William B. Upholt

A buoyant method for the determination of the superhelix density of closed circular DNA.

Abstract A convenient method for the determination of the superhelix density of a closed circular DNA has been developed and tested with a series of closed DNAs with different superhelix densities and molecular weights. The method is based on the superhelix density-dependent binding affinity of closed DNA for the intercalating dye, ethidium bromide. The superhelix density is calculated from the observed separation between nicked and closed DNA in a cesium chlorideethidium bromide density gradient centrifuged under specified conditions in the preparative ultracentrifuge. The linear relation between the separations and the superhelix density obtained in this study is in good agreement with that predicted by Bauer & Vinograd (1970b).

Sedimentation velocity behavior of closed circular SV40 DNA as a function of superhelix density, ionic strength, counterion and temperature

Abstract Ten closed SV40 DNAs with superhelix densities (superhelical turns per ten base pairs) ranging from −0.007 to −0.085 have been prepared. This family of DNAs has been used to examine the effects of superhelix density on the sedimentation velocity behavior of closed SV40 DNA at high ionic strength in cesium chloride and sodium chloride solutions. The sedimentation coefficient increases as the absolute value of the superhelix density, ¦ σ o ¦, rises from a low value to 0.019, then decreases to a local minimum at 0.035, and finally increases steadily as ¦ σ o ¦ rises to 0.085. Examination of several of these DNAs in the electron microscope has suggested a plausible explanation for the observed non-monotonic variation of S with σ o . The sedimentation velocity-ethidium bromide titrations of these DNAs (Gray, Upholt & Vinograd, 1971) have been converted from the primary S 20 0 .∗ versus c data, in which S 20 0 .∗ is the standard sedimentation coefficient still uncorrected for the buoyant effect of bound ethidium chloride and c is the free ethidium bromide concentration, to the more meaningful S 20, w 0 versus σ form, with the aid of the coefficients in the expression for the free energy of superhelix formation developed by Bauer & Vinograd (1970a). The resultant curves form a family that is approximately superimposable on the curve for S 20, w 20 versus σ 0 in the absence of ethidium bromide. Similarly transformed sedimentation velocitydye titrations for viral PM2 DNA (6 × 10 6 daltons) and a high superhelix-density λb 2 b 5 c DNA (25 × 10 6 daltons) have the same general character and contain both a local maximum and a local minimum. The results of a study of the dependence of the sedimentation coefficient of selected SV40 DNAs upon ionic strength, the nature of the cation and temperature are consistent with the previously reported effects of these variables (Wang, 1969a) on the rotation angle of the base pairs along the helix axis.

N-Quaternary Compounds. Part LVI. 3-Hydroxyquinoline-2(lH)-thiones and Their N-Vinylation.

Synthese de quinoleinediol-2,3 et conversion en dihydro-1,2 thiazolo [3,2-a] quinoleinioolate-4; scission de cette betaine en N- et S-vinyl quinoleines isomeres

Cell-Free Synthesis of Cartilage Specific Proteins

Publisher Summary Cartilage is characterized by the presence of extensive extracellular matrix largely composed of chondroitin sulfate proteoglycan, type II collagen, and hyaluronic acid. This chapter describes the use of differentiating cultures to study the expression of cartilage specific genes. Antisera have been raised in rabbits against proteoglycan core protein (anti-PGS) prepared by the treatment of proteoglycan subunit with testicular hyaluronidase to remove chondroitin sulfate chains. These antisera have been used to study nascent polypeptide chains on polysomes obtained from differentiating cultures as well as the products synthesized in the wheat germ cell-free protein synthesizing system under the direction of polysomes or mRNA.