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21.
N Chaly M Cadrin J G Kaplan D L Brown 《Biology of the cell / under the auspices of the European Cell Biology Organization》1988,63(1):9-17
Assembly of active nuclei in lymphocytes stimulated by mitogen is paralleled by the elaboration of a structurally and biochemically complex nuclear matrix (NM). To examine the dynamics of individual NM polypeptide components during blastogenesis, we have applied immunofluorescence labelling with anti-NM antibodies to concanavalin A-stimulated mouse splenocytes. Whereas peripherin and PI2 antigens did not reorganize during stimulation, labelling of PI1 and small nuclear ribonucleoprotein (snRNP) antigens increased markedly in intensity and redistributed in concert with the previously reported NM restructuring. Double-labelling showed, furthermore, that snRNPs and the internal staining component of PI1 were largely co-localized. As an approach to studying the role of RNA and RNA synthesis in NM organization, we have further examined the effects of the inhibitor of RNA synthesis, 5,6-dichloro-1-beta-D-ribofuranosyl benzimidazole (DRB), on NM antigen distribution. The rapid inhibition of 3H-uridine incorporation by DRB was accompanied by coordinate aggregation of snRNPs and of the internal PI1 component into large, brightly stained patches. Both 3H-uridine incorporation levels and antigen localization were readily reversed upon removal of DRB. We conclude that NM antigens behave independently during nuclear and NM assembly and that NM organization, as reflected by NM antigen distribution, is modulated by con A- and DRB-induced alterations in RNA synthesis. We propose, furthermore, that the PI1 antigen plays a role in RNA metabolism, and is possibly involved in RNA transport to the nuclear periphery. 相似文献
22.
Cloning and sequencing of the genes encoding the large and the small subunits of the H2 uptake hydrogenase (hup) of Rhodobacter capsulatus 总被引:12,自引:0,他引:12
Michèle Leclerc Annette Colbeau Béatrice Cauvin Paulette M. Vignais 《Molecular & general genetics : MGG》1988,214(1):97-107
Summary The structural genes (hup) of the H2 uptake hydrogenase of Rhodobacter capsulatus were isolated from a cosmid gene library of R. capsulatus DNA by hybridization with the structural genes of the H2 uptake hydrogenase of Bradyrhizobium japonicum. The R. capsulatus genes were localized on a 3.5 kb HindIII fragment. The fragment, cloned onto plasmid pAC76, restored hydrogenase activity and autotrophic growth of the R. capsulatus mutant JP91, deficient in hydrogenase activity (Hup-). The nucleotide sequence, determined by the dideoxy chain termination method, revealed the presence of two open reading frames. The gene encoding the large subunit of hydrogenase (hupL) was identified from the size of its protein product (68108 dalton) and by alignment with the NH2 amino acid protein sequence determined by Edman degradation. Upstream and separated from the large subunit by only three nucleotides was a gene encoding a 34 256 dalton polypeptide. Its amino acid sequence showed 80% identity with the small subunit of the hydrogenase of B. japonicum. The gene was identified as the structural gene of the small subunit of R. capsulatus hydrogenase (hupS). The R. capsulatus hydrogenase also showed homology, but to a lesser extent, with the hydrogenase of Desulfovibrio baculatus and D. gigas. In the R. capsulatus hydrogenase the Cys residues, (13 in the small subunit and 12 in the large subunit) were not arranged in the typical configuration found in [4Fe–4S] ferredoxins. 相似文献
23.
Rhizome dynamics and resource storage in Phragmites australis 总被引:6,自引:1,他引:5
Wilhelm Granéli Stefan E. B. Weisner Mark D. Sytsma 《Wetlands Ecology and Management》1992,1(4):239-247
Seasonal changes in rhizome concentrations of total nonstructural carbohydrates (TNC), water soluble carbohydrates (WSC), and mineral nutrients (N, P and K) were monitored in two Phragmites australis stands in southern Sweden. Rhizome biomass, rhizome length per unit ground area, and specific weight (weight/ length ratio) of the rhizomes were monitored in one of the stands.Rhizome biomass decreased during spring, increased during summer and decreased during winter. However, changes in spring and summer were small (< 500 g DW m-2) compared to the mean rhizome biomass (approximately 3000 g DW m–2). Winter losses were larger, approximately 1000 g DW m-2, and to a substantial extent involved structural biomass, indicating rhizome mortality. Seasonal changes in rhizome length per unit ground area revealed a rhizome mortality of about 30% during the winter period, and also indicated that an intensive period of formation of new rhizomes occurred in June.Rhizome concentrations of TNC and WSC decreased during the spring, when carbohydrates were translocated to support shoot growth. However, rhizome standing stock of TNC remained large (> 1000 g m–2). Concentrations and standing stocks of mineral nutrients decreased during spring/ early summer and increased during summer/ fall. Only N, however, showed a pattern consistent with a spring depletion caused by translocation to shoots. This pattern indicates sufficient root uptake of P and K to support spring growth, and supports other evidence that N is generally the limiting mineral nutrient for Phragmites.The biomass data, as well as increased rhizome specific weight and TNC concentrations, clearly suggests that reloading of rhizomes with energy reserves starts in June, not towards the end of the growing season as has been suggested previously. This resource allocation strategy of Phragmites has consequences for vegetation management.Our data indicate that carbohydrate reserves are much larger than needed to support spring growth. We propose that large stores are needed to ensure establishment of spring shoots when deep water or stochastic environmental events, such as high rhizome mortality in winter or loss of spring shoots due to late season frost, increase the demand for reserves. 相似文献
24.
At the end of the spring 1987 growing season, the mycoparasite Sporidesmium sclerotivorum was applied at 0, 0.2, 2 or 20 kg ha‐1 to lettuce plants infected with Sclerotinia minor. Disease incidence was monitored in the same plots for five subsequent crops (three fall and two spring crops) without additional application of either pathogen or mycoparasite. Logistic growth curves were fitted to the data to describe disease progression over time for each inoculum level within each of the five crops. Within each crop, increasing the quantity of mycoparasite inoculum resulted in positive horizontal displacement of the curve with respect to time. As quantities of inoculum of S. sclerotivorum increased, inflection points of the disease progress curves increased at a decreasing rate. Thus, additional mycoparasite inoculum resulted in ever‐smaller increases in inflection point, and after a certain threshold level of mycoparasite inoculum (< 0.2 kg ha‐1), increases in inflection point did not result in meaningful increases in harvestable lettuce. Maximum rates of disease increase were not different among the treatments within each crop, but were different between crops. Maximum rates of disease increase averaged 3.4, 3.4, 2.1, 3.6 and 1.5% day‐1 for the fall 1987, spring 1988, fall 1988, spring 1989, and fall 1989, respectively. At all inoculum levels, the fall epidemics began later after planting than the spring epidemics. 相似文献
25.
Disulfide bridges in tomato pectinesterase: variations from pectinesterases of other species; conservation of possible active site segments. 总被引:3,自引:0,他引:3
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Analysis of tomato pectinesterase by carboxymethylation, with and without reduction, shows that the enzyme has two intrachain disulfide bridges. Analysis of fragments obtained from the native enzyme after digestion with pepsin identified bridges connecting Cys-98 with Cys-125, and Cys-166 with Cys-200. The locations of disulfide bridges in tomato pectinesterase are not identical to those in three distantly related pectinesterases (18-33% residue identities) from microorganisms. However, one half-Cys (i.e., Cys-166) position is conserved in all four enzymes. Sequence comparisons of the overall structures suggest a special importance for three short segments of the entire protein. One segment is at the N-terminal part of the tomato pectinesterase, another in the C-terminal portion near the distal end of the second disulfide loop, and the third segment is located in the central part between the two disulfide bridges. The latter segment, encompassing only 40 residues of the entire protein, appears to high-light a functional site in a midchain segment. 相似文献
26.
Genetic control of the mitochondrial form of superoxide dismutase in hexaploid wheat 总被引:3,自引:0,他引:3
Extracts of mature grains of a large number of aneuploid derivatives of Triticum aestivum cv. Chinese Spring and of the members of five wheat-alien chromosome addition series were subjected to isoelectric focusing in polyacrylamide gels in order to study the genetic control of superoxide dismutase (SOD). Evidence was obtained that homologous structural genes for the mitochondrial form of SOD are located in the long arms of the homoeologous group 2 chromosomes of Chinese Spring and in chromosome 2R of Secale cereale cv. Imperial. The SOD gene loci located in chromosomes 2A, 2B, 2D, and 2R were designated Sod-A1, Sod-B1, Sod-D1, and Sod-R1, respectively. Chromosome-arm pairing data indicate that 2DL is not homoeologous to either 2AS or 2BL. The results of this study suggest, however, that 2BL is partially homoeologous to both 2AL and 2DL.Technical article No. 21074 of the Texas Agricultural Experiment Station. This work was supported by USDA Grant 83-CRCR-1-1322 to GEH. 相似文献
27.
This contribution illustrates the advantages of some chromophoric and fluorophoric carbohydrate derivatives such asp-nitrophenyl (pNO2Phe) or 4-methylumbelliferyl (MeUmb) glycosides andN-dansylgalactosamine in studies of the binding equilibrium and kinetics with some plant lectins. The methods used involve
continuous titrations of changes in ligand or protein absorption and ligand fluorescence, including substitution titrations
as well as stopped-flow, temperature-jump or pressure-jump relaxation kinetics.
When monitored by temperature-jump relaxation, binding of MeUmbαGal to the bloodgroup A specific lectin GSAI-A4 fromGriffonia simplicifolia is a simple bimolecular association with parametersk
+
= 9.4 × 104 M-1 s-1 andk
-1 = 5.3 s-1 at 23°C, but binding to the GSAI-B4 lectin is biphasic.
The complementarity of the peanut agglutinin binding site with Galβ1 → 3GalNAc that occurs in manyO-glycoproteins follows from enthalpic considerations and also from the value of the dissociation-rate parameterk
-1 = 0.24 s-1 of the MeUmbβGalβl → 3GalNAc.lectin complex. This value, obtained by stopped-flow kinetics is 100 times smaller than for other mono-and disaccharides
investigated. The binding mechanism is simple and the derivatisation of Galβ1 → 3GalNAc does not affect the affinity to a considerable degree.
The binding preference of tetravalentsoybean agglutinin for MeαGalNAc over MeαGal by a factor of 25 is mainly of enthalpic origin with an additional 7 kJ mol-1; the NAc group causes perturbation of a tryptophanyl residue, evidenced by protein difference absorption spectrometry. In
the glycosides, a large aglycon likeβpNO2 Phe orβMeUmb hardly affects the affinity of SBA but a largeN-dansyl group increases the affinity by a factor 20 as compared to GalNAc. The 10-fold increase in carbohydrate-specificN-dansylgalactosamine fluorescence, together with a very favourable entropic contribution point at the presence of a hydrophobic
region in the vicinity of the carbohydrate-binding site. The dissociation-rate parameter of the MeUmbβGalNAc SBA complex is slower than for any reported monosaccharide-lectin complex: 0.4 s-1.
The divalent lectin fromErythrina cristagalli preferentially binds the Galβ1 → 4GlcNAc structure that occurs in manyN-glycoproteins. The combining site was mapped thermodynamically with carbohydrates ranging from mono-to pentasaccharides as
derived fromN-glycoproteins. Here, N-dansylgalactosamine was used as a fluorescent indicator ligand in substitution titrations. When Galβ1 → 4GlcNAc was linkedα1 → 2 orα1 → 6 to Man, the binding enthalpy and entropy remained practically constant. Application of stopped flow kinetics and pressure-jump
relaxation withN-dansylgalactosamine gave mono-exponential signal changes with a concentration dependence corresponding tok
+ = 4.8 x 104 M-1 s-1
k
- = 0.4 to 0.66 s-1 and a change in reaction volume of+7ml/mol. 相似文献
28.
Mutant forms of staphylococcal nuclease with altered patterns of guanidine hydrochloride and urea denaturation 总被引:19,自引:0,他引:19
Eleven mutant forms of staphylococcal nuclease with one or more defined amino acid substitutions have been analyzed by solvent denaturation by using intrinsic fluorescence to follow the denaturation reaction. On the basis of patterns observed in the value of m--the rate of change of log Kapp (the apparent equilibrium constant between the native and denatured states) with denaturant concentration--these proteins can be grouped into two classes. For class I mutants, the value of m with guanidine hydrochloride is less than the wild-type value and is either constant or increases slightly with increasing denaturant; the value of m with urea is also less than wild type but shows a marked increase with increasing denaturant concentration, often approaching but never exceeding the wild-type value. For class II mutants, m is constant and is greater than wild type in both denaturants, with the increase being consistently larger in guanidine hydrochloride than in urea. When double or triple mutants are constructed from members of the same mutant class, the change in m is usually the sum of the changes produced by each mutation in isolation. One plausible explanation for these altered patterns of denaturation is that chain-chain or chain-solvent interactions in the denatured state have been modified--interactions which appear to involve hydrophobic groups. 相似文献
29.
Structural proteins and the characteristics of infectious flacherie virus (IFV) purified from the silkworm, Bombyx mori, are described. The purified IFV had four major structural proteins, which were detected only in high concentration gels of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and a few minor ones. Molecular weights of the major proteins were 35,200 (VP 1), 33,000 (VP 2), 31,200 (VP 3), and 11,600 (VP 4), and numbers per virion were 62, 57, 54, and 31, respectively. Amino acid compositions of VP 1, VP 2, and VP 3 were similar to each other but that of VP 4 was somewhat different. By isoelectric focusing and two-dimensional electrophoresis, high resolution of the structural proteins was obtained with silver staining. The isoelectric points of the four major proteins were determined as 7.7(VP 1), 6.7(VP 2), 4.8(VP 3), and 5.5(VP 4). This work is the first report on insect picornaviruses that presents some discriminative properties of each viral protein that was compared to those of mammalian picornaviruses. 相似文献
30.
Robert L. Uffen Annette Colbeau Pierre Richaud Paulette M. Vignais 《Molecular & general genetics : MGG》1990,221(1):49-58
Summary
Rhodocyclus gelatinosus grew photosynthetically in the light and consumed H2 at a rate of about 665 nmol/min per mg protein. The uptake-hydrogenase (H2ase) was found to be membrane bound and insensitive to inhibition by CO. The structural genes of R. gelatinosus uptake-H2ase were isolated from a 40 kb cosmid gene library of R. gelatinosus DNA by hybridization with the structural genes of uptake-H2ase of Bradyrhizobium japonicum and Rhodobacter capsulatus. The R. gelatinosus genes were localized on two overlapping DNA restriction fragments subcloned into pUC18. Two open reading frames (ORF1 and ORF2) were observed. ORF1 contained 1080 nucleotides and encoded a 39.4 kDa protein. ORF2 had 1854 nucleotides and encoded a 68.5 kDa protein. Amino acid sequence analysis suggested that ORF1 and ORF2 corresponded to the small (HupS) and large (HupL) subunits, respectively, of R. gelatinosus uptake-H2ase. ORF1 was approximately 80% homologous with the small, and ORF2 was maximally 68% homologous with the large subunit of typical membrane-bound uptake-H2ases. 相似文献