Function of glycosyltransferase genes involved in urdamycin A biosynthesis.
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BACKGROUND: Urdamycin A, the principle product of Streptomyces fradiae Tu2717, is an angucycline-type antibiotic. The polyketide-derived aglycone moiety is glycosylated at two positions, but only limited information is available about glycosyltransferases involved in urdamycin biosynthesis. RESULTS: To determine the function of three glycosyltransferase genes in the urdamycin biosynthetic gene cluster, we have carried out gene inactivation and expression experiments. Inactivation of urdGT1a resulted in the predominant accumulation of urdamycin B. A mutant lacking urdGT1b and urdGT1c mainly produced compound 100-2. When urdGT1c was expressed in the urdGT1b/urdGT1c double mutant, urdamycin G and urdamycin A were detected. The mutant lacking all three genes mainly accumulated aquayamycin and urdamycinone B. Expression of urdGT1c in the triple mutant led to the formation of compound 100-1, whereas expression of urdGT1a resulted in the formation of compound 100-2. Co-expression of urdGT1b and urdGT1c resulted in the production of 12b-derhodinosyl-urdamycin A, and co-expression of urdGT1a, urdGT1b and urdGT1c resulted in the formation of urdamycin A. CONCLUSIONS: Analysis of glycosyltransferase genes of the urdamycin biosynthetic gene cluster led to an unambiguous assignment of each glycosyltransferase to a certain biosynthetic saccharide attachment step. (+info)
Altered mRNA expression of glycosyltransferases in human colorectal carcinomas and liver metastases.
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BACKGROUND/AIMS: Biosynthesis of carbohydrate structures is tissue specific and developmentally regulated by glycosyltransferases such as fucosyltransferases, sialyltransferases, and N-acetylgluco- saminyltransferases. During carcinogenesis, aberrant glycosylation leads to the development of tumour subpopulations with different adhesion properties. Therefore alterations in glycosyltransferase mRNA expression in colorectal carcinomas were examined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). METHODS: Colorectal carcinoma specimens were classified and characterised according to the WHO/UICC system. Expression of fucosyltransferases FT-I, FT-III, FT-IV, FT-V, FT-VI, and FT-VII, sialyltransferases ST3Gal-I, ST3Gal-III, ST3Gal-IV, and ST6Gal-I, beta1,4-galacto- syltransferase, and beta1,6-Nacetylgluco- saminyltransferase V (GNT-V) was screened simultaneously in extracts of 22 homogenised tumour specimens by RT-PCR and compared with corresponding mucosa from each patient. Also 12 adenomas and 17 liver metastases of colorectal carcinomas were examined. RESULTS: GNT-V expression was enhanced in colorectal adenomas (p = 0.039), carcinomas (p<0.001), and liver metastases of colorectal carcinomas (p<0.001). Also, expression of fucosyltransferase FT-IV was increased in colorectal adenomas (p = 0.039) and carcinomas (p<0. 001). In addition, fucosyltransferase FT-I (p<0.001) and sialyltransferases ST6Gal-I (p = 0.004) and ST3Gal-III (p = 0.001) showed increased expression in carcinoma specimens. On the other hand, fucosyltransferase FT-III was less abundantly expressed in carcinomas exhibiting distant metastases (p = 0.046) and in highly invasive tumours (p = 0.041). CONCLUSIONS: Glycosyltransferase mRNA expression is significantly altered in colorectal adenomas and carcinomas isolated from surgical specimens. RT-PCR determination of specific glycosyltransferases may be helpful for earlier detection of carcinomas and for tumour prognosis. (+info)
Subcellular localization and processing of the lytic transglycosylase of the conjugative plasmid R1.
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Protein P19 encoded by the conjugative resistance plasmid R1, is essential for efficient conjugative DNA transfer and infection by the pilus-specific RNA phage R17. Based on sequence homologies P19 belongs to a family of lysozyme-like virulence factors which are found in type III and type IV secretion systems. In this report we describe the processing and subcellular localization of P19. Pulse-chase experiments were used to demonstrate the processing of P19 by the signal peptidase I of Escherichia coli. Translocation of P19 across the inner membrane was shown by gene 19-phoA fusions. Cell fractionation studies of P19 expressing cells showed the presence of P19 in the membrane compartment. P19 was solubilized with the detergent Sarkosyl indicating an inner membrane localization. Using sucrose density gradient centrifugation to separate inner and outer membranes, P19 was found in both membrane fractions. Taken together, our data suggest that mature P19 is a periplasmic protein which may be attached to the proposed membrane-spanning DNA transport complex. (+info)
Retroviral transfection of Madin-Darby canine kidney cells with human MDR1 results in a major increase in globotriaosylceramide and 10(5)- to 10(6)-fold increased cell sensitivity to verocytotoxin. Role of p-glycoprotein in glycolipid synthesis.
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Retroviral infection of the Madin-Darby canine kidney (MDCK) renal cell line with human MDR1 cDNA, encoding the P-glycoprotein (P-gp) multidrug resistance efflux pump, induces a major accumulation of the glycosphingolipid (GSL), globotriaosylceramide (Galalpha1-4Galbeta1-4glucosylceramide-Gb(3)), the receptor for the E. coli-derived verotoxin (VT), to effect a approximately million-fold increase in cell sensitivity to VT. The shorter chain fatty acid isoforms of Gb(3) (primarily C16 and C18) are elevated and VT is internalized to the endoplasmic reticulum/nuclear envelope as we have reported for other hypersensitive cell lines. P-gp (but not MRP) inhibitors, e.g. ketoconazole or cyclosporin A (CsA) prevented the increased Gb(3) and VT sensitivity, concomitant with increased vinblastine sensitivity. Gb(3) synthase was not significantly elevated in MDR1-MDCK cells and was not affected by CsA. In MDR1-MDCK cells, synthesis of fluorescent N-[7-(4-nitrobenzo-2-oxa-1,3-diazole)]-aminocaproyl (NBD)-lactosylceramide (LacCer) and NBD-Gb(3) via NBD-glucosylceramide (GlcCer) from exogenous NBD-C(6)-ceramide, was prevented by CsA. We therefore propose that P-gp can mediate GlcCer translocation across the bilayer, from the cytosolic face of the Golgi to the lumen, to provide increased substrate for the lumenal synthesis of LacCer and subsequently Gb(3). These results provide a molecular mechanism for the observed increased sensitivity of multidrug-resistant tumors to VT and emphasize the potential of verotoxin as an antineoplastic. Two strains (I and II) of MDCK cells, which differ in their glycolipid profile, have been described. The original MDR1-MDCK parental cell was not specified, but the MDR1-MDCK GSL phenotype and glycolipid synthase activities indicate MDCK-I cells. However, the partial drug resistance of MDCK-I cells precludes their being the parental cell. We speculate that the retroviral transfection per se, or the subsequent selection for drug resistance, selected a subpopulation of MDCK-I cells in the parental MDCK-II cell culture and that drug resistance in MDR1-MDCK cells is thus a result of both MDR1 expression and a second, previously unrecognized, component, likely the high level of GlcCer synthesis in these cells. (+info)
Multiple phosphorylation events regulate the activity of the mannitol transcriptional regulator MtlR of the Bacillus stearothermophilus phosphoenolpyruvate-dependent mannitol phosphotransferase system.
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D-mannitol is taken up by Bacillus stearothermophilus and phosphorylated via a phosphoenolpyruvate-dependent phosphotransferase system (PTS). Transcription of the genes involved in mannitol uptake in this bacterium is regulated by the transcriptional regulator MtlR, a DNA-binding protein whose affinity for DNA is controlled by phosphorylation by the PTS proteins HPr and IICB(mtl). The mutational and biochemical studies presented in this report reveal that two domains of MtlR, PTS regulation domain (PRD)-I and PRD-II, are phosphorylated by HPr, whereas a third IIA-like domain is phosphorylated by IICB(mtl). An involvement of PRD-I and the IIA-like domain in a decrease in affinity of MtlR for DNA and of PRD-II in an increase in affinity is demonstrated by DNA footprint experiments using MtlR mutants. Since both PRD-I and PRD-II are phosphorylated by HPr, PRD-I needs to be dephosphorylated by IICB(mtl) and mannitol to obtain maximal affinity for DNA. This implies that a phosphoryl group can be transferred from HPr to IICB(mtl) via MtlR. Indeed, this transfer could be demonstrated by the phosphoenolpyruvate-dependent formation of [(3)H]mannitol phosphate in the absence of IIA(mtl). Phosphoryl transfer experiments using MtlR mutants revealed that PRD-I and PRD-II are dephosphorylated via the IIA-like domain. Complementation experiments using two mutants with no or low phosphoryl transfer activity showed that phosphoryl transfer between MtlR molecules is possible, indicating that MtlR-MtlR interactions take place. Phosphorylation of the same site by HPr and dephosphorylation by IICB(mtl) have not been described before; they could also play a role in other PRD-containing proteins. (+info)
Dynamic expression of lunatic fringe during feather morphogenesis: a switch from medial-lateral to anterior-posterior asymmetry.
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Expression of Lunatic fringe mRNA was studied during feather morphogenesis and showed three stages of dynamic expression pattern. (1) Lunatic fringe was first expressed in the epithelium as a ring bordering the feather primordium when it was initially induced. (2) Shortly after, it showed a polarized pattern, first toward the lateral side of the feather primordium and then made a 90 degrees C switch toward the posterior side of the short bud. It then becomes weakly expressed in the long bud stage. (3) Finally, it is expressed in the marginal plate epithelia of feather filaments. In contrast, Radical fringe is weakly expressed in the feather bud, but is also present in the marginal plate epithelia of feather filaments. (+info)
Dynamic Lunatic fringe expression is correlated with boundaries formation in developing mouse teeth.
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The formation of boundaries is a fundamental organizing principle during development. The Notch signalling pathway regulates this developmental patterning mechanism in many tissues. Recent data suggest that Notch receptors are involved in boundary determination during odontogenesis. It remains, however, uncertain if other components of the Notch pathway are also important for compartmental lineage restrictions in teeth. Here we report on the expression of the Lunatic fringe gene, which encodes a secreted signalling molecule regulating the Notch pathway, during the development of mouse teeth. Lunatic fringe is expressed in both epithelial and mesenchymal components of the developing molar. The expression pattern of Lunatic fringe in the epithelium is complementary to that of the Notch receptors. Lunatic fringe is asymmetrically expressed in the incisor epithelium during its antero-posterior rotation. This expression pattern defines the lingual comportment of the incisor epithelium whereas the labial comportment is defined by Notch2 expression. (+info)
Characterization of XET-related genes of rice.
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To elucidate the mechanism of internodal elongation in rice (Oryza sativa L.), we analyzed genes encoding xyloglucan endotransglycosylase (XET), a cell wall-loosening enzyme essential for cell elongation. Four rice XET-related (XTR) genes, OsXTR1, OsXTR2, OsXTR3, and OsXTR4, were isolated and their expression patterns in rice plants determined. The expression of the four XTR genes showed different patterns of organ specificity and responses to several plant hormones. OsXTR1 and OsXTR3 were up-regulated by gibberellin and brassinosteroids, whereas OsXTR2 and OsXTR4 showed no clear response to these hormones. Expression of the four XTR genes was also investigated in elongating internodes at different developmental stages. OsXTR1 and OsXTR3 were preferentially expressed in the elongating zone of internodes, while OsXTR2 and OsXTR4 were expressed in nodes and in the divisional and elongating zones of internodes. In three genetic mutants with abnormal heights, the expression of OsXTR1 and OsXTR3 correlated with the height of the mutants, whereas no such correlation was observed for OsXTR2 and OsXTR4. Based on these observations, we discuss the roles that OsXTR1 and OsXTR3 may play in internodal elongation in rice. (+info)