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S Millward

Publications and source records attributed to S Millward.

At least 37 records · Page 2Linked to original sources
PubMed1984

mRNA discrimination in extracts from uninfected and reovirus-infected L-cells.

Uncapped reovirus mRNA extracted at late times from infected L-cells is preferentially translated in extracts from infected L-cells. However, translation of this uncapped, late, reovirus mRNA in extracts from infected cells is sensitive to inhibition by the cap analog m7GTP . These results imply that reovirus infection does not induce a transition from cap-dependent to cap-independent translation. Nevertheless, the results of in vitro translational competition experiments between L-cell mRNA and late viral mRNA were consistent with the view that reovirus does induce an alteration in the cap-dependent translational apparatus of L-cells. The reduced efficiency of translation of a variety of capped mRNAs in extracts from infected cells is also consistent with this notion. We further conclude that a factor exists in reovirus-infected L-cells that specifically stimulates translation of uncapped reovirus mRNAs.

Animals↗
PubMed1981

In vitro translation in reovirus- and poliovirus-infected cell extracts. Effects of anti-cap binding protein monoclonal antibody.

Monoclonal antibodies directed against rabbit reticulocyte cap binding proteins were used as probes to study cap-dependent translation initiation in mock-infected and virus (poliovirus or reovirus)-infected cells. The antibodies inhibited capped mRNA translation, but did not have any effect on the translation of naturally uncapped mRNAs in uninfected cell-free extracts prepared from either L-cells or HeLa cells. Cell-free extracts prepared from either reovirus-infected L-cells or poliovirus-infected HeLa cells did not support capped mRNA translation. However, translation of naturally uncapped mRNAs in the latter extracts was comparable to that observed in extracts from uninfected cells. Thus, translation of mRNAs in extracts from either reovirus- or poliovirus-infected cells proceeds most probably via a cap-independent mechanism, since it is not inhibited by antibodies directed against cap binding proteins.

Animals↗
PubMed1981

Similar sets of terminal oligonucleotides from reovirus double-stranded RNA and viral messenger RNA synthesized in vitro.

Reovirus genomic double-stranded RNA (dsRNA) and viral messenger RNA synthesized in vitro were labeled by periodate oxidation and [3H]borohydride reduction. The 3H label was incorporated into 3'-terminal C residues and 5'-terminal N7-methylguanosine residues. Analysis of ribonuclease digests of the 3H-labeled RNA indicated that the minus strands of dsRNA contained the common 3'-terminal sequence ... PypPupGpC and that the plus strands contained heterogeneous sequence at both the 5' termini and 3'termini that corresponded to sequences at the 5' termini and 3' termini of mRNA. These results suggest that the plus strands of dsRNA and mRNA synthesized in vitro are essentially identical sets of molecules.

Base Sequence↗
PubMed1980

Reovirus-induced modification of cap-dependent translation in infected L cells.

The translational apparatus in cell-free extracts prepared from L cells infected with reovirus undergoes a time-dependent transition from cap dependence to cap independence. Extracts from uninfected L cells translate capped reovirus mRNA at high efficiency and synthesize the expected three size classes of reovirus polypeptides, and the translation is sensitive to m7G(5')ppp. This same extract translates uncapped mRNA at a much lower efficiency. In contrast, extracts from infected L cells translate uncapped reovirus mRNA at high efficiency and synthesize the correct three size classes of polypeptides, and the translation is not sensitive to inhibition by m7G(5')ppp. Infected cell extracts translate capped mRNA at reduced efficiency (a,proximately 25%), the translation is not sensitive to inhibition by m7G(5')ppp, and the correct three size classes of viral polypeptides are not synthesized. These observations may explain how reovirus takes over the host translational apparatus.

Animals↗
PubMed1980

mRNA capping enzymes are masked in reovirus progeny subviral particles.

We examined the enzyme activities associated with progeny subviral particles isolated from L-cells infected with reovirus at 12 h postinfection. Activities normally present in reovirus cores were also found to be present in the progeny subviral particles, with the exception of the capping enzymes. The methylase and guanyl transferase activities, which constitute the capping system, were present in a masked form that could be activated by chymotrypsin digestion. The appearance of these progeny subviral particles in infected cells coincided with the time when mRNA synthesis was maximal, suggesting that viral mRNA synthesized at later times is uncapped.

Adenosine Triphosphatases↗
PubMed1980

Reovirus progeny subviral particles synthesize uncapped mRNA.

Reovirus progeny subviral particles were isolated from L-cells at late times postinfection. It has been shown (D. Skup and S. Millward, J. Virol. 34: 490--496, 1980) that these progeny subviral particles have masked capping enzymes, indicating that mRNA synthesized by these particles should be uncapped. When progeny subviral particles were used for mRNA synthesis in vitro, they failed to incorporate the beta-phosphate of [beta-32P]GTP into the 5' terminal. Direct analysis of reovirus mRNA synthesized by progeny subviral particles in the presence of either [alpha-32P]GTP or [alpha-32P]CTP indicated that the 5' terminal was uncapped, having the structure pGpC... The implications of this finding to the reovirus replicative cycle are discussed.

Chymotrypsin↗
PubMed1978

Nucleotide sequences at the 5' termini of reovirus mRNA's.

During in vitro synthesis of reovirus mRNA by viral cores, methyl groups from S-adenosylmethionine are incorporated only into 5'-terminal cap structures, i.e., m7GpppGmCp.... Thus, mRNA synthesized in the presence of S-adenosyl-[methyl-3H]methionine is 3H labeled specifically at the 5' terminus. This circumstance was exploited in the determination of 5'-terminal nucleotide sequences. Seven 5'-terminal fragments derived by complete RNase T1, digestion of methyl-3Hlabeled mRNA were partially degraded with RNase T2, and the products were separated by electrophoresis-homochromatography. From the patterns formed by the methyl-3H-labeled RNase T2 products, the sequences of the seven RNase T1-generated fragments were deduced. All seven fragments started with the sequence m7GpppGmCUA, after which the sequences diverged, with a tendency to be either U-rich or A-rich. Their chain lengths ranged from 7 to 10 nucleotides (excluding the m7G residue), and none of them contained an initiator AUG triplet. The sequences obtained support the hypothesis that virion-associated oligonucleotides arise through abortive transcription of the viral genome. There is no apparent 5'-terminal sequence feature distinctive of early versus late mRNA species within the small-mRNA size class.

Base Sequence↗
PubMed1977

5' Terminal noncoding sequence heterogeneity in reovirus mRNA.

The nucleotide sequences of the mRNAs of reovirus appear to diverge near the 5' termini. Ribonuclease T1 digestion of methylated mRNA synthesized in vitro yielded seven different 5' terminal fragments of the form m7G5'pp5' GmpCpUp(Np)nGp. Chain length analysis showed that the parameter "n" in this structural formula assumes the values 3, 4 and 5.

Base Sequence↗
PubMed1977

Highly efficient translation of messenger RNA in cell-free extracts prepared from L-cells.

Micrococcal nuclease was used to eliminate endogenous protein synthesis in extracts prepared from L cells. The nuclease can be inhibited subsequently with 2'-deoxythymidine-3', 5'-diphosphate. Nuclease-treated extracts primed with exogenous reovirus mRNA, synthesized full length polypeptides with linear kinetics for almost two hours leading to stimulation of the order of 10(4) times over endogenous background. On the average, between 40 and 50 molecules of polypeptide were synthesized per molecule of mRNA.

Animals↗
PubMed1976

Methylated constituents of poly(A)- and poly(A)+ polyribosomal RNA of sea urchin embryos.

We have examined the poly(A)- and poly(A)+ polyribosomal (Ps) RNAs of developing sea urchin embryos to see whether they contain the capped structures previously observed in the poly(A)+ cytoplasmic RNA (mRNA) of a variety of eucaryotic organisms. We have found that both classes of Ps RNA contain 7-methylguanosine linked to the RNA by a 5'-5' triphosphate bridge. Capped structures derived from either class of Ps RNA appear to be of the type m7G5'ppp5'NmpNp, with only a single sugar-methylated nucleoside per cap, and we have estimated that both classes of Ps RNA are capped to the same extent. The 5' terminal 2'-0-methylated nucleosides have been identified and quantitated. The relative amounts are Am = 78%, Gm = 14%, Cm = 5%, Um = 3% for poly(A)- Ps RNA, and Am = 78%, Gm = 15%, Cm = 5%, Um = 2% for poly (A)+ Ps RNA. In addition, both classes of Ps RNA contain 2-4 internal residues of N6-methyladenylic acid per molecule.

Adenosine↗
PubMed1975

m7G5'ppp5'GmptcpUp at the 5' terminus of reovirus messenger RNA.

In the presence of S-adenosyl methionine the 5' terminal guanosine residue of in vitro synthesized reovirus mRNA becomes methylated at the 2'-OH position. In addition, 7-methyl guanylic acid is condensed covalently at the 5' terminus resulting in the formation of a 5' to 5' triphosphate bridge. Analysis of the 5' terminal sequence of methylated reovirus mRNA revealed that it has the structure m7G5'ppp5'GmpCpUp.

Base Sequence↗
PubMed1974

In vitro methylation of nascent reovirus mRNA by a virion-associated methyl transferase.

Chymotrypsin-derived cores, but not virions, catalyze the transfer of methyl groups from S-adenosyl methionine (SAM) to nascent mRNA synthesized in vitro by the core polymerase. The reaction requires Mg(++), and is dependent on the presence of all 4 ribonucleoside triphosphates (rNTPs). Methylation proceeds optimally at 51 degrees C. All ten species of mRNA become methylated during transcription and it is estimated that one methyl residue is incorporated per RNA chain. Experiments designed to determine the location of the methylated nucleotide clearly demonstrate that methylation occurs exclusively at the 5' ends of nascent mRNA.

Chymotrypsin↗
PubMed1974

Control of transcription of the reovirus genome.

The ten double-stranded RNA segments of the reovirus genome are not transcribed at equal frequencies until later times during the course of infection. When an inhibitor of protein synthesis such as cycloheximide was added to infected cells at the beginning of infection, only four of the ten segments were transcribed. By two hr post infection, five and possibly seven of the segments were being transcribed. By four hr post infection all ten segments were being transcribed but not yet at equal relative frequencies. The transcription pattern at intermediate (4 hr) and late (10 hr) times were verified without using cycloheximide. A repressor present in the host cell may be responsible for controlling transcription of the reovirus genome.

Animals↗