A procedure for the isolation of deoxyribonucleic acid from micro-organisms

We constructed a series of recombinant genomes which directed expression of the enzyme chloramphenicol acetyltransferase (CAT) in mammalian cells. The prototype recombinant in this series, pSV2-cat, consisted of the beta-lactamase gene and origin of replication from pBR322 coupled to a simian virus 40 (SV40) early transcription region into which CAT coding sequences were inserted. Readily measured levels of CAT accumulated within 48 h after the introduction of pSV2-cat DNA into African green monkey kidney CV-1 cells. Because endogenous CAT activity is not present in CV-1 or other mammalian cells, and because rapid, sensitive assays for CAT activity are available, these recombinants provided a uniquely convenient system for monitoring the expression of foreign DNAs in tissue culture cells. To demonstrate the usefulness of this system, we constructed derivatives of pSV2-cat from which part or all of the SV40 promoter region was removed. Deletion of one copy of the 72-base-pair repeat sequence in the SV40 promoter caused no significant decrease in CAT synthesis in monkey kidney CV-1 cells; however, an additional deletion of 50 base pairs from the second copy of the repeats reduced CAT synthesis to 11% of its level in the wild type. We also constructed a recombinant, pSV0-cat, in which the entire SV40 promoter region was removed and a unique HindIII site was substituted for the insertion of other promoter sequences.

Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells.

After infection and a prolonged incubation period, the scrapie agent causes a degenerative disease of the central nervous system in sheep and goats. Six lines of evidence including sensitivity to proteases demonstrate that this agent contains a protein that is required for infectivity. Although the scrapie agent is irreversibly inactivated by alkali, five procedures with more specificity for modifying nucleic acids failed to cause inactivation. The agent shows heterogeneity with respect to size, apparently a result of its hydrophobicity; the smallest form may have a molecular weight of 50,000 or less. Because the novel properties of the scrapie agent distinguish it from viruses, plasmids, and viroids, a new term "prion" is proposed to denote a small proteinaceous infectious particle which is resistant to inactivation by most procedures that modify nucleic acids. Knowledge of the scrapie agent structure may have significance for understanding the causes of several degenerative diseases.

http://science.sciencemag.org/content/sci/216/4542/136.full.pdf

Novel proteinaceous infectious particles cause scrapie

New M13 vectors for cloning.

Allen, R. J. L. (1940). Biochem. J. 34, 858. Bartlett, P. D., Grimmett, P., Beers, L. & Shelata, S. (1956). J. biol. Chem. 218, 419. Burton, K. (1956). Biochem. J. 62, 315. Caiger, P., Morton, R. K., Jarrett, I. G. & Filsell, 0. H. (1962). Biochem. J. 85, 351. Filsell, 0. H., Jarrett, I. G., Atkinson, M. R., Caiger, P. & Morton, R. K. (1963). Biochem. J. 89, 92. Fishkin, A. F. & Lata, G. F. (1958). Endocrinology, 63, 162. Franz, J. M. & Lata, G. F. (1957). Endocrinology, 60, 602. Gallagher, C. H. (1959). Au8t. J. agric. Res. 10, 854. Gallagher, C. H. & Buttery, S. H. (1959). Biochem. J. 72, 575. Handschumacher, R. E., Mueller, G. C. & Strong, F. M. (1951). J. biol. Chem. 189, 335. Holdsworth, E. S., Neville, E., Nader, C., Jarrett, I. G. & Filsell, 0. H. (1963). Biochim. biophy8. Acta (in the Press). Jarrett, I. G. & Filsell, 0. H. (1958). Aust. J. exp. Biol. med. Sci. 36, 433. Jarrett, I. G. & Filsell, 0. H. (1960). Aust. J. exp. Biol. med. Sci. 38, 347. Jarrett, I. G., Jones, G. B. & Potter, B. J. (1964). Biochem. J. 90, 189. Jarrett, I. G. & Potter, B. J. (1952). Au8t. J. exp. Biol. med. Sci. 30, 207. Kaplan, N. 0. & Lipmann, F. (1948). J. biol. Chem. 174,37. Klein, H. & Lipmann, F. (1953). J. biol. Chem. 203, 101. Krebs, H. A. & Kornberg, H. L. (1957). Ergebn. Physiol. 49, 212. McCandless, E. L. & Dye, J. A. (1950). Amer. J. Physiol. 162, 434. McCarthy, R. D., Shaw, J. C. & Lakshmanan, S. (1958). Proc. Soc. exp. Biol., N. Y., 99, 560. McLean, P. (1958). Biochim. biophys. Acta, 30, 303. Novelli, G. D. (1953). Phy8iol. Rev. 33, 525. Olson, R. E. & Kaplan, N. 0. (1948). J. biol. Chem. 175, 515 Schneider, W. C. (1945). J. biol. Chem. 161, 293. Thompson, M. & Mayer, J. (1962). Amer. J. Physiol. 202, 1005.

Estimation of the molecular weights of proteins by Sephadex gel-filtration.

Covalently closed circular duplex DNA’s are now known to be widespread among living organisms. This DNA structure, originally identified in polyoma viral DNA,1’2 has been assigned to the mitochondrial DNA’s in ox3 and sheep heart,4 in mouse and chicken liver,3 and in unfertilized sea urchin egg.5 The animal viral DNA’s—polyoma, SV40,6 rabbit7 and human8 papilloma—the intracellular forms of the bacterial viral DNA’s—φX174,9, 10 lambda,11, 12 M13,13 and P2214—and a bacterial plasmid DNA, the colicinogenic factor E2,15 have all been shown to exist as closed circular duplexes. Other mitochondrial DNA’s16, 17 and a portion of the DNA from boar sperm18 have been reported to be circular, but as yet have not been shown to be covalently closed.

A dye-buoyant-density method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells.

This communication presents a new method for the study of the molecular weight and partial specific volume of macromolecules, with some illustrations based on results with deoxyribonucleic acid (DNA) and several viruses. The method involves observation of the equilibrium distribution of macromolecular material in a density gradient itself at equilibrium. The density gradient is established by the sedimentation of a low-molecular-weight solute in a solution subject to a constant centrifugal field.

https://www.pnas.org/content/pnas/43/7/581.full.pdf

Equilibrium sedimentation of macromolecules in density gradients.

The interaction of closed circular DNA with intercalative dyes. I. The superhelix density of SV40 DNA in the presence and absence of dye

The major part of the DNA from polyoma virus has been shown to consist of circular base-paired duplex molecules without chain ends1–3 The intertwined circular form accounts for the ease of renaturation4 of this DNA and the failure of the strands to separate in strand-separating solvents1–3

Jerome Vinograd

Papers

A dye-buoyant-density method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells.

Equilibrium sedimentation of macromolecules in density gradients.

The interaction of closed circular DNA with intercalative dyes. I. The superhelix density of SV40 DNA in the presence and absence of dye

The twisted circular form of polyoma viral DNA.

The determination of density distributions and density gradients in binary solutions at equilibrium in the ultracentrifuge1