Rabbit Reticulocyte Lysate Cell-free System Research Articles

Regulation of synthesis of citrate synthase in regreening Euglena gracilis.

Exposure of dark-grown Euglena to white or red light, but not blue light, produced a twofold increase in the specific activity of citrate synthase. A 400-fold purification of mitochondrial citrate synthase (subunit Mr = 44000) was achieved from cells of Euglena gracilis by affinity chromatography on ATP-activated agarose. Antisera, raised against the homogeneously pure enzyme, were used to demonstrate that the increase in citrate synthase activity on exposure of dark-grown cells to light resulted from an increase in citrate synthase protein. Anti-(citrate synthase) was used to detect precursor citrate synthase resulting from the translation of total polyadenylated RNA from Euglena in a cell-free rabbit reticulocyte lysate system. Citrate synthase mRNA was found to be present in cells at all stages of regreening. However, extraction and translation of polyadenylated RNA from free polysomes isolated from darkgrown and regreening cells demonstrated that appreciable translation of citrate synthase mRNA was only occurring in regreening cells.

In vitro morphogenesis of foot-and-mouth disease virus.

Foot-and-mouth disease virion RNA is translated efficiently and completely in a rabbit reticulocyte lysate cell-free system. Treatment of cell-free lysates with monospecific serum prepared against the individual viral structural proteins or with monoclonal antibodies prepared against the inactivated virus or against a viral structural protein precipitated all of the structural proteins, suggesting that structural protein complexes were formed in vitro. Sucrose gradient analysis of the cell-free lysate indicated that complexes sedimenting at 5, 14, 60 to 70, and ca. 110S were assembled in vitro. Structural proteins VP0, VP1, and VP3 were the major polypeptides found in these complexes. The material sedimenting at 110S, i.e., containing VP0, VP1, and VP3, was precipitated by a 140S-specific monoclonal antibody but not by a 12S subunit-specific monoclonal antibody, suggesting that this capsid structure contained at least one epitope present on the intact virus.

Cleavage of newly translated NADPH-cytochrome P-450 reductase in a rabbit reticulocyte lysate cell-free system in the absence of exogenous membranes

In a rabbit reticulocyte lysate cell-free system and using double antibody immunoprecipitation method, newly translated rat liver NADPH-cytochrome P-450 reductase (E.C. 1.6.2.4) was shown to be cleaved in the absence of exogenous membranes. Reductase having a M r=78,000 was shown to be converted to a M r=67,000 upon incubation with either lysate or antisera. Peptide maps of 78,000 dalton reductase and 67,000 dalton protein were identical except for three additional peptides present in the 78,000 dalton reductase map. The cleavage activity associated with the antisera could be prevented by using the IgG fraction, while that associated with the lysate was inhibited by using the protease inhibitors leupeptin, pepstatin or bestatin.

Biosynthesis of rabbit haptoglobin: Chemical evidence for a single chain precursor

The primary translation product of the mRNA for rabbit haptoglobin was obtained from a rabbit reticulocyte lysate cell-free system by immunoprecipitation with an antiserum that was directed to the β chain of haptoglobin. Analysis of the translation product by gel electrophoresis and by protein sequencing analysis identified a single polypeptide of M r 41 000. Sequence analysis established a signal region of 18 residues that was immediately followed by the α chain sequence. These results give strong evidence that haptoglobin is initially synthesized as a single chain composed of a signal peptide followed by α and β chain regions, respectively.

Protein synthesis directed by polyadenylated and non-polyadenylated RNA isolated from membrane-bound and free polysomes of Tetrahymena pyriformis.

Free and membrane-bound polysomes were prepared from the protozoa Tetrahymena pyriformis using a procedure which gives good recovery and practically no cross-contamination. Polysomes are intact as analysed by sedimentation analysis. Poly(A)-rich RNA and poly(A)-free RNA, isolated from both populations of polysomes, show similar electrophoretic patterns. These RNAs were translated in the rabbit reticulocyte lysate cell-free system and the translation products were analysed by one-dimensional and two-dimensional gel electrophoresis. The most striking differences were found in the two-dimensional electrophoretic analysis namely: (a) a group of polypeptides (10) is synthesized mainly on membrane-bound polysomes, (b) a second abundant group is synthesized mainly in free polysomes (c) and a third class of polypeptides is synthesized on both kinds of polysomes. Poly(A)-free RNAs, isolated from free polysomes, are also able to promote synthesis of some polypeptides. The results are discussed taking into account the fact that T. pyriformis is a non-secretory cell.

Control of type I collagen synthesis: evidence for pretranslational coordination of pro alpha 1 (I) and pro alpha 2 (I) chain synthesis in embryonic chick bone.

The normal type I collagen molecule contains two alpha 1 (I) chains and one alpha 2 (I) chain. In embryonic chick calvaria, the two-chains are synthesized in a 2:1 ratio, and total polysomes from this tissue contain twice as much mRNA for pro alpha 1 (I) as for pro alpha 2 (I). To further investigate the mechanism by which synthesis may be coordinated, RNA isolated from various cell fractions of embryonic chick calvaria was translated in a rabbit reticulocyte lysate cell-free system. The procollagen chain products were separated by gel-electrophoresis and densitometrically quantitated from autoradiograms of the gels. Total cellular RNA, total cytoplasmic RNA, and polysomal RNA each directed the synthesis of pro alpha 1 (I) and pro alpha 2 (I) in a proportion of 2:1, whereas no procollagen mRNA activity was found in nonpolysomal cytoplasmic RNA. These results indicate that in the chick bone cells, all compartments contain twice as much pro alpha 1 (I) mRNA as pro alpha (I) mRNA, and that virtually all procollagen mRNA in the cytoplasm in polysome-bound. The coordination of procollagen chain synthesis thus presumably occurs at a pretranslational level, through differential rates of formation and/or degradation of the two mRNAs.

Cell-free translation of human liver apolipoprotein AI and AII mRNA. Processing of primary translation products.

Human liver apolipoprotein AI and A II poly(A+) mRNA has been translated in the cell-free rabbit reticulocyte lysate system. The structures of the two primary translation products of these two main protein components of human serum high-density lipoprotein (HDL) have been characterized. The products of the synthesis in vitro are preproapolipoproteins. The signal sequence (pre-sequence) of the primary translation product of human apo AI mRNA consists of 18 amino acids, that of apo AII of 17 amino acids. The cotranslational translocation into dog microsomal vesicles is associated with the cleavage of these sequences by the signal peptidase releasing the proapolipoproteins AI and AII, both extended by an N-terminal hexapeptide. Preproapolipoprotein AII is synthesized in its monomeric form consisting of 100 amino acids. Pro-apo AII is present in the vesicles of the endoplasmic reticulum also as monomer. Sequencing of the radiolabelled signal sequences of both pre-forms revealed their strongly hydrophobic nature. Despite the high affinity of HDL-apolipoproteins for complex lipids their secretion requires these hydrophobic signal sequences for translocation. Internal recognition sequences in the native apoproteins are not responsible for the transmembrane transport.

Translational competition between related virus RNA species in cell-free systems.

The competition between the RNAs from two tobacco mosaic virus (TMV) strains for translation in rabbit reticulocyte lysate and wheat germ cell-free systems was investigated. The virus translational products were distinguished by the size difference of the I2 polypeptides. The RNAs of the two strains were translated simultaneously if added to the translation system together at the beginning of the reaction. Each of the TMV RNAs could inhibit translation of the other when the reaction was initiated by the RNA of one strain and that of the other strain was added after active translation had commenced. In contrast, translation of alfalfa mosaic virus (AMV) RNA was not appreciably inhibited when its RNA was added after the start of translation of TMV RNA; the addition of TMV RNA to an AMV RNA-initiated reaction also resulted in independent synthesis of the products of both virus RNAs.

Inhibitory effect of alpha-fetoprotein on protein synthesis in a reticulocyte lysate cell-free system.

The inhibitory effect of human alpha-fetoprotein on the protein synthesis (incorporation of [3H]leucine into the total protein fraction) weas observed in a rabbit reticulocyte lysate cell-free system. The incorporation was inhibited to 50% of the control level by 14-16 microM alpha-fetoprotein, and decreased to about 15% at a concentration higher than 40 microM. An almost identical dose dependence was obtained between fetal alpha-fetoprotein and hepatoma-derived alpha-fetoprotein. This inhibitory effect on the protein synthesis was due to the interference of alpha-fetoprotein to the 40 S initiation complex formation from the ternary complex (eukaryotic initiation factor 2.GTP.Met-tRNAfMet). In contrast human serum albumin purified from fetal cord sera did not exhibit this inhibition under the same conditions. These results indicate that alpha-fetoprotein may function as a regulator of the protein synthesis in the fetal stage.

A family of proteins accumulating in ectoderm of sea urchin embryos specified by two related cDNA clones

Two cDNA clones, pSpec 1 and pSpec 2, had been selected previously as corresponding to transcripts greatly enriched in ectoderm of pluteus-stage larvae of the sea urchin, Stronglyocentrotus purpuratus. The two cDNA clones, corresponding to the 3′ ends of polysomal RNAs, have similar but distinct restriction maps. Messenger RNAs hybrid selected by these two cDNA clones were translated in a rabbit reticulocyte lysate cell-free system into the same set of 10 acidic proteins having molecular weights of 14–17,000 daltons. These in vitro products have positions identical or similar on two-dimensional gels to a group of proteins whose synthesis in vivo is highly enriched in the ectoderm. The 1.5- and 2.2-kb transcripts corresponding to pSpec 1 and pSpec 2 increase in prevalence by at least 100-fold during embryonic development. The rates of synthesis of these ectoderm proteins also increase by over 100-fold, though the patterns of change are distinct. It is likely that pSpec 1 and pSpec 2 correspond to mRNAs which are part of a small family of genes coding for the group of similar ectoderm proteins. These proteins and their mRNAs are enriched in ectoderm by the early gastrula stage.

Cell-free translation of a biologically active, antigen-specific suppressor T cell factor.

In vitro synthesis of an antigen-specific T cell suppressor factor (TsF) has been accomplished by using partially purified poly(A)-containing RNA in a rabbit reticulocyte lysate cell-free translation system. The poly(A)-containing mRNA was isolated from a cloned T cell hybridoma that constitutively produces a TsF specific for the synthetic polypeptide antigen poly-(LGlu60LAla30LTyr10) (GAT). The RNA was fractionated by size and translated in vitro. The 16S RNA fraction stimulated synthesis of a biologically active protein that specifically suppressed both the GAT-specific antibody response by spleen cells in vitro and the proliferation response to GAT by lymph node T cells from GAT-primed mice. Further, the suppressor factor had a binding site for GAT, a determinant encoded by the I subregion of the major histocompatibility complex (MHC), and an apparent Mr 19,000 estimated by functional assays on protein separated by NadodSO4/polyacrylamide gel electrophoresis. These results indicate that virtually no posttranslational modifications (other than proteolytic cleavage) are necessary to obtain biologically active TsF. Hence, the presence of carbohydrate or other chemical groups does not contribute to either the serological properties of GAT-TsF or its biological properties.

Translationally mediated changes in patterns of protein synthesis during maturation of starfish oocytes

When meiotic maturation of primary oocytes of the starfish Asterias forbesi is induced by 1-methyladenine, rapid and striking changes in the pattern of protein synthesis detectable by electrophoresis occur after germinal vesicle breakdown. These include a decline in relative labeling with [ 35S]methionine of several polypeptides synthesized in the oocyte, and increased labeling and new appearance of several polypeptides. Fertilization does not result in other detectable changes. The population of total mRNA translatable in a rabbit reticulocyte lysate cell-free system does not change, but the distribution of mRNAs between polysomes and the postribosomal supernatant reflects the changes observed in vivo. Thus these changes are regulated at the translational level. A review of the literature indicates that translationally mediated changes in patterns of protein synthesis during maturation of oocytes may be a widespread phenomenon.

Phosphorylation of ribosomal protein S6 in the Ehrlich ascites tumor cell: Lack of effect of phosphorylation upon ribosomal function in vitro

The incorporation of 32P i into the ribosomal protein S6, which is a component of the 40 S ribosomal subunit, was measured in Ehrlich ascites tumor cells growing in suspension culture. During a 2-h incubation period, incorporation was 10-fold greater into S6 of cells growing exponentially in complete medium than in cells in which protein synthesis and growth were inhibited by omission of glutamine from the medium. Since the labeling of other phosphoproteins was not similarly depressed in glutamine-deprived cells, the decreased labeling most likely reflects a specific decrease in phosphate incorporation into S6 rather than a lower phosphate pool specific activity. The cycling of ribosomes is inhibited in glutamine-deprived cells, with an accumulation of monomeric ribosomes indicating an inhibition of protein synthesis initiation. To assess whether the decreased phosphorylation of S6 in the ribosomes of glutamine-deprived cells was responsible for their decreased ability to initiate protein synthesis and enter polyribosomes, a mixture of 14C-labeled ribosomes from rapidly growing cells and 3H-labeled ribosomes from glutamine-deprived cells was added to a rabbit reticulocyte lysate cell-free protein-synthesizing system in which ribosomes cycle actively. The 14 C 3 H ratio was determined in polyribosomes as a measure of the relative ability of the two kinds of ribosomes to initiate protein synthesis. The 14 C 3 H ratio in polyribosomes was found to be identical to the input of labeled ribosomes, indicating an equal ability of ribosomes from growing cells and glutamine-deprived cells to function in initiation. Control studies indicated lack of significant dephosphorylation of S6 during purification of ribosomes and during incubation in the reticulocyte lysate. Thus, a functional difference between more and less phosphorylated ribosomes could not be demonstrated in this system.

In vitro translation of cutinase mRNA: Evidence for a precursor form of an extracellular fungal enzyme

The extracellular fungal enzyme, cutinase, has been synthesized in vitro in a rabbit reticulocyte lysate cell-free translation system primed with poly(A) + mRNA isolated from glucose-grown Fusarium solani f. s. pisi in which cutinase synthesis was induced with cutin hydrolysate. Optimum translation rates were obtained with 4–10 μg of mRNA, 40–80 m m K +, and 0.49 m m Mg 2+. Total translation products of mRNA from induced and noninduced cultures showed no observable differences, except for a protein of 25–27K daltons which was found only when the mRNA from the induced culture was used. Immunoprecipitation of the translation products with antibodies prepared against cutinase resulted in the recovery of a 25.5K-dalton peptide. This peptide was obtained only when the translation mixture was primed with mRNA from the induced culture. Translation of the mRNA in a wheat germ cell-free system also gave a 25.5K-dalton peptide. This primary translation product was 2100 daltons larger than the mature extracellular enzyme. It is concluded that cutinase is synthesized as a precursor which is processed by proteolysis and glycosylation before excretion.

Translation of coxsackievirus B RNAs in a rabbit reticulocyte lysate: characterization of the genome RNA, reaction conditions for translation, and analysis of the products.

The genomic RNA of coxsackievirus B5 was characterized, and the RNAs from B4 and B5 viruses were translated in a micrococcal nuclease-treated rabbit reticulocyte lysate cell-free system to characterize the products. The viral RNA, native or denatured, sedimented as a sharp symmetrical peak between 45S and 28S marker rRNAs; its estimated molecular weight was 2.53×106. It was polyadenylated and contained about 7,440 nucleotides which could code for proteins with a total molecular weight of approximately 273,000. At saturating concentrations a five- to sevenfold stimulation in methionine incorporation over background was obtained when translating either B4 or B5 RNA. The translation directed by RNA was inhibited by pactamycin, aurintricarboxylic acid, puromycin, or cycloheximide (inhibitors of initiation and elongation) and by RNase. 7-Methylguanosine-5′-monophosphate, a cap analog which inhibits protein synthesis directed by capped messengers, did not inhibit this translation, suggesting that B5 RNA may not be capped. At least 10 polypeptides with apparent molecular weights of 31,000 to 98,000 were synthesized in the presence of either RNA; many of these comigrated with polypeptides synthesized in the virus-infected HeLa cells. A polypeptide (MW 36,000) of B4 RNA-directed products, which comigrated with a similar one from the virus-infected cells, also comigrated with an authentic capsid protein VP2 of the virus. The products of B4 RNA-directed synthesis were different from B5 RNA-directed products: the former contained at least five polypeptides of MW 31,000 to 45,000, while the latter contained only one (31,000). Antiserum to B5 virus capsid proteins precipitated several cell-free polypeptides with molecular weights of 52,000 to 92,000, suggesting that an immunologic relationship may exist between cell-free products and the capsid proteins. Comparison of partial proteolytic digests of cell-free products with molecular weights of 69,000 to 92,000 and authentic capsid proteins also suggests strongly that the products may contain some sequences of B5 virus capsid proteins.

The synthesis of Ricinus communis agglutinin, cotranslational and posttranslational modification of agglutinin polypeptides.

Polyadenylated RNA isolated from the endosperm tissue of maturing castor bean seeds was translated in a cell-free rabbit reticulocyte lysate system. Rabbit antibodies raised against Ricinus communis agglutinin were used to identify nascent agglutinin chains. In contrast to the authentic agglutinin polypeptides with molecular weights of 31000 (A chains) and 37000 (glycosylated B chains), immunoreactive translational products of Mr 33500 and 59000 were observed. The inclusion of canine pancreatic microsomes in the translational system resulted in the cotranslational segregation of these immunoreactive products into the lumen of the vesicles and their modification, to molecular weights of 32000 and 66000--69000 respectively. These cotranslational size modifications resulted from the cleavage of leader sequences and, in the case of the larger product, concomittant core glycosylation, 32000-Mr and 66000--69000-Mr proteins were also observed amongst the immunoreactive products initially formed during the labelling of intact endosperm tissue in vivo, together with 37000-Mr and 39000-Mr proteins. Pulse-chase experiments showed that 66000--69000-Mr proteins slowly disappeared while the smaller proteins were further cleaved to chains of Mr 31000 (authentic A chain), 34000 and 37000 (authentic glycosylated B chains). It was concluded that R. communis agglutinin polypeptides were synthesized in precursor form, possibly as a 'giant' precursor in the case of the B chain, on membrane-bound polysomes. Cotranslational translocation across the endoplasmic reticulum membrane was accompanied by proteolysis to remove leader sequences and, where appropriate, core glycosylation. Even after cotranslational processing agglutinin polypeptides were still in precursor form. Processing to authentic size appeared to occur posttranslationally.

Control of fatty acid synthetase mRNA levels in rat liver by insulin, glucagon, and dibutyl cyclic AMP

The quantity of translatable fatty acid synthetase mRNA in liver of rats subjected to different hormonal states was determined with a rabbit reticulocyte lysate cell-free translation system. Both membrane-free polysomal and total cellular poly (A)-containing RNA were translated. The level of translatable fatty acid synthetase mRNA was 11-fold or more lower in livers of diabetic rats than in similar animals treated with insulin. In contrast, both glucagon and dibutyl cyclic AMP caused a 3-fold reduction over controls in the amount of translatable fatty acid synthetase mRNA in livers of animals refed a fat-free diet for 12 hr. These changes are consistent with the previously reported alterations in the relative rates of fatty acid synthetase synthesis measured in vivo. This suggests that the changes in the amount of fatty acid synthetase that occur in liver in response to the above hormonal changes are primarily due to changes in the amount of mRNA coding for this enzyme.

Isolation of polysomes from larval and adult Drosophila melanogaster

A method has been developed for the isolation of polysomes from adult Drosophila melanogaster. The profiles are not degraded by endogenous nucleases at 4°C, but are sensitive to added ribonuclease A. Cycloheximide added to the preparation does not alter the profile, indicating that chain elongation leading to loss of polysomes is not occurring under these conditions. Resuspension of the polysome pellets in high-salt buffer leads to dissociation of inactive ribosomes which is reversible. The addition of N-ethylmaleiide, cytidine 5′-monophosphoric acid, diethyl sulfate, and polyvinylsulfate does not improve the profiles. The profiles are dependent on added magnesium ion. Polyadenylated RNA was extracted from the polysomes using the guanidinium chloride method and oligo(dT)-cellulose chromatography. The RNA was translated in a rabbit reticulocyte lysate cell-free system and the translation products were analyzed by polyacrylamide gel electrophoresis.

Synthesis in vitro of corticosteroid-binding globulin from rat liver messenger ribonucleic acid.

To show that corticosteroid-binding globulin (CBG), a plasma glycoprotein, is synthesized in the liver as well as to develop a tool with which to study the hormonal factors which control its synthesis, we undertook to translate rat CBG mRNA in vitro. Rat liver mRNA was translated in the presence of [35S]methionine in both a rabbit reticulocyte lysate (cell-free) System and a Xenopus laevis oocyte (whole cell) system. A monospecific antibody against highly purified rat CBG was used to precipitate a single 35S-labeled product from the mixture of newly synthesized hepatic proteins in the two systems. In the rabbit reticulocyte lysate system, the protein migrated with an apparent molecular weight of 41,000 on sodium dodecyl sulfate-gel electrophoresis; CBG isolated from rat serum migrated as a doublet with apparent molecular weights of 56,000 and 62,500. Since proteins synthesized by a cell-free system are not glycosylated, the difference in molecular weight can probably be accounted for by the carbohydrate content of plasma CBG. Translation of rat liver mRNA in a Xenopus laevis oocyte system again resulted in immunoprecipitation of a single 35S-labeled protein, but this time with an apparent molecular weight of 53,000 on sodium dodecyl sulfate-gel electrophoresis; this probably represents glycosylated CBG and corresponds to the apparent molecular weight of one of the species isolated from rat plasma.

MRNA for the rat uterine estrogen-induced protein. Translation in vitro and regulation by estrogen.

Poly(A)-rich RNA was isolated from uterus and brain of immature rats and translated in the rabbit reticulocyte lysate cell-free protein-synthesizing system. The translation products were analyzed by 1) specific immunoprecipitation using antibodies directed against purified estrogen-induced protein (IP), 2) two-dimensional gel electrophoresis, and 3) partial protease digestion of selected spots from two-dimensional gels. These analyses showed that IP mRNA from both uterus and brain is faithfully translated in the cell-free system. Comparison of translation products derived from uterine mRNA of untreated and 1-h estrogen-treated rats revealed a specific increase in the ability of mRNA from treated animals to direct the synthesis of IP. Thus, estrogen stimulation of IP synthesis results, at least partly, from increased uterine concentrations of IP mRNA.