scholarly journals EARLY EVENTS IN LYMPHOCYTE TRANSFORMATION BY PHYTOHEMAGGLUTININ

1972 ◽  
Vol 53 (3) ◽  
pp. 635-641 ◽  
Author(s):  
Beatriz G. T. Pogo
Keyword(s):  
Protein Synthesis ◽  
Rna Polymerase ◽  
Lymphocyte Transformation ◽  
Enzymatic Activities ◽  
Initial Increase ◽  
Nuclear Fraction ◽  
Isolated Nuclei ◽  
Polymerase Iii ◽  
Inhibition Of Protein Synthesis ◽  
Polymerase I

The DNA-dependent RNA polymerase activities of isolated nuclei from lymphocytes were examined after stimulation with phytohemagglutinin (PHA). The nuclear fraction was prepared with Mg++ or Mn++ to distinguish between polymerase I (nucleolar) and polymerase II (nucleoplasmic). Distinction between polymerases II and III was obtained by the addition of α-amanitin to the reaction mixture. The results indicated that within 15 min after exposure to PHA the activity of polymerase I increased. Polymerase II activity increased after 1 hr. The enhancement was linear for 6 hr and then leveled off for the subsequent 48 hr. Small increase in polymerase III activity was observed at 48 hr. Inhibition of protein synthesis at the time of exposure to PHA did not prevent the increase in activities during the initial 6 hr. These results imply that the initial increase in enzymatic activities is dependent upon preexisting polymerase molecules and/or factors.

scholarly journals Polymerase III transcription factor B activity is reduced in extracts of growth-restricted cells.

1988 ◽  
Vol 8 (2) ◽  
pp. 1001-1005 ◽  
Author(s):  
J Tower ◽  
B Sollner-Webb
Keyword(s):  
Rna Polymerase ◽  
Stationary Phase ◽  
Rna Polymerase Iii ◽  
Control Cell ◽  
Rna Polymerase I ◽  
Rdna Transcription ◽  
Down Regulation ◽  
Cell Extracts ◽  
Polymerase Iii ◽  
Polymerase I

Extracts of cells that are down-regulated for transcription by RNA polymerase I and RNA polymerase III exhibit a reduced in vitro transcriptional capacity. We have recently demonstrated that the down-regulation of polymerase I transcription in extracts of cycloheximide-treated and stationary-phase cells results from a lack of an activated subform of RNA polymerase I which is essential for rDNA transcription. To examine whether polymerase III transcriptional down-regulation occurs by a similar mechanism, the polymerase III transcription factors were isolated and added singly and in pairs to control cell extracts and to extracts of cells that had reduced polymerase III transcriptional activity due to cycloheximide treatment or growth into stationary phase. These down-regulations result from a specific reduction in TFIIIB; TFIIIC and polymerase III activities remain relatively constant. Thus, although transcription by both polymerase III and polymerase I is substantially decreased in extracts of growth-arrested cells, this regulation is brought about by reduction of different kinds of activities: a component of the polymerase III stable transcription complex in the former case and the activated subform of RNA polymerase I in the latter.

Polymerase III transcription factor B activity is reduced in extracts of growth-restricted cells

1988 ◽  
Vol 8 (2) ◽  
pp. 1001-1005 ◽  
Author(s):  
J Tower ◽  
B Sollner-Webb
Keyword(s):  
Rna Polymerase ◽  
Stationary Phase ◽  
Rna Polymerase Iii ◽  
Control Cell ◽  
Rna Polymerase I ◽  
Rdna Transcription ◽  
Down Regulation ◽  
Cell Extracts ◽  
Polymerase Iii ◽  
Polymerase I

Extracts of cells that are down-regulated for transcription by RNA polymerase I and RNA polymerase III exhibit a reduced in vitro transcriptional capacity. We have recently demonstrated that the down-regulation of polymerase I transcription in extracts of cycloheximide-treated and stationary-phase cells results from a lack of an activated subform of RNA polymerase I which is essential for rDNA transcription. To examine whether polymerase III transcriptional down-regulation occurs by a similar mechanism, the polymerase III transcription factors were isolated and added singly and in pairs to control cell extracts and to extracts of cells that had reduced polymerase III transcriptional activity due to cycloheximide treatment or growth into stationary phase. These down-regulations result from a specific reduction in TFIIIB; TFIIIC and polymerase III activities remain relatively constant. Thus, although transcription by both polymerase III and polymerase I is substantially decreased in extracts of growth-arrested cells, this regulation is brought about by reduction of different kinds of activities: a component of the polymerase III stable transcription complex in the former case and the activated subform of RNA polymerase I in the latter.

scholarly journals ISOLATION AND BIOCHEMICAL CHARACTERIZATION OF NUCLEI FROM CHICK EMBRYO LIVER

1971 ◽  
Vol 50 (2) ◽  
pp. 385-398 ◽  
Author(s):  
Genevieve S. Incefy ◽  
Attallah Kappas
Keyword(s):  
Electron Microscope ◽  
Chick Embryo ◽  
Rna Polymerase ◽  
Biochemical Characterization ◽  
Nuclear Fraction ◽  
Dna Recovery ◽  
Isolated Nuclei ◽  
Sucrose Solutions ◽  
Embryo Liver

A procedure is described for the isolation of enzymatically active nuclei from chick embryo liver. It consists of the homogenization of the pooled tissue in 0.32 M sucrose-3 mM MgCl2 followed by a slow centrifugation. The resulting nuclear pellet is then purified further in a discontinuous density gradient composed of sucrose solutions containing Mg2+ ions, the lower portion of the gradient being 2.2 M sucrose-1 mM MgCl2. Based on DNA recovery, the nuclear fraction isolated by the procedure described contained an average of 62% of the nuclei in the original filtered homogenate. Light and electron microscope examinations showed that 90% of the isolated nuclei were derived from hepatocytes. They appeared intact with well preserved nucleoplasmic and nucleolar components, nuclear envelope, and pores. The isolated nuclei were quite pure, having a very low level of cytoplasmic contamination as indicated by cytoplasmic enzyme marker activities and electron microscope studies. The nuclear fraction consisted of 19.9% DNA, 6.2% RNA, 74% protein, the average RNA/DNA ratio being 0.32. Biosynthetic activities of the two nuclear enzymes NAD-pyrophosphorylase and DNA-dependent RNA polymerase were preserved. The specific activities of these enzymes were: NAD-pyrophosphorylase, 0.049 µmoles nicotinamide adenine dinucleotide (NAD) synthesized/min per mg protein; Mg2+ activated RNA polymerase, 4.3 µµmoles UMP-2-C14 incorporated into RNA/µg DNA per 10 min; and Mn2+-(NH4)2SO4 activated RNA-polymerase, 136 µµmoles UMP-2-C14 incorporated into RNA/µg DNA per 45 min.

scholarly journals Cross Talk between tRNA and rRNA Synthesis inSaccharomyces cerevisiae

2001 ◽  
Vol 21 (1) ◽  
pp. 189-195 ◽  
Author(s):  
Jean-François Briand ◽  
Francisco Navarro ◽  
Olivier Gadal ◽  
Pierre Thuriaux
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Rna Polymerase I ◽  
Rrna Synthesis ◽  
Northern Blot Hybridization ◽  
Permissive Temperature ◽  
Wild Type ◽  
Polymerase Iii ◽  
Polymerase I

ABSTRACT Temperature-sensitive RNA polymerase III (rpc160-112and rpc160-270) mutants were analyzed for the synthesis of tRNAs and rRNAs in vivo, using a double-isotopic-labeling technique in which cells are pulse-labeled with [33P]orthophosphate and coextracted with [3H]uracil-labeled wild-type cells. Individual RNA species were monitored by Northern blot hybridization or amplified by reverse transcription. These mutants impaired the synthesis of RNA polymerase III transcripts with little or no influence on mRNA synthesis but also largely turned off the formation of the 25S, 18S, and 5.8S mature rRNA species derived from the common 35S transcript produced by RNA polymerase I. In the rpc160-270mutant, this parallel inhibition of tRNA and rRNA synthesis also occurred at the permissive temperature (25°C) and correlated with an accumulation of 20S pre-rRNA. In the rpc160-112 mutant, inhibition of rRNA synthesis and the accumulation of 20S pre-rRNA were found only at 37°C. The steady-state rRNA/tRNA ratio of these mutants reflected their tRNA and rRNA synthesis pattern: therpc160-112 mutant had the threefold shortage in tRNA expected from its preferential defect in tRNA synthesis at 25°C, whereas rpc160-270 cells completely adjusted their rRNA/tRNA ratio down to a wild-type level, consistent with the tight coupling of tRNA and rRNA synthesis in vivo. Finally, an RNA polymerase I (rpa190-2) mutant grown at the permissive temperature had an enhanced level of pre-tRNA, suggesting the existence of a physiological coupling between rRNA synthesis and pre-tRNA processing.

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