Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.2.1.13 (glyceraldehyde-3-phosphate dehydrogenase)
6,511 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have isolated and characterized a plasmid (pChug 20.1) that contains the cDNA of a nuclear uracil DNA glycosylase (UDG) gene isolated from normal human placenta. This cDNA directed the synthesis of a fusion protein (Mr 66,000) that exhibited UDG activity. The enzymatic activity was specific for a uracil-containing polynucleotide substrate and was inhibited by a glycosylase antibody or a beta-galactosidase antibody. Sequence analysis demonstrated an open reading frame that encoded a protein of 335 amino acids of calculated Mr 36,050 and pI 8.7, corresponding to the Mr 37,000 and pI 8.1 of purified human placental UDG. No homology was seen between this cDNA and the UDG of herpes simplex virus, Escherichia coli, and yeast; nor was there homology with the putative human mitochondrial UDG cDNA or with a second human nuclear UDG cDNA. Surprisingly, a search of the GenBank data base revealed that the cDNA of UDG was completely homologous with the 37-kDa subunit of human glyceraldehyde-3-phosphate dehydrogenase. Human erythrocyte glyceraldehyde-3-phosphate dehydrogenase was obtained commercially in its tetrameric form. A 37-kDa subunit was isolated from it and shown to possess UDG activity equivalent to that seen for the purified human placental UDG. The multiple functions of this 37-kDa protein as here and previously reported indicate that it possesses a series of activities, depending on its oligomeric state. Accordingly, mutation(s) in the gene of this multifunctional protein may conceivably result in the diverse cellular phenotypes of Bloom syndrome.
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PMID:A human nuclear uracil DNA glycosylase is the 37-kDa subunit of glyceraldehyde-3-phosphate dehydrogenase. 192 5

This study presents the first evidence that the 5' promoter region of the Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase gene (G-3-PD) promoter will permit expression of an adjacent foreign gene. The S. cerevisiae G-3-PD promoter was linked to the herpes simplex virus--thymidine kinase (HSV-TK) gene in a shuttle plasmid capable of autonomous replication in both yeast and Escherichia coli. Since the HSV-TK gene promoter is not functional in yeast, yeast cells containing these plasmids will express the HSV-TK gene and synthesize thymidine kinase only if the yeast promoter fragment is fused to the HSV-TK gene in the proper orientation. The 5' flanking sequences necessary for the expression of heterologous eukaryotic genes in S. cerevisiae are discussed.
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PMID:Control of Herpes simplex virus thymidine kinase gene expression in Saccharomyces cerevisiae by a yeast promoter sequence. 632 18

The herpes simplex virus type 1 (HSV-1) UL41 gene product, virion host shutoff (vhs), has homologs among five alphaherpesviruses (HSV-1, HSV-2, pseudorabies virus, varicella-zoster virus, and equine herpesvirus 1), suggesting a role for this protein in neurotropism. A mutant virus, termed UL41NHB, which carries a nonsense linker in the UL41 open reading frame at amino acid position 238 was generated. UL41NHB and a marker-rescued virus, UL41NHB-R, were characterized in vitro and tested for their ability to replicate in vitro and in vivo and to establish and reactivate from latency in a mouse eye model. As demonstrated by Western blotting (immunoblotting) and Northern (RNA) blotting procedures, UL41NHB encodes an appropriately truncated vhs protein and, as expected for a vhs null mutant, fails to induce the degradation of cellular glyceraldehyde-3-phosphate dehydrogenase mRNA. The growth of UL41NHB was not significantly altered in one-step growth curves in Vero or mouse C3H/10T1/2 cells but was impaired in corneas, in trigeminal ganglia, and in brains of mice compared with the growth of KOS and UL41NHB-R. As a measure of establishment of latency, quantitative DNA PCR showed that the amount of viral DNA within trigeminal ganglia latently infected with UL41NHB was reduced by approximately 30-fold compared with that in KOS-infected ganglia and by 50-fold compared with that in UL41NHB-R-infected ganglia. Explant cocultivation studies revealed a low reactivation frequency for UL41NHB (1 of 28 ganglia, or 4%) compared with that for KOS (56 of 76, or 74%) or UL41NHB-R (13 of 20 or 65%). Taken together, these results demonstrate that vhs represents a determinant of viral pathogenesis.
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PMID:Role of the virion host shutoff (vhs) of herpes simplex virus type 1 in latency and pathogenesis. 747 89

Herpes simplex virus type 1 (HSV-1) infection induces the selective shut-off of host protein synthesis, other than ribosomal proteins, and the successive synthesis of viral proteins. Because viral mRNAs persist in the cytoplasm after viral protein synthesis has been inhibited, we hypothesized that viral gene expression may be under translational control. Expression of genes encoding immediate early ICP27, early DBP and late US11 proteins, together with glyceraldehyde-3-phosphate dehydrogenase (GAPDH), was monitored over the course of infection at the level of mRNA and protein synthesis. After an efficient synthesis beginning with the appearance of successive viral mRNAs in the cytoplasm, synthesis of viral proteins was shut off similarly to the synthesis of GAPDH. This shut-off was not achieved by mRNA degradation but by progressive shifts of viral mRNAs from large polyribosomes to smaller ones, then to 40S ribosomal subunits. Transient expression of the UL41 gene alone, directing synthesis of virion-associated host shut-off (VHS) protein, induced efficient mRNA degradation, but did not impair recruitment of the remaining GAPDH and beta-actin mRNAs into polyribosomes. These results indicate that HSV-1 induces a selective repression of initiation of mRNA translation which is probably the main cause of the shut-off of viral protein synthesis, and which contributes to the repression of host protein synthesis. VHS protein is not directly involved in this repression, at least in the absence of other viral proteins.
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PMID:Translational control of viral and host protein synthesis during the course of herpes simplex virus type 1 infection: evidence that initiation of translation is the limiting step. 982 Jan 53

Herpes simplex virus type 1 (HSV-1) induces prominent shifts in the rates of transcription of host cellular genes of relevance for the outcome of the viral infection. The quantitative analysis of transcription may be obscured by virus-induced alterations in the levels of RNA encoded by cellular housekeeping genes that are used commonly for normalisation of real time reverse transcription PCR (RT-qPCR). In the present study, we analysed beta-actin, GAPDH and 18S rRNA for their usefulness in normalisation of RT-qPCR analysis of the transcription of the HSV-1 gamma gB-1 gene and FUT5, a cellular gene induced by viral infection. The transcription of these genes was monitored in a TaqMan-based real time RT-PCR system over a 24h interval of virus infection of human embryonic lung fibroblasts. The levels of gB-1 and FUT5 RNA were normalised via difference in the threshold cycle (deltaC(t)) values relative to each and one of the housekeeping genes or calculated in relation to the number of infected cells without any further normalisation. The levels of RNA encoded by beta-actin or GAPDH were found to vary by several orders of magnitude during HSV-1 infection, introducing large errors in the estimation of the gB-1 and FUT5 RNA levels. In contrast, the variation of C(t) values for 18S rRNA was less than one cycle during 24h period of HSV-1 infection. The FUT5 and gB-1 RNA figures obtained by DeltaC(t) normalisation relative 18S rRNA were identical to those calculated in relation to the number of infected cells. These data recommend 18S rRNA for normalisation in HSV-1-infected human cells but discourage the use of beta-actin and GAPDH RNA for this purpose. By applying these procedures, it was shown that the transcription of FUT5 was increased by 50-fold 5-24h after HSV-1 infection and 200-fold by the inhibition of viral DNA replication in HSV-infected cells.
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PMID:Real time PCR for monitoring regulation of host gene expression in herpes simplex virus type 1-infected human diploid cells. 1508 3

Herpes simplex virus (HSV) ICP27 is an essential and multifunctional regulator of gene expression that modulates the synthesis and maturation of viral and cellular mRNAs. Processes that are affected by ICP27 include transcription, pre-mRNA splicing, polyadenylation, and nuclear RNA export. We have examined how ICP27 influences the expression of the essential HSV tegument protein and transactivator of immediate-early gene expression VP16. We monitored the effects of ICP27 on the levels, nuclear export, and polyribosomal association of VP16 mRNA and on the amount and stability of VP16 protein. Deletion of ICP27 reduced the levels of VP16 mRNA without altering its nuclear export or the stability of the encoded protein. However, the translational yield of the VP16 mRNA produced in the absence of ICP27 was reduced 9- to 80-fold relative to that for wild-type infection, suggesting a defect in translation. In the absence of ICP27, the majority of cytoplasmic VP16 mRNA was not associated with actively translating polyribosomes but instead cosedimented with 40S ribosomal subunits, indicating that the translational defect is likely at the level of initiation. These effects were mRNA specific, as polyribosomal analysis of two cellular transcripts (glyceraldehyde-3-phosphate dehydrogenase and beta-actin) and two early HSV transcripts (thymidine kinase and ICP8) indicated that ICP27 is not required for efficient translation of these mRNAs. Thus, we have uncovered a novel mRNA-specific translational regulatory function of ICP27.
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PMID:Control of VP16 translation by the herpes simplex virus type 1 immediate-early protein ICP27. 1576 13

We reported that varicella-zoster virus (VZV) causes a delayed host shutoff during its replicative cycle. VZV open reading frame 17 (ORF17) is the homologue of the herpes simplex virus (HSV) UL41 gene encoding the virion host shutoff (vhs) protein which is responsible for the shutoff effect observed in HSV-infected cells. In the present study, we demonstrated that ORF17 is expressed as a late protein during the VZV replicative cycle in different infected permissive cell lines which showed a delayed shutoff of cellular RNA. A cell line with stable expression of VZV ORF17 was infected with VZV. In these cells, VZV replication and delayed host shutoff remained unchanged when compared to normal infected cells. ORF17 was not capable of repressing the expression of the beta-gal reporter gene under the control of the human cytomegalovirus immediate-early gene promoter or to inhibit the expression of a CAT reporter gene under the control of the human GAPDH promoter, indicating that ORF17 has no major function in the VZV-mediated delayed host shutoff. To determine whether other viral factors are involved in the host shutoff, a series of cotransfection assays was performed. We found that the immediate-early 63 protein (IE63) was able to downregulate the expression of reporter genes under the control of the two heterologous promoters, indicating that this viral factor can be involved in the VZV-mediated delayed host shutoff. Other factors can be also implicated to modulate the repressing action of IE63 to achieve a precise balance between the viral and cellular gene expression.
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PMID:The varicella-zoster virus-mediated delayed host shutoff: open reading frame 17 has no major function, whereas immediate-early 63 protein represses heterologous gene expression. 1603 98

The herpes simplex virus 1 ORF U(L)41 encodes a protein (virion host shutoff or vhs) associated with selective degradation of mRNA early in infection. Some mRNAs, exemplified by GAPDH or beta-actin mRNAs, are degraded rapidly. Others, for example IEX-1 mRNA, are degraded in two stages: whereas the 3' domain disappears rapidly, a large 5' domain fragment of the mRNA lingers for several hours. Still a third, exemplified by tristetraprolin mRNA, is not degraded, allowing its protein product to accumulate in infected cells. Here we report the following: (i) a GST-vhs protein produced in Escherichia coli, solubilized and purified to homogeneity acts as bona fide endoribonuclease when tested on in vitro transcribed IEX-1 probes. A GST-vhs protein in which three key vhs amino acids were replaced with alanines, solubilized and purified by the same protocol, had no enzymatic activity. (ii) The number of fragments generated by cleavage of a truncated IEX-1 RNA by vhs appears to be small; the cleavage sites are centered at or near the AU-rich elements located at the 3' untranslated region of the mRNA. A truncated RNA containing only the IEX-1 coding domain was cleaved numerous times. (iii) In cells infected at high multiplicity and exposed to a large number of particles per cell, the vhs protein accumulated within 3 h after infection, in small uniform cytoplasmic granules raising the possibility that vhs colocalizes with tristerapolin, a protein induced after infection, in structures involved in degradation of RNA.
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PMID:The U(L)41 protein of herpes simplex virus 1 degrades RNA by endonucleolytic cleavage in absence of other cellular or viral proteins. 1647 41

The herpes simplex virus (HSV) virion host shutoff (Vhs) protein is an endoribonuclease that accelerates decay of many host and viral mRNAs. Purified Vhs does not distinguish mRNAs from nonmessenger RNAs and cuts target RNAs at many sites, yet within infected cells it is targeted to mRNAs and cleaves those mRNAs at preferred sites including, for some, regions of translation initiation. This targeting may result in part from Vhs binding to the translation initiation factor eIF4H; in particular, several mutations in Vhs that abrogate its binding to eIF4H also abolish its mRNA-degradative activity, even though the mutant proteins retain endonuclease activity. To further investigate the role of eIF4H in Vhs activity, HeLa cells were depleted of eIF4H or other proteins by transfection with small interfering RNAs (siRNAs) 48 h prior to infection or mock infection in the presence of actinomycin D. Cellular mRNA levels were then assayed 5 h after infection. In cells transfected with an siRNA for the housekeeping enzyme glyceraldehyde-3-phosphate dehydrogenase, wild-type HSV infection reduced beta-actin mRNA levels to between 20 and 30% of those in mock-infected cells, indicative of a normal Vhs activity. In contrast, in cells transfected with any of three eIF4H siRNAs, beta-actin mRNA levels were indistinguishable in infected and mock-infected cells, suggesting that eIF4H depletion impeded Vhs-mediated degradation. Depletion of the related factor eIF4B did not affect Vhs activity. The data suggest that eIF4H binding is required for Vhs-induced degradation of many mRNAs, perhaps by targeting Vhs to mRNAs and to preferred sites within mRNAs.
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PMID:Small interfering RNAs that deplete the cellular translation factor eIF4H impede mRNA degradation by the virion host shutoff protein of herpes simplex virus. 1844 41

The virion host shutoff protein product of the U(L)41 gene of herpes simplex virus 1 is an endoribonuclease that selectively degrades mRNAs during the first hours after infection. Specifically, in contrast to the events in uninfected cells or cells infected with a mutant lacking the RNase, in wild-type virus-infected cells mRNA of housekeeping genes exemplified by GAPDH is degraded rapidly, whereas mRNAs containing AU elements are cleaved and the 5' cleavage product of these RNAs persists for many hours. We report that in wild-type virus-infected cells there was a rapid increase in the number and size of processing bodies (P-bodies). These P-bodies were also preset in cycloheximide (CHX)-treated cells but not in either treated or untreated uninfected cells or cells infected with the RNase minus mutant. Additional studies revealed that polyribosomes extracted from cytoplasm of wild-type virus-infected cells treated with CHX and displayed in sucrose gradients contained ribosome-loaded, truncated AU-rich mRNAs lacking the 3' UTR and poly(A) tails. The results suggest that the virion RNase is bound to polyribosomes by virtue of the reported association with translation machinery and cleaves the RNAs 5' to the AU elements. In contrast to the slow degradation of the of the residual 5' domain, the 3' UTR of the AU-rich mRNA and the GAPDH mRNA are rapidly degraded in wild-type virus-infected cells.
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PMID:The virion-packaged endoribonuclease of herpes simplex virus 1 cleaves mRNA in polyribosomes. 1958 46


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