Assignment of the RNA-specific adenosine deaminase gene (Adar) to mouse chromosome 3F2 by in situ hybridization.
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Adenosine Deaminase↗
Biomedical subjects
C X George
Publications and source records attributed to C X George.
Structure and structure-function studies of lipid/plasmid DNA complexes.
Recent synchrotron-based X-ray diffraction studies have enabled us to comprehensively solve the self-assembled structures in mixtures of cationic liposomes (CLs) complexed with linear lambda-DNA. In one case the CL-DNA complexes were found to consist of a higher ordered multilamellar structure (labeled L(alpha)C with DNA sandwiched between cationic bilayer membranes. The membrane charge density is found to control the DNA interaxial spacing with high densities leading to high DNA compaction between lipid bilayers. A second self-assembled structure (labeled H(II)C) consists of linear DNA strands coated by cationic lipid monolayers and arranged on a 2D hexagonal lattice. In this paper we report on a combined X-ray diffraction and optical microscopy study of CLs complexed with functional supercoiled plasmid DNA. We describe the self-assembled structures in cell culture medium for both a high transfectant complex (DOTAP/DOPE, phiDOPE = 0.72) and a low transfectant complex (DOTAP/DOPC, (phiDOPC = 0.72). Fluorescence optica microscopy shows two distinct interactions between these two types of complexes and mouse fibroblast L-cells, demonstrating the existence of a correlation between structure and transfection efficiency.
Animals↗
Alternative splice variants of the human PKR protein kinase possessing different 5'-untranslated regions: expression in untreated and interferon-treated cells and translational activity.
The double-stranded RNA-dependent protein kinase PKR is an interferon-inducible enzyme that possesses antiviral and antiproliferative activities. We examined expression of PKR transcripts in human placenta tissue and cultured human amnion U cells. Alternative exon 2 structures were identified and characterized that possess different functional activities. Cloning and sequence analyses of 5'-RACE cDNAs from human placenta established a linkage between exon 1 and three alternative exon 2 structures that constitute, together with part of exon 3, the 5'-untranslated region of the PKR mRNA. The alternative splice variants of exon 2 were designated Ex2alpha (83 nucleotides), Ex2beta (167 nucleotides), and Ex2gamma (401 nucleotides). All three exon 2 variants were present in placenta tissue. However, only the Ex2alpha and Ex2beta forms were detectable in the amnion U cell line. Nuclease protection analysis revealed that the Ex2beta form was slightly more abundant than the Ex2alpha form, in both placenta tissue and U cells. Interferon treatment of U cells increased the level of both Ex2alpha and Ex2beta RNA by approximately 5-fold. The translational activities, measured in a luciferase reporter assay, of RNA transcripts possessing the Ex2alpha and Ex2beta forms of the PKR 5'-UTR were comparable to each other and more efficient than those with the Ex2gamma form.
5' Untranslated Regions↗
Human RNA-specific adenosine deaminase ADAR1 transcripts possess alternative exon 1 structures that initiate from different promoters, one constitutively active and the other interferon inducible.
RNA-specific adenosine deaminase (ADAR1) catalyzes the deamination of adenosine to inosine in viral and cellular RNAs. Two size forms of the ADAR1 editing enzyme are known, an IFN-inducible approximately 150-kDa protein and a constitutively expressed N-terminally truncated approximately 110-kDa protein. We have now identified alternative exon 1 structures of human ADAR1 transcripts that initiate from unique promoters, one constitutively expressed and the other IFN inducible. Cloning and sequence analyses of 5'-rapid amplification of cDNA ends (RACE) cDNAs from human placenta established a linkage between exon 2 of ADAR1 and two alternative exon 1 structures, designated herein as exon 1A and exon 1B. Analysis of RNA isolated from untreated and IFN-treated human amnion cells demonstrated that exon 1B-exon 2 transcripts were synthesized in the absence of IFN and were not significantly altered in amount by IFN treatment. By contrast, exon 1A-exon 2 transcripts were IFN inducible. Transient transfection analysis with reporter constructs led to the identification of two functional promoters, designated PC and PI. Exon 1B transcripts were initiated from the PC promoter whose activity in transient transfection reporter assays was not increased by IFN treatment. The 107-nt exon 1B mapped 14.5 kb upstream of exon 2. The 201-nt exon 1A that mapped 5.4 kb upstream of exon 2 was initiated from the interferon-inducible PI promoter. These results suggest that two promoters, one IFN inducible and the other not, initiate transcription of the ADAR1 gene, and that alternative splicing of unique exon 1 structures to a common exon 2 junction generates RNA transcripts with the deduced coding capacity for either the constitutively expressed approximately 110-kDa ADAR1 protein (exon 1B) or the interferon-induced approximately 150-kDa ADAR1 protein (exon 1A).
Adenosine Deaminase↗
Characterization of the 5'-flanking region of the human RNA-specific adenosine deaminase ADAR1 gene and identification of an interferon-inducible ADAR1 promoter.
The double-stranded RNA-specific adenosine deaminase (ADAR1) is inducible by interferon (IFN) and is implicated in the editing of viral RNAs during lytic and persistent infection. We have now isolated and characterized human genomic clones that contain the promoter region required for transcription of the ADAR1 gene. Rapid amplification of cDNA 5'-ends (5'-RACE) identified additional upstream exon 1 sequence that was localized on P1-phage and lambda-phage genomic clones by Southern gel-blot analysis and sequence analysis. A Northern gel-blot analysis using a probe corresponding to the 5'-RACE exon 1 sequence and adjacent exon 2 sequence detected a major RNA transcript of approximately 6.7kb that was IFN-inducible in human amnion U cells. Transient transfection assays, using chloramphenicol acetyltransferase (CAT) as the reporter in constructs possessing various 5'-flanking fragments of the ADAR1 gene, led to the identification of a functional TATA-less promoter that directed IFN-inducible transcription of CAT. Sequence determination and deletion analysis of the promoter region revealed a consensus copy of the IFN-Stimulated Response Element (ISRE) involved in IFN inducibility that was flanked by a Kinase Conserved Sequence (KCS)-like element previously found to be unique to the human and mouse PKR gene promoters. A 63-bp minimal promoter fragment possessing the KCS-like and ISRE elements was sufficient to drive IFN-inducible transcription.
Adenosine Deaminase↗
Binding of the protein kinase PKR to RNAs with secondary structure defects: role of the tandem A-G mismatch and noncontiguous helixes.
The human interferon-induced double-stranded RNA (dsRNA)-activated protein kinase (PKR) is an antiviral agent that is activated by long stretches of dsRNA. PKR can also be activated or repressed by a series of cellular and viral RNAs containing non-Watson-Crick motifs. PKR has a dsRNA-binding domain (dsRBD) that contains two tandem copies of the dsRNA-binding motif (dsRBM). In vitro selection experiments were carried out to search for RNAs capable of binding to a truncated version of PKR containing the dsRBD. RNA ligands were selected by binding to His6-tagged proteins and chromatography on nickel(II) nitrilotriacetic acid agarose. A series of RNAs was selected that bind either similar to or tighter than a model dsRNA stem loop. Examination of these RNAs by a variety of methods, including sequence comparison, free-energy minimization, structure mapping, boundary experiments, site-directed mutagenesis, and footprinting, revealed protein-binding sites composed of noncontiguous helices. In addition, selected RNAs contained tandem A-G mismatches (5'AG3'/3'GA5'), yet bound to the truncated protein with affinities similar to duplexes containing only Watson-Crick base pairs. The NMR structure of the tandem A-G mismatch in an RNA helix (rGGCAGGCC)2 reveals a global A-form helix with minor perturbations at the mismatch [Wu, M., SantaLucia, J., Jr., and Turner, D. H. (1997) Biochemistry 36, 4449-4460]. This supports the notion that dsRBM-containing proteins can bind to RNAs with secondary structure defects as long as the RNA has an overall A-form geometry. In addition, selected RNAs are able to activate or repress wild-type PKR autophosphorylation as well as its phosphorylation of protein synthesis initiation factor eIF-2, suggesting full-length PKR can bind to and be regulated by RNAs containing a tandem A-G mismatch.
Base Sequence↗
Interaction of the human protein kinase PKR with the mouse PKR homolog occurs via the N-terminal region of PKR and does not inactivate autophosphorylation activity of mouse PKR.
The RNA-dependent protein kinase (PKR) is implicated in the antiviral and antiproliferative actions of interferon. Mutant forms of human PKR display a transdominant behavior when expressed in transfected cells. The potential for the human PKR protein to physically interact with the mouse PKR homolog has therefore been examined. The yeast two-hybrid system was used to probe the association between mouse and human PKR proteins as measured by activation of two Gal4-responsive reporter genes, HIS3 and IacZ. Expression of full-length wild-type mouse PKR(1-515)WT as a Gal4 fusion protein did not exhibit the growth suppression phenotype in yeast characteristic of wild-type human PKR(1-551)WT. Coexpression of mouse PKR(1-515)WT as a Gal4 DNA-binding domain fusion with either the catalytic-deficient human PKR(1-551) K296R mutant, the RNA-binding-deficient human PKR(1-551)K64E/K296R double mutant, or wild-type mouse PKR(1-515)WT as full-length PKR-Gal4 activation domain fusions resulted in activation of the HIS3 and lacZ reporters. The N-terminal RNA-binding region of human PKR, both WT and the K64E RNA-binding-deficient mutant, also interacted with mouse PKR(1-515)WT sufficiently to activate the reporters but the human catalytic region did not. Mouse and human full-length PKR proteins expressed as glutathione S-transferase (GST) fusions in Escherichia coli were purified on Sepharose beads. Using GST-PKR fusion chromatography, direct physical interaction between the mouse and human PKR homologs was established. Intraspecies PKR interactions were more efficient than interspecies PKR interactions, and interactions between RNA-binding-sufficient PKR proteins were more efficient than those involving an RNA-binding mutant as measured by binding to GST-PKR protein Sepharose beads. The N-terminal region of human PKR within amino acids 1-184 was sufficient for binding mouse PKR. Purified mouse full-length PKR(1-515)WT GST fusion protein retained kinase activity on Sepharose beads, but the activity was not impaired by association with either the full-length or the N-terminal region of human PKR.
Animals↗
Functionally distinct double-stranded RNA-binding domains associated with alternative splice site variants of the interferon-inducible double-stranded RNA-specific adenosine deaminase.
The double-stranded RNA-specific adenosine deaminase (ADAR) is an interferon-inducible RNA-editing enzyme implicated in the site-selective deamination of adenosine to inosine in viral RNAs and cellular pre-mRNAs. We have isolated and characterized human genomic clones of the ADAR gene and cDNA clones encoding splice site variants of the ADAR protein. Southern blot and sequence analyses revealed that the gene spans about 30 kilobase pairs and consists of 15 exons. The codon phasing of the splice site junctions of exons 3, 5, and 7 that encode the three copies of the highly conserved RNA-binding R-motif (RI, RII, and RIII) was exactly conserved and identical to those R-motif exons of the interferon-inducible RNA-dependent protein kinase. Alternative splice site variants of the 1226-amino acid ADAR-a protein, designated b and c, were identified that differed in exons 6 and 7. ADAR-b was a 5'-splice site variant that possessed a 26-amino acid deletion within exon 7; ADAR-c was a 3'-splice site variant that possessed an additional 19-amino acid deletion within exon 6. The wild-type ADAR-a, -b, and -c proteins all possessed comparable double-stranded RNA-specific adenosine deaminase activity. However, mutational analysis of the R-motifs revealed that the exon 6 and 7 deletions of ADAR-b and -c variants altered the functional importance of each of the three R-motifs.
Adenosine Deaminase↗
The PKR protein kinase--an interferon-inducible regulator of cell growth and differentiation.
Post-translational modifications such as protein phosphorylation provide an important mechanism by which the functional activity of proteins can be controlled and, hence, biological processes regulated. Interferons (IFN) are a multigene family of cytokines that can profoundly affect a wide variety of functions in animal cells including virus replication, cell growth and differentiation, and the immune response. Changes in protein phosphorylation mediated by the IFN-inducible, RNA-dependent protein kinase (PKR) are implicated in the control of cell proliferation mediated by IFNs. Our knowledge of the structure, regulation and function of PKR will be summarized in this brief review, with focus on those aspects of protein phosphorylation and interferon action involving PKR that are central to the roles of the enzyme in the control of cell growth and proliferation.
Animals↗
Characterization of the heparin-mediated activation of PKR, the interferon-inducible RNA-dependent protein kinase.
Heparin can substitute for double-stranded (ds) RNA in the autophosphorylation and activation of the interferon-inducible, RNA-dependent elF-2 alpha protein kinase (PKR). We have used heparin oligosaccharides of defined lengths to examine the heparin-mediated activation of human PKR. Heparin oligosaccharide with 8 sugar residues was nearly as efficient as 16-residue heparin (Hep-16) in mediating the activation of PKR autophosphorylation, whereas 6-residue heparin was a poor activator. When examined in combination, Hep-16 and dsRNA did not act synergistically in activating PKR autophosphorylation. The RNA-binding activity of recombinant PKR, measured with adenovirus VA RNA, was competed by poly(rl):poly(rC) but not by Hep-16. When the catalytically inactive, histidine-tagged mutant PKR protein [His-PKR(K296R)] was examined as a substrate for purified wild-type PKR, the intermolecular phosphorylation of His-PKR(K296R) was efficiently catalyzed by dsRNA-activated PKR but not by heparin-activated PKR. However, elF-2 alpha phosphorylation was catalyzed by both heparin-and dsRNA-activated PKR. Preincubation of PKR with Hep-16 in the absence of ATP blocked subsequent autophosphorylation mediated either by Hep-16 or dsRNA, whereas preincubation with dsRNA either alone or in combination with Hep-16 did not impair subsequent autophosphorylation. Neither Hep-16 nor dsRNA caused a detectable degradation of PKR during preincubation or subsequent autophosphorylation of PKR. These results suggest that, while both dsRNA and heparin are capable of activating PKR autophosphorylation, the structural and functional basis of PKR activation differs for these two classes of polyanionic biomolecules.
Animals↗
The interferon-inducible, double-stranded RNA-specific adenosine deaminase gene (DSRAD) maps to human chromosome 1q21.1-21.2.
The interferon-inducible double-stranded RNA-specific adenosine deaminase is an RNA-modifying enzyme implicated in the generation of biased hypermutations viral RNAs and the site-selective editing of mammalian mRNAs of neural origin. The gene for the dsRNA-specific adenosine deaminase has been mapped by fluorescence in situ hybridization (FISH) of genomic clones to a single locus on human chromosome 1 bands q21.1-21.2. Simultaneous multicolor FISH including lambda clones and yeast artificial chromosomes showed a localization of the gene in band 1q21 centromeric of D1S1705.
Adenosine Deaminase↗
Studies on the antiviral activity of inorganic heteropolyanions against Semliki forest virus in vivo and vaccinia virus in vitro.
Seven new inorganic heteropolyanions were tested for their antiviral activity. One of these, sodium 12-tungstoborate, was found to protect mice from Semliki forest virus and mouse embryo fibroblast monolayers from vaccinia virus infections. In mice these heteropolyanions exhibited no synergistic antiviral activity with the interferon inducing mycoviral double-stranded RNA.
Animals↗
Mechanism of interferon action. Expression of reovirus S3 gene in transfected COS cells and subsequent inhibition at the level of protein synthesis by type I but not by type II interferon.
The effect of interferon on the expression of the reovirus serotype 1 Lang strain S3 gene was examined in simian COS cells transfected with the expression vector pSVS3 containing the S3 gene under the control of the SV40 late promoter. When COS cells were treated with type I interferon-alpha 24 hr after transfection, the synthesis of the reovirus S3-encoded sigma NS polypeptide was inhibited about 10-fold as compared to that in untreated control cells. By contrast, under the same conditions, neither the plasmid DNA copy number nor the S3 gene mRNA levels were significantly affected by interferon treatment. Type II interferon-gamma, unlike the type I interferons-alpha, did not affect the rate of synthesis of polypeptide sigma NS in pSVS3-transfected cells.
Animals↗
Biosynthesis of reovirus-specified polypeptides. Molecular cDNA cloning and nucleotide sequence of the reovirus serotype 1 Lang strain s2 mRNA which encodes the virion core polypeptide sigma 2.
Human reovirus serotype 1 Lang strain s2 mRNA, which encodes the virion inner capsid core polypeptide sigma 2, was cloned as a cDNA:mRNA heteroduplex in Escherichia coli using phage M13. A complete consensus nucleotide sequence was determined. The Lang strain s2 mRNA is 1331 nucleotides in length and possesses an open reading frame with a coding capacity of 335 amino acids, sufficient to account for a sigma 2 polypeptide of 37,682 daltons. Comparison of the serotype 1 Lang s2 sequence derived from cDNA clones of s2 mRNA with the serotype 3 Dearing S2 sequence derived from cDNA clones of the S2 dsRNA genome segment reveals 86 percent homology at the nucleotide level. The predicted sigma 2 polypeptides of the Lang and Dearing strains display 98 percent homology at the amino acid level. Of 147 silent nt differences in the translated region, 136 were in the third base position of codons.
Amino Acid Sequence↗
Biosynthesis of reovirus-specified polypeptides. Molecular cDNA cloning and nucleotide sequence of the reovirus serotype 1 Lang strain s3 mRNA which encodes the nonstructural RNA-binding protein sigma NS.
Human reovirus serotype 1 Lang strain s3 mRNA, which encodes the nonstructural RNA-binding polypeptide sigma NS, was cloned as a cDNA:mRNA heteroduplex in Escherichia coli using phage M13. A complete consensus nucleotide sequence was determined. The Lang strain s3 mRNA is 1198 nucleotides in length and possesses an open reading frame with a coding capacity of 366 amino acids, sufficient to account for a sigma NS polypeptide of 41,179 daltons. Comparison of the serotype 1 (Lang) s3 sequence with the serotype 3 (Dearing) s3 sequence reveals 86.8 percent homology at the nucleotide level. The predicted sigma NS polypeptides of the Lang and Dearing strains display 97 percent homology at the amino acid level.
Amino Acid Sequence↗
Enhancement in anti-Semliki Forest virus activity of ds RNA by a muramyl dipeptide.
Antiviral activity of an interferon-inducing mycoviral ds RNA against Semliki Forest virus infection was considerably enhanced by N-palmitoylmuramyl-L-alanyl-D-isoglutamine (PMDP), a new muramyl dipeptide. This enhancement in activity was not due to increased production of interferon, but resulted probably from a PMDP-induced increase in nonspecific resistance to infection. These results indicate that a combined treatment with an interferon inducer and muramyl dipeptide may prove highly useful to control effectively viral infections.
Acetylmuramyl-Alanyl-Isoglutamine↗
Combined treatment of mice with Panax ginseng extract and interferon inducer. Amplification of host resistance to Semliki forest virus.
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Animals↗
Antiviral activity in plants of a mycoviral double-strained RNA from Trichothecium roseum.
Hexagonal virus-like particles (VLPs) measuring 45 nm across were detected in mycelial extracts from Trichothecium roseum Himachal strain, the source fungus for the production of T-poly (Trichothecium polysaccharide), a known inhibitor of plant viruses. VLPs were found to contain double-stranded RNA (ds RNA) and the purified ds RNA was capable of inhibiting tobacco mosaic virus infection in Nicotiana glutinosa plants. Active preparations of T-poly were found to contain traces of ds RNA, probably of mycoviral origin.
Mitosporic Fungi↗