mRNA-dependent Reticulocyte Lysate Research Articles

Development of a muscle actin specified by maternal and zygotic mRNA in ascidian embryos

In this investigation, we characterize the embryonic and adult actins and describe the embryonic expression of a muscle actin in the ascidian Styela. Two-dimensional polyacrylamide gel electrophoresis showed that embryos, tadpole larvae, and adult organs contain three major and two minor isoforms of actin. Two of the major isoforms, which are present in the mantle, branchial sac, alimentary tract, and gonads of adults and in eggs, embryos, and heads and tails of tadpoles, are likely to be cytoplasmic actins. The third major isoform, which was enriched in the mantle and branchial sac of adults and localized primarily in the tails of tadpoles, is a muscle actin. The muscle actin isoform was not detected in eggs and early embryos. Radioactivity incorporation studies showed that the cytoplasmic actins were synthesized throughout early development, but muscle actin synthesis was first detected between the 16- and 64-cell stages, 2–3 hr after fertilization. Two lines of evidence indicate that embryonic muscle actin synthesis is directed in part by maternal mRNA. First, poly(A) + RNA isolated from unfertilized eggs directed the synthesis of muscle actin in an mRNA-dependent reticulocyte lysate. Second, muscle actin was synthesized in anucleate egg fragments. Arguments are also presented that muscle actin synthesis is not directed exclusively by maternal mRNA. It is concluded that embryonic and adult Styela exhibit actin heterogeneity, that one of the actin isoforms is a muscle actin, and that the muscle actin is synthesized during embryogenesis under the direction of maternal and zygotic mRNA.

Cell-free translation of a chimeric eucaryotic-procaryotic message yields functional chloramphenicol acetyltransferase

A vaccinia virus (VV) recombinant containing the DNA sequences encoding the bacterial chloramphenicol acetyltransferase (CAT) gene was constructed. The ability of the chimeric VV:CAT transcript to be translated in vitro into enzymatically active enzyme was assessed. Addition of mRNA isolated from the cytoplasm of VV:CAT infected cells to a mRNA-dependent reticulocyte lysate resulted in the synthesis of high levels of enzymatically active CAT. These results suggest that this assay may be used in concert with physical assays to study the expression and stability of chimeric transcripts in virus-infected cells.

Inhibition of polypeptide chain initiation in Daudi cells by interferons. Evidence that activity of initiation factor eIF-2 and availability of mRNA are unimpaired

The accompanying paper [McNurlan & Clemens (1986) Biochem. J. 237, 871-876] shows that the inhibition of proliferation of Daudi cells by human interferons is associated with impairment of the overall rate of protein synthesis. We have examined whether two of the mechanisms which are believed to control translation in interferon-treated virus-infected cells may be responsible for the inhibition of protein synthesis during the antiproliferative response in these uninfected cells. Although the rate of polypeptide chain initiation is lower in interferon-treated Daudi cells, as indicated by the disaggregation of polysomes, there is no significant inhibition of activity of initiation factor eIF-2 or of [40 S . Met-tRNAf] initiation complex formation in cell extracts. The phosphorylation state of the alpha subunit of eIF-2 remains unaltered. There is no major decrease in mRNA content as a proportion of total RNA up to 4 days of interferon treatment, as judged by poly(A) content, although the amount of total mRNA/10(6) cells eventually declines. The mRNA present in extracts from interferon-treated cells remains translatable when added to an mRNA-dependent reticulocyte lysate system. We conclude that neither the interferon-inducible eIF-2 protein kinase pathway nor the 2',5'-oligo(adenylate)-ribonuclease L pathway are responsible for the inhibition of polypeptide chain initiation. Rather, the data suggest impairment at the level of formation of [80 S ribosome X mRNA] initiation complexes.

Modulation of the expression of poliovirus proteins in reticulocyte lysates

The translation of poliovirus RNA into specific viral proteins in mRNA-dependent reticulocyte lysates (MDLs) was found to be highly dependent on individual lysate preparations. Under optimal conditions. the first polypeptide detected was always P3-1b (formerly NCVP 1b). the product of the 3′ portion of the poliovirus genome; the formation of P1-1a (formerly NCVP 1a) followed as shown by time-course and pulse-chase experiments. However, some lysates synthesized little or no P1-1a despite their ability to synthesize P3-1b and to translate normally other cellular and viral mRNAs. When an MDL competent in synthesizing P1-1a was diluted ca. twofold, while maintaining optimal concentrations of salts, tRNA, DTT, creatine phosphate, and amino acids, P1-1a formation was virtually eliminated, while the synthesis of P3-1b, presumably as a consequence of a more downstream initiation, was maintained. The synthesis of P1-1a in a diluted MDL was restored, and P3-1b synthesis suppressed, by the addition of a S 10 fraction prepared from uninfected or virus-infected HeLa cells. Nuclease treatment and dialysis of the S 10 fraction did not inhibit its activity. These findings indicate that individual MDLs either possess limiting quantities of, or occasionally are deficient in, a factor(s) that promotes the utilization of the presumed 5′ proximal initiation site (the AUG at nucleotide position 781–783) and that a homologous factor(s) exists in HeLa cells. The implication of these findings for the strategy of poliovirus replication is discussed.

The induction of α1-acid glycoprotein by methylmercury

1. 1. The incorporation of [ 14C]leucine into protein was measured in liver preparations and blood of rats following the s.c. administration of methylmercury hydroxide (24 mg/kg body wt) or turpentine (5.0 ml/kg body wt). 2. 2. The translatability of the RNA obtained from polysomes in an mRNA-dependent reticulocyte lysate was elevated significantly in the preparations derived from the treated rats compared to control rats. 3. 3. Immunoprecipitation of the labelled translation products or of serum proteins showed that the mRNA activity and the synthesis of α 1-acid glycoprotein, an acute phase reactant, was elevated by the methylmercury treatment as well as by the turpentine-induced inflammatory response.

Translation of muscle mRNA in rats following acute exposure to Pb 2+ or Cd 2+

1. The hindleg muscle of rats was studied 2 days following the i.p. administration of 0.5 mg Pb 2+ 100g body wt or 0.12mg Cd 2+ 100g body wt or both Pb 2+ and Cd 2+. 2. The incorporation of [ 14C]leucine into proteins was measured using mRNA obtained from the muscle polysomes. 3. The translatability of this poly(A) + RNA in a mRNA-dependent reticulocyte lysate was elevated similarly in each of the preparations from heavy metal treated rats compared to control rats. 4. Evidence for increased mRNA activity for glyceraldehyde-3-phosphate dehydrogenase and actin was obtained.

Further characterization of L-β-hydroxyacid dehydrogenase from Drosophila

L-β-Hydroxyacid dehydrogenase ( L-β-hydroxyacid--NAD-oxidoreductase, EC 1.1.1.45) of Drosophila is composed of two, identical subunits with a molecular weight of approx. 33 300. The enzyme was purified 938-fold from Drosophila melanogaster. An isoelectric point of 8.6 was determined for L-β-hydroxyacid dehydrogenase. An amino acid analysis was conducted of the purified enzyme. A single subunit was obtained by SDS-gel electrophoresis of the purified enzyme. Translation of larval and adult mRNA in a mRNA-dependent reticulocyte lysate, followed by immune precipitation using anti- L-β-hydroxyacid dehydrogenase IgG revealed a single L-β-hydroxyacid dehydrogenase subunit of 33 300. Larval and adult proteins were the same size. The enzyme does not appear to be subjected to substantial post-translational modifications.

Ethanol-glucocorticoid regulation of hepatic glucose-6-phosphate dehydrogenase

The effect of ethanol, alone and in combination with glucocorticoid and insulin, on glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49) was studied in primary cultures of rat hepatocytes maintained in a chemically defined medium. Maintenance of hepatocytes from fasted animals in a culture medium devoid of hormones and ethanol resulted in a 2.5-fold increase in G6PDH activity in 48 hr. Parallel cultures treated with glucocorticoid and insulin or glucocorticoid, insulin and ethanol stimulated enzyme activity 6- and 9-fold, respectively in 48 hr. Treatment with ethanol for 48 hr potentiated basal and glucocorticoid plus insulin-induced enzyme activity 1.4-fold. The activity of G6PDH mRNA, estimated by cell-free translation of hepatic mRNA in a mRNA-dependent reticulocyte lysate and by RNA dot-blot hybridization, was compared with enzyme activity and relative rate of G6PDH synthesis. The increases in enzyme activity observed in response to glucocorticoid and insulin or ethanol, alone or in combination with glucocorticoid and insulin, were paralleled by comparable increases in the rate of synthesis and mRNA levels of G6PDH. The results of this study show that the glucocorticoids, insulin and ethanol interact to stimulate the synthesis of G6PDH primarily by increasing the concentration of G6PDH mRNA.

Phytoalexin synthesis in soybean cells: Elicitor induction of phenylalanine ammonia-lyase and chalcone synthase mRNAs and correlation with phytoalexin accumulation

A glucan elicitor from cell walls of the fungus Phytophthara megasperma f. sp. glycinea, a pathogen of soybean ( Glycine max), induced large and rapid increases in the activities of enzymes of general phenylpropanoid metabolism, phenylalanine ammonia-lyase, and of the flavonoid pathway, acetyl-CoA carboxylase and chalcone synthase, in suspensioncultured soybean cells. The changes in phenylalanine ammonia-lyase and chalcone synthase activities were correlated with corresponding changes in the mRNA activities encoding these enzymes, as determined by enzyme synthesis in vitro in a mRNA-dependent reticulocyte lysate. The time courses of the elicitor-induced changes in mRNA activities for both enzymes were very similar with respect to each other. Following the onset of induction, the two mRNA activities increased significantly at 3 h, reached highest levels at 5 to 7 h, and subsequently returned to low values at 10 h. Similar degrees of induction of mRNA activities and of the catalytic activities of phenylalanine ammonia-lyase and chalcone synthase were observed in response to three diverse microbial compounds, the glucan elicitor from P. megasperma, xanthan, an extracellular polysaccharide from Xanthomonas campestris, and endopolygalacturonase from Aspergillus niger. However, whereas the glucan elicitor induced the accumulation of large amounts of the phytoalexin, glyceollin, in soybean cells, endopolygalacturonase induced only low, albeit significant, amounts; xanthan did not enhance glyceollin accumulation under the conditions of this study. This result might imply that enzymes other than phenylalanine ammonia-lyase or chalcone synthase exert an important regulatory function in phytoalexin synthesis in soybean cells.

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.

N protein alone satisfies the requirement for protein synthesis during RNA replication of vesicular stomatitis virus.

Genomic replication of the negative-strand RNA viruses is dependent upon protein synthesis. To examine the requirement for protein synthesis in replication, we developed an in vitro system that supports the genome replication of defective interfering particles of the negative-strand rhabdovirus vesicular stomatitis virus (VSV), as a function of protein synthesis (Wertz, J. Virol. 46:513-522, 1983). The system consists of defective interfering nucleocapsid templates and an mRNA-dependent reticulocyte lysate to support protein synthesis. We report here an analysis of the requirement for individual viral proteins in VSV replication. Viral mRNAs purified by hybridization to cDNA clones were used to direct the synthesis of individual proteins in the in vitro system. By this method, it was demonstrated that the synthesis of the VSV nucleocapsid protein, N, alone, resulted in the replication of genome-length RNA by both defective interfering intracellular nucleocapsids and virion-derived nucleocapsids. Neither the viral phosphoprotein, NS, nor the matrix protein, M, supported RNA replication. The amount of RNA replication for a given amount of N protein was the same in reactions in which either all of the VSV proteins or only N protein were synthesized. In addition, RNA replication products synthesized in reactions containing only newly made N protein assembled with the N protein to form nucleocapsids. These results demonstrate that the major nucleocapsid protein (N) can by itself fulfill the requirement for protein synthesis in RNA replication and allow complete replication, i.e., initiation and elongation, as well as encapsidation of genome-length progeny RNA.

Inhibition of protein synthesis in reticulocyte lysates by a double-stranded RNA component in HeLa mRNA

Double-stranded RNA (dsRNA) inhibits protein synthesis initiation in rabbit reticulocyte lysates by the activation of a latent dsRNA-dependent cAMP-independent protein kinase which phosphorylates the α-subunit of the eukaryotic initiation factor eIF-2. In this study, we describe a dsRNA-like component which is present in preparations of HeLa mRNA (poly A +) isolated from total cytoplasmic RNA. The inhibitory species in the HeLa cytoplasmic mRNA was detected by (a) its ability to inhibit protein synthesis with biphasic kinetics in reticulocyte lysates translating endogenous globin mRNA, and (b) by the inefficient translation of HeLa cytoplasmic mRNA in a nuclease-treated mRNA-dependent reticulocyte lysate. The inhibitory component was characterized as dsRNA by several criteria including (i) the ability to activate the lysate dsRNA-dependent eIF-2α kinase (dsI); (ii) the prevention of both dsI activation and inhibition of protein synthesis by high levels of dsRNA or cAMP; (iii) the reversal of inhibition by eIF-2; and (iv) the inability to inhibit protein synthesis in wheat germ extracts which lack latent dsI. By the same criteria, the putative dsRNA component(s) appears to be absent from preparations of HeLa mRNA isolated exclusively from polyribosomes.

Differential in vitro translation and independent in vivo regulation of mRNA's for subunits of ligandin

Synthesis of both subunits (Y a and Y b) of ligandin in equal amounts was observed when poly(A) + mRNA isolated from the post-mitochondrial fraction was translated in an in vitro wheat-germ system and the products were immunoprecipitated by monospecific antibody to ligandin and analyzed by SDS-polyacrylamide gel electrophoresis and fluorography. When the Mg 2+ or K + concentrations were increased in the in vitro wheat-germ system the ratio of synthesis of Y b Y a subunits was 3. With a mRNA-dependent reticulocyte lysate, the synthesis of Y a subunits was 20–30% higher than Y b subunits. At a fixed K + and Mg 2+ concentration, the ratio of incorporation of [ 35S]methionine into Y b Y a subunits remained 1 and 0.7 in wheat-germ and reticulocyte lysate systems, respectively, up to 60 min. When poly(A) + mRNA was fractionated on a 5–20% sucrose gradient, ligandin mRNA was present in fractions having a peak sedimentation value of 11 S. When poly(A) + mRNA was fractionated by gel electrophoresis, fractions enriched in mRNA for each subunit were obtained. By administration of [ 3H]leucine followed by determination of radioactivity in ligandin and total proteins by immunoprecipitation and trichloroacetic acid precipitation, respectively, synthesis of the Y a subunits was selectively stimulated by phenobarbital administration. When poly(A) + mRNA from liver of rats administered phenobarbital was translated in vitro a selective increase in the mRNA content of Y a subunits was observed. When poly(A) + RNA from testes was translated in the wheat-germ system and products analyzed, Y b subunits were the predominant subunit (> 90%) synthesized, reflecting the subunit composition of testicular ligandin. These results suggest that in spite of the close sequence homology between the two subunits of ligandin, there are separate mRNA's for each subunit which are independently regulated.

Assembly of Procollagen mRNA Translation Products into Pepsin-Resistant Structures

Proteolytic digestion was used to probe the conformation of the preproα chains synthesized by a mRNA-dependent reticulocyte lysate from chicken calvarial RNA. Pepsin-resistant α1- and α2-like chains were recovered even from translation reactions that were not preincubated below the reported Tm of the unhydroxylated triple helix. The pepsin-resistant structures were stable to thermal denaturation at 45 °C and a fraction remained resistant to peptic digestion at 30 °C. Interchain disulfide bonds did not appear to be required for the formation or thermal stability of these structures. Pepsin resistance is normally interpreted as evidence for a triple-helical conformation. Therefore, these results suggest that the in vitro synthesized preproα chains contain the requisite information to associate in register for correct helix folding. The unusual thermal stability of these structures is not understood, but this may indicate assembly into higher orders of structure.

Translation of mRNA from rat kidney following acute exposure to lead

1. 1. Two days after the administration of Pb 2+ (0.54 mg/100 body wt) to rats. the kidneys were removed and homogenate fractions obtained. 2. 2. The machinery for peptide synthesis exhibited increased activity in the kidney preparations from lead-treated rats. 3. 3. The poly(A) + RNA was obtained from the polysome fraction and translated in a mRNA-dependent reticulocyte lysate system. 4. 4. The translatability of the poly(A) + RNA was similar using the preparations obtained from control and lead-treated rats except for proteins of approx molecular weight of 50,000 which contained increased amounts [ 14C]leucine in the fractions derived from lead-treated rats. 5. 5. The results are compared with those after acute Cd 2+ toxicity where poly(A) + RNA exhibited increased translatability for low molecular weight metallothioneins.

Glial fibrillary acidic protein synthesized in vitro using messenger RNA from a human glioma cell line.

Messenger RNA (mRNA) extracted from a continuous human glioma cell line grown in culture or as a solid tumor was translated in an mRNA-dependent reticulocyte lysate system. Translation products labeled with [35S]methionine were immunoprecipitated with antiserum specific for glial fibrillary acidic (GFA) protein, separated by one- and two-dimensional polyacrylamide gel electrophoresis and analyzed fluorographically. Immunoprecipitates from both cell culture and tumor mRNA translations had a molecular weight of 49,000 daltons, consistent with GFA protein extracted from human tissue. In two dimensions, the 49,000-dalton band resolved into two to three spots at pH 5.7-5.9, the isoelectric point of GFA protein. Minor lower molecular weight products were detected in fluorographs of heavily overloaded gels or in film exposed for extended periods of time. These data indicate that the GFA protein produced by this glioma cell line is chemically and immunologically similar to normal human GFA protein, which suggests that the primary phenotypic expression of GFA protein in this tumor cell line is not altered by the neoplastic process.

Cell-free translation and regulation of Candida tropicalis catalase messenger RNA.

To gain information on metabolic control and peroxisome biogenesis in Candida tropicalis growing on n-alkanes, cell-free translation of catalase (H2O2:H2O2 oxidoreductase, EC 1.11.1.6), a general marker enzyme of peroxisomes, was performed. The level of catalase activity in alkane-grown cells was approximately 9-fold and 27-fold higher than that in ethanol-grown and glucose-grown cells, respectively. Immunochemical titration experiments with rabbit antiserum against the purified peroxisomal catalase from alkane-grown C. tropicalis indicated that the remarkable variation in the enzyme activity level on different carbon sources was ascribable to a corresponding change in the amount of the enzyme protein. When cell-free translation was carried out with the mRNA-dependent reticulocyte lysate system, total RNA prepared from alkane-grown cells was shown to direct the synthesis of catalase subunit in vitro. The identity of the cell-free translation product was ascertained by the following evidence: (a) the translation product was immuno-reactive with specific antibody to catalase and competed effectively with the authentic enzyme for immunoprecipitation; (b) it possessed a molecular weight indistinguishable from that of authentic catalase subunit (Mr 54000); (c) its peptide fragments formed by partial digestion with Staphylococcus aureus V8 protease were identical with those from the authentic enzyme. With the use of the cell-free translation system, it was indicated that the significant change in the amount of catalase protein on different carbon sources nearly paralleled that in the activity of the mRNA encoding the enzyme.

The effect of triiodothyronine on the cardiac mRNA

Cardiac hypertrophy was induced in rats by daily injections of L-triiodothyronine (0.2 mg/kg body wt, s.c., 1 to 3 days). Regression of hypertrophy was studied after 3 days of injection for a period of 1 to 16 days. Cardiac RNA was isolated by phenolextraction, mRNA by affinity chromatography of cardiac total RNA on oligo-dT-cellulose. In addition, cardiac mRNA was also quantitated by hybridization to 3H-Poly(U). After 3 days of T 3-treatment, the extractable amount of cardiac total RNA increased from 759 ± 181 μg/g heart (controls) to 980 ± 119 μg/g heart (T 3-rats), and that of mRNA from 24.7 ± 2.0 (controls) to 39.4 ± 7.0 (T 3-rats), increases of 29.1% (total RNA) and 59.5% (mRNA). 16 days after cessation of T 3-treatment, total RNA and mRNA levels had returned to normal. The increase in cardiac mRNA in T 3-rats was investigated in further detail by in vitro translation of the isolated mRNA in the mRNA-dependent reticulocyte lysate system. Incorporation of 35S-methionine into total protein was identical for cardiac mRNA from control and T 3-rats. Separation of the in vitro synthesized proteins on SDS-Polyacrylamide-Slabgels revealed the complete synthesis of actin, troponin-T, tropomyosin, myoglobin and myosin-light-chains 1 and 2. The mRNAs coding for these proteins represented identical fractions of total mRNA in control and T 3-rats. This leads to the conclusion that T 3 had stimulated the synthesis of all of these specific mRNAs in an unselective manner. From our data and from the results of other authors we conclude that changes in cardiac mRNA levels are the major regulatory factor in causing changes in cardiac protein synthesis rates leading to cardiac hypertrophy in hyperthyroidism.

Initiation reactions in the mRNA-dependent reticulocyte lysate

Reticulocyte lysates depleted of mRNA by digestion with micrococcal nuclease still show an unexpectedly high rate of formation of 80 S initiation complexes. Formation of these complexes is sensitive to all inhibitors of the normal protein synthesis initiation process tested. Such lysates contain high concentrations of mRNA fragments which can be utilized for initiation, with which exogenous mRNA must compete. As a consequence of this competition, mRNAs that are weak initiators may be translated poorly by this system even at low exogenous mRNA concentrations.

41] Cell-free translation of elastin mRNAs

To cover fully the system for effective cell-free translations of elastin mRNAs, the methodology is divided into four sections dealing with (1) isolation of RNA and preparation of mRNA, (2) preparation of the mRNA-dependent reticulocyte lysate and optimal conditions for the cell-free translation of elastin mRNAs, (3) identification of tropoelastins in a cell-free translation assay, and (4) quantitation of tropoelastins in the cell-free translation system. It is important to note that the discussion of the cell-free synthesis of elastin involves the synthesis of two proteins designated tropoelastins a and b. All the evidence to date suggests strongly that these two tropoelastins are separate gene products. Consequently, the methodologies described are geared not only for the production of soluble elastin, but also for the separate identification of tropoelastins a and b.