Artículos de revistas: "Insertion mutagenesi"

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Skamaki, Kalliopi, Stephane Emond, Matthieu Chodorge, John Andrews, D. Gareth Rees, Daniel Cannon, Bojana Popovic, Andrew Buchanan, Ralph R. Minter y Florian Hollfelder. "In vitro evolution of antibody affinity via insertional scanning mutagenesis of an entire antibody variable region". Proceedings of the National Academy of Sciences 117, n.º 44 (16 de octubre de 2020): 27307–18. http://dx.doi.org/10.1073/pnas.2002954117.

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We report a systematic combinatorial exploration of affinity enhancement of antibodies by insertions and deletions (InDels). Transposon-based introduction of InDels via the method TRIAD (transposition-based random insertion and deletion mutagenesis) was used to generate large libraries with random in-frame InDels across the entire single-chain variable fragment gene that were further recombined and screened by ribosome display. Knowledge of potential insertion points from TRIAD libraries formed the basis of exploration of length and sequence diversity of novel insertions by insertional-scanning mutagenesis (InScaM). An overall 256-fold affinity improvement of an anti–IL-13 antibody BAK1 as a result of InDel mutagenesis and combination with known point mutations validates this approach, and suggests that the results of this InDel mutagenesis and conventional exploration of point mutations can synergize to generate antibodies with higher affinity.

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Herod, Morgan R., Eleni-Anna Loundras, Joseph C. Ward, Fiona Tulloch, David J. Rowlands y Nicola J. Stonehouse. "Employing transposon mutagenesis to investigate foot-and-mouth disease virus replication". Journal of General Virology 96, n.º 12 (1 de diciembre de 2015): 3507–18. http://dx.doi.org/10.1099/jgv.0.000306.

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Probing the molecular interactions within the foot-and-mouth disease virus (FMDV) RNA replication complex has been restricted in part by the lack of suitable reagents. Random insertional mutagenesis has proven an excellent method to reveal domains of proteins essential for virus replication as well as locations that can tolerate small genetic insertions. Such insertion sites can subsequently be adapted by the incorporation of commonly used epitope tags, facilitating their detection with commercially available reagents. In this study, we used random transposon-mediated mutagenesis to produce a library of 15 nt insertions in the FMDV non-structural polyprotein. Using a replicon-based assay, we isolated multiple replication-competent as well as replication-defective insertions. We adapted the replication-competent insertion sites for the successful incorporation of epitope tags within FMDV non-structural proteins for use in a variety of downstream assays. Additionally, we showed that replication of some of the replication-defective insertion mutants could be rescued by co-transfection of a ‘helper’ replicon, demonstrating a novel use of random mutagenesis to identify intergenomic trans-complementation. Both the epitope tags and replication-defective insertions identified here will be valuable tools for probing interactions within picornavirus replication complexes.

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Coyote-Maestas, Willow, David Nedrud, Steffan Okorafor, Yungui He y Daniel Schmidt. "Targeted insertional mutagenesis libraries for deep domain insertion profiling". Nucleic Acids Research 48, n.º 2 (20 de noviembre de 2019): e11-e11. http://dx.doi.org/10.1093/nar/gkz1110.

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Abstract Domain recombination is a key principle in protein evolution and protein engineering, but inserting a donor domain into every position of a target protein is not easily experimentally accessible. Most contemporary domain insertion profiling approaches rely on DNA transposons, which are constrained by sequence bias. Here, we establish Saturated Programmable Insertion Engineering (SPINE), an unbiased, comprehensive, and targeted domain insertion library generation technique using oligo library synthesis and multi-step Golden Gate cloning. Through benchmarking to MuA transposon-mediated library generation on four ion channel genes, we demonstrate that SPINE-generated libraries are enriched for in-frame insertions, have drastically reduced sequence bias as well as near-complete and highly-redundant coverage. Unlike transposon-mediated domain insertion that was severely biased and sparse for some genes, SPINE generated high-quality libraries for all genes tested. Using the Inward Rectifier K+ channel Kir2.1, we validate the practical utility of SPINE by constructing and comparing domain insertion permissibility maps. SPINE is the first technology to enable saturated domain insertion profiling. SPINE could help explore the relationship between domain insertions and protein function, and how this relationship is shaped by evolutionary forces and can be engineered for biomedical applications.

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Carlson, Corey M., Adam J. Dupuy, Sabine Fritz, Kevin J. Roberg-Perez, Colin F. Fletcher y David A. Largaespada. "Transposon Mutagenesis of the Mouse Germline". Genetics 165, n.º 1 (1 de septiembre de 2003): 243–56. http://dx.doi.org/10.1093/genetics/165.1.243.

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Abstract Sleeping Beauty is a synthetic “cut-and-paste” transposon of the Tc1/mariner class. The Sleeping Beauty transposase (SB) was constructed on the basis of a consensus sequence obtained from an alignment of 12 remnant elements cloned from the genomes of eight different fish species. Transposition of Sleeping Beauty elements has been observed in cultured cells, hepatocytes of adult mice, one-cell mouse embryos, and the germline of mice. SB has potential as a random germline insertional mutagen useful for in vivo gene trapping in mice. Previous work in our lab has demonstrated transposition in the male germline of mice and transmission of novel inserted transposons in offspring. To determine sequence preferences and mutagenicity of SB-mediated transposition, we cloned and analyzed 44 gene-trap transposon insertion sites from a panel of 30 mice. The distribution and sequence content flanking these cloned insertion sites was compared to 44 mock insertion sites randomly selected from the genome. We find that germline SB transposon insertion sites are AT-rich and the sequence ANNTANNT is favored compared to other TA dinucleotides. Local transposition occurs with insertions closely linked to the donor site roughly one-third of the time. We find that ∼27% of the transposon insertions are in transcription units. Finally, we characterize an embryonic lethal mutation caused by endogenous splicing disruption in mice carrying a particular intron-inserted gene-trap transposon.

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Idnurm, Alexander, Jennifer L. Reedy, Jesse C. Nussbaum y Joseph Heitman. "Cryptococcus neoformans Virulence Gene Discovery through Insertional Mutagenesis". Eukaryotic Cell 3, n.º 2 (abril de 2004): 420–29. http://dx.doi.org/10.1128/ec.3.2.420-429.2004.

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ABSTRACT Insertional mutagenesis was applied to Cryptococcus neoformans to identify genes associated with virulence attributes. Using biolistic transformation, we generated 4,300 nourseothricin (NAT)-resistant strains, of which 590 exhibited stable resistance. We focused on mutants with defects in established virulence factors and identified two with reduced growth at 37°C, four with reduced production of the antioxidant pigment melanin, and two with an increased sensitivity to nitric oxide (NO). The NAT insertion and mutant phenotypes were genetically linked in five of eight mutants, and the DNA flanking the insertions was characterized. For the strains with altered growth at 37°C and altered melanin production, mutations were in previously uncharacterized genes, while the two NO-sensitive strains bore insertions in the flavohemoglobin gene FHB1, whose product counters NO stress. Because of the frequent instability of nourseothricin resistance associated with biolistic transformation, Agrobacterium-mediated transformation was tested. This transkingdom DNA delivery approach produced 100% stable nourseothricin-resistant transformants, and three melanin-defective strains were identified from 576 transformants, of which 2 were linked to NAT in segregation analysis. One of these mutants contained a T-DNA insertion in the promoter of the LAC1 (laccase) gene, which encodes a key enzyme required for melanin production, while the second contained an insertion in the promoter of the CLC1 gene, encoding a voltage-gated chloride channel. Clc1 and its homologs are required for ion homeostasis, and in their absence Cu+ transport into the secretory pathway is compromised, depriving laccase and other Cu+-dependent proteins of their essential cofactor. The NAT resistance cassette was optimized for cryptococcal codon usage and GC content and was then used to disrupt a mitogen-activated protein kinase gene, a predicted gene, and two putative chloride channel genes to analyze their contributions to fungal physiology. Our findings demonstrate that both insertional mutagenesis methods can be applied to gene identification, but Agrobacterium-mediated transformation is more efficient and generates exclusively stable insertion mutations.

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Roseman, R. R., E. A. Johnson, C. K. Rodesch, M. Bjerke, R. N. Nagoshi y P. K. Geyer. "A P element containing suppressor of hairy-wing binding regions has novel properties for mutagenesis in Drosophila melanogaster." Genetics 141, n.º 3 (1 de noviembre de 1995): 1061–74. http://dx.doi.org/10.1093/genetics/141.3.1061.

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Abstract P elements are widely used as insertional mutagens to tag genes, facilitating molecular cloning and analyses. We modified a P element so that it carried two copies of the suppressor of Hairy-wing [su(Hw)] binding regions isolated from the gypsy transposable element. This transposon was mobilized, and the genetic consequences of its insertion were analyzed. Gene expression can be altered by the su(Hw) protein as a result of blocking the interaction between enhancer/silencer elements and their promoter. These effects can occur over long distances and are general. Therefore, a composite transposon (SUPor-P for suppressor-P element) combines the mutagenic efficacy of the gypsy element with the controllable transposition of P elements. We show that, compared to standard P elements, this composite transposon causes an expanded repertoire of mutations and produces alleles that are suppressed by su(Hw) mutations. The large number of heterochromatic insertions obtained is unusual compared to other insertional mutagenesis procedures, indicating that the SUPor-P transposon may be useful for studying the structural and functional properties of heterochromatin.

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Murray, Gerald L., Viviane Morel, Gustavo M. Cerqueira, Julio Croda, Amporn Srikram, Rebekah Henry, Albert I. Ko et al. "Genome-Wide Transposon Mutagenesis in Pathogenic Leptospira Species". Infection and Immunity 77, n.º 2 (1 de diciembre de 2008): 810–16. http://dx.doi.org/10.1128/iai.01293-08.

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ABSTRACT Leptospira interrogans is the most common cause of leptospirosis in humans and animals. Genetic analysis of L. interrogans has been severely hindered by a lack of tools for genetic manipulation. Recently we developed the mariner-based transposon Himar1 to generate the first defined mutants in L. interrogans. In this study, a total of 929 independent transposon mutants were obtained and the location of insertion determined. Of these mutants, 721 were located in the protein coding regions of 551 different genes. While sequence analysis of transposon insertion sites indicated that transposition occurred in an essentially random fashion in the genome, 25 unique transposon mutants were found to exhibit insertions into genes encoding 16S or 23S rRNAs, suggesting these genes are insertional hot spots in the L. interrogans genome. In contrast, loci containing notionally essential genes involved in lipopolysaccharide and heme biosynthesis showed few transposon insertions. The effect of gene disruption on the virulence of a selected set of defined mutants was investigated using the hamster model of leptospirosis. Two attenuated mutants with disruptions in hypothetical genes were identified, thus validating the use of transposon mutagenesis for the identification of novel virulence factors in L. interrogans. This library provides a valuable resource for the study of gene function in L. interrogans. Combined with the genome sequences of L. interrogans, this provides an opportunity to investigate genes that contribute to pathogenesis and will provide a better understanding of the biology of L. interrogans.

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Combier, Jean-Philippe, Delphine Melayah, Colette Raffier, Régis Pépin, Roland Marmeisse y Gilles Gay. "Nonmycorrhizal (Myc¯) Mutants of Hebeloma cylindrosporum Obtained Through Insertional Mutagenesis". Molecular Plant-Microbe Interactions® 17, n.º 9 (septiembre de 2004): 1029–38. http://dx.doi.org/10.1094/mpmi.2004.17.9.1029.

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Polyethylene glycol-mediated transformation of protoplasts was used as a method for insertional mutagenesis to obtain mutants of the ectomycorrhizal fungus Hebeloma cylindrosporum impaired in symbiotic ability. Following restriction enzyme-mediated integration or conventional plasmid insertion, a library of 1,725 hygromycin-resistant monokaryotic transformants was generated and screened for the symbiotic defect, using Pinus pinaster seedlings as host plants. A total of 51 transformants displaying a dramatically reduced mycorrhizal ability were identified. Among them, 29 were nonmycorrhizal (myc¯), but only 10 of them had integrated one or several copies of the transforming plasmid in their genome. Light and scanning electron microscopy observations of pine roots inoculated with myc¯ mutants suggested that we selected mutants blocked at early stages of interaction between partners or at the stage of Hartig net formation. Myc¯ mutants with plasmid insertions were crossed with a compatible wild-type monokaryon and allowed to fruit. Monokaryotic progenies were obtained in three independent crosses and were analyzed for symbiotic activity and plasmid insertion. In all three progenies, a 1:1 myc¯:myc+ segregation ratio was observed, suggesting that each myc¯ phenotype resulted from a single gene mutation. However, for none of the three mutants, the myc¯ phenotype segregated with any of the plasmid insertions. Our results support the idea that master genes, the products of which are essential for symbiosis establishment, do exist in ectomycorrhizal fungi.

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Qin, Aiping, Aimee M. Tucker, Andria Hines y David O. Wood. "Transposon Mutagenesis of the Obligate Intracellular Pathogen Rickettsia prowazekii". Applied and Environmental Microbiology 70, n.º 5 (mayo de 2004): 2816–22. http://dx.doi.org/10.1128/aem.70.5.2816-2822.2004.

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ABSTRACT Genetic analysis of Rickettsia prowazekii has been hindered by the lack of selectable markers and efficient mechanisms for generating rickettsial gene knockouts. We have addressed these problems by adapting a gene that codes for rifampin resistance for expression in R. prowazekii and by incorporating this selection into a transposon mutagenesis system suitable for generating rickettsial gene knockouts. The arr-2 gene codes for an enzyme that ADP-ribosylates rifampin, thereby destroying its antibacterial activity. Based on the published sequence, this gene was synthesized by PCR with overlapping primers that contained rickettsial codon usage base changes. This R. prowazekii-adapted arr-2 gene (Rparr-2) was placed downstream of the strong rickettsial rpsL promoter (rpsLP ), and the entire construct was inserted into the Epicentre EZ::TN transposome system. A purified transposon containing rpsLP-Rparr-2 was combined with transposase, and the resulting DNA-protein complex (transposome) was electroporated into competent rickettsiae. Following selection with rifampin, rickettsiae with transposon insertions in the genome were identified by PCR and Southern blotting and the insertion sites were determined by rescue cloning and inverse PCR. Multiple insertions into widely spaced areas of the R. prowazekii genome were identified. Three insertions were identified within gene coding sequences. Transposomes provide a mechanism for generating random insertional mutations in R. prowazekii, thereby identifying nonessential rickettsial genes.

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Upadhyaya, Narayana M., Xue-Rong Zhou, Qian-Hao Zhu, Kerrie Ramm, Limin Wu, Andrew Eamens, Ramani Sivakumar et al. "An iAc/Ds gene and enhancer trapping system for insertional mutagenesis in rice". Functional Plant Biology 29, n.º 5 (2002): 547. http://dx.doi.org/10.1071/pp01205.

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We evaluated a two-component transposon iAc/Ds system for generating a library of insertional mutants in rice. The constructs used have gene or enhancer trapping properties, plasmid rescue and T-DNA/Ds launching pad reporter facilities. Mutagenic iAc/Ds lines were produced by three methods: crossing iAc and Ds containing lines; co-transformation with iAc and Ds constructs; and super-transformation of iAc transgenic calli with Ds constructs. First and second generation screening populations, derived from crosses (F2 and F3) or double transformation (DtT1 and DtT2), were analysed for stable insertion lines containing Ds transposed to locations unlinked to iAc. The average frequencies of putative stable insertion (PSI) lines in the F2, DtT1, F3 and DtT2 populations were 6.61, 5.58, 11.47 and 7.05% respectively, with large variations in these frequencies in screening populations derived from different mutagenic lines. Further analyses indicated that 41, 33, 65 and 64% of the PSI lines, respectively, have Ds transposed to locations unlinked to the original Ds launching pad. Using the plasmid rescue system, sequences flanking Ds from 137 PSI lines were obtained. Sixty-eight of these lines had unique insertions in genomic regions, of which 18 were known sequences. Because the average frequency of proven stable insertion lines in any of our screening populations has been less than 5%, we suggest that additional features should be incorporated in this two-component iAc/Ds system to increase the screening efficiency, and to make it suitable for large-scale insertional mutagenesis and determination of gene function in rice.

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Kwiatkowski, Boguslaw A. y Robert E. Richard. "Identification of Genes Cooperating with Mpl Signaling Using Retroviral Insertional Mutagenesis." Blood 114, n.º 22 (20 de noviembre de 2009): 4558. http://dx.doi.org/10.1182/blood.v114.22.4558.4558.

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Abstract Abstract 4558 The thrombopoietin receptor (Mpl) regulates the proliferation of hematopoietic progenitors, their differentiation into mature megakaryocytes, and formation of platelets. Mutations in the Mpl gene have been shown to be associated with myeloproliferative neoplasms (MPN). We attempted to identify genes cooperating with Mpl signaling that could provide cell survival and proliferative advantage using MSCV based retroviral insertional mutagenesis coupled with an inducible Mpl signaling system. The retroviral Mpl vectors used in our screening system were based on the well-described retroviral vector MGIFM. That vector carried a bacterial shuttle plasmid coding for the Neo gene and a bacterial origin of replication in its 3′ untranslated region, and also coding sequences of a drug dependent, dimerizable, fusion protein containing the cytoplasmic domain of Mpl. This vector can interrupt gene structure through insertion, and its intact long terminal repeats can activate adjacent genes. The shuttle plasmid allows nonbiased recovery of proviral genomic integration sites (RIS). The retroviral Mpl vectors were used to transduce the human leukemia cell line K562. Selection for Mpl signaling dependent cells required blocking of the endogenous transforming Bcr-Abl kinase with imatinib and supplying the dimerizer drug, AP20187. Similar selection approach was applied in another leukemia cell line, a cytokine dependent UT7 cells, after transduction depriving them of exogenous cytokines and supplying the AP20187. In multiple independent transductions only a few percent of the transduced cells survived in the presence of AP20187, as assessed by the GFP expression, which was co-expressed with the Mpl fusion gene through an internal ribosome entry site. The surviving cells in long term culture were dependent on Mpl signaling and were sensitive to a Jak2 inhibitor, AG490. A wide variation in the mean fluorescent intensity of the surviving cells (from 37.5 to 238.8) in independent transductions indicated that a high level of Mpl expression was not the critical factor of selection. Transduction conditions were designed so that each experiment started with 30,000 independent insertion events from which selection of an Mpl-dependent cell population could be derived. Thirty six independent transductions have been performed with a presumed ∼ 1.89 × 106 independent insertions in the initial non-selected, cell population. After selection, 668 retroviral insertion sites (RIS) have been recovered that represent 203 independent insertion events. Within each independent transduction, a small number of RISs were identified suggesting selection of insertion events that result in a proliferative advantage. This was not observed in an independent experiment in K562 cells, in which the cells were not switched to Mpl dependent growth. Insertion events that occurred in close proximity are considered to indicate a potential cooperating mutation that results in a proliferative and/or survival advantage to the cell. Insertional events in several instances fell in regions of known frequent chromosomal aberrations in MPN. The distribution of insertions appears to be non-random. Many sites are adjacent to genes that have been identified as regulators of apoptosis, proliferation and myeloid development (data not shown). We are validating this conclusion by performing a parallel screening of RIS in multiple (40) populations of independent transductions of the K562 and UT7 cells transduced with retrovirus/Mpl vector without selection on AP20187. This screening will provide sufficient data to statistically compare the outcome of the two sets of data, AP20187- selected versus non selected cell populations. To date, we have a number of candidate genes. The Discs large homolog-associated protein 1 (DLGAP1) gene is implicated secondary to three independent insertion events at 18p11.3. All insertions are in the fourth intron. This protein interacts with a known tumor suppressor and is being investigated as a possible novel hematopoietic regulator. Disclosures: No relevant conflicts of interest to declare.

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Appelt, Jens U., Frank A. Giordano, Marcel Zimmermann, Stephan Weinhard, Nadja Grund, Agnes Hotz-Wagenblatt, W. Jens Zeller, Heike Allgayer, Stefan Fruehauf y Stephanie Laufs. "Genes Involved in Acute Leukemias Are Favored Targets of HIV Vector Integration". Blood 110, n.º 11 (16 de noviembre de 2007): 3738. http://dx.doi.org/10.1182/blood.v110.11.3738.3738.

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Abstract Insertional mutagenesis and development of leukemia following retroviral gene therapy has created intense interest in assessing the safety of viral vectors for further gene therapy trials. Using the gtsg.org database we analyzed more than 14,900 different viral integration sites of ASLV, FIV, FV, HIV, MLV and SIV based vectors in terms of insertions into fragile sites, cancer genes, transcription factor binding sites, CpG islands, and repetitive elements (SINE, LINE, LTR elements). When we compared these data with our newly generated random set, containing 1,000,000 random integrations, we discovered that the gene density on fragile sites strongly correlates to the HIV vector insertion frequency. Furthermore, we report a up to a five fold increased frequency of HIV, MLV and SIV insertions in cancer genes. The majority of cancer genes preferentially hit by HIV viruses were found associated to acute leukemias, while MLV and SIV vector insertion sites are seen more evenly spread over the cancer gene repertoire. When analyzing different cell entities, it turned out that CD34+ hematopoetic stem cells had highest rates of intragenic insertions and hosted significantly more HIV and FV insertions in cancer genes than other cell types, such as HeLa, T cells, 293T cells, macrophages, fibroblasts, or SupT1 cells.

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Kustikova, Olga, Zhixiong Li, Christopher Baum y Boris Fehse. "Insertion Sites of Retroviral Gene-Marking Vectors Influence the Contribution of Individual, Non-Malignant Cell Clones to Long-Term Hematopoiesis." Blood 104, n.º 11 (16 de noviembre de 2004): 293. http://dx.doi.org/10.1182/blood.v104.11.293.293.

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Abstract Replication-defective gammaretroviral vectors have successfully been used for gene marking and therapy studies. Recently, we and others have shown that proto-oncogene upregulation due to retroviral vector insertion may trigger malignant transformation of engineered cells. We have now investigated whether insertional mutagenesis may also influence the contribution of individual cell clones to normal long-term hematopoiesis. Therefore, using LM-PCR we retrieved retroviral insertion sites from hematopoietic cells of mice exhibiting progression to oligoclonal or monoclonal hematopoiesis upon serial bone marrow transplantation. From 28 mice analyzed (12 primary, 16 secondary) we obtained a total of 67 insertions that matched the murine genome. In samples harvested from primary recipients seven months after transplantation of gene-marked cells a number of intriguing hits (HoxB4, Evi1, Ly78, Ccnd3, Pip5k2a) was found obviously already reflecting selection for long-term repopulating ability. Strikingly, almost all clones analyzed five months after serial transplantation revealed hits in genes with an established or potential role in the self-renewal process of hematopoietic stem cells (e.g., Vegfa, HoxB4, CyclinD3, Evi1) or other genes involved in the regulation of cell survival (Ly78, Pip5k2a, Irf2bp, Cflar). Two (Evi1, Ccnd3) among the 13 insertions associated with serial repopulation activity were already established common integration sites (CIS) in the context of mouse leukemias elicited by replication-competent retroviruses (RTCGD database). Using RT-PCR transcriptional activation involving fusion transcripts originating from the retroviral LTR into exons could be detected for the two genes (HoxB4, Vegfa) tested. Interestingly, Evi1 was the only gene targeted several times - with distinct insertions in 5 different clones. However, despite the fact that Evi1 was also the only established proto-oncogene found to be marked, four of the clones detected in primary recipients were lost upon serial transplantation. This indicates that insertion into the Evi1 locus, although having the potential to promote engraftment and/or survival of a given clone, was not sufficient for malignant transformation. Thus our data demonstrate a delicate regulatory balance following insertional mutagenesis by replication-deficient retroviral vectors. These findings have major implications for interpreting results from diagnostic gene marking experiments, for gene therapy, and the discovery of genes regulating stem cell turnover. (The first two authors contributed equally to this work)

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Coyote-Maestas, Willow, David Nedrud, Steffan Okorafor, Yungui He y Daniel Schmidt. "Targeted insertional mutagenesis libraries for deep domain insertion profiling". Nucleic Acids Research 48, n.º 2 (4 de diciembre de 2019): 1010. http://dx.doi.org/10.1093/nar/gkz1155.

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Bokhoven, Marieke, Sam L. Stephen, Sean Knight, Evelien F. Gevers, Iain C. Robinson, Yasuhiro Takeuchi y Mary K. Collins. "Insertional Gene Activation by Lentiviral and Gammaretroviral Vectors". Journal of Virology 83, n.º 1 (22 de octubre de 2008): 283–94. http://dx.doi.org/10.1128/jvi.01865-08.

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ABSTRACT Gammaretroviral and lentiviral vectors are promising tools for gene therapy, but they can be oncogenic. The development of safer vectors depends on a quantitative assay for insertional mutagenesis. Here we report a rapid, inexpensive, and reproducible assay which uses a murine cell line to measure the frequency of interleukin-3 (IL-3)-independent mutants. Lentiviral and gammaretroviral vectors cause insertional mutagenesis at similar frequencies; however, they use different mechanisms. Human immunodeficiency virus (HIV)-based vectors generate mutants by insertion only into the growth hormone receptor (Ghr) locus. The HIV enhancer/promoter is active in the absence of the HIV Tat protein in this locus, and an HIV/Ghr spliced transcript expresses GHR and cells respond to GH. Deletion of the enhancer/promoter in a self-inactivating HIV-based vector prevents this mechanism of insertional mutagenesis. In contrast, gammaretroviral vectors insert into other loci, including IL-3 and genes identified as common insertion sites in the Retroviral Tagged Cancer Gene Database (RTCGD).

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Narberhaus, Franz y Sylvia Balsiger. "Structure-Function Studies of Escherichia coli RpoH (σ32) by In Vitro Linker Insertion Mutagenesis". Journal of Bacteriology 185, n.º 9 (1 de mayo de 2003): 2731–38. http://dx.doi.org/10.1128/jb.185.9.2731-2738.2003.

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ABSTRACT The sigma factor RpoH (σ32) is the key regulator of the heat shock response in Escherichia coli. Many structural and functional properties of the sigma factor are poorly understood. To gain further insight into RpoH regions that are either important or dispensable for its cellular activity, we generated a collection of tetrapeptide insertion variants by a recently established in vitro linker insertion mutagenesis technique. Thirty-one distinct insertions were obtained, and their sigma factor activity was analyzed by using a groE-lacZ reporter fusion in an rpoH-negative background. Our study provides a map of permissive sites which tolerate linker insertions and of functionally important regions at which a linker insertion impairs sigma factor activity. Selected linker insertion mutants will be discussed in the light of known sigma factor properties and in relation to a modeled structure of an RpoH fragment containing region 2.

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Gorski, Lisa y Dale Kaiser. "Targeted Mutagenesis of ς54 Activator Proteins in Myxococcus xanthus". Journal of Bacteriology 180, n.º 22 (15 de noviembre de 1998): 5896–905. http://dx.doi.org/10.1128/jb.180.22.5896-5905.1998.

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ABSTRACT Myxococcus xanthus DNA segments related to the highly conserved central sequence of ς54 activator proteins have been investigated. A genetic technique designed to inactivate a gene that encodes such an activator by inserting a plasmid-borne internal fragment of the putative gene has been tested. When the internal fragment inserted by homologous recombination into the corresponding chromosomal locus, the expected duplication of the gene was observed by Southern hybridization. The single restriction fragment characteristic of each segment was replaced in the insertion strains by two hybridizing fragments, and one of these fragments hybridized with the kanamycin resistance gene of the plasmid vector. The combined molecular weights of the two fragments from the insertion strains were equal to the molecular weight of the original fragment plus the expected molecular weight contributed by the vector. In the duplication, one copy is expected to have an N-terminal deletion and the other copy is expected to have a C-terminal deletion. In most cases, the net result should be loss of activator function. If an activator is essential for vegetative growth, then it should not be possible to obtain the insertion strain by plasmid integration. Indeed, integrants for three of the segments were not obtained in repeated trials; however, a plausible explanation for these results other than lethality can be offered. Of the seven insertions validated by Southern hybridization, four strains exhibited defects in the development of fruiting bodies. One of these failed to develop in submerged culture, though it developed normally on agar. The other three showed arrested development of fruiting bodies, each at a morphologically different stage of aggregation. One of the mutants may be defective in the reception pathway of A-signal.

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Grech, Leanne, Daniel C. Jeffares, Christoph Y. Sadée, María Rodríguez-López, Danny A. Bitton, Mimoza Hoti, Carolina Biagosch et al. "Fitness Landscape of the Fission Yeast Genome". Molecular Biology and Evolution 36, n.º 8 (11 de mayo de 2019): 1612–23. http://dx.doi.org/10.1093/molbev/msz113.

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Abstract The relationship between DNA sequence, biochemical function, and molecular evolution is relatively well-described for protein-coding regions of genomes, but far less clear in noncoding regions, particularly, in eukaryote genomes. In part, this is because we lack a complete description of the essential noncoding elements in a eukaryote genome. To contribute to this challenge, we used saturating transposon mutagenesis to interrogate the Schizosaccharomyces pombe genome. We generated 31 million transposon insertions, a theoretical coverage of 2.4 insertions per genomic site. We applied a five-state hidden Markov model (HMM) to distinguish insertion-depleted regions from insertion biases. Both raw insertion-density and HMM-defined fitness estimates showed significant quantitative relationships to gene knockout fitness, genetic diversity, divergence, and expected functional regions based on transcription and gene annotations. Through several analyses, we conclude that transposon insertions produced fitness effects in 66–90% of the genome, including substantial portions of the noncoding regions. Based on the HMM, we estimate that 10% of the insertion depleted sites in the genome showed no signal of conservation between species and were weakly transcribed, demonstrating limitations of comparative genomics and transcriptomics to detect functional units. In this species, 3′- and 5′-untranslated regions were the most prominent insertion-depleted regions that were not represented in measures of constraint from comparative genomics. We conclude that the combination of transposon mutagenesis, evolutionary, and biochemical data can provide new insights into the relationship between genome function and molecular evolution.

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Pearson, Melanie M. y Eric J. Hansen. "Identification of Gene Products Involved in Biofilm Production by Moraxella catarrhalis ETSU-9 In Vitro". Infection and Immunity 75, n.º 9 (11 de junio de 2007): 4316–25. http://dx.doi.org/10.1128/iai.01347-06.

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ABSTRACT Moraxella catarrhalis ETSU-9 was subjected to random transposon insertion mutagenesis to identify genes encoding products involved in the ability of the organism to form biofilms in vitro. Screening of approximately 3,000 transposon insertion mutants in the crystal violet-based biofilm assay system yielded six mutants that exhibited greatly reduced abilities to form biofilms. Three of these mutants had transposon insertions in the uspA2H gene, which encodes a surface protein previously shown to be involved in the ability of M. catarrhalis to both attach to human cell lines in vitro and resist killing by normal human serum. Random insertion mutagenesis of the uspA2H gene, involving the introduction of a 15-nucleotide fragment encoding 5 amino acids, was used to attempt to identify the domain(s) necessary for biofilm formation. Most of these insertions adversely affected biofilm formation, whereas the abilities of these same mutants to attach to Chang conjunctival epithelial cells in vitro were usually not reduced. Gain-of-function experiments showed that introduction of the M. catarrhalis ETSU-9 uspA2H gene into Escherichia coli conferred biofilm formation ability on this recombinant strain. Two of the other three M. catarrhalis ETSU-9 transposon insertion mutants that had greatly reduced abilities to form biofilms were shown to have insertions in genes encoding products predicted to be directly or indirectly involved in cell wall metabolism.

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20

Begun, Jakob, Costi D. Sifri, Samuel Goldman, Stephen B. Calderwood y Frederick M. Ausubel. "Staphylococcus aureus Virulence Factors Identified by Using a High-Throughput Caenorhabditis elegans-Killing Model". Infection and Immunity 73, n.º 2 (febrero de 2005): 872–77. http://dx.doi.org/10.1128/iai.73.2.872-877.2005.

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ABSTRACT Staphylococcus aureus is an important human pathogen that is also able to kill the model nematode Caenorhabditis elegans. We constructed a 2,950-member Tn917 transposon insertion library in S. aureus strain NCTC 8325. Twenty-one of these insertions exhibited attenuated C. elegans killing, and of these, 12 contained insertions in different genes or chromosomal locations. Ten of these 12 insertions showed attenuated killing phenotypes when transduced into two different S. aureus strains, and 5 of the 10 mutants correspond to genes that have not been previously identified in signature-tagged mutagenesis studies. These latter five mutants were tested in a murine renal abscess model, and one mutant harboring an insertion in nagD exhibited attenuated virulence. Interestingly, Tn917 was shown to have a very strong bias for insertions near the terminus of DNA replication.

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21

Colegio, Oscar R., Thomas J. Griffin, Nigel D. F. Grindley y Jorge E. Galán. "In Vitro Transposition System for Efficient Generation of Random Mutants of Campylobacter jejuni". Journal of Bacteriology 183, n.º 7 (1 de abril de 2001): 2384–88. http://dx.doi.org/10.1128/jb.183.7.2384-2388.2001.

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ABSTRACT Campylobacter jejuni is the most common cause of food-borne illnesses in the United States. Despite the fact that the entire nucleotide sequence of its genome has recently become available, its mechanisms of pathogenicity are poorly understood. This is in part due to the lack of an efficient mutagenesis system. Here we describe an in vitro transposon mutagenesis system based on theStaphylococcus aureus transposable element Tn552 that allows the efficient generation of insertion mutants of C. jejuni. Insertions occur randomly and throughout the entire bacterial genome. We have tested this system in the isolation of nonmotile mutants of C. jejuni. Demonstrating the utility of the system, six nonmotile mutants from a total of nine exhibited insertions in genes known to be associated with motility. An additional mutant had an inactivating insertion in sigma 54, implicating this transcription factor in flagellum regulation. The availability of this efficient system will greatly facilitate the study of the mechanisms of pathogenesis of this important pathogen.

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22

Kraiß, Anita, Stefan Schlör y Joachim Reidl. "In Vivo Transposon Mutagenesis inHaemophilus influenzae". Applied and Environmental Microbiology 64, n.º 12 (1 de diciembre de 1998): 4697–702. http://dx.doi.org/10.1128/aem.64.12.4697-4702.1998.

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ABSTRACT In order to devise an in vivo insertion mutagenesis scheme forHaemophilus influenzae, a novel set of transposons has been constructed. These are Tn10-based minitransposons carried on pACYC184- and pACYC177-based replicons, which are suitable for in vivo transposition in H. influenzae. The transposon delivery system was designed to contain an H. influenzae-specific uptake signal sequence which facilitates DNA transformation into H. influenzae. The following mini-Tn10 elements have been made suitable for specific tasks in H. influenzae: (i) Tn10d-cat, which can be used to generate chloramphenicol-selectable insertion mutations; (ii) Tn10d-bla, an ampicillin-selectable translational fusion system allowing the detection of membrane or secreted proteins; and (iii) Tn10d-lacZcat, a chloramphenicol-selectablelacZ transcriptional fusion system. For the rapid identification of the transposon insertions, a PCR fragment enrichment method was developed. This report demonstrates that this in vivo mutagenesis technique is a convenient tool for the analysis of biochemical and regulatory pathways in the human pathogen H. influenzae.

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23

Chen, Wenbiao, Shawn Burgess, Greg Golling, Adam Amsterdam y Nancy Hopkins. "High-Throughput Selection of Retrovirus Producer Cell Lines Leads to Markedly Improved Efficiency of Germ Line-Transmissible Insertions in Zebra Fish". Journal of Virology 76, n.º 5 (1 de marzo de 2002): 2192–98. http://dx.doi.org/10.1128/jvi.76.5.2192-2198.2002.

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ABSTRACT Vesicular stomatitis virus glycoprotein G-pseudotyped mouse retroviral vectors have been used as mutagens for a large-scale insertional mutagenesis screen in the zebra fish. To reproducibly generate high-titer virus stocks, we devised a method for rapidly selecting cell lines that can yield high-titer viruses and isolated a producer cell line that yields virus at a high titer on zebra fish embryos. Virus produced from this line, designated GT virus, is nontoxic following injection of zebra fish blastulae and efficiently infects embryonic cells that give rise to the future germ line. Using GT virus preparations we generated roughly 500,000 germ line-transmissible proviral insertions in a population of 25,000 founder fish in about 2 months. The GT virus contains a gene trap, and trap events can be detected in the offspring of almost every founder fish. We discuss potential applications of this highly efficient method for generating germ line-transmissible insertions in a vertebrate

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24

Burns, Kathleen H. "Comprehensive Mapping of Transposon Insertions in Human Hematopoietic Neoplasias." Blood 114, n.º 22 (20 de noviembre de 2009): 1103. http://dx.doi.org/10.1182/blood.v114.22.1103.1103.

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Abstract 1103 Poster Board I-125 Our genomes are replete with mobile DNAs, many of which are retrotransposons that have accumulated over time by “copy-and-paste” mechanisms involving reverse transcription of RNA intermediates. Subsets of human transposable elements have been recently active or remain active today, resulting in many insertional polymorphisms in modern populations. In vitro studies in human tumor cell lines have unequivocally shown that expressed retrotransposons can generate new insertions and potentiate large scale genomic rearrangements. Though normally transposon sequences are highly methylated and thus stably suppressed in somatic cells, loss of methylation has been described in some malignant states, including chronic lymphocytic leukemia (CLL), multiple myeloma (MM), and ‘blast crisis’ phase chronic myeloid leukemia (CML). This has led to speculation that derepressed transposons contribute to clonal evolution of these pathologies, though experimental evidence for this has been lacking due to an inability to detect new genomic insertions. In collaboration with Jef Boeke's laboratory, I have developed an array-based transposon insertion profiling method (TIP-chip) for mapping mobile retrotransposons in the human genome. Early application of this technology in leukemia patients and leukemia cell lines shows numerous novel insertions of L1 LINEs, AluYb SINEs, and HERV-K transposons, including several insertions in genes known to be involved in leukemogenesis. We expect the technology will add a new dimension to our understanding of the human genome, including genetic predispositions to cancer development, and will enable tests of the hypothesis that insertional mutagenesis by endogenous transposons is a driving force in hematopoietic malignancies. Disclosures No relevant conflicts of interest to declare.

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25

Yesilkaya, Hasan, Jeremy W. Dale, Norval J. C. Strachan y Ken J. Forbes. "Natural Transposon Mutagenesis of Clinical Isolates of Mycobacterium tuberculosis: How Many Genes Does a Pathogen Need?" Journal of Bacteriology 187, n.º 19 (1 de octubre de 2005): 6726–32. http://dx.doi.org/10.1128/jb.187.19.6726-6732.2005.

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ABSTRACT Transposable elements can affect an organism's fitness through the insertional inactivation of genes and can therefore be used to identify genes that are nonessential for growth in vitro or in animal models. However, these models may not adequately represent the genetic requirements during chains of human infection. We have therefore conducted a genome-wide survey of transposon mutations in Mycobacterium tuberculosis isolates from cases of human infection, identifying the precise, base-specific insertion sites of the naturally occurring transposable element IS6110. Of 294 distinct insertions mapped to the strain H37Rv genome, 180 were intragenic, affecting 100 open reading frames. The number of genes carrying IS6110 in clinical isolates, and hence apparently not essential for infection and transmission, is very much lower than the estimates of nonessential genes derived from in vitro studies. This suggests that most genes in M. tuberculosis play a significant role in human infection chains. IS6110 insertions were underrepresented in genes associated with virulence, information pathways, lipid metabolism, and membrane proteins but overrepresented in multicopy genes of the PPE family, genes of unknown function, and intergenic sequences. Population genomic analysis of isolates recovered from an organism's natural habitat is an important tool for determining the significance of genes or classes of genes in the natural biology of an organism.

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26

Golden, Neal J., Andrew Camilli y David W. K. Acheson. "Random Transposon Mutagenesis ofCampylobacter jejuni". Infection and Immunity 68, n.º 9 (1 de septiembre de 2000): 5450–53. http://dx.doi.org/10.1128/iai.68.9.5450-5453.2000.

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ABSTRACT Genetic studies of Campylobacter jejuni have been limited due to the lack of a transposon mutagenesis method. Here, we describe a novel technique for random transposon mutagenesis using amariner-based transposon into C. jejuni strain 480. Insertions were random, as demonstrated by Southern blot analysis and insertional junction sequencing. We have demonstrated, for the first time, random in vivo transposon mutagenesis of C. jejuni.

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27

Liu, Ru-Juan, Min Tan, Dao-Hai Du, Bei-Si Xu, Gilbert Eriani y En-Duo Wang. "Peripheral insertion modulates the editing activity of the isolated CP1 domain of leucyl-tRNA synthetase". Biochemical Journal 440, n.º 2 (14 de noviembre de 2011): 217–27. http://dx.doi.org/10.1042/bj20111177.

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A large insertion domain called CP1 (connective peptide 1) present in class Ia aminoacyl-tRNA synthetases is responsible for post-transfer editing. LeuRS (leucyl-tRNA synthetase) from Aquifex aeolicus and Giardia lamblia possess unique 20 and 59 amino acid insertions respectively within the CP1 that are crucial for editing activity. Crystal structures of AaLeuRS-CP1 [2.4 Å (1 Å=0.1 nm)], GlLeuRS-CP1 (2.6 Å) and the insertion deletion mutant AaLeuRS-CP1Δ20 (2.5 Å) were solved to understand the role of these insertions in editing. Both insertions are folded as peripheral motifs located on the opposite side of the proteins from the active-site entrance in the CP1 domain. Docking modelling and site-directed mutagenesis showed that the insertions do not interact with the substrates. Results of molecular dynamics simulations show that the intact CP1 is more dynamic than its mutant devoid of the insertion motif. Taken together, the data show that a peripheral insertion without a substrate-binding site or major structural role in the active site may modulate catalytic function of a protein, probably from protein dynamics regulation in two respective LeuRS CP1s. Further results from proline and glycine mutational analyses intended to reduce or increase protein flexibility are consistent with this hypothesis.

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28

Hamilton, Holly L., Kevin J. Schwartz y Joseph P. Dillard. "Insertion-Duplication Mutagenesis ofNeisseria: Use in Characterization of DNA Transfer Genes in the Gonococcal Genetic Island". Journal of Bacteriology 183, n.º 16 (15 de agosto de 2001): 4718–26. http://dx.doi.org/10.1128/jb.183.16.4718-4726.2001.

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ABSTRACT We created plasmids for use in insertion-duplication mutagenesis (IDM) of Neisseria gonorrhoeae. This mutagenesis method has the advantage that it requires only a single cloning step prior to transformation into gonococci. Chromosomal DNA cloned into the plasmid directs insertion into the chromosome at the site of homology by a single-crossover (Campbell-type) recombination event. Two of the vectors contain an erythromycin resistance gene, ermC, with a strong promoter and in an orientation such that transcription will proceed into the cloned insert. Thus, these plasmids can be used to create insertions that are effectively nonpolar on the transcription of downstream genes. In addition to the improved ermC, the vector contains two copies of the neisserial DNA uptake sequence to facilitate high-frequency DNA uptake during transformation. Using various chromosomal DNA insert sizes, we have determined that even small inserts can target insertion mutation by this method and that the insertions are stably maintained in the gonococcal chromosome. We have used IDM to create knockouts in two genes in the gonococcal genetic island (GGI) and to clone additional regions of the GGI by a chromosome-walking procedure. Phenotypic characterization oftraG and traH mutants suggests a role for the encoded proteins in DNA secretion by a novel type IV secretion system.

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29

de Ridder, Jeroen, Anthony Uren, Jaap Kool, Marcel Reinders y Lodewyk Wessels. "Detecting Statistically Significant Common Insertion Sites in Retroviral Insertional Mutagenesis Screens". PLoS Computational Biology 2, n.º 12 (2006): e166. http://dx.doi.org/10.1371/journal.pcbi.0020166.

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30

de Ridder, Jeroen, Anthony Uren, Jaap Kool, Marcel J. T. Reinders y Lodewyk F. A. Wessels. "Detecting Statistically Significant Common Insertion Sites in Retroviral Insertional Mutagenesis Screens". PLoS Computational Biology preprint, n.º 2006 (2005): e166. http://dx.doi.org/10.1371/journal.pcbi.0020166.eor.

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31

Mutaqin, Kikin, Jana L. Comer, Astri C. Wayadande, Ulrich Melcher y Jacqueline Fletcher. "Selection and characterization ofSpiroplasma citrimutants by random transposome mutagenesis". Canadian Journal of Microbiology 57, n.º 6 (junio de 2011): 525–32. http://dx.doi.org/10.1139/w11-026.

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Phytopathogenic spiroplasmas can multiply in vascular plants and insects. A deeper understanding of this dual-host life could be furthered through the identification by random mutagenesis of spiroplasma genes required. The ability of the EZ::TN™ <DHFR-1> Tnp transposome™ system to create random insertional mutations in the genome of Spiroplasma citri was evaluated. The efficiency of electroporation-mediated transformation of S. citri BR3-3X averaged 28.8 CFUs/ng transposome for 109spiroplasma cells. Many transformants appearing on the selection plates were growth impaired when transferred to broth. Altering broth composition in various ways did not improve their growth. However, placing colonies into a small broth volume resulted in robust growth and successful subsequent passages of a subset of transformants. PCR using primers for the dihydrofolate reductase gene confirmed the transposon’s presence in the genomes of selected transformants. Southern blot hybridization and nucleotide sequencing suggested that insertion was random within the chromosome and usually at single sites. The insertions were stable. Growth rates of all transformants were lower than that of the wild-type S. citri, but none lost the ability to adhere to a Circulifer tenellus (CT-1) cell line. The EZ::TN™ <DHFR-1> Tnp transposome™ system represents an additional tool for genetic manipulation of the fastidious spiroplasmas.

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32

Bergerson, Rachel J. S., Lara S. Collier, Tony Cox, Wendy A. Hudson, Raha Allaei, Linda Wolff, John H. Kersey, David J. Adams y David A. Largaespada. "A Screen for Mll-AF9 Cooperating Mutations in Leukemogenesis Using MLV-Based Mutagenesis." Blood 108, n.º 11 (16 de noviembre de 2006): 1417. http://dx.doi.org/10.1182/blood.v108.11.1417.1417.

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Abstract Human translocations involving the MLL gene are associated with a variety of myeloid and lymphoid leukemia. The MLL-AF9 translocation is common in acute myeloid leukemia (AML). C57BL/6 mice with a Mll-AF9 knock-in mimic the phenotype observed in human patients with this translocation: 75% develop AML but only after a preleukemic phase and prolonged latency. A minority of these mice develop acute lymphoblastic leukemia (ALL) (Dobson, et al. EMBO J.1999, 18(13): 3564). This model provides a system for understanding the evolution of AML initiated by a MLL fusion oncoprotein, including the identification of cooperating genetic events required for AML induction. The recombinant retrovirus MOL4070LTR (M4070) induces AML in some strains of mice (Wolff, et al. J Virol.2003, 77(8): 4965). We hypothesized that this virus could cooperate with the MLL-AF9 oncoprotein to accelerate AML by acting as an insertional mutagen, providing second “hits” in leukemia progression. We bred Mll-AF9 heterozygous C57BL/6 males to 129/SvJ wild type (WT) females, and injected the 280 offspring at 3 days of age with either M4070 virus (212) or a control supernatant (68). All mice were genotyped and observed for disease progression. Infected Mll-AF9/+ mice succumb to disease with a significantly reduced latency period when compared to virus treated WT mice (p < .0001) and uninfected Mll-AF9/+ mice (p < .0001), indicating that M4070 infection causes significant leukemia acceleration in these mice. The gross pathology and preliminary analysis of the surface immunophentoype by flow cytometry and Southern blot indicate infected Mll-AF9/+ animals present with primarily myeloid leukemia while infected WT animals present with lymphoid leukemia. To determine what genes, when mutated by M4070 insertion, can cooperate with Mll-AF9, retroviral insertion sites have been cloned from over 140 diseased tissues from our accelerated leukemia animals using a shotgun-based, linker-mediated, cloning protocol. More than 1,000 independent insertions have been isolated and are being analyzed to identify common insertion sites (CIS). So far, we have found several CIS enriched in Mll-AF9/+ AML accelerated by M4070 insertion. We plan to verify nearby genes for cooperation with MLL-AF9 in AML development using human microarray data and mouse modeling.

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33

Ruzin, Alexey, David Keeney y Patricia A. Bradford. "AcrAB Efflux Pump Plays a Role in Decreased Susceptibility to Tigecycline in Morganella morganii". Antimicrobial Agents and Chemotherapy 49, n.º 2 (febrero de 2005): 791–93. http://dx.doi.org/10.1128/aac.49.2.791-793.2005.

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ABSTRACT Transposon mutagenesis of a clinical isolate of Morganella morganii, G1492 (tigecycline MIC of 4 μg/ml), yielded two insertion knockout mutants for which tigecycline MICs were 0.03 μg/ml. Transposon insertions mapped to acrA, which is constitutively overexpressed in G1492, suggesting a role of the AcrAB efflux pump in decreased susceptibility to tigecycline in M. morganii.

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34

Dalby, B., A. J. Pereira y L. S. Goldstein. "An inverse PCR screen for the detection of P element insertions in cloned genomic intervals in Drosophila melanogaster." Genetics 139, n.º 2 (1 de febrero de 1995): 757–66. http://dx.doi.org/10.1093/genetics/139.2.757.

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Abstract We developed a screening approach that utilizes an inverse polymerase chain reaction (PCR) to detect P element insertions in or near previously cloned genes in Drosophila melanogaster. We used this approach in a large scale genetic screen in which P elements were mobilized from sites on the X chromosome to new autosomal locations. Mutagenized flies were combined in pools, and our screening approach was used to generate probes corresponding to the sequences flanking each site of insertion. These probes then were used for hybridization to cloned genomic intervals, allowing individuals carrying insertions in them to be detected. We used the same approach to perform repeated rounds of sib-selection to generate stable insertion lines. We screened 16,100 insert bearing individuals and recovered 11 insertions in five intervals containing genes encoding members of the kinesin superfamily in Drosophila melanogaster. In addition, we recovered an insertion in the region including the Larval Serum Protein-2 gene. Examination by Southern hybridization confirms that the lines we recovered represent genuine insertions in the corresponding genomic intervals. Our data indicates that this approach will be very efficient both for P element mutagenesis of new genomic regions and for detection and recovery of "local" P element transposition events. In addition, our data constitutes a survey of preferred P element insertion sites in the Drosophila genome and suggests that insertion sites that are mutable at a rate of approximately 10(-4) are distributed every 40-50 kb.

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35

Cleveland, Susan M. y Utpal P. Dave. "Insertional Activation of GLI2 in Adult T-Cell Leukemia/Lymphoma." Blood 110, n.º 11 (16 de noviembre de 2007): 4149. http://dx.doi.org/10.1182/blood.v110.11.4149.4149.

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Abstract Retroviruses induce cancer by integrating into the cellular genome and activating oncogenes or inactivating tumor suppressor genes. Human T-cell Leukemia Virus type 1 (HTLV-1), a complex retrovirus, induces Adult T-cell Leukemia/Lymphoma (ATLL) after a latency of over 30 years and in only 5% of carriers. The long latency and incomplete penetrance is similar to how slow transforming retroviruses induce cancer in mice and imply multiple oncogenic “hits” need to accumulate for clinically apparent disease. Insertional mutagenesis may be one mechanism by which ATLL develops. We used splinkerette-PCR to clone and map insertion sites from an HTLV-1 infected T-cell line, Hut-102. We identified an HTLV-1 insertion 5′ of the GLI2 gene, formerly known as Tax-Helper-Protein-1. We found GLI2 was up-regulated by promoter insertion. Interestingly, we found GLI2 protein occupied the HTLV-1 Long Terminal Repeat. The effect of GLI2 expression on viral expression was investigated by knockdown of GLI2 in Hut-102 cells. Our results show that retroviral insertional mutagenesis can be an important mechanism in HTLV-1-induced leukemias and lymphomas.

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36

Maier, Tamara M., Roger Pechous, Monika Casey, Thomas C. Zahrt y D. W. Frank. "In Vivo Himar1-Based Transposon Mutagenesis of Francisella tularensis". Applied and Environmental Microbiology 72, n.º 3 (marzo de 2006): 1878–85. http://dx.doi.org/10.1128/aem.72.3.1878-1885.2006.

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ABSTRACT Francisella tularensis is the intracellular pathogen that causes human tularemia. It is recognized as a potential agent of bioterrorism due to its low infectious dose and multiple routes of entry. We report the development of a Himar1-based random mutagenesis system for F. tularensis (HimarFT). In vivo mutagenesis of F. tularensis live vaccine strain (LVS) with HimarFT occurs at high efficiency. Approximately 12 to 15% of cells transformed with the delivery plasmid result in transposon insertion into the genome. Results from Southern blot analysis of 33 random isolates suggest that single insertions occurred, accompanied by the loss of the plasmid vehicle in most cases. Nucleotide sequence analysis of rescued genomic DNA with HimarFT indicates that the orientation of integration was unbiased and that insertions occurred in open reading frames and intergenic and repetitive regions of the chromosome. To determine the utility of the system, transposon mutagenesis was performed, followed by a screen for growth on Chamberlain's chemically defined medium (CDM) to isolate auxotrophic mutants. Several mutants were isolated that grew on complex but not on the CDM. We genetically complemented two of the mutants for growth on CDM with a newly constructed plasmid containing a nourseothricin resistance marker. In addition, uracil or aromatic amino acid supplementation of CDM supported growth of isolates with insertions in pyrD, carA, or aroE1 supporting the functional assignment of genes within each biosynthetic pathway. A mutant containing an insertion in aroE1 demonstrated delayed replication in macrophages and was restored to the parental growth phenotype when provided with the appropriate plasmid in trans. Our results suggest that a comprehensive library of mutants can be generated in F. tularensis LVS, providing an additional genetic tool to identify virulence determinants required for survival within the host.

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37

Lee, Myeong S., Chaok Seok y Donald A. Morrison. "Insertion-Duplication Mutagenesis inStreptococcus pneumoniae: Targeting Fragment Length Is a Critical Parameter in Use as a Random Insertion Tool". Applied and Environmental Microbiology 64, n.º 12 (1 de diciembre de 1998): 4796–802. http://dx.doi.org/10.1128/aem.64.12.4796-4802.1998.

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ABSTRACT To examine whether insertion-duplication mutagenesis with chimeric DNA as a transformation donor could be valuable as a gene knockout tool for genomic analysis in Streptococcus pneumoniae, we studied the transformation efficiency and targeting specificity of the process by using a nonreplicative vector with homologous targeting inserts of various sizes. Insertional recombination was very specific in targeting homologous sites. While the recombination rate did not depend on which site or region was targeted, it did depend strongly on the size of the targeting insert in the donor plasmid, in proportion to the fifth power of its length for inserts of 100 to 500 bp. The dependence of insertion-duplication events on the length of the targeting homology was quite different from that for linear allele replacement and places certain limits on the design of mutagenesis experiments. The number of independent pneumococcal targeting fragments of uniform size required to knock out any desired fraction of the genes in a model genome with a defined probability was calculated from these data by using a combinatorial theory with simplifying assumptions. The results show that efficient and thorough mutagenesis of a large part of the pneumococcal genome should be practical when using insertion-duplication mutagenesis.

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38

Greenberg, Kathleen E., Li Li, David Huso, Linda Wolff y Donald Small. "Retroviral Insertional Mutagenesis Reveals Genes That Cooperate with FLT3-ITD in Leukemogenesis." Blood 114, n.º 22 (20 de noviembre de 2009): 1960. http://dx.doi.org/10.1182/blood.v114.22.1960.1960.

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Abstract Abstract 1960 Poster Board I-983 FLT3 is a receptor tyrosine kinase that plays an important role in hematopoiesis and is one of the most mutated genes in acute myeloid leukemia (AML). The internal tandem duplication (ITD) mutation, which causes autophosphorylation and constitutive activation of the receptor, has been found in up to 35% of AML and 3% of ALL patients. Our lab has created and described a FLT3ITD/wt knock-in mouse with the mutation expressed under the endogenous promoter. These mice develop myeloproliferative disorder (MPD) with a median time to disease of 52 weeks, but do not progress to acute leukemia, indicating that an additional genetic event is required for leukemogenesis. We have used the MOL4070LTR retrovirus in a retroviral insertional mutagenesis (RIM) screen to identify genes that can cooperate with the FLT3/ITD to cause leukemia. FLT3ITD/wt mice were injected with the retrovirus at birth and 78% developed leukemia with a median time to disease of 18 weeks (50% B-ALL/lymphoma, 18% AML, 10% biphenotypic leukemia). By comparison, retrovirus-injected FLT3wt/wt mice developed leukemias with a much longer median time to disease of 69 weeks. The decreased latency in the retrovirally-infected FLT3ITD/wt mice implies that the viral integrations have created mutations that collaborate with FLT3/ITD signaling to generate leukemia. To identify the genes responsible, we cloned the insertion sites using an inverse PCR technique. 343 integration sites were cloned from 25 FLT3ITD/wt mice that developed leukemia, 29 of which were found in two or more mice. These common insertion sites (CIS) are considered putative cooperating genes. The most common insertion, found in 5 mice, involved the Evi1 gene, with the virus inserting approximately 17kb upstream of the gene in three of the mice or approximately 100kb upstream of the gene in the other two mice. All of the mice showed overexpression of Evi1 by quantitative RT-PCR. This is an interesting collaborating gene because its overexpression has been described in AML and predicts a poor prognosis in patients, but mouse models have shown that, like FLT3/ITD, it is not leukemogenic on its own. Though singularly insufficient, together these two mutations may cooperate to cause leukemia. Another putative cooperating gene found in the screen is Ring1a. Insertions upstream of the gene (22kb, 96kb and 209kb) were found in 3 mice displaying myeloid-associated disease, and in all cases caused overexpression of Ring1a, as seen by quantitative RT-PCR. Strong expression of Ring1a, part of the PRC1 polycomb repressor complex, has been observed in several cancer types but not in myeloid leukemia; however, another member of PRC1, Bmi-1, seems to play an extensive role in myeloid leukemia. Integrations near Ring1a have not previously been described in the Retrovirus Tagged Cancer Gene Database (RTCGD mm8), indicating a specific cooperation with FLT3/ITD and a role in the disease. With these studies we have shown that additional mutations cooperate with the FLT3/ITD to cause leukemia and we have identified several possible collaborating genes, including Evi1 and Ring1a. Disclosures: No relevant conflicts of interest to declare.

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39

Hudson, P., T. S. Gorton, L. Papazisi, K. Cecchini, S. Frasca y S. J. Geary. "Identification of a Virulence-Associated Determinant, Dihydrolipoamide Dehydrogenase (lpd), in Mycoplasma gallisepticum through In Vivo Screening of Transposon Mutants". Infection and Immunity 74, n.º 2 (febrero de 2006): 931–39. http://dx.doi.org/10.1128/iai.74.2.931-939.2006.

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ABSTRACT To effectively analyze Mycoplasma gallisepticum for virulence-associated determinants, the ability to create stable genetic mutations is essential. Global M. gallisepticum mutagenesis is currently limited to the use of transposons. Using the gram-positive transposon Tn4001mod, a mutant library of 110 transformants was constructed and all insertion sites were mapped. To identify transposon insertion points, a unique primer directed outward from the end of Tn4001mod was used to sequence flanking genomic regions. By comparing sequences obtained in this manner to the annotated M. gallisepticum genome, the precise locations of transposon insertions were discerned. After determining the transposon insertion site for each mutant, unique reverse primers were synthesized based on the specific sequences, and PCR was performed. The resultant amplicons were used as unique Tn4001mod mutant identifiers. This procedure is referred to as signature sequence mutagenesis (SSM). SSM permits the comprehensive screening of the M. gallisepticum genome for the identification of novel virulence-associated determinants from a mixed mutant population. To this end, chickens were challenged with a pool of 27 unique Tn4001mod mutants. Two weeks postinfection, the birds were sacrificed, and organisms were recovered from respiratory tract tissues and screened for the presence or absence of various mutants. SSM is a negative-selection screening technique whereby those mutants possessing transposon insertions in genes essential for in vivo survival are not recovered from the host. We have identified a virulence-associated gene encoding dihydrolipoamide dehydrogenase (lpd). A transposon insertion in the middle of the coding sequence resulted in diminished biologic function and reduced virulence of the mutant designated Mg 7.

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Nunes, Alvaro, Vandana Thathy, Thomas Bruderer, Ali A. Sultan, Ruth S. Nussenzweig y Robert Ménard. "Subtle Mutagenesis by Ends-in Recombination in Malaria Parasites". Molecular and Cellular Biology 19, n.º 4 (1 de abril de 1999): 2895–902. http://dx.doi.org/10.1128/mcb.19.4.2895.

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ABSTRACT The recent advent of gene-targeting techniques in malaria (Plasmodium) parasites provides the means for introducing subtle mutations into their genome. Here, we used the TRAPgene of Plasmodium berghei as a target to test whether an ends-in strategy, i.e., targeting plasmids of the insertion type, may be suitable for subtle mutagenesis. We analyzed the recombinant loci generated by insertion of linear plasmids containing either base-pair substitutions, insertions, or deletions in their targeting sequence. We show that plasmid integration occurs via a double-strand gap repair mechanism. Although sequence heterologies located close (less than 450 bp) to the initial double-strand break (DSB) were often lost during plasmid integration, mutations located 600 bp and farther from the DSB were frequently maintained in the recombinant loci. The short lengths of gene conversion tracts associated with plasmid integration intoTRAP suggests that an ends-in strategy may be widely applicable to modify plasmodial genes and perform structure-function analyses of their important products.

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KING, Steven C., Liaoyuan A. HU y Amy PUGH. "Induction of substrate specificity shifts by placement of alanine insertions within the consensus amphipathic region of the Escherichia coli GABA (γ-aminobutyric acid) transporter encoded by gabP". Biochemical Journal 376, n.º 3 (15 de diciembre de 2003): 645–53. http://dx.doi.org/10.1042/bj20030595.

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The Escherichia coli GABA (γ-aminobutyric acid) permease GabP is a prototypical APC (amine/polyamine/choline) super-family transporter that has a CAR (consensus amphipathic region) containing multiple specificity determinants, ostensibly organized on two helical surfaces, one hydrophobic [SHS (sensitive hydrophobic surface)] and the other hydrophilic [SPS (sensitive polar surface)]. To gauge the functional effects of placing alanine insertions at close intervals across the entire GabP CAR, 64 insertion variants were constructed. Insertions, particularly those in the SHS and the SPS, were highly detrimental to steady-state [3H]GABA accumulation. TSR (transport specificity ratio) analysis, employing [3H]nipecotic acid and [14C]GABA, showed that certain alanine insertions were associated with a specificity shift (i.e. a change in kcat/Km). An insertion (INS Ala-269) located N-terminal to the SHS increased specificity for [3H]nipecotic acid relative to [14C]GABA, whereas an insertion (INS Ala-321) located C-terminal to the SPS had the opposite effect. Overall, the results are consistent with a working hypothesis that the GabP CAR contains extensive functional surfaces that may be manipulated by insertion mutagenesis to alter the specificity (kcat/Km) phenotype. The thermodynamic basis of TSR analysis provides generality, suggesting that amino acid insertions could affect specificity in many other transporters, particularly those such as the E. coli phenylalanine permease PheP [Pi, Chow and Pittard (2002) J. Bacteriol. 184, 5842–5847] that have a functionally significant CAR-like domain.

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Winterberg, Kelly M., John Luecke, Amanda S. Bruegl y William S. Reznikoff. "Phenotypic Screening of Escherichia coli K-12 Tn5 Insertion Libraries, Using Whole-Genome Oligonucleotide Microarrays". Applied and Environmental Microbiology 71, n.º 1 (enero de 2005): 451–59. http://dx.doi.org/10.1128/aem.71.1.451-459.2005.

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ABSTRACT Complete genome sequences in combination with global screening methods allow parallel analysis of multiple mutant loci to determine the requirement for specific genes in different environments. In this paper we describe a high-definition microarray approach for investigating the growth effects of Tn5 insertions in Escherichia coli K-12. Libraries of insertion mutants generated by a unique Tn5 mutagenesis system were grown competitively in defined media. Biotin-labeled runoff RNA transcripts were generated in vitro from transposon insertions in each population of mutants. These transcripts were then hybridized to custom-designed oligonucleotide microarrays to detect the presence of each mutant in the population. By using this approach, the signal associated with 25 auxotrophic insertions in a 50-mutant pool was not detectable following nine generations of growth in glucose M9 minimal medium. It was found that individual insertion sites could be mapped to within 50 bp of their genomic locations, and 340 dispensable regions in the E. coli chromosome were identified. Tn5 insertions were detected in 15 genes for which no previous insertions have been reported. Other applications of this method are discussed.

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Bergerson, Rachel J., Lara S. Collier, Raha Allaei, Kevin A. Silverstein, Anne-Francoise F. Lamblin, Linda Wolff, Scott C. Kogan, John H. Kersey, David J. Adams y David A. Largaespada. "Novel Mll-AF9 Cooperating Genes Identified in a Leukemogenesis Screen Using Retroviral Mutagenesis." Blood 110, n.º 11 (16 de noviembre de 2007): 825. http://dx.doi.org/10.1182/blood.v110.11.825.825.

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Abstract Human translocations involving the MLL gene are associated with a variety of myeloid and lymphoid leukemia. The MLL-AF9 translocation is common in acute myeloid leukemia (AML). C57BL/6 mice with a Mll-AF9 knock-in mimic the phenotype observed in human patients with this translocation: most develop AML but only after a preleukemic phase and prolonged latency. A minority of these mice develop acute lymphoblastic leukemia (ALL) (Dobson, et al. EMBO J. 1999, 18(13)). This model provides a system for understanding the evolution of AML initiated by a MLL fusion oncoprotein, including the identification of cooperating genetic events required for AML induction. The recombinant retrovirus MOL4070LTR (M4070) induces AML in some strains of mice (Wolff, et al. J Virol. 2003, 77(8)). We hypothesized that this virus could cooperate with the MLL-AF9 oncoprotein to accelerate AML by acting as an insertional mutagen, providing second hits in leukemia progression. We bred Mll-AF9 heterozygous C57BL/6 males to 129/SvJ wild type (WT) females, and injected the 280 offspring at 3 days of age with either M4070 virus (212) or a control supernatant (68). All mice were genotyped and observed for disease progression. Infected Mll-AF9/+ mice succumb to disease with a significantly reduced latency period when compared to virus treated WT mice (p < .0001) and uninfected Mll-AF9/+ mice (p < .0001), indicating that M4070 infection causes significant leukemia acceleration in these mice. Infected Mll - AF9 /+ Mice Succumb to Disease More Rapidly Than All Other Groups Infected Mll-AF9/+ Mice Succumb to Disease More Rapidly Than All Other Groups The gross pathology and analysis of the surface immunophentoype by flow cytometry and Southern blot indicate infected Mll-AF9/+ animals present with primarily myeloid leukemia while infected WT animals present with lymphoid leukemia. Retroviral insertion sites were cloned from 169 of our accelerated leukemia tissues using a shotgun-based, linker-mediated, cloning protocol to identify the genes most frequently mutated in these animals. From more than 20,000 sequence reads, over 4,700 independent proviral insertions were isolated and have been analyzed to identify 88 common insertion sites (CIS) present in at least 3 mice. We have found several CIS enriched in Mll-AF9/+ AMLs accelerated by M4070 insertion. We are examining the expression of CIS-associated genes in human AML with MLL gene translocations using human microarray data and in our murine leukemia samples using qRT-PCR to verify the viral insertions altered the expression of the candidate gene. We intend to test some of these genes for cooperation with MLL-AF9 in AML development using mouse transplantation assays. We are currently transducing bone marrow from Mll-AF9/+ mice with a retrovirus encoding a candidate gene to observe disease progression compared to controls.

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Garfinkel, David J., M. Joan Curcio, Susan D. Youngren y Nancy J. Sanders. "The biology and exploitation of the retrotransposon Ty in Saccharomyces cerevisiae". Genome 31, n.º 2 (15 de enero de 1989): 909–19. http://dx.doi.org/10.1139/g89-162.

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Retrotransposons are a widely distributed group of eukaryotic mobile genetic elements that transpose through an RNA intermediate. The element is transcribed into RNA, and this RNA is reverse transcribed into a DNA copy capable of insertion into many different chromosomal locations. Maturation of proteins and reverse transcription take place within noninfectious intracellular viruslike particles. We have studied the element Ty, which is found dispersed in the genome of the yeast Saccharomyces cerevisiae. The frequency of Ty element transposition is normally quite low but can be greatly increased by expressing an element from a strong promoter. We have used the ability to control the level of Ty transposition to investigate the functions of Ty proteins, the regulation of Ty transposition, and the exploitation of Ty elements as insertional mutagens in yeast. The information gained from these experiments should be applicable to the study of retrotransposons found in multicellular organisms.Key words: yeast, Saccharomyces cerevisiae, transposons, Ty elements, mutagenesis.

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45

Mandal, Ajeet, Antresh Kumar, Ashutosh Singh, Andrew M. Lynn, Khyati Kapoor y Rajendra Prasad. "A key structural domain of the Candida albicans Mdr1 protein". Biochemical Journal 445, n.º 3 (13 de julio de 2012): 313–22. http://dx.doi.org/10.1042/bj20120190.

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A major multidrug transporter, MDR1 (multidrug resistance 1), a member of the MFS (major facilitator superfamily), invariably contributes to an increased efflux of commonly used azoles and thus corroborates their direct involvement in MDR in Candida albicans. The Mdr1 protein has two transmembrane domains, each comprising six transmembrane helices, interconnected with extracellular loops and ICLs (intracellular loops). The introduction of deletions and insertions through mutagenesis was used to address the role of the largest interdomain ICL3 of the MDR1 protein. Most of the progressive deletants, when overexpressed, eliminated the drug resistance. Notably, restoration of the length of the ICL3 by insertional mutagenesis did not restore the functionality of the protein. Interestingly, most of the insertion and deletion variants of ICL3 became amenable to trypsinization, yielding peptide fragments. The homology model of the Mdr1 protein showed that the molecular surface-charge distribution was perturbed in most of the ICL3 mutant variants. Taken together, these results provide the first evidence that the CCL (central cytoplasmic loop) of the fungal MFS transporter of the DHA1 (drug/proton antiporter) family is critical for the function of MDR. Unlike other homologous proteins, ICL3 has no apparent role in imparting substrate specificity or in the recruitment of the transporter protein.

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46

Lanckriet, A., L. Timbermont, L. J. Happonen, M. I. Pajunen, F. Pasmans, F. Haesebrouck, R. Ducatelle, H. Savilahti y F. Van Immerseel. "Generation of Single-Copy Transposon Insertions in Clostridium perfringens by Electroporation of Phage Mu DNA Transposition Complexes". Applied and Environmental Microbiology 75, n.º 9 (6 de marzo de 2009): 2638–42. http://dx.doi.org/10.1128/aem.02214-08.

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ABSTRACT Transposon mutagenesis is a tool that is widely used for the identification of genes involved in the virulence of bacteria. Until now, transposon mutagenesis in Clostridium perfringens has been restricted to the use of Tn916-based methods with laboratory reference strains. This system yields primarily multiple transposon insertions in a single genome, thus compromising its use for the identification of virulence genes. The current study describes a new protocol for transposon mutagenesis in C. perfringens, which is based on the bacteriophage Mu transposition system. The protocol was successfully used to generate a single-insertion mutant library both for a laboratory strain and for a field isolate. Thus, it can be used as a tool in large-scale screening to identify virulence genes of C. perfringens.

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Thomy, Julie, Frederic Sanchez, Marta Gut, Fernando Cruz, Tyler Alioto, Gwenael Piganeau, Nigel Grimsley y Sheree Yau. "Combining Nanopore and Illumina Sequencing Permits Detailed Analysis of Insertion Mutations and Structural Variations Produced by PEG-Mediated Transformation in Ostreococcus tauri". Cells 10, n.º 3 (17 de marzo de 2021): 664. http://dx.doi.org/10.3390/cells10030664.

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Ostreococcus tauri is a simple unicellular green alga representing an ecologically important group of phytoplankton in oceans worldwide. Modern molecular techniques must be developed in order to understand the mechanisms that permit adaptation of microalgae to their environment. We present for the first time in O. tauri a detailed characterization of individual genomic integration events of foreign DNA of plasmid origin after PEG-mediated transformation. Vector integration occurred randomly at a single locus in the genome and mainly as a single copy. Thus, we confirmed the utility of this technique for insertional mutagenesis. While the mechanism of double-stranded DNA repair in the O. tauri model remains to be elucidated, we clearly demonstrate by genome resequencing that the integration of the vector leads to frequent structural variations (deletions/insertions and duplications) and some chromosomal rearrangements in the genome at the insertion loci. Furthermore, we often observed variations in the vector sequence itself. From these observations, we speculate that a nonhomologous end-joining-like mechanism is employed during random insertion events, as described in plants and other freshwater algal models. PEG-mediated transformation is therefore a promising molecular biology tool, not only for functional genomic studies, but also for biotechnological research in this ecologically important marine alga.

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Dorsey, Caleb W., Andrew P. Tomaras y Luis A. Actis. "Genetic and Phenotypic Analysis of Acinetobacter baumannii Insertion Derivatives Generated with a Transposome System". Applied and Environmental Microbiology 68, n.º 12 (diciembre de 2002): 6353–60. http://dx.doi.org/10.1128/aem.68.12.6353-6360.2002.

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ABSTRACT Acinetobacter baumannii is a metabolically versatile pathogen that causes severe infections in compromised patients. However, little is known about the genes and factors involved in its basic physiology and virulence properties. Insertion mutagenesis was used to initiate the identification and characterization of some of these factors and genes in the prototype strain 19606. The utilization of the pLOFKm suicide delivery vector, which harbors a suicide mini-Tn10 derivative, proved to be unsuccessful for this purpose. The EZ::TN 〈R6Kγori/KAN-2〉 Tnp transposome system available from Epicentre was then used in conjunction with electroporation to generate isogenic insertional derivatives of A. baumannii 19606. Replica plating showed that 2% of the colonies that grew after electroporation on agar plates without antibiotics also grew in the presence of 40 μg of kanamycin per ml. DNA hybridization proved that all of the kanamycin-resistant derivatives contained the EZ::TN 〈R6Kγori/KAN-2〉 insertion element, which was mapped to different genomic locations. Replica plating on Simmons citrate agar and microtiter plate-plastic tube assays identified growth- and biofilm-defective derivatives, respectively. The location of the insertion in several of these derivatives was determined by self-ligation of NdeI- or EcoRI-digested genomic DNA and electroporation of Escherichia coli TransforMax EC100D (pir +). Sequence analysis of the recovered plasmids showed that some of the A. baumannii 19606 growth-defective derivatives contain insertions within genes encoding activities required for the generation of energy and cell wall components and for the biosynthesis of amino acids and purines. A gene encoding a protein similar to the GacS sensor kinase was interrupted in four derivatives, while another had an insertion in a gene coding for a hypothetical sensor kinase. A. baumannii 19606 derivatives with defective attachment or biofilm phenotypes had insertions within genes that appear to be part of a chaperone-usher transport system described for other bacteria. DNA hybridization experiments showed that the presence of strain 19606 genes encoding regulatory and attachment or biofilm functions is widespread among other A. baumannii clinical isolates.

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Bergerson, Rachel J. y David A. Largaespada. "What gene have I ID'ed?" Blood 111, n.º 2 (15 de enero de 2008): 471–72. http://dx.doi.org/10.1182/blood-2007-10-118067.

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Retroviral insertional mutagenesis screens have identified of dozens of potential leukemia/lymphoma genes in mice and rats. Sauvageau and colleagues suggest that proviral insertions may affect the expression of multiple nearby genes in leukemia cells, and that the genes affected may be cell-type dependent.

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Hu, Jingqiong, Theotonius Gomes, Andrea Ferris, Gabriel Renaud, Paul C. Hendrie, Allen E. Krouse, Robert E. Donahue et al. "Distinctive Integration Profile of Avian Sarcoma Leukosis Virus Vectors in Rhesus Long-Term Repopulating Cells." Blood 110, n.º 11 (16 de noviembre de 2007): 198. http://dx.doi.org/10.1182/blood.v110.11.198.198.

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Abstract Insertional mutagenesis continues to be a major concern in hematopoietic stem cell gene therapy. Non-conventional gene transfer vectors with more favorable integration features in comparison to conventional retrovirus and lentivirus vectors are being pursued. The avian sarcoma leukosis virus (ASLV) has been reported to have an unbiased integration pattern in cell lines, but this vector system has not previously been investigated for transduction of repopulating hematopoietic cells. We investigated the efficiency and integration profile of this vector in the clinically-relevant rhesus macaque autologous transplantation model. Using an ASLV derived RCAS (Replication Competent, ALV LTR with A Splice acceptor) vector, we could obtain transduction efficiencies of up to 33% ex vivo in rhesus CD34+ cells. We have been able to transplant two rhesus macaques with ASLV-transduced autologous CD34+ cells and achieve long-term gene marking levels of 1–3% as far as 18 months post-transplant. Using an optimized linear-amplification mediated PCR (LAM-PCR), we have been able to identify so far close to 300 unique insertion sites in the two animals. Here we reported for the first time a systematic analysis of 239 ASLV insertion sites identified in rhesus long-term repopulating cells. Out of 239 unique insertions identified in 4–18 months post-transplant granulocytes and lymphocytes, 99 (41.1%) have landed within RefSeq gene coding regions. 14 (5.8%) have landed within 5kb upstream of RefSeq genes, indicating no preference of inserting into transcription start site. 10 (4.2%) have landed within 5kb downstream of RefSeq genes, which is comparable to random insertions. No insertion into the Mds1-Evi1 locus has been identified to date, at any time point, which is in striking contrast to significant overrepresentation of this insertion site for MLV vectors in the same transplantation model. No preference into CpG islands was found. We further employed a rapid and quantitative assay for measuring neighboring gene activation by vector provirus LTR enhancers using a luciferase assay. LTR from vectors are cloned into the pACT5 plasmid, upstream of a minimal promoter-IRES-luciferease cassette, allowing measurement of neighboring gene activation by a simple luciferase assay. RCAS vectors produced no detectable luciferase activation, both in the forward and reverse orientations, suggesting lack of enhancer activity in the LTRs in mammalian cells, in contrast to significant enhancer activity and read-through transcription from MLV vectors measured in the same assay. Our findings indicate that ASLV derived RCAS vectors have a potentially safer integration pattern in comparison to commonly used MLV and HIV derived vectors. Furthermore, ASLV LTRs do not have significant promoter and enhancer activity in mammalian cells, which provide a second safety feature of the system. Therefore, these vectors should be further explored for hematopoietic stem cell gene transfer purposes.

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