Search PubMed⌕ Search

Biomedical subjects

B Moss

Publications and source records attributed to B Moss.

At least 415 records · Page 23Linked to original sources
PubMed1981

Post-transcriptional modifications of mRNA. Purification and characterization of cap I and cap II RNA (nucleoside-2'-)-methyltransferases from HeLa cells.

The existence in HeLa cell extracts of two separate RNA (nucleoside-2')-methyltransferases involved in the modification of mRNA was established using assays that specifically measure the conversion of cap O [m7G(5')pppNpN-] to cap I [m7G(5')pppNmpN-] and cap I to cap II [m7G(5')pppNmpNm-]. Cap II methyltransferase activity was found almost exclusively in cytoplasmic fractions while cap I methyltransferase activity was also found in the nucleus, its apparent biological site of action. The two enzymes were purified by DEAE-cellulose and phosphocellulose chromatography and their optimal reaction conditions were determined. The substrate specificity of cap I methyltransferase was examined with particular regard to information that would help elucidate the natural order of capping and methylation was drawn from data presented here. Both purine and pyrimidine nucleosides in the N position of M7G(5')pppN- were methylated by purified cap I methyltransferase.

Cell Nucleus↗
PubMed1981

Distinctive nucleotide sequences adjacent to multiple initiation and termination sites of an early vaccinia virus gene.

Poxviruses, unlike other DNA viruses, replicate in the cytoplasm of infected cells and use their own system of transcription. Examination on one early mRNA synthesized in vivo and in vitro indicated that it has multiple closely spaced 5' and 3' ends. A remarkable 88% AT-rich 60 bp DNA sequence was found immediately upstream of the initiation of transcription sites. Although DNA sequences that bear some homology to Pribnow and Hogness boxes are present, additional recognition sequences located further upstream of procaryotic and eucaryotic initiation sites are absent. A possible initiation of translation codon occurs about 50 nucleotides from the 5' end of the message. The transcript terminates near or within a hexanucleotide CTATTC that is tandemly repeated four times. Sequences similar to those regulating termination of transcription in procaryotes or poly (A) addition in eucaryotes were not found, suggesting that poxviruses have evolved unique recognition signals.

Cytoplasm↗
PubMed1981

Instability and reiteration of DNA sequences within the vaccinia virus genome.

The sequence arrangement within the nontranscribed portion of the inverted terminal repetition of the vaccinia virus genome exists in quasi-stable and unstable forms that are not distinguishable on the basis of viral infectivity. The unstable forms, which composed about 20% of a serially passaged stock of virus, were recognized by terminal heterogeneity on restriction endonuclease analysis. Instead of a single terminal fragment from each end of the genome, an array of eight or more fragments differing in size by 1650-base-pair increments was detected. This feature was not eliminated by repeated plaque purification, indicating that the population of DNA molecules with various numbers of reiterations can rapidly evolve from the DNA of a single virus particle. However, at each successive round of plaque purification, about 20% of the unstable isolates revert back to the more stable form. Stable forms are characterized by the presence of a set of 13-17 tandem 70-base-pair repeats on each side of a 435-base-pair intervening sequence near both ends of the genome. In contrast, the unstable forms possess sets of tandem repeats and intervening sequences that alternate many times in series. The transition between the two genomic forms and the evolution of the unstable form appear to be mediated by recombinational events.

Base Sequence↗
PubMed1981

Hybridization selection and cell-free translation of mRNA's encoded within the inverted terminal repetition of the vaccinia virus genome.

Early polypeptides encoded within the 10,000-base pair terminally repeated region of the vaccinia virus genome were mapped by cell-free translation of mRNA that was selected by hybridization to restriction fragments and to separated strands of a recombinant lambda phage. The results, which were confirmed by hybrid arrest of translation, indicated that polypeptides of 7,500 (7.5K), 19,000 (19K), and 42,000 (42K) daltons mapped at approximately 3.2 to 4.3, 6.5 to 7.2, and 7.2 to 8.3 kilobase pairs from the end of the genome, respectively. mRNA's for the 42K and 7.5K polypeptides were transcribed towards the end of the genome, whereas mRNA for the 19K polypeptide was transcribed in the opposite direction. Including polyadenylic acid tails, the lengths of the mRNA's for the 7.5K, 19K, and 42K polypeptides, determined by gel electrophoresis of denatured RNA, hybridization selection, and cell-free translation, were approximately 1,200, 680, and 1,280 nucleotides, respectively. mRNA's for the 42K and 19K polypeptides were only about 100 nucleotides longer than the minimums required to code for their respective polypeptides, whereas mRNA for the 7.5K polypeptide contained 900 nucleotides of untranslated sequence. This long untranslated portion of the latter mRNA was probably located near the 3' end, because this gene was only inactivated by high doses of UV irradiation. This small target size also excluded certain models for RNA processing involving formation of the mRNA's for the 42K and 7.5K polypeptides from a common promoter. Rabbitpox virus, which has an inverted terminal repetition approximately half that of vaccinia virus, was also shown to encode mRNA's that hybridized to the cloned terminal segment of vaccinia virus DNA.

Bacteriophage lambda↗
PubMed1981

In vitro transcription of the inverted terminal repetition of the vaccinia virus genome: correspondence of initiation and cap sites.

Specific RNAs synthesized in vitro by vaccinia virus cores were analyzed with the aid of DNA from the terminal 9,000 base pairs of the genome that was cloned in phage lambda, pBR322, and the single-stranded phage fl. Three mRNA's coding for polypeptides with molecular weights of 7,500 (7.5K), 19K, and 42K were shown to have sizes and map positions similar to those described for mRNA's made early in infection. A previously undescribed transcript made in vivo and in vitro, with a 5' end at about 8.7 kilobase pairs from the end of the genome, was also detected. After chemical removal of the terminal 7-methylguanosine residue, the 5' ends of the RNAs were specifically labeled by enzymatic capping and the mapped by gel electrophoresis of nuclease-resistant RNA.DNA hybrids, as well as by hybridization of the end-labeled RNA to immobilized DNA restriction fragments. Analysis of the purified cap structures demonstrated that three of the mRNA's have both m7G(5')pppAm and, m7G(5')pppGm ends, indicating some degree of terminal heterogeneity. The fourth transcript has exclusively m7G(5')pppAm ends. By synthesizing RNA in the presence of [beta-32P]GTP, it could be shown that cap sites correspond to sites of initiation of RNA synthesis.

Bacteriophage lambda↗
PubMed1981

Transcriptional and translational mapping of a 6.6-kilobase-pair DNA fragment containing the junction of the terminal repetition and unique sequence at the left end of the vaccinia virus genome.

The penultimate EcoRI fragments from the left and right ends of the vaccinia virus genomes were cloned in phage lambda. Heteroduplex analysis and comparison of restriction fragments indicated that the inverted terminal repetition extended 780 base pairs (bp) beyond the EcoRI site or about 9,800 bp from each end of the genome. Detailed physical, transcriptional, and translational maps of the 6,600-bp left penultimate EcoRI fragment were prepared so as to extend previous maps of the 9,000-bp terminal EcoRI fragment. Polypeptides with molecular weights of 6,000 (6K polypeptide), 13,000, 19,000, 21,000, and 60,000 were synthesized in a reticulocyte cell-free system programmed with immediate early RNA (made in the presence of cycloheximide) or early RNA (made in the presence of cytosine arabinoside) and selected by hybridization to immobilized recombinant DNA. A 22K polypeptide was detected as a translation product of late RNA that hybridized to this DNA fragment. A variety of biochemical procedures were used to size and map the mRNA's. Of the five messages that hybridized to this 6,600-bp EcoRI fragment, only the one for the 21K polypeptide was encoded within the inverted terminal repetition and hybridized to the rightward-reading DNA strand. (Three additional early polypeptides were encoded within the first 9,000 bp of the inverted terminal repetition.) The remaining early polypeptides were encoded within the unique portion of the penultimate EcoRI fragment and were transcribed from the leftward-reading strand. Additional high-molecular-weight early RNAs of unknown function were also detected; however, there was no evidence indicating that mature mRNA's were spliced.

Base Sequence↗
PubMed1981

Extension of the transcriptional and translational map of the left end of the vaccinia virus genome to 21 kilobase pairs.

Physical, transcriptional, and translational maps of an EcoRI fragment located between 15,800 and 20,600 base pairs from the left end of the vaccinia virus genome were prepared. Major polypeptides with molecular weights of 14,000 (14K polypeptide), 32,000 and 38,000 were synthesized in a reticulocyte cell-free system programmed with immediate early RNA made in the presence of cycloheximide and selected by hybridization to lambda recombinant DNA containing the EcoRI fragment. With early RNA made in the presence of cytosine arabinoside, an inhibitor of DNA replication, the polypeptide pattern was similar except for quantitative differences in which less 38K polypeptide was detected as a translation product. With late RNA, isolated 6 h after infection without inhibitors, only traces of the early translation products were found and a new 40K polypeptide was detected. The size of the mRNA's for the 14K, 32K, and 38K polypeptides were determined to be approximately 760,880, and 1,150 nucleotides, respectively, by several independent procedures. Several large early RNAs not shown to code for any additional translation products were also detected. The size of the late message for the 40K polypeptide varied from 920 to 3,100 nucleotides. This heterogeneity appeared to be a general property of vaccinia virus late mRNA's. No evidence of RNA splicing was obtained by analysis of RNA-DNA hybrids after nuclease S1 treatment. Further analyses using separated recombinant DNA strands and restriction fragments indicated that all mRNA's were encoded by the leftward-reading DNA strand and at least two were overlapping. Since early and late mRNA's were encoded by the same DNA strand, the possibility of temporal regulation by transcriptional strand switching was eliminated. In conjunction with previous studies, a transcriptional map of the left 20,600 base pairs of the vaccinia virus genome was derived.

DNA Restriction Enzymes↗
PubMed1981

Deletion of a 9,000-base-pair segment of the vaccinia virus genome that encodes nonessential polypeptides.

Deletions contained within the genomes of unstable and stable variants of vaccinia virus (strain WR) were analyzed. Restriction endonuclease mapping and hybridization to specific 32P-labeled DNA probes indicated that more than 6 X 10(6) daltons of DNA were deleted from the variants. In each case, the deletion occurred on the left side of the genome and started very close to the junction of the inverted terminal repetition and unique sequence. Both variants also contained a new SstI side on the right side of the genome. Hybridization selection and cell-free translation experiments indicated that these variants lost the ability to synthesize at least eight early mRNA's mapping within the deleted region. Although the deleted DNA was not essential for replication of the WR strain of vaccinia virus under laboratory conditions of infection, it presumably has a defined role under other circumstances. This conclusion was based on the conservation within the Elstree strain of vaccinia, the Utrecht strain of rabbitpox, and the Brighton strain of cowpox virus of sequences homologous to the deleted DNA. Moreover, mRNA's that hybridized to the deleted vaccinia virus DNA segment and encoded similar size polypeptides were made in cells infected with rabbitpox and cowpox viruses.

Base Sequence↗
PubMed1980

Purification and characterization of a DNA-dependent RNA polymerase from vaccinia virions.

A DNA-dependent RNA polymerase has been extracted from vaccinia virions and purified to near homogeneity as judged by glycerol gradient sedimentation and polyacrylamide gel electrophoresis. The native enzyme has a molecular weight of approximately 500,000 and can be dissociated into putative subunits of 140,000, 137,000, 37,000, 35,000, 31,000, 22,000, and 17,000 daltons. Activity was dependent on all four ribonucleoside triphosphates, Mn2+, and a DNA template. Optimal activity occurred at pH 7.9 in the presence of 90 mM KCl or 40 mM (NH4)2SO4. All single-stranded DNAs tested served as templates. By contrast, linear double-stranded DNAs were not effectively transcribed and very low activity was obtained with circular supercoiled DNAs which contain small single-stranded regions. The synthetic alternating copolymer poly(dA-dT), which forms a completely base-paired structure, also was not transcribed, whereas poly(dA,dT) and other random copolymers served as templates. Of the four homopolydeoxribonucleotides, only poly(dC) and poly(dT) were transcribed, suggesting that initiation specifically occurs with purine ribonucleotides. In support of this, higher Km values were obtained for GTP and ATP (333 and 80 micro M, respectively) than for UTP and CTP (22 and 12 micro M, respectively) using a DNA template. The RNA polymerase was inhibited by polyanions but was resistant to rifampicin and alpha-amanitin.

Base Composition↗
PubMed1980

Inverted terminal repetition in vaccinia virus DNA encodes early mRNAs.

Vaccinia virus DNA contains a long inverted terminal repetition of MW approximately 6.8 x 10(6). A fragment of MW 6.3 X 10(6) from this repetition has been cloned in coliphage lambda and used to isolate RNA from virus-infected cells. Electron microscopy indicates that early RNAs are transcribed from the repeated sequence and cell-free translation shows that the RNAs code for polypeptides.

Bacteriophage lambda↗
PubMed1980

Purification and characterization of mRNA guanylyltransferase from HeLa cell nuclei.

GTP:mRNA guanylyltransferase, an enzyme that catalyzes the transfer of a GMP residue from GTP to the 5' end of RNA to form a cap structure identified as G(5')pppN-, has been isolated from HeLa cell nuclei. The enzyme has been purified approximately 1000-fold and separated by column chromatography (using DEAE-cellulose, phosphocellulose, Cibacron blue-agarose, and GTP-agarose) from a variety of other activities, including RNA triphosphatase and mRNA (guanine-7)methyltransferase. The reaction product was identified by its resistance to Penicillium nuclease and alkaline phosphatase, sensitivity to venom phosphodiesterase, and electrophoretic and chromatographic mobilities relative to authentic standards. Optimal enzyme activity was obtained at pH 7.5 in the presence of Mn2+ or Mg2+, GTP, and an appropriate acceptor polyribonucleotide. The enzyme was inhibited by elevated concentrations of salt and by sulfhydryl-binding reagents but was unaffected by S-adenosylmethionine or S-adenosylhomocysteine. A molecular weight of 48,500 was estimated by sucrose gradient centrifugation of purified enzyme.

Cell Nucleus↗
PubMed1980

Modification of the 5' end of mRNA. Association of RNA triphosphatase with the RNA guanylyltransferase-RNA (guanine-7-)methyltransferase complex from vaccinia virus.

Purified RNA guanylyltransferase . RNA (guanine-7-)methyltransferase complex from vaccinia virus contains RNA triphosphatase activity. The latter activity, which removes the gamma-phosphate from triphosphate-ended polyribonucleotides, co-chromatographed with the capping and methylating enzyme complex on seven different ion exchange or affinity columns and co-sedimented with the complex on a glycerol gradient. On a molar basis, the RNA triphosphatase was about 100 times more active than the associated RNA guanylyl-transferase. When the purified enzyme complex was incubated with poly(A) containing a 5'-triphosphate, removal of the gamma-phosphate preceded capping. Furthermore, there was no significant difference in the rate or extent of capping 5'-diphosphate- or 5'-triphosphate-ended poly(A). Physical association of the three enzymatic activities appears to be an efficient mechanism for carrying out the following successive steps in cap formation: (formula: see text).

Acid Anhydride Hydrolases↗
PubMed1980

Tandem repeats within the inverted terminal repetition of vaccinia virus DNA.

A tandemly repeated sequence within the genome of vaccinia virus is cut to fragments of approximately 70 bp by Hinf I, Taq I or Mbo II. The 70 bp repetition was localized within the much larger (10,300 bp) inverted terminal repetition by restriction analysis of cloned DNA fragments and by hybridization of the purified 70 bp repeat to vaccinia virus DNA restriction fragments. The molar abundance of the 70 bp fragment corresponds to a 30 fold repetition at each end of the genome. The repeating restriction endonuclease sites were mapped by agarose gel electrophoresis of partial Hinf I digests of the terminally labeled cloned DNA fragment. The first of 13 repetitive Hinf I sites occurred approximately 150 bp from the end of the cloned DNA. After an intervening sequence of approximately 435 bp, a second series of 17 repetitive Hinf I sites occurred. The DNA between the two blocks of repetitions has a unique sequence containing single Dde I, Alu I and Sau 3A sites. Tandem repeats within the inverted terminal repetition could serve to accelerate self-annealing of single strands of DNA to form circular structures during replication.

Base Sequence↗
PubMed1980

Expression of the vaccinia virus genome: analysis and mapping of mRNAs encoded within the inverted terminal repetition.

We have investigated the organization of transcriptional units within a 9000 bp segment of the terminally repeated region of the DNA genome of vaccinia virus, which uses its own enzyme system to synthesize mRNA within the cytoplasm of infected cells. RNA splicing, which has been demonstrated for DNA viruses that replicate within the nucleus of infected cells, does not appear to be involved in the formation of these first vaccinia virus mRNAs to be examined. Three immediate early mRNAs, approximately 1050, 600 and 1100 nucleotides long, were mapped between 3.21 and 4.24, 6.54 and 7.16, and 7.20 and 8.23 kb from the end of the genome, respectively. The direction of transcription was toward the end of the genome for the two larger mRNAs and in the opposite direction for the smallest one. Additional minor RNAs, which were larger in size, were mapped between and to the same DNA strand as the mRNAs of 1050 and 1100 nucleotides. No evidence for interrupted genes was obtained by nuclease S1 analysis after hybridization of RNA to labeled DNA. In addition, the 5' ends of the mRNAs, which were specifically labeled by in vitro capping, hybridized to DNA adjacent to the body of the message.

DNA, Recombinant↗