RESUMO
Genetic instability due to stalled replication forks is thought to underlie a number of human diseases, such as premature ageing and cancer susceptibility syndromes. In addition, site-specific stalling occurs at some genetic loci. A detailed understanding of the topology of the stalled replication fork gives a valuable insight into the causes and mechanisms of replication stalling. The method described here allows mapping of the position of the 3'-end of the nascent leading or lagging strand at the replication fork, stalled at a site-specific barrier. The replicating DNA is purified, digested with restriction enzymes, and enriched by BND-cellulose chromatography. The DNA is separated on a sequencing gel, transferred to a membrane, and hybridised to a strand-specific probe. The data obtained using this method allow determining the position of the 3'-end of the nascent strand at a stalled fork with a one-nucleotide resolution.
Assuntos
Replicação do DNA/genética , Mapeamento de Nucleotídeos/métodos , Sítios de Ligação/genética , Cromatografia DEAE-Celulose , DEAE-Celulose/análogos & derivados , DNA Fúngico/biossíntese , DNA Fúngico/genética , DNA Fúngico/isolamento & purificação , Eletroforese em Gel de Poliacrilamida , Técnicas de Sonda Molecular , Hibridização de Ácido Nucleico , Schizosaccharomyces/genética , Schizosaccharomyces/crescimento & desenvolvimento , Schizosaccharomyces/metabolismoRESUMO
Glycogen phosphorylase (GPase) from the body wall of the lugworm Arenicola marina (Annelida, Polychaeta) probably exists as a phospho-dephospho hybrid (GPase ab). The hybrid was identified by phosphorylation of purified lugworm GPase b (unphosphorylated form) with rabbit muscle GPase kinase and [gamma-32P]ATP. The completeness of phosphorylation was checked on DEAE-Sephacel. Only one GPase form was eluted. Its 32P incorporation was determined to 0.52 +/- 0.08 mol 32P/100,000 x g protein (n = 4). This GPase ab produced by in vitro phosphorylation has shown similar dependences on AMP and caffeine as GPase extracted from the body wall of the lugworm. Its reversible conversion with endogenous phosphatase and kinase to GPase b has also been demonstrated while a completely phosphorylated form (GPase a) was not detected neither in vivo nor in vitro. Lugworm GPase ab has shown a 2.4-fold higher specific activity as GPase b. The Km for P(i) was 16 mmol/l in absence and 13 mmol/l in presence of AMP. Half maximum activation by AMP was reached at 9 mumol/l. IMP up to 10 mmol/l did not activate and ATP up to 4 mmol/l did not inhibit GPase ab in absence of AMP.
Assuntos
Hipóxia/enzimologia , Fosforilases/fisiologia , Poliquetos/enzimologia , Monofosfato de Adenosina/metabolismo , Monofosfato de Adenosina/farmacologia , Animais , Soluções Tampão , Cromatografia por Troca Iônica , DEAE-Celulose/análogos & derivados , Ativação Enzimática/efeitos dos fármacos , Inosina Monofosfato/farmacologia , Cinética , Radioisótopos de Fósforo , Fosforilases/antagonistas & inibidores , Fosforilases/isolamento & purificação , Fosforilação , Fluoreto de Sódio/farmacologiaRESUMO
The ascitic form of a chemically-induced pancreatic ductal adenocarcinoma in the Syrian golden hamster was very bloody and indistinguishable from blood macroscopically. Unlike blood, the bloody fluid remained unclotted at room temperature. To explore the possibility of presence of anticoagulants, we mixed 40% cell-free fluid with 60% normal human plasma and tested the clottability of the mixture with standard techniques. Plasma containing the fluid showed markedly prolonged activated partial thromboplastin time (APTT), thrombin time (TT) and recalcification time (RCT), and normal prothrombin time (PT) and reptilase time (RT). Comparing the prolongation of APTT of samples containing the fluid to those containing a commercial heparin, the fluid contained an anticoagulant activity equivalent to 0.436 +/- 0.03 unit heparin per ml (mean +/- SEM, n = 14). In addition to prolonging the APTT, TT and RCT, the fluid also inhibited the clotting and amidolytic activities of thrombin. "Heparsorb" had nearly completely neutralized the anticoagulant activity in fluid samples, while protamine sulfate was only partially effective. Incubation of fluid with pronase or phospholipase did not affect its anticoagulant activity; incubation with heparinase had only a minimal effect. Electrophoresis of an alkali digested fluid on cellulose acetate revealed the presence of heparan sulfate. The native ascitic fluid also contained other hemostatic components including platelets, fibrinogen and antithrombin III, but their concentrations were much lower than in blood. Apparently, heparan sulfate in the neoplastic effusion is largely responsible for the bloody ascites tumor remaining unclotted.