RNA synthesis in isolated nuclei of lactating mammary cells in presence of unmodified and /nercuiy-labeled CTP

Isolated nuclei of lactat ing mouse mammary gland were capable of supporting DNA-dependent RNA synthesis in vitro in presence of unmodified and mercurated CTP (Hg-CTP) at high ionic condition at 25°C. In presence of unmodified CTP, [H]UMP incorporation into RNA increased l inear ly upto 180 min. The kinetic pattern of the reaction and the rate of RNA synthesis were essential ly simi lar when CTP was replaced by Hg-CTP. Both in unmodified and Hg-CTP containing reactions, 70-80% of RNA synthesis was inhibi ted by a-amanitin. Presence of poly(A) in a small portion of the in vitro synthesized messenger-like RNA was detectable by oligo(dT) cellulose chromatography. Both poly(A) and poly(A)" RNAs sedimented with a clear peak around 15S region in a formamide-sucrose denaturing gradient. The Hg-RNA after separation from endogenous nuclear RNA by SH-agarose a f f i n i t y column chromatography also sedimented around 15S region in a formamide-sucrose gradient. The Hg-RNA synthesized in the isolated mammary cel l nuclei in vitro should now permit monitoring hormonal regulation of specif ic gene (casein) transcript ion in the mammary cel ls by molecular hybridization of the Hg-RNA with cDNA to casein mRNA. INTRODUCTION The synergistic actions of prolactin and a glucocort icoid (hydrocortisone or corticosterone) are required for lactogenesis, including casein synthesis, and in recent years elucidation of the mechanisms involved in th is multiple hormone regulated specif ic gene expression in the mammary ce l ls has been of considerable in te res t 1 " 4 . Studies in our laboratory have demonstrated that an enriched glucocort icoid environment in the post-partum mothers is conducive to the maintenance of larger ribosomal aggregates, active in casein synthesis in the lactat ing mammary gland. We have also reported that the glucocorticoid in presence of prolact in is required for the induction as well as maintenance of the casein mRNA i t s e l f 5 " 7 . The same series of studies fur ther revealed that ce l lu lar concentration of the casein mRNA is also modulated by the intensity of suckl ing, a stimulus known to regulate the p i tu i ta ry prolact in release by indirect act ivat ion of the hypothalamo-hypophyseal con t ro l 8 . These observations thus raise the question concerning the discrete ro le of the glucocorticoid and © Information Retrieval Limited 1 Falconberg Court London W1V5FG England 4463 Nucleic Acids Research the polypeptide hormones on the t ranscr ipt ional regulat ion of the mRNA for the mi l k -p ro te in . Elucidat ion of the above question requires a c e l l f r e e RNA synthesis system, which permits re l iab le monitoring of t ranscr ip t ion of the spec i f i c mRNA fo r the milk p ro te in . Widnell and Tata f i r s t reported that isolated nuclei of l i v e r ce l l s in a short term c e l l f r e e RNA synthesis system can support a predominant synthesis of DNA-like RNA at high ionic condit ions. Recently, a few reports have i n d i cated that f a i t h f u l RNA synthesis in isolated nuclei at high ionic condit ion can be sustained fo r an extended period of t ime 1 0 " 1 2 . Moreover, reports also ind icate that isolated nuclei and chromatin can support synthesis of mercurated RNA (Hg-RNA) in vitro in presence of mercury-labeled nucleotide (Hg-CTP or Hg-UTP) in the reaction m ix tu re 1 3 " 1 7 . The technical f e a s i b i l i t y of separation of the Hg-RNA synthesized in vitro from pre-exist ing excess nuclear RNA by SHagarose a f f i n i t y column chromatography appears to provide the tool for monitoring spec i f ic gene t ranscr ip t ion in vitro-'. In th is report we present the resul ts of studies on a predominantly RNA-polymerase I I directed extended RNA synthesis system, derived from isolated nuclei of lac ta t ing mammary ce l l s and also the synthesis o f Hg-RNA in a s imi lar in vitro model. MATERIALS AND METHODS Isolat ion of Nuclei The methods described by Yu was used with some modifications to su i t the conditions of the lactat ing mammary t issue. Lactating (10-11 day) BALB/c mice nursing 6-8 pups were k i l l ed by cervical dislocat ion; mammary glands were excised and stored at -80°C. Frozen mammary tissue was pulverized in presence of l iqu id nitrogen and then homogenized in 2.5 vol of 2.3 M sucrose containing 3.3 mM CaCl2The homogenate was di luted 8 fo ld with the same solut ion, f i l t e r e d through four layers of cheese cloth and centrifuged at 50,000 xg for 60 min at 4°C. The nuclear pel let was suspended in 0.32 M sucrose-30% glycerol and centrifuged again at 700 xg for 10 min at 4°C. The f inal nuclear pe l le t was suspended in sucrose-glycerol solution and stored at -80°C. ONA was estimated by diphenylamine react ion . In Vitro RNA Synthesis The standard reaction mixture in 100 yl volume contained 100 mM Tris-HCl (pH 8.1); 10 mM 2-mercaptoethanol; 4 mM MnCl2; 0.18 M (NHiJzSO., (pH 8.1); 0.6 mM each of ATP, GTP, CTP and 0.1 mM [H]UTP (1 nCi/0.01 pmol, New England Nuclear Corp., Boston). The reaction was started by adding the nuclei (10-20