Induction of uridine kinase in rat peripheral blood lymphocytes: dependence on mitogen specificity and effect of pyrimidine analogs.
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Biomedical subjects
Publications and source records attributed to D E Schumm.
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A cell-free system is described which permits a significant and prolonged synthesis of RNA in isolated rat liver nuclei, under conditions previously demonstrated to support normal nuclear processing and transport of both rRNA and mRNA. The system contains cytosol but not (NH4)2SO4 or other non-physiological components. Evidence is presented for cytosol factors which stimulate ribosomal, and to a lesser degree, non-ribosomal RNA synthesis.
Putative regulatory macromolecules, which may be useful in diagnosis or tumor detection, were identified in the peripheral blood plasma of tumor-bearing animals. We monitored the components by measuring their ability to stimulate messenger RNA (mRNA) release from isolated nuclei in a cell-free system of rat liver nuclei in fortified homologous cytosol. This in vitro test system exhibited near-normal in vivo nuclear RNA restriction. When added to the assay at a protein concentration of 3.0 mg/ml, dialyzed plasma from rats or mice with chemically induced transplantable or primary tumors stimulated mRNA release from 87% to more than 300% over control plasma from normal rats. Plasma from partially hepatectomized rats stimulated only 26% over control plasma. The test system derived from rat liver seemed to permit the monitoring of plasma from other species. Available evidence, particularly relating to tumor-host-interaction, suggests, but does not prove, that regulatory components are released from the tumor cells to the circulation.
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Treatment of rats with Cordycepin (3'-deoxyadenosine), an inhibitor of nuclear poly(A) synthesis, selectively decreased the energy and cytosol-dependent release of a portion of the mRNA species from the isolated hepatic nuclei in a cell-free system by approximately 40%. An analysis of the differential binding of the transported mRNA containing poly(A) tracts to nitrocellulose filters, and to cellulose or poly(U)-Sepharose columns suggested that the poly(A) tracts in the mRNA transported from the isolated hepatic nuclei of Cordycepin-treated rats, are decreased in size. This size decrease was confirmed through an analysis of the average size of the poly(A) tracts, released from the messengers by ribonuclease activity, on sucrose density gradients.
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Following a 3 hour in vivo labelling of cytoplasmic RNA in rat liver with orotic acid-6-14C under conditions where ribosomal RNA synthesis was suppressed, the proportion of labelled messenger-like RNA released to the cytoplasm which contained polyadenylate (poly(A)) tracts was about 3.0 times higher in the livers of juvenile (50 day) as compared to adult (180 day) rats. This discrepancy was confirmed in a cell-free system which consisted of isolated prelabelled nuclei in fortified cytosol. Thus under conditions where approximately 80% of the released labelled RNA was messenger-like, the proportion of polyadenylated labelled RNA transported to the homologous cytosol was 3.4-fold greater in the systems derived from juvenile as compared to adult rat liver. Through comparisons of homologous and heterologous systems it was determined that the age-dependent change in the metabolism of polyadenylated messenger RNA resides in the nucleus and not in the cytoplasm. This change, furthermore does not involve the known ATP-dependence of nuclear RNA release. Rather it must involve other age-dependent changes in the processing or transport of polyadenylated messenger RNA.