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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Sequence-Independent, Single-Primer Amplification (SISPA) is a primer initiated technique that requires target sequence modification to achieve the non-selective logarithmic amplification of heterogeneous DNA populations. The method contrasts with the polymerase chain reaction (PCR), and its modified approaches, that have as their objective the amplification of unique or homologous sequences. SISPA requires the directional ligation of an asymmetric linker/primer oligonucleotide onto the target population of blunt ended DNA molecules. The common end sequence allows one strand of the double-stranded linker/primer to be used in repeated rounds of annealing, extension and denaturation in the presence of thermostable Taq DNA polymerase. The linker/primers contain restriction endonuclease sites to facilitate the molecular cloning of as little as 1 pg of starting material after amplification. SISPA is especially useful when the nucleotide sequence of the desired molecule is both unknown and present in limited amounts making its recovery by standard cloning procedures technically difficult. These conditions are present in the initial isolation and cloning of previously uncharacterized viral genomes. The application and quantitation of SISPA is described, together with its utility in the cloning and recovery of low abundance genetic sequences, as illustrated here with the hepatitis C virus.
Mol Cell Probes 1991 Dec
PMID:Sequence-independent, single-primer amplification (SISPA) of complex DNA populations. 166 49

The nucleotide sequences of cDNAs (275 base-pairs) in the non-structural protein 5 regions of Japanese isolates of hepatitis C virus (HCV-J) from the plasma of 11 patients with non-A, non-B hepatitis and the livers of five patients with hepatocellular carcinoma were analyzed. Approximately 14 to 17% of nucleotide sequences of the HCV-Js examined differed from that of the original isolate in the United States (HCV-US). Furthermore, 2.5 to 11% sequence diversity was found among the HCV-Js. The nucleotide sequences of the HCV-Js showed characteristic common differences from that of HCV-US, although they also showed some random substitutions. Plural HCV-J genomes were found in two of the cDNAs derived from liver specimens, and a deletion of 102 nucleotides was found in the cDNA derived from one plasma specimen. These results suggest that HCV-J is a strain different from the HCV-US and that mutation of the viral genome occurs at as high a frequency as in that of the human immunodeficiency virus.
Mol Biol Med 1990 Dec
PMID:Sequence diversity of hepatitis C viral genomes. 196 17

The behavior of hepatitis C virus (HCV) infection with regards to type and number of HCV genotypes (tested with genotype-specific nested polymerase chain reaction) was evaluated in 60 patients with anti-HCV-positive chronic active hepatitis without cirrhosis [17 untreated and 43 subjects undergoing single or repeat courses of interferon (IFN) therapy] during a mean follow-up period of 76 +/- 18 months. In untreated patients (2 genotype I, 6 genotype II, 9 mixed infections) 4 out of 9 mixed infections selected for genotype II at the end of follow-up. Of the 43 treated patients 10 were long-term responders with histological remission, 6 were short-term responders, and 22 did not respond. Fifteen of the latter patients received another course of IFN therapy, and only 3 patients responded. Eight of the 10 responders had infection with a single genotype (4 gt I, 3 gt II, 3 gt III). After IFN therapy, all but 2 patients cleared the HCV infection. The responders to the second IFN course (1 gt I, 1 gt II, 1 gt III) remained viremic. Of the short-term responders, 2/6 patients had genotype II and 4 had a mixed infection (3 gt II +/- I and 1 gt II +/- III); gt III became prevalent in the latter in all but one patient. Of the nonresponders 18/24 had more than one genotype, 5 were genotype II at baseline and one had genotype I. At the end of the follow-up period 15/18 with mixed infection had selected for gt II (P < 0.01 vs. untreated patient).(ABSTRACT TRUNCATED AT 250 WORDS)
J Mol Med (Berl) 1995 May
PMID:Selection of more pathogenic hepatitis C virus genotype II during long-term follow-up of interferon-treated patients. 754 26

The purpose of this study was to compare the serological analysis for hepatitis C infection using the recombinant immunoblot assay (RIBA) to the direct in situ localization of the hepatitis C viral genome using the reverse transcriptase (RT) in situ PCR technique. Concurrent sera and liver biopsies from 42 patients with clinical and histologic evidence of chronic liver disease were studied. Antibodies against hepatitis C specific antigens were demonstrated by RIBA in the sera of 39/42 (92%), and PCR amplified viral cDNA was detected in the biopsies of 21 (54%) of the 39 seropositive patients. The detection rate using standard in situ hybridization for the tissues known to be viral positive with RT in situ PCR was 9/21 (42%). It is concluded that approximately one-half of patients with chronic hepatitis and serologic evidence of hepatitis C infection will not have virus detectable in their liver biopsy even with a highly sensitive PCR-based technique.
Diagn Mol Pathol 1995 Jun
PMID:Comparison of serologic analysis and in situ localization of PCR-amplified cDNA for the diagnosis of hepatitis C infection. 755

Multicentre quality control studies demonstrated that optimization and standardization of HCV RNA reverse transcription polymerase chain reaction (RT-PCR) amplification techniques were possible: thus, a nested HCV RNA RT-PCR assay was described as a reliable tool for the detection of hepatitis C viral RNA. Besides this, another procedure, the Amplicor HCV RNA qualitative test, a standardized RT-PCR assay, became available. In order to assess the relationship between seropositivity and potential infectivity and to compare both RT-PCR assays, all patients undergoing maintenance hemodialysis were evaluated at hospital de Meaux (Meaux, France) during one year. We conclude that both assays are equally sensitive and that acid guanidinium thiocyanate based methods, which are used to prepare RNA prior to PCR, are more efficient than the usual phenol extraction protocols. Care should be taken with sera from hemodialyzed patients as the presence of inhibitors of the PCR has been demonstrated during the course of HCV RNA testing.
Cell Mol Biol (Noisy-le-grand) 1995 Jul
PMID:Detection of hepatitis C viral RNA: comparison of Amplicor single PCR and nested set PCR processes with HCV serology for hemodialyzed patients. 758 Aug 52

Translation extracts were prepared from various strains of Saccharomyces cerevisiae. The translation of mRNA molecules in these extracts were cooperatively enhanced by the presence of 5'-terminal cap structures and 3'-terminal poly(A) sequences. These cooperative effects could not be observed in other translation systems such as those prepared from rabbit reticulocytes, wheat germ, and human HeLa cells. Because the yeast translation system mimicked the effects of the cap structure and poly(A) tail on translational efficiency seen in vivo, this system was used to study cap-dependent and cap-independent translation of viral and cellular mRNA molecules. Both the 5' noncoding regions of hepatitis C virus and those of coxsackievirus B1 conferred cap-independent translation to a reporter coding region during translation in the yeast extracts; thus, the yeast translational apparatus is capable of initiating cap-independent translation. Although the translation of most yeast mRNAs was cap dependent, the unusually long 5' noncoding regions of mRNAs encoding cellular transcription factors TFIID and HAP4 were shown to mediate cap-independent translation in these extracts. Furthermore, both TFIID and HAP4 5' noncoding regions mediated translation of a second cistron when placed into the intercistronic spacer region of a dicistronic mRNA, indicating that these leader sequences can initiate translation by an internal ribosome binding mechanism in this in vitro translation system. This finding raises the possibility that an internal translation initiation mechanism exists in yeast cells for regulated translation of endogenous mRNAs.
Mol Cell Biol 1994 Nov
PMID:Cap-dependent and cap-independent translation by internal initiation of mRNAs in cell extracts prepared from Saccharomyces cerevisiae. 793 46

Hepatocellular carcinoma (HCC) is one of the most frequent malignancies in humans and in most cases a consequence of chronic infection of the liver by hepatotropic viruses (Hepatitis B Virus (HBV) and possibly Hepatitis C Virus (HCV)). Formation of HCC results from a stepwise process involving different preneoplastic lesions that reflect multiple genetic events, like protooncogene activation, tumor suppressor gene inactivation, and growth factor over- or reexpression. Recent investigations have gained new insights into how these factors are activated and may interact. In addition, improved knowledge of the molecular biology of HBV has led to better understanding of its pleiotropic effects on induction and progression in hepatocarcinogenesis.
Virchows Arch B Cell Pathol Incl Mol Pathol 1993
PMID:Current pathogenetic and molecular concepts in viral liver carcinogenesis. 809 24

From a blood serum of patients with chronic posttransfusional non-A, non-B hepatitis the genomic RNA of hepatitis C virus (HCV) was isolated. Using RT-PCR (reverse transcription-polymerase chain reaction) there were synthesized and cloned cDNA fragments, representing 3 regions of the genome of a new virus isolate (HCV-R): 5'-nontranslating region, a core gene and a part of the nonstructural region NS3/NS4. Analysis of the nucleotide and of the amino acid sequences of a core and NS3/NS4 regions revealed significant difference between isolates from Russia (HCV-R) and from Japan (HCV-J). Nucleotide sequence homology between them was 90.0-90.87%, while homology between Russian and American isolates (USA-PT) complised 95.27-97.32%. No essential variations were found in the nucleotide sequences of 5'-nontranslating region of all three HCV isolates.
Mol Gen Mikrobiol Virusol
PMID:[Determination of the nucleotide sequence of the Russian variant of the hepatitis C virus]. 813 50

Molecular evolutionary analyses were carried out to elucidate the phylogenetic relationships, the evolutionary rate, and the divergence times of hepatitis C viruses. Using the nucleotide sequences of the viruses isolated from various locations in the world, we constructed phylogenetic trees. The trees showed that strains isolated from a single location were not necessarily clustered as a group. This suggests that the viruses may be transferred with blood on a worldwide scale. We estimated the evolutionary rates at synonymous and nonsynonymous sites for all genes in the viral genome. We then found that the rate (1.35 x 10(-3) per site per year) at synonymous sites for the C gene was much smaller than those for the other genes (e.g., 6.29 x 10(-3) per site per year for the E gene). This indicates that a special type of functional constraint on synonymous substitutions may exist in the C gene. Because we found an open reading frame (ORF) with the C gene region, the possibility exists that synonymous substitutions for the C gene are constrained by the overlapping ORF whose reading frame is different from that of the C gene. Applying the evolutionary rates to the trees, we also suggest that major groups of hepatitis C viruses diverged from their common ancestor several hundred years ago.
J Mol Evol 1994 Jan
PMID:Reduction of synonymous substitutions in the core protein gene of hepatitis C virus. 815 15

Substitution rates were estimated for the coding and noncoding regions of the hepatitis delta virus (HDV). The estimated rates of synonymous substitution in HDV were lower than the rates of substitution at non-synonymous sites and in the noncoding region. HDV has lower synonymous substitution rates than the hepatitis C virus, though both are RNA viruses. The relatively low rate of synonymous substitution in HDV may be due to a strong preference of G and C nucleotides at third codon positions. Variation in substitution rate among HDV lineages may be correlated with the clinical development of the HDV-induced hepatitis. The phylogenetic tree inferred for 24 HDV strains reveals similarities between lineages isolated from the same geographic region.
J Mol Evol 1995 Dec
PMID:Substitution rates in hepatitis delta virus. 858 17


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