Showing papers on "Gel electrophoresis published in 1992"

Single-cell gel electrophoresis applied to the analysis of UV-C damage and its repair in human cells.

Abstract: DNA breaks in eukaryotic cells can be detected by alkaline electrophoresis of cells embedded in agarose. DNA containing breaks extends in the direction of the anode forming an image resembling the tail of a comet. We have adapted this procedure of single cell gel electrophoresis (SCGE) for studying DNA damage and repair induced by UV-C-radiation, using HeLa cells. UV-C itself does not induce DNA breakage, and though cellular repair of UV-C damage produces DNA breaks as intermediates, these are too short-lived to be detected by SCGE. Incubation of UV-C-irradiated cells with the DNA synthesis inhibitor aphidicolin causes accumulation of incomplete repair sites to a level readily detected by SCGE even after doses as low as 0·5 J m−2 and incubation for as little as 5 min. We have also used SCGE to study UV-C-dependent incision, repair synthesis and ligation in permeable cells. Finally, we have incubated permeable cells, after UV-C-irradiation, with exogenous UV endonuclease, examining the consequent breaks bo...

Hydrolytic elimination of a mutagenic nucleotide, 8-oxodGTP, by human 18-kilodalton protein: sanitization of nucleotide pool.

Abstract: 8-Oxoguanine nucleotide can pair with cytosine and adenine nucleotides at almost equal efficiencies. Once 8-oxodGTP is formed in the cellular nucleotide pool, this mutagenic nucleotide is incorporated into DNA and would cause transversion mutations. The MutT protein of Escherichia coli possesses enzyme activity to hydrolyze 8-oxodGTP to the corresponding nucleoside monophosphate and thus may be responsible for preventing the occurrence of such mutations. Here we show that the human cell has an enzyme specifically hydrolyzing 8-oxodGTP in a fashion similar to that seen with MutT protein. The human 8-oxodGTPase has been found in cell-free extracts from Jurkat cells and purified > 400-fold. Analyses by gel filtration and gel electrophoresis revealed that the molecular mass of the native form of human 8-oxodGTPase is 18 kDa. Mg2+ ion is required for the enzyme action and the optimum pH for the reaction is pH 8.0. The enzyme hydrolyzes 8-oxodGTP to 8-oxodGMP with a Km value of 12.5 microM. dGTP and dATP are also degraded to dGMP and dAMP, respectively, with Km values 70 times greater than that for 8-oxodGTP. dTTP and dCTP are not hydrolyzed. These properties of the human 8-oxodGTPase are similar to those observed with the E. coli MutT protein, suggesting that the function of protecting the genetic information from the threat of endogenous oxygen radicals is widely distributed in organisms.

Characterization and primary structure of the poly(C)-binding heterogeneous nuclear ribonucleoprotein complex K protein.

Abstract: At least 20 major proteins make up the ribonucleoprotein (RNP) complexes of heterogeneous nuclear RNA (hnRNA) in mammalian cells. Many of these proteins have distinct RNA-binding specificities. The abundant, acidic heterogeneous nuclear RNP (hnRNP) K and J proteins (66 and 64 kDa, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) are unique among the hnRNP proteins in their binding preference: they bind tenaciously to poly(C), and they are the major oligo(C)- and poly(C)-binding proteins in human HeLa cells. We purified K and J from HeLa cells by affinity chromatography and produced monoclonal antibodies to them. K and J are immunologically related and conserved among various vertebrates. Immunofluorescence microscopy with antibodies shows that K and J are located in the nucleoplasm. cDNA clones for K were isolated, and their sequences were determined. The predicted amino acid sequence of K does not contain an RNP consensus sequence found in many characterized hnRNP proteins and shows no extensive homology to sequences of any known proteins. The K protein contains two internal repeats not found in other known proteins, as well as GlyArgGlyGly and GlyArgGlyGlyPhe sequences, which occur frequently in many RNA-binding proteins. Overall, K represents a novel type of hnRNA-binding protein. It is likely that K and J play a role in the nuclear metabolism of hnRNAs, particularly for pre-mRNAs that contain cytidine-rich sequences.

Properties and localization of DNA methyltransferase in preimplantation mouse embryos: implications for genomic imprinting.

Abstract: Preimplantation mouse embryos contain very high levels of DNA methyltransferase activity. We show here that the form of DNA methyltransferase (DNA MTase) in early embryos differs from the form found in other cells and tissues by a slightly higher mobility on gel electrophoresis. Levels of DNA MTase were found to be very high throughout preimplantation development even though levels of 5-methylcytosine (m5C) in nuclear DNA are known to undergo a substantial decline in the same period. Confocal laser scanning microscopy of mouse embryos stained with DNA MTase-specific antibodies showed striking developmentally regulated changes in the distribution of DNA MTase. From the oocyte stage to the four-cell-stage, most DNA MTase was concentrated in peripheral cytoplasm, and nuclei did not contain detectable DNA MTase. In four- and eight-cell embryos, DNA MTase was seen in cytoplasmic granules; and in eight-cell embryos, DNA MTase was also present in large amounts in nuclei. Nuclei of blastocysts stained only faintly, whereas the cytoplasmic granules remained prominent. Paradoxically, DNA MTase was found to be at its highest levels in nuclei at a developmental stage where levels of m5C in DNA are decreasing most rapidly. Changes in methylation patterns in preimplantation embryos are therefore proposed to be under the control of unidentified regulatory factors rather than DNA MTase itself; these regulatory factors could be members of the group that contains the products of the Ssm-1 and Imp-1 genes, which are involved in the regulation of genomic imprinting.

Purification and characterization of phytase from Bacillus subtilis (natto) N-77

Abstract: An extracellular phytase from Bacillus subtilis (natto) N-77 was purified 322-fold to homogeneity with the specific activity of 8.7 units per mg protein by ultrafiltration, and a combination of Sephadex G-100 and DEAE-Sepharose CL-6B column chromatographies. The molecular weight of the purified enzyme was estimated to be 36 kDa on gel filtration and 38 kDa on SDS-polyacrylamide gel electrophoresis, suggesting that the native enzyme is a monomeric protein. The enzyme had the isoelectric point of pH 6.25, and Ca2+ requirement for the production and activity, the Km value of 0.5 mM, and the activation energy of 9.87 kcal/mol for sodium phytate. The enzyme proved to be fairly specific for phytate and was most active at pH 6.0–6.5 and 60°C. Its activity was greatly inhibited by reagents and metal ions such as EDTA, Zn2+, Cd2+, Ba2+, Cu2+, Fe2+, and Al3+.

Capillary array electrophoresis using laser-excited confocal fluorescence detection

Abstract: Capillary electrophoresis (CE) has found widespread application in analytical and biomedical research, and the scope and sophistication of CE is still rapidly advancing. Gel-filled capillaries have been employed for the rapid separation and analysis of synthetic polynucleotides, DNA sequencing fragments, and DNA restriction fragments. Open-tube capillary electrophoresis has attained subattomole detection levels in amino acid separations 14 and proven its utility for the separation of proteins, viruses, and bacteria. Separation of the optical isomers of dansyl amino acids has also been successfully demonstrated. Micellar electrokinetic capillary chromatography, isoelectric focusing, and on-column derivatization can all be performed on CE columns, demonstrating the utility of capillary electrophoresis as an analytical and micropreparative tool. 29 refs., 6 figs., 1 tab.

Assembly of recombinant human immunodeficiency virus type 1 capsid protein in vitro.

Abstract: The capsid protein (CA) (p24) of human immunodeficiency virus (HIV) type 1 expressed in Escherichia coli and purified to greater than 90% homogeneity was used to examine assembly in vitro and to probe the nature of interactions involved in the formation of capsid structures. The protein was detected in dimeric and oligomeric forms as indicated by molecular size measurements by gel filtration column chromatography, sedimentation through sucrose, and nondenaturing gel electrophoresis. Chemical cross-linking of CA molecules was observed with several homobifunctional reagents. Oligomer size was dependent on cross-linker concentration and exhibited a nonrandom pattern in which dimers and tetramers were more abundant than trimers and pentamers. Oligomers as large as dodecamers were detected in native polyacrylamide gels. These were stable in solutions of high ionic strength or in the presence of nonionic detergent, indicating that strong interactions were involved in oligomer stabilization. Limited tryptic digestion converted the putative dodecamers to octamers, suggesting that a region involved in CA protein multimerization was exposed in the structure. This region was mapped to the central portion of the protein. The recombinant CA proteins assembled in vitro into long rodlike structures and were disassembled into small irregular spheres by alterations in ionic strength and pH. The observation that assembly and disassembly of purified HIV type 1 CA protein can be induced in vitro suggests an approach for identifying possible control mechanisms involved in HIV viral core assembly.

Production of mitogen-contamination free alginates with variable ratios of mannuronic acid to guluronic acid by free flow electrophoresis.

Abstract: Commercial alginates consisting of variable homopolymeric regions of β-D-mannuronic acid and α-L-guluronic acid, interspaced with regions of alternating blocks, are potent stimulators of macrophages and lymphocytes. Therefore, inflammatory reactions and fibrotic overgrowth of the beads result if Langerhans islets are encapsulated in raw alginate hydrogel beads (cross-linked with divalent cations). The result is random failure of the islets some time after transplantation. Analysis of raw alginates by using free flow electrophoresis demonstrated that commercial alginates contained at least 10–20 fractions (characterized by different electrophoretic mobilities) which showed mitogenic activity. These fractions could be quantitatively separated from the alginic acids by free flow electrophoresis on a preparative scale. The purified alginates cross-linked with Ca2+ ions exhibited no mitogenic reactions as proved by an in vitro assay. In addition, examination of purified Ba2+ alginate beads implanted intraperitoneally in rats or mice for three weeks showed no fibrotic overgrowth in contrast to implants made from unpurified alginate.

Purification of core-binding factor, a protein that binds the conserved core site in murine leukemia virus enhancers.

Abstract: The Moloney murine leukemia virus causes thymic leukemias when injected into newborn mice. A major genetic determinant of the thymic disease specificity of the Moloney virus genetically maps to two protein binding sites in the Moloney virus enhancer, the leukemia virus factor b site and the adjacent core site. Point mutations introduced into either of these sites significantly shifts the disease specificity of the Moloney virus from thymic leukemia to erythroleukemia (N. A. Speck, B. Renjifo, E. Golemis, T. Frederickson, J. Hartley, and N. Hopkins, Genes Dev. 4:233-242, 1990). We have purified several polypeptides that bind to the core site in the Moloney virus enhancer. These proteins were purified from calf thymus nuclear extracts by selective pH denaturation, followed by chromatography on heparin-Sepharose, nonspecific double-stranded DNA-cellulose, and core oligonucleotide-coupled affinity columns. We have achieved greater than 13,000-fold purification of the core-binding factors (CBFs), with an overall yield of approximately 19%. Analysis of purified protein fractions by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis reveals more than 10 polypeptides. Each of the polypeptides was recovered from an SDS-polyacrylamide gel, and those in the molecular size range of 19 to 35 kDa were demonstrated to have core-binding activity. The purified CBFs were shown by DNase I footprint analyses to bind the core site in the Moloney virus enhancer specifically, and also to core motifs in the enhancers from a simian immunodeficiency virus, the immunoglobulin mu chain, and T-cell receptor gamma-chain genes.

Screening for mutations by RNA single-strand conformation polymorphism (rSSCP): comparison with DNA-SSCP

Abstract: Single-strand conformation polymorphism (SSCP) is a simple method for detecting the presence of mutations in a segment of DNA, but the fraction of all mutations detected is unclear We have evaluated SSCP for the detection of single-base mutations in the factor IX gene Multiple conditions were examined including electrophoresis temperature, electrophoresis buffer concentration, acrylamide to bis-acrylamide ratio, and water-cooled versus fan-cooled gel apparatuses Depending on conditions, 10-11 of 12 known mutations were detected in a 183 bp segment whereas only 11-14 of 22 known mutations were detected in a 307 bp segment We hypothesized that single stranded RNA should have a larger repertoire of secondary structure because shorter hairpins form stable duplexes and the 2' hydroxyl group is available for sugar-base and sugar-sugar hydrogen bonds By incorporating phage promoter sequences into PCR primers, RNA-SSCP (rSSCP) could be compared directly with standard DNA SSCP rSSCP was generally superior to SSCP, especially for the 307 bp segment In addition, the abundance of transcript produced as a result of rSSCP allows the rapid, nonradioactive detection of mutations by staining the gel with ethidium bromide To gauge the utility of the method in a prospective manner, a blinded study was performed in which SSCP, rSSCP, and direct genomic sequencing were compared in 28 patients with hemophilia B A total of 26 kb of factor IX genomic sequence was examined in nine regions ranging from 180 to 497 nucleotides of factor IX sequence Sequence changes at 20 different sites were detected by direct genomic sequencing; 70% of these were detected by rSSCP while only 35% were detected by SSCP