Two distinct DNA ligases from Drosophila melanogaster embryos

Embryos of Drosophila melanogaster contain two distinct DNA ligases (DNA ligase I and II). DNA ligase I was eluted at 0.2 M KCl and DNA ligase II at 0.6 M KCl on phosphocellulose column chromatography. The former was rich in early developing embryos and its activity decreased during embryonic development. The latter was found constantly throughout the developing stages of embryos. DNA ligase I existed in a cytoplasmic fraction and DNA ligase II is concentrated in nuclei. Both enzymes ligate 5'-phosphoryl and 3'-hydroxyl groups in oligo(dT) in the presence of poly(dA). DNA ligase II is also able to join oligo(dT)(poly(rA). Both enzymes require ATP and Mg2+ for activity. The Km for ATP is 2.7 X 10(-6) M for DNA ligase I, and 3.0 X 10(-5) M for DNA ligase II. DNA ligase I requires dithiothreitol and polyvinyl alcohol, but DNA ligase II does not. Both enzymes are inhibited in the presence of N-ethylmaleimide. DNA ligase I is active at a low salt concentration (0-30 mM KCl), but DNA ligase II is active at high salt concentrations (50-100 mM). DNA ligase I is more labile than DNA ligase II. The molecular masses of DNA ligase-AMP adducts were determined as 86 and 75 kDa for DNA ligase I, and as 70 (major protein) and 90 kDa (minor protein) for DNA ligase II under denaturing conditions. A sedimentation coefficient of 4.2 S was observed for DNA ligase II. Consequently, Drosophila DNA ligase I and II are quite similar to mammalian DNA ligase I and II. Drosophila DNA ligase I and a DNA ligase by B.A. Rabin et al. [(1986) J. Biol. Chem. 261, 10637-10645] seem to be the same enzyme.     

cDNA cloning and amino acid sequence of bovine brain 2',3'-cyclic-nucleotide 3'-phosphodiesterase

2',3'-Cyclic-nucleotide 3'-phosphodiesterase (EC 3.1.4.37) has been widely used as a marker for myelin-oligodendrocytes in the central nervous system. Evidence has been provided that the enzyme is identical with one of the Wolfgram proteins of central nervous system myelin. The amino acid sequence of bovine 2',3'-cyclic-nucleotide 3'-phosphodiesterase was determined by both protein and cDNA sequence analyses. Protein sequence analysis was done on bovine elastase 2',3'-cyclic-nucleotide 3'-phosphodiesterase, a low molecular weight enzyme obtained by solubilization with pancreatic elastase (EC 3.4.21.36) (Nishizawa, Y., Kurihara, T., and Takahashi, Y. (1980) Biochem. J. 191, 71-82; Kurihara, T., Nishizawa, Y., Takahashi, Y., and Odani, S. (1981) Biochem. J. 195, 153-157). Based on the carboxyl-terminal sequence of bovine elastase 2',3'-cyclic-nucleotide 3'-phosphodiesterase, synthetic oligodeoxyribonucleotides were prepared and used as probes for screening a cDNA library of bovine brain. A cDNA of 2305 base pairs was obtained and sequenced, and the complete amino acid sequence of bovine 2',3'-cyclic-nucleotide 3'-phosphodiesterase was deduced. Bovine 2',3'-cyclic-nucleotide 3'-phosphodiesterase deduced contains 400 amino acids including initiation methionine and has a molecular weight of 44,850. Bovine elastase 2',3'-cyclic-nucleotide 3'-phosphodiesterase corresponds to the 236 amino acids of bovine 2',3'-cyclic-nucleotide 3'-phosphodiesterase. RNA blot analysis revealed a single-species mRNA of about 2600 bases.     

Quantitative study of tissue collagen metabolism

A procedure for the quantification of various parameters of metabolism of collagen in fibrotic mouse liver has been developed. The method involves derivatization of hydroxyproline, a marker of collagen, with dansyl chloride, high-performance liquid chromatography of the derivative on an octadecyl C-18 column, and its detection by fluorescence. This assay improves upon existing procedures in several respects: It extends the analysis so that not only the collagen content of the tissue but also the metabolism of collagen is determined at levels found intracellularly. It is sensitive enough to quantify 0.1-10 nmol of hydroxyproline, and it includes three major amino acids (hydroxyproline, glycine, and proline) of collagen and two assay controls; it generates information on both the purity and quantity of collagen in each assay. The determination of specific activity of intracellular free [14C]proline, which is the precursor of protein-bound hydroxyproline, defines the specific activity of [14C]hydroxyproline of collagen converted from precursor residues of [14C]proline by the action of prolyl hydroxylase. The specific activity of [14C]hydroxyproline can be used for the evaluation of collagen synthesis and secretion and intracellular and extracellular degradation of the newly synthesized and secreted collagen in the tissue. The determination of specific activities of [14C]hydroxyproline and [14C]proline and of the ratio of [14C]hydroxyproline to [14C]proline of newly secreted collagen provides information concerning the extent of hydroxylation of [14C]proline residues of newly synthesized collagen.     

Optimum conditions for electric pulse-mediated gene transfer to mammalian cells in suspension

A pulse-generating machine which delivers exponentially decaying pulses over broad range of pulse lengths was used to determine the optimum pulse conditions for gene transfer to FM3A cells. In the transformation of tk- cells with pTK1, a single pulse of 100-2000 microseconds gave a high transformation frequency at 1.5-6 kV/cm and room temperature, the highest transformation frequency obtained being 3 X 10(-3). As the suspension buffer for cells exposed to the pulse, Saline G was better than PBS(-) for obtaining a large number of transformants because it ensured high cell viability.     

Resting spore formation ofLeptocylindrus danicus (Bacillariophyceae) during short time-scale upwelling and its significance as predicted by a simple model

Resting spore formation during short time-scale upwelling and its significance were investigated in the field and by a simple theoretical model. Field observations of spore formation ofLeptocylindrus danicus were made off Izu Peninsula, Japan. A rapid increase in ratio of resting spore to vegetative cell numbers indicated thatL. danicus formed resting spores quickly as a response to nutrient depletion in the upwelled water, although only a very low number of resting spores was found in the upwelling. A simple model was constructed to investigate the possible advantages of spore formation during short time-scale upwelling. This showed that there is a critical time-scale for resting spore formation to be advantageous. The nutrient depletion period of the upwelling off Izu was shorter than the critical time-scale determined by the model. Rapid-sinking of resting spores may increase further the critical time-scale, unless spores return with upwelling water. For short time-scale upwelling, the vegetative cell may be better suited than the resting spore for enduring a short period of nutrient depletion. Contribution from Shimoda Marine Research Center, University of Tsukuba, No. 475.     

Methyl iodide, a potent inducer of the adaptive response without appreciable mutagenicity in E. coli

Methyl iodide (MeI), a very weak mutagen, induced the adaptive response in E. coli to a similar extent to those induced by potently mutagenic methylating agents. MeI potentiated the mutagenicity of a methylating mutagen, N-methyl-N-nitrosourea, by its co-treatment. These results might give indication that MeI directly methylates O6-methylguanine-DNA methyltransferase resulting in induction of the adaptive response and depletion of the repair capacity of enzyme.     

Porcine polymorphonuclear leukocyte NADPH-cytochrome c reductase generates superoxide in the presence of cytochrome b559 and phospholipid

NADPH-cytochrome c reductase and cytochrome b559 were purified from the membrane fraction of phorbol myristate acetate-stimulated porcine polymorphonuclear leukocytes. The highly purified reductase oxidized NADPH and generated superoxide when combined with partially purified cytochrome b559 in the presence of phosphatidylcholine. In the same system, but under the anaerobic condition, the reductase was found to reduce cytochrome b559.     

Fibrinolysis by urokinase endowed with magnetic property

The activated magnetic modifier was synthesized from magnetite, alpha, omega-dicarboxymethylpoly(oxyethylene) and N-hydroxysuccinimide (Biochem. Biophys. Res. Commun., 145, 908-914, 1987). Urokinase was directly coupled with the activated magnetic modifier to obtain magnetic urokinase. The magnetic urokinase dispersed in saline and exerted high fibrinolytic activity (13.8 X 10(4) IU/mg protein), and was readily recovered from saline by magnetic force of 250 Oe. By applying magnetic force, the urokinase was attracted at our will and local fibrinolysis was achieved on fibrin gel in a petri dish.     

Structural analysis of N-linked oligosaccharides by a combination of glycopeptidase, exoglycosidases, and high-performance liquid chromatography

A simple, sensitive, and rapid method for the analysis of structures of N-linked carbohydrates is reported. The method involves four steps: preparation of carbohydrate chains from glycopeptides by N-oligosaccharide glycopeptidase digestion; derivatization of the reducing ends of carbohydrate chains with a fluorescent reagent, 2-aminopyridine, by using sodium cyanoborohydride; separation of oligosaccharide derivatives by reverse-phase high-performance liquid chromatography; and structural analysis of oligosaccharides by sequential exoglycosidase digestion. The elution positions of 50 standard oligosaccharide derivatives were determined by HPLC. The structure of an unknown oligosaccharide can be characterized by comparison of its elution position with those of the standard compounds. The method was applied to elucidate the structures of oligosaccharides in the myeloma IgG protein, Yot.