Biomedical subjects

G W Both

Publications and source records attributed to G W Both.

84 records · Page 5Linked to original sources

PubMed1976

Dependence of translation on 5'-terminal methylation of mRNA.

Many eukaryotic cell and viral mRNAs contain 5'-terminal m7G(5')ppp(5')N. Methylation is important for the in vitro translation of vesicular stomatitis virus and reovirus mRNAs. Unmethylated viral mRNAs are methylated by cell-free extrascts of wheat germ and L cells to form 5'-terminal structures of the type, m7GpppN and m7GpppNm, respectively. Rabbit globin mRNA also contains 5'-terminal 7-methylguanosine (m7G). Removal of the m7G by beta-elimination decreases translation. The efficient binding of mRNA to ribosomes is dependent on the presence of 5'-terminal m7G.

Animals↗

PubMed1975

mRNA methylation and protein synthesis in extracts from embryos of brine shrimp, Artemia salina.

Cell-free protein-synthesizing extracts prepared from the brine shrimp, Artemia salina, translate methylated mRNAs. Reovirus unmethylated mRNA is inactive as a template when methylation is prevented by the inhibitor, S-adenosylhomocysteine. A salina mRNAs from both undeveloped and developed embryos contain 5'-terminal 7-methylguanosine in an inverted 5'-5' linkage through three phosphate groups to the rest of the polynucleotide chain. Removal of the 7-methylguanosine by beta elimination converts the mRNA from an active form to one inactive in protein synthesis in extracts of A. salina or wheat germ. Extracts of undeveloped and developed embryos methylate reovirus unmethylated mRNA at the 5' ends to form 5'-terminal structures of the type, m7G(5')ppp(5')G and m7G(5')ppp(5')Gm.

Animals↗

PubMed1975

5'-Terminal 7-methylguanosine in eukaryotic mRNA is required for translation.

Unmethylated reovirus and VSV mRNAs are specifically methylated to form 5'-terminal structures of the type, m-7-G(5')ppp(5')N by protein synthesising extracts prepared from wheat germ and mouse L cells. Reticulocyte mRNA also contains 5'-terminal m-7-G. MRNAs having 5'-terminal m-7-G stimulate protein synthesis in vitro. Removal of m-7-G by beta-elimination abolishes translation of the mRNAs.

Animals↗

PubMed1975

Ribosome binding to reovirus mRNA in protein synthesis requires 5' terminal 7-methylguanosine.

Purified reovirus synthesizes in vitro a mixture of mRNA molecules that contain 5' terminal structures of the type ppG..., GpppG..., and m7GpppGm, the relative amount of each depending upon the presence of S-adenosylmethionine (SAM) or S-adenosylhomocysteine (SAH) in the transcription incubation mixture. Reovirus mRNAs containing 5' termini of the type ppG... and GpppG... can be specifically modified by wheat germ extracts in the presence of SAM to yield the structure m7GpppG...(Muthukrishnan et al., 1975). The mRNA methylase activity is ribosome-independent and is recovered almost entirely in the high speed supernatant fraction of wheat germ extracts. Its activity is inhibited by aurintricarboxylic acid. Ribosome binding experiments with reovirus mRNA and wheat germ extract indicate that only those mRNA molecules containing 5' terminal m7G are capable of participating in the initiation of protein synthesis and subsequent polysome formation. mRNA with unmethylated 5' termini do not form a complex with 40S ribosomal subunits. Discrimination between unmethylated and methylated reovirus mRNA, active in protein synthesis, apparently occurs at or before the formation of 40S-mRNA complexes. T1 or pancreatic RNAase digestion of methylated mRNA bound to 80S ribosomes yields 5' terminal fragments of apparent chain lengths about 32 and 36 nucleotides, respectively. A portion of the T1 RNAase-resistant fragments rebinds to ribosomes to form a nuclease-resistant complex. In contrast, the shorter 5' terminal oligonucleotide m7GpppGmpCpUp(Np)3Gp derived by RNAase T1 digestion of purified reovirus mRNA does not bind to ribosomes. The results suggest that 5' terminal m7G may function as the primary recognition signal for ribosome binding and that wheat germ ribosomes bind very close to the 5' termini of some of the species of methylated reovirus mRNAs.

Aurintricarboxylic Acid↗

PubMed1975

Synthesis of all the gene products of the reovirus genome in vivo and in vitro.

Sixteen virus-specific polypeptides have been resolved in reovirus-infected mouse L cells by using SDS-polyacrylamide slab gel electrophoresis and autoradiography. Of these, ten have been designated as primary products of the genome by the following criteria: they are present in lysates of infected cells labeled for a short time; they co-migrate on SDS-polyacrylamide slab gels with polypeptides synthesized in cell-free-extracts of wheat germ in response to purified viral mRNA; and their molecular weights correspond to the values expected if all ten reovirus mRNA species are monocistronic. Reovirus mRNA species lack 3' poly(A) but are translated into proteins of the expected size. The pattern of synthesis of the primary gene products observed in vitro mimicks that observed in reovirus-infected cells suggesting that the structure of the mRNA may profoundly influence its translation. The results further indicate that there is little, if any, exclusively regulatory information in the reovirus genome since both in vivo and in vitro, transcripts of the ten genome segments direct the synthesis of ten polypeptides that presumably correspond to the primary gene products. The expression of the reovirus genome thus appears to be complete.

Electrophoresis, Polyacrylamide Gel↗

PubMed1975

Translation and identification of the mRNA species synthesized in vitro by the virion-associated RNA polymerase of vesicular stomatitis virus.

Vesicular stomatitis virus messenger RNA has been transcribed in vitro from the viral genome by the virion-associated RNA polymerase in quantities suitable for translation. Wheat germ cell-free extracts programmed with the isolated in vitro 12-18S RNA fraction synthesize polypeptides similar to the viral N, NS, and M proteins, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and tryptic peptide mapping of the in vitro products and the viral marker polypeptides. In addition, the RNA synthesized in vitro also codes for a protein of molecular weight 63,000 which may be a nonglycosylated form of the viral glycoprotein G. The 12-18S RNA has been partially separated into individual messenger species and these have been identified by the proteins for which they code. There are four monocistronic messenger species in the in vitro 12-18S RNA and the coding capacity of three of these molecules agrees with the estimated molecular weight of the polypeptide assigned to it.

Animals↗

PubMed1975

Methylation-dependent translation of viral messenger RNAs in vitro.

Methylated reovirus and vesicular stomatitis virus mRNAs, synthesized in vitro in the presence of S-adenosylmethionine by the virion-associated polymerases (RNA nucleotidyltransferases, EC 2.7.7.6), stimulate protein synthesis by wehat germ extracts to a greater extent than unmethylated mRNAs. Addition of S-adenosylmethionine to a cell-free extract programmed with unmethylated mRNA stimulates protein synthesis and results in methylation of the mRNA. An inhibitor of mRNA methylation. S-adenosylhomocysteine, blocks translation of unmethylated, but not of methylated, mRNAs. Aurintricarboxylic acid, which inhibits polypepetide chain initiation, also prevents mRNA methylation by wheat germ extracts. In contrast, sparsomycin, which inhibits polypeptide chain elongation, does not reduce mRNA methylation. The results indicate that methylation of viral mRNA is required for translation in vitro and suggest that mRNA methylation occurs at the initiation step of protein synthesis.

Aurintricarboxylic Acid↗

PubMed1975

Translation and identification of the viral mRNA species isolated from subcellular fractions of vesicular stomatitis virus-infected cells.

The cytoplasm of vesicular stomatitis virus (VSV)-infected BHK cells has been separated into a fraction containing the membrane-bound polysomes and the remaining supernatant fraction. Total poly(A)-containing RNA was isolated from each fraction and purified. A 17S class of VSV mRNA was found associated almost exclusively with the membrane-bound polysomes, whereas 14,5S and 12S RNAs were found mostly in the postmembrane cytoplasmic supernatant. Poly(A)-containing VSV RNA synthesized in vitro by purified virus was resolved into the same size classes. The individual RNA fractions isolated from VSV-infected cells or synthesized in vitro were translated in cell-free extracts of wheat germ, and their polypeptide products were compared by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. The corresponding in vivo and in vitro RNA fractions qualitatively direct the synthesis of the same viral polypeptides and therefore appear to contain the same mRNA species. By tryptic peptide analysis of their translation products, the in vivo VSV mRNA species have been identified. The 17S RNA, which is compartmentalized on membrane-bound polysomes, codes for a protein of molecular weight 63,000 (P-63) which is most probably a nonglycosylated form of the viral glycoprotein, G. Of the viral RNA species present in the remaining cytoplasmic supernatant, the 14.5S RNA codes almost exclusively for the N protein, whereas the 12S RNA codes predominantly for both the NS and M proteins of the virion.

Amino Acids↗

PubMed1973