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At least 1,369 records · Page 76Linked to original sources
PubMed1992

Agroinfection of transgenic plants leads to viable cauliflower mosaic virus by intermolecular recombination.

Intermolecular reconstitution of a plant virus has been detected in whole plants in a system using a defective cauliflower mosaic virus genome and transgenic host plants containing the missing viral gene. The information for the gene VI protein of the virus was integrated into the chromosome of host Brassica napus plants and leaves of these plants were inoculated with Agrobacterium tumefaciens containing the complementing viral sequences. In several cases, upper leaves contained replicating viral DNA which was able to incite CaMV symptoms on turnip plants. The sequence of the resultant recombinant viral molecules suggested that both DNA and RNA recombination events may have been involved in the production of functional virus, one event being gene targeting of the T-DNA.

Containment of Biohazards↗
PubMed1995

Resistance of Spiroplasma citri Lines to the Virus SVTS2 Is Associated with Integration of Viral DNA Sequences into Host Chromosomal and Extrachromosomal DNA.

Spiroplasmavirus SVTS2, isolated from Spiroplasma melliferum TS2, produces plaques when inoculated onto lawns of Spiroplasma citri M200H, a derivative of the type strain Maroc R8A2. S. citri strains MR2 and MR3, originally selected as colonies growing within plaques on a lawn of M200H inoculated with SVTS2, were resistant to SVTS2. Genomic DNA fingerprints and electrophoretic protein profiles of M200H, MR2, and MR3 were similar, but three proteins present in M200H were missing or significantly reduced in both resistant lines. None of these three polypeptides reacted with antiserum against S. citri membrane proteins, indicating that they probably are not surface-located virus receptors. Electroporation with SVTS2 DNA produced 1.5 x 10(sup5) transfectants per (mu)g of DNA in M200H but none in MR2 or MR3, suggesting that resistance may result from inhibition of viral replication. The digestion patterns of the extrachromosomal double-stranded (ds) DNA of these lines were similar. Three TaqI fragments of MR2 extrachromosomal DNA that were not present in M200H extrachromosomal DNA hybridized strongly to an SVTS2 probe, and two of these fragments plus an additional one hybridized with the MR3 extrachromosomal DNA, indicating that a fragment of SVTS2 DNA was present in the extrachromosomal ds DNA of MR2 and MR3 but not of M200H. When the restricted genomes of all three lines were probed with SVTS2 DNA, strong hybridization to two EcoRI fragments of chromosomal MR2 and MR3 DNA but not M200H DNA indicated that SVTS2 DNA had integrated into the genomes of MR2 and MR3 but not of M200H. When MR3 extrachromosomal ds DNA containing a 2.1-kb SVTS2 DNA fragment was transfected into M200H, the transformed spiroplasmas were resistant to SVTS2. These results suggest that SVTS2 DNA fragments, possibly integrated into the chromosomal or extrachromosomal DNA of a previously susceptible spiroplasma, may function as viral incompatibility elements, providing resistance to superinfection by SVTS2.

Journal Article↗
PubMed1995

A permanent glial precursor cell line, immortalized with the adenovirus E1A gene, undergoes apoptosis in restrictive growth conditions.

We have previously described some differentiation properties of the ERD.1.1 cell line, obtained after transfer and integration of the adenovirus-5 E1A gene. Depending on the growth conditions, these cells differentiate towards the astrocyte or early oligodendrocyte differentiation pathway. However, in growth restrictive conditions, we observed dying cells that detached from the monolayer constituted of differentiating cells. This led us to examine the characteristics of the dying cells. The study of the low-molecular-weight DNA and the ultrastructural examination of chromatin showed that these cells were undergoing apoptosis. The results also suggest that the expression of an integrated polyoma middle T gene can partly save the cells from apoptosis.

Adenovirus E1A Proteins↗
PubMed1996

Simplified plasmid rescue of host sequences adjacent to integrated proviruses.

We have previously described a Moloney murine leukemia retroviral (MoMLV) vector useful for the generation of anchored long-range maps of complex mammalian genomes. We now report the development of a modified vector carrying the ColE1 origin of replication and the chloramphenicol-resistance (CmR) gene to facilitate the recovery of genomic sequences adjacent to integrated proviruses. We demonstrate the utility of this new vector for the rescue in plasmid form of a 6-kb human fragment containing portions of an Alu element adjacent to the proviral 3'-LTR from an infected human primary fibroblast clone. We generated a sequence-tagged site (STS) from the derived sequence outside the Alu element, and used a somatic cell hybrid mapping panel to assign this STS to human chromosome 10 by a polymerase chain reaction-based assay. We suggest that this new CmR vector will be useful for recovering sequences of genes interrupted by proviral insertion, to generate directional maps with respect to the inserted provirus using the single cleavage sites within the vector, and to generate site-specific STS for defining and mapping the integration site.

Animals↗
PubMed1998

Targeted integration of a recombinant globin gene adeno-associated viral vector into human chromosome 19.

Transfer of a globin gene into stem cells along with the regulatory elements required to achieve high level expression in maturing erythroid cells would provide effective gene therapy for Cooley's Anemia. We have explored the use of recombinant adeno-associated viral (rAAV) vectors for this purpose. A vector designated rHS32A gamma*3'RE that contains regulatory elements from the locus control and flanking regions, integrates as a stable head-to-tail concatamer in erythroleukemia cells at a high multiplicity of infection and exhibits high level, regulated gamma globin gene expression. Inducible expression of the non-structural Rep proteins of wild-type AAV in HeLa cells transduced with rAAV vectors does not increase overall integration frequency, but targeted integration of rHS32A gamma*'3'RE into human chromosome 19 was documented.

Chromosome Mapping↗
PubMed1993

Evidence that retroviruses integrate into post-replication host DNA.

We have studied the question of whether a retrovirus integrates into the chromosomal DNA of the host cell before or after the DNA is replicated during the S phase of the cell cycle. We have infected single NIH-3T3 cells with BAG, a replication-incompetent retroviral vector which encodes the lacZ gene, then observed the clones derived from these cells to discover whether all the cells carry a copy of the proviral DNA. We have discovered that only half of the progeny of an infected cell carries a copy of the provirus. This indicates that the virus only integrates into post-replication DNA. We discuss the implications of this result for applications of retroviruses, such as gene therapy and cell lineage, which use them as vehicles for gene transfer into stem cells.

3T3 Cells↗
PubMed1981

Concentration of human respiratory syncytial virus using ammonium sulfate, polyethylene glycol or hollow fiber ultrafiltration.

Human respiratory syncytial virus was concentrated by polyethylene glycol or ammonium sulfate precipitation as well as by hollow ultrafiltration. Recoveries obtained were respectively 49.4%, 47.7%, and 75.2%; however, further analysis of these results by resuspension experiments showed that all the infectivity could be recovered from the different concentrates. The protein content of polyethylene glycol concentrates was much lower than those of ammonium sulfate or hollow fiber ultrafiltration. Electron microscopy revealed that the morphological integrity of virus particles was unaffected by the concentration methods used. Purified virus was obtained when polyethylene glycol concentrates were centrifuged through two successive density gradients.

Ammonium Sulfate↗
PubMed1995

Characterization of an open reading frame involved in site-specific integration of filamentous phage Cf1t from Xanthomonas campestris pv. citri.

Cf1t is a single-stranded DNA filamentous phage; a 1.9-kb segment of DNA from Cf1t was found to be responsible for site-specific integration into Xanthomonas campestris pv. citri (XW47), in the absence of any Xanthomonas origin of replication. Deletion analysis and introduction of amber stop codons into this fragment from Cf1t revealed an open reading frame (ORF344) which was involved in the integration function. The predicted amino-acid sequence of ORF344 bears no homology with conserved sequences of the integrase family.

Amino Acid Sequence↗
PubMed2003

Screening of CHO cell clones expressing histidine-tagged major S hepatitis B surface protein using a semi-quantitative PCR protocol.

Different mammalian cells have been used successfully for the expression of the hepatitis B surface antigen (HBsAg). The patterns of expression of the HBsAg seem to depend on the cell type and the number of gene copies integrated into cellular genome. The expression of an HBsAg fused to histidine tag (His-HBsAg), had not been reported in mammalian cells. This paper describes a semi-quantitative polymerase chain reaction (PCR) employed to investigate the patterns of expression of His-HBsAg in stably transfected Chinese hamster ovary (CHO) cell clones. pcDNA4CR20, a mammalian expressing vector, encoding His-HBsAg, was constructed. The correlation between His-HBsAg expression and the number of integrated copies of the HBsAg gene into cell clones genome was evaluated by the semi-quantitative PCR, with limit dilutions of the genomic DNA as template. The results show a positive correlation between the expression levels of His-HBsAg with the number of the HBsAg gene copies integrated into CHO cell clones. The approach of a semi-quantitative PCR proved to be a good and non-expensive alternative strategy to analyze the patterns of expression of integrated genes into CHO cells genome.

Animals↗
PubMed2000

Single active site catalysis of the successive phosphoryl transfer steps by DNA transposases: insights from phosphorothioate stereoselectivity.

The transposase family of proteins mediate DNA transposition or retroviral DNA integration via multistep phosphoryl transfer reactions. For Tn10 and phage Mu, a single active site of one transposase protomer catalyzes the successive transposition reaction steps. We examined phosphorothioate stereoselectivity at the scissile position for all four reaction steps catalyzed by the Tn10 transposase. The results suggest that the first three steps required for double-strand cutting at the transposon end proceed as a succession of pseudo-reverse reaction steps while the 3' end of the transposon remains bound to the same side of the active site. However, the mode of substrate binding to the active site changes for the cut transposon 3' end to target DNA strand joining. The phosphorothioate stereoselectivity of the corresponding steps of phage Mu transposition and HIV DNA integration matches that of Tn10 reaction, indicating a common mode of substrate-active site interactions for this class of DNA transposition reactions.

Bacteriophage mu↗
PubMed1995

Reversal of Mu gem2ts-induced mutations.

Mutations induced by the integration of a Mu gem2ts mutant prophage can revert at frequencies around 1 x 10(-6), more than 10(4)-fold higher than that obtained with Mu wild-type. Several aspects characterize Mu gem2ts precise excision: (i) the phage transposase is not involved; (ii) the RecA protein is not necessary; and (iii) revertants remain lysogenic with the prophage inserted elsewhere in the host genome. In addition, prophage re-integration seems to be non-randomly distributed, whereas Mu insertion into the host genome is a transposition event without any sequence specificity. In this paper, we describe that the site of re-integration somehow depends on the original site of insertion. Two alternative models are proposed to explain the strong correlation between donor and receptor sites.

Bacteriophage mu↗
PubMed2002

Integrated human papillomavirus type 16 is frequently found in cervical cancer precursors as demonstrated by a novel quantitative real-time PCR technique.

In contrast to cervical cancer, integration of human papillomavirus (HPV) DNA into the host genome has been considered a rare event in cancer precursor lesions (cervical intraepithelial neoplasia [CIN]). With our new real-time PCR method, we demonstrated that integrated HPV type 16 (HPV16) is already present in CIN lesions. The physical state of HPV16 and the viral load were simultaneously detected. A unique region of the E2 open reading frame (ORF) that is most often deleted during HPV16 integration is targeted by one set of PCR primers and a probe, and another set targets the E6 ORF. In episomal form, both targets should be equivalent, while in integrated form, the copy numbers of E2 would be less than those of E6. The method was tested with DNAs from 31 cervical lesions (non-CIN to CINIII) from 24 women prospectively followed up for 10 years. This report presents viral load and integration results from the largest series of CIN lesions described to date. Only one sample contained exclusively episomal HPV16 DNA, and this lesion regressed spontaneously. Samples from another patient, with only integrated HPV16, rapidly progressed from CINI to CINIII in 2 years. In all other patients, episomal and integrated forms of HPV16 DNA were found to coexist. Rapid progression of the CIN lesions was closely associated with a heavy load of integrated HPV16. Thus, the method described here is a very sensitive tool with which to assess the physical state of HPV, which is useful in predicting disease progression.

DNA, Viral↗
PubMed1996

Detection of integrated high risk human papillomavirus in adenoid cystic carcinoma of the uterine cervix.

AIM: To investigate the role of human papillomavirus (HPV) in adenoid cystic carcinoma of the uterine cervix. METHODS: Eleven archival, paraffin wax embedded specimens were analysed by non-isotopic in situ hybridisation (NISH) for HPV types 6, 11, 16, 18, 31, and 33 using digoxigenin labelled probes. The polymerase chain reaction (PCR) was carried out on each of the cases using consensus primers to HPV. RESULTS: A total of eight adenoid cystic carcinomas harboured the HPV genome by NISH, of which five were PCR positive. Integrated HPV 16 DNA was demonstrated in seven of the eight NISH positive cases. One adenoid cystic carcinoma showed integrated HPV 31. HPV DNA was not detected in the three remaining cases. CONCLUSIONS: Integrated high risk HPV genome, in particular type 16, is associated with this uncommon type of primary cervical cancer.

Aged↗
PubMedDate not supplied

[Cervical intraepithelial neoplasia (CIN) and human papillomavirus (HPV) infection].

Many studies have shown a strong correlation between CIN and HPV infection. Molecular biology has allowed identification of types of HPV which seem to be connected, more frequently than others, to dysplastic lesions. Physical state of HPV-genome seems to play an important role in the development of cervical cancer. In this study the HPV-genome has been searched in tissue specimens obtained from 34 women affected by CIN II and III. All patients underwent laser conization. Immediately before treatment, colposcopically directed biopsies of the cervical lesion and of the areas with no colposcopically apparent disease were taken and on these samples, HPV-DNA has been searched, isolated and analysed for HPV types and physical state. Histologic examination on cones showed 6 cases of CIN II (3 with HPV), 24 cases of CIN III (14 with HPV), 1 microinvasive carcinoma and 3 with no residual lesion. Southern blot analysis detected HPV-DNA in 4 cases of CIN II (16.7%) and in 20 cases of CIN III (70.6%). In 50% of CIN II and 85% of CIN III HPV 16 DNA has been found and in the remaining 50% of CIN II and 15% of CIN III HPV 31 DNA has been detected. All CIN II and 14 cases of CIN III showed episomal HPV-DNA. Integrated HPV-DNA has been found in 3 cases of CIN III and the other 3 cases of CIN III showed both integrated and episomal HPV-genome. Integrated form has been noticed only for HPV 16 type. In no case of colposcopically normal tissue has HPV-DNA been found. These data seem to confirm the strong correlation between HPV 16 type, which often has integrated form, and CIN III strengthening the hypothesis of its potential oncogenic action.

Adult↗
PubMedDate not supplied

HPRT mutations, TCR gene rearrangements, and HTLV-1 integration sites define in vivo T-cell clonal lineages.

HPRT mutations in vivo in human T-lymphocytes are useful probes for mechanistic investigations. Molecular analyses of isolated mutants reveal their underlying mutational changes as well as the T-cell receptor (TCR) gene rearrangements present in the cells in question. The latter provide temporal reference points for other perturbations in the in vivo clones as well as evidence of clonal relationships among mutant isolates. Immunological studies and investigations of genomic instability have benefited from such analyses. A method is presented describing a T-cell lineage analysis in a patient with HTLV-1 infection. Lineage reconstruction of an in vivo proliferating HPRT mutant clone allows timing of the integration event to a postthymic differentiated cell prior to the occurrence of HPRT mutations.

Cell Differentiation↗
PubMed1975

Molecular pathogenesis of systemic lupus erythematosus.

The study of patients with systemic lupus erythematosus revealed the close association of the disease with measles--or a related virus. High titres of antibodies to measles virus were found in patients that correlated with the course of the disease. Immunofluorescence tests revealed measles virus or a related antigen in lupus-affected tissues. Inclusion bodies consisting of paramyxovirus-like ribonucleoprotein structures were regularly detected in both affected tissues and leukocytes. Molecular hybridization of measles virus RNA with DNA from the affected tissues showed that DNA transcripts of measles or a closely related virus are integrated in the cellular nuclear DNA. Possible pathogenetic mechanisms of the disease are discussed.

Adolescent↗
PubMed1992

Differences in the integration pattern and episomal forms of human papillomavirus type 16 DNA found within an invasive cervical neoplasm and its metastasis.

Human papillomavirus (HPV) type 16 DNA was found in three separate neoplastic lesions within a female patient. The physical state of the viral DNA in each lesion was determined by two-dimensional agarose gel electrophoresis. The primary cervical tumor contained large amounts of several distinct episomal forms as well as integrated HPV DNA. Metastatic tumor tissue found in the vagina had greatly reduced levels of episomal DNA and a viral DNA integration pattern that was different from that of the primary tumor. The vulvar carcinoma in situ had what appears to be free and integrated forms of viral DNA. The results show that although metastatic tissue retained HPV DNA, further rearrangements of the integrated viral DNA pattern found in the primary tumor may occur with a dramatic decrease of episomal forms during malignant progression.

Carcinoma, Squamous Cell↗