182 results on '"J B Clements"'
Search Results
2. Herpes Simplex Virus IE63 (ICP27) Protein Interacts with Spliceosome-Associated Protein 145 and Inhibits Splicing prior to the First Catalytic Step
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Sarah Wadd, J B Clements, Helen E. Bryant, Angus I. Lamond, and Saul J. Silverstein
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Spliceosome ,RNA Splicing ,viruses ,Immunology ,Exonic splicing enhancer ,Prp24 ,Herpesvirus 1, Human ,Biology ,Heterogeneous ribonucleoprotein particle ,Microbiology ,Catalysis ,Heterogeneous-Nuclear Ribonucleoproteins ,Immediate-Early Proteins ,Splicing factor ,Protein splicing ,Virology ,Humans ,Binding Sites ,Alternative splicing ,RNA-Binding Proteins ,Molecular biology ,Virus-Cell Interactions ,Ribonucleoproteins ,Insect Science ,RNA splicing ,Spliceosomes ,HeLa Cells - Abstract
The multifunctional herpes simplex virus type 1 (HSV-1) protein IE63 (ICP27) interacts with the essential pre-mRNA splicing factor, spliceosome-associated protein 145 (SAP145), and in infected cells IE63 and SAP145 colocalize. This interaction was reduced or abrogated completely using extracts from cells infected with IE63 viral mutants, with mutations in IE63 KH and Sm homology domains, which do not exhibit host shutoff or inhibit splicing. In the presence of IE63, splicing in vitro was inhibited prior to the first catalytic step and the B/C complex formed during splicing was shifted up in mobility and reduced in intensity. With the use of splicing extracts, IE63 and SAP145 both comigrated with the B/C complex, suggesting that they interact within this complex to inhibit B/C complex formation or conversion. The inhibition of splicing may facilitate the export of viral or cellular transcripts, possibly via other protein partners of IE63. These data provide important new insights into how IE63 influences pre-mRNA processing during HSV-1 infection. more...
- Published
- 2001
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Catalog
3. Herpes simplex virus type 1 immediate early protein IE63 shuttles between nuclear compartments and the cytoplasm
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J B Clements and A Phelan
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Cell Nucleus ,Cytoplasm ,Nuclear cap-binding protein complex ,Biological Transport ,Herpes Simplex ,Herpesvirus 1, Human ,Biology ,Heterogeneous ribonucleoprotein particle ,Virology ,Molecular biology ,Immediate early protein ,Immediate-Early Proteins ,RNA splicing ,Humans ,snRNP ,Nuclear protein ,Nuclear export signal ,Small nuclear ribonucleoprotein ,HeLa Cells - Abstract
Herpes simplex virus type 1 (HSV-1) immediate early protein IE63, an essential nuclear protein, is pleiotropic in function and, at the post-transcriptional level, inhibits RNA splicing, interacts with cellular splicing small nuclear ribonucleoprotein particles (snRNPs), binds RNA and prevents the nucleocytoplasmic transport of intron-containing mRNAs. Here it is reported that IE63 is a nucleocytoplasmic shuttle protein able to travel from snRNP- and RNA-rich nuclear foci to the cytoplasm, where it accumulates during actinomycin D treatment. This newly identified property suggests that IE63 facilitates nuclear export of HSV-1 transcripts, in addition to retaining intron-containing transcripts in the nucleus. The mechanism by which IE63 controls RNA export has yet to be defined. more...
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- 1997
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4. Regulation of herpes simplex virus poly (A) site usage and the action of immediate-early protein IE63 in the early-late switch
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James I. Dunlop, J B Clements, F McGregor, and A Phelan
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Gene Expression Regulation, Viral ,RNA Splicing ,Blotting, Western ,Molecular Sequence Data ,Immunology ,Herpesvirus 1, Human ,Biology ,medicine.disease_cause ,Microbiology ,Immediate early protein ,Cell Line ,Immediate-Early Proteins ,Cricetinae ,Virology ,Chlorocebus aethiops ,Gene expression ,medicine ,Animals ,Humans ,RNA, Messenger ,RNA Processing, Post-Transcriptional ,Vero Cells ,Gene ,Cell Nucleus ,mRNA Cleavage and Polyadenylation Factors ,Regulation of gene expression ,Cleavage stimulation factor ,Messenger RNA ,Base Sequence ,RNA-Binding Proteins ,Ribonucleoprotein, U2 Small Nuclear ,Molecular biology ,Up-Regulation ,Herpes simplex virus ,Insect Science ,RNA, Viral ,Poly A ,Gene Deletion ,Small nuclear ribonucleoprotein ,HeLa Cells ,Research Article - Abstract
The essential herpes simplex virus type 1 (HSV-1) immediate-early IE63 (ICP27) is pleiotropic in function, promoting the switch from the early to late phase of virus gene expression, and has effects on the posttranscriptional processes of mRNA splicing and 3' processing. We have investigated the role of IE63 in the regulation of viral mRNA 3' processing and of late gene expression. Our in vitro 3' processing studies demonstrated that HSV-1 infection induces an activity, which requires IE63 gene expression, responsible for an observed increase in 3' processing of selected HSV-1 poly(A) sites. Processing efficiencies at the poly(A) sites of two late genes, UL38 and UL44, shown to be inherently weak processing sites, were increased by the IE63-induced activity. In contrast, 3' processing at the poly(A) sites of selected immediate-early and early genes, stronger processing sites, was unaffected by IE63 expression. UV cross-linking experiments demonstrated that HSV infection caused enhanced binding of protein factors, including the 64-kDa component of cleavage stimulation factor (CstF), to poly(A) site RNAs from virus genes of all temporal classes and that this enhanced binding required expression of IE63. By immunofluorescence, the homogeneous pattern of the 64-kDa CstF protein distribution became slightly clumped with infection, whereas the splicing small nuclear ribonucleoprotein particles were recognized into a highly punctate distribution away from the sites of virus transcription. This effect could create an increase in the relative concentration of 3' processing factors available to pre-mRNAs. Western blot (immunoblot) analysis showed that IE63 was required for expression of several true late genes and for the efficient and timely expression of the UL29 and UL42 early genes, integral components on the viral DNA synthesis machinery. Our data are consistent with two effects of IE63 on late gene regulation: firstly, a stimulation of pre-mRNA 3' processing and, secondly, as a requirement for expression of functions necessary for viral DNA synthesis. more...
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- 1996
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5. Characterization of the novel protein kinase activity present in the R1 subunit of herpes simplex virus ribonucleotide reductase
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J B Clements, J Conner, and J Cooper
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Adenosine ,Herpesvirus 2, Human ,Molecular Sequence Data ,Immunology ,Herpesvirus 1, Human ,Biology ,Mitogen-activated protein kinase kinase ,Microbiology ,SH3 domain ,MAP2K7 ,Virology ,Ribonucleotide Reductases ,Amino Acid Sequence ,Carbon Radioisotopes ,c-Raf ,Kinase activity ,Protein kinase A ,Sequence Deletion ,Base Sequence ,Sequence Homology, Amino Acid ,Cyclin-dependent kinase 2 ,Affinity Labels ,Precipitin Tests ,Molecular biology ,Mutagenesis, Insertional ,Oligodeoxyribonucleotides ,Biochemistry ,Insect Science ,biology.protein ,Cyclin-dependent kinase 9 ,Protein Kinases ,Research Article - Abstract
We have compared the protein kinase activities of the R1 subunits from herpes simplex virus types 1 (HSV-1) and 2 (HSV-2) ribonucleotide reductase following expression in Escherichia coli. Autophosphorylation activity was observed when kinase assays were performed with immunoprecipitated R1 or proteins purified to homogeneity, and the activity was stimulated by the basic protein protamine. Transphosphorylation of histones or calmodulin by purified or immunoprecipitated HSV-1 and HSV-2 R1 was not observed, and our results suggest that the activities of these two proteins are similar. We further characterized the protein kinase activity of HSV-1 R1 by producing insertion and deletion mutants constructed with a plasmid expressing R1 amino acids 1 to 449. C-terminal deletion analysis identified the catalytic core of the enzyme as comprising residues 1 to 292, and this polypeptide will be useful for structural determinations by X-ray crystallography. Insertion of a 4-amino-acid sequence at sites within the protein kinase domain identified regions essential for activity; insertions at residues 22 and 112 completely inactivated activity, and an insertion at residue 136 reduced activity sixfold. Similar insertions at residues 257, 262, 292, and 343 had no effect on activity. The ATP analog 5'-fluorosulfonylbenzoyladenosine, which covalently modifies conventional eukaryotic kinases at an essential lysine residue within the active site, did label HSV R1, but this labelling occurred outside the N-terminal domain. These data indicate that the HSV R1 kinase is novel and distinct from other eukaryotic protein kinases. more...
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- 1995
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6. Ribonucleotide reductase of herpesviruses
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J. Conner, J. B. Clements, and Howard S. Marsden
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Infectious Diseases ,Ribonucleotide reductase ,Biochemistry ,Virology ,Biology - Published
- 1994
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7. In Vitro Systems to Analyze HSV Transcript Processing
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A, Phelan and J B, Clements
- Abstract
During lytic virus replication, herpes simplex virus (HSV) exhibits a closely regulated pattern of viral gene expression and of DNA replication, resulting in virion production (1). Broadly, HSV genes can be divided into immediate early, early, and late categories based on the kinetics of their expression. The five immediate early genes are expressed in the absence of prior viral protein synthesis although their expression is stimulated by a viral tegument protein. Two immediate early proteins are essential for virus replication in vitro and act at the transcriptional (IE 175) and posttranscriptional (IE63) levels to regulate early and late gene expression. Throughout infection, mRNA is synthesized using cellular RNA poly-merase II, which is modified by the action of an immediate early protein (2). more...
- Published
- 2011
8. A herpes simplex virus type 1 immediate-early gene product, IE63, regulates small nuclear ribonucleoprotein distribution
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A Phelan, Maria Carmo-Fonseca, J B Clements, Angus I. Lamond, and J McLaughlan
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Multidisciplinary ,viruses ,Fluorescent Antibody Technique ,Gene Expression ,DNA virus ,Herpesvirus 1, Human ,Biology ,Ribonucleoproteins, Small Nuclear ,Transfection ,Molecular biology ,Antibodies ,Immediate early protein ,Immediate-Early Proteins ,Gene product ,Gene expression ,RNA splicing ,Humans ,snRNP ,Cycloheximide ,Genes, Immediate-Early ,Small nuclear ribonucleoprotein ,Research Article ,HeLa Cells ,Ribonucleoprotein - Abstract
Herpes simplex virus 1 (HSV-1), a nuclear replicating DNA virus, has 73 identified genes of which only 4 contain introns. For this reason the virus probably makes only minimal use of the cellular RNA-splicing machinery. Antigens associated with the small nuclear ribonucleoprotein particles (snRNPs) that are subunits of splicing complexes have been reported to redistribute in the nucleus and become concentrated into the intranuclear structures, the interchromatin granules, after HSV-1 infection [Martin, T. E., Barghusen, S. C., Leser, G. P. & Spear, P. G. (1987) J. Cell Biol. 105, 2069-2082]. We observe this snRNP redistribution upon HSV-1 infection, in which the widespread snRNP staining pattern changes to a restricted punctate distribution with a concomitant loss of coiled bodies in HSV-1-infected cells. We show here that expression of the immediate-early (IE) subset of HSV-1 genes is necessary and sufficient for snRNP redistribution. Using a series of HSV-1 mutants in different IE genes, we have established that specifically the product of the viral IE63 (ICP27) gene is essential for this effect, and transfection experiments revealed that IE63 expression alone can cause the snRNP redistribution. Further, we show that the IE63 gene product colocalizes with the redistributed snRNP in the nucleus. The snRNP redistribution caused by HSV-1 infection resembles the effect seen after inhibition of transcription in uninfected cells. In HSV-1-infected cells, however, the snRNP redistribution is under the control of viral IE gene products and occurs during active virus gene transcription. more...
- Published
- 1993
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9. Herpes simplex virus IE63 acts at the posttranscriptional level to stimulate viral mRNA 3' processing
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J B Clements, Colin Loney, John McLauchlan, Rozanne M. Sandri-Goldin, and A Phelan
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Chloramphenicol O-Acetyltransferase ,Gene Expression Regulation, Viral ,Transcription, Genetic ,Immunology ,Mutant ,Biology ,Transfection ,medicine.disease_cause ,Microbiology ,Virus ,Cell Line ,Virology ,RNA Precursors ,medicine ,Animals ,Humans ,Simplexvirus ,RNA, Messenger ,RNA Processing, Post-Transcriptional ,Gene ,Cell Nucleus ,Messenger RNA ,Reporter gene ,RNA ,Molecular biology ,Recombinant Proteins ,Kinetics ,Herpes simplex virus ,Insect Science ,RNA, Viral ,Immediate early gene ,Research Article ,HeLa Cells - Abstract
We have shown previously that a novel herpes simplex virus-induced activity, LPF, selectively increases RNA 3'-end processing at the poly(A) site of a late virus gene (J. McLauchlan, S. Simpson, and J. B. Clements, Cell 59:1093-1105, 1989). Here, our in vivo and in vitro analyses both demonstrate that LPF is induced during early stages of virus infection. Studies of virus mutants indicate that expression of the immediate-early IE63 gene is required for induction of this activity. The selective effects on 3' processing displayed in the presence of IE63 provide direct evidence that IE63 can influence this posttranscription process. This extends previous studies which reported increases in reporter gene activity with certain poly(A) sites by IE63 (R. M. Sandri-Goldin and G. E. Mendoza, Genes Dev. 6:848-863, 1992). more...
- Published
- 1992
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10. An autophosphorylating but not transphosphorylating activity is associated with the unique N terminus of the herpes simplex virus type 1 ribonucleotide reductase large subunit
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Furlong J, J Cooper, J B Clements, and J Conner
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Macromolecular Substances ,Protein subunit ,Restriction Mapping ,Immunology ,Mutant ,Biology ,medicine.disease_cause ,Microbiology ,Adenosine Triphosphate ,Virology ,Ribonucleotide Reductases ,Escherichia coli ,medicine ,Simplexvirus ,Cloning, Molecular ,Phosphorylation ,Protein kinase A ,Sequence Deletion ,Autophosphorylation ,Molecular biology ,Recombinant Proteins ,N-terminus ,Ribonucleotide reductase ,Herpes simplex virus ,Biochemistry ,Insect Science ,Autoradiography ,Electrophoresis, Polyacrylamide Gel ,Phosphorus Radioisotopes ,Research Article - Abstract
We report on a protein kinase function encoded by the unique N terminus of the herpes simplex virus type 1 (HSV-1) ribonucleotide reductase large subunit (R1). R1 expressed in Escherichia coli exhibited autophosphorylation activity in a reaction which depended on the presence of the unique N terminus. When the N terminus was separately expressed in E. coli and partially purified, a similar autophosphorylation reaction was observed. Importantly, transphosphorylation of histones and of proteins in HSV-1-infected cell extracts was also observed with purified R1 and with truncated R1 mutants in which most of the N terminus was deleted. Ion-exchange chromatography was used to separate the autophosphorylating activity of the N terminus from the transphosphorylating activity of an E. coli contaminant protein kinase. We propose a putative function for this activity of the HSV-1 R1 N terminus during the immediate-early phase of virus replication. more...
- Published
- 1992
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11. A negative regulatory element in the human papillomavirus type 16 genome acts at the level of late mRNA stability
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J B Clements, I M Kennedy, and J K Haddow
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Chloramphenicol O-Acetyltransferase ,Untranslated region ,Molecular Sequence Data ,Immunology ,Negative regulatory element ,Regulatory Sequences, Nucleic Acid ,Biology ,Microbiology ,Genome ,Cell Line ,Open Reading Frames ,Virology ,Humans ,Coding region ,RNA, Messenger ,Papillomaviridae ,Genetics ,Messenger RNA ,Base Sequence ,Stop codon ,Tumor Virus Infections ,Open reading frame ,Regulatory sequence ,Insect Science ,DNA, Viral ,Poly A ,Research Article - Abstract
A negative regulatory element present in the human papillomavirus type 16 genome has been characterized. Deletion analysis has localized the 5' end of the element to the late region of the genome at the extreme 3' end of the coding region of the L1 open reading frame, around the L1 stop codon, with the element extending into the L1 3' untranslated region. For the cell lines used, the element's function was independent of cell type, tissue, or species of origin, unlike papillomavirus infection, which is very dependent on such factors. By using an mRNA decay assay, we have determined that polyadenylated RNA containing the element is much less stable than polyadenylated RNA lacking the element. This indicates that the element acts as an mRNA instability element. The significance of A-rich, GU-rich, and AUG-rich sequences for the functioning of this human papillomavirus type 16 instability element is discussed. more...
- Published
- 1991
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12. Interaction between Herpes Simplex Virus Type 1 IE63 Protein and Cellular Protein p32
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Sarah Wadd, Joy Kean, Helen E. Bryant, David A. Matthews, J B Clements, Sheila V. Graham, W. C. Russell, and J. Scott
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Heterogeneous nuclear ribonucleoprotein ,viruses ,RNA Splicing ,Immunology ,Molecular Sequence Data ,Herpesvirus 1, Human ,Biology ,Protein Serine-Threonine Kinases ,Heterogeneous ribonucleoprotein particle ,Microbiology ,Immediate early protein ,Heterogeneous-Nuclear Ribonucleoproteins ,Immediate-Early Proteins ,Heterogeneous-Nuclear Ribonucleoprotein K ,Mitochondrial Proteins ,Virology ,Humans ,Amino Acid Sequence ,Nuclear protein ,Nuclear export signal ,Casein Kinase II ,Ribonucleoprotein ,Nuclear Proteins ,Molecular biology ,Precipitin Tests ,Virus-Cell Interactions ,body regions ,Gene Products, rev ,Ribonucleoproteins ,Insect Science ,RNA splicing ,Carrier Proteins ,HeLa Cells - Abstract
The herpes simplex virus type 1 (HSV-1) immediate-early gene IE63 (ICP27), the only HSV-1 regulatory gene with a homologue in every mammalian and avian herpesvirus sequenced so far, is a multifunctional protein which regulates transcriptional and posttranscriptional processes. One of its posttranscriptional effects is the inhibition of splicing of viral and cellular transcripts. We previously identified heterogeneous nuclear ribonucleoprotein (hnRNP) K and casein kinase 2 (CK2) as two protein partners of IE63 (H. Bryant et al., J. Biol. Chem. 274:28991–28998, 1999). Here, using a yeast two-hybrid assay, we identify another partner of IE63, the cellular protein p32. Confirmation of this interaction was provided by coimmunoprecipitation from virus-infected cells and recombinant p32 binding assays. A p32-hnRNP K-CK2 complex, which required IE63 to form, was isolated from HSV-1-infected cells, and coimmunoprecipitating p32 was phosphorylated by CK2. Expression of IE63 altered the cytoplasmic distribution of p32, with some now colocalizing with IE63 in the nuclei of infected and transfected cells. As p32 copurifies with splicing factors and can inhibit splicing, we propose that IE63 together with p32, possibly with other IE63 partner proteins, acts to disrupt or regulate pre-mRNA splicing. As well as contributing to host cell shutoff, this effect could facilitate splicing-independent nuclear export of viral transcripts. more...
- Published
- 2000
13. Nuclear sites of herpes simplex virus type 1 DNA replication and transcription colocalize at early times postinfection and are largely distinct from RNA processing factors
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James I. Dunlop, Arvind H. Patel, J B Clements, Nigel D. Stow, and A Phelan
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DNA Replication ,Transcription, Genetic ,viruses ,Immunology ,Eukaryotic DNA replication ,Biology ,Microbiology ,DNA replication factor CDT1 ,Replication factor C ,Control of chromosome duplication ,Virology ,Humans ,Simplexvirus ,Cell Nucleus ,DNA replication ,Herpes Simplex ,Licensing factor ,Viral replication ,Insect Science ,DNA, Viral ,biology.protein ,Origin recognition complex ,RNA, Viral ,Research Article ,HeLa Cells - Abstract
We have visualized the intracellular localization of herpes simplex virus (HSV) type 1 replication and transcription sites in infected HeLa cells by using direct labelling methods. The number of viral transcription foci increases in a limited way; however, the number of replication sites increases in a near-exponential manner throughout infection, and both replication and transcription sites are found buried throughout the nuclear interior. Simultaneous visualization of viral transcription and replication foci shows that the two processes colocalize at early times, but at later times postinfection, there are additional sites committed solely to replication. This contrasts with the situation in adenovirus-infected cells in which, throughout replication, sites of transcription are adjacent to but do not colocalize with sites of viral DNA replication. The data for an increase in HSV transcription sites suggest an initial phase of replication of input genomes which are then transcribed. Sites of HSV replication colocalize with viral DNA replication and packaging proteins but are largely distinct from the punctate distribution of small nuclear ribonucleoprotein particles. Very high multiplicities of infection have shown an upper limit of some 18 viral transcription foci per nucleus, suggesting cellular constraints on transcription site formation. Use of virus replication mutants confirms that the labelled foci are sites of viral RNA and DNA synthesis; in the absence of viral DNA replication functions, no replication foci and only a limited number of transcription foci were present. Absence of a packaging function had no apparent effect on transcription or replication site formation, illustrating that DNA packaging is not a prerequisite for ongoing DNA synthesis. Further, the essential HSV protein IE63 is required for efficient replication site formation at later times postinfection but is not required for transcription foci formation. more...
- Published
- 1997
14. Immediate early protein IE63 of herpes simplex virus type 1 binds RNA directly
- Author
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A. Ingram, James I. Dunlop, A. Phelan, and J B Clements
- Subjects
Binding Sites ,Binding protein ,RNA Splicing ,RNA-dependent RNA polymerase ,RNA ,RNA-binding protein ,Herpesvirus 1, Human ,Biology ,Non-coding RNA ,Virology ,Molecular biology ,Recombinant Proteins ,Post-transcriptional modification ,Immediate-Early Proteins ,Escherichia coli ,Humans ,Signal recognition particle RNA ,Binding site ,Poly A ,Protein Binding - Abstract
The herpes simplex virus type 1 (HSV-1) immediate early protein IE63 acts post-transcriptionally to affect RNA 3'-processing and splicing. Functional domains such as the RGG box and zinc-finger motifs potentially provide the protein with RNA binding capacity. Here, IE63 protein expressed in E. coli, purified by affinity chromatography and used in RNA binding assays, demonstrated similar binding to RNA substrates containing poly(A) sites from different temporal classes of HSV-1 genes, RNA containing splice site recognition sequences and RNA containing no recognized processing motifs. Competition binding assays showed that IE63 binding could be competed out, suggesting that IE63 binds RNA weakly. HSV-1 infection results in an increase or stabilization in vitro of protein binding to poly(A) site-containing RNAs; IE63 is required for this effect. RNA binding assays combining purified IE63 with protein from mock-infected and HSV-1 infected nuclear extracts demonstrated no effect on protein-RNA binding patterns. more...
- Published
- 1996
15. Herpes simplex virus type 1 protein IE63 affects the nuclear export of virus intron-containing transcripts
- Author
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A Phelan, J B Clements, and James I. Dunlop
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Immunology ,Biology ,medicine.disease_cause ,Microbiology ,Immediate early protein ,Immediate-Early Proteins ,Virology ,medicine ,Humans ,Simplexvirus ,Nuclear export signal ,Cellular localization ,In Situ Hybridization ,Cell Nucleus ,Small Nuclear Ribonucleoprotein Particle ,Intron ,Biological Transport ,Herpes Simplex ,Molecular biology ,Cell nucleus ,medicine.anatomical_structure ,Herpes simplex virus ,Insect Science ,Mutation ,Trans-Activators ,Small nuclear ribonucleoprotein ,HeLa Cells ,Research Article - Abstract
Using in situ hybridization labelling methods, we have determined that the herpes simplex virus type 1 immediate-early protein IE63 (ICP27) affects the cellular localization of virus transcripts. Intronless transcripts from the IE63, UL38, and UL44 genes are rapidly exported to and accumulate in the cytoplasm throughout infection, in either the presence or absence of IE63 expression. The intron-containing transcripts from the IE110 and UL15 genes, while initially cytoplasmic, are increasingly retained in the nucleus in distinct clumps as infection proceeds, and the clumps colocalize with the redistributed small nuclear ribonucleoprotein particles. Infections with the IE63 mutant virus 27-lacZ demonstrated that in the absence of IE63 expression, nuclear retention of intron-containing transcripts was lost. The nuclear retention of UL15 transcripts, which demonstrated both nuclear and cytoplasmic label, was not as pronounced as that of the IE110 transcripts, and we propose that this is due to the late expression of UL15. Infections with the mutant virus 110C1, in which both introns of IE110 have been precisely removed (R.D. Everett, J. Gen. Virol. 72:651-659, 1991), demonstrated IE110 transcripts in both the nucleus and the cytoplasm; thus, exon definition sequences which regulate viral RNA transport are present in the IE110 transcript. By in situ hybridization a stable population of polyadenylated RNAs was found to accumulate in the nucleus in spots, most of which were separate from the small nuclear ribonucleoprotein particle clumps. The IE63 protein has an involvement, either direct or indirect, in the regulation of nucleocytoplasmic transport of viral transcripts, a function which contrasts with the recently proposed role of herpes simplex virus type 1 Us11 in promoting the nuclear export of partially spliced or unspliced transcripts (J.-J. Diaz, M. Duc Dodon, N. Schaerer-Uthurraly, D. Simonin, K. Kindbeiter, L. Gazzolo, and J.-J. Madjar, Nature [London] 379:273-277, 1996), the significance of which is discussed. more...
- Published
- 1996
16. Bringing core biopsy into a surgical practice
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S M, Roe, M P, Sumida, R P, Burns, M S, Greer, and J B, Clements
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Stereotaxic Techniques ,Biopsy, Needle ,Humans ,Breast Neoplasms ,Female ,Breast ,Ultrasonography, Mammary ,Ultrasonography, Interventional - Abstract
Minimally invasive diagnostic techniques in evaluating patients with breast disease have been increasingly utilized and accepted by physicians and patients over recent years. The incorporation of stereotactic core needle biopsy and ultrasound-guided core needle biopsy into the office practice of evaluating patients with breast disease by our surgical faculty has been met favorably. These procedures are readily learned by surgeons. The judicious use of these procedures is evidenced by the malignancy rate of core biopsies of 16 per cent, identical to the historical rate for needle localization assisted excisional biopsy at our institution. Core breast biopsy expedites definitive diagnosis and optimizes patient convenience. Reimbursement is highly variable, and active physician participation in negotiating with payors to insure that costs are met is essential. more...
- Published
- 1996
17. Cell type and cell state determine differential in vitro growth of non-neurovirulent ICP34.5-negative herpes simplex virus types 1 and 2
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Alasdair Roderick Maclean, J B Clements, J Podlech, S. M. Brown, and Harland J
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Cell type ,Genes, Viral ,Herpesvirus 2, Human ,Blotting, Western ,Herpesvirus 1, Human ,Biology ,medicine.disease_cause ,Kidney ,Virus ,Herpesviridae ,Cell Line ,Tissue culture ,Viral Proteins ,Species Specificity ,Virology ,Cricetinae ,medicine ,Animals ,Neurons ,Mutation ,Virulence ,Phenotype ,Kinetics ,Herpes simplex virus ,Cell culture - Abstract
The herpes simplex virus (HSV) gene RL1 encodes the protein ICP34.5, which is a specific neurovirulence factor. Null mutants in RL1 fail to replicate in the central nervous system of mice and are therefore totally non-neurovirulent. Additionally, they fail to replicate in neurons of the peripheral nervous system, although they are capable of establishing and reactivating from a latent infection. As the precise function of ICP34.5 in HSV-neuronal interactions is unknown, we have studied the role of ICP34.5 in vitro by examining in detail the phenotypes of RL1-negative viruses in two defined tissue culture systems. The first was mouse embryo fibroblast 3T6 cells, in which RL1-negative mutants are impaired and the in vivo phenotype is mimicked. This impairment is amplified when the cells are in the stationary state. The second was mouse embryo testicular carcinoma F9 cells which, in the undifferentiated state, provide a reversal of phenotype; wild-type virus fails to grow but RL1-negative virus replicates efficiently. Differentiation results in the ability to support wild-type virus growth. The stage at which the replication cycle is blocked plus the role of cellular factors is addressed in both tissue culture systems. Evidence is provided that cell type and cell state are crucial to ICP34.5-cellular interaction and hence, based on these parameters, ICP34.5 can be defined as a host-range determinant. Identification of cellular proteins that specifically interact with or are homologues of ICP34.5 may lead to the identification of neuron-specific proteins that have a similar role. more...
- Published
- 1994
18. Penetrating carotid artery injury with associated neurologic deficit
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C P, Major, W B, Brock, J B, Clements, and D E, Barker
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Carotid Arteries ,Adolescent ,Humans ,Female ,Hemiplegia ,Wounds, Gunshot ,Carotid Artery Injuries - Published
- 1994
19. Purification and characterization of the herpes simplex virus type 1 ribonucleotide reductase small subunit following expression in Escherichia coli
- Author
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John McLauchlan, Lankinen H, Mistry A, Howard S. Marsden, Weir M, J B Clements, McGarrity A, Furlong J, and Joe Conner
- Subjects
Macromolecular Substances ,Protein subunit ,Blotting, Western ,Genes, Fungal ,Genetic Vectors ,Protomer ,Biology ,medicine.disease_cause ,Open Reading Frames ,Virology ,Gene expression ,Protein purification ,Ribonucleotide Reductases ,medicine ,Escherichia coli ,T7 RNA polymerase ,Simplexvirus ,Amino Acids ,Cloning, Molecular ,DNA-Directed RNA Polymerases ,Chromatography, Ion Exchange ,Molecular biology ,Recombinant Proteins ,Molecular Weight ,Kinetics ,Ribonucleotide reductase ,Isoelectric point ,Electrophoresis, Polyacrylamide Gel ,T-Phages ,Isoelectric Focusing ,medicine.drug ,Plasmids - Abstract
The herpes simplex virus type 1 (HSV-1) gene encoding the ribonucleotide reductase (RR) small subunit (R2) was cloned as an unfused and intact open reading frame into a T7 RNA polymerase expression system in Escherichia coli. The expressed product was recovered from bacteria in soluble form and constituted 7% of the soluble protein. Protein purification yielded 3·5 mg of 95% pure R2 per litre of bacterial culture. The correct composition of the purified protein was verified by amino acid analysis and N-terminal sequencing. The isoelectric point of the protein was 5·3. Atomic emission spectroscopy indicated that the iron content of the E. coli-expressed R2 was 0·2 to 0·5 atoms of iron per R2 protomer as compared with a theoretical maximum value of 2. The E. coli-expressed HSV-1 R2 existed as a combination of a stable dimer and monomer. Combination of the E. coli-expressed R2 with the E. coli-expressed large subunit (R1) gave an active holoenzyme. Thus, the T7 expression system provides a rich source of enzymically active HSV-1 RR. more...
- Published
- 1991
20. A single amino acid substitution in the large subunit of herpes simplex virus type 1 ribonucleotide reductase which prevents subunit association
- Author
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Hilkka Lankinen, I. Nikas, Howard S. Marsden, Anne Cross, J B Clements, and A. J. Darling
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Macromolecular Substances ,Protein Conformation ,Protein subunit ,Mutant ,Molecular Sequence Data ,Biology ,medicine.disease_cause ,Serine ,Structure-Activity Relationship ,Virology ,Ribonucleotide Reductases ,medicine ,Simplexvirus ,Asparagine ,Amino Acid Sequence ,Peptide sequence ,Mutation ,Base Sequence ,Molecular Structure ,Nucleic acid sequence ,Temperature ,Molecular biology ,Ribonucleotide reductase ,Biochemistry ,Oligopeptides - Abstract
The herpes simplex virus type 1 temperature-sensitive (ts) mutant ts1207 does not induce detectable levels of ribonucleotide reductase activity at the non-permissive temperature (NPT, 39.5 degrees C). The ts lesion prevents the association of the enzyme's large (RR1) and small (RR2) subunits to give an active holoenzyme and maps within the gene specifying RR1. Here, it is shown that the ts mutant phenotype is due to the substitution of an asparagine for the wild-type (wt) serine at RR1 position 961, which is located within a region highly conserved between herpesviral and cellular RR1 subunit polypeptides. This ts1207 asparagine is predicted to alter a wt alpha-helix to a beta-strand. We have used synthetic oligopeptides, corresponding to the wt amino acid sequence of the mutation site, and antisera raised against them to determine whether this region is involved in subunit association. Neither the oligopeptides nor the antisera inhibit the enzyme activity, or the reconstituted activity formed by mixing intact RR2 and RR1 subunits present in partially purified extracts of cells infected at the NPT with ts1207 or ts1222 (an HSV-1 mutant with a lesion in the RR2 subunit), respectively. We infer from these results that the site of the mutation is unlikely to be positioned at the surface of RR1 and hence is probably not directly involved in subunit association. We suggest that the mutation site identifies an important RR1 region whose alteration in ts1207 changes the structure of a contact region(s) positioned at the RR1/RR2 interface. more...
- Published
- 1990
21. Blunt intestinal trauma
- Author
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D F, Fisher, M S, Greer, W L, Russell, and J B, Clements
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Adult ,Intestines ,Male ,Accidents, Occupational ,Humans ,Abdominal Injuries ,Wounds, Nonpenetrating - Published
- 1990
22. A 3′ co-terminus of two early herpes simplex virus type 1 mRNAs
- Author
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J B Clements and John McLauchlan
- Subjects
Nuclease ,Base Sequence ,Transcription, Genetic ,Polyadenylation ,RNA Splicing ,Hybridization probe ,DNA, Recombinant ,DNA Restriction Enzymes ,Biology ,medicine.disease_cause ,Genome ,Molecular biology ,DNA sequencing ,Virus ,Herpes simplex virus ,Complementary DNA ,DNA, Viral ,Genetics ,biology.protein ,medicine ,Simplexvirus ,RNA, Messenger ,Cloning, Molecular ,Research Article - Abstract
A 3' co-terminus of two early herpes simplex virus type 1 mRNAs has been identified using the nuclease -S1 mapping procedure with cloned virus DNA probes. These mRNAs (5.0 kb and 1.2 kb), located within the genome region 0.56-0.60, are unspliced and are transcribed rightwards on the prototype genome orientation. The position of their 3' ends has been located on the virus DNA sequence and lies downstream from the polyadenylation signal 5'-AATAAA-3'. This hexanucleotide sequence also was present in the complementary DNA strand and was shown to be the polyadenylation signal for a leftwards-transcribed late mRNA. The abundance within the cytoplasm of the 5.0 kb and 1.2 kb mRNAs was investigated. Results indicated that these mRNAs were regulated in concert. It is suggested that sequences at the 3' co-terminus may be involved in their regulation. more...
- Published
- 1982
- Full Text
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23. A Partial Denaturation Map of Herpes Simplex Virus Type 1 DNA: Evidence for Inversions of the Unique DNA Regions
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J. B. Clements and H. Delius
- Subjects
Perchlorates ,Dna evidence ,Biology ,Nucleic Acid Denaturation ,medicine.disease_cause ,Virology ,Genome ,chemistry.chemical_compound ,Herpes simplex virus ,chemistry ,DNA, Viral ,medicine ,Nucleic Acid Conformation ,Simplexvirus ,Molecule ,Denaturation (biochemistry) ,DNA - Abstract
Summary Partial denaturation maps of 30 HSV-1 DNA molecules have been obtained using a procedure designed to avoid possible hydrolysis of the DNA at alkalilabile bonds. From the denaturation pattern of the long unique DNA region these molecules were divided into two groups comprised of 16 and 14 molecules. Histogram plots relating the percentage denaturation to position on the DNA for these two groups were aligned in a manner appropriate to the HSV-1 genome model. It was apparent that these groups had the orientation of the long region inverted with respect to each other. Similarly, from the denaturation maps of the short unique region, the molecules were divided into two groups each comprising 15 molecules. Alignment of the histogram plots of these groups indicated that the orientation of the short region was inverted in one group relative to the other. These partial denaturation data confirm the presence of four HSV-1 genome arrangements resulting from the possible combinations of inversions of the two unique DNA regions. more...
- Published
- 1976
- Full Text
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24. Components required forin vitrocleavage and polyadenylation of eukaryotic mRNA
- Author
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J B Clements, John McLauchlan, Claire Moore, and S Simpson
- Subjects
Cell Nucleus ,Cleavage factor ,Cleavage stimulation factor ,Messenger RNA ,Base Sequence ,Polyadenylation ,RNA ,Cleavage and polyadenylation specificity factor ,Biology ,Molecular biology ,Post-transcriptional modification ,Biochemistry ,Transcription (biology) ,RNA Precursors ,Genetics ,Humans ,Simplexvirus ,RNA, Messenger ,Poly A ,HeLa Cells ,Plasmids ,Protein Binding - Abstract
We have studied in vitro cleavage/polyadenylation of precursor RNA containing herpes simplex virus type 2 poly A site sequences and have analyzed four RNA/protein complexes which form during in vitro reactions. Two complexes, A and B, form extremely rapidly and are then progressively replaced by a third complex, C which is produced following cleavage and polyadenylation of precursor RNA. Substitution of ATP with cordycepin triphosphate prevents polyadenylation and the formation of complex C however a fourth complex, D results which contains cleaved RNA. A precursor RNA lacking GU-rich downstream sequences required for efficient cleavage/polyadenylation fails to form complex B and produces a markedly reduced amount of complex A. As these GU-rich sequences are required for efficient cleavage, this establishes a relationship between complex B formation and cleavage/polyadenylation of precursor RNA in vitro. The components required for in vitro RNA processing have been separated by fractionation of the nuclear extract on Q-Sepharose and Biorex 70 columns. A Q-Sepharose fraction forms complex B but does not process RNA. Addition of a Biorex 70 fraction restores cleavage activity at the poly A site but this fraction does not appear to contribute to complex formation. Moreover, in the absence of polyethylene glycol, precursor RNA is not cleaved and polyadenylated, however, complexes A and B readily form. Thus, while complex B is necessary for in vitro cleavage and polyadenylation, it may not contain all the components required for this processing. more...
- Published
- 1988
- Full Text
- View/download PDF
25. The association of herpes simplex virus with squamous carcinoma of the cervix, and studies of the virus thymidine kinase gene
- Author
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J. B. Clements, R. P. Eglin, N M Wilkie, and P. G. Sanders
- Subjects
Serotype ,Inverted repeat ,Uterine Cervical Neoplasms ,In situ hybridization ,Adenocarcinoma ,Biology ,Cervical intraepithelial neoplasia ,medicine.disease_cause ,Thymidine Kinase ,Virus ,medicine ,Humans ,Simplexvirus ,Cervix ,General Environmental Science ,General Engineering ,Nucleic Acid Hybridization ,medicine.disease ,Virology ,Squamous carcinoma ,Herpes simplex virus ,medicine.anatomical_structure ,DNA, Viral ,Carcinoma, Squamous Cell ,RNA, Viral ,General Earth and Planetary Sciences ,Female - Abstract
Herpes simplex virus (HSV) is an endemic human virus characterized by latent infection of sensory ganglia, with reactivation leading to peripheral lesions in a proportion of cases. The virus genome (145 500 base pairs) comprises two segments, L and S, bounded by inverted repeat regions of approximately 10500 base pairs (TR L and IR L ) and 6000 base pairs (IR s and TR s ) respectively. A direct terminal repeat of about 300 base pairs is also present in an inverted form at the junction between L and S. Numerous indirect studies have suggested an association of the venereal serotype (HSV-2) with squamous carcinoma of the cervix (Nahmias et al . 1970; Thomas & Rawls 1978). Direct evidence has been notably lacking, but the presence of HSV RNA in cervical neoplasms has recently been reported (Jones et al . 1979; McDougall et al . 1980), with in situ hybridization to tritiated HSV DNA. In collaboration with Dr Frank Sharp of the Department of Midwifery, Glasgow University, we have now completed a major screening of cervical neoplasms by the in situ technique. Patients referred to Dr Sharp’s clinic for colposcopic examination were swabbed to detect intracervical infectious HSV. Punch biopsy samples (approximately 5 mm in section) were taken for pathological examination and categorization as non-neoplastic cervical intraepithelial neoplasia (c. i. n.) or squamous carcinoma. Parallel punches were snap-frozen and thin sections (7 µm) mounted on glass slides, fixed and then hybridized to in vitro 125 I-labelled HSV-2, Ad2, Ad5 or phage lambda DNA. Biopsies of primary adenocarcinomas of the cervix were treated in the same way. After washing and autoradiography, results are expressed as background-corrected mean grain counts per field (mean of ten fields per sample, 3.6 x 10 –2 mm 2 per field; ca . 20 cells per field). more...
- Published
- 1980
- Full Text
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26. Immediate-early mRNA-2 of herpes simplex viruses types 1 and 2 is unsplced: conserved sequences around the 5′ and 3′ termini correspond to transcription regulatory signals
- Author
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J B Clements, Frazer J. Rixon, Andrew J. Easton, and J L Whitton
- Subjects
Genes, Viral ,Transcription, Genetic ,viruses ,Kidney ,Homology (biology) ,DNA sequencing ,Cell Line ,Conserved sequence ,Transcription (biology) ,Cricetinae ,Genes, Regulator ,Genetics ,Animals ,Simplexvirus ,RNA, Messenger ,Gene ,Repetitive Sequences, Nucleic Acid ,Base Composition ,Messenger RNA ,Nuclease ,Base Sequence ,biology ,DNA Restriction Enzymes ,Exonuclease VII ,biology.protein ,Nucleic Acid Conformation - Abstract
Nuclease S1 and exonuclease VII analyses of immediate-early (IE) mRNA-2 of herpes simplex viruses types 1 and 2 (HSV-1, HSV-2) show them to be unspliced and of similar length. The DNA sequences around the 5' and 3' termini have been determined. Comparison of the sequences around the 5' ends reveals several common features. (1) Four discrete blocks of upstream homology which are precisely colinear with respect to the 5' termini of the mRNAs; the blocks include the 'TATA' box, a G-C rich sequence and a sequence (AATTAAATACAT) which may be involved in the coordinate induction of the IE class of genes. (2) Several copies of the sequence CCCCGCCC, found in different upstream positions in HSV-1 and HSV-2, which may be important in the expression of a wide variety of eukaryotic genes. (3) Potential hairpin structures in the region of the 5' termini which are present at similar locations in HSV-1 and HSV-2. Sequence comparison around the 3' termini of IEmRNA-2 reveals high homology at the proposed C-terminus of the polypeptide. more...
- Published
- 1983
- Full Text
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27. ARMORIAL BOOK-STAMPS AND THEIR OWNERS
- Author
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H. J. B. Clements
- Subjects
Arts and Humanities (miscellaneous) ,Library and Information Sciences - Published
- 1939
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28. Purification and Characterization of Bovine Enteroviruses
- Author
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J. B. Clements, S. J. Martin, and M. D. Johnston
- Subjects
Sucrose ,History ,viruses ,Cesium ,Buoyant density ,Centrifugation ,Picornaviridae ,Biology ,Tritium ,Kidney ,medicine.disease_cause ,Virus ,Cell Line ,Phosphates ,Education ,Sepharose ,Chlorides ,Cricetinae ,Culture Techniques ,Virology ,Centrifugation, Density Gradient ,medicine ,Animals ,Amino Acids ,Uridine ,Enterovirus ,Carbon Isotopes ,Phosphorus Isotopes ,RNA ,Sedimentation ,Centrifugation, Zonal ,Computer Science Applications ,Sedimentation coefficient ,Microscopy, Electron ,Virus type ,Chromatography, Gel ,RNA, Viral ,Cattle ,Research Article - Abstract
Summary Three serologically distinct bovine enteroviruses (vg-5-27; vc-65-182; vp-7-19) grew in BHK21 cells to concentrations of approximately 5 × 108 p.f.u./ml. A purification procedure was used which involved sedimentation and gel-filtration through a Sepharose 2 B column. The virus particles were spherical (diameter of 27 nm.), had a buoyant density of 1.34 g./ml. in CsCl and a sedimentation coefficient of approximately 165s. The RNA of each of the virus types had a sedimentation coefficient of 35s in sucrose-gradients and the base compositions were similar, being characterized by high A (30%) and low U (21%). more...
- Published
- 1970
- Full Text
- View/download PDF
29. Red Light Induction of Gibberellin Synthesis in Leaves
- Author
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D. J. Carr, D. M. Reid, and J. B. Clements
- Subjects
chemistry.chemical_compound ,Multidisciplinary ,Phytochrome ,Chemistry ,Botany ,Etiolation ,Biophysics ,food and beverages ,Gibberellin ,Irradiation ,Red light ,Gibberellic acid - Abstract
GIBBERELLIC acid is known to mimic the morphogenetic effects of red light1 on certain plants. Brian2 has suggested that gibberellins are synthesized in plants after the absorption of red light and Kohler3 has shown that there is a significant increase in the content of gibberellic acid in etiolated peas after 24 h of irradiation with red light. This and other work4 suggests that phytochrome in the Pfr form (after irradiation with red light) initiates synthesis, or at least brings about increases in the content of gibberellin in plants and seeds. Many responses of phytochrome, however, can be observed a few hours (or in some cases in less than 1 h (ref. 5)) after illumination with red light. Thus to explain effects of phytochrome in terms of the action of gibberellin, it is necessary to show that gibberellins are synthesized during or very soon after the end of a period of red irradiation. This is what we have tried to do. more...
- Published
- 1968
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30. TRIALS IN NYASALAND OF INTRODUCED PINES
- Author
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J. B. Clements
- Subjects
Forestry ,Biology - Published
- 1938
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31. RNA and Protein Synthesis; Prerequisites of Red Light-induced Gibberellin Synthesis
- Author
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J. B. Clements and D. M. Reid
- Subjects
Light ,Color ,Cycloheximide ,Biology ,chemistry.chemical_compound ,Protein biosynthesis ,Anilides ,Plant Proteins ,chemistry.chemical_classification ,Messenger RNA ,Multidisciplinary ,Phytochrome ,Herbicides ,food and beverages ,RNA ,Gibberellins ,Radiation Effects ,Chloramphenicol ,Enzyme ,chemistry ,Biochemistry ,Etiolation ,Dactinomycin ,Gibberellin ,Carbamates ,Edible Grain - Abstract
IT has been shown that the synthesis of mRNA and protein is a prerequisite for phytochrome -mediated synthesis of anthocyanin1,2, and that irradiation of plant tissue with red light stimulates the production of several enzymes3,4. We recently demonstrated that red light irradiation of segments from etiolated barley leaves would quickly induce the synthesis of gibberellin-like substances5. Subsequently we have investigated the possibility that this red light-induced gibberellin synthesis requires the formation of enzymes and RNA before the production of the gibberellins themselves. more...
- Published
- 1968
- Full Text
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32. Virus specified enzyme activity and RNA species in herpes simplex virus type 1 transformed mouse cells
- Author
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J. B. Clements, J. C. M. Macnab, B. Perbal, and A. T. Jamieson
- Subjects
Thymidine kinase activity ,Ultraviolet Rays ,viruses ,Phosphotransferases ,RNA ,Viral transformation ,Biology ,medicine.disease_cause ,Virology ,Molecular biology ,Thymidine Kinase ,Virus ,Cell Line ,Mice ,Herpes simplex virus ,Cell Transformation, Neoplastic ,Antigen ,Lytic cycle ,Thymidine kinase ,medicine ,Animals ,RNA, Viral ,Simplexvirus - Abstract
Summary LMTK- cells infected with u.v.-irradiated herpes simplex virus type 1 have been selected for the presence of the enzyme thymidine kinase. These cells have an altered morphology compared to the control cells and contain herpes-specific antigens in their cytoplasm. The thymidine kinase activity present in these cells has been shown, on the basis of a number of biochemical properties, to be identical to the herpes virus specified deoxypyrimidine kinase found during lytic infection of this virus. In addition it has been possible to detect herpes simplex-specific RNA sequences in the transformed cells and this occurs in both the polyadenylated and non-polyadenylated cytoplasmic and nuclear fractions. more...
- Published
- 1976
33. Process of infection with bacteriophage phiX174. XXXVII. RNA metabolism in phiX174-infected cells
- Author
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R L Sinsheimer and J B Clements
- Subjects
Immunology ,RNA-dependent RNA polymerase ,Biology ,medicine.disease_cause ,Virus Replication ,Microbiology ,Coliphages ,chemistry.chemical_compound ,Virology ,medicine ,Escherichia coli ,RNA, Messenger ,Gel electrophoresis ,Messenger RNA ,Dimethyl sulfoxide ,DNA Viruses ,RNA ,Nuclease protection assay ,Molecular biology ,Molecular Weight ,RNA, Bacterial ,chemistry ,Insect Science ,RNA, Viral ,DNA ,Research Article - Abstract
The RNA produced in vivo from bacteriophage phiX174 DNA has been analyzed by polyacrylamide-agarose gel electrophoresis and sedimentation in dimethyl sulfoxide gradients, and the results of Hayashi and Hayashi (1970) have been confirmed and extended. An efficient procedure for recovery of RNA from gels, followed by a hybridization assay, has indicated the presence in infected cells of 18 distinct RNA species with sizes up to and greater than the unit (viral) length. The sizes of phiX mRNA's were similar irrespective of whether material was analyzed on gels or in dimethyl sulfoxide gradients. When virus-induced RNA was detected by a double-label method, seven additional low-molecular weight species were observed on gels and the resolution of dimethyl sulfoxide gradients was enhanced. The present results lend support to aspects of the model of Hayashi and Hayashi (1970) for the generation of these discrete mRNA species; an alternative model is also discussed. more...
- Published
- 1975
34. A modular system for the assay of transcription regulatory signals: the sequence TAATGARAT is required for herpes simplex virus immediate early gene activation
- Author
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D F Gaffney, John McLauchlan, J B Clements, and J L Whitton
- Subjects
Chloramphenicol O-Acetyltransferase ,Genes, Viral ,Transcription, Genetic ,viruses ,Simian virus 40 ,Biology ,Transfection ,Chloramphenicol acetyltransferase ,Transactivation ,Acetyltransferases ,Gene expression ,Genes, Regulator ,Genetics ,Escherichia coli ,Humans ,Simplexvirus ,Enhancer ,Promoter Regions, Genetic ,Gene ,Regulation of gene expression ,Terminator Regions, Genetic ,Base Composition ,Base Sequence ,DNA Restriction Enzymes ,Molecular biology ,Terminator (genetics) ,Genes ,Immediate early gene ,HeLa Cells - Abstract
A modular system for assaying the activity of transcriptional regulatory signals based on herpes simplex virus (HSV) promoter and terminator sequences linked to the bacterial chloramphenicol acetyltransferase (CAT) gene has been used to study activation of HSV immediate early (IE) gene expression. Insertion of the SV40 72 base pair (bp) repeat increased mRNA levels by 15-fold thus demonstrating the ability of the HSV IE promoter to respond to a heterologous enhancer. A fragment containing part of the intergenic region located between HSV-2 immediate early (IE) genes-3 and -4/-5 increased mRNA levels by 5-fold in response to transactivation by an HSV virion structural polypeptide. The HSV activator fragment increased mRNA levels by 2-fold in the absence of transactivation indicating that cellular proteins are involved in IE gene expression. From HSV-1/HSV-2 DNA sequence comparisons we previously proposed that a DNA sequence, consensus TAATGARAT, present upstream of all HSV-1 and HSV-2 IE genes was required for the co-ordinate induction of IE genes. We show here that a synthetic oligonucleotide containing TAATGARAT conferred the ability to stimulate CAT activity only on transactivation: two copies of TAATGARAT stimulated expression by 2-fold while six copies gave an 8-fold increase. This activation, which was not dependent on orientation of the TAATGARAT sequence, directly demonstrates that TAATGARAT is a component of the IE gene activation sequence. more...
- Published
- 1985
35. Orientation of herpes simplex virus type 1 immediate early mRNA's
- Author
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John McLauchlan, D J McGeoch, and J B Clements
- Subjects
Genes, Viral ,Biology ,medicine.disease_cause ,Virus ,chemistry.chemical_compound ,RNA polymerase ,Complementary DNA ,Genetics ,medicine ,Simplexvirus ,RNA, Messenger ,Repeated sequence ,Messenger RNA ,Base Sequence ,Nucleic Acid Hybridization ,Promoter ,DNA Restriction Enzymes ,Virology ,Molecular biology ,Molecular Weight ,Herpes simplex virus ,chemistry ,DNA, Viral ,Poly A ,DNA ,Research Article - Abstract
We have determined the orientation of 4 immediate early (IE) mRNA's on the herpes simplex virus type 1 genome by mapping cDNA's complementary to the 3'-termini of messages. These IE mRNA's are transcribed by a pre-existing cell RNA polymerase, and we propose a model which allows their synthesis from a circular template using a single virus promoter region. The promoter region, which is located in the two repetitive DNA regions which flank the short unique region of the virus genome, may serve to initiate bidirectional transcription of these IE mRNA's. more...
- Published
- 1979
36. DNA sequence homology between two co-linear loci on the HSV genome which have different transforming abilities
- Author
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John McLauchlan and J B Clements
- Subjects
Genes, Viral ,Transcription, Genetic ,TATA box ,Locus (genetics) ,Biology ,General Biochemistry, Genetics and Molecular Biology ,Conserved sequence ,Viral Proteins ,Transcription (biology) ,Consensus sequence ,Simplexvirus ,Amino Acid Sequence ,RNA, Messenger ,Codon ,Molecular Biology ,Gene ,Peptide sequence ,Genetics ,AU-rich element ,General Immunology and Microbiology ,Base Sequence ,General Neuroscience ,Cell Transformation, Viral ,Molecular biology ,DNA, Viral ,Nucleic Acid Conformation ,Research Article - Abstract
A transcription unit at the herpes simplex virus (HSV) type 2 transforming region, mtr-2 (map coordinates 0.580-0.625), comprises two early, unspliced mRNAs of 4.5 kb and 1.2 kb which are 3' co-terminal; a region including that specifying the 1.2-kb mRNA has been sequenced. The putative translated portions of these two mRNAs do not overlap and this feature, together with the arrangement of the mRNAs, is similar to the apparently equivalent co-linear HSV-1 locus which however does not transform. A putative stem and loops structure containing a TATA box is located upstream from the 5' terminus of the 1.2-kb mRNA within the translated portion of the 4.5-kb mRNA. Evidence for the generation of this structure by intra-strand reassociation under our hybridisation conditions has been obtained and possibilities are that it may function as a transcription-activated promoter or as an RNA polymerase pause site. A comparison of the equivalent HSV-2 and HSV-1 regions reveals a conserved sequence downstream from the 3' co-terminus which is present at a similar location in many eukaryotic genes (consensus sequence YGTGTTYY). The overall sequence conservation at this transcription unit is high except for regions located at: (1) the untranslated leaders of the 1.2-kb mRNAs; (2) the N termini of the polypeptides specified by the HSV-2/HSV-1 1.2-kb mRNAs; (3) the intergenic region beyond the 3' co termini. Regions (2) and (3) are located within a transforming fragment of HSV-2. The possible significance of these data for HSV-mediated cell transformation is discussed. more...
- Published
- 1983
37. Virus transcript mapping studies in cells infected with temperature-sensitive mutants of herpes simples virus type 1
- Author
-
R J, Watson and J B, Clements
- Subjects
Time Factors ,Genes, Viral ,Transcription, Genetic ,Mutation ,Temperature ,Chromosome Mapping ,Nucleic Acid Hybridization ,RNA, Viral ,Simplexvirus ,Cell Transformation, Viral - Abstract
Nuclear and cytoplastic transcripts, synthesized in cells infected with six DNA-negative temperature-sensitive (ts) mutants of HSV-1 (ts B, ts D, ts E, ts K, ts S and ts T) under non-permissive conditions, were isolated and hybridized to unlabelled fragments of HSV-1 DNA, generated by restriction endonuclease digestion and immobilized on to nitrocellulose membranes by the method of Southern (1975). In this way it has been possible to map those regions of the HSV-1 genome represented by stable transcrips in cells infected with these mutants and compare them with those regions transcribed in cells infected with the wild-type virus at early and late times post infection (before and after viral DNA replication) and in the presence of DNA- and protein-synthesis inhibitors. Viral transcription in ts D, ts T and ts K-infected cells is restricted, the patterns of hybridization being similar, but not identical to that observed with immediate early RNA. Since these three mutants fall into two complementation groups, these experiments suggest that at least two viral products are required for the switch-on of early transcripts. In contrast, transcript mapping with the other early mutants (ts B, ts E and ts S) has shown a much less restricted transcriptional pattern, the pattern obtained resembling that with early, rather than late RNA. more...
- Published
- 1978
38. Human papillomavirus type 16 DNA from a vulvar carcinoma in situ is present as head-to-tail dimeric episomes with a deletion in the non-coding region
- Author
-
J B Clements, J. C. M. Macnab, I. M. Kennedy, and S. Simpson
- Subjects
DNA polymerase II ,DNA, Recombinant ,Molecular cloning ,Virus Replication ,law.invention ,law ,Virology ,Humans ,Genomic library ,Papillomaviridae ,biology ,Base Sequence ,Vulvar Neoplasms ,DNA replication ,Gene Amplification ,DNA, Neoplasm ,Molecular biology ,Restriction enzyme ,DNA, Viral ,Recombinant DNA ,biology.protein ,Human genome ,Female ,In vitro recombination ,Carcinoma in Situ ,Plasmids - Abstract
Summary A number of genital cancer biopsy samples were screened for the presence of human papillomavirus type 16 (HPV-16) DNA sequences. One of these samples (a vulvar carcinoma in situ) was found to contain more than 100 copies of HPV-16 DNA sequences per cell. Using this tumour DNA, a genomic library was constructed in bacteriophage lambda and the library was screened for recombinant phage containing HPV-16 sequences. Five recombinant phage clones were isolated and their DNA was analysed by restriction endonuclease digestion and blot hybridization. All five recombinants contained two copies of the HPV-16 genome present in a head-to-tail arrangement. The data are consistent with the presence of HPV-16 sequences in the tumour DNA arranged as genomic dimers in a circular episomal configuration. The HPV-16 genomes contained a deletion within the non-coding region, a region which includes the viral origin of DNA replication and transcriptional control sequences. Possible consequences of this deletion for viral replication and transcription are discussed. more...
- Published
- 1987
39. RNA and protein synthesis in herpesvirus-infected cells
- Author
-
J. Hay and J. B. Clements
- Subjects
Peptide Biosynthesis ,Transcription, Genetic ,viruses ,Disease ,Biology ,medicine.disease_cause ,Virus Replication ,Virus ,Viral Proteins ,Virology ,Culture Techniques ,Herpesvirus infection ,medicine ,Protein biosynthesis ,Simplexvirus ,Gene ,Base Sequence ,RNA ,Defective Viruses ,Herpes simplex virus ,Protein Biosynthesis ,DNA, Viral ,RNA, Viral ,RNA Polymerase II ,Function (biology) - Abstract
Introduction. Herpesviruses infect a wide range of vertebrates and are important human and animal pathogens: in a number of cases infection persists for very long periods, in a latent form, with occasional episodes of overt typical disease. In addition, several herpesviruses have been shown to be the causative agent of tumour formation in their normal hosts (e.g. Lucke frog tumour virus) or in related species (e.g. herpesvirus saimiri). Indirect evidence exists for the involvement of Epstein-Barr virus and herpes simplex virus type 2 (HSV-2) in human cancers. Thus there exist good reasons for the pursuit and identification of the mechanisms of herpesvirus infection and replication. From the molecular virological standpoint, herpesviruses also offer an important system for study. This large virus has the potential to code for about one hundred genes and the identification of these gene products, their function and their control poses many interesting questions. more...
- Published
- 1977
40. Hybrid plasmids containing an active thymidine kinase gene of Herpes simplex virus 1
- Author
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Werner Boll, Ned Mantei, J. B. Clements, D. Lonsdale, N. M. Wilkie, and Charles Weissmann
- Subjects
Genes, Viral ,Transcription, Genetic ,viruses ,EcoRI ,DNA, Recombinant ,Biology ,medicine.disease_cause ,Thymidine Kinase ,Plasmid ,Genetics ,medicine ,Escherichia coli ,Simplexvirus ,Gene ,Base Sequence ,Nucleic Acid Hybridization ,DNA Restriction Enzymes ,Cell Transformation, Viral ,Virology ,Molecular biology ,PBR322 ,Restriction site ,Herpes simplex virus ,Deoxyribonuclease BamHI ,Thymidine kinase ,biology.protein ,Plasmids - Abstract
The gene for the thymidine kinase (TK) of Herpes simplex virus type 1 (HSV-1) is located in the KpnI m and BamHI p fragments of the genome (Wigler et al., Cell 11, 223-232 (1977)). These fragments have been inserted into the EcoRI and BamHI sites, respectively, of plasmid pBR322, and propagated in E.coli. The TK gene contained in the recombinant plasmids was shown to be biologically active when introduced into TK- mouse L cells. Detailed restriction site maps of the BamHI p fragment have been constructed and the approximate location of the TK gene has been determined. Mouse cells transformed with cloned HSV-1 tk+ DNA produced HSV-1-specific thymidine kinase; superinfection with HSV-1 tk- virus increased the level of TK activity tenfold, suggesting that the BamHI p sequences present in transformed cells respond to virus-encoded regulatory gene product(s). more...
- Published
- 1979
41. Detection of RNA complementary to herpes simplex virus DNA in human cervical squamous cell neoplasms
- Author
-
R P, Eglin, F, Sharp, A B, MacLean, J C, Macnab, J B, Clements, and N M, Wilkie
- Subjects
Histocytochemistry ,Biopsy ,Nucleic Acid Hybridization ,Uterine Cervical Neoplasms ,Antibodies, Viral ,DNA, Viral ,Carcinoma, Squamous Cell ,Autoradiography ,Humans ,RNA ,Simplexvirus ,Female ,RNA, Neoplasm ,Probability - Abstract
Nonneoplastic and neoplastic cervical biopsy specimens were examined by in situ hybridization to 125I-labeled DNA of herpes simplex virus (HSV), adenovirus, and bacteriophage lambda DNA's, and quantitative hybridization data were obtained using a Video Image Analyser. HSV-specific RNA was detected in 72% of cervical intraepithelial neoplasia, 60% of squamous cervical carcinomas, 2% of nonneoplastic cervices, and 9% of primary adenocarcinomas of the cervix. None of the tissues gave positive hybridization with adenovirus or lambda DNA probes. In paired biopsies of cervical intraepithelial neoplasia and nonneoplastic epithelium from 29 individuals, HSV-specific RNA was detected only in the epithelium of the neoplastic sample and not in the nonneoplastic control. Infectious HSV-2 was isolated from a low proportion (2%) of both ectocervical swabs and cell-free tissue extracts of patients examined, suggesting that the HSV-specific RNA detected in squamous cell neoplasms was not due to overt infections. more...
- Published
- 1981
42. Analysis of herpesvirus DNA substructure by means of restriction endonucleases
- Author
-
J. B. Clements, Rita Cortini, and N. M. Wilkie
- Subjects
Genetics ,Base Sequence ,EcoRI ,DNA Restriction Enzymes ,Biology ,Haemophilus influenzae ,Restriction fragment ,Cell Line ,Molecular Weight ,chemistry.chemical_compound ,Restriction enzyme ,Restriction map ,chemistry ,Genes ,Models, Chemical ,Virology ,DNA, Viral ,biology.protein ,Simplexvirus ,Amplified fragment length polymorphism ,Restriction digest ,Restriction fragment length polymorphism ,DNA - Abstract
Summary The mol. wt. and molar ratios of the Hind III and Hpa I fragments of HSV-1 DNA and the Eco RI fragments of HSV-2 DNA have been determined. Results obtained suggest that DNA isolated from both HSV-1 and HSV-2 consists of molecules with four different sequence arrangements which are present in similar amounts. Our explanation of the cleavage patterns of these four genome arrangements with the different restriction enzymes is presented. Some of the possible implications of these four genome arrangements for genetic recombination are discussed. more...
- Published
- 1976
43. Structural features of ribonucleotide reductase
- Author
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J B Clements, I Nikas, William R. Taylor, Andrew J. Davison, and John McLauchlan
- Subjects
Herpesvirus 3, Human ,Herpesvirus 4, Human ,Ribonucleotide ,viruses ,Molecular Sequence Data ,Biology ,Biochemistry ,Homology (biology) ,chemistry.chemical_compound ,Structural Biology ,Sequence Homology, Nucleic Acid ,Ribonucleotide Reductases ,Simplexvirus ,Amino Acid Sequence ,Binding site ,Promoter Regions, Genetic ,Molecular Biology ,Peptide sequence ,Genetics ,chemistry.chemical_classification ,Base Sequence ,Biological Evolution ,Amino acid ,Open reading frame ,Ribonucleotide reductase ,chemistry ,DNA, Viral ,DNA - Abstract
Herpes simplex virus type 1 (HSV-1) encodes a ribonucleotide reductase which comprises two polypeptides with sizes of 136,000 (RR1) and 38,000 mol. wt. (RR2). We have determined the entire DNA sequence specifying HSV-1 RR1 and have identified two adjacent open reading frames in varicella-zoster virus (VZV) which have homology to HSV RR1 and RR2; the predicted sizes for the VZV RR1 and RR2 polypeptides are 87,000 and 35,000 mol. wt. respectively. Amino acid comparisons with RR1 and RR2 polypeptides from other organisms indicate that HSV-1 RR1 contains a unique N-terminal domain which is absent from other RR1 polypeptides apart from HSV-2 RR1. These N-terminal amino acid sequences are poorly conserved between HSV-1 and HSV-2 in contrast to the remainder of the protein which shows greater than 90% homology. Polypeptide structural predictions suggest that the HSV-1 N-terminal domain may be separated into two regions, namely, a beta-sheet structure followed by a nonstructured area. Across the remainder of RR1 and RR2, comparisons also reveal blocks of amino acids conserved between the different ribonucleotide reductases, and these may be important for enzyme activity. From predictions on the structure of these conserved blocks, we have proposed that the location of a substrate binding site within RR1 is centered on three conserved glycine residues in a region which is predicted to adopt a beta-sheet/turn/alpha-helical structure; this approximates to the structure for ADP nucleotide binding folds. Finally, we propose that the promoters for the HSV and Epstein-Barr virus (EBV) RR2 transcripts have evolved by separate evolutionary routes. more...
- Published
- 1986
44. Organization of the herpes simplex virus type 1 transcription unit encoding two early proteins with molecular weights of 140000 and 40000
- Author
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J B Clements and John McLauchlan
- Subjects
Genetics ,Nuclease ,biology ,Base Sequence ,Transcription, Genetic ,medicine.disease_cause ,Virology ,Molecular biology ,Exonuclease VII ,DNA sequencing ,Single-stranded binding protein ,Molecular Weight ,genomic DNA ,Viral Proteins ,Herpes simplex virus ,Transcription (biology) ,DNA, Viral ,biology.protein ,medicine ,Coding region ,RNA, Viral ,Simplexvirus ,RNA, Messenger ,Cloning, Molecular - Abstract
Summary The 5′ ends of two early herpes simplex virus type 1 mRNAs have been identified by nuclease S1 and exonuclease VII analysis using cloned virus DNA probes. These mRNAs (5.0 kb and 1.2 kb), located within the genome region between map coordinates 0.56 and 0.60, are unspliced and share a 3′ terminus. Genomic DNA at the 5′ ends has been sequenced and the 5′ termini have been located on the virus DNA sequence. The DNA sequence has revealed signals involved in the initiation of transcription of both mRNAs, and the 5′ end of the 1.2 kb mRNA is encoded within the internal sequences of the 5.0 kb mRNA. The probable translational initiation codons for the polypeptides specified by these mRNAs have been identified, and the results indicate that the coding regions of the two mRNAs do not overlap. more...
- Published
- 1983
45. Separation and characterization of herpes simplex virus type 1 immediate-early mRNA's
- Author
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R J Watson, C M Preston, and J B Clements
- Subjects
Polyadenylation ,Immunology ,Cycloheximide ,Biology ,medicine.disease_cause ,Kidney ,Microbiology ,Virus ,Cell Line ,chemistry.chemical_compound ,Nucleic acid thermodynamics ,Viral Proteins ,Virology ,Cricetinae ,medicine ,Protein biosynthesis ,Animals ,Simplexvirus ,RNA, Messenger ,Messenger RNA ,Cell-Free System ,Nucleic Acid Hybridization ,Molecular biology ,Herpes simplex virus ,chemistry ,Insect Science ,Protein Biosynthesis ,DNA, Viral ,RNA, Viral ,Poly A ,DNA ,Research Article - Abstract
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. more...
- Published
- 1979
46. The structure and biological properties of herpes simplex virus DNA
- Author
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J. B. Clements, N. M. Wilkie, J. H. Subak-Sharpe, and J. C. M. Macnab
- Subjects
Simplexvirus ,food.ingredient ,DNA, Single-Stranded ,Herpes simplex virus DNA ,Biology ,medicine.disease_cause ,Biochemistry ,Coliphages ,Cell Line ,food ,Cytopathogenic Effect, Viral ,Biological property ,Genetics ,medicine ,Dna viral ,Molecular Biology ,Gene ,Mutation ,Temperature ,DNA Restriction Enzymes ,Virology ,Molecular Weight ,Cell Transformation, Neoplastic ,Genes ,Cell culture ,DNA, Viral - Published
- 1975
47. Popliteal artery injury associated with tibial fracture in a five year old
- Author
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J B, Cofer, R P, Burns, and J B, Clements
- Subjects
Male ,Tibial Fractures ,Child, Preschool ,Joint Dislocations ,Humans ,Popliteal Artery ,Thrombosis ,Wounds, Nonpenetrating - Published
- 1986
48. Painful edema of the arm after insertion of single-needle subclavian vein dialysis catheters: pathogenesis and treatment
- Author
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J B Clements, J Yium, J Watlington, and G R Coates
- Subjects
Male ,medicine.medical_specialty ,Percutaneous ,medicine.medical_treatment ,Hemodialysis Catheter ,Pain ,Constriction, Pathologic ,Subclavian Vein ,Catheters, Indwelling ,Blood vessel prosthesis ,Renal Dialysis ,Edema ,medicine ,Humans ,Pain Management ,Vascular Diseases ,Dialysis ,Aged ,Aged, 80 and over ,business.industry ,General Medicine ,Middle Aged ,medicine.disease ,Blood Vessel Prosthesis ,Stenosis ,Evaluation Studies as Topic ,cardiovascular system ,Arm ,Female ,Radiology ,medicine.symptom ,Complication ,business ,Subclavian vein - Abstract
The percutaneous subclavian vein hemodialysis catheter, which is widely used for vascular access in patients with renal failure, can give rise to several complications. Asymptomatic stenosis or occlusion of the subclavian vein due to these catheters can lead to painful arm edema if further dialysis accesses are constructed in the ipsilateral arm. This complication can lead to loss of valuable dialysis fistulas or grafts. We present six cases of this complication, along with management alternatives, which include conservative elevation, ligation of shunts, and surgical bypass of stenosed veins. Awareness of this complication should encourage early evaluation of patients who have had subclavian catheters and guide future placement of dialysis shunts. To preserve dialysis access sites, we advise an individualized approach to each patient who develops this painful arm edema. more...
- Published
- 1988
49. Studies on Leaf Unrolling in Barley
- Author
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D. J. Carr, J. B. Clements, and R. Menhenett
- Subjects
Horticulture ,Cell expansion ,Leaf width ,Coleoptile ,Phytochrome ,Seedling ,Red light ,Biology ,biology.organism_classification ,Action spectrum - Abstract
The first leaf of a grass seedling is normally folded in the form of a scroll inside the coleoptile (Duval-Jouve, 1875). In light-grown plants the leaf unrolls as it emerges from the tip of the coleoptile. When grass seedlings are grown in the dark unrolling does not occur. Unrolling of the leaf is due to differential cell expansion largely within the mesophyll (Burstrom, 1942). Dark-grown leaves are roughly circular in cross-section with the margins considerably overlapping each other. When unrolling occurs, the diameter of the cross-section increases and it bears a weakly sigmoidal relationship to the length of the inner arc of the leaf (Virgin, 1962). However, this lack of linearity does not seriously impair quantitative estimates of the degree of unrolling based on measurements of the projected leaf width. Virgin (1962) determined the action spectrum for light-induced unrolling of the first leaf of wheat seedlings and found it to be a process promoted by red light (c. 660 nm), the promoting effect being nullified by far-red illumination (c.730 nm). This evidence, which we and others (e.g. Wagne, 1964, 1965) have extended to barley seedlings, identifies the pigment system involved as phytochrome. A considerable number of morphogenetic phenomena (Mohr, 1969) including the unbending of the plumular hook of dark-grown legume seedlings, a process analogous in some ways to grass leaf unrolling, are known to be mediated by light absorbed in phytochrome. Most of these responses are developmental events involving irreversible steps in differentiation. more...
- Published
- 1972
- Full Text
- View/download PDF
50. Evidence for large strands of ribonucleic acid induced by a bovine enterovirus
- Author
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J. B. Clements and S. J. Martin
- Subjects
Sucrose ,Time Factors ,Virus Cultivation ,Size-exclusion chromatography ,Kidney ,Tritium ,Virus Replication ,Virus ,Cell Line ,Sepharose ,chemistry.chemical_compound ,Ribonucleases ,Virology ,Cricetinae ,Centrifugation, Density Gradient ,Animals ,Ribonuclease ,Serotyping ,Uridine ,Enterovirus ,Carbon Isotopes ,biology ,Molecular mass ,RNA ,Electrophoresis, Disc ,Molecular biology ,Molecular Weight ,Electrophoresis ,Biochemistry ,chemistry ,Acrylamide ,biology.protein ,Chromatography, Gel ,Dactinomycin ,RNA, Viral ,Cattle - Abstract
Summary RNA from purified bovine enterovirus (serotype VG-5-27) was fractionated by sedimentation on sucrose gradients, gel filtration through Sepharose 2B and electrophoresis in acrylamide-agarose gels. Virus RNA was single stranded with a molecular weight of approximately 2.8 × 106. The RNA obtained from virus infected cells was separated into replicative intermediate and single-stranded molecules by gel filtration through Sepharose 2B. Electrophoresis of the replicative intermediate fraction on acrylamide-agarose gels gave two peaks of radioactivity, whereas after ribonuclease treatment only a single peak was present. Electrophoresis of the single-stranded molecules on acrylamide gels showed that a portion of these had slower mobilities than RNA from purified virus. This single-stranded fraction contained molecules with molecular weights in the range 5.6 to 2.8 × 106. Continuous labelling experiments showed that this size range was a function of time after infection. The existence of single strands of RNA longer than RNA found in purified virus was interpreted as supporting a cyclic model for RNA replication. more...
- Published
- 1971
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