Activity of DNA-dependent RNA polymerases in rabbit mammary gland during lactogenesis (original) (raw)
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Rapidly-Labelled Rna in the Mouse Mammary Gland Before and During Lactation
Journal of Endocrinology, 1973
SUMMARY Linear sucrose density gradient analysis showed that the synthesis of rapidly-labelled high molecular weight RNA was virtually absent in the mammary glands of virgin mice. The rapidly-labelled RNA was first evident in the mammary gland in pregnancy and was also present during lactation. The bulk of this newly-made nuclear RNA sedimented as 45S and 32S fractions after a 15-min [3H]uridine pulse period in vivo. No labelled 18S RNA was detectable in the nuclear fraction after the 15-min pulse but it was present in the cytoplasm, suggesting that the 18S RNA migrates to the cytoplasm almost immediately after its formation. Thirty minutes after injection of [3H]uridine, the initial radioactivity of the 45S region migrated to the 32S fraction and a labelled 28S peak was also present in the cytoplasmic RNA at 60 min, suggesting that the processed 28S ribosomal RNA in the mammary gland began to migrate to the cytoplasm between 30 and 60 min after the nuclear synthesis of the precurso...
Measurements of DNA and RNA in mammary gland homogenates by the ethidium bromide technique
Analytical Biochemistry, 1975
A rapid method of determining simultaneously DNA and RNA in mammary gland homogenates using the ethidium bromide technique is discussed. The method utilizes a quantitative extraction of DNA and RNA with 2.0 M sodium chloride, SDS. and EDTA at pH 8.0. Assays of mammary gland RNA and DNA using previously published methods were compared with determinations using the ethidium bromide technique. While the fluorescence method gave lower values for RNA when compared to those obtained using the orcinol or absorbancy ratio (OD 260 nm/280 nm). DNA measurements agreed well with the values determined by the diphenylamine technique. Extinction coefficient data for total mammary gland RNA isolated using a modified phenol extraction procedure are also presented.
European Journal of Biochemistry, 1979
Poly(A) polymerase activities were measured from intact rabbit uterine nuclei and chromatin after a single dose of progesterone, and following a 5-day treatment with progesterone alone or in combination with estradiol. A single intravenous dose of progesterone (5 mg/kg) to estrogen-primed rabbits elicited an early increase in both bound and free nuclear poly(A) polymerase activities, the peak activity (about 40 % over the controls) being reached 30-60 min posttreatment. The bound enzyme activity seemed to exhibit a second rise that occurred 12 h after progesterone administration. A 5-day treatment of estrous rabbits with progesterone (1 mg/kg) brought about a sixfold elevation in the bound poly(A) polymerase and a twofold increase in the free nucleoplasmic enzyme activity. A corresponding activation also occurred in the chromatin-associated poly(A) polymerase. Concomitant administration of estradiol (50 pg/kg) with progesterone significantly depressed progesterone-elicited increases in the poly(A) polymerases. Changes in the nuclear RNA polymerase I and TI were minor during a long-term progesterone treatment, while the total chromatin template activity, as measured in a transcription system in vitro using wheat germ RNA polymerase 11, was significantly reduced after a 5-day progesterone administration. When estradiol was given along with progesterone, it counteracted the effect of progesterone on the chromatin template. These results suggest that progesterone exerts in the rabbit uterus posttranscriptional actions on the metabolism and processing of mRNA species. Moreover, the antiprogestational action of estrogens was already noted in the regulation of enzymes responsible for completion of mature mRNA synthesis
European Journal of Biochemistry, 1980
Endometrical nuclei, prepared from rabbits subjected to different hormonal treatments, were used for the cell-free synthesis of RNA. Optimal conditions for the incorporation of [3H]UMP into RNA are described, leading to the synthesis of relatively undegraded RNA molecules. Under these conditions there is virtually no initiation of new RNA chains in vitro, and RNA chain elongation is inhibited up to 6004 by low concentrations of a-amanitin and up to 90% by actinomycin D. The synthesis of RNA is slightly inhibited in the presence of Hg-CTP and monothioglycerol, but newly synthesized mercurated RNA can be efficiently separated from endogenous RNA upon chromatography on sulfhydryl-Sepharose under stringent conditions. The RNA synthesized in vitro by endometrial nuclei from pseudopregnant rabbits contains RNA sequences transcribed from the uteroglobin gene, as demonstrated by hybridization to an excess of purified preuteroglobin cDNA. In endometrial cells from pseudopregnant animals the number of RNA polymerase I1 molecules transcribing the uteroglobin gene is 12-fold higher than in control animals, demonstrating that at least part of the hormonally induced accumulation of preuteroglobin mRNA is due to an increased rate of transcription of the uteroglobin gene.
Journal of Experimental Zoology, 1981
Kinetics of accumulation of total and poly(A)-containing RNA have been measured during growth of the mouse oocyte. Total RNA from oocytes isolated a t discrete stages of growth was determined by two independent microassays. The fully-grown oocyte contained about 0.60 ng of RNA. Kinetics of accumulation of total RNA with respect to oocyte volume were biphasic. Small, growing oocytes (about 30 pl) contained about 0.20 ng of RNNoocyte. The amount of RNA increased in a quasi-linear fashion until oocyte volume was about 160 pl, at which point there was about 0.57 ng of RNAIoocyte. Thus oocytes about 65% of their final volume had accumulated about 95% of the total amount of RNA present in the fully-grown oocyte. The relative amount of poly(A)-containing RNA in oocytes of various size was determined by in situ hybridization of rH]poly(U) to ovarian sections from juvenile mice of known age, followed by autoradiography. The kinetics of accumulation of poly(A)-containing RNA were similar to those of total RNA, oocytes about 70% of their final volume had accumulated about 95% of the amount of poly(A)-containing RNA present in the fully-grown oocyte. The poly(A)-containing RNA resided predominantly in the cytoplasm and no obvious cytoplasmic localization was observed. Kinetics of accumulation of total RNA, which is mainly ribosomal, and poly(A)-containing RNA were consistent with levels of RNA polymerases I and
Cancer Letters, 1988
In the present study the effect of all-transretinoic acid (RA) on nuclear RNA polymerase activity in N-methyl-N-nitrosourea (MNU)-induced mammary tumors was investigated. Three experimental protocols were used. (I) The tumor mince was incubated with 1 fl RA for 30 min at 3OOC; the RNA polymerase actiuity was measured in the purified nuclei and compared with control nuclei. (2) In order to evaluate the influence of retinoic binding protein on enzyme activity, mammary tumor nuclei were incubated with RA bound cytosolic retinoic acid binding protein complex (RA-CRABP) at 2!S°C for 30 min. This step allows the complex to translocate into the nuclei. The enzyme activity in these nuclei was compared with the nuclei pre-incubated with buffer or cytosol. (3) Finally, the influence of the addition of RA-CRABP complex directly into the RNA polymerase reaction mixture was determined and compared with appropriate controls. Results indicated that the RNA polymerase activity in the nuclei of RA treated tissue as well as in the nuclei subsequent to the translocation step was significantly reduced. However, the
An improved method for the rapid isolation of RNA from tissue with high ribonuclease content
Analytical Biochemistry, 1976
Investigations of the synthesis of pancreatic RNA from ruminants and rodents, such as the rat, guinea pig, and mouse, are handicapped by the prodigious concentration of pancreatic RNase in these species (1). In developmental studies the difficulty is compounded by the small amount of embryonic tissue available (2,3). During the course of an investigation of ribosomal RNA metabolism in the embryonic rat pancreas, we found that published RNA isolation procedures yielded only degraded preparations from both embryonic and adult tissues. We therefore devised a method for preparing undegraded ribosomal pancreatic RNA from tissues containing 1 to 5 pg RNase/mg homogenate protein. We also increased the sensitivity of an existing method for gel electrophoresis of RNA. This study establishes the feasibility of polyacrylamide gel electrophoresis as an analytical tool in the study of stable RNA in embryonic and adult mammalian tissue containing high levels of RNase. MATERIALS Ammonium persulfate and Cyanogum 41 were purchased from Fischer Scientific Co. Stains-All and 3-dimethylaminopropionitrile were purchased from Eastman Organic Chemicals. Agarose was purchased from Kinman Optical Co. L-arginine from Sigma Chemical Co., formamide from Aldrich Chemical Co. L-methionine from Nutrition Biochemicals Corp. ,, and polyvinyl sulfate from General Biochemicals. Fetal calf serum, Eagle's minimal essential medium (4) containing Hanks' balanced salt solution (5), L-glutamine, and antibiotics were obtained from Grand Island Biological Co. [5,3H]Uridine, 25 Ci/mmol and [4,5-3H]L-leucine, 25 Ci/mmol. were obtained from New England Nuclear Corp. Filters (Ultrathin HA, 0.22 pm) were products of the Millipore Filter Corp. Culturing Procedures METHODS Pregnant rats (Spartan Research, Haslett, Mich.) were killed by cervical dislocation followed by decapitation. Adult tissue and embryonic pancreatic rudiments were dissected in Earle's balanced salt solution (6), then were transferred to Eagle's minimal essential medium and cut into pieces approximately 1 mm in diameter, corresponding to 5-15 pg protein. From 50 to 150 pg of tissue pieces were placed on sterile 33-mm filters.