Abnormal recovery of DNA replication in ultraviolet-irradiated cell cultures of Drosophila melanogaster which are defective in DNA repair

Summary Cell cultures prepared from embryos of a control stock of Drosophila melanogaster respond to ultraviolet light with a decline and subsequent recovery both of thymidine incorporation and in the ability to synthesize nascent DNA in long segments. Recovery of one or both capacities is absent or diminished in irradiated cells from ten nonallelic mutants that are defective in DNA repair and from four of five nonallelic mutagen-sensitive mutants that exhibit normal repair capabilities. Recovery of thymidine incorporation is not observed in nine of ten DNA repair-defective mutants. On the other hand, partial or complete recovery of incorporation is observed in all but one repair-proficient mutagen-sensitive mutant.Irradiated cells from two mutants that display no excision capacity exhibit a gradual arrest of thymidine incorporation within 20 h after the initial decline. This arrest of incorporation is not observed in mutants exhibiting only partial defects in excision repair.Recovery of the ability to synthesize nascent DNA in long segments is normal in cells from the two mutants that display no excision capacity, indicating that recovery does not depend upon the excision of pyrimidine dimers from cellular DNA. Recovery of that ability is not observed, however, in cells from one partially excision-defective mutant, two of three postreplication repair-defective mutants, two of four mutants defective in both excision and postreplication repair, and one of five repair-proficient mutagen-sensitive mutants. These results indicate that recovery of normal DNA replication in irradiated Drosophila cells depends upon the activity of several functions.Abbreviation used UV ultraviolet light — principal wavelength 254 nm     

Genetic analysis of X-linked mutagen-sensitive mutants of Drosophila melanogaster

Mutants at 2 new loci which control mutagen-sensitivity are described. Mutants at both loci are female-sterile and are hypersensitive to killing by MMS; neither increases the frequency of sex-linked recessive lethals. A screen of previously described female-sterile and meotic mutants has revealed that a number of these are also sensitive to mutagens. In addition, several new mutants have been identified on the basis of sensitivity to either HN2 or MMS. An anlysis of complementation data suggests that all of the X-linked genes controlling sensitivity to MMS may now have been identified. Among the new mei-41 alleles are mutants which show verly little meiotic nondisjunction or loss. Cytogenetic mapping of previously known mutants is also described. The mutants mus(1)104^D1 and mei-41^D5 are located in th eregion 14B13±?14D1,2 on the polytene chromosome map, and they map very close to each other genetically. Cytogenetically mus(1)101^D1 is between salivary chromosome bands 12A6,7 and 12D3, mus(1)103^D1 is between bands 12A1,2 and 12A6,7, and mus(1)-109^A1 is in section 8F3-9A2.     

A physico-chemical study of heparin: Evidence for a calcium-induced co-operative conformational transition

The conformations of heparin in aqueous solution in the presence of sodium, potassium, magnesium and calcium cations were studied using circular dichroism, optical rotation, nuclear magnetic resonance and equilibrium dialysis. Potassium and magnesium cations, when added to sodium heparinate solutions, cause small chiroptical changes. Binding of calcium ions gives rise to large changes in both optical rotation and circular dichroism. This is indicative of a major change in chain conformation, which is also manifest in ¹³C and ¹H n.m.r.⁴Equilibrium dialysis suggests one mole of calcium bound per mole of tetrasaccharide, which n.m.r. indicates to be appropriately sulphated iduronateglucosamine-iduronate-glucosamine. The calcium is chelated by two iduronate carboxyl groups. Proton-proton coupling constants, determined by convolution difference spectroscopy and Carr-Purcell sequences, indicate that, over the temperature range 285 to 353 K, the iduronate ring is best described as ¹C₄(l) and the glucosamine residue as ⁴C₁(d) for both sodium and calcium forms.The conformational change induced by calcium is ascribed to rotation around the glycosidic linkages. The binding process is co-operative and the binding constant of 10³ to 10⁴m^?1 is biologically significant. The findings are consistent with intramolecular binding. Hence, this study represents the first report of a polysaccharide undergoing a cation-induced intramolecular disorder-order process. The authors postulate that a function of the post-polymerization epimerization of d-glucuronate to l-iduronate is the attainment of the precise geometry required for co-operative calcium binding with consequent modulation of the flexibility of the tetrasaccharide units.     

Effects of glucagon and insulin on liver lysosomes

Recent experimental evidence has been obtained, principally in the laboratory of Glenn Mortimore, that hepatic lysosomes can act as a pool of amino acids during fasting. This pool is generated through $\text{autophagy}$, whereby intracellular proteins are somehow captured by the lysosomes and then rapidly hydrolyzed to free amino acids by the lysosomal proteinases. Two important metabolic fates of these lysosomal digestive products can be: 1) conversion of the glucogenic amino acids into glucose, and 2) conversion of trimethyl-lysine into carnitine. The latter metabolite is required to transfer fatty acids to the mitochondrial site of β-oxidation. Most interesting is the observation that glucagon appears to induce lysosomal autophagy and the resulting degradation of intracellular proteins by decreasing the size of amino acid pools in the perfused liver. This effect of the hormone may be directed at the single amino acid glutamine, since adding it alone to the perfusate can prevent the increase in autophagy caused by glucagon. Insulin also rapidly inactivates hepatic autophagy and its ensuing proteolysis. The $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ for the rate of los of autophagic vocuoles from the insulin-treated liver (or animal) is approximately 8 min. Thus, glucagon and insulin actively control intracellular protein catabolism that takes place within hepatic lysosomes, and this regulation by the two hormones may be one of their major molecular effects on gluconegenesis in the liver.     

Properties of a novel ATPase enzyme in chromaffin granules

Membranes were isolated from mitochondria and chromaffin granules of bovine adrenal medullae. The cross-contamination between the two membranes was examined by comparing the radioactive bands on autoradiograms of gels after phosphorylation of the membranes with [-³²P]-ATP and decoration with [¹²⁵I]concanavalin A and [¹²⁵I]protein A with antibody that was raised against chromaffin-granule membranes. It was found that the membranes cross-contaminated each other by less than 10%. The technique of immunodecoration with antibodies against subunits of proton-ATPases from yeast mitochondria, spinach chloroplasts, andE. coli membranes was used for quantitative estimation of proton-ATPase complexes in chromaffin granules and mitochondrial membranes. It was found that chromaffin-granule membranes contain less than 10% of the amount of proton-ATPase complex in mitochondrial membranes. The specific ATPase activity of chromaffin-granule membranes was on the order of 30 to 50% of the mitochondrial membranes. The ATPase activity of the chromaffin-granule membranes was more sensitive to 4-acetamido-4-isothiocyano-2,2-disulfonic acid stilbene and 4-chloro-7-nitrobenzofurazan. It was much less sensitive than the mitochondrial membranes to antibody against subunit of proton-ATPase fromE. coli membranes. After solubilization of chromaffin-granule membranes by octyglucoside and cholate and subsequent centrifugation on sucrose gradient, two different ATPase enzymes were separated. The heavier enzyme was identical to the mitochondrial-ATPase complex, while the lighter enzyme was identified as a novel ATPase, which might be responsible for the special properties of the ATPase activity of chromaffin-granule membranes.Abbreviations DCCD dicyclohoxylcarbodiimide - NBD-Cl 4-chloro-7-nitrobenzofurazan - SITS 4-acetamido-4-isothiocyano-2,2-disulfonic acid stilbene - SDS sodium dodecyl sulfate - MES 2-(N-morpholino)ethane sulfonic acid - FITC fluorescein isothiocyanate     

Guanosine-5′-diphosphate-3′-diphosphate inhibits the in, vitro synthesis of β-lactamase from pBR322 DNA

The $\text{in}\text{,}\text{vitro}$ synthesis of β-lactamase directed by pBR322 DNA is inhibited by guanosine-5′-diphosphate-3′-diphosphate.     

Lysosomal pool of free-amino acids

Free (non-protein) amino acids were measured in whole rat liver and in unmodified lysosomes which were prepared from rat liver by the technique of free-flow electrophoresis. Significant intralysosomal pools of threonine, serine, valine, cystine, methionine, isoleucine, leucine, tyrosine, phenylalanine, lysine and arginine were found. No efflux occurred from rat liver lysosomes in isotonic buffered sucrose at 0°C, but all amino acids showed various degrees of efflux at 20⁰ and 37⁰.     

Mutants partially defective in excision repair at five autosomal loci in Drosophila melanogaster

Primary cell cultures derived from mutants in seventeen different genes were analyzed for their ability to excise pyrimidine dimers from DNA. Five of these mutagen-sensitive mutants [mus(2)205^A1, mus(3)302^D1, mus(3) 304^D3, mus(3)306^D1, mus(3)308^D2] display a significantly reduced excision capacity relative to control cultures. In addition, two of the five [mus(3)306^D1, mus(3)308^D2] are defective in the accumulation of single-strand breaks normally seen after ultraviolet irradiation. This study, therefore, brings the total number of Drosophila mutants known to be defective in excision repair to seven. The results are discussed relative to other genetic and biochemical properties of these mutants. This work is dedicated to Professor W. Beermann whose own contributions were instrumental in focusing a modern analysis of the eukaryotic genome on the diptera. Those of us who benefitted so much from his personal guidance recognize that we did so as a result of some sacrifice on his part. One of Boyd's contemporaries in Tübingen once remarked: “It's terrifying to think what Professor Beermann could do if he were in the lab full time.”     

The effect of the hypocholesteremic drug, AY 9944 on the synthesis of bile salts in rat liver

1. The compound trans-1,4 bis-(2-dichlorobenzylaminomethyl)cyclohexane dihydrochloride (AY9944) blocks cholesterol synthesis at a late stage. This leads to a decrease in cholesterol and accumulation of cholesta-5,7-diene-3-beta-ol (7-dehydrocholesterol) in tissues and plasma. 2. The effect of AY9944 on bile salt synthesis in rat liver was studied. The synthesis of conjugated cholic and chenodeoxycholic acids was measured in hepatocytes isolated from rats 2 h, 24 h and 48 h after administration of a single oral dose of AY9944. Production of the two bile salts was inhibited by 70-80% in hepatocytes from AY9944-treated as compared to untreated animals. 3. When AY9944 was added to the incubation medium in vitro of hepatocytes prepared from untreated rats the synthesis of conjugated cholic and chenodeoxycholic acids was not inhibited during the first hour of incubation, probably because of the presence of endogenous cholesterol. However when hepatocytes from untreated rats were incubated with AY9944 for periods of 2 h or longer, bile salt production was decreased markedly. 4. Bile salt synthesis is stimulated when rats are subjected to total biliary drainage for 24 h. The effect of AY9944 on this stimulation was studied. The content of conjugated cholic and chenodeoxycholic acid in the bile was measured as an indicator of bile salt synthesis. 5. In control animals the rate of secretion of biliary bile salts began to increase after about 24 h of total biliary drainage and reached a maximum after approximately 36 h. A single oral dose of AY9944 given 2 h after the start of total biliary drainage delayed and reduced this response. 6. The results show that inhibition of cholesterol synthesis by AY9944 resulting in the replacement of cholesterol by 7-dehydrocholesterol decreases but does not completely prevent bile salt synthesis.