The 3′ noncoding region (3′ NCR) of the hepatitis C virus (HCV) genome contained in viral particles was analyzed by an RNA linker ligation followed by reverse transcription-polymerase chain reaction. Sequence analysis of the amplified fragment from four strains, including different genotypes 1b, 2b, 3a, and 3b indicated that the 3′ NCR is composed of between 200 and 235 nts. The sequence of the 3′ NCR consists of a type-specific region (immediately following the termination codon), a poly(U) stretch, a C(U)n-repeat, and highly conserved region termed the core element. The poly(U) stretch and C(U)n-repeat regions varied in length and in sequence among different genotypes. Core elements having putative secondary structure consisted of 98 or 100 nts and were highly conserved in all genotypes. Most of the nt changes found in different genotypes did not affect the secondary structure of the core elements, suggesting that this region may play an important role in replication, stabilization of the HCV RNA, and/or packaging of the genome. Most of the HCV-1b strains carried two U residues at the 3′ end of the core element, while the minor HCV-1b strains had no U residues, demonstrating that there are two variants in type 1b strains. Amplification of the core element using linker-primed cDNA was comparable with that using the 3′ proximal core element-primed cDNA, indicating that the 3′ end of HCV genome was terminated by an OH group.

Aldehyde dehydrogenase with a low Michaelis constant (Km), ALDH2, is a major enzyme involved in the conversion of acetaldehyde, a toxic metabolite of ethanol, into acetic acid in the liver. Inherited deficiency of ALDH2 activity is found in half of Japanese, and is characterized by "Oriental flushing" after alcohol consumption. The aim of the present study is to evaluate the influence of the genetic polymorphism in alcohol metabolism on the sensitivity to the pressor effect of alcohol. Genotypes of ALDH2 were determined in 403 middle-aged Japanese men using genomic DNA extracted from white blood cells. Two hundred and forty-three (60%) of the subjects were shown to be homozygotes for the normal ALDH2 gene, 25 (6%) of the subjects were homozygotes for the mutant ALDH2 gene, and the remaining 135 (33%) were heterozygotes. None of the homozygotes for the mutant gene drank enough to show the pressor effect of alcohol. Elevations of blood pressure associated with increasing alcohol consumption or with elevations of serum gamma-glutamyl transpeptide (GTP) level were not different between the other two ALDH2-genotypes. It can be concluded that polymorphism in the ALDH2-genotype found in Japanese men does not affect the individual sensitivity to the pressor effect of alcohol.

Hepatitis C virus (HCV) type K3a (type 3a), which represents a minor genotype in Europe, the U.S.A. and Asia, appears to be significantly distributed throughout Australia and Brazil. We amplified the HCV-K3a/650 genome by reverse transcription polymerase chain reaction in ten overlapping fragments and determined the nucleotide sequences. The total sequence was 9454 bases in length and contained an open reading frame of 3021 amino acids, which is 10 or 11 amino acids longer than in HCV type 1 and 12 amino acids shorter than the sequence of type 2. These differences were due to the different lengths of both the putative envelope protein E2 and the NS5A regions, whose nucleotide lengths differ between types 1 and 2 also. Phylogenetic analysis of the putative core region and a portion of NS5B encoding the Gly-Asp-Asp motif indicated that HCV-K3a closely matched the corresponding type 3a group. The deletion and addition of amino acids in both E2 and NS5A may be associated with their pathobiological features.

Hepatitis C virus is a positive single-strand RNA virus distantly related to flaviviruses. Therefore RNA replicase, an RNA-dependent RNA polymerase, may be essential for the replication of hepatitis C virus, as well as other RNA viruses. In this study we synthesized the recombinant polypeptide (HCV-NS5 antigen) with a 576 bp cDNA encoding a part of the NS5 region of the HCV genome that has the Gly-Asp-Asp motif. The antibody against this polypeptide was obtained from rabbit serum. In Western-blot analysis with NS5 IgG HCV antibody, an 84-kD protein was clearly detected as a single band in the microsomal fraction but not in the nuclear and mitochondrial fractions or in the cytosol fraction. Immunohistochemically, HCV-NS5 antigen was clearly stained in the cytoplasm of hepatocytes but not in the nucleus or cell membrane. Moreover, as determined on immunoelectron microscopy, HCV-NS5 antigen was demonstrated with fine granular distribution along the endoplasmic reticulum but not in other organelles, including the nucleus and mitochondria. Immunoreaction in other cell types was negative. These results indicate that replication of HCV may occur only in hepatocytes and that HCV-NS5 may be produced in the endoplasmic reticulum of these cells. HCV-NS5 antigen was stained only in the livers of hepatitis C virus-positive patients but not in sections from patients with chronic type B hepatitis or alcoholic fibrosis. In chronic type C liver disease, the overall detection rate of HCV-NS5 antigen was 56% (33% in chronic persistent hepatitis, 52% in chronic active hepatitis and 86% in cirrhosis).(ABSTRACT TRUNCATED AT 250 WORDS)

We identified a new homozygous missense mutation His373 --> Leu in the CYP17 gene of two sisters with 17alpha-hydroxylase deficiency with an elevated plasma aldosterone concentration by sequencing their genomic DNAs amplified by polymerase chain reaction. Using polymerase chain reaction-based site-directed mutagenesis, we prepared a DNA that encoded the Leu373 mutant protein. COS-1 cells transfected with the mutant DNA, despite having an RNA hybridizable to the P450c17 cDNA, did not show 17alpha-hydroxylase and 17,20-lyase activities. Also, the cells were devoid of 11beta-hydroxylase and aldosterone synthase activities. To examine the mechanism by which the single amino acid change His373 --> Leu eliminates activity, we expressed N-terminally modified P450c17 proteins with and without the Leu373 Mutation in Escherichia coli and performed spectral studies. Membrane preparations from E. coli cells expressing the wild-type form of the modified enzyme showed an absorption peak at 449 nm upon addition of carbon monoxide in the reduced state and produced characteristic substrate-induced difference spectra, whereas those from the cells expressing the mutant form did not show these spectral changes. The 17alpha-hydroxylase and 17,20-lyase activities were observed only in E. coli cells expressing the wild-type enzyme. These results show that the His373 --> Leu mutant does not incorporate the heme prosthetic group properly and suggest a critical role of His373 in heme binding.

We recently classified the hepatitis C virus (HCV) into 4 types (HCV-PT, -K1, -K2a and -K2b) according to differences in nucleotide sequences. It was found that HCV-PT, the prototype reported from the U.S.A., was rare in Japan, suggesting that distribution of HCV genotypes may be different in various countries. The prevalence of HCV genotypes was therefore compared in different countries. Genotyping of HCV was performed by slot-blot hybridization analysis using cDNA probes specific to each type of HCV or by restriction fragment length polymorphism analysis. In 121 Japanese non-cancer patients, the prevalence of HCV genotypes was 77.7% for HCV-K1, 16.5% for HCV-K2a and 5.0% for HCV-K2b. HCV-PT was detected in only 1 patient (0.8%). The prevalence in 43 Japanese hepatocellular carcinoma (HCC) patients was 74.4% for HCV-K1, 18.6% for HCV-K2a and 4.7% for HCV-K2b. HCV-PT was found in only 1 sample. In 19 European non-cancer patients, HCV-PT was found in 42.1% and HCV-K1 was found in 52.6%. HCV-K2 was not found. All 7 samples from European HCC patients were HCV-K1, indicating a significantly higher prevalence than in non-cancer patients. In 13 Brazilian non-cancer patients, the distribution pattern was similar to that of the Europeans. In 10 samples from the U.S.A., HCV-PT was found in 70% and HCV-K2 was found in 1 sample. In 18 Chinese non-cancer patients, HCV-K1 was found in 44.4%, HCV-K2a in 50.0% and HCV-K2b in 5.6% HCV-PT was not found. Two samples from Chinese HCC patients were HCV-K1.(ABSTRACT TRUNCATED AT 250 WORDS)

Autophosphorylation of a DNA-activated protein kinase (DNA-PK) in Raji Burkitt's lymphoma cells generated a band that corresponded to a phosphoprotein of about 300 kDa on SDS/PAGE. This band corresponds to a 300-350-kDa DNA-PK found previously in HeLa cells. In addition to the 300-kDa phosphoprotein, the band of a highly phosphorylated 58-kDa protein was detected by SDS/PAGE of partially purified DNA-PK preparations after the phosphorylation reaction in the presence of double-stranded DNA. This phosphoprotein was specifically immunoprecipitated by phosphoprotein nor detectable activities of other kinases, phosphorylated recombinant c-Myc proteins in the presence of DNA. The c-Myc phosphorylation by DNA-PK was markedly stimulated by relaxed, double-stranded DNA, but neither by single-stranded DNA nor by RNA. Phosphopeptide mapping and phosphoamino acid analysis indicated that DNA-PK phosphorylates c-Myc in vitro at several serine residues.

1. Manual alignment of amino acid sequences of mammalian S-adenosylmethionine-dependent methyltransferases of known sequence revealed the presence of 2 homologous regions. 2. The sequence of the region at the C-terminal side is unique to mammalian methyltransferases, and in guanidinoacetate methyltransferase this sequence occurs at residues 159-165. 3. Mutagenesis of 5 conserved residues in this sequence did not affect the catalytic activity but altered tryptic susceptibility at Arg20.

Hepatitis C virus (HCV) genomes were recently detected in biological materials, and variations of nucleotide sequences were reported. In the present study, typing of the HCV genomes was performed in 91 HCV-RNA-positive patients and the clinical features of patients with different types of HCV were compared. From the nucleotide sequences of the cDNA fragments, HCV can be divided into at least two types: HCV-K1-PT and HCV-K2. All cDNAs amplified from 91 patients were hybridized with cDNA probes of either HCV-K1-PT or HCV-K2. HCV-K1-PT was found in about 80% of the patients, and HCV-K2 was found in about 20% of the patients. These results indicate that types of HCV are limited to two types, i.e., K1-PT and K2, and the major type is HCV-K1-PT, at least in Japan. Detection rate of antibodies to C-100-3 protein were not different between the patients having HCV-K1-PT and HCV-K2, indicating that the antibodies may develop in HCV-related patients without relation to the types of the HCV genomes. Prevalence of the two types of HCV were nearly the same in various forms of NANB-related liver disease. However, the prevalence was somewhat different in alcoholic liver disease. HCV-K2 was found in patients younger than the patients with HCV-K1-PT. Frequency of a history of blood transfusion tended to be lower and the initial response to interferon treatment was clearly better in patients having HCV-K2 versus patients having HCV-K1-PT. These results suggest the possibility that clinical features due to HCV-K1 may be somewhat different from those due to HCV-K1-PT. However, the number of patients examined was too small to allow a definite conclusion, indicating a necessity for further study with a larger number of patients.

Although human DNA polymerase beta (DNA pol beta) shows 96% identity with rat DNA pol beta at the amino acid level, it is weakly expressed in Escherichia (E.) coli relative to the rat enzyme. The mechanism of this suppression was investigated. Pulse-chase protein labeling and steady state mRNA analysis showed that mature human DNA pol beta protein is relatively stable in E. coli and the levels of human and rat DNA pol beta mRNA were comparable indicating that the human DNA pol beta expression is suppressed at the translational level. By systematic expression analysis of a number of chimeric genes composed of human and rat cDNAs, two strong translational suppression regions were mapped in the human DNA pol beta mRNA; one was named TSR-1, corresponding to CGG encoding arginine (arg) at position 4 and the other, termed TSR-2, is located between codons 153 and 199. Since substitution of the rat Arg-4 codon with synonymous codons showed strong effects upon the expression level, we propose that the arg codon at the N-terminal coding region plays a role in modulating expression.

The sequence Gly-Asp-Met-Asp, spanning positions 189-192 of rat DNA polymerase-beta, is similar to the sequence motif Gly-Asp-Thr-Asp that is highly conserved in a number of replicative DNA polymerases from eukaryotic cells, viruses, and phages. The role of this sequence in the catalytic function of rat DNA polymerase-beta was investigated by individually changing each amino acid in this region by site-directed mutagensis. The mutant enzymes DE190 and DE192, in which aspartic acid residues at positions 190 and 192, respectively, were replaced by glutamic acid, showed about 0.1% activity of the wild-type enzyme. On the other hand, the replacement of Gly-189 by alanine or Met-191 by isoleucine or threonine only slightly affected the enzyme activity. A gel mobility shift assay showed that DNA complexes with enzyme DE190 and especially with DE192 were less stable than the corresponding complex with the wild-type enzyme. Kinetic analysis with these mutant enzymes indicate that their K(m)'s for primer DNA were about 10-fold higher than that of the wild type, while K(m)'s for deoxyribonucleoside triphosphate were not changed. Since neither DE190 nor DE192 had any significant alteration in secondary structure, our results suggest that both Asp-190 and Asp-192 are located in the active site and are involved in the interaction of DNA polymerase-beta with primer.

Recently, we reported that hepatitis C virus (HCV) can be classified genetically into two types, HCV-K1 and HCV-K2, which show 67% and 71% identity at the nucleotide and amino acid sequence levels in a 340 bp region which encodes the NS5 gene Gly-Asp-Asp motif. To develop a rapid method to classify the genomes of HCV isolates, we identified restriction fragment length polymorphisms (RFLPs) in reverse transcriptase-polymerase chain reaction products encoding a portion of the NS5 gene. AluI and AccII enabled HCV to be classified into the K1 and K2 types, and Sau96I enabled classification into the K1 type, and the K2a and K2b subtypes. These RFLPs also generally allow Japanese isolates to be distinguished from the prototype (PT, an isolate from the U.S.A.), which is a K1 type. Sequence analysis of the 5'-untranslated regions of Japanese isolates revealed near identity between the K1 type and PT, and 93 to 94% identity between the K1 and K2 types, indicating that there are type K1- and K2-specific RFLPs in this region. Our results suggest that the nucleotide sequences of the K1 and K2 types are different throughout the HCV genome. The incidence of HCV types K1, K2a and K2b, and PT in 50 samples was 74%, 16%, 8% and 2%, respectively.

Three members of the myb gene family have been identified in human cDNA libraries c-myb, A-myb, and B-myb. We compared the DNA binding properties of the B-myb and c-myb proteins (B-MYB and c-MYB) using bacterially synthesized B-MYB and c-MYB in DNase I footprinting. B-MYB bound to most of the c-MYB binding sites examined, including the c-MYB binding site, MBS-I, in the simian virus (SV) 40 enhancer, in which the most frequent sequence was CCTAACTG. The MBS-I site was an enhancer element dependent on B-MYB and c-MYB in a co-transfection assay that used the B-myb or c-myb expression plasmid. Some sites in the SV40 genome, including the MBS-BI site, had high affinity with B-MYB but little or no affinity with c-MYB, in which the most frequent sequence was AGAAANPyrG. The MBS-BI site was an enhancer element dependent on B-MYB and a very weakly dependent on c-MYB. Our results showed that B-MYB is a transcriptional activator, like c-MYB, and that although B-MYB and c-MYB have similar sequence specificity for DNA binding some sequences were recognized by B-MYB preferentially.