ABSTRACT
A new class of glutathione transferases has been discovered by analysis of the expressed sequence tag data base and sequence alignment. Glutathione S-transferases (GSTs) of the new class, named Omega, exist in several mammalian species and Caenorhabditis elegans. In humans, GSTO 1-1 is expressed in most tissues and exhibits glutathione-dependent thiol transferase and dehydroascorbate reductase activities characteristic of the glutaredoxins. The structure of GSTO 1-1 has been determined at 2.0-A resolution and has a characteristic GST fold (Protein Data Bank entry code ). The Omega class GSTs exhibit an unusual N-terminal extension that abuts the C terminus to form a novel structural unit. Unlike other mammalian GSTs, GSTO 1-1 appears to have an active site cysteine that can form a disulfide bond with glutathione.
Subject(s)
Glutathione Transferase/chemistry , Glutathione Transferase/metabolism , Amino Acid Sequence , Animals , Base Sequence , Caenorhabditis elegans/enzymology , Crystallography, X-Ray , Female , Glutathione Transferase/genetics , Humans , Isoenzymes/chemistry , Isoenzymes/genetics , Isoenzymes/metabolism , Kinetics , Male , Mammals , Models, Molecular , Molecular Sequence Data , Phylogeny , Protein Conformation , Protein Structure, Secondary , Sequence Tagged Sites , Substrate Specificity , Transcription, GeneticSubject(s)
DNA/isolation & purification , Genetic Vectors , Magnetics , Nucleic Acid Conformation , Base Composition , Binding Sites , DNA/chemistry , DNA/metabolism , Hydrogen Bonding , Hydrogen-Ion Concentration , Microspheres , Oligonucleotides/metabolism , Plants/genetics , Plasmids , Pyrimidines/metabolismABSTRACT
Repetitive DNA sequences were detected directly on somatic metaphase chromosome spreads from soybean root tips using fluorescent in situ hybridization. Methods to spread the forty small metaphase chromosomes substantially free of cellular material were developed using protoplasts. The specific DNA probe was a 1.05 kb internal fragment of a soybean gene encoding the 18S ribosomal RNA subunit. Two methods of incorporating biotin residues into the probe were compared and detection was accomplished with fluorescein-labeled avidin. The rDNA probe exhibits distinct yellow fluorescent signals on only two of the forty metaphase chromosomes that have been counterstained with propidium iodide. This result agrees with our previous analyses of soybean pachytene chromosomes showing that only chromosome 13 is closely associated with the nucleolus organizer region. Fluorescent in situ hybridization with the rDNA probe was detected on three of the forty-one metaphase chromosomes in plants that are trisomic for chromosome 13.
Subject(s)
Chromosomes/chemistry , Glycine max/genetics , Nucleic Acid Hybridization , Biotin , DNA Probes , DNA, Ribosomal/analysis , Fluorescence , Metaphase , RNA, Ribosomal, 18S/genetics , Repetitive Sequences, Nucleic Acid , TrisomyABSTRACT
Products from the hydrolysis of phosphatidylinositol 4,5-bisphosphate (IP3) can increase and/or potentiate cAMP accumulation in a variety of cells. Antibody to surface immunoglobulins activates IP3 hydrolysis in B-lymphocytes. In this study we have examined whether anti-Ig also stimulated and/or potentiated increases in the cAMP levels of splenocytes from athymic nude mice. Furthermore, since TPA potentiates anti-Ig-induced DNA synthesis and cAMP modulates DNA synthesis, the effects of TPA on any anti-Ig-induced changes in cAMP were also studied. Antibody (25 micrograms/ml) stimulated a rapid ris in cAMP which increased from 250 fmol/10(6) cells to 400 fmol/10(6) cells within 1 min and then subsided to 310 fmol/10(6) cells by 10 min. TPA (96 nM) suppressed the anti-Ig-induced cAMP accumulation at 1 min by 60%, but potentiated the forskolin (114 microM)-induced rise by 151%. Two other activators of protein kinase C, dioctanoylglycerol (5 microM), and anti-Ig (25 micrograms/ml), also potentiated the forskolin response by 198% and 52%, respectively. These results suggest that modulation of the adenylate cyclase system by anti-Ig may act in concert with cytokines and/or prostaglandins secreted by other lymphoid cells to define the state of proliferation or differentiation in B-cells.
