Analysis by multiplex PCR of the physical status of human papillomavirus type 16 DNA in cervical cancers.
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Integration of human papillomavirus (HPV) DNA occurs early in cancer development and is an important event in malignant transformation of cervical cancer. Integration of HPVs preferentially disrupts or deletes the E2 open reading frame, which results in the loss of its expression. The preferential disruption of the E2 gene causes the absence of the E2 gene sequences in the PCR product following integration. Twenty-two carcinomas positive for HPV type 16 (HPV-16) DNA were first tested for the disruption of the E2 gene by PCR. A specific fragment of the E2 gene was not amplified in 10 cases, suggesting integration of HPV DNA into the host genome. Next, multiplex PCR for the HPV E2 and E6 genes was carried out in the remaining 12 cases. Copy numbers of both genes should be equivalent in episomal forms, while the E2 gene copy number will be smaller than that for E6 following the preferential disruption of the E2 gene in concomitant forms. Although relative ratios of HPV E2 to E6 PCR products (E2/E6 ratios) ranged from 1.40 to 2.34 in 10 of 12 cases, multiplex PCR products from 2 cases displayed extremely low ratios of 0.69 and 0.61. Southern blot hybridization with an HPV-16 probe revealed that only in these two cases was both episomal and integrated HPV DNA being carried simultaneously. Thus, multiplex PCR for the E2 and E6 genes of HPV-16 DNA following PCR for the E2 gene can distinguish the pure episomal form from a mixed form of episomal and integrated HPV DNA. Clinical application of this technique will help researchers to understand the implication of the integration of HPV DNA for cervical carcinogenesis and cervical cancer progression. (+info)
Adult T-cell leukemia/lymphoma in which the pathohistological diagnosis was identical to that of Ki-1 positive anaplastic large cell lymphoma.
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A 65-year-old man developed severe lumbago and a loss of appetite two months before presentation. A computerized tomograph at admission revealed soft tissue masses destroying the Th12, L4 and L5 vertebral bones. We diagnosed the lesions to be metastatic bone tumors, but the primary focus could not be determined. Just after the irradiation treatment, abnormal lymphocytes were detected in the peripheral blood cells. Under the suspicion of adult T-cell leukemia/ lymphoma (ATL), we thus performed a lymph node biopsy. The specimens were histologically composed of Ki-1 positive anaplastic large cell lymphoma (ALCL). The lymphoma cells demonstrated a biclonal integration of HTLV-1 proviral DNA. After 6 cycles of chemotherapy, the patient has demonstrated a partial and favorable remission from ATL. (+info)
Adeno-associated virus (AAV) site-specific integration: formation of AAV-AAVS1 junctions in an in vitro system.
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An in vitro system to study the mechanism of site-specific integration of adeno-associated virus (AAV) was developed. This system is based on two substrates, a linear or circular AAV donor and a circular acceptor containing the preintegration locus AAVS1. In the presence of HeLa extract and the His-Tag-purified Rep68 protein, specific covalent junctions between AAV and AAVS1 were formed and detected by PCR. The majority of the junctions were located within the Rep binding site of both the AAV and the AAVS1 substrates, underlining the involvement of the Rep protein. A limited amount of replication and the presence of nuclear factors promoted the efficiency of the reaction. The process was ATP-dependent, indicating that the helicase activity of Rep may be important in the formation of the junctions. According to current models of integration, the formation of the junctions would represent a first step in the process of AAV integration. This step could be crucial for the site specificity of the recombination event that leads to the integration of AAV into human chromosome 19 in vivo. (+info)
Identification of a common site of provirus integration in radiation leukemia virus-induced T-cell lymphomas in mice.
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The BL/VL(3) Kaplan radiation leukemia virus (RadLV-VL(3)) is a nondefective retrovirus that induces T cell lymphomas in several strains of mice. By using DNA probes derived from RadLV/VL(3) provirus-flanking sequences cloned from the BL/VL(3) cell line, we identified a DNA region rearranged in 5 of 19 tumors analysed (25%). All proviruses were integrated in the same 5'-to-3' orientation in a small DNA region called Kis1 (Kaplan integration site 1). This region was localized on distal mouse chromosome 2 in a region not previously identified as important to lymphomagenesis. The cells rearranged at the Kis1 locus represent a clonal subpopulation of the clonal tumor masses examined, indicating a probable role of Kis1 in tumor progression. (+info)
Recurrent integration of human papillomaviruses 16, 45, and 67 near translocation breakpoints in new cervical cancer cell lines.
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Progressive chromosomal changes and integration of human papillomavirus (HPV) sequences mark the development of invasive cervical cancer. Chromosomal localization of HPV integration is essential to the study of genomic regions involved in HPV-induced pathogenesis. Yet, the available information about HPV integration loci is still limited, especially with respect to different HPV types. We have established cell lines from five cervical cancers with HPV-16, HPV-45, and HPV-67. We have determined HPV integration sites and karyotype abnormalities by using the multicolor combined binary ratio-fluorescence in situ hybridization method (Tanke et al.) with 24 chromosome-specific paints in combination with full-length HPV DNA probes. All cell lines were cytogenetically abnormal, and exhibited numerical and structural chromosomal deviations. HPV sequences were integrated at various (segments of) chromosomes. Duplicate integration sites were seen in all multiploid cell lines, suggesting that viral integration had preceded chromosomal endoreduplication. HPV-16 was found near the t(3p14.1-14.3;14) breakpoint in cervical squamous cell carcinoma (CSCC)-7 and mainly in episomal form in CSCC-1. HPV-45 was integrated near 3q26-29 in cervical (adeno or adenosquamous) carcinoma (CC)-8 and near 1q21-23 as well as near the t(1q21;22q13) breakpoint in CC-10A and CC-10B variant lines. HPV-67 was localized near the breakpoint of t(3p23-26;13q22-31) in CC-11. Southern blot analysis showed that, except for CSCC-1, the physical state of HPV in the cell lines was the same as in the original tumor lesions. This set of six cervical cancer cell lines included three lines with HPV-45, a major non-Western high-risk HPV type, the first reported HPV-67-positive cell line, and two cell lines with integrated and episomal HPV-16 DNA, respectively. The novel combined binary ratio-fluorescence in situ hybridization technique enabled us to simultaneously map chromosomal rearrangements and HPV integration sites, thereby revealing recurrent integration near translocation junctions for all of these HPV types in the cell lines from three of the five primary tumors. The detection of multiple HPV integration sites at rearranged chromosomes at such high frequency in cervical cancer-derived cells may reflect events that are relevant to the development of cervical cancer. (+info)
Integrated pararetroviral sequences define a unique class of dispersed repetitive DNA in plants.
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Although integration of viral DNA into host chromosomes occurs regularly in bacteria and animals, there are few reported cases in plants, and these involve insertion at only one or a few sites. Here, we report that pararetrovirus-like sequences have integrated repeatedly into tobacco chromosomes, attaining a copy number of approximately 10(3). Insertion apparently occurred by illegitimate recombination. From the sequences of 22 independent insertions recovered from a healthy plant, an 8-kilobase genome encoding a previously uncharacterized pararetrovirus that does not contain an integrase function could be assembled. Preferred boundaries of the viral inserts may correspond to recombinogenic gaps in open circular viral DNA. An unusual feature of the integrated viral sequences is a variable tandem repeat cluster, which might reflect defective genomes that preferentially recombine into plant DNA. The recurrent invasion of pararetroviral DNA into tobacco chromosomes demonstrates that viral sequences can contribute significantly to plant genome evolution. (+info)
TPW22, a lactococcal temperate phage with a site-specific integrase closely related to Streptococcus thermophilus phage integrases.
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The temperate phage TPW22, induced from Lactococcus lactis subsp. cremoris W22, and the evolutionarily interesting integrase of this phage were characterized. Phage TPW22 was propagated lytically on L. lactis subsp. cremoris 3107, which could also be lysogenized by site-specific integration. The attachment site (attP), 5'-TAAGGCGACGGTCG-3', of phage TPW22 was present on a 7.5-kb EcoRI fragment, a 3.4-kb EcoRI-HindIII fragment of which was sequenced. Sequence information revealed the presence of an integrase gene (int). The deduced amino acid sequence showed 42 and 28% identity with integrases of streptococcal and lactococcal phages, respectively. The identities with these integrase-encoding genes were 52 and 45%, respectively, at the nucleotide level. This could indicate horizontal gene transfer. A stable integration vector containing attP and int was constructed, and integration in L. lactis subsp. cremoris MG1363 was obtained. The existence of an exchangeable lactococcal phage integration module was suggested. The proposed module covers the phage attachment site, the integrase gene, and surrounding factor-independent terminator structures. The phages phiLC3, TP901-1, and TPW22 all have different versions of this module. Phylogenetically, the TPW22 Int links the phiLC3 lactococcal integrase with known Streptococcus thermophilus integrases. (+info)
Integration of hepadnavirus DNA in infected liver: evidence for a linear precursor.
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DNA of the avian hepadnavirus, duck hepatitis B virus, was found to be integrated at low abundance into the cellular DNA extracted from the livers of infected ducklings. The frequency of integration was estimated to be at least one viral genome per 10(3) to 10(4) cells by 6 days postinfection. The structures of virus-cell junctions determined by sequencing were compared with those of virus-virus junctions formed by nonhomologous recombination between the ends of linear viral DNA forms. This comparison allowed us to conclude that linear viral DNA was the preferential form used as an integration substrate. Potential factors promoting viral DNA integration during chronic infection are discussed. (+info)