Dean L. Engelhardt

Peptide coding capacity of polysomal and non-polysomal messenger RNA during growth of animal cells

Abstract The major peptides encoded by cytoplasmic RNA preparation translated in vitro in extracts of wheat germ were displayed by two-dimensional electrophoresis on polyacrylamide gels. With this assay system polysomal and non-polysomal RNA preparations were found to differ in coding capacity. These differences tended to be greater in RNA preparations from stationary phase cells than in those from exponential phase cells. These differences were maintained when the concentrations of potassium and magnesium were above or below the optimal concentrations for incorporation. Most of the messenger RNA activities preferentially in the post-polysomal region could be driven into the polysomal region in the presence of cycloheximide. We conclude that these measurements are valid measurements of concentrations of individual functional mRNA species in these RNA preparations.

Effects of the tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate on newly synthesized proteins in mouse epidermis

Abstract We have analyzed the effects of treatment of mouse epidermis with the potent tumor promoter TPA on the profile of newly synthesized proteins. TPA was applied to the skin of the intact mouse, and either 3 or 24 hr later skin fragments were pulse-labeled in vitro with 35 S-methionine for 4 hr. The epidermal proteins were extracted and separated by two-dimensional gel electrophoresis. Over 200 individual proteins were resolved in acidic gels. At least 10 of these showed major (by a factor of 5 or more) increases or decreases in response to TPA; eight of these appear to be keratin proteins. Two-dimensional gel profiles of basic proteins synthesized by mouse epidermis resolved over 100 individual proteins. Only one of these showed a significant change in response to TPA. This 41 kd protein increased more than 100-fold within 24 hr after the application of TPA. Treatment of mouse skin with mezerein, a plant diterpene structurally related to TPA, produces an almost identical change in the pattern of proteins produced. Four agents that induce hyperplasia but are not potent tumor promoters, ethylphenylpropiolate, acetic acid, turpentine oil and the Ca ++ ionophore A23187, modulate the synthesis of only three of the keratin proteins. Thus the changes in protein profiles induced by TPA and mezerein are not simply the consequence of hyperplasia. In addition, application to mouse skin of a glucocorticoid that is a potent inhibitor of tumor promotion inhibits most of the changes in protein profiles induced by TPA. Taken together, these results indicate that TPA and mezerein induce early and marked changes in the profile of specific epidermal proteins. It seems likely that some of these changes are directly related to the process of tumor promotion.

Alterations in the protein synthetic apparatus of cells infected with herpes simplex virus

Abstract The effect of infection with herpes simplex virus (HSV) on the rate of protein synthesis, the distribution of ribosomes, and the time required to complete nascent peptides (transit time) in either actively growing or stationary phase Vero cells was examined. It was shown that the rate of protein synthesis per polysomal ribosome decreased continuously throughout the early portion of the infection cycle. This occurred despite an increase in the accumulation of ribosomes in polysome-like structures that accompanies the onset of synthesis of HSV-specified proteins. Transit-time measurements demonstrated that the rate of polypeptide elongation was not altered after infection. These data are discussed in light of a model that suggests that polysome-like structures are present but not functioning (or functioning very poorly) in HSV infected-cells.