Isolation and in Vitro Translation of the Messenger RNA Coding for Pancreatic Amylase*

RNA prepared from dog pancreas polysomes or microsomes directs the synthesis of pancreas-specific proteins in heterologous cell-free translation systems. A translation product, approximately 1500 daltons larger than authentic amylase, corresponding to pancreatic amylase was identi-fied by immunoprecipitation with anti-amylase y-globulin and tryptic peptide analysis. We suggest that this larger form of amylase is an amylase precursor. Using amylase immunoprecipitation of reticulocyte translation reactions as an assay, we have shown that greater than 99% of the mRNA for amylase is associated with polysomes bound to the endoplasmic reticulum. Electrophoresis of pancreatic mRNA preparations in formamide-containing polyacrylamide gels and subsequent translation of the fractions have shown that amylase mRNA is of a discrete size with a mobility equivalent to that of 18 S ribosomal RNA, and therefore significantly larger than required to code solely for the amino acid sequence of the amylase precursor. The vertebrate exocrine pancreas is readily amenable analysis of the mechansims of differential gene expression. secre- a limited defined

RNA prepared from dog pancreas polysomes or microsomes directs the synthesis of pancreas-specific proteins in heterologous cell-free translation systems. A translation product, approximately 1500 daltons larger than authentic amylase, corresponding to pancreatic amylase was identified by immunoprecipitation with anti-amylase y-globulin and tryptic peptide analysis. We suggest that this larger form of amylase is an amylase precursor.
Using amylase immunoprecipitation of reticulocyte translation reactions as an assay, we have shown that greater than 99% of the mRNA for amylase is associated with polysomes bound to the endoplasmic reticulum. Electrophoresis of pancreatic mRNA preparations in formamide-containing polyacrylamide gels and subsequent translation of the fractions have shown that amylase mRNA is of a discrete size with a mobility equivalent to that of 18 S ribosomal RNA, and therefore significantly larger than required to code solely for the amino acid sequence of the amylase precursor.
The vertebrate exocrine pancreas is readily amenable to analysis of the mechansims of differential gene expression. Acinar cells are highly specialized for the synthesis and secretion of a limited number of well defined enzymes and proenzymes utilized in digestion (l-4); greater than 80% of the protein synthesis of the gland is required for the synthesis of 12 to 20 proteins. During acinar cell development these proteins accumulate in lo4 to 10g-fold excess over nondifferentiated levels (2,5). These pancreas-specific proteins accumulate during a small development interval, but not in a strictly coordinate manner. The accumulation profiles of three pairs of proteins (amylase and chymotrypsinogen, specific lipase and procarboxypeptidase A, trypsinogen and procarboxypeptidase B) appear independently synchronous. Others (ribonuclease and nonspecific lipase) accumulate independently.
The presence of dexamethasone selectively enhances the synthesis of amylase and procarboxypeptidase B (6). Furthermore, the relative proportions of the various proteins change after birth (5). These results indicate that the synthesis of individual or subsets of the secretory proteins are regulated independently. * This work was supported by Grant BMS72-02222 from the National Science Foundation.
$ Postdoctoral Fellow of the American Cancer Society. The isolation and characterization of the exocrine protein messenger RNAs is a requisite first step in the analysis of the regulation of expression of the genes coding for the secretory proteins. However, the isolation of polysomes or RNA from many vertebrate pancreases is hampered by high levels of ribonuclease. This is not a problem with dog, cat, rabbit, man, and certain avian species since the pancreases of these animals do not produce significant amounts of RNase (7). Indeed, Dickman and Bruenger (8) (Fig. 1C). A final yield of 2 to 3% of the total microsomal RNA was obtained after a second passage of the RNA (

Identification of in Vitro Translation
Products -Poly(A+l RNA was assayed for its capacity to direct protein synthesis (messenger RNA activity) in both the wheat germ and reticulocyte cell-free systems. Only the results for translation in the reticulocyte system are reported, similar results were obtained with a cell-free system derived from wheat germ. The translation products were analyzed concurrently with a reticulocyte control (without exogenous poly(A+) RNA) by SDS-polyacrylamide gel electrophoresis.
The results shown in Fig. 2 demonstrate that the poly(A+) pancreas RNA elicited the synthesis of several polypeptide species not observed in the control lysate; these polypeptides could easily be discriminated from the peptides (largely globin and a 65,OOOdalton protein) synthesized in the control reticulocyte lysate ( Fig. 2A, Column 2) presence of dog poly(A+) RNA at 20 pg/ml, approximately 30% of the total incorporation was attributable to synthesis of pancreas-specific polypeptides. A polypeptide of M, = 57,500, slightly larger than pancreatic amylase (M, = 56,000), is a major translation product. Identical bands of similar intensity were observed when total membrane-bound polysomal RNA was translated (data not shown). Thus, affinity chromatography on oligo(dT)-cellulose results in the retention of all major pancreas mRNAs.
In Vitro Synthesis of Amylase -The synthesis of the polypeptide corresponding to amylase was further characterized. A y-globulin fraction containing anti-amylase activity was used to specifically precipitate amylase and related peptides. The data in Fig. 2B show that only amylase polypeptides were precipitated from W-labeled pancreatic secretory proteins by this y-globulin preparation.
A radioactive polypeptide was observed coincident with authentic purified amylase, the major protein synthesized by the exocrine pancreas. The broad amylase band is due to overloading; as a result, the minor components are emphasized. The minor bands of slightly lower molecular weight present in the immunoprecipitate appear to be proteolytic fragments of amylase rather than other nonspecifically precipitated proteins; these polypeptides do not co-migrate with other secretory proteins, and polypeptides of identical mobility are generated upon storage of purified amylase.
The 57,500-dalton polypeptide synthesized in vitro was identified by specific amylase immunoprecipitation (Fig. 2B) Table I. Passage of total membranebound polysomal RNA through oligo(dT)-cellulose bound 87% of the amylase message activity as assayed by specific immunoprecipitation in the reticulocyte system. A second passage of the bound RNA enriched the amylase message activity 17-fold relative to total membrane-bound RNA. In this experiment, 3.2% of the total RNA and 87% of the amylase mRNA activity was obtained in the poly(A+) fraction. Thus, a 27-fold purification is predicted; assuming no loss of message activity during The RNA from each gel fraction was translated in reticulocyte translation assays and amylase immunoprecipitable radioactivity was determined. the procedure.
The discrepancy between the predicted and actual purification values may be due to inactivation of onethird of the amylase RNA during purification, or perhaps to the requirement of a ribosomal RNA for efficient translation as suggested by Kabat (31).

Resolution of Pancreatic mRNA Activities by Formamide-Polyacrylamide Electrophoresis
Resolution of pancreatic mRNAs was performed by electrophoresis in polyacrylamide gels containing formamide to reduce artifacts caused by aggregation and secondary structure. The absorbance profile of poly(A+) RNA after electrophoresis is displayed in Fig. 6. The profile of the RNA is similar when analyzed under nondenaturing conditions (see Fig. ID). To assay for amylase RNA activity, the gel was sliced, and the RNA was extracted and translated in the reticulocyte system (see "Materials and Methods").
Arnylase synthesis directed by fractioned poly(A+) RNA is shown by the histogram in Fig. 6 In the reticuloctye cell-free translation system, the isolated RNA directed the synthesis of a limited number of discrete polypeptides. The number and relative size of these translation products approximate those of pancreatic secretory proteins isolated from zymogen granules or from pancreatic secretions. We are continuing an investigation to determine whether all major translation products are related to the pancreatic secretory proteins.
Two independent criteria indicate that pancreas mRNA directs the synthesis of amylase in heterologous cell-free translation systems. First, a monospecific antibody prepared against purified dog pancreatic amylase selectively precipitates an in vitro translation product approximately the same size as amylase. Second, the methionine-labeled tryptic peptide profile of the in vitro synthesized product is identical to that of authentic amylase. Although this does not confirm identity of the molecules (since not all tryptic peptides are labeled with methionine), it does indicate extensive homology between the synthetic product and amylase.
The in vitro synthesized product is larger than authentic amylase. Consistent differences in electrophoretic mobility in SDS suggest the synthesized product is approximately 1500 daltons larger. The mobility difference remains apparent when both preparations are carboxymethylated prior to electrophoresis. 2 We have not demonstrated a precursor/product relationship; however, the evidence is consistent with the hypothesis that the amylase-like molecule synthesized in vitro is a precursor of the enzyme found in zymogen granules.
The in vitro synthesis of putative dog pancreas secretory protein precursors has also been reported by Devillers-Thiery et al. (30). Larger in vitro translation products have been reported for other secretory proteins in cell-free systems which lack a membrane-bound processing activity. Human placental lactogen (33,34), immunoglobulin heavy ( (37,43) that the additional NH,terminal sequence of secretory protein precursors functions to bind the nascent chain to the endoplasmic reticulum and commits the polypeptide to transport into the cisternae of the endoplasmic reticulum as a first step to secretion. The NH,terminal addition is hydrolyzed during transit into the cisternae (43). The presence of a secretion-specific peptide segment on the nascent chain may explain the specificity of binding to the endoplasmic reticulum of those polysomes synthesizing proteins for export.
We have also obtained evidence that the putative amylase precursor has altered functional properties2 Very small amounts of dog pancreatic amylase can be specifically precipitated by the addition of carrier pig pancreatic amylase and stoichiometric amounts of glycogen as described by Levitzki et al. (49). Amylase molecules and a limit dextrin of the glycogen, each possessing more than one combining site, form an extensive lattice structure that can be collected by brief centrifugation.
The putative amylase precursor is not precipitated under these conditions. This suggests that glycogen binding is blocked by the additional peptide sequence present in in vitro synthesized amylase.
Loss of catalytic activity may be another important biological consequence of the additional sequence in the polypeptide. Thus, the specificity of binding of ribosomes to the endoplasmic reticulum need not be so stringent. If an occasional polysome for a secretory protein were not attached to the endoplasmic reticulum, translation would produce a catalytically inactive molecule which would not interfere with intracellular functions. A catalytically inactive precursor would be an obvious biological advantage for such digestive enzymes as amy-2 R. J. MacDonald, A. E. Przybyla, and W. J. Rutter, unpublished observations. lase, RNase, DNase, and perhaps other secretory molecules not synthesized as inactive zymogens.
The various mRNA species present in the pancreas poly(A+) fraction can be partially resolved by electrophoresis in acrylamide gels. Resolution is indicated not only by the dispersion of optical density (Fig. 6), but also by the analysis of translation products of the separated mRNA.3 In these experiments, there is a direct correlation between the size of the RNA and the size of the translation product. As a consequence of the considerable size range of the pancreatic mRNAs, and the high concentration of the mRNA for amylase, it is feasible to obtain amylase mRNA only slightly contaminated with other mRNAs.
This identification and preliminary characterization of amylase mRNA and further identification of the mRNAs for the remaining secretory proteins will afford a basis for investigations of transcriptional and post-transcriptional control mechanisms operative during acinar cell differentiation.
In particular, it is now possible to use the reticulocyte translation system and specific immunoprecipitation to measure the changing levels of amylase mRNA during pancreatic differentiation.