1,721,050 research outputs found
GENOMIC STRUCTURE OF C-KI-RAS PROTOONCOGENE OF THE HERMAPHRODITIC FISH RIVULUS-MARMORATUS (TELEOSTEI, RIVULIDAE)
No full textThe ras homologue of the rivulid fish Rivulus marmoratus was isolated and characterized by screening about 3.0 x 10(5) genomic clones from a Rivulus genomic library using human c-Ha-ras probe. When this clone was partially sequenced focusing on the region coding exons, it showed 97.5% amino acid homology to the human c-Ki-ras gene. The Rivulus c-Ki-ras gene spans about 6.3 kb and consists of five exons including the alternative splicing exon 4a/4b. The exon-intron boundaries of Rivulus c-Ki-ras gene coincided with the GT/AG rule of consensus splice acceptor and donor sequences as in mammalian c-Ki-ras genes. Amino acid sequence analysis of some domain regions of the Rivulus c-Ki-ras gene revealed 100% identity to mammalian c-Ki-ras gene. This report is the first that elucidate the entire structure of c-Ki-ras in a fish
Hepatitis C virus NS5A protein is phosphorylated by casein kinase II
No full textHepatitis C virus (HCV) has a positive-strand RNA genome that encodes a polyprotein, which is posttranslationally processed by cellular and viral proteinases into three structural and six non-structural (NS) proteins. The NS5A protein, expressed in mammalian cells, exists as two phosphorylated forms of 56 kDa and 58 kDa. In this study, we provide evidence for a stable association between NS5A and a protein kinase from rat-1 cells by affinity to immobilized glutathione-S-transferase (GST)-NS5A fusion protein. This protein kinase was associated through the N-terminus of NS5A and was not regulated by cell cycle. The GST-NS5A was also phosphorylated in vitro by the purified casein kinase II (CKII), a member of the CMCG kinase family. Since CKII and the NS5A-associated protein kinase have the same molecular size and property by In-gel kinase assay and an inhibitor treatment test, we conclude that HCV NS5A protein is phosphorylated by CKII. (C) 1999 Academic Press
Organization, primary structure, and evolution of histone H2A and H2B genes of the fission yeast Schizosaccharomyces pombe
No full textThe two yeast histone H2A genes encode similar protein subtypes
No full textThe sequence of the two histone H2A genes in the yeast Saccharomyces cerevisiae have been determined. These genes encode two histone H2A subtypes which are 131 amino acids in length but differ at 2 amino acid positions: an Ala→Thr and a Thr →Ala change at positions 124 and 125. Thus, the two histone H2A subtypes have identical amino acid compositions. The coding regions of the two H2A genes are homologous at 369 of 393 bases (94%), with all but 2 of the 24 changes being silent. There is only 30% homology in the 5 flanking sequences of the two H2A genes. Like other eukaryotic histone genes, the yeast H2A genes are not interrupted by intervening sequences. When the yeast H2A histones are compared to those from other eukaryotes, there is at least 80% homology in amino acid sequence
The sen1(+) gene of Schizosaccharomyces pombe, a homologue of budding yeast SEN1, encodes an RNA and DNA helicase
No full textTwo polynucleotide-dependent ATPases, 95 and 181 kDa in size, have been purified to near homogeneity from cell-free extracts of Schizosaccharomyces pombe. Despite their size differences, their biochemical properties were strikingly similar. Both enzymes were capable of unwinding RNA and DNA duplexes in keeping with their ability to hydrolyze ATP in the presence of either ribo- or deoxyribopolynucleotide, In addition, they were capable of unwinding DNA/RNA or RNA/DNA hybrid duplexes and translocated in the 5' to 3' direction. These results strongly indicate that they are closely related to each other. Determination of the partial amino acid sequence of the 95-kDa enzyme revealed that it is encoded by the sen1(+) gene, an S. pombe homologue of yeast SEN1, a protein essential for the processing of small nucleolar RNA, transfer RNA, and ribosomal RNA. The molecular weight of the S. pombe Sen1 protein (SpSen1p) predicted from the sen1(+) open reading frame was 192.5 kDa, suggesting that the 181-kDa enzyme is likely to be a full-length protein, whereas the 95-kDa polypeptide has arisen by proteolysis. In accord with this possibility, polyclonal antibodies specific to the C-terminal region of sen1(+) cross-reacted with both 95- and 181-kDa polypeptides. We discuss the biochemical activities associated with SpSen1p and their relevance to the apparently divergent functions ascribed to the yeast Sen1 protein in RNA metabolism
Hepatitis C virus genotypes in Korea and their relationship to clinical outcome in type C chronic liver diseases.
No full textOBJECTIVES: The relationship between HCV genotype and the development of more serious liver disease has not been clearly established. This study was to investigate the distribution pattern of HCV genotypes in Korea and their relationship to the viremic level and to progression of chronic liver disease. METHODS: Study population was 217 patients with type C chronic liver disease. They were divided into 4 groups; 83 patients with near-normal ALT (group 1), 64 patients with elevated ALT (group 2), 20 patients with decompensated liver cirrhosis (group 3) and 50 patients with hepatocellular carcinoma (group 4). HCV genotypes were determined by reverse transcription polymerase chain reaction (RT-PCR) using mixed primer sets, and then the fidelity of genotyping was confirmed by cloning and sequencing. HCV RNA concentration was measured by quantitative competitive RT-PCR for 23 patients in group 2. RESULTS: The genotypes could be determined in 166 (76%) out of 217 patients. Type 1b and type 2a were predominantly occurring over the other types in somewhat similar frequency (45% and 51%, respectively). The genotype distribution of type 1b and 2a among four different groups showed 42% and 54% in group 1, 49% and 45% in group 2, 53% and 47% in group 3 and 41% and 57% in group 4; thus there was no significant difference in genotype distribution among 4 different disease groups. However, the viremia levels in patients with genotype 1b infection were significantly higher than those with genotype 2a. CONCLUSION: Genotype 2a infection is as prevalent as genotype 1b in Korea, and genotype 2a infection may pose no less risk for progression of disease despite lower replication level than genotype lb infection
The arginine-1493 residue in QRRGRTGR1493G motif IV of the hepatitis C virus NS3 helicase domain is essential for NS3 protein methylation by the protein arginine methyltransferase 1
No full textThe NS3 protein of hepatitis C virus (HCV) contains protease and RNA helicase activities, both of which are likely to be essential for HCV propagation. An arginine residue present in the arginine-glycine (RG)-rich region of many RNA-binding proteins is posttranslationally methylated by protein arginine methyltransferases (PRMTs). Amino acid sequence analysis revealed that the NS3 protein contains seven RG motifs, including two potential RG motifs in the 1486-QRRGRTGRG-1494 motif IV of the RNA helicase domain, in which arginines are potentially methylated by PRMTs. Indeed, we found that the full-length NS3 protein is arginine methylated in vivo. The full-length NS3 protein and the NS3 RNA helicase domain were methylated by a crude human cell extract. The purified PRMT1 methylated the full-length NS3 and the RNA helicase domain, but not the NS3 protease domain. The NS3 helicase bound specifically and comigrated with PRMT1 in vitro. Mutational analyses indicate that the Arg(1493) in the QRR(1488)GRTGR(1493)G region of the NS3 RNA helicase is essential for NS3 protein methylation and that Arg(1488) is likely methylated. NS3 protein methylation by the PRMT1 was decreased in the presence of homoribopolymers, suggesting that the arginine-rich motif IV is involved in RNA binding. The results suggest that an arginine residue(s) in QRXGRXGR motif IV conserved in the virus-encoded RNA helicases can be posttranslationally methylated by the PRMT1
Interaction of E1 and hSNF5 proteins stimulates replication of human papillomavirus DNA
No full textMammalian viruses often use components of the host's cellular DNA replication machinery to carry out replication of their genomes, which enables these viruses to be used as tools for characterizing factors that are involved in cellular DNA replication. The human papillomavirus (HPV) E1 protein is essential for replication of the virus DNA(1-3). Here we identify the cellular factor that participates in viral DNA replication by using a two-hybrid assay(4) in the yeast Saccharomyces cerevisiae and E1 protein as bait. Using this assay, we isolated Ini1/hSNF5 (ref. 5), a component of the SWI/SNF complex which facilitates transcription by altering the structure of chromatin(6). In vitro binding and immunoprecipitation confirmed that E1 interacts directly with Ini1/hSNF5. Transient DNA-replication assay revealed that HPV DNA replication is stimulated in a dose-dependent manner by addition of Ini1/hSNF5, and that Ini1/hSNF5 antisense RNA blocks the replication of HPV DNA. Amino-acid substitution at residues that are conserved among E1 proteins prevented the E1-Ini1/hSNF5 interaction and reduced DNA replication of HPV in vivo. Our results indicate that Ini1/hSNF5 is required for the efficient replication of papillomavirus DNA and is therefore needed, either alone or in complex with SWI/SNF complex, for mammalian DNA replication as well
Identification of a cell surface 30 kDa protein as a candidate receptor for Hantaan virus
No full textCellular receptors play an important role in virus pathogenesis. As a first step in virus infection, viruses attach to specific receptors on the surface of cells; Hantaan virus infects susceptible cells by attaching to a receptor located on the cell surface. To date, the identity of the Hantaan virus host cell receptor remains unknown. To determine the protein on the cell surface to which Hantaan virus binds, a virus overlay protein-binding assay was performed with radiolabelled virus. A 30 kDa (30K) protein was identified as a putative receptor for Hantaan virus. The specificity of virus interactions with this protein was demonstrated with a competition assay using unlabelled Hantaan virus and poliovirus. Unlabelled Hantaan virus inhibited the binding of radiolabelled Hantaan virus to this 30 kDa protein, whereas poliovirus did not. A polyclonal antibody against the 30K protein blocked binding of Hantaan virus to Vero-E6 cells and, consequently, virus infection. Blocking with the anti-30K antibody reduced virus infection of cells by 70%. These data strongly suggest that the 30 kDa surface protein is a putative receptor for Hantaan virus
Mutational analysis of the hepatitis C virus RNA helicase
No full textThe carboxyl-terminal three-fourths of the hepatitis C virus (HCV) NS3 protein has been shown to possess an RNA helicase activity, typical of members of the DEAD box family of RNA helicases. In addition, the NS3 protein contains four amino acid motifs conserved in DEAD box proteins. In order to inspect the roles of individual amino acid residues in the four conserved motifs (AXXXXGKS, DECH, TAT, and QRRGRTGR) of the NS3 protein, mutational analysis was used in this study. Thirteen mutant proteins were constructed, and their biochemical activities were examined. Lys1235 in the AXXXXGKS motif was important for basal nucleoside triphosphatase (NTPase) activity in the absence of polynucleotide cofactor. a serine in the X position of the DEXH motif disrupted the NTPase and RNA helicase activities. Alanine substitution at His1318 of the DEXH motif made the protein possess high NTPase activity. In addition, we now report inhibition of NTPase activity of NS3 by polynucleotide cofactor. Gln1486 was indispensable for the enzyme activity, and this residue represents a distinguishing feature between DEAD box and DEXH proteins. There are four Arg residues in the QRRGRTGR motif of the HCV NS3 protein, and the second, Arg1488, was important for RNA binding and enzyme activity, even though it is less well conserved than other Arg residues. Arg1490 and Arg1493 were essential for the enzymatic activity. As the various enzymatic activities were altered by mutation, the enzyme characteristics were also changed
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