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1.
Loss and acquisition of duck hepatitis B virus integrations in lineages of LMH-D2 chicken hepatoma cells. 总被引:5,自引:1,他引:4
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Hepatocellular carcinoma is the culmination of a series of genetic events which progressively alter the phenotype of a hepatocyte toward malignancy. Hepadnaviral DNA integrations are agents of genetic change which can promote the process of hepatocarcinogenesis. We previously characterized episomally derived duck hepatitis B virus (DHBV) integrations in LMH-D2 cells that replicate wild-type DHBV. In an effort to understand how integrations function as agents of progressive genetic change, we have studied integrations of DHBV DNA in three lineages of LMH-D2 cells through three generations of subclones. Our data have established several features of the integration process. First, single and multiple integrations occur continuously through successive cell generations. Second, the integration frequency can vary dramatically in subclones of the same cell line. Third, integrations can be lost from successive generations of cells and loss of an integration can be accompanied by loss of cellular DNA associated with the integration. Finally, certain subclones which acquire greater plating efficiency have been distinguished by unique new integration patterns. These results provide a basis for DHBV integrations to function as activators of protooncogenes, as well as agents of the loss of tumor suppressor genes during hepatocellular carcinogenesis. 相似文献
2.
Increase in the frequency of hepadnavirus DNA integrations by oxidative DNA damage and inhibition of DNA repair. 总被引:2,自引:1,他引:1
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Persistent hepadnavirus infection leads to oxidative stress and DNA damage through increased production of toxic oxygen radicals. In addition, hepadnaviral DNA integrations into chromosomal DNA can promote the process of hepatocarcinogenesis (M. Feitelson, Clin. Microbiol. Rev. 5:275-301, 1992). While previous studies have identified preferred integration sites in hepadnaviral genomes and suggested integration mechanisms (M. A. Buendia, Adv. Cancer Res. 59:167-226, 1992; C. E. Rogler, Curr. Top. Microbiol. Immunol. 168:103-141, 1991; C. Shih et al., J. Virol. 61:3491-3498, 1987), very little is known about the effects of agents which damage chromosomal DNA on the frequency of hepadnaviral DNA integrations. Using a recently developed subcloning approach to detect stable new integrations of duck hepatitis B virus (DHBV) (S. S. Gong, A. D. Jensen, and C. E. Rogler, J. Virol. 70:2000-2007, 1996), we tested the effects of increased chromosomal DNA damage induced by H2O2, or of the disturbance in DNA repair due to the inhibition of poly(ADP-ribose) polymerase (PARP), on the frequency of DHBV DNA integrations. Subclones of LMH-D21-6 cells, which replicate DHBV, were grown in the presence of various H2O2 concentrations and exhibited up to a threefold increase in viral DNA integration frequency in a dose-dependent manner. Moreover, inhibition of PARP, which plays a role in cellular responses to DNA breakage, by 3-aminobenzamide (3-AB) resulted in a sevenfold increase in the total number of new DHBV DNA integrations into host chromosomal DNA. Removal of either H2O2 or 3-AB from the culture medium in a subsequent cycle of subcloning was accompanied by a reversion back towards the original lower frequency of stable DHBV DNA integrations for LMH-D21-6 cells. These data support the hypothesis that DNA damage sites can serve as sites for hepadnaviral DNA integration, and that increasing the number of DNA damage sites dramatically increases viral integration frequency. 相似文献
3.
Topoisomerase I-mediated integration of hepadnavirus DNA in vitro. 总被引:14,自引:4,他引:10
Hepadnaviruses integrate in cellular DNA via an illegitimate recombination mechanism, and clonally propagated integrations are present in most hepatocellular carcinomas which arise in hepadnavirus carriers. Although integration is not specific for any viral or cellular sequence, highly preferred integration sites have been identified near the DR1 and DR2 sequences and in the cohesive overlap region of virion DNA. We have mapped a set of preferred topoisomerase I (Topo I) cleavage sites in the region of DR1 on plus-strand DNA and in the cohesive overlap near DR2 and have tested whether Topo I is capable of mediating illegitimate recombination of woodchuck hepatitis virus (WHV) DNA with cellular DNA by developing an in vitro assay for Topo I-mediated linking. Four in vitro-generated virus-cell hybrid molecules have been cloned, and sequence analysis demonstrated that Topo I can mediate both linkage of WHV DNA to 5'OH acceptor ends of heterologous DNA fragments and linkage of WHV DNA into internal sites of a linear double-stranded cellular DNA. The in vitro integrations occurred at preferred Topo I cleavage sites in WHV DNA adjacent to the DR1 and were nearly identical to a subset of integrations cloned from hepatocellular carcinomas. The end specificity and polarity of viral sequences in the integrations allows us to propose a prototype integration mechanism for both ends of a linearized hepadnavirus DNA molecule. 相似文献
4.
Selenium derivatization and crystallization of DNA and RNA oligonucleotides for X-ray crystallography using multiple anomalous dispersion 总被引:3,自引:1,他引:2
We report here the solid phase synthesis of RNA and DNA oligonucleotides containing the 2′-selenium functionality for X-ray crystallography using multiwavelength anomalous dispersion. We have synthesized the novel 2′-methylseleno cytidine phosphoramidite and improved the accessibility of the 2′-methylseleno uridine phosphoramidite for the synthesis of many selenium-derivatized DNAs and RNAs in large scales. The yields of coupling these Se-nucleoside phosphoramidites into DNA or RNA oligonucleotides were over 99% when 5-(benzylmercapto)-1H-tetrazole was used as the coupling reagent. The UV melting study of A-form dsDNAs indicated that the 2′-selenium derivatization had no effect on the stability of the duplexes with the 3′-endo sugar pucker. Thus, the stems of functional RNA molecules with the same 3′-endo sugar pucker appear to be the ideal sites for the selenium derivatization with 2′-Se-C and 2′-Se-U. Crystallization of the selenium-derivatized oligonucleotides is also reported here. The results demonstrate that this 2′-selenium functionality is suitable for RNA and A-form DNA derivatization in X-ray crystallography. 相似文献
5.
'Immunomers'--novel 3'-3'-linked CpG oligodeoxyribonucleotides as potent immunomodulatory agents
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Yu D Kandimalla ER Bhagat L Tang JY Cong Y Tang J Agrawal S 《Nucleic acids research》2002,30(20):4460-4469
Oligodeoxyribonucleotides containing CpG dinucleotides (CpG DNAs) are currently being evaluated as novel immunomodulators in clinical trials. Recently, we showed that an accessible 5′ end is required for immunostimulatory activity and blocking the 5′ end of CpG DNA by conjugation of certain ligands abrogates immunostimulatory activity. Based on these results, we designed and synthesized 3′–3′-linked CpG DNAs that contained two or more identical CpG DNA segments, referred to here as ‘immunomers’. The use of solid support bearing diDMT-glyceryl-linker permitted convenient synthesis of immunomers with both segments synthesized simultaneously, giving better yields and purity. The in vitro and in vivo studies suggest that as a result of accessibility to two 5′ ends for recognition, immunomers show an enhanced immunostimulatory activity compared with linear CpG DNAs. We also studied the suitability of a number of different linkers for attaching the two segments of immunomers. A C3-linker was found to be optimal for joining the two segments of immunomers. Incorporation of multiple linkers between the two segments of immunomers resulted in different cytokine profiles depending on the nature and number of linkers incorporated. Additionally, the length of immunomer also plays a significant role in inducing immune responses. An immunomer containing 11 nt in each segment showed the highest activity and an 11mer linear CpG DNA failed to stimulate an immune response. These results suggest that immunomers have several advantages over conventional linear CpG DNAs for immunomodulatory activity studies. 相似文献
6.
Differential Selection of Cells with Proviral c-myc and c-erbB Integrations after Avian Leukosis Virus Infection
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Min Gong Helen L. Semus Kelly J. Bird Brian J. Stramer Alanna Ruddell 《Journal of virology》1998,72(7):5517-5525
Avian leukosis virus (ALV) infection induces bursal lymphomas in chickens after proviral integration within the c-myc proto-oncogene and induces erythroblastosis after integration within the c-erbB proto-oncogene. A nested PCR assay was used to analyze the appearance of these integrations at an early stage of tumor induction after infection of embryos. Five to eight distinct proviral c-myc integration events were amplified from bursas of infected 35-day-old birds, in good agreement with the number of transformed bursal follicles arising with these integrations. Cells containing these integrations are remarkably common, with an estimated 1 in 350 bursal cells having proviral c-myc integrations. These integrations were clustered within the 3′ half of c-myc intron 1, in a pattern similar to that observed in bursal lymphomas. Bone marrow and spleen showed a similar number and pattern of integrations clustered within 3′ c-myc intron 1, indicating that this region is a common integration target whether or not that tissue undergoes tumor induction. While all tissues showed equivalent levels of viral infection, cells with c-myc integrations were much more abundant in the bursa than in other tissues, indicating that cells with proviral c-myc integrations are preferentially expanded within the bursal environment. Proviral integration within the c-erbB gene was also analyzed, to detect clustered c-erbB intron 14 integrations associated with erythroblastosis. Proviral c-erbB integrations were equally abundant in the bone marrow, spleen, and bursa. These integrations were randomly situated upstream of c-erbB exon 15, indicating that cells carrying 3′ intron 14 integrations must be selected during induction of erythroblastosis. 相似文献
7.
Rous sarcoma virus (RSV) integration in vivo: a CA dinucleotide is not required in U3, and RSV linear DNA does not autointegrate 总被引:1,自引:1,他引:0
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The sequences required for integration of retroviral DNA have been analyzed in vitro. However, the in vitro experiments do not agree on which sequences are required for integration: for example, whether or not the conserved CA dinucleotide in the 3′ end of the viral DNA is required for normal integration. At least a portion of the problem is due to differences in the experimental conditions used in the in vitro assays. To avoid the issue of what experimental conditions to use, we took an in vivo approach. We made mutations in the 5′ end of the U3 sequence of the Rous sarcoma virus (RSV)-derived vector RSVP(A)Z. We present evidence that, in RSV, the CA dinucleotide in the 5′ end of U3 is not essential for appropriate integration. This result differs from the results seen with mutations in the U5 end, where the CA appears to be essential for proper integration in vivo. In addition, based on the structure of circular viral DNAs smaller than the full-length viral genome, our results suggest that there is little, if any, integrase-mediated autointegration of RSV linear DNA in vivo. 相似文献
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A novel multiplex quantitative DNA array based PCR (MQDA-PCR) for quantification of transgenic maize in food and feed 总被引:12,自引:1,他引:11
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We have developed a novel multiplex quantitative DNA array based PCR method (MQDA-PCR). The MQDA-PCR is general and may be used in all areas of biological science where simultaneous quantification of multiple gene targets is desired. We used quantification of transgenic maize in food and feed as a model system to show the applicability of the method. The method is based on a two-step PCR. In the first few cycles bipartite primers containing a universal 5′ ‘HEAD’ region and a 3′ region specific to each genetically modified (GM) construct are employed. The unused primers are then degraded with a single-strand DNA-specific exonuclease. The second step of the PCR is run containing only primers consisting of the universal HEAD region. The removal of the primers is essential to create a competitive, and thus quantitative PCR. Oligo nucleotides hybridising to internal segments of the PCR products are then sequence specifically labelled in a cyclic linear signal amplification reaction. This is done both to increase the sensitivity and the specificity of the assay. Hybridisation of the labelled oligonucleotides to their complementary sequences in a DNA array enables multiplex detection. Quantitative information was obtained in the range 0.1–2% for the different GM constructs tested. Seventeen different food and feed samples were screened using a twelve-plex system for simultaneous detection of seven different GM maize events (Bt176, Bt11, Mon810, T25, GA21, CBH351 and DBT418). Ten samples were GM positive containing mainly mixtures of Mon810, Bt11 and Bt176 DNA. One sample contained appreciable amounts of GA21. An eight-plex MQDA-PCR system for detection of Mon810, Bt11 and Bt176 was evaluated by comparison with simplex 5′ nuclease PCRs. There were no significant differences in the quantifications using the two approaches. The samples could, by both methods, be quantified as containing >2%, between 1 and 2%, between 0.1 and 1%, or <0.1% in 43 out of 47 determinations. The described method is modular, and thus suited for future needs in GM detection. 相似文献
12.
The composition of the 3′terminal, 5′terminal and 5′penultimate nucleotides of the oligonucleotides released by spleen acid DNase and snail acid DNase from five `repetitive' DNAs (guinea pig, mouse and crab satellite DNAs, yeast mitochondrial DNA and poly (dAT:dAT)) and four `eukaryotic' DNAs (calf thymus, guinea pig and mouse liver DNAs, and yeast nuclear DNA) have been investigated and found to deviate in characteristic ways from those expected for bacterial DNAs having comparable base compositions. The deviation patterns obtained represent a novel way of characterizing and comparing different DNAs on the basis of the frequency of the nucleotide sequences they contain. 相似文献
13.
Reverse DNA oligonucleotide synthesis (i.e. from 5′→3′) is a strategy that has yet to be exploited fully. While utilized previously for the construction of alternating 3′-3′- and 5′-5′-linked antisense oligonucleotides, the use of nucleoside 5′-phosphoramidites has not generally been used for the elaboration of (modified) oligonucleotides. Presently, the potential of reverse oligonucleotide synthesis for the facile synthesis of 3′-modified DNAs is illustrated using a phosphoramidite derived from tyrosine. The derived oligonucleotide was shown to have chromatographic and electrophoretic properties identical with the modified oligonucleotide resulting from the proteinase K digestion of the vaccinia topoisomerase I–DNA covalent complex. The results confirm the nature of the structure previously assigned to this product, and establish the facility with which proteinase K is able to complete the digestion of the polypeptide backbone of the DNA oligonucleotide-linked topoisomerase I. 相似文献
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Pre-P Is a Secreted Glycoprotein Encoded as an N-Terminal Extension of the Duck Hepatitis B Virus Polymerase Gene
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Feng Cao Catherine A. Scougall Allison R. Jilbert John E. Tavis 《Journal of virology》2009,83(3):1368-1378
The duck hepatitis B virus (DHBV) pregenomic RNA is a bicistronic mRNA encoding the core and polymerase proteins. Thirteen AUGs (C2 to C14) and 10 stop codons (S1 to S10) are located between the C1 AUG for the core protein and the P1 AUG that initiates polymerase translation. We previously found that the translation of the DHBV polymerase is initiated by ribosomal shunting. Here, we assessed the biosynthetic events after shunting. Translation of the polymerase open reading frame was found to initiate at the C13, C14, and P1 AUGs. Initiation at the C13 AUG occurred through ribosomal shunting because translation from this codon was cap dependent but was insensitive to blocking ribosomal scanning internally in the message. C13 and C14 are in frame with P1, and translation from these upstream start codons led to the production of larger isoforms of P. We named these isoforms “pre-P” by analogy to the pre-C and pre-S regions of the core and surface antigen open reading frames. Pre-P was produced in DHBV16 and AusDHBV-infected duck liver and was predicted to exist in 80% of avian hepadnavirus strains. Pre-P was not encapsidated into DHBV core particles, and the viable strain DHBV3 cannot make pre-P, so it is not essential for viral replication. Surprisingly, we found that pre-P is an N-linked glycoprotein that is secreted into the medium of cultured cells. These data indicate that DHBV produces an additional protein that has not been previously reported. Identifying the role of pre-P may improve our understanding of the biology of DHBV infection. 相似文献
16.
Adam Mani Ido Braslavsky Rinat Arbel-Goren Joel Stavans 《Nucleic acids research》2010,38(6):2036-2043
Homologous recombination plays pivotal roles in DNA repair and in the generation of genetic diversity. To locate homologous target sequences at which strand exchange can occur within a timescale that a cell’s biology demands, a single-stranded DNA-recombinase complex must search among a large number of sequences on a genome by forming synapses with chromosomal segments of DNA. A key element in the search is the time it takes for the two sequences of DNA to be compared, i.e. the synapse lifetime. Here, we visualize for the first time fluorescently tagged individual synapses formed by RecA, a prokaryotic recombinase, and measure their lifetime as a function of synapse length and differences in sequence between the participating DNAs. Surprisingly, lifetimes can be ∼10 s long when the DNAs are fully heterologous, and much longer for partial homology, consistently with ensemble FRET measurements. Synapse lifetime increases rapidly as the length of a region of full homology at either the 3′- or 5′-ends of the invading single-stranded DNA increases above 30 bases. A few mismatches can reduce dramatically the lifetime of synapses formed with nearly homologous DNAs. These results suggest the need for facilitated homology search mechanisms to locate homology successfully within the timescales observed in vivo. 相似文献
17.
Prassolov A Hohenberg H Kalinina T Schneider C Cova L Krone O Frölich K Will H Sirma H 《Journal of virology》2003,77(3):1964-1976
All hepadnaviruses known so far have a very limited host range, restricted to their natural hosts and a few closely related species. This is thought to be due mainly to sequence divergence in the large envelope protein and species-specific differences in host components essential for virus propagation. Here we report an infection of cranes with a novel hepadnavirus, designated CHBV, that has an unexpectedly broad host range and is only distantly evolutionarily related to avihepadnaviruses of related hosts. Direct DNA sequencing of amplified CHBV DNA as well a sequencing of cloned viral genomes revealed that CHBV is most closely related to, although distinct from, Ross' goose hepatitis B virus (RGHBV) and slightly less closely related to duck hepatitis B virus (DHBV). Phylogenetically, cranes are very distant from geese and ducks and are most closely related to herons and storks. Naturally occurring hepadnaviruses in the last two species are highly divergent in sequence from RGHBV and DHBV and do not infect ducks or do so only marginally. In contrast, CHBV from crane sera and recombinant CHBV produced from LMH cells infected primary duck hepatocytes almost as efficiently as DHBV did. This is the first report of a rather broad host range of an avihepadnavirus. Our data imply either usage of similar or identical entry pathways and receptors by DHBV and CHBV, unusual host and virus adaptation mechanisms, or divergent evolution of the host genomes and cellular components required for virus propagation. 相似文献
18.
Therapeutic small interfering RNAs (siRNAs) have attracted a lot of interest both in basic biomedical sciences as well as in translational medicine. Apart from their therapeutic efficacy adverse effects of siRNAs must be addressed. The generation of stable mRNA cleavage fragments and the translation of N-truncated proteins induced by antisense oligodeoxynucleotides (ASOs) have been reported. Similar to ASOs, siRNAs are considered to function via an antisense mechanism that promotes the cleavage of the target mRNA. To further investigate whether the stable mRNA cleavage fragments also occur in siRNA we constructed a short hairpin RNA (shRNA) expression plasmid, pshRNA794, containing the same sequence reported in experiments using ASOs which directly targeted the overlapping region of the pre-genomic mRNA (pgmRNA) and sub-genomic mRNA (sgmRNA) of duck hepatitis B virus (DHBV). The shRNA resulted in a 70.9% and 69.9% reduction of the DHBV mRNAs in LMH and HuH-7 cells, respectively. In addition a 70% inhibition of the DHBV DNA level was observed. Interestingly, 3′-mRNA cleavage fragments were detected in LMH but not in HuH-7 cells. Taken together, our findings demonstrate that the ASO sequence was also effective in siRNA. Importantly, our results provide direct evidence that stable 3′-mRNA fragments were generated by siRNA in cells with high levels of DHBV replication. Whether these can cause adverse RNAi effects needs to be explored further. 相似文献
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A unique pattern of intrastrand anomalies in base composition of the DNA in hypotrichs 总被引:1,自引:0,他引:1
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The 50 non-coding bases immediately internal to the telomeric repeats in the two 5′ ends of macronuclear DNA molecules of a group of hypotrichous ciliates are anomalous in composition, consisting of 61% purines and 39% pyrimidines, A>T (ratio of 44:32), and G>C (ratio of 17:7). These ratio imbalances violate parity rule 2, according to which A should equal T and G should equal C within a DNA strand and therefore pyrimidines should equal purines. The purine-rich and base ratio imbalances are in marked contrast to the rest of the non-coding parts of the molecules, which have the theoretically expected purine content of 50%, with A = T and G = C. The ORFs contain an average of 52% purines as a result of bias in codon usage. The 50 bases that flank the 5′ ends of macronuclear sequences in micronuclear DNA (12 cases) consist of ~50% purines. Thus, the 50 bases in the 5′ ends of macronuclear sequences in micronuclear DNA are islands of purine richness in which A>T and G>C. These islands may serve as signals for the excision of macronuclear molecules during macronuclear development. We have found no published reports of coding or non-coding native DNA with such anomalous base composition. 相似文献