Three cDNA fragments located within NS5 region of HCV were synthesized by RT using viral RNA extracted from blood sample of Korean patient as a template. The cDNAs were amplified by PCR, cloned into the T-vector, and the nucleotide sequences were determined. Comparative a analysis of the nucleotide and amino acid sequence of NS5 cDNAs showed that it is closely related with HCV type 1b. The cloned NS5 cDNA showed 91-94% homology at the nucleotide sequence level and 96-98% homology at the amino acid sequence level with several strains of the HCV type 1b. The NS5 cDNAs were subcloned into E. coli expression vectors to construct pRSETA5-1, pTHAN5-1, pRSETC5-2, pRSETBB1, pRESTCB1 and PRSETB-H3. Expression of the NS5 proteins was achieved by inducing the promoter with isopropyl-thio-P-D-galactoside (IPTG) and confirmed by SDS-polyacrylamide gel electrophoresis. The NS5 proteins were immunoreactive against sera from Korean hepatitis C patients in Western blot analysis. Among the recombinant NS5 proteins, pRSETA5-1 plasmid derived protein, coded from aa2022 to aa2521 of HCV polyprotein, showed the strongest immunoreactivity against sera from Korean hepatitis C patients in immunoblot analysis. These results suggest that NS5 proteins would be useful as an antigen for detection of antibody against HCV in the blood samples.
Amino Acid Sequence ; Base Sequence ; Blotting, Western ; Clone Cells* ; Cloning, Organism* ; DNA, Complementary ; Electrophoresis ; Hepacivirus* ; Hepatitis C* ; Hepatitis* ; Humans ; Plasmids ; Polymerase Chain Reaction ; RNA, Viral

PURPOSE: The incidence of salivary gland tumor is approximately 2% among all head and neck tumors, of which malignant cases account for only about 5%. Much research has been performed in order to clarify the mechanism of oncogene activation, however salivary gland tumors remain understudied. We performed this study in order to characterize the ras gene in these tumors. MATERIALS AND METHODS: We treated white rats with 7, 12-dimethylbenz[a]anthracene (DMBA) and confirmed the occurrence of salivary gland tumors after ten to thirty weeks. Isolated genomic DNAs from tumor tissues were added to NIH 3T3 cells. In order to detect Ha-ras mutations, we performed a two-step PCR-RFLP and 7analyzed the mutated sequences. RESULTS: We induced salivary gland tumors by DMBA treatment in white rats. Isolated DNAs from the tumor tissues transformed the NIH 3T3 cells. Point mutations were observed in codons 12 and 61 of the Ha-ras oncogene. The total frequency of point mutations was 13.9% in DMBA-induced salivary gland tumors in rats. CONCLUSION: Our results demonstrate that a variety of cancers ras oncogene mutations were also found in salivary gland tumors. We confirmed that a point mutation of the Ha-ras oncogene in a DMBA-induced salivary gland tumor occurs at a frequency of 13.9%.
9,10-Dimethyl-1,2-benzanthracene* ; Animals ; Codon ; DNA ; Genes, ras* ; Head ; Incidence ; Neck ; NIH 3T3 Cells ; Oncogenes ; Point Mutation ; Rats* ; Salivary Glands*

A new type of human calicivirus (HuCV) showing the classic cup-shaped surface morphology was identified in the stool sample from a child with symptoms of acute gastroenteritis in Seoul, Korea (SK virus). Genomic RNA was extracted directly from the stool sample, and the nucleotide sequence of 3.2 kb of the 3' end of SK virus was determined from cDNA. This region spanned sequences from the RNA-dependent RNA polymerase (RDRP) region in the open reading frame 1 (ORF1) to the 3' poly A tail. The non-structural and capsid protein coding sequences were fused in a single ORF as observed in Manchester type (Genogroup III). However, ORF2 of Manchester virus was missing in SK virus. In RDRP region, SK virus showed amino acid and nucleotide identities of 74-75% and 68-69% respectively, with those of Manchester virus, while showed 34-46% and 55-60% identities respectively with those of other human caliciviruses. However, capsid protein of SK virus showed a partial (29-46%) amino acid identity with those of other caliciviruses including Manchester type. The closest resemblance in amino acid (97-99%) and nucleotide sequence (85-86%) identities were found in RDRP region with Vanderbijlpark and Pretoria isolates recently found in South Africa. These results suggest that SK virus together with Vanderbijlpark and Pretoria isolates belong to a new type different from Manchester virus.
Amino Acid Sequence ; Base Sequence ; Calicivirus/ultrastructure ; Calicivirus/isolation & purification* ; Calicivirus/genetics* ; Child ; Cloning, Molecular ; DNA, Complementary/genetics ; DNA, Complementary/chemistry ; Feces/virology ; Genome, Viral ; Genotype ; Human ; Korea ; Microscopy, Electron ; Molecular Sequence Data ; Open Reading Frames ; RNA, Viral/isolation & purification ; RNA, Viral/genetics ; Sequence Alignment ; Sequence Analysis, DNA ; Sequence Homology, Amino Acid