Biosynthesis of salivary proteins in the parotid gland of the subhuman primate, Macaca fascicularis. Cell-free translation of the mRNA for a proline-rich glycoprotein and partial amino acid sequence and processing of its signal peptide

The major anionic proline-rich proteins in the parotid and submandibular secretions of subhuman primates and man perform the important biological function of inhibiting crystal growth of calcium phosphate salts from saliva, which is supersaturated with calcium phosphate salts, thereby preventing excess deposition of hydroxylapatite on tooth surfaces. The present work was initiated as a first step towards investigating proline-rich protein biosynthesis in parotid glands using the subhuman primate, Macaca fascicularis, as a model system. RNA was isolated from macaque parotid glands and separated into poly(A)-enriched and poly(A)-deficient fractions by chromatography on oligo(dT)-cellulose. The mRNAs in both fractions promoted incorporation of radiolabeled amino acids into polypeptides in an mRNA-dependent reticulocyte lysate translation system. Five major proline-rich polypeptides were detected and one of these was shown to be the in vitro precursor of the major anionic macaque proline-rich protein (MPRP), which is the structural and functional counterpart of the major anionic proline-rich proteins in the parotid and submandibular secretions of man (Oppenheim, F.G., Offner, G.D., and Troxler, R.F. (1982) J. Biol. Chem. 257, 9271-9282). Radiosequencing of the material in anti-MPRP immune precipitates showed that the in vitro precursor of MPRP contained an 18-residue signal peptide. The in vitro precursor of MPRP was processed in dog pancreas vesicles to a form with a lower apparent Mr and with an NH2-terminal amino acid sequence identical to that of native MPRP. The phenylthiohydantoin derivatives of Ala and Ile were detected at residue 9 and those of Val and Met were detected at residue 16 of the signal peptide. This indicated that the in vitro precursor of MPRP, which migrated electrophoretically as a single band in anti-MPRP immune precipitates, contained two different in vitro polypeptides derived from two different mRNAs. These results are discussed in the context of the genetic polymorphism among the major anionic proline-rich proteins in the parotid and submandibular secretions of man.     

Polyadenylated mRNA from the photosynthetic procaryote Rhodospirillum rubrum.

Total cellular RNA extracted from Rhodospirillum rubrum cultured in butyrate-containing medium under strict photosynthetic conditions to the stationary phase of growth has been fractionated on an oligodeoxythymidylic acid-cellulose column into polyadenylated [poly(A)+] RNA and poly(A)- RNA fractions. The poly(A)+ fraction was 9 to 10% of the total bulk RNA isolated. Analysis of the poly(A)+ RNA on a denaturing urea-polyacrylamide gel revealed four sharp bands of RNA distributed in heterodisperse fashion between 16S and 9S. Similar fractionation of the poly(A)- RNA resulted in the separation of 23, 16, and 5S rRNAs and 4S tRNA. Poly(A)+ fragments isolated after combined digestion with pancreatic A and T1 RNases and analysis by denaturing gel electrophoresis demonstrated two major components of 80 and 100 residues. Alkaline hydrolysis of the nuclease-resistant, purified residues showed AMP-rich nucleotides. Through the use of snake venom phosphodiesterase, poly(A) tracts were placed at the 3' end of poly(A)+ RNA. Stimulation of [3H]leucine incorporation into hot trichloroacetic acid-precipitable polypeptides in a cell-free system from wheat germ primed by the poly(A)+ RNA mixture was found to be 220-fold higher than that for poly(A)- RNAs (on a unit mass basis), a finding which demonstrated that poly(A)+ RNAs in R. rubrum are mRNAs. Gel electrophoretic analysis of the translation mixture revealed numerous 3H-labeled products including a major band (Mr, 52,000). The parent protein was precipitated by antibodies to ribulose bisphosphate carboxylase-oxygenase and comprised 6.5% of the total translation products.     

Infidelity of translation of encephalomyocarditis viral RNA with tRNA from human malignant trophoblastic cells.

We have investigated tRNA from the human malignant trophoblastic cells (BeWo cell) and human chorionic tissue for the translation of specific mRNAs, in a tRNA-dependent protein synthesizing system from Ehrlich ascites cells. BeWo cell tRNA and chorionic tRNA supported oviduct mRNA or encephalo-myocarditis (EMC) viral RNA directed amino acid incorporation into polypeptides equally effectively. Polypeptides synthesized with oviduct mRNA and tRNA from both sources were identical upon sodium dodecylsulfate polyacrylamide gel electrophoresis. But the EMC RNA directed polypeptides synthesized with BeWo cell tRNA were different from those synthesized with chorionic tRNA. A polypeptide (molecular weight 58,000) was apparently not synthesized and the synthesis of a faster moving component (molecular weight, 14,000) was enhanced when BeWo cell tRNA was used. These results imply a functional difference in tRNA from human malignant cells compared to their normal counterpart.     

Basic proline-rich proteins of murine parotid glands. Induction of mRNA by isoprenaline and post-secretion processing

Five major basic polypeptides with characteristics typical of proline-rich proteins, accumulated in parotid glands after long term isoprenaline treatment of Balb C mice. They were studied by two-dimensional gel electrophoresis and designated B1 degree, B2' degrees, B2 degrees, B3 degrees and B4 degrees on the basis of pI-dependent mobility. They were not observed in the glands of normal mice and were precipitated when glands were homogenized in 10% trichloroacetic acid unlike the three isoprenaline-induced proline-rich proteins of murine parotid glands reported previously. Isoprenaline induced six proline-rich in vitro translation products which were absent normally. Four of these species had pI-dependent mobilities almost identical to B1 degree, B2 degrees, B3 degrees and B4 degrees, indicating not only precursor/product relationships, but also that isoprenaline induced the accumulation of the proteins by regulating the mRNA. Identical salivary counterparts of the basic glandular proline-rich proteins were not detected whereas a series of smaller and more basic isoprenaline-induced polypeptides were observed in saliva (major speices B1s-B4s). The glandular proline-rich proteins were secreted from parotid tissue in vitro and the data indicate that proline-rich proteins are synthesised as precursors and processed into salivary form in the parotid glands after secretion. The relationships between the B-type in vitro translation products, parotid gland precursors and salivary proteins were also confirmed immunologically.     

Characterization and in vitro translation of polyadenylated messenger ribonucleic acid from Neurospora crassa.

Ribonucleic acid (RNA) extracted from Neurospora crassa has been fractionated by oligodeoxythymidylic acid [oligo(dT)]-cellulose chromatography into polyadenylated messenger RNA [poly(A) mRNA] and unbound RNA. The poly(A) mRNA, which comprises approximately 1.7% of the total cellular RNA, was further characterized by Sepharose 4B chromatography and polyacrylamide gel electrophoresis. Both techniques showed that the poly(A) mRNA was heterodisperse in size, with an average molecular weight similar to that of 17S ribosomal RNA (rRNA). The poly(A) segments isolated from the poly(A) mRNA were relatively short, with three major size classes of 30, 55, and 70 nucleotides. Gel electrophoresis of the non-poly(A) RNA indicated that it contained primarily rRNA and 4S RNA. The optimal conditions were determined for the translation of Neurospora mRNA in a cell-free wheat germ protein-synthesizing system. Poly(A) mRNA stimulated the incorporation of [14C]leucine into polypeptides ranging in size from 10,000 to 100,000 daltons. The RNA that did not bind to oligo(dT)-cellulose also stimulated the incorporation of [14C]leucine, indicating that this fraction contains a significant concentration of mRNA which has either no poly(A) or very short poly(A) segments. In addition, the translation of both poly(A) mRNA and unbound mRNA was inhibited by 7-methylguanosine-5'-monophosphate (m7G5'p). This is preliminary evidence for the existence of a 5'-RNA "cap" on Neurospora mRNA.     

RNA synthesis in cultured human placenta

The in vitro synthesis of RNA in the human placental tissue, incubated in organ culture, was investigated. We followed the synthesis of the poly A(-) and poly A(+) RNA fractions, and investigated the distribution of the newly synthesized RNA among the subcellular fractions isolated from first and third trimester placentas.The poly A(-) RNA was the major fraction of the RNA synthesized in vitro. The incorporation of [³H]uridine into the poly A(+) RNA fraction was very low.As protein synthesis occurred during the entire incubation period, we suggest the presence of a pool of mRNA molecules in the form of mRNP particles.     

Structure and function of rat liver polysome populations. II. Characterization of polyadenylate-containing mRNA associated with subpopulations of membrane-bound particles

Poly(A)+RNA fractions prepared from free and loosely and tightly membrane-bound polysome populations (poly(A)+RNAfree, poly(A)+RNAloose, and poly(A)+RNAtight) were used to drive cDNA in homologous and heterologous hybridization reactions. A large fraction by mass of sequences was shared among the three poly(A)+RNA populations, but shared sequences exhibited distinct frequency distributions within the different populations. 13-15 in vitro translation products of poly(A)+RNAfree and poly(A)+RNAloose detected by gel electrophoresis were shared. Most of these were produced in different relative quantities by the two RNA populations. Five or six higher mol wt polypeptides were produced by poly(A)+RNAloose that were not detected as products of either poly(A)+free or poly(A)+RNAtight. We suggest that loosely bound polysomes may not be artifactually derived as reflected in their quantitatively distinct poly(A)+RNA population. Two tightly membrane-bound RNP fractions were prepared from rat liver on the basis of their release from or retention on purified rough microsomes or a crude membrane fraction after in vitro disaggregation of polysomes with high-salt and puromycin. Homologous and heterologous hybridizations involving their poly(A)+RNA fractions revealed that a large portion by mass of sequences was shared but that these sequences exhibited distinct frequency distributions in the two fractions. The RNA fractions produced exhibited distinct frequency distributions in the two fractions. The RNA fractions produced an identical set of in vitro translation products but individual polypeptides were produced in different relative quantities. This indicates that the two RNP fractions do not arise by any random artifactual process and suggests that they may represent functionally distinct populations.     

Translational properties of a non-polyadenylated oligo(U)-containing RNA fraction from wheat embryos

A non-polyadenylated oligo(U)-containing RNA (poly(A)- . oligo(U)+ RNA) fraction was isolated from wheat embryo cytoplasm and its properties were compared with those of polyadenylated RNA (poly(A)+ RNA) from the same source. Both RNA preparations were highly heterogeneous and effectively stimulated [14C]leucine incorporation in a wheat germ cell-free translation system. Electrophoretic patterns of the translation products appearing in the non-polyadenylated RNA- and polyadenylated RNA-supplemented translation assays, respectively, differed from each other. The non-polyadenylated RNA-specific translation products included, in particular, a series of high molecular weight polypeptides. It is concluded that a specific class of non-polyadenylated oligo(U)-containing mRNA species (other than histone mRNAs) occurs in the wheat embryo cells.     

Messenger RNA of human immune interferon: isolation and partial characterization

Suspension cultures of total and lymphocyte-enriched peripheral white blood cells were used as a source for the preparation of mRNA for human γ-interferon. The cells were lysed and total RNA was extracted with the phenol method. A poly(A)-rich RNA fraction was isolated by affinity chromatography on oligo(dT)-cellulose and further purified on a preparative sucrose gradient. The RNA preparations were translated by oocytes into (a) protein(s) that showed biological activity in the interferon-assay. On sucrose gradient centrifugation the translatable fractions of the RNA migrated with a sedimentation coefficient of approximately 15 S, a value compatible with the molecular weight of human γ-interferon. The translation product was further characterized by serological cross reactivity with purified γ-interferon in neutralization reactions.     

The biogenesis of the cyanellae of Cyanophora paradoxa. III. In vitro synthesis of cyanellar polypeptides using separated cytoplasmic and cyanellar RNA

RNA from Cyanophora paradoxa was separated into cytoplasmic and cyanellar fractions by using a combination of subcellular fractionation and oligo-dT chromatography. In vitro translation of the separated cytoplasmic and cyanellar RNAs in a rabbit reticulocyte lysate system in the presence of [35S]methionine resulted in the incorporation of radiolabel into electrophoretically distinct sets of polypeptides. Monospecific and polyspecific antibodies that react with cyanellar polypeptides were used to probe the in vitro translation products by indirect immunoprecipitation by using Staphylococcus protein A conjugated to Sepharose beads. The results indicate that linker polypeptide L1 of the phycobilisome, the gamma subunit of coupling factor CF1, and subunit II of PS I are synthesized in the cytoplasm as precursor molecules that are 5-8 kDa larger than their mature sizes. Antibodies directed against the psbA gene product (the D1 protein) precipitated a polypeptide found in the translation products of the cyanellar RNA-directed reactions, which is about 1.5 kDa larger than the mature protein.     

Gene activity during germination of spores of the fern, Onoclea sensibilis. Cell-free translation analysis of mRNA of spores and the effect of alpha-amanitin on spore germination

Poly(A)-RNA fractions of dormant, dark-imbibed (non-germinating) and photoinduced (germinating) spores of Onoclea sensibilis were poor templates in the rabbit reticulocyte lysate protein synthesizing system, but the translational efficiency of poly(A)+RNA was considerably higher than that of unfractionated RNA. Poly(A)+RNA isolated from photoinduced spores had a consistently higher translational efficiency than poly(A)+RNA from dark-imbibed spores. Analysis of the translation products by one-dimensional polyacrylamide gel electrophoresis showed no qualitative differences in the mRNA populations of dormant, dark-imbibed, and photoinduced spores. However, poly(A)+RNA from dark-imbibed spores appeared to encode in vitro fewer detectable polypeptides at a reduced intensity than photoinduced spores. A DNA clone encoding the large subunit of maize ribulose bisphosphate carboxylase hybridized at strong to moderate intensity to RNA isolated from dark-imbibed spores, indicating the absence of mRNA degradation. Although alpha-amanitin did not inhibit the germination of spores, the drug prevented the elongation of the rhizoid and protonemal initial with a concomitant effect on the synthesis of poly(A)+RNA. These results are consistent with the view that some form of translational control involving stored mRNA operates during dark-imbibition and photoinduced germination of spores.     

The structural genes for three Drosophila glue proteins reside at a single polytene chromosome puff locus.

The polytene chromosome puff at 68C on the Drosophila melanogaster third chromosome is thought from genetic experiments to contain the structural gene for one of the secreted salivary gland glue polypeptides, sgs-3. Previous work has demonstrated that the DNA included in this puff contains sequences that are transcribed to give three different polyadenylated RNAs that are abundant in third-larval-instar salivary glands. These have been called the group II, group III, and group IV RNAs. In the experiments reported here, we used the nucleotide sequence of the DNA coding for these RNAs to predict some of the physical and chemical properties expected of their protein products, including molecular weight, amino acid composition, and amino acid sequence. Salivary gland polypeptides with molecular weights similar to those expected for the 68C RNA translation products, and with the expected degree of incorporation of different radioactive amino acids, were purified. These proteins were shown by amino acid sequencing to correspond to the protein products of the 68C RNAs. It was further shown that each of these proteins is a part of the secreted salivary gland glue: the group IV RNA codes for the previously described sgs-3, whereas the group II and III RNAs code for the newly identified glue polypeptides sgs-8 and sgs-7.     

Protein synthesis in chloroplasts. VIII. Differential synthesis of chloroplast proteins during spinach leaf development

Excised primary leaves of spinach (Spinacia oleracea) incorporate [35S]-methionine into a number of chloroplast polypeptides. The ratio of incorporation of isotope into the large subunit of ribulose bisphosphate carboxylase relative to a thylakoid polypeptide (peak D) decreases during leaf development in whole leaves; this changing pattern of incorporation is also observed in isolated chloroplasts where these two polypeptides are the major products of protein synthesis. Chloroplast RNA prepared from developing leaves was translated in a reticulocyte lysate extract to yield full-length carboxylase large subunit and peak D polypeptides. The fidelity of translation of these two polypeptides was checked by partial protease digestion. Changes in the synthesis of the large subunit of the carboxylase and peak D in developing leaves are reflected in changes in the amount of translatable mRNA for these two polypeptides.     

Molecular cloning of human intestinal mucin cDNAs. Sequence analysis and evidence for genetic polymorphism

A human small intestine lambda gt11 cDNA library was screened using antisera prepared against the deglycosylated protein backbone of human colon cancer xenograft mucin. Three cDNAs were isolated from this screening, designated SMUC 40-42. These cDNAs were all found to contain tandem repeats of 69 nucleotides which encoded a threonine- and proline-rich protein consensus sequence of PTTTPITTTTTVTPTPTPTGTQT. RNA blots probed with one of these cDNAs, SMUC 41, exhibited large, polydisperse hybridization bands at approximately 7,600 bases. Band intensities were strongest when human small intestine, colon, and colon cancer poly(A)+ RNA was used. In vitro translation of poly(A)+ RNA from human small intestine, colon, and colon cancer cells produced a 162,000-dalton peptide that was immunoprecipitated with antibodies to deglycosylated mucin. SMUC 41 was also used to probe DNA blots, which indicated the presence of restriction fragment length polymorphisms in the intestinal mucin gene. These findings may be important in assessing the abnormal mucins found associated with several human diseases.     

Cell-free synthesis of rat liver zinc-thioneins.

The induction of rat liver zinc-thioneins mRNA was studied in a wheat germ cell-free translation system. Liver poly A rich polysomal RNA was isolated from rats which had been injected with zinc sulfate 5 h previously. These RNA preparations stimulated the incorporation of [35S]cystine into trichloroacetic acid insoluble proteins when assayed in the cell-free synthetic system. The translation products were characterized by Sephadex G-75 chromatography in 8 M urea--50 mM beta-mercaptoethanol, by disc gel electrophoresis in 4 M area--Tris-glycine buffer (pH 9.2), and by peptide fingerprinting with pepsin. These results were identical with authentic rat liver zinc-thioneins. The zinc-thioneins mRNA activity in the control rats, however, was minimal. The stimulation in zinc-thioneins synthesis observed in the cell-free synthesis was similar to the increased synthesis of these polypeptides in vivo.     

Heat-shock protein synthesis in Chlamydomonas reinhardi. Translational control at the level of initiation of a poly(A)-rich-RNA coded 22-KDa protein in a cell-free system

The effect of temperature on the in vitro translation of control and heat-shock poly(A)-rich RNA, obtained from Chlamydomonas reinhardi cells, incubated for 2 h at 25 degrees C respectively, was studied using the wheat-germ translation system. Incubation of the cells at 42 degrees C induces the synthesis of RNAs coding for several heat-shock proteins, including a 22-kDa major polypeptide as well as several proteins of 45-94 kDa, as demonstrated by run-off translation of polyribosomes isolated from intact cells. However, the high-molecular-mass heat-shock proteins are poorly translated in the wheat-germ system. The poly(A)-rich RNA coding for the 22-kDa heat-induced polypeptide has an apparent sedimentation coefficient higher than that expected from the molecular mass of its translation product, and was preferentially translated in vitro at temperatures above 31 degrees C as compared with pre-existing RNAs. Raising the temperature of translation, slightly inhibited (10%) the runoff translation of polyribosomes isolated from intact cells. However, when initiation was carried out in vitro for a short time at increasing temperatures and translation continued at 25 degrees C in the presence of aurintricarboxylic acid, the 22-kDa heat-shock polypeptides was preferentially translated. Aurintricarboxylic acid did not significantly inhibit incorporation of [35S]methionine when added to polyribosomes isolated from control or heat-shocked cells. From the above data we conclude that the translation of the 22-kDa heat-shock protein is controlled in vitro at the initiation level.     

Histone messenger RNAs of the mouse testis

A 6-12S RNA fraction has been isolated following sucrose gradient fractionation of mouse testis RNA, and further resolved into poly A+ and poly A- RNA fractions by oligo-(dt)-cellulose chromatography. Polyacrylamide gel electrophoresis of products formed in a reticulocyte lysate-dependent cell-free translation system has enabled identification of histone variants, H1t, H2S, H2A . X, an H4-like protein and a low Mr protein (presumably TP and/or protamine). Cell-free synthesis of a number of these histone variants appears to be directed by poly A+ mRNAs.     

Cell-free synthesis of tumor-type poly(A) polymerase

Previous studies in this laboratory suggested that in adult liver, either the gene for the tumor-type poly(A) polymerase is poorly transcribed or the mRNA for this enzyme is largely not expressed. To test these possibilities, total RNA from rat liver and Morris hepatoma 3924A RNA were isolated by using a guanidine thiocyanate method; poly(A+) RNA and poly(A-) RNA were separated by oligo(dT)-cellulose chromatography and used for translation in a rabbit reticulocyte lysate system. After in vitro translation, the products were immunoprecipitated with either purified anti-tumor poly(A) polymerase antibodies or control immunoglobulins. When the polypeptides translated from poly(A+) or poly(A-) hepatoma RNA were precipitated with immune sera, a unique [35S]methionine-labeled 35-kilodalton (kDa) protein was observed. This band was not apparent when control serum was used for the immunoprecipitation. The radiolabeled 35-kDa polypeptide was not evident when the products were incubated with highly purified tumor nuclear poly(A) polymerase prior to immunoprecipitation. Prior incubation of the translation products with bovine serum albumin instead of poly(A) polymerase had no effect on the immunoprecipitation. This 35-kDa protein was not apparent when liver poly(A+) RNA was used to direct translation. These data demonstrate that (a) the tumor enzyme is not synthesized as a precursor, (b) tumor mRNA, but not normal liver mRNA, contains detectable sequences coding for tumor-type poly(A) polymerase, and (c) poly(A) polymerase mRNA also exists as a poly(A-) population.     

The synthesis of freezing-point-depressing protein of the winter flounder Pseudopleuronectusamericanus in Xenpuslaevis oocytes

The serum of the winter flounder $\text{Pseudopleuronectus}$$\text{americanus}$ contains a freezing-point-depressing protein of a molecular weight approximately 10,000 with 60% alanine in its composition. When injected into Xenopus o?cyte, a 6–10 S, poly A-rich RNA preparation isolated from the fish liver polysomes stimulated 3–4 fold the incorporation of [³H] alanine into 10% trichloroacetic acid-soluble, non-dialysable proteins. Analysis of the protein fractions showed a translation product similar in molecular weight and electrophoretic mobility to flounder freezing-point-depressing protein. These observations indicated that the 6–10 S RNA from the flounder contained mRNA for the synthesis of flounder's freezing-point-depressing protein.