Transcriptionally active RNA polymerases from Morris hepatomas and rat liver. Elucidation of the mechanism for the preferential increase in the tumour RNA polymerase I

Duceman, Barry W.; Jacob, Samson T.

doi:10.1042/bj1900781

Cited by 26 publications

(11 citation statements)

References 32 publications

(34 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…It is known that tumors have a higher rate of rRNA precursor synthesis; compared to liver, the nucleolus of the Walker tumor has a 10-fold higher rate of 45S RNA synthesis (22,23). Interestingly, in the Morris hepatoma 3924A from which the nuclear extract was prepared for the present studies, the level of chromatin-associated RNA polymerase I is about 10 times higher than that of the corresponding enzyme from normal liver (11). Although the RNA synthetic activity may not always correlate with RNA polymerase I activity (7,24), alteration in the rate of rRNA synthesis might at least, in part, be due to modifications in the regulatory factors associated with RNA polymerase I, which may exert a positive role in accurate transcription of rDNA.…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Accurate initiation of rat ribosomal RNA gene transcription using a fractionated nuclear extract from normal liver and a hepatoma.

Kurl¹,

Jacob²

1985

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

Nuclear extracts obtained from normal rat liver and from Morris hepatoma 3924A were fractionated by DEAE-Sephadex chromatography. The fraction eluted with 175 mM (NH4)2SO4 (DE-B), which contains >90% of RNA polymerase I activity, supported accurate transcription of cloned rat rDNA. A similar fraction obtained from the cytosol had all of the factors required for rDNA transcription. However, its transcriptional activity was at most one-sixth that of the corresponding nuclear fraction, as determined by the amount of protein needed to produce a similar quantity of the transcript. Unfractionated nuclear or cytosol preparations did not yield an accurate transcript. Optimal KCI and magnesium concentrations for rDNA transcription were 60 mM and 5-7.5 mM, respectively. The extent of transcriptional activity was in the following order: hepatoma nuclear fraction DE-B > whole cell extract derived from rat mammary adenocarcinoma cells >> normal liver fraction DE-B. The hepatoma preparation produced at least 10 times the amount of transcript produced by the corresponding liver nuclear preparation. Transcriptional activity was proportional to the levels of RNA polymerase I and to the rate of rRNA synthesis in these tissues.The development of cell-free systems (1-3) has facilitated studies on the transcription of specific genes by class I, II, and III RNA polymerases (reviewed in refs. 4 and 5). These systems have provided the means to investigate the promoter regions for several genes and to identify the transcriptional regulatory factors. Either a whole-cell extract (3) or an S-100 fraction (2) has been used for these studies. A major disadvantage with cell extracts is the large number of cells needed to identify and characterize factors involved in the transcriptional process (6). This problem could be circumvented if tissue extracts could be prepared that would accurately transcribe purified genes by specific RNA polymerases. Methods that are used for preparing whole-cell extracts have not been extended to tissues probably because of the relatively higher ribonuclease levels in tissues.Since transcription occurs in the nucleus, most factors required for transcription must be found in this organelle. Consequently, a transcriptional system derived from the nuclei will have a distinct advantage over other systems. Over many years, the enzymology and subunit structure of RNA polymerase I and the modifications of its activity and/or amount in response to physiological and pathological stimuli have been studied in our laboratory (7-13). Recently, we have initiated studies on the identification of accessory factors required for transcription of rDNA by class I RNA polymerase. In the course of these studies, we prepared a nuclear extract from tissues that, upon a single chromatographic step, yields a fraction capable of accurate transcription of rDNA. The activity of the fractionated nuclear extract with respect to rDNA transcription was proportional to the rRNA synthetic activity of the tissue from which the initial nuclea...

show abstract

Section: Discussionmentioning

confidence: 99%

“…Various fractions were tested for their ability to transcribe cloned rat rDNA linearized with Xho I (11). The assay conditions were those described by Miller and SollneriWebb (19), except that a-amanitin was used at a concentration of 150 4g/ml, which completely inhibited RNA polymierases II atnd III.…”

Section: Methodsmentioning

confidence: 99%

Accurate initiation of rat ribosomal RNA gene transcription using a fractionated nuclear extract from normal liver and a hepatoma.

Kurl¹,

Jacob²

1985

Proc. Natl. Acad. Sci. U.S.A.

Self Cite

View full text Add to dashboard Cite

show abstract

“…Casein kinase II-mediated phosphorylation of topoisomerases I (20, 21) and II (17) has been shown to increase catalytic activity -3-fold, and in both instances only serine is phosphorylated. Enzyme activities of RNA polymerases I (24) and II (25) and poly(A) polymerase (26) have been shown to increase 2-to 5-fold, 3-fold, and 3-to 8-fold, respectively, after phosphorylation. Processivity, or chain elongation, is increased for RNA polymerase I (27), while initiation frequency is increased for poly(A) polymerase (26) and RNA polymerase II (28).…”

Section: Discussionmentioning

confidence: 99%

Phosphorylation of a high molecular weight DNA polymerase alpha.

Donaldson¹,

Gerner²

1987

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Anti-human DNA polymerase a murine IgG SJK-287-38 [Tanaka, S., Hu, S.-Z., Wang, T. S. These data suggest that fluctuation in DNA polymerase a activity is not due to changes in DNA polymerase a protein content, or nuclear translocation, but that another mode of enzyme regulation, such as posttranslational modification, might be involved.Detailed investigations of posttranslational alterations of DNA polymerase a have not been carried out for several reasons. DNA polymerase a shows heterogeneity with respect to both size (4, 5) and oligoenzyme association (6). Furthermore, DNA polymerase a exists in low abundance, with estimates of 20,000-60,000 molecules per cell (7). Recent development of immunological probes to DNA polymerase a provides a means to ask whether posttranslational phosphorylation/dephosphorylation modifications of this enzyme occur in vivo. In this study, we asked whether DNA polymerase a was phosphorylated in exponentially growing rat embryonic cells expressing pp60src, the tyrosine-specific protein kinase and transforming function of Rous sarcoma virus and, if so, whether the enzyme activity was altered by phosphorylation/dephosphorylation reactions. MATERIALS AND METHODSCell Culture. Rat-l(tsLA24/RSV) cells (rat embryonic fibroblasts infected with a Rous sarcoma virus mutant that is temperature-sensitive for the transformed phenotype) were grown in monolayer culture at the permissive temperature (35°C) as described (8). Hybridoma cells producing monoclonal antibody to DNA polymerase a (SJK-287-38) were obtained from the American Type Culture Collection and grown as directed.For labeling exponentially growing cultures with [32P]orthophosphate, cells were seeded at 6 x 106 cells per 150-mm dish (Falcon). Forty hours later, the cells were labeled for 6 hr at 350C with [32P]orthophosphate (HCl-free, ICN) at 1.0 mCi/ml (1 Ci = 37 GBq) in 5.0 ml of phosphate-free McCoy's SA medium plus 10% fetal bovine serum per plate. Cellular proteins were radiolabeled by incubating cultures for 6 hr in 5 ml of methionine-free McCoy's SA medium containing 10% fetal bovine serum and 100 ,tCi of [35S]methionine (1000 Ci/mmol, New England Nuclear) per ml.DNA Polymerase a Activity Assay. The incorporation of [methyl-3H]dTTP (77.0 Ci/mmol, New England Nuclear) into bovine pancreatic DNase I-activated salmon sperm DNA (Worthington) was measured as described (9). One unit of enzyme activity is defined as 1 nmol of [3H]dTMP incorporated into product per hour at 37°C.Neutralization and Immunoprecipitation of Rat-1(tsLA24/ RSV) DNA Polymerase a by Anti-Human Polymerase a IgG SJK-287-38. Highly purified monoclonal antibody to human DNA polymerase a was prepared as described (10,11).Cells were swelled in 50 mM Tris-HCl, pH 7.5/1 mM EDTA/5 mM MgCI2/1 mM 2-mercaptoethanol/1 mM phenylmethylsulfonyl fluoride with 10% (vol/vol) glycerol, or without glycerol, for 30 min on ice, disrupted by sonication, and then centrifuged at 31,000 x g for 30 min at 40C. After centrifugation in sucrose gradients, fractions were adjusted to 0...

show abstract

“…Since the specific radioactivity of RNA is increased more than that of UTP, there are strong indications of an enhanced RNA synthesis (Bucher & Swaffield, 1969;Yngner et al, 1979a). This interpretation is supported by the fact that the activities of RNA polymerases I, II and III are elevated at 18 h after partial hepatectomy (Organtini et al, 1975;Yu, 1975b;Duceman & Jacob, 1980). Polymerase I is responsible for the synthesis of pre-rRNA, polymerase II produces hnRNA, and polymerase III catalyses the synthesis of pre-tRNA and 5S RNA.…”

mentioning

confidence: 96%

“…Numerous observations have been published dealing with different biochemical aspects of liver regeneration after partial hepatectomy. The regenerating rat liver is often used as a model for the study of molecular events during proliferation (Bresnick, 1971;Bucher & Malt, 1971;Lesch & Reutter, 1975;Lewan et al, 1977) and as a control tissue in studies of hepatoma growth (Weber et al, 1965;Lea et al, 1974;Duceman & Jacob, 1980). There is an enhanced uptake and incorporation of labelled orotic acid into rat liver RNA early after partial hepatectomy (Bresnick, 1971;Bucher & Malt, 1971).…”

mentioning

confidence: 99%

RNA synthesis in regenerating mouse liver evaluated by incorporation of [methyl-14C]methionine and by determination of RNA polymerase activity

Ljungquist¹,

Yngner²,

Lewan³

et al. 1984

Biochemical Journal

View full text Add to dashboard Cite

The synthesis of RNA during mouse liver regeneration was studied by two different methods at 24 and 48 h after partial hepatectomy. Total chromatin-bound RNA polymerase activity showed an increase of 32% at 24 h after partial hepatectomy. At 48 h a slight increase in total activity was also observed in regenerating liver, but the difference was not significant. The increase in total RNA polymerase activity was due to a rise in RNA polymerase I plus III activity. This enzyme activity was increased at both 24 and 48 h. The increase was 57% at 24 h and 51% at 48 h. When [methyl-14C]methionine was used for labelling of methyl groups in rRNA, there was an increased specific radioactivity of this class of RNA at both 24 h and 48 h. The increases were 263 and 103% at 24 and 48 h respectively. Thus both methods revealed an increased synthesis of rRNA during mouse liver regeneration. The results are discussed in relation to previous results from this laboratory [Yngner, Carlberg, Lewan & Engelbrecht (1979) Hoppe-Seyler's Z. Physiol. Chem. 360, 1069-1074; Yngner, Engelbrecht, Lewan & Annerfeldt (1979) Biochem. J. 178, 1-8; Yngner, Bengtsson, Carlberg, Engelbrecht & Wieslander (1980) Exp. Cell. Biol. 48, 393-403], which have shown that the incorporation of orotic acid or uridine into RNA is not increased in mouse liver regenerating after partial hepatectomy.

show abstract

Transcriptionally active RNA polymerases from Morris hepatomas and rat liver. Elucidation of the mechanism for the preferential increase in the tumour RNA polymerase I

Cited by 26 publications

References 32 publications

Accurate initiation of rat ribosomal RNA gene transcription using a fractionated nuclear extract from normal liver and a hepatoma.

Accurate initiation of rat ribosomal RNA gene transcription using a fractionated nuclear extract from normal liver and a hepatoma.

Phosphorylation of a high molecular weight DNA polymerase alpha.

RNA synthesis in regenerating mouse liver evaluated by incorporation of [methyl-14C]methionine and by determination of RNA polymerase activity

Contact Info

Product

Resources

About