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Identification and separation of components of calf thymus DNA using a CsCl-netropsin density gradient
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Calf thymus DNA containing satellite components of various densities was used as a model to study the effect of netropsin on the density of DNA in a CsCl gradient. The binding of netropsin resulted in a decrease in density which depended upon the quantity of netropsin added and on the average composition of the DNA. Differences in density of DNA components were higher in CsCl - netropsin gradients than in simple CsCl gradients. By use of netropsin a main band and four satellite bands could be differentiated in calf thymus DNA. Satellite DNA's were isolated using preparative CsCl - netropsin gradient centrifugation and were characterised by density and homogeneity in native and in reassociated state. Two of the satellite components, with densities of 1.722 and 1.714 g/cm3, are probably of homogenous sequence, the other two components of densities 1.709 and 1.705 g/cm3 appear to be heterogeneous. 相似文献
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Euglena gracilis chloroplast DNA has an average buoyant density of 1.685 gm/cm3, corresponding to 25 mol% G . C base pairs. To test for base compositional heterogeneity within this 130 kilobase pairs (kbp) genome, previously mapped restriction endonuclease fragments were isolated, and characterized by equilibrium buoyant density centrifugation. The chloroplast DNA can be characterized as containing two major buoyant density components. A segment of 17 kbp, representing 13% of the genome and containing the rRNA genes is 43--44 mol% G . C. The remaining 113 kbp, accounting for 87% of the genome, has an average 20--21 mol% G . C content. 相似文献
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High resolution melting profiles of different mammalian DNAs are presented. Melting curves of various mammalian DNAs were compared with respect to the degree of asymmetry, first moment, transition breath and Tmi of individual subtransitions. Quantitative comparison of the shape of all melting curves was made. Correlation between phylogenetical relations among mammals and shape of the melting profiles of their DNAs was demonstrated. The difference between multi-component heterogeneity of mammalian DNAs found by optical melting analysis and sedimentation in CsCl-netropsin density gradient is also discussed. 相似文献
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Size heterogeneity of EBV and mitochondrial DNAs in Burkitt''s lymphoma lines. 总被引:1,自引:1,他引:1
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A simple, reproducible affinity chromatography method has been adapted for separation of high molecular weight supercoiled circular molecules from mammalian cells. Electron microscopic analysis of EB viral DNA obtained by this method, from the non-producer Burkitt's lymphoma line Raji, revealed monomer-sized viral molecules only. In contrast, the EB viral episomes from recently established human producer lines BL-8 and LY91 were very heterogeneous in size, some being considerably smaller and others much larger than the monomeric DNA. The former are probably related to defective viral species in the B-cell population, but the origin of the latter are as yet unclear. All cell lines contained both monomers and concatemers of mitochondrial DNA; among the latter, molecules apparently greater than 100 kb were observed in the population. 相似文献
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The metabolism of heterogeneous subpopulations of low density lipoprotein (LDL) apoB100 was examined in three normolipidemic and two familial combined hyperlipidemic subjects. Autologous radioiodinated plasma LDL (1.019 less than d less than 1.063 g/ml) were injected into each subject and the disappearance and appearance of radiolabeled lipoproteins into various LDL subpopulations were examined using density gradient ultracentrifugation. Eleven to 13 fractions (-320 microliter each) were collected within LDL defined uniquely in each subject. In all subjects, the disappearance of radiolabeled LDL from plasma was biexponential. However, changes with time in the distribution of radiolabeled LDL among the various LDL density subpopulations revealed complex metabolic behavior that differed among the subjects. When the relationships between density and kinetic characteristics were examined in more detail by following the disappearance of individual fractions defining LDL in each subject, the data suggested that: 1) the kinetic behavior of the LDL fractions was more complex than suggested by the disappearance of radiolabeled LDL from plasma: 2) certain fractions within specific density ranges were kinetically similar; 3) distinct differences in the disappearance curves among the fractions occurred within narrow density ranges; and 4) precursor-product relationships were seen among specific LDL density fractions and varied from subject to subject. These studies underscore the complexities of plasma LDL apoB-100 metabolism. More detailed characterizations of the kinetic behavior of various LDL subpopulations should help in our understanding of the origin(s) and potential physiological consequences of different LDL subpopulations. 相似文献
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Studies on the heterogeneity of mammalian beta-glucuronidases. 总被引:1,自引:0,他引:1
R T Dean M Messer 《Comparative biochemistry and physiology. B, Comparative biochemistry》1976,54(1):107-110
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An apurinic/apyrimidinic (AP) specific endonuclease from mouse plasmacytoma cells (line MPC-11), was observed to cleave apurinic sites in oligonucleotides 9, 11, 12, 39 and 40 nucleotides in length. However, the enzyme failed to cleave AP-sites in two oligonucleotides 7 nucleotides in length. The maximum rates of digestion observed on these short single-stranded DNA (ssDNA) fragments were approximately 1/30 of the rates observed on double-stranded DNA (dsDNA). In studies using the Maxam-Gilbert DNA sequencing analysis, apurinic sites in purine-rich regions were preferentially cleaved in dsDNA but not in ssDNA, indicating that the enzyme has a sequence preference on dsDNA. These results suggest that some sites on DNA might be more efficiently repaired than others. 相似文献
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Denaturing gradient gel electrophoresis (DGGE) is based upon the different melting behaviors of DNA molecules in a chemical denaturant gradient according to their sequences. This technique has recently become a widespread tool to detect mutations. The introduction of a GC-clamp enables the detection of most single base differences between two DNA molecules. As a test system we have applied the polymerase chain reaction (PCR) in combination with DGGE to detect a number of mutations in the mouse H2Kb DNA sequence. A wide variety of spontaneous in vivo mutations of this haplotype have been reported in the C57BL/6J mouse strain and are clustered in the alpha 1 and alpha 2 domains. The combination of PCR and DGGE revealed almost all base changes present in the H2Kb mutants used. However, most of the PCR products of these mutants showed melting behavior which is not easily predicted. We suggest that in addition to current simple theory, which considers that the migration of a DNA molecule in a denaturing gradient depends primarily on its initial melting behavior, additional factors such as secondary structure in partially melted molecules may play a role and can be used to detect mutations. 相似文献
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A bulk electrophoretic method is described for the separation of mammalian cells in Ficoll-sucrose density gradients. The fractionation of cells is performed in a simple commercially available apparatus (Buchler Poly-Prep) which should facilitate the application of the technique in various laboratories. Details of the experimental procedure are presented along with typical model separations of erythrocytes from various species and mouse lymphocytes. 相似文献
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S H Quarfordt R S Jain L Jakoi S Robinson F Shelburne 《Biochemical and biophysical research communications》1978,83(3):786-793
Ubiquitin was first found in nuclei in protein A24 where its carboxyl terminal is covalently bound to histone 2A by an isopeptide linkage (Goldknopf, I. L. and Busch, H. (1977) Proc. Natl. Acad. Sci. USA , 864–868). Two-dimensional polyacrylamide gel electrophoresis of the 0.4 N H2SO4 soluble proteins from fractionated rat liver chromatin showed that protein A24 and histones H1, H2A, H2B, H3 and H4 were present in fractions P1 and P2 and markedly diminished in relative amounts in fraction S2. Conversely, a spot designated Ub was found in fraction S2 along with an increased amount and number of non-histone proteins. The Ub spot was not found in chromatin fractions P1 and P2. Ub was identified as ubiquitin by migration on two-dimensional gels and after purification by preparative polyacrylamide gel electrophoresis by its methionine NH2-terminal amino acid and its amino acid composition. 相似文献
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The intramolecular base composition heterogeneity of human DNA has been investigated by electron microscopic observations of partially denatured structures and by equilibrium solution thermal denaturation techniques. DNA sequences having an average length of less than 2000 base pairs are found to be heterogeneous in base composition. These heterogeneous sequences occupy a minimum of 67 to 81% of the human genome. 相似文献
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Purification and biochemical heterogeneity of the mammalian SWI-SNF complex. 总被引:35,自引:1,他引:35
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W Wang J Ct Y Xue S Zhou P A Khavari S R Biggar C Muchardt G V Kalpana S P Goff M Yaniv J L Workman G R Crabtree 《The EMBO journal》1996,15(19):5370-5382
We have purified distinct complexes of nine to 12 proteins [referred to as BRG1-associated factors (BAFs)] from several mammalian cell lines using an antibody to the SWI2-SNF2 homolog BRG1. Microsequencing revealed that the 47 kDa BAF is identical to INI1. Previously INI1 has been shown to interact with and activate human immunodeficiency virus integrase and to be homologous to the yeast SNF5 gene. A group of BAF47-associated proteins were affinity purified with antibodies against INI1/BAF47 and were found to be identical to those co-purified with BRG1, strongly indicating that this group of proteins associates tightly and is likely to be the mammalian equivalent of the yeast SWI-SNF complex. Complexes containing BRG1 can disrupt nucleosomes and facilitate the binding of GAL4-VP16 to a nucleosomal template similar to the yeast SWI-SNF complex. Purification of the complex from several cell lines demonstrates that it is heterogeneous with respect to subunit composition. The two SWI-SNF2 homologs, BRG1 and hbrm, were found in separate complexes. Certain cell lines completely lack BRG1 and hbrm, indicating that they are not essential for cell viability and that the mammalian SWI-SNF complex may be tailored to the needs of a differentiated cell type. 相似文献
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The behavior of E. coli ribosomes during sedimentation on sucrose gradients is predicted under a variety of conditions by computer simulations. Since numerous recent kinetic studies indicate equilibration in times short compared to the time of sedimentation, these simulations assume that the system attains local reaction equilibrium at every point in the gradient at all times. For any type of homogeneous equilibrating ribosome population, governed by a single formation constant at one atmosphere pressure for 70S couples, no more than two clearly defined zones will be resolved, although the presence of large dissociating effects due to pressure gradients in high speed experiments will spread the subunit zone. Normally the pattern will consist of a 30S zone and a so-called “70S” zone, which is in reality a mixture of 70S couples and 30S and 50S subunits in local equilibrium. The greater the dissociation into subunits, the more the “70S” zone will be slowed below the nominal rate of 70 Svedberg units. If ribosomes have been collected from the “70S” zone in several successive cycles of purification, the repeated deletion of resolved 30S subunits can result in a preparation with so large a molar excess of 50S subunits that the ensuing sucrose density gradient sedimentation pattern may exhibit a “70S” zone followed by zone of 50S subunits, insteadof a zone of 30S subunits. Our most important conclusion is that whenever a well-resolved 50S zone is present in a sucrose density gradient sedimentation experiment on E. coli ribosomes, in addition to a 30S and a “70S” zone, under conditions where ribosomes and subunits should be in reversible equilibrium, the preparation must be microheterogeneous, containing a mixture of “tight” and “loose” couples. Moreover in such cases the content of large subunits in the 50S zone must be derived entirely from “loose” couples whereas the 30S zone must contain small subunits derived from both “tight” and “loose” couples. Sedimentation patterns predicted for various mixtures of “tight” and “loose” couples display all the major characteristics of published experimental patterns for E. coli ribosomes, including the partial or complete resolution into three zones, depending on rotor velocity and level of Mg2+. 相似文献
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Changes along a disturbance gradient in the density and composition of propagule banks in floodplain aquatic habitats 总被引:7,自引:0,他引:7
This study used germination methods to examine the density, species composition and functional composition of propagule banks in a series of riverine wetland aquatic habitats subject to varying degrees of hydrological and management-related disturbance. Under permanent inundation (the conditions prevailing at most sites during the growing season) propagule germination and species richness was low, with floodplain perennials and helophytes particularly affected. Densities of floodplain annuals were largely maintained through continued germination of a few flooding tolerant species. On damp mud (conditions associated with hydrological instability) total seedling number and species richness increased significantly, but species richness of germinating hydrophytes declined. Mean seedling density at 0–0.1m depth was 15450 ± 4400 m–2, reaching a maximum (162 050 m–2) in temporary backwaters. Annual (e.g., Lindernia dubia, Cyperus fuscus) and facultative ruderal species (e.g., Lythrum salicaria and Alisma plantago-aquatica) predominated. Vertical zonation of the propagule bank was weakly developed. The numbers of individuals and species germinating varied significantly between sites. The seasonal, most intensely disturbed sites (temporary backwaters) supported a numerically large, species-rich propagule bank based on floodplain annuals, while the permanent, less disturbed sites (ditches and an oxbow pond) had a small, species-poor propagule bank composed of hydrophytes and helophytes supplemented by allochthonous seed inputs. Sites intermediate on the gradient had a propagule bank dominated by facultative amphibious, ruderal hydrophytes. The composition of the seed bank and the established vegetation was most similar at the heavily disturbed sites where the seed bank was maintained by vigorously fruiting annuals and supplemented by inputs from temporary habitats upstream. At permanent sites much of the propagule bank composition could be accounted for by inputs of floodborne seed from the immediately adjacent floodplain. The established vegetation at such sites appeared to be maintained mainly by vegetative propagation with recruitment from the propagule bank likely only after severe disturbance. The potential contribution of functionally diverse propagule banks to sucessional processes within fluvially dynamic floodplain aquatic habitats is emphasised. 相似文献