Thymine lesions produced by ionizing radiation in double-stranded DNA

A DNA glycosylase which catalyzes the release of thymine residues damaged by ring saturation, fragmentation, or ring contraction from double-stranded DNA has been used to characterize such base derivatives in gamma-irradiated DNA. It is shown by chromatographic analysis that irradiation of DNA in neutral solution generates the ring-saturated forms cis-thymine glycol, trans-thymine glycol, and a monohydroxydihydrothymine, probably 6-hydroxy-5,6-dihydrothymine. The latter compound is only observed after irradiation under hypoxic conditions. The ring-contracted thymine derivative 5-hydroxy-5-methylhydantoin is also formed, and it is the major lesion after irradiation of DNA under O2. Ring-fragmented products such as methyltartronylurea were only generated in small quantities. Isolation and analysis of the DNA from gamma-irradiated human cells also revealed the formation of ring-saturated thymine derivatives, but 5-hydroxy-5-methylhydantoin was not found in this case.     

Initiation of DNA replication in Escherichia coli: RNase H-deficient mutants do not require the dnaA function

Summary A series of temperature-resistant revertants were isolated from strains of Escherichia coli K12 carrying a temperature-sensitive mutation in the dnaA gene. Four independent revertants were found which still carry the original ts mutation. The ability of these strains to grow at high temperature is due to a suppressor mutation, called sin. All four sin mutations are located between the genes metD and proA on the genetic map of E. coli, which suggests that they all affect the same gene. The sin suppressors, which were isolated for their ability to suppress one dnaA mutation, are also able to suppress three other temperature-sensitive dnaA mutations, but they are not able to suppress mutations in either of the two genes dnaB or dnaC. The sin suppressors alone do not confer any particular phenotype on bacteria, but they are deficient in the enzyme RNase H. On the basis of these findings we propose that the function of the dnaA protein is to protect a DNA-RNA hybrid at the origin of replication against RNase H.     

Autoaggregating Yersinia enterocolitica express surface fimbriae with high surface hydrophobicity

For 13 strains of Yersinia enterocolitica, there was a good correlation between the production of the broad-spectrum, mannose-resistant Yersinia haemagglutinin (MR/Y HA), the presence of fimbriae and high surface hydrophobicity. Each of these characters was expressed in cultures grown at low (less than 32 degrees C) but not at high (Greater than 35 degrees C) temperatures.     

Binding of platelet factor 4 to heparin oligosaccharides.

Heparin fractions of differing Mr (7800-18 800) prepared from commercial heparin by gel filtration and affinity chromatography on immobilized anti-thrombin III had specific activities when determined by anti-Factor Xa and anti-thrombin assays that ranged from 228 to 448 units/mg. The anti-Factor Xa activity of these fractions could be readily and totally neutralized by increasing concentrations of platelet factor 4 (PF4). That these fractions bound to immobilized PF4 was indicated by the complete binding under near physiological conditions of 3H-labelled unfractionated commercial heparin. An anti-thrombin III-binding oligosaccharide preparation (containing predominantly eight to ten saccharide units), prepared by degradation of heparin with HNO2 had high (800 units/mg) anti-Factor Xa, but negligible anti-thrombin, specific activity. The anti-Factor Xa activity of this material could not be readily neutralized by PF4, and the 3H-labelled oligosaccharides did not completely bind to immobilized PF4. A heterogeneous anti-thrombin III-binding preparation containing upwards of 16 saccharides had anti-thrombin specific activity of just less than one-half the anti-Factor Xa specific activity. This material was completely bound to immobilized PF4 and was eluted with similar concentrations of NaCl to those that were required to elute unfractionated heparins from these columns. Furthermore, increasing concentrations of PF4 neutralized the anti-Factor Xa activity of this material in a manner similar to that of unfractionated heparin. It is concluded that heparin oligosaccharides require saccharide units in addition to the anti-thrombin III-binding sequence in order to fully interact with PF4.     

Degradation of heparin in mouse mastocytoma tissue

1. Heparin was prepared from mouse mastocytoma tissue by mild procedures, including extraction of mast-cell granules with 2m-potassium chloride, precipitation of the extracted polysaccharide with cetylpyridinium chloride from 0.8m-potassium chloride and finally digestion of the isolated material with testicular hyaluronidase. The resulting product (fraction GE(H)) represented approx. 40% of the total heparin content of the tissue. 2. Fraction GE(H) was fractionated by gel chromatography on Sepharose 4B into three subfractions, with average molecular weights ( M(w)) of approx. 60000-70000 (highly polydisperse material), 26000 and 9000 respectively. Treatment of each of the subfractions with alkali or with papain did not affect their behaviour on gel chromatography. Amino acid and neutral sugar analyses indicated that the two low-molecular-weight fractions consisted largely of single polysaccharide chains lacking the carbohydrate-protein linkage region. It was suggested that these heparin molecules had been degraded by an endopolysaccharidase. 3. Pulse labelling in vivo of mastocytoma heparin with [(35)S]sulphate showed initial labelling of large molecules followed by a progressive shift of radioactivity toward fractions of lower molecular weight. Further, heparin-depolymerizing activity was demonstrated by incubating (35)S-labelled heparin in vitro with a mastocytoma 10000g-supernatant fraction. Appreciable degradation of the polysaccharide occurred, as demonstrated by gel chromatography. In contrast, no depolymerization was observed on subjecting (14)C-labelled chondroitin sulphate to the same procedure.     

The distribution of sulphate residues in the chondroitin sulphate chain

1. Electrophoresis of chondroitin sulphate, before and after partial degradation with testicular hyaluronidase, revealed charge heterogeneity of the degraded but not of the intact polymer. 2. Hyaluronidase-treated chondroitin sulphate was fractionated by gel chromatography. Two subfractions which were essentially monodisperse with regard to molecular weight (values of 8600 and 4800, respectively) were separated further by chromatography on Dowex 1. The resulting subfractions differed considerably with respect to their sulphate/disaccharide molar ratios. 3. Amino acid and neutral-sugar analyses of the Dowex 1 subfractions showed that the less sulphated fragments contained the carbohydrate-protein linkage region, whereas the high-sulphated fragments essentially lacked this constituent. It was concluded that chondroitin sulphate contains relatively less sulphate in the vicinity of the carbohydrate-protein linkage region than in the more peripheral portion of the polysaccharide chain.     

Distribution of sulphate and iduronic acid residues in heparin and heparan sulphate

1. A method was developed for determination of the uronic acid composition of heparin-like glycosaminoglycans. Polymers or oligosaccharides are degraded to monosaccharides by a combination of acid hydrolysis and deamination with HNO₂. The resulting uronic acid monosaccharides (accounting for about 70% of the uronic acid contents of the starting materials) are isolated and converted into the corresponding aldono-1,4-lactones, which are separated by g.l.c. The calculated ratios of glucuronic acid/iduronic acid are reproducible within 5%. 2. Samples of heparin from pig intestinal mucosa (molar ratio of sulphate/disaccharide unit, 2.40) and heparan sulphate from human aorta (sulphate/disaccharide ratio, 0.46) were subjected to uronic acid analysis. l-Iduronic acid constituted 77% and 19% respectively of the total uronic acid contents. 3. The correlation between the contents of sulphate and iduronic acid indicated by this finding also applied to the fractionated deamination products of the two polymers. The sulphated fragments varied in size from disaccharide to octasaccharide (or larger) and showed sulphate/disaccharide molar ratios in the range of 0.05–2.0. The proportion of iduronic acid increased with increasing ester sulphate contents of the oligosaccharides. 4. Previous studies on the biosynthesis of heparin in a cell-free system have shown that l-iduronic acid residues are formed by C-5 epimerization of d-glucuronic acid units at the polymer level; the process requires concomitant sulphation of the polymer. The results obtained in the present structural study conform to these findings, and suggest further that similar mechanisms may operate in the biosynthesis of heparan sulphate. The epimerization reaction appears to be linked to the sulphation of hydroxyl groups but does not seem to require sulphation of the target uronic acid residues. The significance of sulphamino groups in relation to the formation of iduronic acid is unknown.