Electrophoretic separation of in vitro translation products on giant two-dimensional gels allows detailed analysis of cellular mRNAs

The in vitro translation products of mRNA pretreated with methylmercuric hydroxide were examined by giant two-dimensional gel electrophoresis. In addition to increasing overall translational efficiency approximately 2.5-fold, methylmercuric hydroxide selectively increases the translation of mRNAs coding for higher molecular mass (greater than 45 kDa) proteins, allowing the routine resolution of 1500 [35S]methionine-labeled proteins. This yields 3 to 4-fold the number of translation products seen with smaller size two-dimensional gels. With this method we compare thymus cell proteins synthesized in vivo with the products of in vitro translation of mRNA recovered from thymus cells. Fifty-eight percent of the translation products are qualitatively the same as proteins synthesized in vivo (similar Mr, pI, and neighboring proteins), with 64% of these also being quantitatively similar (less than 5-fold difference). A comparison of thymus mRNA in vitro translation products with those coded for by mRNA from liver reveals only 32% qualitative similarity, with 63% of these also being quantitatively similar. These results are discussed in relation to predictions of mRNA abundance and complexity based on DNA:RNA hybridization data. Giant two-dimensional gel separations of in vitro translation products appear to be useful for detecting less abundant cellular mRNAs, including those that may be regulated by hormones or other physiological mediators.     

In vitro translation of epidermal RNA from different anatomical regions and metamorphic stages of Hyalophora cecropia

Epidermal RNA isolated from different anatomical regions and metamorphic stages of Hyalophora cecropia was translated in vitro with commercial wheat germ and rabbit reticulocyte systems. The translation products were analyzed by 2D gel electrophoresis. The two systems yielded identical products if canine microsomal membranes were added to remove signal peptides from the reticulocyte products. The endogenous processing by the wheat germ extract occurred even in the presence of protease inhibitors. Some of the processed translation products co-migrated with unlabeled cuticular protein standards. All of the major cuticular proteins could be identified, but only when translations were carried out with RNA from epidermis underlying cuticle containing these proteins. Hence, cuticular protein distribution is due to differential synthesis and not to differential extractability. For larval abdominal RNA, most of the major translation products did not co-migrate with known larval cuticular proteins or with proteins synthesized and retained by the epidermis. These unknown products were lower in apparent molecular weight than the cuticular proteins. Their identity remains unknown; they may be premature translation products, but altering translation conditions did not reduce their abundance.     

Acyl-Coa oxidase and hydratase-dehydrogenase, two enzymes of the peroxisomal beta-oxidation system, are synthesized on free polysomes of clofibrate-treated rat liver

We investigated the site of synthesis of two abundant proteins in clofibrate-induced rat hepatic peroxisomes. RNA was extracted from free and membrane-bound polysomes, heated to improve translational efficiency, and translated in the mRNA-dependent, reticulocyte-lysate- cell-free, protein-synthesizing system. The peroxisomal acyl-CoA oxidase and enoyl-CoA hydratase-beta-hydroxyacyl-CoA dehydrogenase 35S- translation products were isolated immunochemically, analyzed by SDS PAGE and fluorography, and quantitated by densitometric scanning. The RNAs coding for these two peroxisomal proteins were found predominantly on free polysomes, and the translation products co-migrated with the mature proteins. As in normal rat liver, preproalbumin and catalase were synthesized mainly by membrane-bound and by free polysomes, respectively. mRNAs for a number of minor 35S-translation products also retained by the anti-peroxisomal immunoadsorbent were similarly found on free polysomes. These results, together with previous data, allow the generalization that the content proteins of rat liver peroxisomes are synthesized on free polysomes, and the data imply a posttranslational packaging mechanism for these major content proteins.     

Variation of Cell-free Translation Profiles Among Pathogenic Strains of Soybean Mosaic Virus

Cell-free translation products from isolates representing soybean mosaic virus (SMV) strains G1 to G7 and G7a, along with several other SMV isolates, were analyzed. SMV RNAs were translated in both rabbit reticulocyte lysates and wheat germ extracts, yielding approximately 20 translation products for each strain from each translation system. Comparison of translation profiles by the presence or absence of proteins allowed for the formation of distinctive groups from each cell-free translation system. Groupings formed by analysis of products from rabbit reticulocyte lysates correlated with pathogenicity; groupings formed by analysis of products from wheat germ extracts had no apparent biological significance.     

Variations in Brain Cortical Polysome Translation Products Among Rats of the Same Strain

Studies were undertaken to determine whether there exist variations among the translation products of polysomes from different brains of animals of the same strain. Polysomes were prepared from individual rat cortices and translated in a reticulocyte protein-synthesizing system containing rabbit reticulocyte factors and L-[35S]methionine; he resulting radioactive proteins were analyzed by two-dimensional polyacrylamide gel electrophoresis autoradiography. Comparison of the autoradiographs revealed that two acidic proteins, A and B, of apparent 54,000 M. W. occur as three phenotype: A only, B only, or A plus B. These proteins were not detectable by Coomassie brilliant blue staining of two-dimensional electrophoretograms of brain protein preparations. Messenger RNA was extracted from pooled cortices and translated in a wheat germ extract, and both A and B proteins were detected among the products of translation. Cyclic AMP affinity chromatography of the translation products of cortical polysomes showed that both A and B proteins bind to cyclic AMP. Our data are consistent with the conclusion that there are qualitative differences in the polysome translation products that bind to cyclic AMP among individual cortices of rats of the same strain.     

In vitro synthesis and assembly of photosystem II proteins of spinach chloroplasts

The synthesis and assembly of photosystem II (PS II) proteins of spinach chloroplasts were investigated in three different in vitro systems, i.e., protein synthesis in isolated chloroplasts (in organello translation), read-out translation of thylakoid-bound ribosomes, and transport of translation products from spinach leaf polyadenylated RNA into isolated chloroplasts. Polyacrylamide gel electrophoresis of labeled thylakoid polypeptides in the presence of sodium dodecyl sulfate revealed that the first two systems were capable of synthesizing the reaction center proteins of PS II (47 and 43 kDa), the herbicide-binding protein, and cytochrome b559. The reaction center proteins synthesized in organello were shown to bind chlorophyll and to assemble properly into the PS II core complex. One of the reaction center proteins translated by the thylakoid-bound ribosomes (47 kDa) was also found to be integrated in situ into the complex but was lacking bound chlorophyll. Incorporation of radioactivity into the three extrinsic proteins of the oxygen-evolution system (33, 24, and 18 kDa) was detected only when intact chloroplasts were incubated with the translation products from polyadenylated RNA, showing that these proteins are coded for by nuclear DNA. The occurrence of a precursor polypeptide 6 kDa larger than the 33-kDa protein was immunochemically detected in the translation products.     

Identification of proteins encoded by the Gazdar murine sarcoma virus genome by in vitro translation and comparison with Moloney murine sarcoma virus 124.

The gene products of Gazdar murine sarcoma virus (Gz-MuSV) were identified by in vitro translation of Gz-MuSV virion RNA. An overlapping set of proteins with approximate molecular weights of 37,000 (37K), 33K, 24K, and 18K were synthesized from the transforming gene of Gz-MuSV, v-mosGz. In addition, Gz-MuSV-specific RNA directed the in vitro synthesis of a 62K gag gene protein and a 37.5K env gene-related product. The Gz-MuSV-specific in vitro translation products were compared with the in vitro translation products of M-MuSV 124, an independent isolate with a similar v-mos gene. This analysis showed that the 62K Gz-MuSV gag gene protein and the 37K, 33K, 24K, and 18K v-mosGz proteins were almost identical to the M-MuSV 124 62K (gag) and 37K, 33K, 24K, and 18K (v-mosMo) proteins that we previously identified and characterized. The 37.5K env gene product from Gz-MuSV does not have a correlate in the M-MuSV 124 translation products. These results were analyzed in the context of expectations based on similarities and differences in genetic organization of these two viral genomes.     

Characterization of the size and 5' cap of messenger RNA encoding neurophysin precursors

The coding activity of bovine hypothalamic poly A+ mRNA for neurophysin I and II immunoreactive proteins was characterized with respect to size and 5' cap. The mRNA was fractionated by methylmercuric hydroxide agarose gel electrophoresis and subsequently translated in vitro in rabbit reticulocyte lysates. Alternatively, mRNA was fractionated by gel exclusion HPLC and translated in wheat germ extracts. Immunoprecipitated translation products were analyzed by gel exclusion HPLC. Neurophysin-immunospecific protein of approximately 17,000 daltons, the size expected for the neuropeptide hormone-neurophysin precursors, was encoded by mRNA species of two size classes. The smaller class of mRNA's was of the size expected from the size of the precursor proteins. The larger class was 5-10 times larger. The low K+ concentration optimum for translation of unfractionated mRNA encoding neurophysin I immunoreactive proteins and the inability of a cap analogue to inhibit this translation suggest that mRNA species encoding neurophysin I-immunoreactive translation products are incompletely capped. By contrast, the mRNA encoding neurophysin II-immunoreactive products appear to contain a normal cap structure.     

Sequence of formation of molecular forms of plasminogen and plasmin-inhibitor complexes in plasma activated by urokinase or tissue-type plasminogen activator.

Total cellular polyadenylated RNA [poly(A)+ RNA] was prepared after guanidinium thiocyanate extraction of frozen brain tissue from age-matched normal and Down's-syndrome (trisomy 21) human foetuses. Poly(A)+ RNA populations were analysed by translation in vitro, followed by two-dimensional gel analysis by using both isoelectric focusing (ISODALT system) and non-equilibrium pH-gradient electrophoresis (BASODALT system) as the first-dimension separation. The relative concentrations of poly(A)+ RNA species coding for seven translation products were significantly altered in Down's syndrome, as determined by both visual comparisons of translation-product fluorograms from normal and Down's-syndrome samples and by quantitative radioactivity determination of individual translation products. The relative concentrations of mRNA species coding for two proteins (68 kDa and 49 kDa) were increased in Down's syndrome and may represent genes located on chromosome 21. The relative concentrations of mRNA species coding for five proteins (37 kDa, 35 kDa, 25.5 kDa, 24.5 kDa, 23 kDa) were decreased in Down's syndrome, these probably representing secondary effects of the trisomy. Six Down's-syndrome-linked translation products (49 kDa, 37 kDa, 33 kDa, 25.5 kDa, 24.5 kDa, 23 kDa) did not migrate with appreciable amounts of cellular proteins on two-dimensional gels and hence may represent either proteins of high turnover rates or those that are post-translationally modified in vivo. One translation product (68 kDa) comigrated with a major cellular protein species, which was identified as a 68 kDa microtubule-associated protein by limited peptide mapping. The significance of these changes is discussed in relation to the mechanisms whereby the Down's-syndrome phenotype is expressed in the human brain.     

Cell-Free Translation of Free and Membrane-Bound Polysomes and Polyadenylated mRNA from Rabbit Brain Following Administration of d-Lysergic Acid Diethylamide In Vivo

Abstract: Free and membrane-bound polysomes and polyadenylated mRNA isolated from rabbit brain were translated in an mRNA-dependent rabbit reticulocyte lysate system. Electrophoretic analysis of the cell-free translation products demonstrated that although most of the nascent proteins were common to both free and membrane-bound brain polysomes, qualitative and quantitative differences were observed. Compared with the results obtained with purified polyadenylated mRNA, the addition of intact polysomes to the cell-free translation assay was more efficient and produced higher molecular weight products. Analysis of the translation products of free and membrane-bound polysomes revealed the appearance of 74K protein following either LSD administration or hyperthermia induced by elevated temperature treatment. The presence of this 74K protein was verified by analysis of the translation products by two-dimensional gel electrophoresis.     

Synthesis of murine mammary tumor viral proteins in vitro

The coding potential of murine mammary tumor viral genomic RNA was investigated by in vitro translation of various size classes of RNAs isolated from the virions. The major products of translation of full-size 35S polyadenylylated virion RNA were gag-related polyproteins of 75,000, 105,000, and 180,000 daltons (P75, P105, and P180, respectively). Studies on the kinetics of translation of these three proteins established that they were synthesized independently and that the smaller proteins were not post-translational cleavage products of the larger proteins. Tryptic peptide mapping showed that almost all of the P75 sequences were contained within P105 and almost all of the P105 sequences were contained within P180. The syntheses of all three proteins were inhibited by m7GTP, indicating that they were translated from capped mRNA's. Although a 24S polyadenylylated RNA had been identified as the intracellular mRNA for env precursor polyprotein, no such protein could be translated from the 24S polyadenylylated RNA isolated from the virions. However, translation of a 14S size class of polyadenylylated virion yielded four prominent proteins of about 36,000, 23,000, 21,000, and 20,000 daltons. These proteins were unrelated to murine mammary tumor viral structural proteins, as suggested from tryptic peptide mapping and immunoprecipitation data. They might be the products of an as-yet-unidentified gene located near the 3' terminus of the murine mammary tumor viral genomic RNA.     

Synthesis of rat liver microsomal cytochrome b5 by free ribosomes

Free and membrane-bound polyribosomes were separated from liver homogenates and characterized by electron microscopy. Using the wheat germ cell-free translation system, total translation products of poly A+RNA extracted from free polyribosomes (poly A+RNAf) showed some correlation to total liver cytosol proteins. In contrast, translation products of poly A+RNA from membrane-bound polyribosomes (poly A+RNAmb) showed some similarity to rat serum. Antibody to purified rat serum albumin immunoprecipitated from only the translation products of poly A+RNAmb a single polypeptide of mol wt 68,000. i.e., 3,000 greater than secreted serum albumin. In contrast, antibody to detergent-extracted cytochrome b5 immunoprecipitated from only the translation products of poly A+RNAf a single polypeptide of mol wt 17,500, identical to that of microsomal cytochrome b5. A consideration of the known properties of cytochrome b5 is consistent with an exclusive site of synthesis on free ribosomes.     

In vivo uptake of mitochondrial malate dehydrogenase from watermelon by mitochondria of Xenopus laevis oocytes

The heterologous in vivo translation system of Xenopus laevis oocytes was used to translate messenger RNA isolated from water-melon cotyledons. Immunocytochemistry was used to localize the translation products in situ within the oocyte. In addition, the translation products were immunoprecipitated from homogenized oocytes, separated on SDS-polyacrylamide electrophoresis and visualized by fluorography. A variety of watermelon proteins encoded in the injected mRNA were translated within the oocytes. Among them was the mitochondrial isoenzyme of malate dehydrogenase (mtMDH). The mtMDH was correctly imported into the mitochondria of the oocytes, as detected by immunocytochemistry.     

Structural proteins and cell-free translation products of total RNA and hybrid-selected RNA from two DNA variants of vaccinia virus.

Two major variants of vaccinia virus, large (L) and small (S), differ by a deletion of 9.7 kilobase pairs. The structural proteins and the translation products of RNA transcribed in vitro from each of these variants were analyzed by gel electrophoresis. Recombinant plasmids were used to select RNA transcribed from the L variant sequences corresponding to the deletion. This RNA yielded translation products indicating that a minimum of 11 polypeptides, including two structural proteins, map within the deletion.     

Adequate system for investigation of translation initiation of the human retrotransposon L1 mRNA in vitro

Retrotransposon L1 codes for a unique dicistronic mRNA which serves both a transposition intermediate and a template for the synthesis of two proteins of this mobile element. According to preliminary data, the translation initiation of both cistrons of L1 occurs by non-canonical mechanisms. When translating the L1 mRNA in rabbit reticulocyte lysate (RRL), a standard system routinely used by many researchers to study mechanisms of translation initiation in eukaryotes, we observed along with expected products a number of polypeptides resulted from aberrant initiation at internal AUG codons. Such products are absent on translation of L1 mRNA in vivo. Addition to the system of a cytoplasmic extract from HeLa cells resulted in disappearance of these abberant products whereas the efficiency of translation of the first cistron remained unchanged. The level of translation of the second cistron became significantly lower. This also made the picture closer to that observed in vivo. These and other experiments allowed us to clearly demonstrate that the new combined cell-free system is much more adequate to study mechanisms of translation initiation than a regular RRL.     

Larger precursors of mitochondrial translation products in Neurospora crassa. Indications for a precursor of subunit 1 of cytochrome c oxidase

Specific labeling in vivo of the formylated N termini of mitochondrial translation products revealed that some mitochondrially synthesized proteins were not labeled this way. As a consequence, it was worthwhile considering that larger precursor proteins of mitochondrial translation products exist. Although we used a rapid isolation procedure, only after 2-h of labeling in the presence of cycloheximide, could three additional mitochondrial translation products (molecular mass 45, 36, and 25 kilodaltons) be detected. Preincubation with cycloheximide indicated that these proteins might be larger precursors which were no longer processed due to the prolonged presence of cycloheximide. To prevent processing of the precursors during isolation, cells of the slime mutant were directly lysed in boiling sodium dodecyl sulphate solution. In this way, the same three additional mitochondrial translation products were detected after a pulse-labeling of 1 min. These proteins behave in a precursor-like fashion. Labeling at 9 degrees C resulted in a partial accumulation of the three additional proteins. Finally protein blots treated with antibodies and 125I-labeled protein A, support the idea that the 45-kDa protein is a precursor of subunit 1 of cytochrome c oxidase; 50-80% of this precursor could be detected in the post-mitochondrial supernatant, indicating that this polypeptide is not tightly bound to the membrane.     

Thylakoid membranes: the translational site of chloroplast DNA-regulated thylakoid polypeptides

Stromal ribosomes and those bound to thylakoid membranes were prepared from intact spinach chloroplasts which were purified on Percoll gradients. The products of read-out translation of these ribosomes supplemented with an Escherichia coli extract were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Striking similarity was found between the polypeptides labeled in the read-out translation of the chloroplastic ribosomes and those synthesized in isolated chloroplasts. Among the polypeptides translated on thylakoid-bound ribosomes, apoprotein of chlorophyll-protein complex I, alpha and beta subunits of coupling factor 1, and 32,000-Da membrane polypeptide were identified from their mobility on the polyacrylamide gel. The large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and other several stromal proteins were translated exclusively from stromal ribosomes. However, when the translation was programmed in cell-free systems from either E. coli, wheat germ, or rabbit reticulocytes by RNAs isolated separately from stroma and thylakoids, no qualitative difference was found between the products from those RNAs. These results suggest that thylakoid-bound ribosomes are the main sites of synthesis of thylakoid proteins and stromal-free ribosomes are that of stromal proteins, and that thylakoids and stroma contain mRNAs for the stromal and the thylakoid proteins, respectively, in a form not functioning in the chloroplasts.     

Cell-free translation of silkmoth chorion mRNAs: Identification of protein precursors and characterization of cloned DNAs by hybrid-selected translation

The secretory silkmoth chorion proteins are synthesized as precursors bearing signal peptides. Precursors are detected upon cell-free translation of chorion mRNAs in the wheat germ system; they are processed into products identical in size to authentic chorion proteins when translation is performed in the presence of microsomal membranes from dog pancreas. Precursors corresponding to specific protein size classes and subclasses are identified by three approaches: comparison of precursors and products encoded by stage-specific mRNAs, comparison of precursors and products encoded by mRNAs specifically hybridizing to individual chorion cDNA clones, and comparison of relative amino acid compositions of precursors and authentic chorion proteins. Translation of stage-specific mRNA preparations indicates that, in general, the developmental changes of in vivo chorion protein synthesis are based on changes in concentrations of the corresponding mRNAs. Characterization of the precursors makes it possible to identify, for any chorion DNA clone, the protein subclass, a member of which is encoded by the clone sequence.     

Detection of a proteolytic activity in the micrococcal nuclease used for preparation of messenger-dependent reticulocyte lysates

Some, but not all, commercial preparations of micrococcal nuclease used to remove endogenous mRNA from reticulocyte lysates were found to contain proteolytic activity. The protease(s) caused a time-dependent cleavage of the polyprotein primary translation products of genome RNA of several plant viruses, but did not affect the translation products of some other virus mRNA. The activity resulted in the production of smaller proteins and was inhibited by zinc ions. Thus the protease(s) mimicked virus-coded proteases and represents a potential artifact in studies of translation products of virus RNA.