scholarly journals Sequence and Insertion Sites of Murine Melanoma-Associated Retrovirus

1999 ◽  
Vol 73 (11) ◽  
pp. 9178-9186 ◽  
Author(s):  
Mengfeng Li ◽  
Xiaojun Huang ◽  
Zhenyu Zhu ◽  
Elieser Gorelik
Keyword(s):  
Insertional Mutagenesis ◽  
Insertion Site ◽  
Biological Significance ◽  
Melanoma Cells ◽  
Full Length ◽  
Noncoding Region ◽  
Chromosome 8 ◽  
Coding Region ◽  
Basic Leucine Zipper ◽  
Insertion Sites

ABSTRACT We previously showed that B16 melanoma cells produce ecotropic melanoma-associated retrovirus (MelARV) which encodes a melanoma-associated antigen recognized by MM2-9B6 monoclonal antibody. The biological significance of MelARV in melanoma formation remains unknown. We found that infection of normal melanocytes with MelARV resulted in malignant transformation. It is likely that MelARV emerged from the defective Emv-2 provirus, a single copy of ecotropic provirus existing in the genome of C57BL/6 mice. In the present study, we cloned and sequenced the full-length MelARV genome and its insertion sites and we completed sequencing of the Emv-2 provirus. Our data show that MelARV has a typical full-length retroviral genome with high homology (98.54%) to Emv-2, indicating a close relationship between both viruses. MelARV probably emerged as a result of recombination between Emv-2 and an endogenous nonecotropic provirus. Some observed differences in the gag and polregions of MelARV might account for the restoration of productivity and infectivity of a novel retrovirus that somatically emerged during melanoma formation. MelARV does not contain any oncogene and therefore might induce transformation by insertional mutagenesis. We sequenced two insertion sites of MelARV. The first insertion site represents the 3′ coding region of the c-maf proto-oncogene at 67.0 centimorgans (cM) on chromosome 8. The c-mafproto-oncogene encodes a basic leucine zipper protein homologous to c-fos and c-jun. Insertion of MelARV in BL6 melanoma cells resulted in the up-regulation of c-maf. It is noteworthy that the Emv-2 provirus is also inserted into a noncoding region at 61.0 cM on the same chromosome 8. The second insertion site is the 3′ noncoding region of the DNA polymerase gamma (PolG) gene on chromosome 7. The expression of PolG was not affected by the MelARV insertion. Further investigation of the biological significance of MelARV in melanoma formation is being undertaken.

Automated Retroviral Insertion Site Sequence Analysis and Mapping Tool Followed by Database Analysis.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 5528-5528
Author(s):  
Stephanie Laufs ◽  
Frank A. Giordano ◽  
Agnes Hotz-Wagenblatt ◽  
Uwe Appelt ◽  
Daniel Lauterborn ◽  
...  
Keyword(s):  
Insertional Mutagenesis ◽  
Integration Site ◽  
Query Sequence ◽  
Cpg Islands ◽  
Insertion Site ◽  
High Throughput Analysis ◽  
Site Analysis ◽  
Conventional Analysis ◽  
Integration Site Analysis ◽  
Insertion Sites

Abstract Increasing use of retroviral vector-mediated gene transfer and recent reports on insertional mutagenesis in mice and humans created intense interest to characterize vector integrations on the genomic level. Techniques to determine insertion sites, mainly based on time consuming manual data processing and compilation, are thus commonly applied in gene therapy laboratories. Since a high variability in processing methods hampers further data comparison, there is an urgent need to systematically process the data arising from such analysis. The obtained sequences from the integration site analysis are judged to be authentic only if the matching part of the genomic query sequence is surrounded by the 5′LTR-sequence on the one side and the adapter-sequence on the other side. Therefore we developed an Integrationseq tool. In this task, different methods for converting the ABI sequence trace files to high quality sequences and for recognizing and deleting the LTR and adaptor parts of the isolated clones were implemented. If neither a primer nor a LTR could be found, the sequence is discarded. If the LTR is found on the complementary strand, the integration sequence is reversed. The remaining sequence between primer and LTR positions are taken as the n integration sequence and written to a sequence output file. We validated the Integrationseq tool using 259 trace files originating from integration site analysis (LM-PCR). Sequences can be trimmed by IntegrationSeq, leading to an increased yield of valid integration sequence detection, which has shown to be more sensitive (100%) than conventional analysis (94.3%) and 15 times faster than conventional analysis, while the specifities are equal (both 100%). Valid integration sequences get further processed with IntegrationMap for automatic genomic mapping. IntegrationMap runs 50 times faster than conventional methods and retrieves detailed information about whether integrations are located in or close to genes, the name of the gene, the exact localization in the transcriptional units and further parameters like the distance from the transcription start site to the integration. Further information, e.g. data about CpG-Islands, LINEs or SINEs, and their distances to the integration is also displayed. Output files generated by the task were found to be 99.8% identical with results retrieved by conventional mapping with the Ensembl alignment tool. Using both tools, IntegrationSeq and IntegrationMap, a validated, fast and standardized high-throughput analysis of insertion sites can be achieved for the first time.

The development of APE-PCR for the cloning of genomic insertion sites of DNA elements

Biologia ◽  
2013 ◽  
Vol 68 (4) ◽  
Author(s):  
Zhongjuan Xu ◽  
Yanli Li ◽  
Zhengwei Mao ◽  
Bin Yin
Keyword(s):  
Insertional Mutagenesis ◽  
Magnetic Particles ◽  
Magnetic Beads ◽  
Genetic Regulation ◽  
Insertion Site ◽  
Primer Extension ◽  
Successful Implementation ◽  
Nano Scale ◽  
Dna Elements ◽  
Insertion Sites

AbstractInsertional mutagenesis is a productive strategy for the exploration of genetic regulation of important biological and pathological processes, such as tumorigenesis. Successful implementation of this strategy depends heavily on an efficient approach to the identification of insertion sites present in the host genome. Here, we have introduced an easy and efficient protocol, called Adenosine-ended Primer Extension Polymerase Chain Reaction (APE-PCR), which represents several advantages, including the Addition technique we previously developed, primer extension approach coupled with biotin-streptavidin based purification, introduction of nano-scale magnetic particles, and digestion of DNA with a combination of enzymes. We have demonstrated that APE-PCR is able to amplify more and larger specific proviral insertion site (PIS)-derived fragments, with a lower non-specific background produced, fewer steps and less DNA samples required, flexibility in choice of restriction enzymes applied, at a lower cost. Replacement of regular magnetic beads with nano-scale ones in the protocol can further increase its power. Moreover, even with small amount of sample DNA, PISs can be recovered and analyzed. Thus, based on the results provided from this study, we believe that APE-PCR represents an efficient method in mapping of PISs and likely, the insertion sites of other types of DNA elements as well.

Reduced Genotoxic Risk Using Foamy Viral Vectors for the Treatment of Canine Leukocyte Adhesion Deficiency

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3744-3744
Author(s):  
Thomas R. Bauer ◽  
Mehreen Hai ◽  
Rima L. Adler ◽  
James M. Allen ◽  
Laura M. Tuschong ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Insertional Mutagenesis ◽  
Leukocyte Adhesion ◽  
Severe Combined Immunodeficiency ◽  
Insertion Site ◽  
Leukocyte Adhesion Deficiency ◽  
Hematopoietic Stem ◽  
Insertion Sites ◽  
Immunodeficiency Disease

Abstract Gammaretroviral vectors used in recent gene therapy clinical trials have lead to several successes, such as in the treatment of X-linked severe combined immunodeficiency disease, but have also resulted in insertional activation of nearby oncogenes, leading to leukemia in four patients. We previously reported the successful treatment of four dogs with canine leukocyte adhesion deficiency (CLAD), a lethal genetic immunodeficiency disease caused by defects in the leukocyte integrin CD18, by transplanting foamy viral (FV) vector (deltaphiMscv-cCD18) - transduced, autologous CD34+ hematopoietic stem cells. To date, more than 2 years post transplant, all four dogs have maintained CD18+ leukocyte levels ranging between 5–10%, completely reversing of the CLAD phenotype, and have no clinical or laboratory evidence of hematological malignancy. To assess the potential genotoxicity of the FV gene therapy in the treatment of CLAD, we compared the insertion sites (ISs) found in the FV vector treated CLAD dogs with ISs found in CLAD dogs treated by gammaretroviral (RV) vectors (PG13/Mscv-cCD18). Insertion sites were identified by DNA sequence analysis of ligation-mediated PCR (LM-PCR) or linear amplification-mediated PCR (LAM-PCR) amplicons and subsequent comparison to the dog genome (canFam 2.0, May 2005). Insertion site analysis was performed for integrations that were in or within 50 kb of Refseq genes (using mouse/human orthologs). Analysis of the ISs revealed a reduced preference for FV vector integrations near transcription start sites compared to RV vector integrations (41% vs. 48%), fewer integrations near potential oncogenes (11% vs. 16%), and fewer integrations within genes in general (41% vs. 52%), in the FV vector treated animals compared to the RV vector treated animals. These clinically relevant data suggest that a reduced insertional mutagenesis potential exists when using FV vectors compared to RV vectors, and support the use of FV vectors in the treatment of human hematopoietic stem cell diseases such as LAD.

scholarly journals Extensive Homologous Recombination among Widely Divergent TT Viruses

2000 ◽  
Vol 74 (16) ◽  
pp. 7666-7670 ◽  
Author(s):  
Michael Worobey
Keyword(s):  
Homologous Recombination ◽  
Virus Genome ◽  
Full Length ◽  
Noncoding Region ◽  
Coding Region ◽  
Tt Virus ◽  
Virus Genomes ◽  
Recombination Breakpoints

ABSTRACT Analyses of a collection of full-length TT virus genomes showed nearly half of them to be recombinant. The results were highly significant and revealed homologous recombination both within and among genotypes, often involving extremely divergent lineages. Recombination breakpoints were significantly more common in the noncoding region of the TT virus genome than in the coding region.

scholarly journals Large-scale discovery of mouse transgenic integration sites reveals frequent structural variation and insertional mutagenesis

2017 ◽  
Author(s):  
Leslie O. Goodwin ◽  
Erik Splinter ◽  
Tiffany L. Davis ◽  
Rachel Urban ◽  
Hao He ◽  
...  
Keyword(s):  
Large Scale ◽  
Structural Variation ◽  
Insertional Mutagenesis ◽  
Insertion Site ◽  
Essential Element ◽  
Mouse Genetics ◽  
High Rate ◽  
Dna Fragments ◽  
Structural Variations ◽  
Insertion Sites

ABSTRACTTransgenesis has been a mainstay of mouse genetics for over 30 years, providing numerous models of human disease and critical genetic tools in widespread use today. Generated through the random integration of DNA fragments into the host genome, transgenesis can lead to insertional mutagenesis if a coding gene or essential element is disrupted, and there is evidence that larger scale structural variation can accompany the integration. The insertion sites of only a tiny fraction of the thousands of transgenic lines in existence have been discovered and reported due in part to limitations in the discovery tools. Targeted Locus Amplification (TLA) provides a robust and efficient means to identify both the insertion site and content of transgenes through deep sequencing of genomic loci linked to specific known transgene cassettes. Here, we report the first large-scale analysis of transgene insertion sites from 40 highly used transgenic mouse lines. We show that the transgenes disrupt the coding sequence of endogenous genes in half of the lines, frequently involving large deletions and/or structural variations at the insertion site. Furthermore, we identify a number of unexpected sequences in some of the transgenes, including undocumented cassettes and contaminating DNA fragments. We demonstrate that these transgene insertions can have phenotypic consequences, which could confound certain experiments, emphasizing the need for careful attention to control strategies. Together, these data show that transgenic alleles display a high rate of potentially confounding genetic events, and highlight the need for careful characterization of each line to assure interpretable and reproducible experiments.

scholarly journals Tn5406, a New Staphylococcal Transposon Conferring Resistance to Streptogramin A and Related Compounds Including Dalfopristin

2002 ◽  
Vol 46 (8) ◽  
pp. 2337-2343 ◽  
Author(s):  
Julien Haroche ◽  
Jeanine Allignet ◽  
Névine El Solh
Keyword(s):  
Staphylococcus Aureus ◽  
Insertion Site ◽  
Single Copy ◽  
Amino Acid Identity ◽  
Plasmid Copy ◽  
Acid Identity ◽  
Abc Protein ◽  
Single Plasmid ◽  
Insertion Sites ◽  
Related Compounds

ABSTRACT We characterized a new transposon, Tn5406 (5,467 bp), in a clinical isolate of Staphylococcus aureus (BM3327). It carries a variant of vgaA, which encodes a putative ABC protein conferring resistance to streptogramin A but not to mixtures of streptogramins A and B. It also carries three putative genes, the products of which exhibit significant similarities (61 to 73% amino acid identity) to the three transposases of the staphylococcal transposon Tn554. Like Tn554, Tn5406 failed to generate target repeats. In BM3327, the single copy of Tn5406 was inserted into the chromosomal att554 site, which is the preferential insertion site of Tn554. In three other independent S. aureus clinical isolates, Tn5406 was either present as a single plasmid copy (BM3318), as two chromosomal copies (BM3252), or both in the chromosome and on a plasmid (BM3385). The Tn5406-carrying plasmids also contain two other genes, vgaB and vatB. The insertion sites of Tn5406 in BM3252 were studied: one copy was in att554, and one copy was in the additional SCCmec element. Amplification experiments revealed circular forms of Tn5406, indicating that this transposon might be active. To our knowledge, a transposon conferring resistance to streptogramin A and related compounds has not been previously described.

Yeast regulatory gene GAL3: carbon regulation; UASGal elements in common with GAL1, GAL2, GAL7, GAL10, GAL80, and MEL1; encoded protein strikingly similar to yeast and Escherichia coli galactokinases

1988 ◽  
Vol 8 (8) ◽  
pp. 3439-3447 ◽  
Author(s):  
W Bajwa ◽  
T E Torchia ◽  
J E Hopper
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Regulatory Gene ◽  
Data Bank ◽  
Noncoding Region ◽  
Striking Similarity ◽  
Coding Region ◽  
Reading Frame ◽  
Carbon Regulation ◽  
Sequence Similarities

GAL3 gene expression is required for rapid GAL4-mediated galactose induction of the galactose-melibiose regulon genes in Saccharomyces cerevisiae. Here we show by Northern (RNA) blot analysis that GAL3 gene expression is itself galactose inducible. Like the GAL1, GAL7, GAL10, and MEL1 genes, the GAL3 gene is severely glucose repressed. Like the MEL1 gene, but in contrast to the GAL1, GAL7, and GAL10 genes, GAL3 is expressed at readily detectable basal levels in cells grown in noninducing, nonrepressing media. We determined the sequence of the S. cerevisiae GAL3 gene and its 5'-noncoding region. Within the 5'-noncoding region of the GAL3 gene, we found two sequences similar to the UASGal elements of the other galactose-melibiose regulon genes. Deletion analysis indicated that only the most ATG proximal of these sequences is required for GAL3 expression. The coding region of GAL3 consists of a 1,275-base-pair open reading frame in the direction of transcription. A comparison of the deduced 425-amino-acid sequence with the protein data bank revealed three regions of striking similarity between the GAL3 protein and the GAL1-specified galactokinase of Saccharomyces carlsbergensis. One of these regions also showed striking similarity to sequences within the galactokinase protein of Escherichia coli. On the basis of these protein sequence similarities, we propose that the GAL3 protein binds a molecule identical to or structurally related to one of the substrates or products of the galactokinase-catalyzed reaction.

scholarly journals Insertion of the βGeo Promoter Trap into the Fem1c Gene of ROSA3 Mice

2004 ◽  
Vol 24 (9) ◽  
pp. 3794-3803 ◽  
Author(s):  
Cassandra L. Schlamp ◽  
Andrew T. Thliveris ◽  
Yan Li ◽  
Louis P. Kohl ◽  
Claudia Knop ◽  
...  
Keyword(s):  
Cell Death ◽  
Ganglion Cells ◽  
Neuronal Cell Death ◽  
Insertion Site ◽  
Neuronal Cell ◽  
Pyramidal Cells ◽  
Coding Region ◽  
Protein Levels ◽  
Molecular Events ◽  
Promoter Trap

ABSTRACT ROSA3 mice were developed by retroviral insertion of the βGeo gene trap vector. Adult ROSA3 mice exhibit widespread expression of the trap gene in epithelial cells found in most organs. In the central nervous system the highest expression of βGeo is found in CA1 pyramidal cells of the hippocampus, Purkinje cells of the cerebellum, and ganglion cells of the retina. Characterization of the genomic insertion site for βGeo in ROSA3 mice shows that the trap vector is located in the first intron of Fem1c, a gene homologous to the sex-determining gene fem-1 of Caenorhabditis elegans. Transcription of the Rosa3 allele (R3) yields a spliced message that includes the first exon of Fem1c and the βGeo coding region. Although normal processing of the Fem1c transcript is disrupted in homozygous Rosa3 (Fem1cR3/R3 ) mice, some tissues show low levels of a partially processed transcript containing exons 2 and 3. Since the entire coding region of Fem1c is located in these two exons, Fem1cR3/R3 mice may still be able to express a putative FEM1C protein. To this extent, Fem1cR3/R3 mice show no adverse effects in their sexual development or fertility or in the attenuation of neuronal cell death, another function that has been attributed to both fem-1 and a second mouse homolog, Fem1b. Examination of βGeo expression in ganglion cells after exposure to damaging stimuli indicates that protein levels are rapidly depleted prior to cell death, making the βGeo reporter gene a potentially useful marker to study early molecular events in damaged neurons.

scholarly journals The Two Drosophila Telomeric Transposable Elements Have Very Different Patterns of Transcription

1999 ◽  
Vol 19 (1) ◽  
pp. 873-881 ◽  
Author(s):  
O. N. Danilevskaya ◽  
K. L. Traverse ◽  
N. C. Hogan ◽  
P. G. DeBaryshe ◽  
M. L. Pardue
Keyword(s):  
Transposable Elements ◽  
Sequence Similarity ◽  
Sequence Divergence ◽  
Full Length ◽  
Coding Region ◽  
Free Standing ◽  
Reading Frame ◽  
Sequence Organization ◽  
Sense Strand ◽  
Cell Fractions

ABSTRACT The transposable elements HeT-A and TARTconstitute the telomeres of Drosophila chromosomes. Both are non-long terminal repeat (LTR) retrotransposons, sharing the remarkable property of transposing only to chromosome ends. In addition, strong sequence similarity of their gag proteins indicates that these coding regions share a common ancestor. These findings led to the assumption that HeT-A andTART are closely related. However, we now find that these elements produce quite different sets of transcripts. HeT-Aproduces only sense-strand transcripts of the full-length element, whereas TART produces both sense and antisense full-length RNAs, with antisense transcripts in more than 10-fold excess over sense RNA. In addition, features of TART sequence organization resemble those of a subclass of non-LTR elements characterized by unequal terminal repeats. Thus, the ancestral gag sequence appears to have become incorporated in two different types of elements, possibly with different functions in the telomere. HeT-Atranscripts are found in both nuclear and cytoplasmic cell fractions, consistent with roles as both mRNA and transposition template. In contrast, both sense and antisense TART transcripts are almost entirely concentrated in nuclear fractions. Also,TART open reading frame 2 probes detect a cytoplasmic mRNA for reverse transcriptase (RT), with no similarity to TARTsequence 5′ or 3′ of the RT coding region. This RNA could be a processed TART transcript or the product of a “free-standing” RT gene. Either origin would be novel. The distinctive transcription patterns of both HeT-A andTART are conserved in Drosophila yakuba, despite significant sequence divergence. The conservation argues that these sets of transcripts are important to the function(s) ofHeT-A and TART.

A comparison of transparent polyurethane and dry gauze dressings for peripheral i.v. catheter sites: rates of phlebitis, infiltration, and dislodgment by patients

1997 ◽  
Vol 6 (5) ◽  
pp. 377-381 ◽  
Author(s):  
KA Tripepi-Bova ◽  
KD Woods ◽  
MC Loach
Keyword(s):  
Direct Observation ◽  
Meta Analysis ◽  
Insertion Site ◽  
Direct Visualization ◽  
Gauze Dressing ◽  
Insertion Sites ◽  
To Receive ◽  
Lower Frequencies

BACKGROUND: Before a meta-analysis by Hoffman et al was published, polyurethane dressings were used at insertion sites for peripheral i.v. catheters at our institution. On the basis of the results of the meta-analysis, we began to use gauze dressings. The change from polyurethane dressings to gauze dressings limited direct observation of the i.v. insertion site, and i.v. catheters were anecdotally reported not to be anchored as securely as before. OBJECTIVES: The purpose of this study was to compare the effects of the use of transparent polyurethane dressings and gauze dressings at insertion sites for peripheral i.v. catheters on the frequency of phlebitis, infiltration, and catheter dislodgment by patients. METHODS: Two hundred twenty-nine patients were randomized to receive either gauze (n = 121) or transparent polyurethane (n = 108) dressings, and observations were recorded. RESULTS: The frequency of catheter dislodgment by the patient was significantly higher (P < .05) in patients with the gauze dressing (15%) than in patients with the transparent polyurethane dressing (6%). A trend toward lower frequencies of phlebitis (1.8% vs 3.3%) and infiltration (17.6% vs 20.7%) was noted in the patients with the transparent polyurethane dressings. DISCUSSION: The clinical advantages of the transparent polyurethane dressings lie in the ease of direct visualization of the i.v. insertion site and the securement of the i.v. catheter. CONCLUSION: At our institution, given the decreased disruption of the i.v. therapy with the transparent polyurethane dressings and the lack of differences in the rates of phlebitis or infiltration with the two types of dressings, we prefer to use transparent polyurethane rather than gauze dressings at insertion sites for peripheral i.v. catheters.

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