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Biomedical subjects

C E Samuel

Publications and source records attributed to C E Samuel.

At least 55 records · Page 3Linked to original sources
PubMed1995

Sequence of the murine interferon-inducible RNA-dependent protein kinase (PKR) deduced from genomic clones.

The gene encoding the RNA-dependent protein kinase (PKR) was cloned from mouse genomic DNA and characterized by restriction mapping, Southern blot analysis and sequencing. The Southern analyses were consistent with the presence of a single copy of the Pkr gene in the mouse haploid genome. The genomic nucleotide (nt) sequence, when compared to that of previously determined cDNA nt sequences, revealed 16 exons encoding the 515-amino-acid PKR.

Amino Acid Sequence↗
PubMed1995

Mechanism of interferon action: RNA-binding activity of full-length and R-domain forms of the RNA-dependent protein kinase PKR--determination of KD values for VAI and TAR RNAs.

The RNA-binding activity of the interferon-inducible, RNA-dependent protein kinase PKR, expressed from the human PKR cDNA, was quantitated using a gel mobility-shift assay. The N-terminal R-domain truncation Wt(1-243) and the full-length catalytic mutant K296R(21-551) were analyzed for their abilities to bind adenovirus VAI RNA, human immunodeficiency virus TAR RNA, and the synthetic homopolymer pI:pC RNA. The N-terminal 243 amino acid residue form of PKR [Wt(1-243)] bound VAI RNA with similar affinity as the 551 amino acid residue full-length catalytic mutant [K296R(1-551)]. The dissociation constant for VAI RNA was approximately 2 x 10(-9) M for both the K296R(1-551) and Wt(1-243) proteins. The K64E mutation significantly impaired the VAI RNA-binding activity as measured with the full-length double-point mutant PKR protein, K64E/K296R(1-551). Using a gel-shift competition assay, the dissociation constants of K296R(1-551) and Wt(1-243) for VAI(1-160) RNA and pI:pC RNA were comparable. By contrast, the dissociation constants of K296R(1-551) and Wt(1-243) for TAR(1-82) RNA were both about 1 x 10(-7) M. These results suggest that the RNA-binding affinity of PKR is approximately 100-fold lower for TAR RNA than for either VAI RNA or pI:pC RNA and that the full-length and N-terminal R-domain forms of PKR bind RNA with similar affinity.

Adenoviridae↗
PubMed1995

Mechanism of interferon action: characterization of the intermolecular autophosphorylation of PKR, the interferon-inducible, RNA-dependent protein kinase.

The interferon-inducible, RNA-dependent protein kinase (PKR) is activated by autophosphorylation, a process mediated by double-stranded RNA. A catalytically deficient, histidine-tagged mutant PKR protein [His-PKR(K296R)] was used as the substrate for characterization of the intermolecular phosphorylation catalyzed by purified wild-type PKR [PKR(Wt)]. The intermolecular autophosphorylation of His-PKR(K296R) by PKR(Wt) was RNA dependent. Excess His-PKR(K296R) substrate inhibited both the auto- and the trans-phosphorylation activities of PKR(Wt). Inhibition of PKR(Wt) by His-PKR(K296R) was relieved by higher concentrations of activator double-stranded RNA. Phosphopeptide analysis revealed that the sites of intermolecular autophosphorylation in His-PKR(K296R) were very similar, if not identical, to the sites that were autophosphorylated in PKR(Wt) and suggest a multiple of four major phosphorylation sites per PKR molecule.

Chromatography, High Pressure Liquid↗
PubMed1995

Expression and regulation by interferon of a double-stranded-RNA-specific adenosine deaminase from human cells: evidence for two forms of the deaminase.

A 6,474-nucleotide human cDNA clone designated K88, which encodes double-stranded RNA (dsRNA)-specific adenosine deaminase, was isolated in a screen for interferon (IFN)-regulated cDNAs. Northern (RNA) blot analysis revealed that the K88 cDNA hybridized to a single major transcript of approximately 6.7 kb in human cells which was increased about fivefold by IFN treatment. Polyclonal antisera prepared against K88 cDNA products expressed in Escherichia coli as glutathione S-transferase (GST) fusion proteins recognized two proteins by Western (immunoblot) analysis. An IFN-induced 150-kDa protein and a constitutively expressed 110-kDa protein whose level was not altered by IFN treatment were detected in human amnion U and neuroblastoma SH-SY5Y cell lines. Only the 150-kDa protein was detected in mouse fibroblasts with antiserum raised against the recombinant human protein; the mouse 150-kDa protein was IFN inducible. Immunofluorescence microscopy and cell fractionation analyses showed that the 110-kDa protein was exclusively nuclear, whereas the 150-kDa protein was present in both the cytoplasm and nucleus of human cells. The amino acid sequence deduced from the K88 cDNA includes three copies of the highly conserved R motif commonly found in dsRNA-binding proteins. Both the 150-kDa and the 110-kDa proteins prepared from human nuclear extracts bound to double-stranded but not to single-stranded RNA affinity columns. Furthermore, E. coli-expressed GST-K88 fusion proteins that included the R motif possessed dsRNA-binding activity. Extracts prepared either from K88 cDNA-transfected cells or from IFN-treated cells contained increased dsRNA-specific adenosine deaminase enzyme activity. These results establish that K88 encodes an IFN-inducible dsRNA-specific adenosine deaminase and suggest that at least two forms of dsRNA-specific adenosine deaminase occur in human cells.

Adenosine Deaminase↗
PubMed1994

Ligand-independent anti-oncogenic activity of the alpha subunit of the type I interferon receptor.

Two interferon (IFN) alpha-regulated genes, IRF1/ISGF2 and PKR/p68 kinase, may function as tumor suppressor genes suggesting that the IFN system may function as a tumor suppressor system. We report that the expression of the alpha subunit of the type I IFN receptor in human K-562 cells had anti-oncogenic effects that include a marked decrease in: (i) cell proliferation rate, (ii) the cell density at which growth arrest normally occurs, and (iii) the tumorigenicity in nude mice. Furthermore, expression of the alpha subunit in K-562 cells induced erythroid differentiation. While most cytokine receptors become activated after binding their corresponding ligands, the overexpression of the alpha subunit has a physiological effect in the absence of its natural ligand, type I IFNs, suggesting a novel function for this type I IFN receptor subunit. The anti-oncogenic effect of the alpha subunit is mediated by a pathway that does not involve two tumor suppressor genes induced by type I IFNs, the transcriptional regulator IFN response factor-1 and the RNA-dependent protein kinase, or the p135tyk2 tyrosine kinase that directly associates and phosphorylates the alpha subunit.

Animals↗
PubMed1994

Mechanism of interferon action: structure of the mouse PKR gene encoding the interferon-inducible RNA-dependent protein kinase.

The gene for the RNA-dependent eIF-2 alpha protein kinase (PKR) was isolated from mouse genomic DNA and characterized. The mouse PKR gene contains 16 exons and spans about 28 kilobase pairs. Exon 1 is untranslated; the AUG translation initiation site is located early in the second exon. Exon 16 includes the UAG translation termination site. ATTAAA polyadenylylation signal, and a putative TA rather than CA 3' cleavage site. Primer extension analysis determined one major as well as multiple minor transcription initiation sites; the major site was 159 bp upstream of the translation initiation site. The complete cDNA of mouse PKR is, therefore, 2334 bp in length excluding the 3' poly(A)+ tail. The PKR gene 5' flanking region was a functional promoter in interferon-treated, transfected cells as measured with chloramphenicol acetyltransferase as the reporter gene. Sequence analysis of the 5' flanking region disclosed numerous potential binding sites for transcription factors including both an ISRE element and a GAS element involved in interferon inducibility; Ets, Myb, MyoD, and E2F sites commonly associated with growth control regulation and differentiation; and NF-kappa B-like sites as well as sites for two types of interleukin 6-activated factors, NF-IL6 and APRF, often associated with acute-phase, immune, and inflammatory response genes.

Animals↗
PubMed1994

Mechanism of interferon action motif I of the interferon-induced, RNA-dependent protein kinase (PKR) is sufficient to mediate RNA-binding activity.

The interferon-induced P1/eIF-2 alpha protein kinase cDNA, designated PKR, was expressed both in Escherichia coli and in transfected monkey COS cells. TrpE-PKR fusion proteins and PKR nonfusion proteins were examined for their RNA-binding activity by Northwestern blot analysis. PKR is a RNA-binding protein that possesses two copies of a highly conserved motif, RI and RII, within the N-terminal region of the protein. Amino acid residues between 11 and 243 of PKR, which includes both copies of the R motif, displayed RNA-binding activity comparable to that of the full-length 551-amino-acid PKR protein. Analysis of substitution and deletion mutant PKR proteins revealed that motif RI was both necessary and sufficient for RNA-binding activity, whereas motif RII was not. Substitution of the highly conserved lysine at position 64 within the RI motif abolished RNA-binding activity, both of full-length PKR and the N-terminal 243-amino-acid truncated PKR. Finally, PKR substitution and deletion mutant cDNAs deficient for kinase function were expressed to much higher levels in transfected monkey cells than was the full-length wild-type PKR cDNA.

Animals↗
PubMed1994

Interferon-induced proteins and their mechanisms of action.

Interferons modulate a number of biological functions including cell growth and differentiation, the immune response, and virus replication. The antiviral activity of interferons (IFNs), the property that led to their discovery, is mediated by multiple cellular proteins. Among the IFN-induced cellular proteins with antiviral activity are the protein kinase PKR, the enzymes of the 2', 5'-oligoadenylate pathway, and the Mx proteins. The antiviral activities of these proteins differ significantly against viruses of the myxoviridae, rhabdoviridae, and picornaviridae virus families.

2',5'-Oligoadenylate Synthetase↗
PubMed1993

Biosynthesis of reovirus-specified polypeptides. Analysis of ribosome pausing during translation of reovirus S1 and S4 mRNAs in virus-infected and vector-transfected cells.

The steady-state distribution of translating ribosomes on the reovirus s1 and s4 mRNAs was determined using a sensitive primer extension inhibition assay and cDNA clones of the reovirus S1 and S4 genes. Positions of significant ribosome pausing included the sites of initiation and termination of translation, both in vivo in reovirus-infected mouse fibroblast L cells and in vitro in rabbit reticulocyte lysates. The patterns of ribosome pausing detected in vivo and in vitro were comparable. Ribosomes were far less uniformly distributed along the polycistronic s1 mRNA than they were along the monocistronic s4 mRNA. During reovirus infection, ribosome pausing was detected at the initiation codons of the s1 mRNA sigma 1 and sigma 1ns open reading frames and the s4 mRNA sigma 3 open reading frame, direct evidence that initiation is a slow step of translation in vivo. Quantitative analysis of reovirus mRNA translation in infected and transfected cells suggested that ribosomes paused longer at an AUG initiation codon present in strong context than one present in weak context. These results suggest that the dynamics of ribosome movement during translation of the reovirus s1 and s4 mRNAs are similar in vivo and in vitro and that the relative ribosome pause at an AUG initiation codon may be affected by the flanking nucleotide context of the AUG codon.

Animals↗
PubMed1993

Biosynthesis of reovirus-specified polypeptides: identification of regions of the bicistronic reovirus S1 mRNA that affect the efficiency of translation in animal cells.

The reovirus S1 gene cDNA was systematically altered by site-directed mutagenesis in an attempt to identify regions important in determining the relative efficiency of translation of the two open reading frames of the bicistronic S1 mRNA. The synthesis of the minor capsid protein sigma 1 encoded by ORF1, extending from AUG14 to UGA1424, and the synthesis of the nonstructural protein sigma 1NS encoded by ORF2, extending from AUG75 to UAG432, were examined in transfected COS cells. Deletion of the 5'-untranslated region upstream of ORF1 AUG14 did not significantly affect either the relative amount, or the ratio, of sigma 1 and sigma 1NS synthesized. Creation of a strong context ORF1 initiation site by substitution of 5'-untranslated region nucleotides flanking AUG14 likewise did not affect sigma 1 synthesis, but sigma 1NS synthesis from ORF2 was decreased about twofold relative to wild-type S1 mRNA. The amount of sigma 1NS synthesis was increased less than twofold either by elimination of the ORF1 AUG14 initiation codon or by termination of sigma 1 synthesis shortly after initiation from AUG14. No sigma 1 synthesis was detected when the ORF1 AUG14 was mutated to a UUG14 codon. When ribosomes which initiated translation at AUG14 were frameshifted at the next codon after AUG14, elongation occurred with comparable efficiency in the sigma 1NS ORF2 and in sigma 1 ORF1. No sigma 1NS synthesis was detected when the ORF2 AUG75 was mutated to an CUG75 codon, and this mutation did not affect the amount of sigma 1 synthesis. sigma 1NS synthesis was not affected by truncation of the 3'-untranslated region or premature termination of sigma 1 synthesis shortly after the ORF2 UAG432. However, truncation of the ORF2 5'-untranslated region at either 6 or 36 nt following the ORF1 AUG14 significantly increased the efficiency of sigma 1NS synthesis. These results indicate that the region of the S1 mRNA immediately downstream of the sigma 1 ORF1 initiation codon AUG14 but well upstream of the sigma 1NS ORF2 initiation codon AUG75 plays a major role in determining the relative efficiency of synthesis of sigma 1 and sigma 1NS from the reovirus bicistronic s1 mRNA.

2-Aminopurine↗
PubMed1993

Mechanism of interferon action: evidence for intermolecular autophosphorylation and autoactivation of the interferon-induced, RNA-dependent protein kinase PKR.

The interferon-induced RNA-dependent protein kinase (PKR) is postulated to have an important regulatory role in the synthesis of viral and cellular proteins. Activation of the enzyme requires the presence of a suitable activator RNA and is accompanied by an autophosphorylation of PKR. Active PKR phosphorylates the alpha subunit of protein synthesis eukaryotic initiation factor 2, resulting in an inhibition of translation initiation. The mechanism of autophosphorylation is not well understood. Here we present evidence that the autophosphorylation of human PKR can involve intermolecular phosphorylation events, i.e., one PKR protein molecule phosphorylating a second PKR molecule. Both wild-type PKR and the point mutant PKR(K296R) synthesized in vitro were phosphorylated, even though PKR(K296R) was deficient in kinase catalytic activity. Phosphorylation of both wild-type PKR and PKR(K296R) was inhibited in the presence of 2-aminopurine. Furthermore, purified human recombinant PKR(K296R) was a substrate for the purified wild-type human PKR kinase. This intermolecular phosphorylation of mutant PKR(K296R) by wild-type PKR was dependent on double-stranded RNA and was inhibited by 2-aminopurine. Finally, PKR mRNA was capable of mediating an autoactivation of wild-type PKR kinase autophosphorylation in vitro.

Adenosine Triphosphate↗
PubMed1992

Mechanism of interferon action: autoregulation of RNA-dependent P1/eIF-2 alpha protein kinase (PKR) expression in transfected mammalian cells.

The expression of a molecular cDNA clone (P1 KIN) of the human RNA-dependent P1/eIF-2 alpha protein kinase (PKR) was examined in transfected monkey cells and in cell-free protein-synthesizing systems. Expression of the wild-type (wt) P1 KIN cDNA, which encodes an active protein kinase, was compared with that of the phosphotransfer catalytic domain II Lys-296-->Arg (K296R) mutant cDNA, which does not encode an active kinase. wt and K296R mutant P1 mRNAs prepared by transcription in vitro with T7 RNA polymerase programmed the cell-free synthesis of P1 ribosome-associated protein with comparable efficiency in the rabbit reticulocyte system. The K296R mutant P1 protein was also efficiently synthesized in vivo in transfected COS monkey cells. However, synthesis of the wt P1 protein was reduced about 30-fold in transfected COS cells as compared with the K296R mutant P1 protein. Cotransfection of wt P1 KIN cDNA with either K296R mutant P1 KIN cDNA or reovirus S4 cDNA greatly reduced the synthesis of K296R mutant P1 protein and reovirus sigma 3 protein, respectively. Although the wt and K296R mutant P1 KIN plasmid expression vectors replicated with comparable efficiencies in COS cells, the steady-state amount of P1 mRNA was about 3-fold less in COS cells transfected with the wt as compared with the K296R mutant P1 KIN cDNA. These results suggest that RNA-dependent P1 protein kinase expression is autoregulated in vivo in transfected mammalian cells primarily at the level of translation by a mechanism that is likely dependent upon catalytically active P1 kinase.

2-Aminopurine↗
PubMed1992

Mechanism of interferon action. cDNA structure and regulation of a novel splice-site variant of the catalytic subunit of human protein kinase A from interferon-treated human cells.

A molecular cDNA clone (KIN27) was isolated that encodes a novel variant of the catalytic subunit C alpha of the human cyclic AMP-dependent protein kinase (PKA). Analysis of PKA genomic sequence data revealed that the KIN27 variant transcripts likely result from alternative splice-site selection of the C alpha gene transcripts. The KIN27 cDNA has therefore been designated C alpha 2. RNA amplification by polymerase chain reaction revealed that KIN27 C alpha 2 splice-site variant RNA and PKA C alpha RNA were both expressed in the amnion U and HeLa human cell lines. C alpha RNA was about 3- to 5-fold more abundant than C alpha 2 RNA. Interferon treatment decreased the steady-state amount of the C alpha 2 RNA relative to C alpha RNA. These results suggest that alternative splicing may contribute to the structural heterogeneity of C subunits expressed in human cells and that interferon may affect this process.

Amino Acid Sequence↗
PubMed1992

Biosynthesis of reovirus-specified polypeptides: ribosome pausing during the translation of reovirus S1 mRNA.

The steady-state distribution of translating ribosomes on the reovirus serotype 1 Lang strain polycistronic s1 mRNA and the monocistronic s4 mRNA was mapped using a sensitive primer extension assay and cDNA clones of the S1 and S4 genes. The distribution of translating ribosomes on the reovirus s1 and s4 mRNAs was not uniform. Major positions of ribosome pausing were detected in both rabbit reticulocyte and wheat germ cell-free protein synthesizing systems programmed with wild-type and site-directed mutant mRNAs. Two of the ribosome pause sites represent initiation and termination, rate-limiting steps of translation. For the polycistronic s1 mRNA, analysis of mutants in which either the sigma 1 ORF1 initiation codon or the sigma 1NS ORF2 initiation codon was eliminated by site-directed mutagenesis unequivocally established the identity of the specific ribosome pauses with specific ORF translational events. Ribosomes were far less uniformly distributed along the overlapping ORF regions of the polycistronic s1 mRNA than they were along the ORF of the monocistronic s4 mRNA. Furthermore, the rate-limiting initiation event at the sigma 1NS ORF2 AUG led to ribosome stacking and elongation arrest in the sigma 1 ORF1. These results begin to provide a conceptual framework for the dynamics of translation of complex as compared to simple viral mRNAs and suggest that ribosome activity may vary at multiple discrete regions on an mRNA.

Amino Acid Sequence↗
PubMed1992

Mechanism of interferon action: cDNA structure, expression, and regulation of the interferon-induced, RNA-dependent P1/eIF-2 alpha protein kinase from human cells.

A molecular cDNA clone (P1 KIN) was isolated that encodes the human RNA-dependent P1/eIF-2 alpha protein kinase. The complete cDNA sequence of the P1 KIN cDNA was determined; the longest open reading frame (ORF) encoded a 551 amino acid protein with a deduced molecular weight of 62055 Da. Transcripts prepared from the P1 KIN cDNA by transcription in vitro with T7 RNA polymerase programmed the cell-free synthesis of a protein indistinguishable by immunoprecipitation and immunoblot gel analyses from the authentic 67-kDa P1 protein synthesized in human U cells treated with interferon (IFN). Furthermore, by use of a sensitive primer extension assay with T7 DNA polymerase, the major site of translation initiation within the deduced ORF of the P1 KIN cDNA was directly identified. Northern RNA gel-blot analysis revealed that the P1 KIN cDNA strongly hybridized to two IFN-induced mRNAs present in both human amnion U cells and HeLa cells; their sizes were 2.5 and 6 kb. Both transcripts were efficiently induced by IFN-alpha, but poorly by IFN-gamma. Polyclonal antibody was prepared against the product of the P1 KIN cDNA expressed in Escherichia coli. In Western blot analysis the antibody recognized a 67-kDa protein induced in human cells by IFN-alpha and, in addition, a 90-kDa protein whose level was not greatly altered by IFN treatment. The IFN-induced 67-kDa protein was found associated with the ribosomal salt-wash fraction of IFN-treated human cells, whereas the 90-kDa protein was predominantly in the S100 soluble fraction. The time course for the induction by IFN-alpha of RNA-dependent protein P1 kinase activity measured by immunoprecipitation was comparable to the time course for protein P1 induction measured by Western immunoblot analysis. The amino acid sequence of P1/eIF-2 alpha protein kinase deduced from the cDNA was 62% identical with the 518-residue murine TIK kinase and contained, within the carboxy-terminal half of the protein, the motifs commonly conserved among protein-serine/threonine kinases. The amino-terminal half of the P1 protein did not possess conserved kinase motifs, but did show extensive homology with vaccinia virus-predicted protein E3L.

Amino Acid Sequence↗
PubMed1992

Mechanism of interferon action: identification of a RNA binding domain within the N-terminal region of the human RNA-dependent P1/eIF-2 alpha protein kinase.

A molecular cDNA clone of the human RNA-dependent P1/eIF-2 alpha protein kinase was expressed in Escherichia coli. Mutant P1 proteins were examined for RNA binding activity by Northwestern blot analysis using the reovirus s1 mRNA, an activator of the kinase; the adenovirus VAI RNA, an inhibitor of kinase activation; or human immunodeficiency virus (HIV) TAR RNA as probe. Analysis of TrpE-P1 deletion mutant fusion proteins revealed that the 11-kDa N-terminal region of the P1 protein bound reovirus s1 mRNA, adenovirus VAI RNA, and HIV TAR RNA. Neither s1 RNA, VAI RNA, nor TAR RNA was bound by truncated P1 proteins which lacked the N-terminal 98 amino acids. Computer analysis revealed that the human protein P1 sequence corresponding to amino acid residues within the N-terminal RNA binding domain displays high homology (greater than 54% identity; 61 to 94% similarity) with two animal virus proteins which possess RNA binding activity (vaccinia virus E3L; rotavirus VP2) and two proteins of unknown function (murine TIK; rotavirus NS34), but which are likely RNA binding proteins.

Amino Acid Sequence↗