Cell-free Translations of Proline-rich Protein mRNAs* with

Treatment of rats the Bf-agonist isoproterenol causes a dramatic increase in a series of proteins rich in proline (proline-rich proteins) in the parotid glands (Muenzer, D. M. J. Biol. Chem. 254, 5623, 5629). These proteins which contain about 43% proline, comprise more than 50% of the total soluble protein of glands of rats treated with isoproterenol for 10 days. Further studies by in vitro translation analysis using the reticulocyte lysate system and labeling with [3H]proline or [ 3 5 S]methionine show definitive changes in patterns of protein synthesis and proline-rich protein mRNAs are highly elevated in treated animals. Analysis of translation products by sodium dodecyl sulfate-polyacrylamide gel electropho- resis showed (1) very little synthesis of proline-rich proteins from poly(A + ) RNA of glands of normal rats, (2) poly(A+) RNA from glands of treated animals syn- thesize mainly proline-rich proteins, (3) translations with [3Hlproline and [ 3 5 S]methionine give identical labeling patterns of proline-rich proteins, and (4) proline- rich proteins from cell-free translations are all precipitated by antibodies to proline-rich proteins. At least six different proline-rich proteins are translated with poly(A + ) mRNA from glands of treated animals. Each of these proteins is likely a translation product of a separate, specific mRNA. The dramatic changes in protein synthesis of rat parotid glands in response to iso- proterenol treatment suggest to us that the parotid

Treatment of rats with the Bf-agonist isoproterenol causes a dramatic increase in a series of proteins rich in proline (proline-rich proteins) in the parotid glands (Muenzer, J., Bildstein, C., Gleason, M., and Carlson, D. M. (1979) J. Biol. Chem. 254, 5623, 5629). These proteins which contain about 43% proline, comprise more than 50% of the total soluble protein of glands of rats treated with isoproterenol for 10 days. Further studies by in vitro translation analysis using the reticulocyte lysate system and labeling with [3H]proline or [ 3 5 S]methionine show definitive changes in patterns of protein synthesis and proline-rich protein mRNAs are highly elevated in treated animals. Analysis of translation products by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed (1) very little synthesis of proline-rich proteins from poly(A + ) RNA of glands of normal rats, (2) poly(A+) RNA from glands of treated animals synthesize mainly proline-rich proteins, (3) translations with [3Hlproline and [ 3 5 S]methionine give identical labeling patterns of proline-rich proteins, and (4) prolinerich proteins from cell-free translations are all precipitated by antibodies to proline-rich proteins. At least six different proline-rich proteins are translated with poly(A + ) mRNA from glands of treated animals. Each of these proteins is likely a translation product of a separate, specific mRNA. The dramatic changes in protein synthesis of rat parotid glands in response to isoproterenol treatment suggest to us that the parotid gland of the isoproterenol-treated rat is an excellent model system to study the overall responsiveness of gene expression to catecholamines.
Regulation of tissue-specific gene expression is poorly understood. Numerous enzymes and proteins of specific functional significance either for development, such as the silkmoth chorion (1, 2), or for later function such as a-amylase (3,4) and ovalbumin (5,6) have been studied in molecular detail. The parotid glands of rats offer an interesting model system for studying the overall effects of catecholamines on gene-expression. Profound morphological effects on rat salivary glands by adrenergic agonists, specifically isoproterenol, were reported in 1961 by Selye et al. (7) and by Brown-Grant (8). Repeated pharmacological doses caused glandular hypertrophy and hyperplasia. Biochemical changes resulting from * This research was supported in part by Public Health Service Grants AM19175 and GM07076. This is Journal Paper 8966 from the Purdue University Agricultural Experiment Station. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. isoproterenol treatment have been described, and especially a dramatic increase in a series of proteins high in proline, or PRP', by Muenzer et al. (9,10). The high proline content of these proteins (43% Pro) and the dramatic increase in amounts, with PRPs comprising greater than 50% of the total glandular soluble protein in isoproterenol-treated rats, prompted us to study the synthesis of these unusual proteins-In this paper we report on the cell-free translation studies and demonstrate that each protein of a series of at least six prolinerich proteins is likely translated from a separate mRNA. Radioactivity was measured by liquid scintillation counting. Aqueous samples were dissolved in 4 ml of Aquasol II (New England Nuclear) and filter papers were suspended in 4 ml of Beckman Readysolv NA.
Isoproterenol Treatment-Rats were treated with isoproterenol-HCI as described previously (9). Treatment was for 10 days except where otherwise indicated.
Antiserum Preparation-Proline-rich proteins were isolated as described previously (9,10). Basic proteins (IPR-1B, through 1B 6 ) from CM-cellulose chromatography were combined and are referred to as "total B-PRP." Rabbits were injected subcutaneous in four places on the back (3 mg of total B-PRP in 1 ml of sterile 140 mM NaCl plus 1 ml of complete Freund's adjuvant). Booster injections of 3 mg of total B-PRP in incomplete adjuvant were administered similarly every week for 2 months. Subsequently, boosters of 1 mg of total B-PRP were given every other week. Peak titer was reached after 2 months.
Antibody Precipitations-Samples were diluted to 0.1 ml in 2% Triton X-100 and 2% sodium deoxycholate. Ten 1l of a solution of 20% serum in 2% Triton-DOC, and where indicated 10 to 20 Itg competing antigen, were added and the mixture incubated at room temperature for 1.5 h. Goat anti-rabbit IgG (0.1 ml, prepared according to directions of supplier, Calbiochem-Behring) was added and the incubation was continued for 1 h. This reaction mixture was layered over a discontinuous gradient of 0.2 ml of 0.5 M sucrose and 0.2 ml of 1.0 M sucrose and centrifuged at 12,000 x g for 4 min. The pellets were washed four times by vigorous shaking in 1.0 M sucrose and then dissolved in 0.3 ml of 10% acetic acid for liquid scintillation counting or in 80 pl of sample buffer (12) for SDS-polyacrylamide gel electrophoresis.
'The abbreviations used are: PRP, proline-rich proteins; SDS, sodium dodecyl sulfate; Ipr used as a prefix, such as Ipr-lB, indicates specific protein fractions isolated from isoproterenol-treated rats (9). procedure reported by Harding et al. (13) using guanidine thiocyanate and mercaptoethanol, except that the pellet obtained after centrifugation through CsC1 was redissolved in the homogenization buffer and then RNA was precipitated with ethanol. 2 This pellet was washed in 70% ethanol and dissolved in water.
Oligo(dT)-cellulose chromatography of RNA was performed as described by Aviv and Leder (14). The polyadenylated RNA fraction was repeatedly passed over the column until retention was greater than 80%. RNA finally retained by the column is referred to as poly(A + ) RNA. In Vitro Translation Analysis-Reticulocyte lysate was prepared according to Darnbrough et al. (15) from rabbits injected with acetylphenylhydrazine. mRNA was translated in vitro as described by Pelham and Jackson (16) using the reticulocyte lysate. Translation incubations in 60 l contained 37.5 #1j of nuclease-treated lysate, 100 mM KCI, 0.5 mM MgCI 2 , 10 mM creatine phosphate, 25 /M hemin, 1 mM ATP, 0.2 mM GTP, 10 Ipg/ml of creatine phosphokinase, and unless otherwise stated, 40 pCi of radiolabeled amino acid, either [ 3 H] proline or [ 35 S]methionine, and 20 pg of total RNA or 2 pg of poly(A+) RNA. Reaction mixtures were incubated 90 min at 30 °C. Aliquots of the incubation mixtures (5 Id) were added to 1 ml of H 2 0; 0.5 ml of 1 M NaOH/0.5 M H 2 0 2 was added and incubated for 15 min at 37 °C to decolorize the hemin. One ml of cold 25% trichloroacetic acid/2.5% phosphotungstic acid was added and the precipitate was harvested on glass fiber filter discs (Whatman 934-AH, 2.4-cm diameter). Filters were washed successively with 10% trichloroacetic acid/1% phosphotungstic acid and 95% ethanol, dried at 80 °C, and counted. The addition of phosphotungstic acid is necessary because PRP are soluble in 15% trichloroacetic acid.
Products of in vitro translations were prepared for analysis by SDS-polyacrylamide gel electrophoresis by removing an aliquot, usually 5-,, and by terminating the reaction by adding a solution of ribonuclease A (0.1 volume of 1 mg/ml in 50 mM sodium acetate, pH 5.0) and incubating for 15 min at 37 °C. The RNase A preparation was boiled for 5 min to denature proteases and DNases before it was added. Polypeptides in the nuclease-treated samples were precipitated with 8 volumes of acetone for 10 min in a dry ice bath or overnight at -20 °C. Precipitate was collected by centrifuging at 12,000 x g for 4 min, dried in vacuo and dissolved in 80 to 100 pl of sample buffer (12) with boiling for 3 min.

SDS-polyacrylamide Gel Electrophoresis-SDS-polyacrylamide
gels (13%, 30, 0.8 acrylamide, bisacrylamide) with 4.75% stacking gels were prepared according to Laemmli (12). Slab gels (1.5 mm x 12 cm x 15 cm) were run at 30 mA per gel until the bromphenol blue dye was 1 cm from the bottom of the gel. Gels were prepared for fluorography as described by Bonner and Laskey (17). Dried gels were exposed to Kodak X-Omat XRP film at -80 °C.
Incubation of Parotid Gland Slices-Two 1 to 2 mm3 pieces of parotid gland (6 to 7 mg each) were incubated in 0.2 ml of Earle's balanced salt solution containing 33 pCi of either [3S]methionine or [ 3 H]proline for 2.5 h at 37 °C in an atmosphere of 5% C0 2 /95% air. The gland pieces were rinsed in medium containing unlabeled amino acids and sonicated 3 to 4 min in sample buffer (2 pieces of gland in 0.1 ml of buffer) with a microprobe. Sonicated samples were boiled immediately for 3 min, centrifuged at 12,000 x g for 2 min and pellets discarded. Soluble fractions were analyzed by SDS-polyacrylamide gel electrophoresis.

RESULTS AND DISCUSSION
Cell-free Translation-The procedure developed by Harding et al. (13) for isolating RNA from cultured embryonic pancreas yielded intact, biologically active RNA from parotid glands of both normal and Ipr-treated rats. A second extraction with guanidine thiocyanate and mercaptoethanol was necessary to remove traces of ribonuclease. Protein synthesis measured by translation of total RNA from Ipr-treated rats was linear for 1 h. Incorporation of [ 35 S]methionine was proportional to RNA concentration up to 10 pg of total RNA and 0.   (9,10). The data presented in Table  I are definitive only if the same proteins, or in this case if proteins with similar proline and methionine compositions, are synthesized. However, native proline-rich proteins do not contain methionine and the proline-rich proteins labeled with [ 3 5 S]methionine in cell-free translation experiments have the 3 5 S-label only in the NH 2 -terminus. 3 The methionine to proline ratio of the cell-free translation proline-rich proteins, calculated from the amino acid compositions and using a molecular weight of 17,000 (10), is about 1:54. Calculation of methionine and proline concentrations in the translation products using specific activities of the two isotopes and assuming little or no contribution from endogenous amino acids gives concentrations of 6 pmol [ 35 S]methionine and 300 pmol [ 3 H]proline, or a ratio of 1:50. It is important to recognize that almost all of the 3 5 S-label and 3 H-label of translation products from mRNAs of Ipr-treated animals are in proline-rich proteins ( Figs. 1 and 2). Only [ 35 S]methionine labels proline-rich proteins with mRNA from normal animals sufficiently to readily detect by fluorography. The labeled immunoprecipitated products of lane 15 (Fig. 2) are very faint; application of larger amounts gave clear evidence of proline-rich protein synthesis. SDSpolyacrylamide gel electrophoresis analysis of translation incubations with normal mRNA and [ 3 H]proline under conditions given here showed no detectable incorporation into proline-rich proteins (gels not shown) whereas only prolinerich proteins were labeled under the same conditions with mRNA from glands of treated animals (Fig. 2, lane 17).
An approximation of temporal changes in mRNA levels resulting from Ipr-treatment was obtained by translating RNA obtained after 4, 6, 8, and 10 days of treatment. Translation products synthesized with these RNA preparations are shown in Fig. 1. Changes in mRNA species are almost complete after 4 days of Ipr-treatment. These results give a general trend of changes in mRNA levels since translation isn't necessarily proportional to relative abundance of mRNAs (16)(17)(18). An