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991.
992.
The external mycelium forms the major part of the absorbing surface of mycorrhized tree roots. Because the macro pore space of acid forest soils is selectively depleted of mobile nutrient cations, it is ecologically important, whether soil hyphae grow into the soil aggregates or not. Seedlings of Norway Spruce (Picea abies (L.) Karst.) with defined mycorrhiza were grown in unsterilized soil cores taken from the A and B-horizon of a limed and an unlimed cambisol on triassic sandstone in the Northern Black Forest, Germany. Water-tension treatments were 10, 30, 160 and 900 hPa. On ground and polished vertical cuts stained with acridine orange, we identified and measured the location of hyphae and characterized their micropedological environment using an image analyzing system. Mean length density varied between 17 m/cm3 and 100 m/cm3 and was independent of aeration parameters. The percentage of hyphae completely embedded in the soil matrix varied between 30% and 8% and decreased significantly with increasing CO2 concentration in the soil air. Of the hyphae in the soil matrix, 70% were located in a 50 m shell around the macro pores. Pair correlation functions show, that the majority of soil hyphae occur in clusters with diameters below 100 m. Between 60% and 80% of randomly chosen circles with 250 m diameters were completely devoid of hyphae. The inefficient opening up of the intra-aggregate space by soil hyphae is explained by the very slow oxygen diffusion between air-filled macro pores and the intra-aggregate space and mechanical restrictions for hyphae growth. 相似文献
993.
PCR bias in ecological analysis: a case study for quantitative Taq nuclease assays in analyses of microbial communities 总被引:3,自引:0,他引:3
Succession of ecotypes, physiologically diverse strains with negligible rRNA sequence divergence, may explain the dominance of small, red-pigmented (phycoerythrin-rich) cyanobacteria in the autotrophic picoplankton of deep lakes (C. Postius and A. Ernst, Arch. Microbiol. 172:69-75, 1999). In order to test this hypothesis, it is necessary to determine the abundance of specific ecotypes or genotypes in a mixed background of phylogenetically similar organisms. In this study, we examined the performance of Taq nuclease assays (TNAs), PCR-based assays in which the amount of an amplicon is monitored by hydrolysis of a labeled oligonucleotide (TaqMan probe) when hybridized to the amplicon. High accuracy and a 7-order detection range made the real-time TNA superior to the corresponding end point technique. However, in samples containing mixtures of homologous target sequences, quantification can be biased due to limited specificity of PCR primers and probe oligonucleotides and due to accumulation of amplicons that are not detected by the TaqMan probe. A decrease in reaction efficiency, which can be recognized by direct monitoring of amplification, provides experimental evidence for the presence of such a problem and emphasizes the need for real-time technology in quantitative PCR. Use of specific primers and probes and control of amplification efficiency allow correct quantification of target DNA in the presence of an up to 10(4)-fold excess of phylogenetically similar DNA and of an up to 10(7)-fold excess of dissimilar DNA. 相似文献
994.
Oligosaccharyltransferase (OST) is an oligomeric protein complex which catalyses the transfer en bloc of Glc3-Man9-GlcNAc2 from Dol-PP to specific asparagine residues in the nascent polypeptide chain. In order to study the function of the pig enzyme subunits, we have cloned OST48, ribophorin I and ribophorin II and characterized these proteins after in vitro translation as well as after expression in COS-1 cells. The individual full-length cDNAs contained open reading frames (ORFs) encoding polypeptides with calculated molecular masses of 48.9[emsp4 ]kDa (OST48), 68.7[emsp4 ]kDa (ribophorin I) and 69.3[emsp4 ]kDa (ribophorin II), respectively. A Kyte and Doolittle hydrophobicity analysis revealed that OST48, ribophorin I and ribophorin II possess a type I membrane topology with the bulk of their polypeptide chains directed towards the ER-lumen. In contrast to OST48, ribophorin I and II contain, respectively, three or two potential N-glycosylation sites of the Asn-Xaa-Thr/Ser type; only one is found to function as the acceptor site in each protein.Transfection of COS-1 cells with vector constructs encoding either OST48, ribophorin I, or a ribophorin I variant tagged with a myc-peptide sequence, resulted in the over-expression of polypeptides whose molecular masses were similar to those calculated from the respective cDNA ORFs. None of these three polypeptides, or ribophorin II, were found to display OST activity when over-expressed alone. By contrast, a modest but reproducible 25% increase of activity was observed when OST48 together with ribophorin I, or OST48 and myc-tagged ribophorin I, were co-expressed, indicating that these two subunits are probably responsible for the catalytic activity in the hetero-oligomeric OST complex. The only modest over-expression of transferase activity suggests that either the dimeric enzyme complex is catalytically unstable, or that the OST48 and ribophorin I polypeptides are unable to fold properly when other subunit components of the hetero-oligomeric OST complex are lacking. OST48 as well as ribophorin I are expressed in COS-1 cells as ER-resident proteins. Whereas OST48 carries a double-lysine motif in the –3/–5 position of its cytosolic C-terminal domain, ribophorin I does not contain recognizable ER-retention information. Replacing the lysine residue in the –3 position by leucine resulted in plasma membrane expression of the OST48-Leu polypeptide, indicating that this sequence motif may be able to influence OST48 localisation. No cell surface staining was observed when OST48-Leu was co-expressed with ribophorin I. This suggests that localisation of OST48 in the ER is mediated by interaction with ribophorin I rather than by the double-lysine motif. 相似文献
995.
Ernst E 《BMJ (Clinical research ed.)》2000,321(7269):1133-1135
996.
997.
998.
The X-ray structure of the NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase from Saccharomyces cerevisiae
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Monzingo AF Breksa A Ernst S Appling DR Robertus JD 《Protein science : a publication of the Protein Society》2000,9(7):1374-1381
Eucaryotes possess one or more NADP-dependent methylene-THF dehydrogenases as part of multifunctional enzymes. In addition, yeast expresses an unusual monofunctional NAD-dependent enzyme, yMTD. We report X-ray structures for the apoenzyme and its complex with NAD+ at 2.8 and 3.0 A resolution, respectively. The protein fold resembles that seen for the human and Escherichia coli dehydrogenase/cyclohydrolase bifunctional enzymes. The enzyme has two prominent domains, with the active site cleft between them. yMTD has a noncanonical NAD-binding domain that has two inserted strands compared with the NADP-binding domains of the bifunctional enzymes. This insert precludes yMTD from dimerizing in the same way as the bifunctional enzymes. yMTD functions as a dimer, but the mode of dimerization is novel. It does not appear that the difference in dimerization accounts for the difference in cofactor specificity or for the loss of cyclohydrolase activity. These functional differences are probably accounted for by minor differences within the tertiary structure of the active site of the monomeric protein. 相似文献
999.
Ernst Freese 《Molecular & general genetics : MGG》1957,88(3):388-406
Summary In crosses involving three allelic pab markers of the same cistron, the recombination frequencies between any two of the markers were such, that a consistent order of the three markers could be established. In these crosses two closely linked markers (0,9% distance, on each side) were also present. The segregation of the pab independent types showed, with respect to the outside markers, a frequent appearance of all four types of outside marker combinations, which in the crossing-over theory would belong to one crossover between the pab markers and 0, 1, or 2 crossovers in the adjacent regions. This deviation from ordinary crossingover expectations, called correlation effect, is confined to very small dimensions of the genome. In the present case to regions of about 0,05% map units.-Inspite of this effect, which sometimes nearly equals out the four types of outside marker combinations, the two recombinant types were, in all present crosses, significantly different, such that the pab markers could be ordered with respect to the outside markers. This ordering is in agreement with that established independently from recombination frequencies. Thus the frequencies of the different types of outside marker combinations are in agreement with the assumption, that the linear genes are built lengthwise into the chromosomal thread, and in disagreement with the assumption that genes or even larger chromosomal parts are sidechains of the chromosome.For this work a subdividing technique has been used by which it was relatively easy to obtain the double mutants between close markers. The same technique can also be applied for the partial selection of double mutants of allelic markers, provided that relatively close markers are available. In determining the linkage relationships it has been found, that me-3 seems to involve a fairly large deletion.
Mit 8 Textabbildungen
Diese Arbeit wurde unterstützt durch Stipendium und Sachbeihilfe der Deutschen Forschungsgemeinschaft und durch ein Stipendium des Damon Runyon Memorial Funds for Cancer Research. Die experimentelle Arbeit wurde ermöglicht durch die Gastfreundlichkeit von Herrn Prof. J. Straub und die großzügige Unterstützung von Herrn Dr. C. Bresch (Botanisches Institut Köln). 相似文献
Mit 8 Textabbildungen
Diese Arbeit wurde unterstützt durch Stipendium und Sachbeihilfe der Deutschen Forschungsgemeinschaft und durch ein Stipendium des Damon Runyon Memorial Funds for Cancer Research. Die experimentelle Arbeit wurde ermöglicht durch die Gastfreundlichkeit von Herrn Prof. J. Straub und die großzügige Unterstützung von Herrn Dr. C. Bresch (Botanisches Institut Köln). 相似文献
1000.