Regression of myocardial hypertrophy. II. RNA synthesis and RNA polymerase activity

Muscle cell RNA synthesis and RNA polymerase activity were measured during the regression of pressure overload induced myocardial hypertrophy in the rat. Peak increases in RNA synthesis occurred during the first 48 h after aortic constriction followed by a delayed increase in RNA polymerase activity which reached a maximum at 72 h. Following relief of pressure overload at 1, 2, 3 or 10 days there was a rapid decline in RNA synthesis and RNA polymerase activity. Both parameters returned to the level of the sham operated rats within 48 h. Neither RNA synthesis nor RNA polymerase activity decreased below the control levels. In view of previous studies demonstrating no increase in RNA degradation during the regression of myocardial hypertrophy, the present data support the concept that regression of myocardial RNA is due to a decrease in synthesis rather than an increase in degradation.     

Glucocorticoid regulation of rat thymus RNA polymerase activity: the role of RNA and protein synthesis.

Treatment of rat thymus cells with the glucocorticoids cortisol and dexamethasone resulted in the stimulation of RNA polymerase B activity within 10 min of steroid addition. This early effect was followed by the inhibition of both RNA polymerase A and B activities. These effects were glucocorticoid-specific and were inhibited by the antiglucocorticoid cortexolone. The inhibitory effect of dexamethasone on RNA polymerase A activity was abolished by prior treatment of the cells with alpha-amanitin, cordycepin or cycloheximide, but cycloheximide was only capable of inhibiting the steroid effect measured at 3 h if added within 10--20 min after steroid addition. Cycloheximide had no effect on the steroid-mediated inhibition of RNA polymerase B activity. Control RNA polymerase A activities were unaffected by the presence of inhibitors of RNA and protein synthesis. It is concluded that the inhibition of ribosomal RNA synthesis by glucocorticoids is dependent on protein synthesis, but that basal RNA polymerase A activity in rat thymus cells is not stringently coupled to protein synthesis.     

DNA synthesis involving a complexes form of DNA polymerase I in extracts of Escherichia coli.

DNA polymerase I (EC 2.7.7.7; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase) has been recovered as a complex of about 390,000 molecular weight. The complex displays an ATP-stimulated DNA-synthesizing activity that prefers native to heat-denatured DNA. Genetic evidence indicates that the recBC enzyme is associated with the polymerase in the complex. Preliminary evidence for complexes involving DNA polymerases II and III is also presented.     

Fidelity of synthesis of preribosomal RNA in isolated nucleoli and nucleolar chromatin.

Comparisons were made of the T1 ribonuclease digests of 32P-labeled nucleolar 45S RNA of intact Novikoff hepatoma cells and the RNA synthesized in vitro by isolated nucleoli. Approximately 200 oligonucleotide spots were found in the two-dimensional chromatogram of 45S nucleolar RNA labeled in vivo, which includes fragments of 18S and 28S rRNA and nonconserved spacer regions; four spots containing 2'-O-methyl nucleotides were not found in the corresponding pattern of RNA labeled in vitro. This high degree of fidelity was retained in the patterns of spots from the RNA produced with nucleolar chromatin as template. This specific expression of rDNA was lost when the nucleolar chromatin was completely deproteinized. Specific spots found in the control patterns were absent and many nonspecific oligonucleotides were found to be labeled. A similar nonspecific chromatogram pattern was found when nucleolar chromatin was transcribed with RNA polymerase (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) of Escherichia coli. These results show that specificity of genetic expression in vitro of isolated chromatin of eukaryotic systems is dependent on the chromatin-associated proteins and the type of RNA polymerase present.     

Mechanism of DNA polymerase I: exonuclease/polymerase activity switch and DNA sequence dependence of pyrophosphorolysis and misincorporation reactions.

Mechanistic features of several processes involved in the idling-turnover reaction catalyzed by the large (Klenow) fragment of Escherichia coli DNA polymerase I have been established. The exonuclease----polymerase activity switch involved in the excision/incorporation mode of idling-turnover occurs without an intervening dissociation of the enzyme from its DNA substrate. Comparative studies on the pyrophosphorolysis kinetics of related DNA substrates indicate a significant dependence of the reaction rate upon the DNA sequence within the duplex region upstream of the primer-template junction. Finally, a gel electrophoretic analysis of the products of the idling-turnover reaction has provided direct evidence for an alternative DNA sequence-dependent misincorporation/excision pathway.     

The relationship between cell size, the activity of DNA polymerase alpha and proliferative activity in human diploid fibroblast-like cell cultures

In kinetic studies with human diploid fibroblast-like (HDFL) cells carried out in heterokaryons and in monokaryons, we have observed a first-order relationship between the level or concentration of DNA polymerase alpha and the rate of initiation of new rounds of DNA synthesis. Because cell size is inversely proportional to the concentration of DNA polymerase alpha and presumably other replication factors, it is inversely related to the initiation of new rounds of DNA synthesis. An inverse relationship between cell size and clonogenic activity was also observed in both unsorted HDFL cells and in HDFL cells sorted on the basis of size. Experimental enlargement of cells by serum deprivation at low density resulted in changes in colony-forming ability that would be predicted by these studies. A causal relationship between the observed increase in cell size with advancing passage level and the loss of proliferative activity is suggested by these studies; in addition, cell size may be a useful biophysical marker for cellular aging.