CHARACTERIZATION OF RNA FROM THE PAROTID GLANDS OF ISOPROTERENOL-TREATED RATS

MARILYN ANN ZIEMER, Purdue University

Abstract

Isoproterenol, a beta-adrenergic agonist and secretagogue, causes the accumulation of proline-rich proteins (PRPs) in the parotid glands of rats. The biosynthesis of these proteins was studied by characterizing their messenger RNAs (mRNAs). RNAs from parotid glands of normal and isoproterenol-treated rats were isolated and translated in vitro. Analysis of the translation products by SDS polyacrylamide gel electrophoresis demonstrated that the major proteins produced by the normal and isoproterenol-treated glands were dramatically different. Amylase, the major secretory product of normal glands, was barely detectable in the products synthesized with RNA from treated rats, while six new major proteins appeared. These new proteins were all heavily labeled by in vitro translation with {('3)H}proline and were precipitated by antibodies to PRPs. Gel electrophoresis of proteins labeled with {('3)H}proline in parotid gland slice cultures showed that these proteins had different mobilities than the in vitro translation products. Because PRPs are secreted, it was expected that the in vitro translation products contained signal peptides. It is postulated that there may be translational control of PRP synthesis and/or extensive post-translational processing, because the differences in mobilities of the in vitro and in vivo synthesized proteins cannot be easily explained by the simple elimination of a signal peptide. Specific probes for the separate PRP mRNAs were prepared by cloning the complementary DNAs to mRNA from isoproterenol-treated rats. The clones have been identified by hybrid-selected translation, a technique which involves hybridization of mRNA to the cloned cDNA followed by in vitro translation of the specifically bound mRNA. One of the three clones which was investigated contains a cDNA which hybridizes PRP mRNA. Surprisingly, two proteins are made by this RNA. Further studies will be required to demonstrate whether or not the cDNA hybridizes two unique mRNAs which bind through extensive sequence homology. This possibility is likely in view of the simple amino acid composition of the PRPs (44% proline, 22% glycine, 22% glutamine) which might give rise to a repeating nucleotide sequence in the mRNAs.

Degree

Ph.D.

Subject Area

Biochemistry

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