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B Moss

Publications and source records attributed to B Moss.

At least 451 records · Page 25Linked to original sources
PubMed1977

Nucleotide sequences at the N6-methyladenosine sites of HeLa cell messenger ribonucleic acid.

Borate gel chromatography was used to separate internal oligonucleotides containing N6-methyladenosine (m6A) from methylated 5'-terminal oligonucleotides of HeLa cell polyadenylylated mRNA. N6-Methyladenosine occurs primarily in two sequences, -G-m6A-C (70%) and -A-m6A-C-(30%). The nucleoside immediately following cytidine may be uridine, cytidine, or adenosine, while guanosine as well as other nucleosides occupy subsequent positions. Each of the four positions preceding the -(G or A)-m6A-C- sequence may be occupied by a pyrimidine or a purine ribonucleoside. Since on a random basis all possible sequences containing -(G or A)-A-C-(U or C or A)- could occur once per 43 nucleotides whereas there is only one m6A residue per thousand nucleotides, then either (1) not all potential sites are methylated, (2) there are multiple unique sequences perhaps methylated by several different enzymes, or (3) there are other unrecognized discriminating factors. The possibility that methylation of adenosine occurs exclusively in the region close to the 5' terminus of the mRNA was considered. However, such a localization was excluded since the majority of m6A residues were not found in 4 to 6S 5'-terminal fragments isolated by borate gel chromatography.

Adenosine↗
PubMed1977

Histone mRNAs contain blocked and methylated 5' terminal sequences but lack methylated nucleosides at internal positions.

Histone mRNA, labeled with 32P or 3H-methionine during the S phase of partially synchronized HeLa cells, was isolated from the polyribosomes and purified as a "9S" component by sucrose gradient sedimentation. We identified two types of 5' terminals, m7G(5')pppNmpN and m7G(5')pppNm-pNmpN, in which the first methylated nucleoside is 7-methylguanosine, the second is either N6, 2'-O-dimethyladenosine, 2'-O-methyladenosine, or 2'-O-methylguanosine, and the third is 2'-O-methyluridine, 2'-O-methylcytidine, or 2'-O-methyladenosine. Approximately 1.7% of the 32P label was present in the 5' terminal structures. Assuming a similar specific radioactivity for all phosphates, this percentage corresponds to an average of one terminal per 335 nucleotides. Histone mRNA differed from bulk polyadenylylated mRNA of HeLa cells in lacking significant amounts of 2'-O-methyluridine or 2'-O-methylcytidine in the second position of the 5' terminal oligonucleotide and in lacking N6-methyladenosine residues at internal positions.

Adenosine↗
PubMed1977

5'-Terminal capping of RNA by guanylyltransferase from HeLa cell nuclei.

A soluble extract prepared from HeLa cell nuclei has been shown to catalyze the 5'-terminal modification of RNA and synthetic polyribonucleotides to form m7G(5')pppA-and m7G(5')-pppG- structures referred to as caps. The reaction involves the transfer of a GMP moiety from GTP to the 5' end of an RNA molecule containing at least two terminal phosphates. Significantly, neither the beta nor the gamma phosphates of GTP are transferred and polynucleotides with no 5'-terminal phosphate or only one are not acceptors. In the absence of methyl donor, G(5')pppA- and G(5')pppG- structures were synthesized, indicating that methylation is not required for guanylylation. Cap formation was considered to occur by the following mechanism: (see article), in which AdoMet is S-adenosylmethionine, AdoHcy is S-adenosylhomocysteine, and (p)ppN- represents either the original 5' end of an RNA molecule or an internal site to which one or more phosphates were added after processing.

Cell Nucleus↗
PubMed1977

Synthesis of mRNA guanylyltransferase and mRNA methyltransferases in cells infected with vaccinia virus.

Guanylyltransferase and methyltransferases that modify the 5'-terminals of viral mRNA's to form the structures m7G(5')pppAm- and m7G(5')pppGm- appear to be synthesized afte- vaccinia virus infection of HeLa cells. Elevations in these enzyme activities were detected within 1 h after virus inoculation and increased 15- to 30-fold by 4 to 10 h. Increases in the guanylyl- and methyltransferase activities were prevented by cycloheximide, an inhibitor of protein synthesis, but not by cytosine arabinoside, an inhibitor of DNA synthesis. The latter results suggest that the mRNA guanylyl- and methyltransferases are "early" or prereplicative viral gene products. The guanylyltransferase and two methyltransferases, a guanine-7-methyltransferase and nucleoside-2'-methyltransferase, were isolated by column chromatography from infected cell extracts and found to have properties similar or identical to those of the corresponding enzyme previously isolated from vaccinia virus cores. In contrast, enzymes with these properties could not be isolated from uninfected cells.

Cycloheximide↗
PubMed1977

5'-Terminal and internal methylated nucleosides in herpes simplex virus type 1 mRNA.

RNA labeled with [methyl-3H]methionine and/or [32P]orthophosphate was isolated from the polyribosomes of herpes simplex virus (HSV) types 1-infected cells and separated into polyadenylylated [poly(A+)]and non-polyadenylylated [poly(A-)] fractions. Virus-specific RNA was obtained by hybridization in liquid to either excess HSV DNA or filters containing immobilized HSV DNA. Analysis in denaturing sucrose gradients indicated that HSV-specific poly(A+) RNA sedimented in a broad peak, with a modal S value of 20. The ratio of [3H]methyl to 32P decreased with increasing size of RNA, suggesting that each RNA chain contains a similar sumber of methyl groups. Further analysis indicated an average of one RNase-resistant structure of the type m7G(5')pppNmpNp or m7G(5')pppNmpNmpNp per 2,780 nucleotides. The following components were identified in the 5'-terminal oligonucleotides of polyribosome-associated HSV-specific poly(A+) and poly(A-) RNA: 7-methylguanosine, N6,2'-O-dimethyladenosine, and the 2'-O-methyl derivatives of guanosine, adenosine, uridine, and denosine, and the 2'-O-methyl derivatives of guanosine, adenosine, uridine, and cytidine. The most common 5'-terminal sequences were m7G(5')pppm6Am and m7G(5')pppGm. An additional modified nucleoside, N6-methyladenosine, was present in an internal position of HSV-specific RNA.

Adenosine↗
PubMed1976

mRNA guanylyltransferase and mRNA (guanine-7-)-methyltransferase from vaccinia virions. Donor and acceptor substrate specificites.

Characterization of the donor and acceptor specificities of mRNA guanylyltransferase and mRNA (guanine-7-)-methyltransferase isolated from vaccinia virus cores has enabled us to discriminate between alternative reaction sequences leading to the formation of the 5'-terminal m7G(5')pppN-structure. The mRNA guanylyltransferase catalyzes the transfer of a residue of GMP from GTP to acceptors which possess a 5'-terminal diphosphate. A diphosphate-terminated polyribonucleotide is preferred to a mononucleoside diphosphate as an acceptor suggesting that the guanylyltransferase reaction occurs after initiation of RNA synthesis. Although all of the homopolyribonucleotides tested (pp(A)n, pp(G)n, pp(I)n, pp(U)n, and pp(C)n) are acceptors for the mRNA guanylyltransferase indicating lack of strict sequence specificity, those containing purines are preferred. Only GTP and dGTP are donors in the reaction; 7-methylguanosine (m7G) triphosphate specifically is not a donor indicating that guanylylation must precede guanine-7-methylation. The preferred acceptor of the mRNA (guanine-7-)-methyltransferase is the product of the guanylyltransferase reaction, a polyribonucleotide with the 5'-terminal sequence G(5')pppN-. The enzyme can also catalyze, but less efficiently methylation of the following: dinucleoside triphosphates with the structure G(5')pppN, GTP, dGTP, ITP, GDP, GMP, and guanosine. The enzyme will not catalyze the transfer of methyl groups to ATP, XTP, CTP, UTP, or to guanosine-containing compounds with phosphate groups in either positions 2' or 3' or in 3'-5' phosphodiester linkages. The latter specificity provides an explanation for the absence of internal 7-methylguanosine in mRNA. In the presence of PPi, the mRNA guanylyltransferase catalyzes the pyrophosphorolysis of the dinucleoside triphosphate G(5')pppA, but not of m7G(5')pppA. Since PPi is generated in the process of RNA chain elongation, stabilization of the 5'-terminal sequences of mRNA is afforded by guanine-7-methylation.

Guanosine Triphosphate↗
PubMed1976

Modification of the 5' terminus of mRNA by an RNA (guanine-7-)-methyltransferase from HeLa cells.

The 5' termini of many viral and cellular mRNAs contain sequences of the type m7G(5")pppNm. An RNA (guanine-7-)-methyltransferase that specifically methylates the 5'-terminal guanosine residue of RNAs ending in the dinucleoside triphosphate G(5')pppN- has been purified from the cytoplasm of HeLa cells. Approximately two-thirds of the methyltransferase activity detected in an assay employing umnethylated vaccinia virus mRNA as acceptor was located in the cytoplasm when cells were disrupted by Dounce homogenization; 30% of the cytoplasmic activity was associated with ribosomes but was removed by washing with 0.5 M KCl. The enzyme was purified 165-fold from the cytoplasm by removing nucleic acid by phase partition followed by ammonium sulfate precipitation and column chromatography on DEAE-cellulose, denatured DNA-agarose, and CM-Sephadex. The partially purified enzyme preparation methylated heterologous tRNAs as well as vaccinia mRNA, but the tRNA methyltransferases could be separated from the mRNA activity by sucrose gradient sedimentation and gel filtration on Sephadex G-200. The product of the partially purified enzyme using vaccinia mRNA as substrate was exclusively 7-methylguanosine located in the terminal dinucleoside triphosphate. In addition to RNAs and synthetic polyribonucleotides terminating in a dinucleoside triphosphate, free G(5')pppG could be methylated but GTP, GDP, and G(5')pppG could not. The enzyme also methylated the dinucleoside diphosphate G(5')pppG but much less efficiently than G(5')pppG. An S20, W of 3.8, a Stokes radius of 3.6 nm, and a molecular weight of 56,000 were obtained from sucrose gradient sedimentation and Sephadex G-200 column chromatography.

Base Sequence↗
PubMed1976

5'-Terminal and internal methylated nucleotide sequences in HeLa cell mRNA.

The 5'-terminal oligonucleotides m7G(5')ppp(5')NmpNp and m7G(5')ppp(5')NmpNmpNp were isolated by DEAE-cellulose column chromatography after enzymatic digestion of 32P- or methyl-3H-labeled poly(A)" HeLa cell mRNA. The recovery of such oligonucleotides indicated that a high percentage of mRNA has blocked termini. The dimethylated nucleoside, N6, O2'-dimethyladenosine (m6Am), as well as the four common 2'-O-methylribonucleosides (Gm, Am, Um, Cm) were present in the second position linked through the triphosphate bridge to 7-methylguanosine (m7G) whereas little m6Am was in the third position. The only internal methylated nucleoside, N6-methyladenosine (m6A), was found exclusively as m6ApC and Apm6ApC after digestion with RNase A, T1, and alkaline phosphatase. Digestion with RNase A and alkaline phat pyrimidines are present in much smaller amounts or absent from this position. These results imply a considerable sequence specificity since there are thousands of different mRNA species in HeLa cells. Our studies are consistent with the following model of HeLa cell mRNA in which Nm may be m6Am, Gm, Cm, Um, or Am and one or more m6A residues are present at an unspecified internal location: m7G(5')ppp(5')Nm-(Nm)---(G or A)-m6A-C---(A)100-200A.

Alkaline Phosphatase↗
PubMed1976

Sequence of methylated nucleotides at the 5'-terminus of adenovirus-specific RNA.

RNA labeled with [methyl-3H] methionine and [14C]uridine was isolated from the cytoplasm of adenovirus-infected cells and purified by poly(U)-Sepharose chromatography and hybridization to filters containing immobilized adeovirus DNA. Analysis by dimethyl sulfoxide-sucrose gradient sedimentation suggested that the major mRNA species were methylated. 7-Methylguanosine was identified at the 5'-terminus of the advenovirus-specific RNA and could be removed by periodate oxidation and beta-elimination. Structures of the type m7G(5')ppp(5')Nm containing the unusual nucleoside N6, O2'-dimethyladenosine, and smaller amounts of 2'-O-methyladenosine were isolated by DEAE-cellulose chromatography after P1 nuclease digestion of the RNA. Evidence for some 5'-terminal sequences, m7G(5')ppp(5')m6AmpNm, with additional 2'-O-methylribonucleosides was also obtained. A base-methylated nucleoside, N6-methyladenosine, is located within the RNA chain and is released as a mononucleotide by alkali hydrolysis.

Adenosine↗
PubMed1975

Purification of mRNA guanylyltransferase and mRNA (guanine-7-) methyltransferase from vaccinia virions.

The sequences m7G(5')pppGm-and m7G(5')pppAm-are located at the 5' termini of vaccinia mRNAs. Two novel enzymatic activities have been purified from vaccinia virus cores which modify the 5' terminus of unmethylated mRNA. One activity transfers GMP from GTP to mRNA and is designated a GTP: mRNA guanylyltransferase. The second activity transfers a methyl group from S-adenosylmethionine to position 7 of the added guanosine and is designated a S-adenosylmethionine: mRNA (guanine-7-)methyltransferase. Advantage was taken of the selective binding of these activities to homopolyribonucleotides relative to DNA to achieve a 200-fold increase in specific activity. The guanylyl- and methyltransferase remained inseparable during chromatography on DNA-agarose, poly(U)-Sepharose, poly(A)-Sepharose, and Sephadex G-200 and during sedimentation through sucrose density gradients suggesting they were associated. A Stokes radius of 5.0 nm, an S20,w of 6.0 and a molecular weight of 127,000 were obtained by gel filtration on Sephadex G-200 and sedimentation in sucrose density gradients. Under denaturing conditions of sodium dodecyl sulfate-polyacrylamide gel electrophoresis two major polypeptides were detected in purified enzyme preparations. Their molecular weights of 95,000 and 31,400 suggested they were polypeptide components of the 127,000 molecular weight enzyme system.

Guanine Nucleotides↗
PubMed1975

Modification of RNA by mRNA guanylyltransferase and mRNA (guanine-7-)methyltransferase from vaccinia virions.

A purified enzyme system isolated from vaccinia virus cores has been shown to modify the 5' termini of viral mRNA and synthetic poly(A) and poly(G) to form the structures m7G(5')pppA- and m7G(5')pppG-. The enzyme system has both guanylyltransferase and methyltransferase activities. The GTP:mRNA guanylyltransferase activity incorporates GMP into the 5' terminus via a 5'-5' triphosphate bond. The properties of this reaction are: (a) of the four nucleoside triphosphates only GTP is a donor, (b) mRNA with two phosphates at the 5' terminus is an acceptor while RNA with a single 5'-terminal phosphate is not, (c) Mg2+ is required, (d) the pH optimum is 7.8, (e) PP1 is a strong inhibitor, and (f) the reverse reaction, namely the formation of GTP from PP1 and RNA containing the 5'-terminal structure G(5')pppN-, readily occurs. The S-adenosylmethionine:mRNA(guanine-7-)methyltransferase activity catalyzes the methylation of the 5'-terminal guanosine. This reaction exhibits the following characteristics: (a) mRNA with the 5'-terminal sequences G(5')pppA- and G(5')pppG- are acceptors, (b) only position 7 of the terminal guanosine is methylated; internal or conventional 5'-terminal guanosine residues are not methylated, (c) the reaction is not dependent upon GTP or divalent cations, (d) optimal activity is observed in a broad pH range around neutrality, (e) the reaction is inhibited by S-adenosylhomocysteine. Both the guanylyltransferase and methyltransferase reactions exhibit bisubstrate kinetics and proceed via a sequential mechanism. The reactions may be summarized: (see article).

Cations, Divalent↗
PubMed1975

Characterization of a polyriboadenylate polymerase from vaccinia virions.

A poly(A) polymerase with a molecular weight of approximately 80,000 containing 51,000 and 35,000 molecular weight subunits, was purified by affinity chromatography from vaccinia virus cores. The enzyme had a pH optimum of about 8.6, was dependent on divalent cations, and had considerably more activity with Mn-2+ than Mg-2+. At equimolar concentrations, other ribonucleoside triphosphates inhibited poly(A) polymerase activity by less than 10%; NaCl was extremely inhibitory at concentrations above 0.1 M. Under standard assay conditions, poly(A) polymerase activity was stimulated more than 10-fold by poly(C), but to small extent or not at all by other homopolyribonucleotides or natural RNA species unless they were first subjected to partial hydrolysis and alkaline phosphatase treatment. The ineffectiveness of most long polyribonucleotides was attributed to enzyme binding to internal regions. Short poly- or oligoribonucleotides prepared from natural or synthetic RNAs, except poly(G), exhibited similar priming abilities, and isotope transfer experiments indicated the covalent attachment of poly(A) to cytidylate, uridylate, and inosinate residues. Experiments with a series of uridylate oligomers indicated that the minimum effective primer length was four to six nucleotides. Partially digested DNA and short poly- and oligodeoxyribonucleotides of dT, dC, and dI, but not of dA and dG, also acted as effective primers for the poly(A) polymerase.

Adenine Nucleotides↗
PubMed1975

Purification of a protein kinase and two phosphate acceptor proteins from vaccinia virions.

A novel protein kinase that requires protamine as an activator to catalyze the phosphorylation of viral acceptor proteins was extracted from vaccinia virus cores with deoxycholate and purified 250-fold by DNA-cellulose and DEAE-cellulose column chromatography. The enzyme has a molecular weight of 62,000 as determined by sucrose gradient sedimentation. Two heat-stable phosphate acceptor proteins were extracted from virus particles with a nonionic detergent and purified by heat treatment, precipitation with organic solvents, and CM-cellulose chromatography. The molecular weights of the phosphate acceptor proteins, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, are 38,500 and 11,700.

Centrifugation, Density Gradient↗