ABSTRACT
In this study we have analysed mouse telomeres by Pulsed Field Gel Electrophoresis (PFGE). A number of specific restriction fragments hybridising to a (TTA-GGG)4 probe in the size range 50-150kb can be detected. These fragments are devoid of sites for most restriction enzymes suggesting that they comprise simple repeats; we argue that most of these are likely to be (TTAGGG)n. Each discrete fragment corresponds to the telomere of an individual chromosome and segregates as a Mendelian character. However, new size variants are being generated in the germ line at very high rates such that inbred mice are heterozygous at all telomeres analysable. In addition we show that specific small (approximately 4-12kb) fragments can be cleaved within some terminal arrays by the restriction enzyme MnII which recognises 5'(N7)GAGG3'. Like the complete telomere-repeat arrays (TRA's) these fragments form new variants at high rates and possibly by the same process. We speculate on the mechanisms that may be involved.
Subject(s)
Chromosomes/ultrastructure , Genetic Variation , Repetitive Sequences, Nucleic Acid , Animals , DNA Probes , DNA Restriction Enzymes/metabolism , Female , Male , Mice , Mice, Inbred C57BL , Mice, Inbred DBA , Molecular Weight , MuridaeABSTRACT
A rapid and efficient procedure has been developed to purify phosphofructokinase from the muscle of the parasitic roundworm, Ascaris suum. The procedure can be accomplished in 1 day with a 420-fold purification and a 60% yield. The enzyme was shown to be homogeneous by two-dimensional electrophoresis, Sepharose 6B column chromatography, and high performance liquid chromatography utilizing a size exclusion column. The subunit molecular weight of the enzyme was found to be 95,000 by electrophoresis in the presence of sodium dodecyl sulfate. In solutions of low ionic strength, the native enzyme aggregated to species of higher molecular weight than did the rabbit muscle phosphofructokinase. In the presence of 0.2 M (NH4)2SO4, the minimum native molecular weight was determined to be 398,000 by high performance liquid chromatography and Sepharose 6B column chromatography. Therefore, the enzyme appears to be a tetramer with identical or near-identical subunits. The apparent isoelectric point of the enzyme was shown to be 7.3 to 7.4 by both column and gel isoelectric focusing. Amino acid analysis revealed a lower number of the aromatic residues Phe, Tyr, and Trp than in the rabbit muscle enzyme and this is in agreement with the lower extinction coefficient of E1%280 nm = 6.5. Analysis of the purified enzyme revealed 7.4 +/- 0.6 mol of phosphate/mol of enzyme.
Subject(s)
Ascaris/enzymology , Phosphofructokinase-1/isolation & purification , Amino Acids/analysis , Animals , Electrophoresis, Polyacrylamide Gel , Isoelectric Focusing , Macromolecular Substances , Molecular Weight , Muscles/enzymology , Rabbits , Species SpecificityABSTRACT
The initial metabolism of fructose, mannose, and galactose in Moniliformis dubius (M. moniliformis; Acanthocephala) was examined following brief aerobic incubations in 14C-labeled substrate. The pattern of incorporation of radioactivity from 14C-fructose and 14C-mannose into intermediates of hexose metabolism was indistinguishable from that reported elsewhere for the initial post-absorptive metabolism of 14C-glucose under comparable conditions; these hexoses were phosphorylated rapidly following their absorption, and much of the radioactivity absorbed as mannose or fructose was recovered in the nonreducing disaccharide trehalose [alpha-D-glucopyranosyl-(1 leads to 1)-alpha-D-glucopyranoside]. 14C-Galactose was phosphorylated less readily than the other hexoses. More than half of the radioactivity absorbed as galactose was incorporated into disaccharide; some of the galactose-derived disaccharide had properties suggesting the presence of a galactosyl moiety. Incorporation of radioactivity from any of the hexoses into glycogen was minimal. The extensive incorporation of hexose moieties into trehalose or a trehalose-like disaccharide rather than glycogen underscores the probable importance of trehalose metabolism to carbohydrate assimilation in Moniliformis. Physiological factors which might favor trehalose biosynthesis over glycogenesis are considered.
Subject(s)
Acanthocephala/metabolism , Fructose/metabolism , Galactose/metabolism , Mannose/metabolism , Moniliformis/metabolism , Absorption , Animals , Biological Transport , Carbon Radioisotopes , Trehalose/metabolism , Uridine Diphosphate Glucose/metabolismABSTRACT
The uptakes of 14C-glucose, -2-deoxyglucose, -mannose, -N-acetylglucosamine, -3-0-methylglucose, -fructose, and -galactose by female Moniliformis dubius were nonlinear, saturable functions of hexose concentration. Kinetic and inhibition studies indicated that glucose and 2-deoxyglucose were absorbed via a single common transport locus. Mannose, N-acetylglucosamine, 3-0-methylglucose, fructose, and galactose (in decreasing order of effectiveness) inhibited the uptake of glucose in a completely competitive manner; their absorptions appeared to be mediated by the glucose transport locus and, to some degree, by one or more additional transport systems. Kinetic studies suggested that the apparent inhibitions of 14C-glucose uptake by maltose and glucose-6-phosphate were due to free glucose liberated through the action of surface hydrolases. The uptake of 14C-glucose was also inhibited by salicin, alpha-methylglucoside, and beta-methylglucoside, but not by pentoses, L-hexoses, sugar alcohols, disaccharides (except maltose), gluconic acid, glucuronic acid, phlorizin, or ouabain. Glucose uptake was not Na+-dependent.
