Synthesis and Characterization of a DNA Complementary to Pre‐uteroglobin mRNA

Abstract

Uteroglobin, a progesterone-induced uterine protein of the rabbit, is synthesized in cell-free systems as a precursor containing 21 additional amino-acids at its N-terminal end. The mRNA for pre-uteroglobin was purified from the membrane-bound polysomes of induced endometrium and used as template for the synthesis of a full copy complementary[c]DNA. Final purification of the cDNA was based on hybridization to the template mRNA up to a rot [ro = concentration of RNA; t = time of hybridization] value of 0.01 M/s and digestion of the non-hybridized cDNA by S1 nuclease. A comparison of the hybridization kinetics of the pre-uteroglobin cDNA and rabbit globin cDNA to their respective templates indicates a nucleotide sequence complexity of 650 for pre-uteroglobin mRNA, in agreement with the values obtained by sucrose gradient centrifugation and polyacrylamide gel electrophoresis in formamide. The melting temperature of the hybrids of pre-uteroglobin cDNA to its template reflects the absence of mismatched sequences. This cDNA was used to quantify pre-uteroglobin mRNA sequences in the endometrial RNA from control animals and from animals treated sequentially with estradiol and progesterone. In agreement with the induction of uteroglobin-synthesizing activity, there is a dramatic increase in the uterine content of pre-uteroglobin mRNA after hormonal treatment. Part of this effect can be accounted for by hormonally induced cell proliferation. When expressed on DNA basis there is a 50-100-fold increase in the cellular content of pre-uteroglobin mRNA following hormonal treatment.