Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Virus Integration”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,531 records · Page 85Linked to original sources
PubMed2002

Specificity in DNA recognition by phage integrases.

The lambda-related (lambdoid) coliphages are related to one another by frequent natural recombination and maintain a high level of functional polymorphism for several activities of the phages. Arguments are presented that the polymorphism of the integration module results from selection (presumably frequency-dependent) for new (not improved) specificities of site recognition. Analysis of phages lambda and HK022 by Weisberg and collaborators previously showed that changes in five noncontiguous amino acids could switch site recognition specificity. Phage 21 and defective element e14, which integrate at the same site, differ in recognition specificity for both core and arm sites. In vitro assays of e14 and 21 insertion and excision confirm this conclusion. Inhibition by ds arm site oligonucleotides defines the sequence specificity more precisely.

Bacteriophage lambda↗
PubMed2003

Transformation of Rhodococcus rhodnii, a symbiont of the Chagas disease vector Rhodnius prolixus, with integrative elements of the L1 mycobacteriophage.

Elimination of vector populations through the use of insecticides is the principal means of controlling Chagas disease. Because of the limitations of insecticide use, we have been developing a new potential method of control, to be used in conjunction with insecticide programs, a method which utilizes genetically modified symbiotic bacteria. These transformed bacteria can express anti-parasitic agents in the gut of the bug where the trypanosomes also are found. Previous studies have shown that it is possible to transform Rhodococcus rhodnii with a shuttle plasmid that contains the gene for cecropin A, an insect anti-microbial peptide. The bacteria expressed this peptide and reduced or eliminated the number of trypanosomes in the bug Rhodnius prolixus [Proc. Natl. Acad. Sci. U.S.A. 94 (1997) 3274]. In an effort to improve efficacy and transformation stability, we have begun using plasmids that contain integrative elements from the L1 mycobacteriophage to insert DNA into the genome of the bacterium. The integrative plasmid pBP5 contains the attachment site (attP) and integrase gene (int) of the L1 mycobacteriophage, an antibiotic resistance gene and the lacZ gene. After transforming R. rhodnii with pBP5, nine positive clones were obtained and six different insertions sites were identified. In each clone, the integrative plasmid is inserted only once, the lacZ gene is expressed intensely and, all clones but one, remained stable for 100 generations of culture in the absence of antibiotic selection. In addition, the construct remains stable throughout the life cycle of the bug. These data demonstrate that L1 mycobacteriophage integrative plasmids are significantly more stable than episomally located plasmids used in previous studies and will be greatly beneficial for use in the transformation of symbiotic bacteria of Chagas disease vectors.

Animals↗
PubMed1999

Integration complexes derived from HIV vectors for rapid assays in vitro.

Of three enzymes encoded by HIV-reverse transcriptase, protease, and integrase-only the first two have been exploited clinically as inhibitor targets. Efforts to develop inhibitors of purified integrase protein have yielded many compounds, but none with clinical utility. A different source of integration activity for studies in vitro is provided by replication intermediates isolated from HIV-infected cells. These preintegration complexes (PICs) can direct integration of the endogenously synthesized viral cDNA into an added target DNA in vitro. Despite their authentic activities, assays of PICs have not been widely used due to technical obstacles, particularly the requirement for handling large amounts of infectious HIV. Here, we describe greatly improved methods for producing PICs using HIV-based vectors that are capable of establishing an integrated provirus but not a spreading infection. We also report the development of a PIC integration assay using DNA-coated microtiter plates, which speeds assays of PIC integration in vitro. We used this method to screen a library of chemicals related to known integrase inhibitors and found a new compound, quinalizarin sulfate, that displayed enhanced activity against PICs.

Anti-HIV Agents↗
PubMed1996

Integration of bacteriophage Mx8 into the Myxococcus xanthus chromosome causes a structural alteration at the C-terminal region of the IntP protein.

Mx8 is a generalized transducing phage that infects Myxococcus xanthus cells. This phage is lysogenized in M. xanthus cells by the integration of its DNA into the host chromosome through site-specific recombination. Here, we characterize the mechanism of Mx8 integration into the M. xanthus chromosome. The Mx8 attachment site, attP, the M. xanthus chromosome attachment site, attB, and two phage-host junctions, attL and attR, were cloned and sequenced. Sequence alignments of attP, attB, attL, and attR sites revealed a 29-bp segment that is absolutely conserved in all four sequences. The intP gene of Mx8 was found to encode a basic protein that has 533 amino acids and that carries two domains conserved in site-specific recombinases of the integrase family. Surprisingly, the attP site was located within the coding sequence of the intP gene. Hence, the integration of Mx8 into the M. xanthus chromosome results in the conversion of the intP gene to a new gene designated intR. As a result of this conversion, the 112-residue C-terminal sequence of the intP protein is replaced with a 13-residue sequence. A 3-base deletion within the C-terminal region had no effect on Mx8 integration into the chromosome, while a frameshift mutation with the addition of 1 base at the same site blocked integration activity. This result indicates that the C-terminal region is required for the enzymatic function of the intP product.

Amino Acid Sequence↗
PubMed2003

Integration and distribution of Lactobacillus johnsonii prophages.

In Lactobacillus johnsonii strain NCC533, two prophages were integrated into tRNA genes and one was disrupted by integration. In a survey, the prophages were restricted to strains sharing an essentially identical restriction pattern. Microarray analysis showed that the prophage DNA represents about 50% of the NCC533 strain-specific DNA.

Animals↗
PubMed1992

The insertion site of the temperate phage HB-746 is located near the phage remnant in the pneumococcal host chromosome.

Combined Southern blot hybridization analyses of DNA digests of several clinical strains of Streptococcus pneumoniae have revealed the presence of a gene (hblR), or part of it, similar to the hbl7 gene coding for the cell wall lytic enzyme of the temperate HB-746 phage. The results confirmed that the genome of HB-746, which contains protein covalently linked to the 5' ends of its DNA, becomes integrated into the host strain 8R1 and showed that both the host and phage attachment sites, attB and attP, lie downstream of the 3' end of the structural region of the hblR and hbl7 genes, respectively. The data reported also highlight some evolutionary relationships between phage and bacteria.

Bacteriophages↗
PubMed1996

A new variant of B1 elements, vB1, identified upstream of a retroviral integration site.

A new variant of the murine B1 retroposon family, vB1, was identified in antisense orientation 165 bases upstream of the integration site of RFB MuLV in infected NIH3T3 fibroblasts. vB1 revealed a characteristic B1 structure, but contained two additional unique repeats. This variant facilitates identification of related vB1 elements which are considered to represent a minor subgroup of B1 elements.

3T3 Cells↗
PubMed2000

Genetic screens in mammalian cells by enhanced retroviral mutagens.

Genetic approaches such as retrovirus-mediated mutagenesis and cDNA expression libraries have contributed greatly to our understanding of signal transduction in mammalian cells. However, previously described methods for retroviral insertional mutagenesis are hindered by low mutagenesis rates and difficulties in cloning mutated genes. cDNA expression library methods are usually cell-type dependent and bias towards abundant and short messages. With the near completion of the genome projects, alternative genetic methods are needed where large numbers of genes can be more easily isolated and biochemically studied. We have developed a novel retrovirus-mediated genetic screening method in cultured cells. To achieve efficient and regulated mutagenesis, we constructed Enhanced Retroviral Mutagen (ERM) vectors that contained several engineered sequences (e.g., an ERM Tag and a splice donor) controlled by a tetracycline-responsive promoter. Endogenous genes can thus be randomly activated and tagged in a conditional system. NIH3T3 cells were used to screen for focus-forming genes using the ERM strategy. We showed that these added sequences increased the screening efficiency by >10-fold, and allowed more direct identification of the genes targeted. Sequence analysis of approximately 10% of the >600 focus clones recovered revealed both known oncogenes and novel factors such as protein kinases and GTP/GDP exchange proteins. The ERM strategy should help to facilitate large-scale gene identification in diverse pathways and integrate both genetic (with the completion of the genome projects) and functional information more readily.

3T3 Cells↗
PubMed2004

Construction of transgenic Drosophila by using the site-specific integrase from phage phiC31.

The phiC31 integrase functions efficiently in vitro and in Escherichia coli, yeast, and mammalian cells, mediating unidirectional site-specific recombination between its attB and attP recognition sites. Here we show that this site-specific integration system also functions efficiently in Drosophila melanogaster in cultured cells and in embryos. Intramolecular recombination in S2 cells on transfected plasmid DNA carrying the attB and attP recognition sites occurred at a frequency of 47%. In addition, several endogenous pseudo attP sites were identified in the fly genome that were recognized by the integrase and used as substrates for integration in S2 cells. Two lines of Drosophila were created by integrating an attP site into the genome with a P element. phiC31 integrase injected into embryos as mRNA functioned to promote integration of an attB-containing plasmid into the attP site, resulting in up to 55% of fertile adults producing transgenic offspring. A total of 100% of these progeny carried a precise integration event at the genomic attP site. These experiments demonstrate the potential for precise genetic engineering of the Drosophila genome with the phiC31 integrase system and will likely benefit research in Drosophila and other insects.

Animals↗
PubMed2006

Early integration of high copy HPV16 detectable in women with normal and low grade cervical cytology and histology.

BACKGROUND: Integration of human papillomavirus (HPV) DNA has been considered a late event in cervical carcinogenesis. However, integrated forms of HPV were recently detected in cancer precursor lesions using a new real time polymerase chain reaction (PCR) to detect the deletions at the 3362-3443 region of HPV16 E2 OBJECTIVE: To study the frequency of HPV16 DNA integration in cervical lesions and compare the sensitivity of an additional upstream region of the E2 ORF (2962-3138) in detecting HPV integration. METHODS: Using the TaqMan based PCR, HPV16 positive DNA samples were analysed in 164 cervical scrapings from women participating in a multicentre screening trial. Biopsy confirmation was available in 62 cases. RESULTS: Primers targeting the 3362-3443 region detected the majority of E2 deletions. In only 23% of the samples was the E2 upstream region equal or better target than the 3362-3443 region. Mixed (episomal/integrated) pattern was the most prevalent physical state of HPV16, also present in PAP smears with normal morphology. Pure integrated form was most prevalent in HSIL and cancer lesions, but also detectable in low grade abnormalities (NSIL, ASC-US, LSIL). Women with only integrated HPV16 were almost 10 years older than those with episomal HPV16. Viral load of integrated HPV16 was related to cytological abnormality (p = 0.003) but not to histology. CONCLUSIONS: Integrated HPV16 is present in low grade cervical lesions, mostly mixed with the episomal form. Women with the pure integrated form of HPV16 are older than those with the other forms.

Adolescent↗
PubMedDate not supplied

Selection with a regulated cell growth switch increases the likelihood of expression for a linked gamma-globin gene.

Several lines of evidence indicate that in vivo drug selection can be used to overcome the low rates of gene transfer and engraftment encountered in many hematopoietic stem cell gene therapy settings. However, whether selection imposed on one transcription cassette effects the likelihood of expression from a second, independent transcription cassette within the same vector has been less well studied. In order to address this issue, we engineered an oncoretrovirus vector to express two separate transcription units: (i) a bicistronic cassette encoding both GFP and a pharmacologically regulated cell growth switch based on the thrombopoietin receptor Mpl; and (ii) a highly position-dependent second cassette encoding human gamma-globin. Studies in cell cultures and in mice transplanted with transduced marrow indicated that selective expansion increased by more than 9-fold the fraction of erythroid cells expressing the linked but separate expression cassette for gamma-globin. This increase was far greater then that observed for the bicistronic GFP gene, and cannot be explained by a simple increase in the fraction of cells containing provirus. These results suggest that selective expansion favors erythroid stem/progenitor cells with provirus integrated at chromosomal sites which are relatively resistant to silencing position effects.

Animals↗
PubMed1988

A transactivating function encoded in the hepatitis B virus X gene is conserved in the integrated state.

Recently we have shown that the hepatitis B virus (HBV) X gene encodes a transactivating factor. Here we report on the construction of a series of HBx expression plasmids which were tested for a transactivating function by cotransfections with a plasmid expressing the CAT gene under control of the SV40 early promoter. One of the plasmids, expressing a HBx specific protein, is shown to transiently transactivate CAT gene expression after cotransfection into the human cell line CC113. Furthermore, a cloned integrated HBV DNA sequence is also shown to transactivate several viral promoters and LTRs. By sequence analyses we can demonstrate that only the HBx ORF is intact and that it is fused to an ORF of at least 228 bp in the flanking cellular DNA. The HBx gene is cotranscribed with the flanking cellular DNA, resulting in a RNA of approximately 10 kb. By subcloning of the integrate we can demonstrate that the presence of the HBx gene and its expression by the HBV enhancer and/or the HBx promoter is required for the transactivating function of the integrated HBV DNA.

Amino Acid Sequence↗
PubMed1983

Germline integration of moloney murine leukemia virus at the Mov13 locus leads to recessive lethal mutation and early embryonic death.

Thirteen mouse substrains genetically transmitting the exogenous Moloney murine leukemia virus (M-MuLV) at a single locus (Mov locus) have been derived previously. Experiments were performed to investigate whether homozygosity at the Mov loci would be compatible with normal development. Animals heterozygous at an Mov locus were mated, and the genotype of the offspring was analyzed. From parents heterozygous at the loci Mov1 to Mov12, respectively, homozygous offspring were obtained with the expected Mendelian frequency. In contrast, no homozygous offspring or embryos older than day 15 of gestation were obtained from parents heterozygous at the Mov13 locus. When pregnant Mov13 females at day 13 and day 14 of gestation were analyzed, approximately 25% of the embryos were degenerated. Genotyping revealed that these degenerated embryos were invariably homozygous and the normal appearing embryos were either heterozygous or negative for M-MuLV. These results suggest that integration of M-MuLV at the Mov13 locus leads to insertion mutagenesis, resulting in embryonic arrest between day 12 and day 13 of gestation. It is possible that the Mov13 locus represents a gene or gene complex involved in the early embryonic development of the mouse.

Animals↗
PubMed1995

The Y chromosome: a graveyard for endogenous retroviruses.

We have isolated 20 different human endogenous retroviruses (ERV) related to ERV3, Hsrirt and Humer 4-1. Phylogeny and the presence of these ERV among different primates were determined by computer and Southern blot analyses. Preferential localization of ERV to the human, chimpanzee and orangutan Y chromosomes among the low-copy-number ERV is demonstrated. The reason for this accumulation of ERV on the strongly heterochromatic Y chromosome is probably mediated by (i) the absence of recombination of the Y chromosome that makes it more difficult for sequences to be lost, and (ii) integration of retroviruses in heterochromatic regions is less harmful to the organism. If ERV located on the Y chromosome are transcribed and translated to peptides, such peptides could be potential HY-antigens.

Animals↗
PubMed2004

Insertion of retroviral vectors in NOD/SCID repopulating human peripheral blood progenitor cells occurs preferentially in the vicinity of transcription start regions and in introns.

Reports on insertional "genotoxicity" in patients have created intense interest in characterizing retroviral vector integrations on the genomic level. The retroviral vector SF91m3 was used for transduction of human peripheral blood progenitor cells (PBPC). These PBPC were transplanted into nonobese diabetic/severe combined immunodeficient mice. A total of 186 retroviral vector integration sites were isolated by ligation-mediated PCR from chimeric mouse bone marrow of five PBPC donors, sequenced, and blasted against the human genome. Preferred integration near the transcription start regions, within CpG islands, and within Alu regions was observed. Detailed analysis of targeted RefSeq genes showed a favored integration within the first intron. Integrations were most common in genes coding for signaling proteins, transcription factors, and kinases. In all genes targeted independently multiple times the respective orientation of the provirus within the gene was identical, indicating integration hot spot regions and similar steric determinants for integration sites. Possible explanations for these findings could be nonrandom vector integration, clonal selection due to gene expression interference, or engraftment issues related to gene insertion in signaling and cell cycle genes. The low frequency of integrations in exons may be reassuring as to the safety of retroviral gene therapy with normal human PBPC.

Alu Elements↗
PubMed1995

Circuit simulation of genetic networks.

Genetic networks with tens to hundreds of genes are difficult to analyze with currently available techniques. Because of the many parallels in the function of these biochemically based genetic circuits and electrical circuits, a hybrid modeling approach is proposed that integrates conventional biochemical kinetic modeling within the framework of a circuit simulation. The circuit diagram of the bacteriophage lambda lysislysogeny decision circuit represents connectivity in signal paths of the biochemical components. A key feature of the lambda genetic circuit is that operons function as active integrated logic components and introduce signal time delays essential for the in vivo behavior of phage lambda.

Bacteriolysis↗
PubMed2004

Retrotransposon-adenovirus hybrid vectors: efficient delivery and stable integration of transgenes via a two-stage mechanism.

It has long been recognized that the mechanisms mediating retrotransposition might be adapted for genomic integration and long-term expression of foreign genes. In particular, long interspersed nuclear elements (LINEs), an abundant class of retrotransposons that are the most active mobile genetic elements in the human genome, have been largely ignored as candidates for development as an integrating vector system because there has been no suitable method for efficiently introducing them into target cells. We have recently developed a LINE-based retrotransposon-adenovirus hybrid vector, in which a helper-dependent adenovirus (HDAd) is utilized as the platform for delivery of a human L1 element and its linked heterologous transgene cassette into the host cell nuclei. While a major drawback to the use of HDAd vectors has been their lack of specific mechanisms to achieve permanent integration into the host genome, the inserted retrotransposon sequences overcome this limitation. The L1-HDAd hybrid thus represents a single vector capable of mediating long-term gene expression by a two-stage mechanism: in the first (adenovirus) stage, the helper-dependent adenovirus serves as a carrier for efficient delivery and transient expression of its encoded L1/transgene cassette, and in the second (retrotransposon) stage, the L1 retro-element and its associated transgene then permanently integrate into the genome of the adenovirus-transduced cells. We propose that this novel retrotransposon-adenovirus hybrid vector system will be useful both as a vehicle for efficient delivery and long-term stable transduction of therapeutic genes, as well as a tool to elucidate aspects of retrotransposon biology that have previously been difficult to study.

Adenoviridae↗