Abstract
The mRNA coding for pre-prolactin (pPRL) of the adult bovine anterior pituitary was purified to 85% homogeneity and used as a template for cDNA synthesis with reverse transcriptase from avian myeloblastosis virus. The cDNA sequences complementary to pPRL mRNA were further purified by employing a limited back-hybridization step and treatment with S1 nuclease. The pPRL cDNA preparation was judged to be homogeneous by comparing its hybridization kinetics to those of ovalbumin and globin mRNA standards and by thermal melt analysis of the pPRL mRNA:cDNA hybrids. Total cellular RNA was extracted from individual bovine fetal pituitaries of either sex, ranging from 90 to 200 days of gestation, and examined for its pPRL mRNA concentration by hybridization with an excess of pPRL cDNA. The hybridization assay was capable of detecting picogram amounts of pPRL mRNA, e.g. amounts less than 0.002% of input total cellular RNA. These results indicated that from 90 to 200 days of gestation, the levels of pPRL mRNA relative to total cellular RNA in bovine fetal pituitaries increase exponentially. This increase in pPRL mRNA occurs to the same extent in either sex, with the most dramatic shift (over 10-fold) occurring between 120 and 145 days of gestation.
Highlights
ThemRNA codingforpre-prolactin of the led to the proposal that the biosynthesis of pituitary polyadultbovineanteriorpituitary was purified to 85% peptide hormones is determined by the cytoplasmic levels of homogeneity and used as a template forcDNA synthe- their respective mRNAs
Partial Purification of pPRL and p G H mRNA-The isolation scheme employed for the partial purification of pPRL
In an earlier paperwe reported that bovine pPRL mRNA could be separated from pGH mRNA by electrophoresis in acid-urea agarose gels ( 2 2 )
Summary
In an earlier paperwe reported that bovine pPRL mRNA could be separated from pGH mRNA by electrophoresis in acid-urea agarose gels ( 2 2 ). Fractions of RNA, taken from across the gradient, were translated in the wheat germ cell-free system in order to determine that pPRL mRNA was resolved from pGHmRNA(data not shown). This partial separation of pPRL and pGHmRNA must, in part, occur as a result of differences in secondary structure between the two mRNAs since pGH mRNA electrophoresesmorerapidly thanpPRLmRNA in acid-ureaor glyoxal-agarose gels (data not shown)W. e purified pPRL and pGHmRNA from each other by isolating their respective
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