Research Interests: Chemistry, Molecular Biology, Medicine, DNA, Molecular, and 15 moreMethylation, DNA methylation, Enzyme, Hydrogen Bonding, Protein Conformation, Mechanism of action, Metabolic pathway, Base Sequence, Antineoplastic Agents, Nucleic Acid Conformation, Methyltransferase, Biochemistry and cell biology, Molecular Structure, Nucleic Acid, and cytidine
Research Interests: Chemistry, Molecular Biology, Catalysis, Kinetics, Enzyme Inhibitors, and 15 moreMagnetic Resonance Spectroscopy, Medicine, DNA, Molecular, Circular Dichroism, Stereochemistry, Enzyme, Active site, Cysteine, Protein Binding, Chemical Modification, Biochemistry and cell biology, binding sites, cytosine, and cytidine
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Human gamete interaction is of fundamental biological importance, yet the molecular interactions between spermatozoa and the zona pellucida are poorly understood. Surprisingly, the role of the polypeptide backbone of zona pellucida... more
Human gamete interaction is of fundamental biological importance, yet the molecular interactions between spermatozoa and the zona pellucida are poorly understood. Surprisingly, the role of the polypeptide backbone of zona pellucida glycoprotein 3 (ZP3), the putative ligand for spermatozoa activation, has been largely overlooked. Purified recombinant human ZP3 was expressed in Escherichia coli as a C-terminal fusion to the dimeric glutathione S-transferase (GST) from Schistosoma japonicum and was shown to induce acrosomal exocytosis in live, capacitated human spermatozoa. The level of exocytosis is comparable with that obtained using purified, glycosylated, recombinant human ZP3 [van Duin, M., Polman, J. E. M., DeBreet, I. T. M., Van Ginneken, K., Bunschoten, H., Grootenhuis, A., Brindle, J. and Aitken, R. J. (1994). Biol Reprod. 51, 607-617]. These data imply that the polypeptide chain of human ZP3 contributes to recognition of spermatozoa during acrosomal exocytosis in vitro.
Research Interests: Biology, Medicine, Western blotting, Biological Sciences, Humans, and 13 moreMale, P-glycoprotein, Biochemical, Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis, Egg Proteins, CHEMICAL SCIENCES, Exocytosis, Acrosome, Glutathione Transferase, Zona Pellucida, Acrosome Reaction, Medical and Health Sciences, and In Vitro Techniques
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Research Interests: Catalysis, Mass Spectrometry, Biology, Medicine, Biological Sciences, and 15 moreEnvironmental Sciences, Polymerase Chain Reaction, Nucleic Acids, Enzyme, Active site, Acetic Acid, Imidazoles, Acrylic Acid, In Vitro Selection, Nucleotides, Oligonucleotides, Nucleic Acid, restriction enzyme, binding sites, and polyphosphates
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We have developed a rapid, accurate, and quantitative method for the detection of methylation differences at specific CpG sites based on bisulfite treatment of DNA followed by primer extension and ion-pair reversed-phase high performance... more
We have developed a rapid, accurate, and quantitative method for the detection of methylation differences at specific CpG sites based on bisulfite treatment of DNA followed by primer extension and ion-pair reversed-phase high performance liquid chromatography (IP RP HPLC). The application of the method is illustrated by analysis of differentially imprinted alleles arising from Prader-Willi and Angelman syndromes. In order to convert unmethylated cytosines to uracil, plasmid and genomic DNA samples were treated with sodium bisulfite and the targeted sequence was then amplified using oligodeoxynucleotide primers specific for the bisulfite-deaminated DNA. The PCR product(s) from this step was used as a template for a primer extension reaction and the products were subsequently analyzed chromatographically using IP RP HPLC. This method eliminates the need to use restriction enzymes to determine the methylation status of the amplicon and circumvents the need for radio labeling for the quantitative measurements. Finally, this method removes the need for nucleotide sequencing because it is not solely reliant on the presence or absence of one or more PCR products, as is the case with related methods.
Research Interests: Genetics, Biology, Genomic Imprinting, Epigenetics, Medicine, and 15 moreDNA, Humans, Polymerase Chain Reaction, DNA methylation, CpG islands, Clinical Sciences, High Pressure Liquid Chromatography, Deamination, Analytical Method, Angelman Syndrome, Imprinted Genes, Base Sequence, Genetic Markers, Molecular Sequence Data, and Human mutation
Abstract: At the chromatin level, methylated CpG dinucleotides are R. Msp1 resistant compared with nonmethylated counterparts. The DNA of two E. coli strains was analyzed following transformationwith bacterial cytosine-5-methyltransferase... more
Abstract: At the chromatin level, methylated CpG dinucleotides are R. Msp1 resistant compared with nonmethylated counterparts. The DNA of two E. coli strains was analyzed following transformationwith bacterial cytosine-5-methyltransferase gene M. Msp1 in ...
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Research Interests: Analytical Chemistry, Fluorescent Dyes and Reagents, DNA, Cell Differentiation, Humans, and 14 morePolymerase Chain Reaction, Analytical, Mutation Detection, High Pressure Liquid Chromatography, DNA binding, Gel electrophoresis, Analytical Biochemistry, Retention Time, Sensitivity and Specificity, "Intercalating Agents", Biochemistry and cell biology, Nucleic Acid, DNA fragmentation, and fluorescent dyes
The inwardly rectifying K+ channel ROMK1 has been implicated as being significant in K+ secretion in the distal nephron. ROMK1 has been shown by immunocytochemistry to be expressed in relevant nephron segments. The development of the... more
The inwardly rectifying K+ channel ROMK1 has been implicated as being significant in K+ secretion in the distal nephron. ROMK1 has been shown by immunocytochemistry to be expressed in relevant nephron segments. The development of the atomic force microscope has made possible the production of high resolution images of small particles, including a variety of biological macromolecules. Recently, a fusion protein of glutathione S-transferase (GST) and ROMK1 (ROMK1-GST) has been used to produce a polyclonal antibody for immunolocalization of ROMK1. We have used atomic force microscopy to examine ROMK1-GST and the native ROMK1 polypeptide cleaved from GST. Imaging was conducted with the proteins in physiological solutions attached to mica. ROMK1-GST appears in images as a particle composed of two units of similar size. Analyses of images indicate that the two units have volumes of approximately 118 nm3, which is close to the theoretical volume of a globular protein of approximately 65 kD...
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Research Interests: mRNA and cDNA library
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AquI DNA methyltransferase, M.AquI, catalyses the transfer of a methyl group from S-adenosyl-L-methionine to the C5 position of the outermost deoxycytidine base in the DNA sequence... more
AquI DNA methyltransferase, M.AquI, catalyses the transfer of a methyl group from S-adenosyl-L-methionine to the C5 position of the outermost deoxycytidine base in the DNA sequence 5'CYCGRG3'. M.AquI is encoded by two overlapping ORFs (termed alpha and beta) instead of the single ORF that is customary for Class II methyltransferase genes. The structural organization of the M.AquI protein sequence is quite similar to that of other bacterial C5-DNA methyltransferases. Ten conserved motifs are also present in the correct order, but only on two polypeptides. We separately subcloned the genes that encode the alpha and beta subunits of M.AquI into expression vectors. The overexpressed His-fusion alpha and beta subunits of the enzyme were purified to homogeneity in a single step by Nickel-chelate affinity chromatography. The purified recombinant proteins were assayed for biological activity by an in vitro DNA tritium transfer assay. The alpha and beta subunits of M.AquI alone have no DNA methyltransferase activity, but when both subunits are included in the assay, an active enzyme that catalyses the transfer of the methyl group from S-adenosyl-Lmethionine to DNA is reconstituted. We also showed that the beta subunit alone contains all of the information that is required to generate recognition of specific DNA duplexes in the absence of the alpha subunit
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Since the discovery that the DNA methyltransferase M.HhaI utilises a base flipping mechanism to expose its target cytosine during catalysis, this phenomenon has been observed in other nucleic acid modifying enzymes. The crystallographic... more
Since the discovery that the DNA methyltransferase M.HhaI utilises a base flipping mechanism to expose its target cytosine during catalysis, this phenomenon has been observed in other nucleic acid modifying enzymes. The crystallographic analyses of such enzyme-DNA complexes have revealed the molecular features of extrahelical base stabilisation, but have been less informative about the flipping process itself.