Oliver E. Brown

Resolution and fractionation of macromolecules by isokinetic sucrose density gradient sedimentation

Abstract A technique for the preparation, analysis, and fractionation of macromolecules using a computer-calculated isokinetic sucrose gradient is described. The variables of density or partial specific volume, initial sucrose concentrations, temperature, and rotors are included in the computer analysis. The results of this technique using rat liver ribosomes, polysomes, and ribosomal RNA are presented.

DNA Polymerase Activities Associated with Smooth Membranes and Ribosomes from Rat Liver and Hepatoma Cytoplasm

Deoxyribonucleic acid polymerase activity that prefers denatured DNA primer concentrates with the smooth membranes during sucrose gradient centrifugation of rat liver and hepatoma cytoplasmic extracts. The activity in this fraction is eightfold higher in hepatoma tissue. Deoxyribonucleic acid polymerase activity that prefers native DNA primer concentrates with the free ribosome fraction from both tissues.

Co-fractionation of "nickase" with rat liver DNA polymerase.

Abstract DNA polymerase partially purified from rat liver nuclei or ribosomes contains alkaline endonuclease that produces single strand breaks (nicks) in rat liver nuclear DNA. The nuclease activity is specific for DNA and is active with both native and denatured DNA as substrates. The endonuclease has maximal activity in the presence of 15 mM Mg 2+ at pH 9.0, but is inactive in the presence of Ca 2+ at concentrations that also inactivate the DNA polymerase. The 3′-OH primer ends produced by the endonuclease may account, in part, for the native DNA primer preference of the polymerase observed in vitro .

Rat hepatoma DNA polymerase: Partial analysis of the in vitro DNA product☆

Abstract Earlier reports from this laboratory have demonstrated differences in primer preference for DNA polymerase isolated from normal rat liver vs. hepatoma. This study reports the initial analysis of DNA products formed following a two hour in vitro incubation of hepatoma DNA polymerase with native or denatured DNA. The radioactive products were analyzed by the nitrocellulose filter technique and by chromatography with hydroxylapatite and Bio-Gel A5. With native DNA primer the product had the properties of high molecular weight (ca. 4×10 6 ) double-stranded DNA. With denatured primer the product had properties intermediate between native and denatured DNA with both high and lower molecular weight fragments.

Pyrophosphate as a selective inhibitor of macromolecule synthesis in normal and leukemic leukocytes

Abstract Pyrophosphate (PPi) was shown to inhibit RNA, DNA and protein synthesis of intact human leukocytes and HeLa cells and to inhibit such enzymes as DNA polymerase and amino acyl synthetase. The results with intact leukocytes and the demonstration in inhibition of bacterial growth suggest that PPi is readily transported into the cell and is not efficiently degraded by pyrophosphatase. A possible regulatory role for PPi is suggested.