gene expression results in dramatic biochemical and physiological changes in the pericarp. The polygalacturonase (PG) gene, unlike many fruit ripening-induced genes, is not activated by the increase in ethylene hormone concentration associated with the onset of ripening. To investigate ethylene concentration-independent gene transcription in ripe tomato fruit, we analyzed the expression of chimeric PG promoter-beta-glucuronidase (GUS) reporter gene fusions in transgenic tomato plants. We determined that a 1.4-kb PG promoter directs ripening-regulated transcription in outer pericarp but not in inner pericarp cells, with a sharp boundary of PG promoter activity located midway through the pericarp. Promoter deletion analysis indicated that a minimum of three promoter regions influence the spatial regulation of PG transcription. A positive regulatory region from -231 to -134 promotes gene transcription in the outer pericarp of ripe fruit. A second positive regulatory region from -806 to -443 extends gene activity to the inner pericarp. However, a negative regulatory region from -1411 to -1150 inhibits gene transcription in the inner pericarp. DNase I footprint analysis showed that nuclear proteins in unripe and ripe fruit interact with DNA sequences within each of these three regulatory regions. Thus, temporal and spatial control of PG transcription is mediated by the interaction of negative and positive regulatory promoter elements, resulting in gene activity in the outer pericarp but not the inner pericarp of ripe tomato fruit. The expression pattern of PG suggests that, although they are morphologically similar, there is a fundamental difference between the parenchymatous cells within the inner and outer pericarp.
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PMID:Positive and negative regulatory regions control the spatial distribution of polygalacturonase transcription in tomato fruit pericarp. 840 Aug 76

The expI-expR locus drives a quorum-sensing system in the phytopathogenic bacterium, Erwinia chrysanthemi. Purified ExpR, an N-acyl homoserine lactone-responsive regulatory protein, binds to the promoter/operator region of the expI and expR genes. DNase I footprinting experiments showed that ExpR protects the regions between -66 and -40 from the P1 transcription initiation site of expl and between -54 and -18 from the expR transcription initiation site P1. The protected region overlaps the two expR promoters, P1 and P2, suggesting that ExpR exerts a negative control on its own gene expression. This assertion is reinforced by the fact that the addition of OHHL dissociates the ExpR-expR DNA complex. In contrast, the location of the ExpR binding site on the expI gene suggests an activator function, as reported for the pel genes. Moreover, ExpR is able to induce DNA bending. In vivo and in vitro studies revealed that CRP functions as an activator of expR expression, but as a repressor of expI transcription. A second level of control of expR and expI occurs through the PecS repressor, a regulator of pectinase synthesis. PecS represses expI expression, while ExpR activates pecS transcription, suggesting the existence of a mutual control between pecS and the expI-expR system in E. chrysanthemi. Regulation of pectinase synthesis in soft rot Erwinia appears to be a complex network of multiple cross-acting regulatory elements. A model that integrates these regulatory elements is proposed.
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PMID:Integration of the quorum-sensing system in the regulatory networks controlling virulence factor synthesis in Erwinia chrysanthemi. 978 78

Erwinia carotovora subsp. carotovora produces extracellular pectate lyase (Pel), polygalacturonase (Peh), cellulase (Cel), and protease (Prt). The concerted actions of these enzymes largely determine the virulence of this plant-pathogenic bacterium. E. carotovora subsp. carotovora also produces HarpinEcc, the elicitor of the hypersensitive reaction. We document here that KdgREcc (Kdg, 2-keto-3-deoxygluconate; KdgR, general repressor of genes involved in pectin and galacturonate catabolism), a homolog of the E. chrysanthemi repressor, KdgREch and the Escherichia coli repressor, KdgREco, negatively controls not only the pectinases, Pel and Peh, but also Cel, Prt, and HarpinEcc production in E. carotovora subsp. carotovora. The levels of pel-1, peh-1, celV, and hrpNEcc transcripts are markedly affected by KdgREcc. The KdgREcc- mutant is more virulent than the KdgREcc+ parent. Thus, our data for the first time establish a global regulatory role for KdgREcc in E. carotovora subsp. carotovora. Another novel observation is the negative effect of KdgREcc on the transcription of rsmB (previously aepH), which specifies an RNA regulator controlling exoenzyme and HarpinEcc production. The levels of rsmB RNA are higher in the KdgREcc- mutant than in the KdgREcc+ parent. Moreover, by DNase I protection assays we determined that purified KdgREcc protected three 25-bp regions within the transcriptional unit of rsmB. Alignment of the protected sequences revealed the 21-mer consensus sequence of the KdgREcc-binding site as 5'-G/AA/TA/TGAAA[N6]TTTCAG/TG/TA-3'. Two such KdgREcc-binding sites occur in rsmB DNA in a close proximity to each other within nucleotides +79 and +139 and the third KdgREcc-binding site within nucleotides +207 and +231. Analysis of lacZ transcriptional fusions shows that the KdgR-binding sites negatively affect the expression of rsmB. KdgREcc also binds the operator DNAs of pel-1 and peh-1 genes and represses expression of a pel1-lacZ and a peh1-lacZ transcriptional fusions. We conclude that KdgREcc affects extracellular enzyme production by two ways: (i) directly, by inhibiting the transcription of exoenzyme genes; and (ii) indirectly, by preventing the production of a global RNA regulator. Our findings support the idea that KdgREcc affects transcription by promoter occlusion, i.e., preventing the initiation of transcription, and by a roadblock mechanism, i.e., by affecting the elongation of transcription.
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PMID:kdgREcc negatively regulates genes for pectinases, cellulase, protease, HarpinEcc, and a global RNA regulator in Erwinia carotovora subsp. carotovora. 1019 3

This study describes an effective method of in situ RT-PCR (RT-ISPCR) that was developed to localize gene expression in plant tissues. This RT-PCR technique was performed on sectioned tissues of female buds of the cucumber GY3 inbred line. The CUS1 gene, encoding the MADS-box type (agamus-like) protein, the expression pattern of which was described earlier, was used as a marker gene for optimisation of steps in the in situ RT-PCR inside the cells. For the identification of RT-PCR products inside the cells of the female buds, they were fixed in FAA solution, embedded in Paraplast Plus and cut into 7 microm thick sections which were dewaxed by immersion in HistoClear and dehydrated with ethanol. They were washed in water, then in 0.02M HCl, 2xSSC and PBS buffer. In the next step of tissue pretreatment, the sections were digested with 1% pectinase. As shown, the pectinase treatment proved to be a crucial step in the tissue preparation procedure to get successful RT-PCR products. After washing in PBS buffer, the sections were digested with protease K followed by incubation with RNase-free DNase I, and subsequently washed in 2xSSC, 1xSSC and 0.5xSSC and finally in DEPC-treated water. Then the sections were covered with 50 microl of the RT-PCR reaction mixture supplemented with 0.5 microM digoxigenin dUTP and sealed with a coverslip. After amplification in situ the PCR products were identified with anti-digoxigenin antibody (Roche Molecular Biochemicals), conjugated with alkaline phosphatase. The data obtained showed that specific signals reflecting CUS1 gene expression were detected in the female flower buds of cucumber. The specificity of the in situ RT-PCR protocol was confirmed by dot blot hybridization of RT-PCR products with CUS1 cDNA probe.
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PMID:A useful protocol for in situ RT-PCR on plant tissues. 1194 46