摘要:

AbstractDNA fragments that contain an active origin of replication generate bubble‐shaped replication intermediates with diverging forks. We describe two methods that use two‐dimensional (2‐D) agarose gel electrophoresis along with DNA sequence information to identify replication origins in natural and artificialSaccharomyces cerevisiaechromosomes. The first method uses 2‐D gels of overlapping DNA fragments to locate an active chromosomal replication origin within a region known to confer autonomous replication on a plasmid. A variant form of 2‐D gels can be used to determine the direction of fork movement, and the second method uses this technique to find restriction fragments that are replicated by diverging forks, indicating that a bidirectional replication origin is located between the two fragments. Either of these two methods can be applied to the analysis of any genomic region for which there is DNA sequence information or an adequate restri

ISSN:0173-0835    

DOI:10.1002/elps.1150190803

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractUnder favorable conditions, native gel electrophoresis allows the resolution of protein‐DNA complexes that differ in stoichiometry, identities of occupied DNA sequences (configuration), and macromolecular conformation. This technique provides a unique opportunity to analyze, in thermodynamic terms, the molecular interactions that govern the equilibrium distributions of species in protein‐DNA mixtures. Here we describe a general theoretical approach to the analysis of electrophoretic band intensities, and provide examples of its application to the analysis of several interacting syst

ISSN:0173-0835    

DOI:10.1002/elps.1150190804

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractThe cytochromecoxidase (COX) holoenzyme is a 13‐subunit complex that carries out the terminal step in the electron transport chain. Three of the subunits, which contain the electron transfer function, are coded by mitochondrial DNA and the other ten subunits by nuclear DNA. Since the holoenzyme contains equivalent amounts of each subunit, we and others have examined transcriptional regulation of COX nuclear subunits to explore whether there is a common basis for co‐regulation. Each gene is seen to have a unique pattern of recognition by regulatory factors; although some factors bind to more than one gene, not all COX genes seem to be regulated by the same set of factors. Current information about the COX promoters that have been examined is summarized, and the relation of promoter regulation to coordinate gene expression is discus

ISSN:0173-0835    

DOI:10.1002/elps.1150190805

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractAluelements are a family of short interspersed repetitive elements (SINEs) found exclusively in primates. These elements are around 300 base pairs long, are found in excess of one million copies per diploid genome, and are dispersed throughout the human genome.Aluelements are scattered by a mechanism called “retrotransposition”. Three independent steps are involved in retrotransposition: transcription of theAlurepetitive element, reverse transcription of theAluRNA and integration of theAlucDNA. The fact thatAluelements retrotranspose so readily suggests that they have a myriad of effects on the genome, mostly by inactivating genes or altering their function. These characteristics ofAlurepetitive elements point to these repetitive DNA fragments as a major driving force for evolution. In addition,Aluelements are known to adopt diverse functions depending on the context of the surrounding genetic material into which they insert. In this article, we review some of the evidence that demonstrates the functional significance ofAlurepe

ISSN:0173-0835    

DOI:10.1002/elps.1150190806

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractOligonucleotide N3′ → P5′ phosphoramidates (3′‐phosphoramidates) are DNA analogs that are presently under investigation as potential therapeutic agents. These compounds may also hold promise as a diagnostic tool. Here, we describe a rapid method for the analysis of single‐stranded RNA fragments utilizing 3′‐phosphoramidate oligonucleotides as probes in conjunction with capillary gel electrophoresis (CGE). 3′‐Phosphoramidate 9‐mers were mixed with complimentary RNA, and CGE was used to monitor duplex formation. Complimentary strands of RNA and 3′‐phosphoramidate formed duplexes that gave unique relative mobilities based on an internal standard. The ability of CGE to discriminate between perfect duplexes and duplexes that contain a base mismatch was also investigated. However, the primary focus of this work was to determine CGE's ability to detect the presence of the 3′‐phosphoramidates/RNA duplex under routine electrophoretic running conditions. Polyacrylamide gel electrophoresis analysis was utiliz

ISSN:0173-0835    

DOI:10.1002/elps.1150190807

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractA ginseng polypeptide (GPP) found in ginseng roots and its modified peptides were tested by capillary zone electrophoresis (CZE) under acidic as well as basic conditions. Modified peptides were synthesized for three purposes: (i) to analyze their functions in the first three acidic amino acid residues, (ii) to analyze their functions in three sequenced glycines, and (iii) to analyze the length of side chains in acidic amino acids. The roles of glycines, acidic amino acids and amino acid side chains in the binding of Mg2+were studied at pH values less than 7.0. The migration times of GPP varied with the pH of various electrophoresis buffers, and electrophoresis patterns were significantly changed between pH 7.0–7.5. Based on the electrophoretic analysis, it was concluded that the binding mechanisms for Mg2+or conformations of GPP changed between low pH and high p

ISSN:0173-0835    

DOI:10.1002/elps.1150190808

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractLectin‐gradient agarose gel affinity electrophoresis was developed for identification of glycoforms of glycoproteins in lectin affinity electrophoresis. Gradation of lectin was done by stacking agarose gel blocks with increasing concentrations of lectin (discontinuous system) and by keeping the plate in a moist chamber at 4°C overnight (continuous system) before electrophoresis. On the visualization of separated glycoform lines, the antibody‐affinity blotting was superior for low concentrations of α‐fetoprotein. Fluoresceine isothiocyanate (FITC) labeling of whole serum proteins, enzyme activity staining for alkaline phosphatase, and prestaining for lipoproteins were also applicable for visualization of proteins at higher concentrations. The conventional Western blotting can not be recommended because of the competition between lectin and glycoproteins in binding to nitrocellulose membrane. Lectin‐gradient affinity electrophoresis also had a wide application for optimization of the condition of lectin affinity electr

ISSN:0173-0835    

DOI:10.1002/elps.1150190809

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractThe commercial gel electrophoresis apparatus with intermittent scanning of the migration path and preparative capacity (HPGE‐1000, LabIntelligence) is applicable to polymer solutions as well as gels. Unresolved rat liver microsomes can be isolated from 11–15% polyvinylpyrrolidone (PVP) solution by means of a syringe. The automated band isolation technique applied under resolving conditions in dilute polymer solutions allowed for the sequential isolation of three microsome components with 85, 76 and 75% recovery, respectively, under strict control of the dimensions of the volumetric collection module of the HPGE‐1000 apparatus. Separations of unlabeled microsomes and sea urchin egg components in dilute polymer solutions have been performed, using detection by “fluorescence reduction”. The unlabeled major component of a sea urchin egg homogenate has been isolated from electrophoresis in 1.5% PVP (Mr= 106) solution in 25–50% yield (0.24–4 μg/8 lanes of the HPGE‐1000 apparatus). However, since separations of both microsomes and sea urchin egg granules in dilute polymer solutions are restricted to a narrow range of polymer concentrations, their retardation coefficients,KR= d(log mobility)/d(polymer concentration), are

ISSN:0173-0835    

DOI:10.1002/elps.1150190810

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractParticle size‐dependent retardation (“molecular sieving”) in electrophoresis can be achieved in polymer solutions or gels. In the semidilute concentration range of polymer solutions, mobility of “rigid, spherical” particles can be predicted from their size (in the size range less than 20 nm radius), the screening length specific for the particular polymer and a constant that can be experimentally determined for a polymer. That constant could be universal for all hydrophilic uncharged polymers. Band spreading in polymer solutions is constant over a range of particle sizes and polymer concentrations and increases beyond that range. Presumably, the critical division between the two ranges occurs when the diameter of the particle exceeds the screening length of the polymer network. Resolution, defined as separation divided by the sum of bandwidths, can thus be predicted for a limited particle size and polymer concentration range. In gels, resolution can be estimated from the slopes and free mobility intercepts of the Ferguson plot. However, the previous computation of resolution needs to be revised in view of the fact that band spreading in gels proceeds in proportion to time, presumably through interaction with the polymer, and not as a function of the square root of time as would be the case if band spreading resulted from

ISSN:0173-0835    

DOI:10.1002/elps.1150190811

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY

摘要:

AbstractWe have developed a density gradient electrophoresis device (DGE) and used it for the preparative separation of various endocytic organelles that are hard to separate by other means. Our separation by DGE of late endosomal vesicles, recycling vesicles, early endosomes and plasma membranes is unmatched. Using the same DGE device, we performed preparative high‐resolution rate zonal separation of proteins using amphoteric buffers as originally described by Bier (Electrophoresis1993,14, 1011–1018). Isoforms of bovine β‐lactoglobulin, human apo‐transferrin, and bovine erythrocyte carbonic anhydrase that have isoelectric points within 0.8 pH units were readily separated even in the absence of nonionic detergents. The DGE apparatus is inexpensive and has unique separation abilities for vesicles and

ISSN:0173-0835    

DOI:10.1002/elps.1150190812

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1998

数据来源: WILEY