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Biomedical subjects

R K Merrifield

Publications and source records attributed to R K Merrifield.

12 recordsLinked to original sources
PubMed1978

The rate of hydrolysis of methyl phosphotriesters in DNA under conditions used in alkaline sucrose gradients.

Methyl phosphotriesters have been introduced into DNA, in vitro, by reaction with N-methyl-N-nitrosourea and the rate of degradation in alkali has been followed by measurements of the mean sedimentation coefficient using an analytical ultracentrifuge. In 0.3 M NaOH/0.7 M NaCl, a solution commonly used in alkaline sucrose gradient experiments, hydrolysis of the methyl phosphotriesters present is complete after 15 h at 20 degrees C, or after 2--3 h at 37 degrees C. In addition to breaks formed by the latter reaction there was a continuous background degradation of the DNA giving rise to 6.3 and 63 breaks/10(6) nucleotides per h at 20 degrees and 37 degrees C, respectively. The problem of obtaining quantitative data on phosphotriester concentrations from results of alkaline sucrose gradient experiments has been discussed.

Centrifugation, Density Gradient↗
PubMed1976

An assay for phosphotriester formation in the reaction of alkylating agents with deoxyribosenucleic acid in vitro and in vivo.

Preliminary studies in vitro using bacteriophage T7-DNA have shown that breaks formed in the DNA on the alkaline hydrolysis of apurinic sites and phosphotriesters can be distinguished from each other by measuring the extent of degradation of the DNA immediately after adding NaOH to 0.1 M and after incubating for 1 h in 0.5 M NaOH. This method has then been applied to the study of the formation and stability of phosphotriesters invivo. Methyl phosphotriesters formed in liver DNA following injection of mice with N-methyl-N-nitrosourea (MNUA) disappear with time (50% in 4-5 days). The concentration of ethyl phosphotriesters in liver DNA formed by injecting mice with N-ethyl-N-nitrosourea (ENUA) does not appear to decrease with time. Results of experiments on injecting methyl methane-sulphonate (MMS), ethyl methanesulphonate (EMS) and dimethyl sulphate (DMS) are also reported. The method described does not require the use of radioactively labelled reagents.

Animals↗
PubMed1974

The reaction of alkylating agents with bacteriophage R17. Biological effects of phosphotriester formation.

The extent of biological inactivation and of the degradation of the RNA after reaction of bacteriophage R17 with ethyl methanesulphonate, isopropyl methanesulphonate and N-ethyl-N-nitrosourea was studied. Formation of breaks in the RNA chain probably results from hydrolysis of phosphotriesters formed in the alkylation reactions. Near neutral pH the ethyl and isopropyl phosphotriesters are sufficiently stable for the kinetics of the hydrolysis reaction to be followed. Results indicate that the rate of hydrolysis increases rapidly as the pH is raised. The evidence shows that a phosphotriester group does not itself constitute a lethal lesion. The extent of phosphotriester formation by the different agents is discussed in terms of reaction mechanism.

Alkylating Agents↗