Kasper Zechel

On the resolution of polypeptides by isoelectric focusing in polyacrylamide gels in the presence of urea and nonidet-p40.

Abstract Three model substances were used to test the resolving power of isoelectric focusing in polyacrylamide gels in the presence of 9 m urea and 2% Nonidet-P40: (1) The α subunit of RNA polymerase from Escherichia coli modified by a single covalently attached adenosine 5′-diphosphate-ribose residue can be clearly resolved from the unmodified subunit. (2) The α subunit of RNA polymerase from a mutant of E. coli in which a single leucine residue is replaced by a histidine residue can be resolved from the wild-type subunit. (3) Muscle actin presumably modified by carboxymethylation with iodoacetate at only one cysteine residue can be separated from the unmodified polypeptide.

Initiation of DNA Synthesis by RNA

Up to the present one of the basic problems for the understanding of DNA replication is how the synthesis of DNA chains is initiated in vivo. So far none of the known DNA polymerases of prokaryotic or eukaryotic origin has been shown to initiate the synthesis of a DNA chain de novo. All DNA polymerases need a primer, which, in its simplest form, may be an oligoribo- or an oligodeoxyribonucleotide having a free 3′-hydroxyl end (Karkas and Chargaff, 1966). This primer terminus can be extended at its 3′-OH end by addition of new nucleotides as directed by the polynucleotide template to which the primer is linked through hydrogen bonds. Although the requirement for a primer has always been realized, earlier studies on DNA synthesis focused more on the mechanism of chain elongation than on the initiation process.

An inexpensive, disposable minispinner for the cultivation of mammalian cells in suspension

Abstract An inexpensive minispinner vessel for the suspension culture of mammalian cell lines in 2 to 20 ml of culture medium is described. It is made from a commereial glass or low-pressure polyethylene scintillation vial equipped with an easy-to-build stir bar assembly suspended from the cap.

Stability differences of muscle F-actin in formamide in the presence of Mg2+ and Ca2+

Muscle G-actin was polymerized by addition of 2 mM Mg2+ or 2 mM Ca2+. Subsequent addition of formamide reduced the specific viscosity of the polymer solution. However, kinetic analysis of this reduction in the presence or absence of 0.1 M KCl revealed differences between F-actin formed in the presence of Mg2+ and F-actin formed in the presence of Ca2+. In the presence of Mg2+ the viscosity dropped instantaneously, reaching within minutes a steady-state level that was constant for many hours. In contrast, in the presence of Ca2+ the high-shear viscosity continued to decrease slowly after an initial drop, and it could take hours until a quasi-equilibrium was obtained. The time was dependent on both formamide and protein concentration. Addition of formamide increased the critical actin concentration in the presence of Ca2+, but not in the presence of Mg2+. This is taken as evidence that in the presence of Ca2+, but not in the presence of Mg2+, formamide causes partial depolymerization of F-actin.