The mRNA for preuteroglobin, a precursor of the hormonally induced protein uteroglobin, has been partially purified from the endometrium of progesterone treated rabbits. The purification procedure starts with total endometrial polysomes and involves treatment with proteinase K and dodecylsulfate, chromatography on oligo(dT)-cellulose, sucrose density gradient centrifugation, electrophoresis in polyacrylamide gels containing dodecylsulfate, and a second absorption to oligo(dT)-cellulose. The final mRNA preparation codes exclusively for preuteroglobin in a wheat germ cell-free system and migrates as a single band in polyacrylamide gels containing 99% formamide. The average length of the poly(A) segment is 60 nucleotides and the translation of the preuteroglobin mRNA is inhibited by m7G(5')ppp(5')A, indicating that it contains a "tcapped" 5'-terminus. Comparison with known standards yields a molecular weight of 200,000 (600 nucleotides) for preuteroglobin mRNA, approximately twice as many nucleotides as required for encoding the 90 aminoacids of its cell-free product.

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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 has been purified from the membrane-bound polysomes of induced endometrium and used as template for the synthesis of a full copy complementary DNA. Final purification of the cDNA was based on hybridization to the template mRNA up to a low value of rot (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 has been 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 uteroglobinsynthesizing 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 a DNA basis there is a 50-100-fold increase in the cellular content of pre-uteroglobin mRNA following hormonal treatment.The synthesis of uteroglobin in rabbit endometrium is regulated by ovarian steroids, and its induction offers a suitable model system for studying the molecular mechanism of steroid hormone action on specific protein synthesis (for review see [I 3 ) . In a recent paper we have reported the partial purification of the mRNA for pre-uteroglobin which can be used as a template for the synthesis of a complementary DNA [2]. This complementary DNA is needed as a probe for the quantification of specific pre-uteroglobin mRNA sequences during hormonal induction, and could also serve to synthesize a double-stranded DNA fragment suitable for DNA recombination experiments.In this paper we describe an improved purification method for pre-uteroglobin mRNA and present our data on the synthesis, purification, and properties of its cDNA. Using this probe in RNA-excess hydridizaAbbreviations. rot, product o f R N A concentration (in moles of nucleotides per liter) and time (in seconds); r0tli2, rot at which the hybridization of a given population of nucleic acid molecules is half completed.Enzymes. S 1 nuclease or single-stranded nuclease (EC 3.1.4.21); deoxyribonuclease I (EC 3.1.4.5); reverse transcriptase or RNAdirected DNA polymerase (EC 2.7.7.7). tion reactions, we show that there is an accumulation of pre-uteroglobin mRNA sequences in endometrial RNA following hormonal induction of uteroglobin synthesis. MATERIALS AND METHODS Materials ~-[~~S]Methionine (600 Ci/mmol) and [3H JdCTP (22 Ci/mmol) were ob...