Messenger RNA for alpha 2u-globulin of rat liver. Purification, partial characterization of the mRNA, and synthesis of a Hae III restriction fragment as its cDNA probe

Abstract

The mRNA for the androgen-dependent hepatic protein, alpha 2u-globulin is normally present in the liver of mature male rats to the extent of about 1% of the total mRNA population. alpha 2u mRNA which was found to migrate as a 14 S band was purified about 18-fold through preparative urea-agarose gel electrophoresis. 32P-Labeled cDNA synthesized with this partially purified alpha 2u mRNA was used as substrate for two restriction endonucleases Hha I and Hae III. Digestion of the cDNA with Hha I failed to reduce its electrophoretic heterogeneity. However, Hae III digestion of the cDNA preparation greatly reduced the molecular complexity and produced several distinct cDNA bands. One of these Hae III fragments (Band A) containing 410 nucleotide residues was extracted from polyacrylamide gel and found to be complementary to alpha 2u mRNA. The identity of this cDNA fragment was established by its ability to inhibit selectively the translation of alpha 2u mRNA in the rabbit reticulocyte cell-free system and by its hybridization kinetics with poly(A)+ hepatic RNA from animals with different rates of alpha 2u synthesis. The relative R0t 1/2 values showed a direct correlation between mRNA sequences complementary to the cDNA fragment (A) and to both translatable alpha 2u mRNA and hepatic level of alpha 2u-globulin in adult male, female, and maturing male rats. Thus, the cDNA fragment containing 410 nucleotide residues generated by the restriction cleavage with Hae III can be used as a convenient probe for identification and quantitation of alpha 2u mRNA under different physiological and experimental conditions.