Coding capacity of poly(A)+ mRNA isolated from mengovirus-infected Ehrlich ascites tumor cells

Total poly(A)⁺ mRNA was isolated from mengovirus-infected Ehrlich ascites tumor cells at various times postinfection and quantitated in a cell-free system derived from uninfected ascites cells. Basic proteins were separated from acidic proteins by carboxymethyl cellulose chromatography. At the end of the infectious cycle, 8 h postinfection, the cellular contents of most mRNAs coding for basic ribosomal proteins are still between 70 and 90 per cent of those measured at the beginning of infection or in uninfected cells. On the basis of this result, the rapid shutoff of host protein synthesis after mengovirus infection of Ehrlich ascites tumor cells cannot be the consequence of the inactivation of host template RNA.     

Isolation and cell-free translation of a human immunoglobulin light chain messenger RNA.

Human myeloma cell line RPMI 8226 synthesizes and secretes a lambda immunoglobulin light chain. Total cellular RNA, obtained from cells grown in culture, was used for the isolation of poly(A)-containing mRNA by oligo(dT)-cellulose chromatography. The poly(A)-containing mRNA was translated in an Ehrlich ascites extract. Immunoprecipitation of the cell-free products showed that a polypeptide chain antigenically related to human lambda chain was synthesized. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that the cell-free product was larger than the secreted light chain. On tryptic digestion the cell-free product and the secreted light chain exhibited essentially identical peptide patterns except for an additional peptide derived from the cell-free product. We conclude that the lambda mRNA from this human myeloma cell line codes for a precursor to the secreted lambda chain as described for immunoglobulin mRNAs from murine plasmacytomas.     

Partial purification of rat alpha-lactalbumin mRNA.

Alpha-lactalbumin messenger RNA was partially purified from RNA extracted from 3-5 day lactating rat mammary glands on a poly(U)-sepharose column followed by sucrose gradient centrifugation. Apha-Lactalbumin mRNA activity was assayed in wheat germ cell-free translational system by immunoprecipitation of the in vitro synthesized protein using specific antiserum prepared against purified rat alpha-lactalbumin. In the purified mRNA preparation alpha-lactalbumin mRNA activity comprised approximately 85% of the total mRNA activity.     

Cellular protein synthesis shutoff by mengovirus: translation of nonviral and viral mRNA''s in extracts from uninfected and infected Ehrlich ascites tumor cells.

The mechanism whereby picornaviruses inhibit host protein synthesis while their own synthetic processes proceed unabated has remained elusive. One of our approaches to this problem was to study the ability of cell-free extracts derived from uninfected and mengovirus-infected Ehrlich ascites tumor cells to translate viral and nonviral mRNA's under various conditions of incubation. Our results indicate that viral messengers (from mengovirus and encephalomyocarditis virus) and cellular messengers [L cell and Ehrlich ascites tumor poly(A)-containing mRNA's, rabbit globin mRNA, and chicken embryo lens crystallin mRNA] are translated equally well in both extracts. We also examined the simultaneous translation of viral and nonviral mRNA's in extracts from uninfected Ehrlich ascites tumor cells. Our results indicate that under certain conditions mengovirus RNA can suppress completely the translation of globin mRNA. The significance of these results in terms of the shutoff of host protein synthesis is discussed.     

The acute effects of N-hydroxy-acetylaminofluorene on rat liver protein synthesis

A single intraperitoneal injection of N-hydroxy-acetylaminofluorene (N-hydroxy-AAF) at a dosage of 30 mg/kg significantly inhibited rat liver protein synthesis within 15 min. Marked alterations in the subcellular distribution of hepatic RNA accompanied the decline in protein synthesis in treated rats. These changes included decreases in nuclear and bound polysomal RNA and increases in free polysomal and non-sedimentable RNA. Heavy polysomal aggregates, both free and bound, were almost completely degraded to monomers and dimers during this period. Sedimentation profiles of total cytoplasmic RNA revealed no evidence of gross RNA breakdown in N-hydroxy-AAF-treated animals. To determine the mechanisms responsible for the inhibition of protein synthesis by N-hydroxy-AAF, cellular components involved in protein synthesis were purified from control and treated animals and examined in two cell-free systems. In a system which measures polypeptide chain elongation and release, the incorporation of amino acids into protein was reduced by 35% using polysomes from N-hydroxy-AAF treated animals compared with controls. By contrast, the function of the pH 5 fraction (containing aminoacylating enzymes and tRNA) from the carcinogen-treated animals was unimpaired. A wheat germ lysate system was used to determine the ability of mRNA to program polypeptide chain initiation and elongation. Cytoplasmic poly(A)⁺ RNA from N-hydroxy-AAF treated rats showed reduced capacity to stimulate protein synthesis in wheat germ lysates compared with similar preparations from DMSO-injected control rats. The rapid inhibition of protein synthesis by N-hydroxy-AAF may be an important contributing factor to other toxic effects of the carcinogen, including the inhibition of rRNA synthesis.     

An investigation of the stability of messenger RNAs in cell-free,translational systems from uninfected and mengovirus-infected Ehrlich ascites tumor cells

The stabilities and translation of Ehrlich ascites tumor cell poly(A)-containing mRNA and mengovirus RNA in fractionated cell-free protein synthesizing systems from uninfected and mengovirus-infected Ehrlich ascites tumor cells were studied. During incubation of the systems about 20% of the input RNA is reduced in size and associated with ribosomes engaged in polypeptide synthesis; the remainder is rapidly degraded by RNases. At the end of active translation, both mRNA and nascent proteins are bound to polysomes which are of the same size as those formed during active protein synthesis. The kinetics of protein synthesis closely follow those of RNA hydrolysis. The stabilities of mengovirus RNA and poly(A)-containing mRNA from Ehrlich ascites tumor cells are the same in both systems.     

Coding capacity of poly(A)+mRNA isolated from mengovirus-infected Ehrlich ascites tumor cells.

Total poly (A)+mRNA was isolated from mengovirus-infected Ehrlich ascites tumor cells at various times postinfection and quantitated in a cell-free system derived from uninfected ascites cells. Basic proteins were separated from acidic proteins by carboxymethyl cellulose chromatography. At the end of the infectious cycle, 8h postinfection, the cellular contents of most mRNAs coding for basic ribosomal proteins are still between 70 and 90 percent of those measured at the beginning of infection or in uninfected cells. On the basis of this result, the rapid shutoff of host protein synthesis after mengovirus infection of Ehrlich ascites tumor cells cannot be the consequence of the inactivation of host template RNA.     

Preformed mRNA in Cotyledons of Ungerminated Seeds of Cicer arietinum L

Polyadenylated RNA was isolated from total RNA extracted from cotyledons of ungerminated or 18-hour-germinated chick-pea seeds by affinity chromatography on oligo(dT)-cellulose. Both poly(A)-containing RNA fractions exhibited a template activity when assayed in two cell-free translation systems, wheat germ extracts, and nuclease-treated reticulocyte lysates. Translation of preformed mRNA from cotyledons of dry seeds was completely abolished in the presence of several inhibitors of polypeptide chain initiation and also in the presence of the two “cap” analogues m⁷ GTP and m⁷ GMP. The patterns of polypeptides synthesized by translation of poly(A)-containing RNAs from cotyledons of ungerminated or 18-hour-germinated seeds, in the wheat germ system, analyzed by electrophoresis and autoradiography, were similar but not identical. It is concluded that cotyledons of dry Cicer arietinum L. seeds contain preformed mRNA.     

Purification of immunoglobulin heavy chain messenger RNA by immunoprecipitation from the mouse myeloma tumor, MOPC-31C.

Immunoglobulin heavy chain mRNA was purified from immunoprecipitated polysomes derived from the mouse myeloma tumor, MOPC-31C. The purified mRNA migrated predominantly as a single band upon polyacrylamide gel electrophoresis in 98% formamide and the molecular weight of this mRNA was calculated to be 700,000. This mRNA was as active as the purified light chain mRNA when it was employed as a template in a cell-free protein synthesizing system from wheat germ. The translation product had a molecular weight of 55,000 daltons, and migrated slightly faster than mature heavy chain upon polyacrylamide gel electrophoresis in sodium dodecylsulfate. The protein synthesized by the direction of this mRNA was shown to yield tryptic peptides corresponding to those derived from the mature heavy chain protein except that one missing peptide was replaced by another additional peptide. DNA complementary to the mRNA was synthesized by RNA-dependent DNA polymerase from avian myeloblastosis virus. Hybridization kinetic analysis between the heavy chain mRNA and its complementary DNA indicated that the RNA was essentially homogenous with rabbit globin mRNA as a standard.     

Cell-free synthesis of putative neurophysin precursors from rat and mouse hypothalamic poly (A)-RNA.

Poly(A)-containing RNA has been isolated from rat and mouse hypothalamic tissue and used to direct the synthesis of polypeptides in cell-free systems derived from wheat germ extract and rabbit reticulocyte lysate in the presence of [³⁵S]-L-cysteine and [³H]-L-proline. Translation products were subjected to immunoprecipitation using an antiserum to rat neurophysin proteins. Following purification of the immunoprecipitates by protein A-Sepharose chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis revealed a single polypeptide species of molecular weight 17,500 derived from both cell-free systems.     

Evidence for distinct mRNAs for ferritin subunits

Poly A enriched RNA from iron loaded HeLa cells and rat liver were translated separately and together in wheat germ lysates to investigate the origins of the H and L subunits of ferritin. Most of the ferritin translated from the HeLa RNA was of the H type, while that from the liver RNA was mostly L type. Mixtures of these RNAs gave $\text{H}\text{L}$ ratios which correlated with the relative amounts of added HeLa and rat RNAs. These results indicate that the H and L subunits of ferritin are not derived by post-translational modification but from distinct mRNA species.     

A transfer RNA-dependent protein synthesizing system from Ehrlich ascites extracts.

An homologous cell-free system from Ehrlich ascites cells was made tRNA dependent by passing both the ribosomes and the ammonium sulfate fraction through DEAE-cellulose columns in 0.30 M KCl. Protein synthesis was routinely stimulated 20-50 fold by the addition of 100 mug/ml of ascites cell tRNA in the presence of oviduct mRNA, ascites mRNA, or encephalomyocarditis RNA.     

Purification and Characterization of a Cytostatic Factor with Anti-Viral Activity from the Bitter Melon

Abstract

We have previously reported that a crude aqueous extract of the bitter melon (Momordica charantia) has both cytostatic and cytotoxic activi-ties ^1,2, and is a competitive inhibitor of guanylate cyclase activity³. This crude preparation kills human leukemic lymphocytes in a dose-dependent manner while not affecting the viability of normal human lymphocytes at these same doses¹.

In this report we describe the purification and characterization of one of these cytostatic factors which also exhibits anti-viral activity.

The partially purified factor was both cytostatic to BHK-21 cells and inhibitory to VSV plaque formation in a dose-dependent manner. This pre-paration was inhibitory to both viral and host cell RNA and protein synthesis as early as 30 min after addition to these samples. As determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), this purified factor is a single component with a molecular weight corresponding to 40,000 daltons.

The factor is sensitive to boiling and to pre-treatments with trypsin, but not ribonuclease (RNAse), or deoxyribonuclease (DNAse).

As determined by radioactive precursor uptake and incorporation studies, the purified factor inhibits both RNA and protein synthesis in intact tissue culture cells and inhibits protein synthesis in a cell-free wheat germ system⁴. DNA synthesis was slightly stimulated.

The purified factor is cytostatic for both BHK-21 and for the IM9 leukemic cell lines for at least 120 h.

The cytostatic component had no effect on cellular cyclic GMP metabolism.     

Induction of translationally active rat liver glutathione S-transferase B messenger RNA by phenobarbital

Liver poly(A⁺)-RNA was isolated from untreated and phenobarbital-treated rats and translated in cell-free systems derived from wheat germ and rabbit reticulocyte lysates. The primary translation product of glutathione S-transferase B was comprised of two nonidentically sized subunits which comigrated on SDS-polyacrylamide gels with the purified glutathione S-transferase B subunits. The level of translatable glutathione S-transferase B mRNA in rat liver was elevated approximately 3 to 4-fold by phenobarbital administration. Our data suggest that chronic phenobarbital administration to rats increases the amount of cytosolic glutathione S-transferase B via an increase in the functional mRNA level encoding for the enzyme.     

Partial amino acid sequence of the preprotein form of the alpha subunit of human choriogonadotropin and identification of the site of subsequent proteolytic cleavage

Messenger RNA isolated from first trimester placentae was translated using radiolabeled amino acids in both the wheat germ and the ascites cell-free systems. The choriogonadotropin α subunit product was purified by immunoprecipitation with a subunit specific antiserum. Its amino acid sequence was partially determined by automated Edman degradation analysis. An NH₂-terminal extension of 24 amino acids was found and its partial sequence is:

The preprotein form of the subunit was cleaved by the addition of microsomal membranes resulting in a homogeneous NH₂-terminal product. Hence, it is unlikely that this processing step accounts for the heterogeneity that has been observed previously in the structure of this region of the subunit.     

On the existence of polyadenylated histone mRNA in Xenopus laevis oocytes

In a variety of systems, histone mRNA has been shown to lack poly(A) (Adesnik and Darnell, 1972;Grunstein et al., 1973). We have found, however, that in Xenopus laevis oocytes, poly(A)-containing mRNA codes for histones, in a wheat germ cell-free system, based on the following criteria: first, co-migration with authentic X. laevis oocyte histones on polyacrylamide gels; second, no detectable incorporation of tryptophan; third, differential incorporation of lysine and methionine into histone fraction H2A; fourth, resistance of histone fraction H2A to cleavage with cyanogen bromide; and fifth, correspondence of tryptic peptide maps of partially purified cell-free products with authentic X. laevis oocyte histone. RNA which directs the synthesis of histones in the cell-free system is retained on oligo(dT)-cellulose, even after denaturation in 80% DMSO at 70°C, thereby demonstrating the covalent attachment of polyadenylic acid sequences to the mRNA. Poly(A)^? RNA (7S–14S fraction) was also found to code for histones using the same criteria. We discuss the significance of the finding that X. laevis oocytes contain two classes of histone mRNA as well as the potential developmental implications of this observation.     

Cell-free translation of chloroplast RNAs in the wheat germ system

Spinach, tobacco and Euglena chloroplast RNAs (cp RNA) can be successfully translated in the wheat germ cell-free system. The in vitro translation products obtained from spinach cp RNA in the wheat germ and in the Escherichia coli system are similar to each other and to that of in organello synthesis, if analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Translation of mixtures of chloroplast and total RNA of leaves reveals that under conditions of mRNA competition the cytoplasmic type of RNA is preferentially translated in the wheat germ system.     

Purification and Characterization of A Cytostatic Factor With Anti-Viral Activity From The Bitter Melon

We have previously reported -that a crude aqueous extract of the bitter melon (Momordica charantia) has both cytostatic and cytotoxic active ties ^1,2, and is a competitive inhibitor of guanylate cyclase activity³. This crude preparation kills human leukemic lymphocytes in a dose-dependent manner while not affecting the viability of normal human lymphocytes at these same doses¹.

In this report we describe the purification and characterization of one of these cytostatic factors which also exhibits anti-viral activity.

The partially purified factor was both cytostatic to BHK-21 cells and inhibitory to VSV plaque formation in a dose-dependent manner. This preparation was inhibitory to both viral and host cell RNA and protein synthesis as early as 30 min after addition to these samples. As determined by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), this purified factor is a single component with a molecular weight corresponding to 40,000 daltons.

The factor is sensitive to boiling and to pre-treatments with trypsin, but not ribonuclease (RNAse), or deoxyribonuclease (DNAse).

As determined by radioactive precursor uptake and incorporation studies, the purified factor inhibits both RNA and protein synthesis in intact tissue culture cells and inhibits protein synthesis in a cell-free wheat germ system. DNA synthesis was slightly stimulated.

The purified factor is cytostatic for both BHK-21 and for the IM9 leukemic cell lines for at least 120 h.

The cytostatic component had no effect on cellular cyclic CMP metabolism.