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Insertional mutagenesis and illegitimate recombination in mycobacteria.

Kalpana, G V; Bloom, B R; Jacobs, W R
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 15/06/1991 EN
Relevância na Pesquisa
26.72%
Mycobacteria, particularly Mycobacterium tuberculosis, Mycobacterium leprae, and Mycobacterium avium, are major pathogens of man. Although insertional mutagenesis has been an invaluable genetic tool for analyzing the mechanisms of microbial pathogenesis, it has not yet been possible to apply it to the mycobacteria. To overcome intrinsic difficulties in directly manipulating the genetics of slow-growing mycobacteria, including M. tuberculosis and bacille Calmette-Guérin (BCG) vaccine strains, we developed a system for random shuttle mutagenesis. A genomic library of Mycobacterium smegmatis was subjected to transposon mutagenesis with Tn5 seq1, a derivative of Tn5, in Escherichia coli and these transposon-containing recombinant plasmids were reintroduced into mycobacterial chromosomes by homologous recombination. This system has allowed us to isolate several random auxotrophic mutants of M. smegmatis. To extend this strategy to M. tuberculosis and BCG, targeted mutagenesis was performed using a cloned BCG methionine gene that was subjected to Tn5 seq1 mutagenesis in E. coli and reintroduced into the mycobacteria. Surprisingly for prokaryotes, both BCG and M. tuberculosis were found to incorporate linear DNA fragments into illegitimate sites throughout the mycobacterial genomes at a frequency of 10(-5) to 10(-4) relative to the number of transformants obtained with autonomously replicating vectors. Thus the efficient illegitimate recombination of linear DNA fragments provides the basis for an insertional mutagenesis system for M. tuberculosis and BCG.

Identification, sequencing, and targeted mutagenesis of a DNA polymerase gene required for the extreme radioresistance of Deinococcus radiodurans.

Gutman, P D; Fuchs, P; Ouyang, L; Minton, K W
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /06/1993 EN
Relevância na Pesquisa
26.67%
Deinococcus radiodurans and other species of the same genus share extreme resistance to ionizing radiation and many other agents that damage DNA. Two different DNA damage-sensitive strains generated by chemical mutagenesis were found to be defective in a gene that has extended DNA and protein sequence homology with polA of Escherichia coli. Both mutant strains lacked DNA polymerase, as measured in activity gels. Transformation of this gene from wild-type D. radiodurans restored to the mutants both polymerase activity and DNA damage resistance. A technique for targeted insertional mutagenesis in D. radiodurans is presented. This technique was employed to construct a pol mutant isogenic with the wild type (the first example of targeted mutagenesis in this eubacterial family). This insertional mutant lacked DNA polymerase activity and was even more sensitive to DNA damage than the mutants derived by chemical mutagenesis. In the case of ionizing radiation, the survival of the wild type after receiving 1 Mrad was 100% while survival of the insertional mutant extrapolated to 10(-24). These results demonstrate that the gene described here encodes a DNA polymerase and that defects in this pol gene cause a dramatic loss of resistance of D. radiodurans to DNA damage.

A large-scale insertional mutagenesis screen in zebrafish

Amsterdam, Adam; Burgess, Shawn; Golling, Gregory; Chen, Wenbiao; Sun, Zhaoxia; Townsend, Karen; Farrington, Sarah; Haldi, Maryann; Hopkins, Nancy
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em 15/10/1999 EN
Relevância na Pesquisa
26.71%
It is estimated that ∼2500 genes are essential for the normal development of a zebrafish embryo. A mutation in any one of these genes can result in a visible developmental defect, usually followed by the death of the embryo or larva by days 5–7 of age. We are performing a large-scale insertional mutagenesis screen in the zebrafish with the goal of isolating ∼1000 embryonic mutations. We plan to clone a significant fraction of the mutated genes, as these are the genes important for normal embryogenesis of a vertebrate. To achieve this goal, we prepared ∼36,000 founder fish by injecting blastula-stage embryos with one of two pseudotyped retroviruses. We estimate that together these fish harbor between 500,000–1,000,000 proviral insertions in their germ lines. The protocol we have devised and the size of our facility allow us to breed ∼80,000–150,000 of these insertions to homozygosity within 2 years. Because a pilot screen conducted earlier in our laboratory revealed that the frequency of mutations obtained with this type of insertional mutagen is 1 embryonic lethal mutation per 70–100 proviral insertions, screening 100,000 insertions should yield at least 1000 mutants. Here we describe the protocol for the screen and initial results with the first of the two retroviral vectors used...

Application of the mini-Mu-phage for target-sequence-specific insertional mutagenesis of the herpes simplex virus genome.

Jenkins, F J; Casadaban, M J; Roizman, B
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /07/1985 EN
Relevância na Pesquisa
26.71%
An earlier technique for insertional mutagenesis of large viral genomes involved the insertion of the thymidine kinase (TK) gene at a specific target site, cotransfection of the fragment carrying the insertion with the intact viral genome, and selection of the progeny for viral recombinants expressing the TK gene. The inserted TK gene could then be replaced by cotransfection of the recombinant DNA with fragments carrying a foreign sequence or a deletion in the target sequence. To enable the probing of larger target domains and facilitate insertional mutagenesis, we extended this technique by insertion of a 2.2-kilobase-pair (kbp) herpes simplex virus 1 (HSV-1) chimeric alpha TK gene into the 7.5-kbp mini-Mu-phage (alpha TK-mini-Mu) and lysogenized Escherichia coli with the helper Mu phage and the alpha TK-mini-Mu. Induction of phage multiplication of the lysogenized E. coli after transformation with plasmids carrying HSV-1 DNA and subsequent infection of E. coli RecA+ lysogenized with Mu phage yielded plasmid populations carrying randomly inserted alpha TK-mini-Mu DNA. Application of this procedure for insertional mutagenesis of the BamHI B fragment, which spans the junction between the unique and reiterated sequences of the L component of viral DNA...

Site Preferences of Insertional Mutagenesis Agents in Arabidopsis

Pan, Xiaokang; Li, Yong; Stein, Lincoln
Fonte: American Society of Plant Biologists Publicador: American Society of Plant Biologists
Tipo: Artigo de Revista Científica
Publicado em /01/2005 EN
Relevância na Pesquisa
26.79%
We have performed a comparative analysis of the insertion sites of engineered Arabidopsis (Arabidopsis thaliana) insertional mutagenesis vectors that are based on the maize (Zea mays) transposable elements and Agrobacterium T-DNA. The transposon-based agents show marked preference for high GC content, whereas the T-DNA-based agents show preference for low GC content regions. The transposon-based agents show a bias toward insertions near the translation start codons of genes, while the T-DNAs show a predilection for the putative transcriptional regulatory regions of genes. The transposon-based agents also have higher insertion site densities in exons than do the T-DNA insertions. These observations show that the transposon-based and T-DNA-based mutagenesis techniques could complement one another well, and neither alone is sufficient to achieve the goal of saturation mutagenesis in Arabidopsis. These results also suggest that transposon-based mutagenesis techniques may prove the most effective for obtaining gene disruptions and for generating gene traps, while T-DNA-based agents may be more effective for activation tagging and enhancer trapping. From the patterns of insertion site distributions, we have identified a set of nucleotide sequence motifs that are overrepresented at the transposon insertion sites. These motifs may play a role in the transposon insertion site preferences. These results could help biologists to study the mechanisms of insertions of the insertional mutagenesis agents and to design better strategies for genome-wide insertional mutagenesis.

Genetic Analysis of Bacillus subtilis spo Mutations Generated by Tn917-Mediated Insertional Mutagenesis

Sandman, Kathleen; Losick, Richard; Youngman, Philip
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /12/1987 EN
Relevância na Pesquisa
26.66%
Mutations that cause sporulation defects (spo mutations) often identify developmentally regulated transcription units or genes whose products are required for the expression of sporulation-specific regulons. We report here the isolation, genetic analysis and phenotypic characterization of spo mutations produced by insertional mutagenesis with transposon Tn917, a form of mutagenesis that facilitates genetic and physical manipulation of mutated genes in many ways. Twenty-four insertional spo mutations were studied in detail. On the basis of transformation-mediated and transduction-mediated linkage analysis and a range of phenotypic tests, these mutations were assigned to 20 distinct loci, at least 9 of which are different from the 40 previously described spo loci. The insertional mutations caused blocks at a variety of different stages of sporulation, and therefore probably identify genes active at different times during sporulation. In addition to increasing substantially the total of known spo loci, we anticipate that this collection will include representatives of many of the temporally regulated sets of genes that comprise the overall program of sporulation-specific gene activation in Bacillus subtilis. Given the kinds of manipulations that are possible with genes disrupted by Tn 917 insertions...

Unique risk factors for insertional mutagenesis in a mouse model of XSCID gene therapy

Shou, Yan; Ma, Zhijun; Lu, Taihe; Sorrentino, Brian P.
Fonte: National Academy of Sciences Publicador: National Academy of Sciences
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
26.71%
Although gene therapy can cure patients with severe combined immunodeficiency (SCID) syndromes, the clinical occurrence of T cell malignancies due to insertional mutagenesis has raised concerns about the safety of gene therapy. Several key questions have remained unanswered: (i) are there unique risk factors for X-linked SCID (XSCID) gene therapy that increase the risk of insertional mutagenesis; (ii) what other genetic lesions may contribute to transformation; and (iii) what systems can be used to test different vectors for their relative safety? To address these questions, we have developed an XSCID mouse model in which both the Arf tumor-suppressor gene and the γc gene were ablated. Gene therapy in this animal model recapitulates the high incidence of integration-dependent, T cell tumors that was seen in the clinical trial. Ligation-mediated PCR analysis showed integration sites near or within established protooncogenes (Chd9, Slamf6, Tde1, Camk2b, and Ly6e), demonstrating that T cell transformation was associated with targeting of oncogene loci; however, no integrations within the Lmo2 locus were identified. The X-SCID background in transplanted cells was required for high rate transformation and was associated with expansion of primitive hematopoietic cells that may serve tumor precursors. This model should be useful for testing safety-modified vectors and for further exploring the risk factors leading to insertional mutagenesis in gene therapy trials.

Tumor suppressor gene identification using retroviral insertional mutagenesis in Blm-deficient mice

Suzuki, Takeshi; Minehata, Ken-ichi; Akagi, Keiko; Jenkins, Nancy A; Copeland, Neal G
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
26.75%
Retroviral insertional mutagenesis preferentially identifies oncogenes rather than tumor suppressor (TS) genes, presumably because a single retroviral-induced mutation is sufficient to activate an oncogene and initiate a tumor, whereas two mutations are needed to inactivate a TS gene. Here we show that TS genes can be identified by insertional mutagenesis when the screens are performed in Blm-deficient backgrounds. Blm-deficient mice, like Bloom syndrome patients, have increased frequencies of mitotic recombination owing to a mutation in the RecQ protein-like-3 helicase gene. This increased mitotic recombination increases the likelihood that an insertional mutation in one allele of a TS gene will become homozygoused by non-sister chromatid exchange and the homozygosity of the insertion provides a marker for identifying the TS gene. We also show that known as well as novel TS genes can be identified by insertional mutagenesis in Blm-deficient mice and identify two JmjC family proteins that contribute to genome stability in species as evolutionarily diverse as mammals and Caenorhabditis elegans.

Insertional mutagenesis identifies genes that promote the immortalization of primary bone marrow progenitor cells

Du, Yang; Jenkins, Nancy A.; Copeland, Neal G.
Fonte: © 2005 by The American Society of Hematology Publicador: © 2005 by The American Society of Hematology
Tipo: Artigo de Revista Científica
Publicado em 01/12/2005 EN
Relevância na Pesquisa
26.71%
Retroviruses can induce hematopoietic disease via insertional mutagenesis of cancer genes and provide valuable molecular tags for cancer gene discovery. Here we show that insertional mutagenesis can also identify genes that promote the immortalization of hematopoietic cells, which normally have only limited self-renewal. Transduction of mouse bone marrow cells with replication-incompetent murine stem cell virus (MSCV) expressing only neo, followed by serial passage in liquid culture containing stem cell factor (SCF) and interleukin-3 (IL-3), produced immortalized immature myeloid cell lines with neutrophil and macrophage differentiation potential in about 50% of the infected cultures. More than half of the lines have MSCV insertions at Evi1 or Prdm16. These loci encode transcription factor homologs and are validated human myeloid leukemia genes. Integrations are located in intron 1 or 2, where they promote expression of truncated proteins lacking the PRDI-BF1-RIZ1 homologous (PR) domain, similar to what is observed in human leukemias with EVI1 or PRDM16 mutations. Evi1 overexpression alone appears sufficient to immortalize immature myeloid cells and does not seem to require any other cooperating mutations. Genes identified by insertional mutagenesis by their nature could also be involved in immortalization of leukemic stem cells...

Retroviral insertional mutagenesis identifies genes that collaborate with NUP98‐HOXD13 during leukemic transformation

Slape, Christopher; Hartung, Helge; Lin, Ying‐Wei; Bies, Juraj; Wolff, Linda; Aplan, Peter D
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/06/2007 EN
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26.71%
The t(2;11)(q31;p15) chromosomal translocation results in a fusion between the NUP98 and HOXD13genes and has been observed in patients with myelodysplastic syndrome (MDS) or acute myeloid leukemia (AML). We previously demonstrated that expression of the NUP98‐HOXD13 (NHD13) fusion gene in transgenic mice results in an invariably fatal MDS; approximately one third of mice die due to complications of severe pancytopenia, and about two thirds progress to a fatal acute leukemia. In the present study, we used retroviral insertional mutagenesis to identify genes that might collaborate with NHD13 as the MDS transformed to an acute leukemia. Newborn NHD13 transgenic mice and littermate controls were infected with the MOL4070LTR retrovirus. The onset of leukemia was accelerated, suggesting a synergistic effect between the NHD13 transgene and the genes neighbouring retroviral insertion events. We identified numerous common insertion sites located near protein‐coding genes, and confirmed dysregulation of a subset of these by expression analyses. Among these genes were Meis1, a known collaborator of HOX and NUP98‐HOX fusion genes, and Mn1, a transcriptional coactivator involved in human leukemia through fusion with the TEL gene. Other putative collaborators included Gata2...

Cell-intrinsic and Vector-related Properties Cooperate to Determine the Incidence and Consequences of Insertional Mutagenesis

Kustikova, Olga S; Schiedlmeier, Bernhard; Brugman, Martijn H; Stahlhut, Maike; Bartels, Stefan; Li, Zhixiong; Baum, Christopher
Fonte: Nature Publishing Group Publicador: Nature Publishing Group
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
26.76%
In gene therapeutic approaches targeting hematopoietic cells, insertional mutagenesis may provoke clonal dominance with potential progress to overt leukemia. To investigate the contribution of cell-intrinsic features and determine the frequency of insertional proto-oncogene activation, we sorted hematopoietic subpopulations before transduction with replication-deficient γ-retroviral vectors and studied the clonal repertoire in transplanted C57BL/6J mice. Progressive clonal dominance only developed in the progeny of populations with intrinsic stem cell potential, where expanding clones with insertional upregulation of proto-oncogenes such as Evi1 were retrieved with a frequency of ~10−4. Longitudinal studies by high-throughput sequencing and locus-specific quantitative PCR showed clones with >50-fold expansion between weeks 5 and 31 after transplantation. In contrast, insertional events in proto-oncogenes did not endow the progeny of multipotent or myeloid-restricted progenitors with the potential for clonal dominance (risk <10−6). Transducing sorted hematopoietic stem cells (HSCs) with self-inactivating (SIN) lentiviral vectors in short-term cultures improved chimerism, and although clonal dominance developed, there was no evidence for insertional events in the vicinity of proto-oncogenes as the underlying cause. We conclude that cell-intrinsic properties cooperate with vector-related features to determine the incidence and consequences of insertional mutagenesis. Furthermore...

High-throughput semiquantitative analysis of insertional mutations in heterogeneous tumors

Koudijs, Marco J.; Klijn, Christiaan; van der Weyden, Louise; Kool, Jaap; ten Hoeve, Jelle; Sie, Daoud; Prasetyanti, Pramudita R.; Schut, Eva; Kas, Sjors; Whipp, Theodore; Cuppen, Edwin; Wessels, Lodewyk; Adams, David J.; Jonkers, Jos
Fonte: Cold Spring Harbor Laboratory Press Publicador: Cold Spring Harbor Laboratory Press
Tipo: Artigo de Revista Científica
Publicado em /12/2011 EN
Relevância na Pesquisa
26.7%
Retroviral and transposon-based insertional mutagenesis (IM) screens are widely used for cancer gene discovery in mice. Exploiting the full potential of IM screens requires methods for high-throughput sequencing and mapping of transposon and retroviral insertion sites. Current protocols are based on ligation-mediated PCR amplification of junction fragments from restriction endonuclease-digested genomic DNA, resulting in amplification biases due to uneven genomic distribution of restriction enzyme recognition sites. Consequently, sequence coverage cannot be used to assess the clonality of individual insertions. We have developed a novel method, called shear-splink, for the semiquantitative high-throughput analysis of insertional mutations. Shear-splink employs random fragmentation of genomic DNA, which reduces unwanted amplification biases. Additionally, shear-splink enables us to assess clonality of individual insertions by determining the number of unique ligation points (LPs) between the adapter and genomic DNA. This parameter serves as a semiquantitative measure of the relative clonality of individual insertions within heterogeneous tumors. Mixing experiments with clonal cell lines derived from mouse mammary tumor virus (MMTV)-induced tumors showed that shear-splink enables the semiquantitative assessment of the clonality of MMTV insertions. Further...

Cancer gene discovery: exploiting insertional mutagenesis

Ranzani, Marco; Annunziato, Stefano; Adams, David J.; Montini, Eugenio
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
26.7%
Insertional mutagenesis has been utilized as a functional forward genetics screen for the identification of novel genes involved in the pathogenesis of human cancers. Different insertional mutagens have been successfully used to reveal new cancer genes. For example, retroviruses (RVs) are integrating viruses with the capacity to induce the deregulation of genes in the neighborhood of the insertion site. RVs have been employed for more than 30 years to identify cancer genes in the hematopoietic system and mammary gland. Similarly, another tool that has revolutionized cancer gene discovery is the cut-and-paste transposons. These DNA elements have been engineered to contain strong promoters and stop cassettes that may function to perturb gene expression upon integration proximal to genes. In addition, complex mouse models characterized by tissue-restricted activity of transposons have been developed to identify oncogenes and tumor suppressor genes that control the development of a wide range of solid tumor types, extending beyond those tissues accessible using RV-based approaches. Most recently, lentiviral vectors (LVs) have appeared on the scene for use in cancer gene screens. LVs are replication defective integrating vectors that have the advantage of being able to infect non-dividing cells...

Inactivation of two haemolytic toxin genes in Aeromonas hydrophila attenuates virulence in a suckling mouse model

Wong, C.; Heuzenroeder, M.; Flower, R.
Fonte: Universidade de Adelaide Publicador: Universidade de Adelaide
Tipo: Artigo de Revista Científica
Publicado em //1998 EN
Relevância na Pesquisa
36.17%
The contribution of two unrelated Aeromonas hydrophila beta-haemolytic toxins to virulence was assessed in a suckling mouse model. The first haemolysin gene, isolated from an A. hydrophila A6 cosmid bank, encoded a potential gene product of 621 amino acids and a predicted molecular size of 69.0 kDa. The inferred amino acid sequence showed 89% identity to the AHH1 haemolysin of A. hydrophila ATCC 7966, and 51% identity to the HlyA haemolysin of Vibrio cholerae EI Tor strain O17. The second haemolysin gene (designated aerA), which encodes aerolysin, a pore-forming toxin, was partially cloned by PCR for the purpose of mutant construction. This PCR product was a 1040 bp fragment from the C-terminal region of aerA. It is proposed that the 69.0 kDa V. cholerae-HlyA-like haemolysin gene be termed hlyA to contrast with the aerA terminology for the aerolysin. A suicide vector was used to inactivate both the hlyA and aerA genes in A. hydrophila A6. When assessed in the suckling mouse model, only the hlyA aerA double mutant showed a statistically significant reduction in virulence--a 20-fold change in LD50 (Scheffe test, P < 0.05). Cytotoxicity to buffalo green monkey kidney cell monolayers and haemolysis on horse blood agar were eliminated only in the hlyA aerA double mutants. This is the first report of cloning and mutagenesis of two unrelated haemolytic toxin genes in the same strain of a mesophilic aeromonad. For A. hydrophila...

The helix-turn-helix motif of the coliphage 186 immunity repressor binds to two distinct recognition sequences

Shearwin, K.E.; Dodd, I.B.; Egan, J.B.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em //2002 EN
Relevância na Pesquisa
36.17%
The CI protein of coliphage 186 is responsible for maintaining the stable lysogenic state. To do this CI must recognize two distinct DNA sequences, termed A type sites and B type sites. Here we investigate whether CI contains two separate DNA binding motifs or whether CI has one motif that recognizes two different operator sequences. Sequence alignment with 186-like repressors predicts an N-terminal helix-turn-helix (HTH) motif, albeit with poor homology to a large master set of such motifs. The domain structure of CI was investigated by linker insertion mutagenesis and limited proteolysis. CI consists of an N-terminal domain, which weakly dimerizes and binds both A and B type sequences, and a C-terminal domain, which associates to octamers but is unable to bind DNA. A fusion protein consisting of the 186 N-terminal domain and the phage {lambda} oligomerization domain binds A and B type sequences more efficiently than the isolated 186 CI N-terminal domain, hence the 186 C-terminal domain likely mediates oligomerization and cooperativity. Site-directed mutation of the putative 186 HTH motif eliminates binding to both A and B type sites, supporting the idea that binding to the two distinct DNA sequences is mediated by a variant HTH motif.; Keith E. Shearwin...

Mutagenesis of the Shigella flexneri autotransporter IcsA reveals novel functional regions involved in IcsA biogenesis and recruitment of host neural Wiscott-Aldrich syndrome protein

May, K.; Morona, R.
Fonte: Amer Soc Microbiology Publicador: Amer Soc Microbiology
Tipo: Artigo de Revista Científica
Publicado em //2008 EN
Relevância na Pesquisa
46.25%
The IcsA (VirG) protein of Shigella flexneri is a polarly localized, outer membrane protein that is essential for virulence. Within host cells, IcsA activates the host actin regulatory protein, neural Wiskott-Aldrich syndrome protein (N-WASP), which in turn recruits the Arp2/3 complex, which nucleates host actin to form F-actin comet tails and initiate bacterial motility. Linker insertion mutagenesis was undertaken to randomly introduce 5-amino-acid in-frame insertions within IcsA. Forty-seven linker insertion mutants were isolated and expressed in S. flexneri Delta icsA strains. Mutants were characterized for IcsA protein production, cell surface expression and localization, intercellular spreading, F-actin comet tail formation, and N-WASP recruitment. Using this approach, we have identified a putative autochaperone region required for IcsA biogenesis, and our data suggest an additional region, not previously identified, is required for N-WASP recruitment.; Kerrie L. May and Renato Morona

Lymphomagenesis in SCID-X1 Mice Following Lentivirus-mediated Phenotype Correction Independent of Insertional Mutagenesis and gamma c Overexpression

Ginn, S.; Liao, S.; Dane, A.; Hu, M.; Hyman, J.; Finnie, J.; Zheng, M.; Cavazzana-Calvo, M.; Alexander, S.; Trasher, A.; Alexander, I.
Fonte: Academic Press Inc Elsevier Science Publicador: Academic Press Inc Elsevier Science
Tipo: Artigo de Revista Científica
Publicado em //2010 EN
Relevância na Pesquisa
46.71%
The development of leukemia as a consequence of vector-mediated genotoxicity in gene therapy trials for X-linked severe combined immunodeficiency (SCID-X1) has prompted substantial research effort into the design and safety testing of integrating vectors. An important element of vector design is the selection and evaluation of promoter-enhancer elements with sufficient strength to drive reliable immune reconstitution, but minimal propensity for enhancer-mediated insertional mutagenesis. In this study, we set out to explore the effect of promoter-enhancer selection on the efficacy and safety of human immunodeficiency virus-1-derived lentiviral vectors in γc-deficient mice. We observed incomplete or absent T- and B-cell development in mice transplanted with progenitors expressing γc from the phosphoglycerate kinase (PGK) and Wiscott–Aldrich syndrome (WAS) promoters, respectively. In contrast, functional T- and B-cell compartments were restored in mice receiving an equivalent vector containing the elongation factor-1-α (EF1α) promoter; however, 4 of 14 mice reconstituted with this vector subsequently developed lymphoma. Extensive analyses failed to implicate insertional mutagenesis or γc overexpression as the underlying mechanism. These findings highlight the need for detailed mechanistic analysis of tumor readouts in preclinical animal models assessing vector safety...

The Impact of CpG Island on Defining Transcriptional Activation of the Mouse L1 Retrotransposable Elements

Lee, Sung-Hun; Cho, Soo-Young; Shannon, M. Frances; Fan, Jun; Rangasamy, Danny
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
36.23%
BACKGROUND L1 retrotransposable elements are potent insertional mutagens responsible for the generation of genomic variation and diversification of mammalian genomes, but reliable estimates of the numbers of actively transposing L1 elements are mostly nonexistent. While the human and mouse genomes contain comparable numbers of L1 elements, several phylogenetic and L1Xplore analyses in the mouse genome suggest that 1,500-3,000 active L1 elements currently exist and that they are still expanding in the genome. Conversely, the human genome contains only 150 active L1 elements. In addition, there is a discrepancy among the nature and number of mouse L1 elements in L1Xplore and the mouse genome browser at the UCSC and in the literature. To date, the reason why a high copy number of active L1 elements exist in the mouse genome but not in the human genome is unknown, as are the potential mechanisms that are responsible for transcriptional activation of mouse L1 elements. METHODOLOGY/PRINCIPAL FINDINGS We analyzed the promoter sequences of the 1,501 potentially active mouse L1 elements retrieved from the GenBank and L1Xplore databases and evaluated their transcription factors binding sites and CpG content. To this end, we found that a substantial number of mouse L1 elements contain altered transcription factor YY1 binding sites on their promoter sequences that are required for transcriptional initiation...

Meiotic chromosome segregation mutants identified by insertional mutagenesis of fission yeast Schizosaccharomyces pombe; tandem-repeat, single-site integrations

Davidson, Mari K.; Young, Nathan P.; Glick, Gloria G.; Wahls, Wayne P.
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
26.71%
Identification of genes required for segregation of chromosomes in meiosis (scm) is difficult because in most organisms high-fidelity chromosome segregation is essential to produce viable meiotic products. The biology of fission yeast Schizosaccharomyces pombe facilitates identification of such genes. Insertional mutagenesis was achieved by electroporation of linear ura4+ DNA into cells harboring a ura4 deletion. Approximately 1000 stable transformants were screened individually for the production of elevated frequencies of aneuploid spore colonies. Twenty-two candidates were subjected to a secondary screen for cytological defects. Five mutants exhibited significant levels of aberrant meiotic chromosome segregation, but were proficient for mating and completion of meiosis. Each mutant's phenotype cosegregated with its respective ura4+ transgene. The mutations were recessive and defined five complementation groups, revealing five distinct genes (scm1, scm2, scm3, scm4 and scm5). Southern blotting revealed single-site integration in each transformant, indicating that insertional mutagenesis is useful for generating single-locus scm mutations linked to a selectable marker. The transgene insertion points were refractory to analysis by inverse-PCR. Molecular and real-time PCR analyses revealed the presence of multiple...

Directed mutation of the Rubisco large subunit of Tobacco influences photorespiration and growth

Whitney, Spencer; von Caemmerer, Susanne; Hudson, G; Andrews, Thomas
Fonte: American Society of Plant Biologists Publicador: American Society of Plant Biologists
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
36.3%
The gene for the large subunit of Rubisco was specifically mutated by transforming the chloroplast genome of tobacco (Nicotiana tabacum). Codon 335 was altered to encode valine instead of leucine. The resulting mutant plants could not grow without atmospheric CO2 enrichment, in 0.3% (v/v) CO2, the mutant and wildtype plants produced similar amounts of Rubisco but the extent of carbamylation was nearly twice as great in the mutants. The mutant enzyme's substrate-saturated CO2-fixing rate and its ability to distinguish between CO2 and O2 as substrates were both reduced to 25% of the wild type's values. Estimates of these parameters obtained from kinetic assays with the purified mutant enzyme were the same as those inferred from measurements of photosynthetic gas exchange with leaves of mutant plants. The Michaelis constants for CO2, O2, and ribulose-1,5-bisphosphate were reduced and the mutation enhanced oxygenase activity at limiting O2 concentrations. Consistent with the reduced CO2 fixation rate at saturating CO2, the mutant plants grew slower than the wild type but they eventually flowered and reproduced apparently normally. The mutation and its associated phenotype were inherited maternally. The chloroplast-transformation strategy surmounts previous obstacles to mutagenesis of higher-plant Rubisco and allows the consequences for leaf photosynthesis to be assessed.