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Biomedical subjects

C M Preston

Publications and source records attributed to C M Preston.

At least 91 records · Page 5Linked to original sources
PubMed1982

mRNA- and DNA-directed synthesis of herpes simplex virus-coded exonuclease in Xenopus laevis oocytes.

Microinjection of herpes simplex virus (HSV)-infected cell mRNA into Xenopus laevis oocytes resulted in the production of a new exonuclease activity. This enzyme strongly resembled the HSV alkaline exonuclease in many biochemical properties, and hybrid-arrested translation studies showed that it was virus coded, mapping at 0.080 to 0.185 genome map units. Exonuclease mRNA had a size and genome location equivalent to the mRNA encoding V185 in reticulocyte lysates, suggesting that V185 is the exonuclease. The enzyme synthesized in oocytes was found to act as an exonuclease in vivo. Two plasmids containing HSV DNA fragments directed the synthesis of exonuclease when microinjected into oocyte nuclei, and this finding enabled the coding and control sequences for this gene to be localized to 0.155 to 0.185 genome map units.

Animals↗
PubMed1981

Transcription and translation of the herpes simplex virus type 1 thymidine kinase gene after microinjection into Xenopus laevis oocytes.

The hybrid plasmid pTK1 consists of the herpes simplex virus type 1 (HSV-1) BamHI p fragment, which contains the thymidine kinase (TK) gene, inserted into the vector pAT 153. When pTK1 DNA was microinjected into nuclei of Xenopus laevis oocytes, functional HSV-1-specific TK was produced, showing that transcription and translation of the gene occurred. Investigation of pTK1-specific RNA by "Southern' blot hybridization revealed that all regions of the hybrid plasmid were transcribed by RNA polymerase II, but sequences present in TK mRNA were most highly represented in stable transcripts.

Animals↗
PubMed1981

Identification and mapping of two polypeptides encoded within the herpes simplex virus type 1 thymidine kinase gene sequences.

mRNA's homologous to the herpes simplex virus type 1 DNA restriction endonuclease fragment BamHI p, which contains the thymidine kinase gene, have been identified and mapped by hybrid-arrested translation and mRNA selection. Such mRNA's, when translated in vitro, directed the synthesis of polypeptides of apparent molecular weights 43,000 (VI43) and 39,000 (VI39). mRNA for enzymatically active thymidine kinase was enriched by more than 20-fold after selection. Mapping was carried out with restriction endonuclease fragments of BamHI p, and locations of the 5' and 3' termini of VI43 mRNA were deduced. Analysis of nucleotide sequences around the 5' terminus revealed several consensus sequences commonly found at the start of eucaryotic mRNA's and which are presumably involved in initiation of transcription by RNA polymerase II. Translation of mRNA's for VI43, VI39, and the thymidine kinase enzyme was arrested only by a 1,170-base-pair region of BamHI p. Since this region is insufficient for adjacent genes, coding sequences for VI43 and VI39 must overlap; the possible relationship of these two polypeptides is discussed. A virus-induced product equivalent to VI39 was detected in infected cells.

Base Sequence↗
PubMed1981

Enhanced mutability associated with a temperature-sensitive mutant of vesicular stomatitis virus.

Temperature-sensitive (ts) mutant tsD1 of vesicular stomatitis virus, New Jersey serotype, is the sole representative of complementation group D. Clones derived from this mutant exhibited three different phenotypes with respect to electrophoretic mobility of the G and N polypeptides of the virion in sodium dodecyl sulfate-polyacrylamide gel. Analysis of non-ts pseudorevertants showed that none of the three phenotypes was associated with the temperature sensitivity of mutant tsD1. Additional phenotypes, some also involving the NS polypeptide, appeared during sequential cloning, indicating that mutations were generated at high frequency during replication of tsD1. Furthermore, mutations altering the electrophoretic mobility of the G, N, NS, and M polypeptides were induced in heterologous viruses multiplying in the same cells as tsD1. These heterologous viruses included another complementing ts mutant of vesicular stomatitis virus New Jersey and ts mutants of vesicular stomatitis virus Indiana and Chandipura virus. Complete or incomplete virions of tsD1 appeared to be equally efficient inducers of mutations in heterologous viruses. Analysis of the progeny of a mixed infection of two complementing ts mutants of vesicular stomatitis virus New Jersey with electrophoretically distinguishable G, N, NS, and M proteins yielded no recombinants and excluded recombination as a factor in the generation of the electrophoretic mobility variants. In vitro translation of total cytoplasmic RNA from BHK cells indicated that post-translational processing was not responsible for the aberrant electrophoretic mobility of the N, NS, and M protein mutants. Aberrant glycosylation could account for three of four G protein mutants, however. Some clones of tsD1 had an N polypeptide which migrated faster in sodium dodecyl sulfate-polyacrylamide gel than did the wild type, suggesting that the polypeptide might be shorter by about 10 amino acids. Determination of the nucleotide sequence to about 200 residues from each terminus of the N gene of one of these clones, a revertant, and the wild-type parent revealed no changes compatible with synthesis of a shorter polypeptide by premature termination or late initiation of translation. The sequence data indicated, however, that the N-protein mutant and its revertant differed from the parental wild type in two of the 399 nucleotides determined. These sequencing results and the phenomenon of enhanced mutability associated with mutant tsD1 reveal that rapid and extensive evolution of the viral genome can occur during the course of normal cytolytic infection of cultured cells.

Animals↗
PubMed1981

Identification of a virus-specific polypeptide associated with a transforming fragment (BglII-N) of herpes simplex virus type 2 DNA.

The BglII N fragment of herpes simplex virus type 2 (HSV-2) DNA (approximately 0.58 to 0.63 map unit) was examined for encoded products. Using plasmid pGZ59, which consists of BglII-N cloned in pAT153, in conjunction with hybrid arrested translation, mRNA selection, and in vitro protein synthesis, we found that the major translated product of this region has an approximate molecular weight of 37,800. By further mapping, coding sequences for this polypeptide were located within the region of BglII-N representing approximately 0.58 to 0.61 genome map unit. To demonstrate immunological specificity, we used staphylococcal A protein immunoprecipitation with rabbit anti-HSV-1 or HSV-2 sera and antigens from HSV-1 or HSV-2 total mRNA translated in vitro and BglII-N-selected mRNA. The results show that the 37,800-dalton polypeptide has HSV-2 immunological specificity, as it is precipitated with anti-HSV-2 sera but not with anti-HSV-1 or control sera.

Antigens, Viral↗
PubMed1979

Control of herpes simplex virus type 1 mRNA synthesis in cells infected with wild-type virus or the temperature-sensitive mutant tsK.

This paper deals with control of mRNA levels, assayed by in vitro translation, in cells infected with herpes simplex virus type 1 (HSV-1). A particularly useful marker has been pyrimidine deoxyribonucleoside kinase (dPyK) mRNA, for which the enzymatically active product can be assayed quantitatively. Cells infected with the HSV-1 temperature-sensitive mutant tsK at the nonpermissive temperature (38.5 degrees C) or with wild-type HSV-1 in the continuous presence of cycloheximide contained no detectable dPyK mRNA. Upon temperature shift-down of tsK-infected cells to 31 degrees C, dPyK mRNA was produced, and this event was inhibited by actinomycin D but not cycloheximide. This result demonstrated that the defective polypeptide in tsK-infected cells was involved in transcription of the dPyK gene and could regain activity at 31 degrees C. Because tsK-infected cells synthesized mainly immediate early polypeptides at 38.5 degrees C, the involvement of this polypeptide class in synthesis of dPyK mRNA was investigated. Analysis of the kinetics of inductions of dPyK mRNA indicated that the temperature-sensitive lesion in tsK lies in an immediate early polypeptide which is directly responsible for activation of the dPyK gene at the transcriptional level.

Cell Line↗
PubMed1979

Separation and characterization of herpes simplex virus type 1 immediate-early mRNA's.

Polyadenylated immediate-early transcripts of herpes simplex virus type 1, made in BHK cells infected and maintained in the presence of cycloheximide, have been separated on denaturing agarose gels containing methyl mercuric hydroxide. Three virus-specific mRNA bands of estimated sizes 4.7, 3.0, and 2.0 kilobases (kb) were detected, and these mRNA's were mapped on the virus genome and also used to direct protein synthesis in vitro. The 4.7- and 3.0-kb mRNA's hybridized predominantly to certain DNA fragments which are located in the short and long repetitive regions of the genome, respectively, whereas the 2.0-kb mRNA's mapped to three discrete regions of the virus DNA. In vitro translation of these separated mRNA size classes indicated that the 3.0-kb mRNA specified the synthesis of virus polypeptide Vmw 110, whereas the 2.0-kb mRNA's specified Vmw 68, 63, and 12. The synthesis of small amounts of Vmw 175 was specified by the 4.7-kb mRNA. In contrast with the mRNA's which specify these other immediate-early polypeptides, that specifying Vmw 12 is much larger than required for its coding sequences.

Animals↗
PubMed1979

Abnormal properties of an immediate early polypeptide in cells infected with the herpes simplex virus type 1 mutant tsK.

Previous studies (R. J. Watson and J. B. Clements, Virology 91:364--379, 1978; C. M. Preston, J. Virol. 29:275--284, 1979) have shown that the herpes simplex virus type 1 (HSV-1) mutant tsK has a temperature-sensitive lesion in an immediate early polypeptide whose function is to induce synthesis of new viral transcripts, including mRNA, for pyrimidine deoxyribonucleoside kinase. The studies presented here examine the properties of immediate early polypeptides in wild-type HSV-1 and tsK-infected cells at 31 and 38.5 degrees C. The overall pattern of immediate early protein synthesis was similar in wild-type HSV-1- and tsK-infected cells when radiolabeled with [35S]methionine or 14C-amino acid mixture. Further investigation, however, revealed two aberrant properties of the polypeptide Vmw 175 in tsK-infected cells at 38.5 degrees C. Upon cell fractionation, large amounts of this polypeptide were recovered in the cytoplasmic fraction, in contrast to tsK-infected cells at 31 degrees C or wild-type HSV-1-infected cells at either temperature. Furthermore, at 38.5 degrees C tsK-induced Vmw 175 was not processed normally to forms of lower electrophoretic mobility. Both of these defects were reversible upon downshift of tsK-infected cells, even in the absence of further protein synthesis, but were not observed in cells infected with a revertant of tsK. Coinfection of tsK-infected cells with wild-type HSV-1 did not alleviate these lesions, suggesting that they resulted from an abnormal Vmw 175 polypeptide rather than from a defective processing enzyme. Temperature upshift of tsK-infected cells caused reversion of Vmw 175 to the mutant form. The progression to synthesis of late polypeptides was also arrested; therefore, a functional lesion was also reversible upon temperature changes between 31 and 38.5 degrees C during the early stages of infection. The identification of a polypeptide with abnormal properties in tsK-infected cells and the demonstration that these properties, and the functional lesion, are reversible may provide an important system for investigation of HSV-1 transcriptional control.

Animals↗
PubMed1978

Cell-free systems for in vitro translation of herpes simplex virus messenger RNA.

A cell-free system active in translation of HSV mRNA was obtained by fractionation of reticulocyte lysates. Endogenous protein synthesis was further reduced by preincubation with micrococcal nuclease, although this treatment did not affect the translation of infected (or uninfected) BHK cell RNA. Polypeptides synthesized by the fractionated reticulocyte cell-free system were very similar to those produced by unfractionated reticulocyte lysates.

Cell Fractionation↗
PubMed1977

Cell-free translation of RNA synthesized in vitro by a transcribing nucleoprotein complex prepared from purified vesicular stomatitis virus.

The RNA species synthesized in vitro by a transcribing nucleoprotein (TNP) complex of vesicular stomatitis virus (VSV) were translated with high efficiency in a fractionated cell-free system derived from reticulocytes. The use of TNP complexes isolated from VSV Indiana, VSV New Jersey, and Chandipura viruses showed that in each case the predominant polypeptides synthesized had electrophoretic mobilities identical to their virion N, NS, and M polypeptides in proportions reflecting those found in infected cells rather than purified virions. A minor polypeptide corresponding to unglycosylated polypeptide G was also observed, but the in vitro synthesis of polypeptide L was not detected. The addition of RNase inhibitor to transcription mixtures markedly increased the rate of RNA synthesis. Furthermore, the messenger activity of the RNA was significantly enhanced. The inclusion of S-adenosyl L-methionine during transcription substantially increased the messenger activity of the product RNA, suggesting a requirement for methylation. Fractionation by oligodeoxythymidylic acid-cellulose chromatography revealed that the RNA required a polyadnylic acid tract for messenger activity.

Cell-Free System↗
PubMed1977

Cell-free synthesis of herpes simplex virus-coded pyrimidine deoxyribonucleoside kinase enzyme.

The incubation of a cell-free protein-synthesizing system prepared from rabbit reticulocytes with cytoplasmic RNA from herpes simplex virus (HSV)-infected cells resulted in increased thymidine kinase activity. This enzyme activity was specifically inhibited by anti-HSV antiserum and was relatively unaffected by TTP, an inhibitor of cellular thymidine kinases. Induction of the new activity was prevented by addition of inhibitors of eucaryotic protein synthesis, and no new activity was detected when RNA from cells infected with pyrimidine deoxyribonucleoside kinase-deficient mutants, instead of wild-type HSV, was added. An increased deoxycytidine kinase activity with similar properties to the HSV-specified enzyme activity was also present in cell-free systems incubated with RNA from HSV-infected cells. Phosphorylation of thymidine and deoxycytidine at 30 degrees C continued for longer than 11 h. The findings are consistent with the accurate synthesis in vitro of enzymically active HSV-specified pyrimidine deoxyribonucleoside kinase.

Animals↗
PubMed1976

A general survey of the proton spin-lattice relaxation-times of some oligo- and poly-saccharide derivatives.

A Fourier-transform method has been used to measure the spin-lattice relaxation-times (T1 values) of the anomeric protons of a selection of oligo- and poly-saccharide derivatives. Although systematic variations are found for the substances of lower molecular weight, these variations are essentially non-existent at higher molecular weights. Data for the disaccharides cellobiose, maltose, lactose, gentiobiose, and melibiose demonstrate that proton T1-values may provide a powerful method for evaluating conformations of oligosaccharides.

Electron Spin Resonance Spectroscopy↗
PubMed1976

The effects of herpes simplex virus type 1 on cellular DNA-dependent RNA polymerase activities.

An investigation of the activity of nuclear RNA polymerase following infection of LS cells with HSV-1 shows a decline in both major activities. This effect is not entirely due to inhibition of cellular protein synthesis, and the effect of alpha-amanitin-sensitive RNA polymerase is mediated by a protein(s) synthesized in the infected cell. Changes in the properties of this RNA polymerase activity include a reduction in the relative UTP/GTP incorporation ratio and an increased sensitivity to inhibition by actinomycin D, indicating that RNA polymerase II is involved in virus transcription.

Amanitins↗