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PubMed1979

Mechanisms of interferon induced transfer of viral resistance between animal cells.

The sequence of events initiated by interferon and leading to the antiviral state were studied as possible sites for the cell-to-cell transfer of interferon induced viral resistance. The possible role of interferon produced by recipient cells was negated by the demonstration of transfer of resistance in the presence of anti-human interferon antibody and under conditions of a single cycle of VSV growth. Transfer of sensitivity of WISH cells to mouse interferon, possibly through transfer of a membrane receptor, seems unlikely since resistance was transferred in the absence of mouse interferon. From kinetic data and the fact that actinomycin D blocked resistance in human cells for 3 h longer than in mouse cells, it seems unlikely that the mouse antiviral protein itself or its mRNA alone is a likely candidate for the transfer of resistance. Thus, by a process of elimination, we suggest that secondary messenger molecules which transmit the interferon signal from the membrane to the nucleus are the effector substance(s) for the transfer process.

Animals↗

PubMed1978

RNA methylation in vaccinia virus-infected chick embryo fibroblasts treated with homologous interferon.

Interferon-pretreatment of vaccinia-infected chick embryo fibroblasts resulted in a greater than 50% decrease in ribose methylation of the penultimate "cap" nucleotide in virus-specific mRNA. However, in contrast to results obtained with cell-free systems, in intact infected cells there was (a) no detectable reduction in methylation of the 5'-ultimate m7G of viral mRNA; (b) a virus specificity of the interferon-induced change in mRNA "CAP"-methylation seems unlikely and (c) analysis of the ribosomal and transfer RNA fractions isolated from interferon-treated and control cells revealed identical patterns of methylated nucleotides. Thus, the interferon-induced change in methylation is specific for mRNA "CAPS".

Cells, Cultured↗

PubMed1977

[Studies on development of the phycomycete Allomyces arbuscula. III. Polysome formation and RNA metabolism during differentiation of gametangia (author's transl)].

Polysomes were isolated both from growing gametophytes of Allomyces arbuscula and from gametangia prepared from mycelia at different periods during gametogenesis. Analysis of polysomes by sucrose gradients showed that ribosomes present in the gametangia monosome pool were shifted into polysomes. This shift was found to be correlated with gametangia differentiation. The ribosome distribution remained virtually unchanged during the early stage of gamete formation. In mature gametes and swarming zygotes a low level of polysomes was detected. Labeling of rRNA by 32PO4 demonstrated a de novo synthesis of monosomes throughout the period of gametangia differentiation. No incorporation of 32PO4 was found to be present in ribosomes prepared from gametangia after onset of gamete formation. On the basis of these labeling experiments it is concluded that radioactivity in polysomes extracted from mature gametes and swarming zygotes can be attributed in part to conserved mRNA. Synchronous formation of gametangia was induced by transferring the vegetative mycelia from growth medium into a low salt buffer. Under these conditions the incorporation of either 32PO4 or 3H-uridine into RNA, particularly into rRNA, was found to be markedly decreased. This obviously indicates a shutdown of RNA synthesis. rRNA from induced mycelia examined by polyacrylamide gel electrophoresis was found to be severely degraded. In contrast to this, rRNA isolated from ribosomes of developing gametangia and from gametes exhibited no degradation products. It is suggested that endonucleases cause rRNA hydrolysis in the hyphal cytoplasm during gametangia differentiation. Ribosomes compartmentalized in gametangia seem to be inaccessible to nucleases during the later process for gametogenesis.

Chytridiomycota↗

PubMed1977

How a single Sindbis virus mRNA directs the synthesis of one soluble protein and two integral membrane glycoproteins.

Previous work has shown that the 26S RNA found in Sindbis-infected chicken embryo fibroblasts encodes the three viral structural proteins, one internal protein, core, and two membrane glycoproteins, E1 and E2. This mRNA has one initiation site; core, E1, and E2 are derived by proteolytic cleavage. Here we show that during infection, the 26S RNA is found mainly in membrane-bound polysomes which synthesize all three virion structural proteins. These polysomes are released from the membrane upon treatment with puromycin and high salt. Newly synthesized core protein is localized on the cytoplasmic side of endoplasmic reticulum membranes, while newly synthesized envelope proteins are sequestered by the lipid bilayer. These results suggest that the nascent glycoproteins, presumably their amino termini, are of major importance in directing the binding of polysomes containing 26S mRNA to endoplasmic reticulum membranes and the subsequent transfer of glycoproteins into the bilayer.

Capsid↗

PubMed1978

Cordycepin. An inhibitor of newly synthesized globin messenger RNA.

The effect of cordycepin (3'-deoxyadenosine) on newly synthesized globin mRNA in cultured mouse fetal liver erythroid cells is investigated. At cordycepin concentrations that do not inhibit amino acid incorporation into acid-precipitable material, the quantity of pulse-labeled (radioactive) globin mRNA nucleotide sequences is reduced by 90%, as compared to adenosine-treated controls. The reduction of radioactivity in globin-specific RNA sequences is greater than the inhibition of total RNA synthesis in experiments in which the labeling times range from 6 to 60 min. Control experiments demonstrate that cordycepin does not reduce the recovery of total cell RNA or steady state (unlabeled) globin mRNA. The hybridization assay used to detect radioactive globin mRNA sequences is independent of the cellular location or the number of 3'-terminal adenylate residues in the mRNA-containing molecules. These data thus indicate that cordycepin inhibits newly synthesized mRNA as effectively as it inhibits ribosomal and transfer RNA synthesis.

Animals↗

PubMed1977

The presence of ovalbumin mRNA coding sequences in multiple restriction fragments of chicken DNA.

Chicken DNA has been digested with restriction enzymes and the size distribution of the DNA fragments containing ovalbumin specific sequences has been examined after separation of the fragments on agarose gels and transfer to nitrocellulose sheets. Hybridisation with terminally 32P-labelled ovalbumin mRNA fragments or with RNA populations transcribed from the DNA of a hybrid plasmid containing ovalbumin sequences was used to locate the DNA fragments coding for ovalbumin. Digestion with enzymes which do not cut within the portion of the ovalbumin gene synthesised from ovalbumin messenger RNA in vitro has shown the presence of several defined fragments carrying ovalbumin specific sequences. Possible explanations of these observations are discussed.

Animals↗

PubMed1976

Preparation and characterization of eukaryotic initiation factor EIF-3. Formation of binary (EIF-3-Met-tRNAf) and ternary (EIF-3-Met-tRNAf-GTP) complexes.

The 133,000 X g supernatant fraction prepared from ascites cells in 20 mM KCl (low CKl supernatant) contained the initiation factors EIF-1 and EIF-2 (and the elongation factore EF-1 and EF-2) but lacked EIF-3; thus, low KCl supernatant could be used to assay for EIF-3. EIF-3 was prepared from a crude initiation factor perparation (a 250 mM KCl extract of ascites cell ribosomes precipitated with 70% saturated ammonium sulfate) by chromatography on DEAE-Sephadex A-50 and hydroxylapatite. The EIF-O had no detectable EIF-1 and little or no EIF-2. Factor EIF-3 was required fro translation of encephalomyocarditis virus RNA. The molecular weight of EIF-3 was estimated by Sephadex G-200 filtration to be 139,000; the sedimentation coefficient was calculated to be about 5.8. EIF-3 formed a binary complex specifically with the initiator tRNA, Met-tRNAf, and if GTP was present the factor formed a ternary complex (EIF-3-Met-tRNAf-GTP). The EIF-3 preparation had no methionyl-tRNA synthetase activity to account for binding. Complex-formation was with eukaryotic Met-tRNAf and no other aminoacyl-tRNA. The binary and ternary complexes were retained quantitatively on Millipore filters (which was the most convenient assay), but they could also be demonstrated by filtration through Sephadex G-100 or by glycerol gradient centrifugation. GTP increased the rate, the amount, and the stability of complex formed; the ration of GTP to Met-tRNAf in the ternary complex appeared to be 1. The binary and the ternary complexes transferred Met-tRNAf to the 40 S ribosomal subunits, but not to 60 S subparticles. The factor-dependent binding of Met-tRNAf to the 40 S subunit did not require mRNA (or GTP). In the presence of 60 S subunits, the initiator tRNA bound to 40 S subunits was not transferred to 80 S ribosomes even if mRNA was added--that reaction may require another initiation factor. Treatment of EIF-3 with N-ethylmaleimide led to loss of its activity in complex formation and in support of the translation of encephalomyocarditis virus RNA. In addition to forming the binary and ternary complexes, and supporting the translation of encephalomyocarditis virus RNA, EIF-3 also increases the number of free ribosomal subunits by either preventing their association or causing dissociation of 80 S couples.

Animals↗

PubMed1975

Alteration in cellular RNAs during the in vitro lifespan of cultured human diploid fibroblasts.

An abrupt concommitant increase in total cellular RNA and protein was observed as cultured human diploid fibroblasts entered the senescent phase of their in vitro lifespan. DNA content remained stable from early to final passages. Fractionation of cellular RNAs by polyacrylamide gel electrophoresis demonstrated an increase in both 28S and 18S ribosomal and 4S transfer RNAs in these senescent cells. Separation of poly(A) RNA (mRNA) by oligo(dT)-cellulose chromatography suggests an increase in this group of RNAs. However, the ratios of 28S to 18S rRNAs, tRNA to rRNA, and mRNA to total cellular RNA were not significantly different in cells before and after senescence, indicating that the overall increases in total cellular RNA was not due to an accumulation of a single RNA class.

Cell Division↗

PubMed1979

Synthesis and translation of mRNA containing 5'-terminal 7-ethylguanosine cap.

Reovirus mRNA synthesis in vitro by the virion-associated RNA polymerase was only slightly (10 to 15%) diminished in the presence of 2 mM S-adenosylethionine. However, methyl group transfer from S-adenosylmethionine (0.05 mM) to the 5'-terminal cap structure, m7GpppGm in this mRNA was markedly inhibited (80%) under these conditions. Replacement of S-adenosylmethionine by S-adenosylethionine (5 mM) yielded mRNAs containing mainly (70%) 5'-terminal e7GpppGe and e7GpppG, but some of the products were unalkylated (5'-GpppG, ppG). The ethylated mRNAs, but not the unalkylated molecules, bound to wheat germ ribosomes and were translated essentially as well as the corresponding methylated mRNAs in wheat germ extracts and in nuclease-treated rabbit reticulocyte lysates. Protein synthesis directed by ethylated mRNAs in wheat germ extract was 80% decreased by 0.1 mM m7GMP. Under conditions of limited initiation, methylated mRNA bound to wheat germ ribosomes preferentially as compared to ethylated mRNA. The results document for the first time the synthesis of ethylated mRNA and support the hypothesis that N7-alkylation of the 5'-guanosine in caps, rather than methylation itself, is important for the enhancing effect of cap on the initiation of eukaryotic protein synthesis.

DNA-Directed RNA Polymerases↗

PubMed1977

Two methyltransferase activities in the purified virions of vesicular stomatitis virus.

In addition to an RNA-dependent RNA polymerase, purified vesicular stomatitis virus contains a methyltransferase activity which transfers the methyl group from the methyl donor, S-adenosyl-L-methionine, to two positions in the 5'-terminal capped structure of the nascent mRNA's synthesized in vitro as 7mG-(5)'ppp(5')Apm... In the present study it is shown that two distinct methyltransferase activities are discernible in the purified virus. The in vitro concentrations of the methyl donor specify the number and location of the methyl groups transferred to the capped 5'-termini of VSV mRNA's. Limited concentrations of the methyl donor result in a single methylation of the penultimate base in the 2'-hydroxyl position, that is, G(5')ppp(5')Apm..., whereas saturating concentrations of the methyl donor methylate the blocking guanosine residue at the 7-position, resulting in the dimethylated cap, 7mG(5')ppp(5')Apm... Pulse-chase experiments demonstrate that the monomethylated cap structure is the precursor substrate for the dimethylated cap. In this respect, vesicular stomatitis virus system is quite distinct from the vaccinia and reovirus systems. Virus purified from different host cells including hamster, mouse, and human contain both methyltransferase activities. The mRNA's containing monomethylated capped structures are poor templates for protein synthesis in vitro.

Kinetics↗

PubMed1978

A change in the stability of globin mRNA during the induction of murine erythroleukemia cells.

The stability of globin mRNA in murine erythroleukemia cells (Friend cells) before and during DMSO-induced differentiation was investigated. Cells were exposed to 3H-uridine for 2 hr and then transferred to medium without the radioactive precursor. The loss of radioactivity in total RNA, poly(A)-containing RNA and globin mRNA was followed. The globin mRNA was isolated using a highly specific globin cDNA column. In uninduced cells and cells early in differentiation, the globin mRNA decays with a half-life of less than 50 hr. After 4 days of induction, the globin mRNA decays with a half-life of 17 hr, demonstrating a change in stability during the induction process. Although the stability of globin mRNA changes during induction, this is not true for total poly(A)-containing RNA. At all times of induction, the poly(A)-containing RNA decays as two populations, one with a half-life of 6 hr and the other with a half-life of 36 hr. The half-life of the rRNA also remains unchanged during differentiation.

Cell Differentiation↗

PubMed1979

Chicken lysozyme gene contains several intervening sequences.

The organization of the chicken lysozyme gene and its neighboring sequences was examined by a comparison of the restriction map of the lysozyme structural gene with the map of the lysozyme gene in genomic DNA. Chicken DNA was cleaved with restriction endonucleases and the DNA fragments were separated by agarose gel electrophoresis. After transfer of the fragments onto nitrocellulose filters, those fragments that contain lysozyme mRNA sequences were detected by hybridization of the filters to labeled probes generated from pls-1, a recombinant plasmid carrying the lysozyme structural gene. This analysis revealed the presence of at least three intervening sequences, two of which interrupt the protein coding region and one of which is located in the 3' untranslated region. When oviduct DNA and sperm DNA were compared, no difference was observed in the size and number of restriction fragments that contain either lysozyme or ovalbumin structural gene sequences.

Animals↗

PubMed1979

Cloning and expression of the yeast galactokinase gene in an Escherichia coli plasmid.

This report describes the construction and isolation of a plasmid, derived from pBR322, which carries a BglII restriction fragment of DNA containing the galactokinase gene from Saccharomyces cerevisiae. This was accomplished by the following procedure: (1) Purified galactokinase mRNA, labelled with 125I, was hybridized to BglII digests of yeast DNA employing Southern's filter transfer technique to identify a restriction fragment containing the galactokinase gene. (2) This fragment was partially purified by agarose gel electrophoresis, ligated into the BamHI site of pBR322 and transformed into Escherichia coli to generate a clone bank containing the galactokinase gene. (3) This bank was screened by in situ colony hybridization with galactokinase mRNA resulting in the identification of a plasmid carrying this gene. This plasmid DNA hybridized with the galactokinase mRNA to the same extent in the presence of absence of a large excess of unlabelled mRNA from cells that were not induced for galactokinase synthesis, while the same amount of unlabelled galactose-induced mRNA reduced the hybridization by 95%. When this plasmid was introduced into an E. coli strain deleted for the galactose operon it caused the synthesis of low levels of yeast galactokinase activity.

DNA, Recombinant↗

PubMed1975

Changes in RNA in relation to growth of the fibroblast. III. Posttranscriptional regulation of mRNA formation in resting and growing cells.

The proportion of hnRNA processed into cytoplasmic mRNA in resting and growing 3T3 and 3T6 cells has been investigated by measuring the efficiency of transfer of nuclear poly(A) to the cytoplasm. When nuclear poly(A) is either labeled continuously with precursor or pulse labeled and then chased with cordycepin, growing cells transfer approximately twice as much of the poly(A) from the nucleus to the cytoplasm as do resting cells. When cells undergo a serum-induced transition from the resting to growing state, the efficiency of poly(A) transfer is increased to that characteristic of growing cells by 3 hr after the addition of the serum. On the other hand, the proportion of hnRNA which is polyadenylated and the total nuclear poly(A) content are the same in resting and growing cells. It appears that the principal means by which the cell regulates its poly(A)+ mRNA content in the growing and resting states is by regulating the efficiency which nuclear poly(A)+ RNA is converted to cytoplasmic mRNA.

Adenine↗

PubMed1976

Functional relationships between a reticulocyte polypeptide-chain-initiation factor (IF-MP) and the translational inhibitor involved in regulation of protein synthesis by haemin.

The rate of initiation of protein synthesis in rabbit reticulocyte lysates is regulated by a translational inhibitor protein which is activated in the absence of added haemin. The effects of this inhibitor on amino acid incorporation are overcome by the protein synthesis initiation factor IF-MP which binds Met-tRNAf in a ternary complex with GTP and which can transfer this complex to small ribosomal subunits. Addition of this factor to haemin-deficient lysates prevents loss of polysomes and regenerates polysomes from 80-S single ribosomes, thus confirming an effect at the level of polypeptide initiation. The ability of the initiation factor to overcome the effects of various concentrations of the translational inhibitor suggests that the inhibitor inactivates the factor catalytically rather than stoichiometrically. In a system in vitro consisting of salt-washed 40-S ribosomal subunits, initiator Met-tRNAf and GTP, the initiation factor IF-MP transfers Met-tRNAf to the subunits in the absence of any other factor or mRNA. Equilibrium buoyant density gradient analysis in CsCl shows that formaldehyde-fixed subunits carrying Met-tRNAf bound under these conditions have a buoyant density approximately 0.02 g/cm3 lower than the bulk of salt-washed subunits, suggesting that approximately 100000 daltons of additional protein are associated with these subunits. This is in marked contrast to the amounts of protein bound to subunits incubated with Met-tRNAf and GTP in the presence of a crude ribosomal salt-wash fraction. The translational inhibitor has no effect on formation of the ternary complex IF-MP-Met-tRNAf-GTP but does impair the factor-catalysed transfer of Met-tRNAf to washed subunits. The possible mechanisms of action of the inhibitor on polypeptide chain initiation are reviewed in the light of these results.

Animals↗

PubMed2025

Effects of light on chloroplast translation in Marchantia polymorpha are similar to those in angiosperms and are not influenced by light-independent chlorophyll synthesis.

Translation of the chloroplast psbA mRNA in angiosperms is activated by photodamage of its gene product, the D1 subunit of photosystem II (PSII), providing nascent D1 for PSII repair. The involvement of chlorophyll in the regulatory mechanism has been suggested due to the regulatory roles of proteins proposed to mediate chlorophyll/D1 transactions and the fact that chlorophyll is synthesized only in the light in angiosperms. We used ribosome profiling and RNA-seq to address whether the effects of light on chloroplast translation are conserved in the liverwort Marchantia (Marchantia polymorpha), which synthesizes chlorophyll in both the dark and the light. As in angiosperms, ribosome occupancy on psbA mRNA decreased rapidly upon shifting plants to the dark and was rapidly restored upon a transfer back to the light, whereas ribosome occupancy on other chloroplast mRNAs changed very little. The results were similar in a Marchantia mutant unable to synthesize chlorophyll in the dark. Those results, in conjunction with pulse-labeling data, suggest that light elicits a plastome-wide activation of translation elongation and a specific increase in psbA translation initiation in Marchantia, as in angiosperms. These findings show that light regulates chloroplast translation similarly in vascular and non-vascular plants, and that constitutive chlorophyll synthesis does not affect light-regulated psbA translation initiation. Additionally, the translational outputs of chloroplast genes are similar in Marchantia and angiosperms but result from differing contributions of mRNA abundance and translational efficiencies. This adds to the evidence that chloroplast mRNA abundance and translational efficiencies co-evolve under selection to maintain protein outputs.

Chloroplasts↗

PubMed1976

DNA-histones, a computer model.

The model of DNA-histones has the following elements: 1. The hydrogen bonds between the complementary nucleotide bases function as informational gates. When the electrons pi of one nucleotide base are excited, an exchange of protons is produced between the two complementary bases. The result is the displacement of the conjugated double bonds which facilitates the inter-molecular transmission of the electronic wave of excitation by electro-magnetic coupling. 2. Each triplet of nucleotide bases of DNA fixes one definite amino acid (as in the genetic code). Between the nucleotide bases and the amino acids there are constituted informational gates, which ensure the circulation of the electronic wave of excitation. 3. An input signal molecule arrives at the receiver gene and unleashes the activity of the enzymes which introduce in the DNA-histones system the electronic wave of excitation. The electronic wave of excitation arises as a result of the break of the high-energy bonds of ATP. Then, the electronic excitation is transmitted to the productor gene where it represents the signal for starting the synthesis of the mRNA.

Adenosine Triphosphate↗

PubMed1978

HeLa cell RNA (2'-O-methyladenosine-N6-)-methyltransferase specific for the capped 5'-end of messenger RNA.

A novel enzyme involved in the post-transcriptional modification of the 5'-end of mRNA has been partially purified from HeLa cells. Termed an S-adenosyl-L-methionine:RNA(2'-O-methyladenosine-N4)-methyltransferase, the enzyme specifically catalyzes the transfer of a methyl group from S-adenosylmethionine to the N6 position of a 2'-O-methyladenosine residue located within the "capped" 5'-end of mRNA. The dimethylated nucleoside, N6,2'-O-dimethyladenosine, is formed as indicated by the following reaction in which m7G(5')pppAm- represents the capped and methylated 5'-end of mRNA: AdoMet + m7G(5')pppAm- leads to AdoHcy + m7G(5')pppm6A7- Greatest activity is obtained with RNA acceptors ending in m7G(5')pppAm-; less activity is found with RNA ending in m7G(5')pppA-; and barely detectable and no activity is found with RNA ending in G(5')pppA- and ppA-, respectively. Furthermore, no activity is found with oligonucleotides such as m7G(5')pppA, m7G(5')pppAm, and m7G(5')pppAmpN indicating that a longer polymer is required. It can be concluded from the substrate specificity of the enzyme that the formation of N6,2'-O-dimethyladenosine follows the biosynthesis of molecules containing m7G(5')pppAm-N-. The molecular weight of the methyltransferase, as determined by sedimentation in sucrose gradients, is approximately 65,000.

Adenosine↗