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At least 1,387 records · Page 77Linked to original sources
PubMed1995

Alterations in physical state and expression of human papillomavirus type 18 DNA following crisis and establishment of immortalized ectocervical cells.

Integration of episomal human papillomavirus (HPV) DNA in infected cervical lesions during malignant progression is frequently observed, but the importance of integration is poorly understood. We have studied immortalization by HPV-18 of human cervical cells as an in vitro model system. Here, the status and expression of HPV-18 DNA in precrisis ectocervical keratinocytes was compared with that in the same cells after crisis and establishment of immortalization. Southern blots revealed, and two-dimensional gel analysis confirmed, that the precrisis culture contained more than 100 copies/cell of episomal HPV-18 DNA and no detectable integrated viral DNA. In contrast, the postcrisis cells contained a low copy number of only integrated viral genome. The Northern blot patterns of E6-E7 and E2/E4 RNA expression were also different. Analysis of RNA by RT-PCR indicated that neither culture expressed the unspliced HPV-18 E6 oncogene present in tumor cell lines and that the precrisis, but not postcrisis, culture expressed the full-length E2 repressor. The two cultures displayed a similar keratinocyte morphology in vitro and a similar low grade dysplasia in vivo and both were non-tumorigenic. These results suggest that, although insufficient for complete malignant conversion, viral DNA integration during crisis is associated with the establishment of an immortalized phenotype in which HPV-18 DNA is integrated and HPV-18 RNA expression is altered.

Adult↗
PubMed2000

Site-specific integration of targeted DNA into animal cell genomes.

Novel genetically engineered retroviral vectors and targeting plasmids are described that enable the site-specific targeting of exogenous DNA into the genomes of cultured animal cells. The protocol involves the transduction of competent cells by a chimeric retroviral vector containing a transcription unit composed of two linked cassettes: an upstream marker gene under the control of the viral 5' LTR; and a downstream reporter trap containing a strong promoter 5' to a 48bp yeast FRT element. When cells containing such integrated units are co-transfected with a plasmid encoding yeast FLP recombinase and a promoterless targeting plasmid containing a reporter cDNA tract 3' to an homologous FRT element, the targeting plasmid recombines at the chromosomally preconfigured FRT site, and a new hemizygous function is introduced into the downstream cassette. These reagents provide a new portable system for site-specific targeting of chemically modified genes into uniform and unique sites in genomically integrated transcription units.

Animals↗
PubMed2001

Instability and site-specific excision of integration-proficient mycobacteriophage L5 plasmids: development of stably maintained integrative vectors.

Integrative vectors expressing foreign genes are used as tools for the development of recombinant vaccines in mycobacteria since it is assumed that these vectors are stably maintained even without antibiotic selection. We here demonstrate that integration-proficient vectors are lost from the mycobacterial genome in high frequency. Loss of integrated vectors occurred in recA+ and in recA-strains, indicating a RecA-independent mechanism. Loss of the integrated vector was prevented when integrase gene function was carried on a separate plasmid that is unable to replicate in mycobacteria, indicating that excision is a function of integrase. By providing attP in cis and integrase function in trans, vectors integrating at the attB site are stably maintained, even when carrying genes that deleteriously affect the host.

Attachment Sites, Microbiological↗
PubMed2002

Genome-wide comparison of differences in the integration sites of interspersed repeats between closely related genomes.

A technique for genome-wide detection of differences in the integration site positions of interspersed repeats in related genomes (DiffIR) is described. The technique is based on a whole- genome selective PCR amplification of the repeats' flanking regions followed by a differential hybridization screening of the arrayed library of the selected amplicons. The technique was successfully applied to the comparison of the integration sites in the human and chimpanzee genomes, allowing us to discover 11 new human-specific integrations of human endogenous retrovirus, K family (HML-2) long terminal repeats.

Animals↗
PubMed1995

Requirement of RNA polymerase III transcription factors for in vitro position-specific integration of a retroviruslike element.

The yeast retroviruslike element Ty3 inserts at the transcription initiation sites of genes transcribed by RNA polymerase III (Pol III). An in vitro integration assay was developed with the use of Ty3 viruslike particles and a modified SUP2 tyrosine transfer RNA (tRNA(Tyr)) gene target. Integration was position-specific and required Ty3 integrase, Pol III transcription factor (TF) IIIB-, TFIIIC-, and Pol III-containing fractions showed that TFIIIB and TFIIIC, together, were sufficient for position-specific Ty3 integration, but not for transcription. This report demonstrates that in vitro integration of a retroelement can be targeted by cellular proteins.

DNA Nucleotidyltransferases↗
PubMed1995

Evi-5, a common site of retroviral integration in AKXD T-cell lymphomas, maps near Gfi-1 on mouse chromosome 5.

We have identified a novel common site of retroviral integration, Evi-5, in AKXD T-cell lymphomas. All proviruses located at Evi-5 are clustered within a 7-kb genomic region and, where determined, are oriented in the same transcriptional direction. Interspecific backcross analysis localized Evi-5 to mouse chromosome 5, where it cosegregated with another common viral integration site, Gfi-1. Gfi-1 encodes a novel zinc finger transcription factor whose expression is thought to be important for interleukin-2 signaling. Physical mapping studies showed that Evi-5 is located approximately 18 kb upstream of Gfi-1, and Southern analysis showed that Gfi-1, like Evi-5, is a common integration site in AKXD T-cell tumors. With one exception, Evi-5 and Gfi-1 integrations were mutually exclusive. Ten of the tumors with Evi-5 or Gfi-1 integrations also harbored viral integrations at other common integration sites causally associated with T-cell disease. These results are consistent with the hypothesis that T-cell lymphomagenesis is a multistep disease and that viral integration at Evi-5 or Gfi-1 is causally associated with this disease process.

Animals↗
PubMed1979

Reversible inhibition of Newcastle disease virus (NDV) haemolysis by bivalent cations.

The capacity of paramyxoviruses for haemolysis (hl) is enhanced after treatment with physical agents and complement (C'). Bivalent cations in the medium inhibit HL. The inhibition is proportional to the molar concentrations, and is graded Ba++ greater than Ca++ greater than Mg++. Substitution of the bivalent cations with K+ and re-incubation leads to reappearance of HL, but in reverse order. It is postulated that bivalent cations inhibit HL mainly by stabilising the virus membrane integrated into the erythrocyte membrane and slowing down the permeability rates. This inhibition is removed on substitution with monovalent cations. The bivalent cations also reversibly inhibit the permeability of the transferred C' lesion.

Barium↗
PubMed1979

Cellular regulation of mammalian sarcoma virus expression: a gene regulation model for oncogenesis.

Investigations aimed at defining cellular functions required for expression of transformation by mammalian sarcoma viruses have led to the isolation of a class of revertants that contain biologically active feline sarcoma virus, yet possess in vitro and in vivo properties of normal cells. The block to expression of the transformed state in these cellular revertants was spontaneously reversible at low frequency. Moreover, infection with certain helper viruses reversed the block at very high efficiency. Helper virus complementation was shown not to be a direct effect of helper virus functions expressed in the initially infected revertant cell. Rather, the helper virus acted indirectly by rescuing sarcoma virus and allowing it to infect and transform another cell within the revertant population. Using biochemical and immunologic techniques, it was possible to demonstrate a specific and very marked reduction in transcriptional and translational products of the sarcoma viral genome in the revertant cells. Findings that the reversal of this block was associated with reacquisition of the transformed phenotype, together with other evidence, suggest that reversion results from cellular transcriptional regulation of the integrated sarcoma virus genome. Reversion in this virus transformation system provides a model for oncogenesis resulting from derepression of cellular genes that possess malignant potential.

Animals↗
PubMed2000

[Human papillomaviruses, cell cycle and cervical cancer].

Human papillomaviruses (HPVs) are associated with a broad spectrum of cutaneous and mucosal lesions. Until now, more than 120 genotypes have been identified. Most HPVs are associated with benign lesions. Nevertheless certain HPV types are frequently found in carcinomas. For instance, HPV 16 and 18 which are frequently associated with cervical cancer, are capable of immortalizing and transforming primary keratinocytes. The mechanism of transformation is linked to the viral genome integration into the cell's DNA, accompanied by an overexpression of the E6 and E7 genes. The viral gene products interact with cellular proteins that regulate the cycle progression. In particular, the E6 protein binds to the p53 and the E7 protein binds to the p105(Rb). The inactivation of both cellular proteins distorts the cell cycle and results in genetic instability and cellular gene alterations. This article reviews the role of the viruses in the carcinogenesis, the genome structure and the gene expression of HPVs. It also addresses the cell cycle regulation with a focus on the role of HPVs in cell transformation.

Cell Cycle↗
PubMed2000

Acetylation of HIV-1 Tat by CBP/P300 increases transcription of integrated HIV-1 genome and enhances binding to core histones.

The HIV-1 Tat protein is required for viral replication and is a potent stimulator of viral transcription. Although Tat has been extensively studied in various reductive paradigms, to date there is little information as to how this activator mediates transcription from natural nucleosomally packaged long terminal repeats. Here we show that CREB-binding protein (CBP)/p300 interacts with the HIV-1 Tat protein and serves as a coactivator of Tat-dependent HIV-1 gene expression on an integrated HIV-1 provirus. The site of acetylation of Tat was mapped to the double-lysine motif in a highly conserved region, (49)RKKRRQ(54), of the basic RNA-binding motif of Tat. Using HLM1 cells (HIV-1(+)/Tat(-)), which contain a single copy of full-length HIV-1 provirus with a triple termination codon at the first AUG of the Tat gene, we find that only wild type, and not K50A, K51A, or K50A/K51A alone or in combination of ectopic CBP/p300, is able to produce full-length infectious virions, as measured by p24 gag ELISAs. Tat binds CBP/p300 in the minimal histone acetyltransferase domain (1253-1710) and the binding is stable up to 0.85 M salt wash conditions. Interestingly, wild-type peptide 41-54, and not other Tat peptides, changes the conformation of the CBP/p300 such that it can acquire and bind better to basal factors such as TBP and TFIIB, indicating that Tat may influence the transcription machinery by helping CBP/p300 to recruit new partners into the transcription machinery. Finally, using biotinylated wild-type or acetylated peptides, we find that acetylation decreases Tat's ability to bind the TAR RNA element, as well as to bind basal factors such as TBP, CBP, Core-Pol II, or cyclin T. However, the acetylated Tat peptide is able to bind to core histones on a nucleosome assembled HIV-1 proviral DNA.

Acetylation↗
PubMed2002

A novel, helper-dependent, adenovirus-retrovirus hybrid vector: stable transduction by a two-stage mechanism.

We have developed a novel vector system that uses a helper-dependent adenoviral vector as a carrier to deliver a fully functional retrovirus vector. The helper-dependent adenovirus (HDAd) can accommodate large inserts, provide high titers, and infect nondividing as well as dividing cells. However, adenoviral DNA is rarely integrated into the host cell genome, and its episomal expression is transient. Therefore we inserted a replication-competent, ecotropic retrovirus vector containing the green fluorescent protein (GFP) reporter gene as a second-stage component. The well-characterized host species tropism of each vector component provided a stringent biological assay system that demonstrates the two-stage transduction mechanism of the hybrid vector, because the adenovirus stage can efficiently transduce human cells but cannot replicate in murine cells, and conversely, the ecotropic retrovirus stage cannot enter human cells but can efficiently proliferate in murine cells, resulting in permanent integration and progressive spread of reporter gene expression.

Adenoviridae↗
PubMed2003

Efficient switching of mycobacteriophage L5-based integrating plasmids in Mycobacterium tuberculosis.

We previously used a mycobacteriophage L5-derived integrating vector to demonstrate that glnE and aroK are essential genes in Mycobacterium tuberculosis by showing that we were unable to excise the integrated vector when it carried the only functional copy of these genes. We tested three systems to replace the integrated copy with alternative alleles. The most efficient method was to transform the strain with a second copy of the integrating vector. Excision of the resident vector and integration of the incoming vector occurred at an extremely high efficiency. This technique will allow us to study the role and functionality of essential genes in this important human pathogen.

Alleles↗
PubMed1999

Localization of HTLV-1 and HIV-1 proviral sequences in chromosomes of persistently infected cells.

Integration sites for HTLV-1 and HIV-1proviruses were detected by FISH on the chromosomes of HTHIV27 cells persistently infected by HIV-1 (strain IIIB). HTLV-1 signals were found on 9 loci of chromosomes 4, 6, 9, 15 and 16. Integration sites of GC-rich HTLV-1 provirus are located in GC-rich isochores, confirming an 'isopycnic' integration, namely an integration in which the GC level of the host sequences around the integration site match the GC level of the provirus. This conclusion is not only derived from the compositional map of human chromosomes, but also from HTLV-1 hybridization on compositional fractions of human DNA. Integration of GC-poor HIV-1 provirus was found on 4 loci of chromosomes 2, 7, 17 and 19. One copy of a complete HIV-1 provirus, which is active, was integrated in H1 isochores, whereas other defective copies were located in GC-poor L isochores. These results are discussed in terms of regional integration of retroviral sequences.

Base Composition↗
PubMedDate not supplied

Synthesis of oligonucleotide-intercalator conjugates capable to inhibit HIV-1 DNA integration.

This investigation is devoted to design of short "switch" oligonucleotides mono- or bi-functionnalized with intercalating agents capable to form a stable triplex with HIV integrase-cognate sequences and inhibit selectively HIV integration. Methods of intercalator incorporation at 5'- and/or 3'-terminal positions or one of the pyrimidine heterocyclic bases are developed.

Base Sequence↗
PubMed1998

Site-specific integration of bacteriophage VWB genome into Streptomyces venezuelae and construction of a VWB-based integrative vector.

The temperate bacteriophage VWB integrates into the chromosome of Streptomyces venezuelae ETH14630 via site-specific integration. Following recombination of the VWB attP region with the chromosomal attB sequence, the host-phage junctions attL and attR are formed. Nucleotide sequence analysis of attP, attB, attL and attR revealed a 45 bp common core sequence. In attB this 45 bp sequence consists of the 3' end of a putative tRNA Arg(AGG) gene with a 3'-terminal CCA sequence which is typical for prokaryotic tRNAs. Phage DNA integration restores the putative tRNA Arg(AGG) gene in attL. However, following recombination the CCA sequence is missing as is the case for most Streptomyces tRNA genes described so far. Adjacent to VWB attP, an ORF encoding a 427 aa protein was detected. The C-terminal region of this protein shows high similarity to the conserved C-terminal domain of site-specific recombinases belonging to the integrase family. To prove the functionality of this putative integrase gene (int), an integrative vector pKT02 was constructed. This vector consists of a 2.3 kb HindIII-SphI restriction fragment of VWB DNA containing attP and int cloned in a non-replicative Escherichia coli vector carrying a thiostrepton-resistance (tsr) gene. Integration of pKT02 was obtained after transformation of Streptomyces venezuelae ETH14630 and Streptomyces lividans TK24 protoplasts. This vector will thus be useful for a number of additional Streptomyces species in which a suitable tRNA gene can be functional as integration site.

Amino Acid Sequence↗
PubMed1999

Use of the integration elements encoded by the temperate lactococcal bacteriophage TP901-1 to obtain chromosomal single-copy transcriptional fusions in Lactococcus lactis.

Previously we showed that only one phage-expressed protein (Orf1), a 425-bp region upstream of the orf1 gene (presumably encoding a promoter), and the attP region are necessary and also sufficient for integration of the bacteriophage TP901-1 genome into the chromosome of Lactococcus lactis subsp. cremoris (B. Christiansen, L. Brondsted, F. K. Vogensen, and K. Hammer, J. Bacteriol. 178:5164-5173, 1996). In this work, a further analysis of the phage-encoded elements involved in integration was performed. Here we demonstrate that even when the orf1 gene is separated from the attP region, the Orf1 protein is able to promote site-specific integration of an attP-carrying plasmid into the attB site on the L. lactis subsp. cremoris chromosome. Furthermore, the first detailed deletion analysis of an attP region of a phage infecting lactic acid bacteria was carried out. We show that a fragment containing 56 bp of the attP region, including the core, is sufficient for the site-specific integration of a nonreplicating plasmid into the chromosome of L. lactis subsp. cremoris when the orf1 gene is donated in trans. The functional 56-bp attP region of TP901-1 is substantially smaller than minimal attP regions identified for other phages. Based on the deletion analysis, several repeats located within the attP region seem to be necessary for site-specific integration of the temperate bacteriophage TP901-1. By use of the integrative elements (attP and orf1) expressed by the temperate lactococcal bacteriophage TP901-1, a system for obtaining stable chromosomal single-copy transcriptional fusions in L. lactis was constructed. Two promoter-reporter integration vectors containing the reporter gene gusA or lacLM, encoding beta-glucuronidase or beta-galactosidase, respectively, were constructed. Immediately upstream of both genes are found translational stop codons in all three reading frames as well as multiple restriction enzyme sites suitable for cloning of the promoter of interest. By transformation of L. lactis subsp. cremoris MG1363 containing the integrase gene on a replicating plasmid, the promoter-reporter integration vectors integrated with a high frequency site specifically into the chromosomal attachment site attB used by bacteriophage TP901-1.

Attachment Sites, Microbiological↗
PubMed1995

Characterization of genetic elements required for site-specific integration of Lactobacillus delbrueckii subsp. bulgaricus bacteriophage mv4 and construction of an integration-proficient vector for Lactobacillus plantarum.

Temperate phage mv4 integrates its DNA into the chromosome of Lactobacillus delbrueckii subsp. bulgaricus strains via site-specific recombination. Nucleotide sequencing of a 2.2-kb attP-containing phage fragment revealed the presence of four open reading frames. The larger open reading frame, close to the attP site, encoded a 427-amino-acid polypeptide with similarity in its C-terminal domain to site-specific recombinases of the integrase family. Comparison of the sequences of attP, bacterial attachment site attB, and host-phage junctions attL and attR identified a 17-bp common core sequence, where strand exchange occurs during recombination. Analysis of the attB sequence indicated that the core region overlaps the 3' end of a tRNA(Ser) gene. Phage mv4 DNA integration into the tRNA(Ser) gene preserved an intact tRNA(Ser) gene at the attL site. An integration vector based on the mv4 attP site and int gene was constructed. This vector transforms a heterologous host, L. plantarum, through site-specific integration into the tRNA(Ser) gene of the genome and will be useful for development of an efficient integration system for a number of additional bacterial species in which an identical tRNA gene is present.

Amino Acid Sequence↗