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11.
High level biosynthesis and secretion of the thermostable hybrid (1-3,1-4)--glucanase H(A16-M) has been achieved inSaccharomyces cerevisiae by means of the yeast vacuolar endoprotease B promoter (PRB1p) and theBacillus macerans (1-3,1-4)--glucanase signal peptide. The N-glycans present on the yeast-secreted H(A16-M), denoted H(A16-M)-Y, were released by endoglycosidase H, and identified by proton NMR spectroscopy to be a homologous series of Man8-13GlcNAc2, although only traces of Man9GlcNAc2 were found. Therefore, processing of N-glycans on H(A16-M)-Y is similar to that on homologous proteins. Most of the N-glycans (88%) were neutral while the remainder were charged due to phosphorylation. Site-directed mutagenesis of Asn to Gln in two of the N-glycosylation sequons, and subsequent analysis of the N-glycans on the yeast-secreted proteins together with analysis of the N-glycans from the individual sites of H(A16-M)-Y suggest the presence of steric hindrance to glycan modification by the glycans themselves. H(A16-M)-Y produced under control of either the yeast protease B or the yeast 3-phosphoglycerate kinase promoter, each in two differentSaccharomyces strains revealed a dependence of N-glycan profile on both strain and culture conditions. The extent of O-glycosylation was found to be nine mannose units per H(A16-M)-Y molecule. An attempt to identify the linkage-sites for the O-glycans by amino acid sequencing failed, suggesting non-stoichiometric or heterogeneous O-glycosylation. The possible modes in which N-glycans might contribute to resistance of H(A16-M)-Y to irreversible thermal denaturation are discussed with respect to structural information available for H(A16-M)-Y.
Abbreviations: AMY,B. amyloliquefaciens (1-3,1-4)--glucanse; MAC,B. macerans (1-3,1-4)--glucanase H(A16-M), H(A36-M), H(A78-M),H(A107-M) and H(A152-M), hybrid (1-3,1-4)--glucanases containing 16, 36, 78, 107 and 152 N-terminal amino acids, respectively, derived from AMY with the remaining amino acids derived from MAC; similar enzyme abbreviations followed by Y, e.g. H(A16-M)-Y, denote the enzymes secreted from yeast cells; PCR, polymerase chain reaction; PGKp, yeast 3-phosphoglycerate kinase promoter; PRB1p, yeast protease B promoter; LB, Luria-Bertani medium; SC, minimal medium; CNBr, cyanogen bromide; Endo Hf, endoglycosidase H fusion protein; PNGase F, peptide:N-glycosidase F; HPAEC; high pH anion exchange chromatography; HVE, high voltage paper electrophoresis; CPY, yeast carboxypeptidase Y. 相似文献
12.
Measurement of cytokine antibodies. Test development 总被引:1,自引:0,他引:1
Several assays have been used for detection of antibodies against cytokines. The choice of assay is greatly dependent on the
intended goal, e.g. detection of naturally occurring antibodies or therapy induced antibodies. The different assays can be
grouped in 2 categories. The interference or indirect assays are based on the detection of the test sample interference with
the biological activity, with detection of the cytokine in EIA or with binding to cellular receptors. In direct assays cytokine
antibodies are detected by binding to solid phase fixed cytokines, followed by incubation with a secondary enzyme-labelled
anti-human Ig antibody or by binding to125I-labelled cytokines in RIA. 相似文献
13.
P. M. Matthews R. M. Brown K. Morten D. Marchington J. Poulton G. Brown 《Human genetics》1995,96(3):261-268
Studies in vitro have shown that a respiratorydeficient phenotype is expressed by cells when the proportion of mtDNA with a disease-associated mutation exceeds a threshold level, but analysis of tissues from patients with mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS) have failed to show a consistent relationship between the degree of heteroplasmy and biochemical expression of the defect. One possible explanation for this phenomenon is that there is variation of heteroplasmy between individual cells that is not adequately reflected by the mean heteroplasmy for a tissue. We have confirmed this by study of fibroblast clones from subjects heteroplasmic for the MELAS 3243 (A G) mtDNA mutation. Similar observations were made with fibroblast clones derived from two subjects heteroplasmic for the 11778 (GA) mtDNA mutation of Leber's hereditary optic neuropathy. For the MELAS 3243 mutation, the distribution of mutant mtDNA between different cells was not randomly distributed about the mean, suggesting that selection against cells with high proportions of mutant mtDNA had occurred. To explore the way in which heteroplasmic mtDNA segregates in mitosis we followed the distribution of heteroplasmy between clones over approximately 15 generations. There was either no change or a decrease in the variance of intercellular heteroplasmy for the MELAS 3243 mutation, which is most consistent with segregation of heteroplasmic units of multiple mtDNA molecules in mitosis. After mitochondria from one of the MELAS 3243 fibroblast cultures were transferred to a mitochondrial DNA-free (0) cell line derived from osteosarcoma cells by cytoplast fusion, the mean level and intercellular distribution of heteroplasmy was unchanged. We interpret this as evidence that somatic segregation (rather than nuclear background or cell differentiation state) is the primary determinant of the level of heteroplasmy. 相似文献
14.
DNA markers for downy mildew resistance genes in sorghum. 总被引:1,自引:0,他引:1
The random amplified polymorphic DNA technique was used to find markers for a downy mildew resistance gene in sorghum. Of the 674 random primers screened for polymorphism, 2 amplified fragments were linked to a downy mildew resistance gene in sorghum line SC414. Utilization of an existing restriction fragment length polymorphism mapping population (IS3620C x BTx623) also revealed two markers that are linked to a different resistance gene in another sorghum line, BTx623. 相似文献
15.
Summary We have tested the acyl-transferase of penicillin producing strains and penicillin-less mutants of Penicillium chrysogenum for their ability to exchange the d--aminoadipic acid side chain of cephalosporin-C with phenoxyacetic acid. We found the reaction in both types of strains. 相似文献
16.
Sonication: A new method for gene transfer to plants 总被引:9,自引:0,他引:9
Sonication is a novel method for gene transfer into plant protoplasts and intact plant cells. The mode of action of ultrasound and its chemical, biochemical and physiological effects are reviewed. The state of the art of acoustic transformation is presented and possible mechanisms are discussed. 相似文献
17.
Abstract. Woody vegetation was studied in 44 sites in Senegal. Species composition and density were analysed with TWINSPAN and Canonical Correspondence Analysis; 16 vegetation types were defined. A supervised, multispectral and multitemporal classification of day and night, dry season NOAA-AVHRR imagery was used to identify their distribution with a classification accuracy ranging from 60–100%. 相似文献
18.
19.
14C-labeled extracellular organic carbon (EOC) released by the phytoplankton in a Danish Estuary was shown immediately to form particles (>0.2m) when the products were added to a natural water sample. About 14%–20% of the added activity could be recovered as particles. Any bacterial assimilation of the extracellular products was thus masked. The abiotic origin of the particulate EOC was verified, and it was shown that the particle formation was due to some factors present in the estuarine water with a nominal diameter >0.2m. Precaution must be taken to avoid misinterpretations in studies concerning carbon flow from algae to bacteria. 相似文献
20.
Synthesis of low molecular weight RNA components A, C and D by polymerase II in alpha-amanitin-resistant hamster cells. 总被引:17,自引:7,他引:10 下载免费PDF全文
In an attempt to establish which RNA polymerase catalyzes the synthesis of the low molecular weight RNA components A, C and D, Ama 1 cells (mutant Chinese hamster cells) were used in experiments with addition of alpha-amanitin. Ama 1 cells contain an altered RNA polymerase II which is 800 times more resistant towards inhibition by alpha-amanitin than the wild type enzyme. Alpha-amanitin (up to 200 microgram/ml) added to these cells does not affect the synthesis of the low molecular weight RNAs A, C and D. These data together with our previous data showing that alpha-amanitin (0.5 - 5.0 microgram/ml) preferentially inhibits the synthesis of A, C and D in normal cells indicate that RNA polymerase II catalyzes the synthesis of the low molecular weight RNA components A, C and D. 相似文献