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PubMed1994

Chronic liver disease and active hepatitis C virus infection in patients with antibodies to this virus.

AIMS: To assess the association between active hepatitis C virus (HCV) infection and liver damage in randomly selected patients with antibodies to the virus. METHODS: Thirty three consecutive subjects with serologically confirmed positivity for antibodies to HCV were studied for the presence of liver and circulating viral sequences by using the reverse transcription polymerase chain reaction (RT-PCR) and specific primers for the 5'-untranslated region (5'-UTR) of the HCV genome. Parallel clinical, biochemical, and histological investigations were carried out in all cases. RESULTS: A comparative virological and histological investigation showed the presence of molecular signs of active viral replication and different degrees of liver damage in all cases. Baseline values of liver and plasma samples from all the patients showed (with one exception) the presence of detectable HCV RNA sequences, despite alanine amino transferase activities being within normal values or within 1.5 times the upper limit of normal in 13 of them. Examination of percutaneous liver biopsy specimens showed the presence of confirmed liver damage (ranging from chronic persistent hepatitis to cirrhosis) in all 33 patients. CONCLUSIONS: Circulating HCV RNA sequences (a direct sign of active HCV infection) are associated with liver damage, even in the absence of clinical or biochemical signs of overt liver disease. Parallel molecular, histological, and clinical follow up of these patients is needed to understand precisely the natural history of HCV infection and for correct clinical management.

Base Sequence↗
PubMed1992

Two upstream elements activate transcription of a major histocompatibility complex class I gene in vitro.

Expression of major histocompatibility complex (MHC) class I genes exhibits unique tissue and developmental specificity. In an effort to study molecular mechanisms of MHC class I gene regulation, an in vitro transcription system has been established. In B cell nuclear extracts a template DNA containing the mouse H-2Ld promoter sequence accurately directed RNA polymerase II-dependent transcription of a G-free cassette. A conserved class I regulatory complex previously shown to moderately enhance promoter activity in vivo enhanced transcription in vitro by 2-3 fold. Much of this enhancement was accounted for by a 40 bp fragment within the complex, which was capable of activating a basal H-2Ld promoter in either orientation. Farther downstream, another element called site B was identified, which independently activated MHC class I transcription in vitro by 2-4 fold. Site B bound a specific nuclear factor(s) through an NF-1 binding site but not through a neighboring CCAAT site. The functional significance of site B in vivo was demonstrated in transfection experiments in which site B enhanced MHC class I promoter activity to a degree comparable to that seen in vitro. With the identification of the two upstream activators, MHC class I genes may serve as a model to study roles of sequence-specific DNA-binding proteins in transcription in vitro.

Animals↗
PubMed1992

Novel major archaebacterial group from marine plankton.

Marine bacteria often dominate the plankton biomass and are responsible for much of the cycling of organic matter, but bacterial diversity is poorly understood because conventional identification methods (requiring culturing) miss about 99% of the organisms. Recent advances permit characterization of microbial communities by analysis of 16S ribosomal RNA gene sequences directly from biomass without the need to culture the organisms; such studies from surface ocean samples have found only eubacteria, not archaebacteria (or Archaea), which are profoundly different. Here we report 16S rRNA sequences obtained from Pacific Ocean bacterioplankton samples collected from depths of 100 m and 500 m. Among these we found sequences only distantly related to those of any organisms previously characterized by 16S rRNA sequences, with similarities to the nearest such relatives (extreme thermophiles) approximately the same as those between animals and plants. We suggest that these sequences are from a previously undescribed archaebacterial group that may have diverged from the ancestors of characterized organisms very early in evolution.

Animals↗
PubMed1999

Involvement of cis-acting repressive element(s) in the 3'-untranslated region of human connective tissue growth factor gene.

To analyze the regulatory mechanism of connective tissue growth factor expression, the 3'-untranslated region (3'-UTR) of CTGF cDNA was amplified from HeLa cell RNA. Direct nucleotide sequencing revealed a single major population in the amplicon, which was nearly identical to other sequences. Subsequently, the effect of the 3'-UTR on gene expression was evaluated. When it was fused downstream of a firefly luciferase gene, the 3'-UTR strongly repressed luciferase gene expression. Interestingly, the repressive effect of the antisense 3'-UTR appeared to be more prominent than that of the sense one. Together with the fact that several consensus sequences for regulatory elements are found in it, these results suggest the involvement of multiple sets of regulatory elements in the CTGF 3'-UTR.

3' Untranslated Regions↗
PubMed1995

Multifocal vulvar intraepithelial neoplasia grade III and multicentric lower genital tract neoplasia is associated with transcriptionally active human papillomavirus.

BACKGROUND: The incidence of vulvar intraepithelial neoplasia Grade III (VIN III) is increasing and is diagnosed at a younger age than previously. VIN III is often multifocal and frequently coexists with multicentric dysplastic lesions in the cervix and vagina. Warty-type VIN III more often has been found to contain human papillomavirus (HPV) DNA than basaloid-type VIN III: The authors performed HPV DNA polymerase chain reaction (PCR) analysis in 48 VIN III biopsies and reverse transcriptase (RT)-PCR in 8 HPV-16 DNA-positive multifocal VIN III biopsies to detect E6/E7 transcripts. METHODS: Human papillomavirus DNA detection and histologic analysis were performed on alternating slides of paraffin embedded biopsies. Polymerase chain reaction was performed with consensus primers, and HPV typing was performed by direct sequencing. Total RNA was isolated from frozen biopsies by centrifuging a guanidinium thiocyanate (GTC) lysate through a cesium chloride (CsCl) cushion. The RT reaction was performed using a 3' primer, located just downstream of the E7 stop codon, and the PCR reaction was performed using the same 3' primer and a 5' primer located just downstream of the E6 start codon. RESULTS: The mean age of the 48 patients was 37.7 years. Eighty-one percent had multifocal VIN III: Sixty-six percent had multicentric neoplasia. Forty-six percent of the biopsies were warty-type, 17% basaloid-type, 35% mixed-type and 2% differentiated-type. Ninety-two percent were HPV-positive and 83% contained HPV-16 DNA. Human papillomavirus DNA was more often present in multifocal VIN III lesions than in unifocal VIN III lesions and also more often in VIN III lesions coexisting with other dysplastic multicentric lesions than in unicentric VIN III lesions. Warty-type VIN III more often contained koilocytes than basaloid-type VIN III: A correlation between different morphologic forms of VIN III and the presence of HPV DNA was not found. Both types of VIN III often coexist in one lesion. In all the RT-PCRs, a 593-base-pair fragment was detected, corresponding to the expected length of the major E6*-E7 mRNA. CONCLUSIONS: The observed high prevalence of transcriptionally active HPV DNA associated with multifocal and multicentric dysplasia suggests a role of HPV in the pathogenesis of these lesions. A positive correlation between different morphologic forms of VIN III and the presence of HPV DNA was not found.

Adult↗
PubMed2004

Absence of c-kit gene mutations in gastrointestinal stromal tumours from neurofibromatosis type 1 patients.

Most sporadic gastrointestinal stromal tumours (GISTs) have somatic c-kit gene mutations that are considered to be causal. Neurofibromatosis type 1 (NF1) is caused by mutations of the NF1 gene and NF1 patients have an increased risk of developing GISTs. Since most neoplasms are considered to develop as a result of the combination of several gene mutations, these findings suggest that GISTs from NF1 patients might have somatic c-kit gene mutations and that sporadic GISTs from non-NF1 patients might have somatic NF1 gene mutations. The present study analysed 29 GISTs from seven NF1 patients for c-kit gene mutations and ten sporadic GISTs from ten non-NF1 patients for NF1 mutations. Exons 9, 11, 13, and 17 of the c-kit gene were amplified and directly sequenced after the extraction of genomic DNA from wax-embedded tissues from 26 GISTs from five NF1 patients. The whole coding region of the c-kit cDNA and the whole coding region of the NF1 cDNA were amplified and directly sequenced after RNA extraction and cDNA synthesis in three fresh GIST tissues from two NF1 patients and ten fresh GIST tissues from ten non-NF1 patients. Of the ten sporadic GISTs, eight had heterozygous mutations at exon 11, and one at exon 9, of c-kit. Heterozygous NF1 gene mutations were detected in GISTs from the two NF1 patients from whom fresh tissues were available. None of the 29 GISTs derived from NF1 patients had detectable c-kit gene mutations and none of the ten GISTs derived from non-NF1 patients had detectable NF1 mutations. These results suggest that the pathogenesis of GISTs in NF1 patients is different from that in non-NF1 patients.

Base Sequence↗
PubMed2003

A patient in whom only hepatitis B virus (HBV) was thought to have been contracted, by kissing, from a same-sex partner coinfected with HBV and human immunodeficiency virus-1.

A 31-year-old homosexual man was admitted to our hospital in August 2001 with liver dysfunction. His diagnosis was acute hepatitis B. He had had a steady partner coinfected with hepatitis B virus (HBV) and human immunodeficiency virus-1 (HIV) from August 2000 to July 2001. Because he knew that the partner was infected with HIV, the sexual relationship had included only deep kissing, with no oral-genital or anal-genital sex. The serum HBV DNA level of the partner was relatively high compared with the serum HIV RNA level. Direct sequencing of the full HBV DNA genome from serum showed that the entire base sequences of the viruses from both patients were of genotype A and identical. HBV infectivity was shown to be stronger than that of HIV in our patient, in whom only HBV was transmitted from a partner coinfected with HBV and HIV. This case highlights the importance of HBV as a sexually transmitted disease.

Acute Disease↗
PubMed1999

A novel in-frame deletion mutation in a case of lactate dehydrogenase (LD) H subunit deficiency showing an atypical LD isoenzyme pattern in serum and erythrocytes.

OBJECTIVE: We report a case showing an atypical lactate dehydrogenase (LD) isoenzyme pattern involving deficiency only of LD-1 and LD-2 in serum and erythrocytes. LD activity in serum from this patient was extremely low, similar to complete LD-H deficiency, and also that in erythrocytes was low. DESIGN: The DNA fragment containing exon 1 through 7 of the LD-H gene were amplified by PCR and directly sequenced. Total RNA was prepared from venous blood and the proportion of LD-H cDNA to total LD cDNA was semiquantified. RESULTS: Genetic analysis by DNA sequencing detected a three base deletion (AAT) at codon 220 of exon 5, which caused a deletion of one asparagine. The present case did not show reduced LD-H expression at the mRNA level in whole blood. Residue 220 is involved in turning beta-J to alpha1-G and is not buried in the interior of the protein. The novel homozygous in-frame deletion mutation at codon 220 may cause a three-dimensional change of the subunit-binding domain.

Adult↗
PubMed1998

The EMBL nucleotide sequence database.

The EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl. html ) constitutes Europe's primary nucleotide sequence resource. DNA and RNA sequences are directly submitted from researchers and genome sequencing groups and collected from the scientific literature and patent applications (Fig. 1). In collaboration with DDBJ and GenBank the database is produced, maintained and distributed at the European Bioinformatics Institute. Database releases are produced quarterly and are distributed on CD-ROM. EBI's network services allow access to the most up-to-date data collection via Internet and World Wide Web interface, providing database searching and sequence similarity facilities plus access to a large number of additional databases.

Base Sequence↗
PubMed1999

The EMBL Nucleotide Sequence Database.

The EMBL Nucleotide Sequence Database (http://www.ebi.ac.uk/embl.html) constitutes Europe's primary nucleotide sequence resource. Main sources for DNA and RNA sequences are direct submissions from individual researchers, genome sequencing projects and patent applications. While automatic procedures allow incorporation of sequence data from large-scale genome sequencing centres and from the European Patent Office (EPO), the preferred submission tool for individual submitters is Webin (WWW). Through all stages, dataflow is monitored by EBI biologists communicating with the sequencing groups. In collaboration with DDBJ and GenBank the database is produced, maintained and distributed at the European Bioinformatics Institute (EBI). Database releases are produced quarterly and are distributed on CD-ROM. Network services allow access to the most up-to-date data collection via Internet and World Wide Web interface. EBI's Sequence Retrieval System (SRS) is a Network Browser for Databanks in Molecular Biology, integrating and linking the main nucleotide and protein databases, plus many specialised databases. For sequence similarity searching a variety of tools (e.g. Blitz, Fasta, Blast etc) are available for external users to compare their own sequences against the most currently available data in the EMBL Nucleotide Sequence Database and SWISS-PROT.

Amino Acid Sequence↗
PubMed2001

RNA replication kinetics, genetic polymorphism and selection in the case of the hepatitis C virus.

We show in a simple theoretical quasispecies model that the replication dynamics of hepatitis C virus and a related model-system, the bovine viral diarrhoea virus, result in an effective reduction of RNA templates in infected cells. Viral fitness does not translate directly into RNA sequence replication efficiency, and hence the abundance of the viral master sequences diminishes over time. Our results suggest that genes not involved in RNA replication accumulate mutations over time because they do not undergo selection during this phase. The selection of viral RNA occurs not only during replication but also during the ensuing stages of the viral life cycle: (i) envelopment of viral RNA and (ii) successful infection of other cells, which also requires functionality of non-replicative genes. In particular, viral fitness requires the ability of the genome to encode structural proteins which do not encounter selective pressure during RNA replication. We conclude by discussing the potential value of antiviral drugs which inhibit selection on parts of the viral genome.

Hepacivirus↗
PubMed2004

Identification of three F5 gene mutations associated with inherited coagulation factor V deficiency in two Chinese pedigrees.

To investigate the molecular defects in two Chinese pedigrees with inherited factor V (FV) deficiency. A 37-year-old male (proband 1) and an 18-month-old boy (proband 2) were diagnosed as inherited coagulation FV deficiency by severely reduced plasma levels of FV activity and antigen. All 25 exons and their flanking sequence of F5 gene were amplified by polymerase chain reaction (PCR) for both probands and the PCR products were directly sequenced. Total RNA was extracted from the peripheral lymphocytes of proband 1 for detecting the changes at mRNA level. The homozygous deletion IVS8 -2A>G was identified in the F5 gene of proband 1 and complementary DNA (cDNA) analysis revealed the abolishment of the canonical splicing site by the mutation and the activation of the cryptic acceptor site 24 bp upstream instead. The insertion introduced eight additional amino acids (AA) into the FV protein. Two heterozygous mutations of F5 gene were discovered in proband 2. The 2238-9del AG in exon 13 introduced a premature termination code at 689 AA and the substitution of G6410 by T in exon 23 lead to the missense mutation Gly2079Val. Three F5 gene mutations, IVS8 -2A>G, 2238-9del AG and G6410T, have been identified in two Chinese pedigree with congenital FV deficiency, respectively.

Adult↗
PubMed1984

Analysis of an influenza A virus mutant with a deletion in the NS segment.

The influenza virus host range mutant CR43-3, derived by recombination from the A/Alaska/6/77 and the cold-adapted and temperature-sensitive A/Ann Arbor/6/60 viruses, has previously been shown to possess a defect in the NS gene. To characterize this defect, nucleotide sequence data were obtained from cloned cDNAs. The CR43-3 NS gene was found to be 854 nucleotides long and to derive from the NS gene of the A/Alaska/6/77 parent virus by an internal deletion of 36 nucleotides. Direct sequencing of RNA 8 of CR43-3 virus confirmed that the deletion in the NS1-coding region was not an artifact that was generated during the cloning procedure. Protein analysis indicated that the NS1 protein of CR43-3 virus was synthesized in equal amounts in the restrictive (MDCK) cells as well as in the permissive (PCK) host cells. Also, indirect immunofluorescence studies of virus-infected cells showed that the NS1 protein of CR43-3 virus, like that of the parent viruses, accumulates in the nuclei of both cell systems. Although no differences in synthesis or localization of the NS1 protein could be detected, a consistent reduction in M1 protein was noted in CR43-3 virus-infected, nonpermissive cells as compared with that of the permissive host. Since analysis of the CR43-3 virus required us to obtain the NS nucleotide sequence of the 1977 isolate A/Alaska/6/77, we were able to compare this sequence with those of corresponding genes of earlier strains. The result of this analysis supports the idea of a common lineage of human influenza A viruses isolated over a 43-year period.

Amino Acid Sequence↗
PubMed2001

Frameshift signal transplantation and the unambiguous analysis of mutations in the yeast retrotransposon Ty1 Gag-Pol overlap region.

The yeast retrotransposon Ty1 encodes a 7-nucleotide RNA sequence that directs a programmed, +1 ribosomal frameshifting event required for Gag-Pol translation and retrotransposition. We report mutations that block frameshifting, which can be suppressed in cis by "transplanting" the frameshift signal to a position upstream of its native location. These "frameshift transplant" mutants transpose with only a modest decrease in efficiency, suggesting that the location of the frameshift signal in a functional Ty1 element may vary. The genomic architecture of Ty1 is such that Gag, Ty1 PR (PR), and the Gag-derived p4 peptide share a common sequence. The functional independence of the movement of the frameshift signal to a new location within the Ty1 element is used to unambiguously attribute the effect of mutations deleterious to transposition in this region of overlapping coding sequences to effects on the Ty1 (PR). This work defines the amino terminus of the Ty1 PR and introduces a new technique for studying viral genome organization.

Amino Acid Sequence↗
PubMed2005

Dominant optic atrophy caused by a novel OPA1 splice site mutation (IVS20+1G-->A) associated with intron retention.

Dominant optic atrophy (DOA) is the most common form of inherited primary optic neuropathy. The purpose of the current study was to report a novel OPA1 splice site mutation and investigate the impact of the mutation on pre-mRNA splicing in a female proband and her father diagnosed with DOA. We evaluated visual acuity, retinal fundi and kinetic visual fields. Color vision phenotypes were determined using the Farnsworth Panel D-15 and the Farnsworth-Munsell 100-hue tests. All 28 coding exons of the OPA1 gene were analyzed with polymerase chain reaction (PCR) amplification and direct sequencing. Total RNA extraction from white blood cells followed by reverse transcription-PCR (RT-PCR) was performed. We identified a novel heterozygous G to A mutation at position +1 of intron 20 (g.IVS20+1G-->A) in both patients. RT-PCR analysis revealed that the first 25 bp from intron 20 plus exon 20 were spliced onto exon 21. No difference in expression of mutant and wild-type transcripts was found within the linear range of amplification. Clinically, both patients exhibited reduced visual acuities, pallor of optic discs, decreased sensitivities of central visual fields and blue-yellow color vision defects. Previously, only one mechanism (skipping of exon) of pre-mRNA splicing defects has been reported among OPA1 splice site mutations. Our study demonstrates that the mechanism of intron retention is a novel type of pre-mRNA splicing defects. The mutant transcript with a premature termination codon is likely to encode a truncated protein, due to a translational frameshift (V672fsX675), that lacks 289 amino acids of the C-terminal end. Therefore, it is suggested that haploinsufficiency underlies DOA in the patients. However, we could not exclude the possibility that the truncated protein has a dominant negative activity because the mutant transcript is insusceptible to nonsense-mediated mRNA decay.

Adult↗
PubMed2005

Synthetic hammerhead ribozymes as therapeutic tools to control disease genes.

Ribozymes are RNA molecules that have the ability to catalyse the cleavage and formation of covalent bonds in RNA strands at specific sites. The "hammerhead" motif, approximately 30-nucleotide long, is the smallest endonucleolytic cis-acting ribozyme structure found in natural circular RNAs of some plant viroids. Hammerhead ribozymes became appealing when it was shown that it is possible to produce trans-acting ribozymes directed against RNA sequences of interest. Since then, gene-tailored ribozymes have been designed, produced and given to cells to knock down the expression of specific genes. At present, this technology has advanced so much that many hammerhead ribozymes are being used in clinical trials. With this work we would provide some guidelines to design efficient trans-acting hammerhead ribozymes as well as review the recent results obtained with them as gene therapy tools.

Animals↗
PubMed2000

Genotypic and phenotypic nevirapine resistance correlates with virological failure during salvage therapy including abacavir and nevirapine.

OBJECTIVE: To study the development of resistance during 8 weeks of salvage therapy with abacavir and nevirapine in combination with other reverse transcriptase inhibitors (RTIs) and protease inhibitors (PIs). METHODS: Samples obtained at baseline and after 8 weeks of therapy from 16 heavily pretreated patients were analysed for genotypic and phenotypic resistance. Genotypic resistance was analysed in cell-associated DNA and plasma HIV-RNA using direct sequencing. Phenotypic resistance was analysed in a PBMC-based assay and in a recombinant virus assay. Plasma viral load was measured at baseline and after 2, 4 and 8 weeks of therapy. RESULTS: The majority of patients was genotypically and phenotypically resistant to lamivudine, abacavir, zidovudine and PIs, whereas 50% of the patients showed resistance to nevirapine at baseline in at least one of the methods used. After 8 weeks of salvage therapy, no additional development of resistance against nucleoside reverse transcriptase inhibitors and PIs could be detected. However, the amount of patients resistant to nevirapine increased to 83%. When the patients were divided into two groups according to baseline resistance against nevirapine, a significantly higher transient reduction in viral load was observed in patients with nevirapine-sensitive HIV at baseline compared to patients with resistant HIV at baseline. CONCLUSION: The transient effect of salvage therapy including abacavir and nevirapine was due to the effect of nevirapine. The lack of effect of abacavir was most likely due to cross-resistance between abacavir and lamivudine/zidovudine used in previous treatment.

Anti-HIV Agents↗