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Добірка наукової літератури з теми "Insertion mutagenesi"

Автор: Grafiati
Опубліковано: 11 березня 2023

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Статті в журналах з теми "Insertion mutagenesi"

1

Skamaki, Kalliopi, Stephane Emond, Matthieu Chodorge, John Andrews, D. Gareth Rees, Daniel Cannon, Bojana Popovic, Andrew Buchanan, Ralph R. Minter, and 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, no. 44 (October 16, 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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2

Herod, Morgan R., Eleni-Anna Loundras, Joseph C. Ward, Fiona Tulloch, David J. Rowlands, and Nicola J. Stonehouse. "Employing transposon mutagenesis to investigate foot-and-mouth disease virus replication." Journal of General Virology 96, no. 12 (December 1, 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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3

Coyote-Maestas, Willow, David Nedrud, Steffan Okorafor, Yungui He, and Daniel Schmidt. "Targeted insertional mutagenesis libraries for deep domain insertion profiling." Nucleic Acids Research 48, no. 2 (November 20, 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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4

Carlson, Corey M., Adam J. Dupuy, Sabine Fritz, Kevin J. Roberg-Perez, Colin F. Fletcher, and David A. Largaespada. "Transposon Mutagenesis of the Mouse Germline." Genetics 165, no. 1 (September 1, 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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5

Idnurm, Alexander, Jennifer L. Reedy, Jesse C. Nussbaum, and Joseph Heitman. "Cryptococcus neoformans Virulence Gene Discovery through Insertional Mutagenesis." Eukaryotic Cell 3, no. 2 (April 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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6

Roseman, R. R., E. A. Johnson, C. K. Rodesch, M. Bjerke, R. N. Nagoshi, and P. K. Geyer. "A P element containing suppressor of hairy-wing binding regions has novel properties for mutagenesis in Drosophila melanogaster." Genetics 141, no. 3 (November 1, 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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7

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, no. 2 (December 1, 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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8

Combier, Jean-Philippe, Delphine Melayah, Colette Raffier, Régis Pépin, Roland Marmeisse, and Gilles Gay. "Nonmycorrhizal (Myc¯) Mutants of Hebeloma cylindrosporum Obtained Through Insertional Mutagenesis." Molecular Plant-Microbe Interactions® 17, no. 9 (September 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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9

Qin, Aiping, Aimee M. Tucker, Andria Hines, and David O. Wood. "Transposon Mutagenesis of the Obligate Intracellular Pathogen Rickettsia prowazekii." Applied and Environmental Microbiology 70, no. 5 (May 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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10

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, no. 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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Дисертації з теми "Insertion mutagenesi"

1

Basiran, Mohd Nazir. "DNA insertion mutagenesis in higher plants." Thesis, University of Leicester, 1988. http://hdl.handle.net/2381/35436.

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2

Themis, Michael. "Retrovirus insertional mutagenesis : experiences at the hprt locus." Thesis, Brunel University, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.307483.

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3

Bokhoven, Marieke Christina. "Measurement of insertional mutagenesis by retroviral and lentiviral vectors." Thesis, University College London (University of London), 2008. http://discovery.ucl.ac.uk/1444113/.

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Retroviral vectors have been successfully used in the clinic for the correction of inherited immunodeficiencies. However, in 2 recent gene therapy trials for X-linked Severe Combined Immunodeficiency, 5 patients developed leukaemia. This was associated with vector integration near cellular proto-oncogenes, leading to their activation a process known as insertional mutagenesis. This thesis describes the development of a cell line assay that allows us to quantify insertional mutagenesis by retroviral and lentiviral vectors and to analyse the mechanisms by which this occurs. The interleukin-3 (IL-3) dependent cell line BCL15 is derived from the mouse bone marrow cell line BAF3 and over- expresses human Bcl2. The frequency at which IL-3 independent mutants are obtained following vector transduction is measured. Lentiviral and retroviral vectors transform BCL15 cells at similar integrant frequencies of 4.3 x 108 and 1.2 x 107, respectively. However, they cause insertional mutagenesis in this assay by different mechanisms. The human immunodeficiency virus-1 (HIV-1)- derived lentiviral vector HV transforms BCL15 cells by insertional activation of the growth hormone receptor (Ghr) gene. An HIV-Ghr fusion transcript was detected. It originates from the HIV-1 5'-LTR it then splices from the HIV-1 major splice donor to the splice acceptor of Ghr exon 2. The mutants express GHR and grow in response to bovine growth hormone in the foetal calf serum of the culture medium. Deletion of the HIV-1 enhancer/promoter in a self-inactivating vector prevents this mechanism of transformation. Retroviral vector transformation of the BCL15 cell line does not occur via Ghr gene activation. Retroviral vectors up- regulate expression of the cytokine IL-3, either by insertion into the IL-3 gene or by insertion into other genes, which may act as upstream activators of IL-3 expression. This assay is a general method to quantitate insertional mutagenesis. It may inform the design of safer vectors and can be used in initial safety testing of (pre-) clinical vectors.
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4

Gan, Shu Uin. "Retroviral insertional mutagenesis of human myeloid HL-60 cells." Thesis, King's College London (University of London), 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.244680.

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5

Gaiano, Nicholas R. (Nicholas Roger). "Insertional mutagenesis in zebrafish using a pseudotyped retroviral vector." Thesis, Massachusetts Institute of Technology, 1997. http://hdl.handle.net/1721.1/43311.

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6

Kong, Jun. "Transposon-mediated insertional mutagenesis in gene discovery and cancer." Thesis, University of Cambridge, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.609377.

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7

Bronchain, Odile Jacqueline. "Insertional mutagenesis through gene trap approaches in Xenopus embryos." Thesis, University of Cambridge, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.620913.

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8

Sutherland, Helen F. "Gene targeting and insertional mutagenesis in embryonic stem cells." Thesis, University of Edinburgh, 1992. http://hdl.handle.net/1842/20231.

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Mutations are important in the study of gene function. Until recently mouse mutants available for study were spontaneous mutations and those derived from chemical or X-ray mutagenesis. Transgenic technology brought about the creation of more mouse mutants through insertional mutagenesis by retroviral agent or microinjected recombinant DNA. The insert may be used as a tag to molecularly clone and characterise the mutated gene. With the advent of embryonic stem (ES) cell technology the number of mutations available for study should grow - both specific and random mutations. ES cells are pluripotent stem cells, that may be manipulated genetically in culture, and yet retain the ability to contribute to normal development and the germ line of a host blastocyst. Thus the phenotypic effects of the introduced mutation may be studied in vivo. Random mutations may be introduced into the ES cell genome by insertional mutagenesis by retroviral agent or by promoter or enhancer trap vectors. Gene targeting by homologous recombination may be used to introduce specific mutations. Hox-2.1 is a mouse homeobox-containing gene, belonging to the family of Hox genes. It has been cloned and mapped to the Hox-2 cluster on chromosome 11. Hox genes are thought to play important roles in mouse development. They may act as regulatory genes, controlling the expression of structural genes at different positions in the developing embryo. In order to study the function of Hox-2.1 in development it would be useful to have a mouse mutant for Hox-2.1. It was the aim of this project to produce a mouse mutant for Hox2.1. The first step towards this aim is the targeting/knock-out of the gene in ES cells. During this project various Hox-2.1 targeting constructs have been designed and built. Replacement vectors, incorporating a promoterless neo or employing the positive-negative selection strategy, were designed to enrich for targeting events. These and two insertion vectors were transfected separately into ES cells. Over 200 selected clones have been screened for disruption of the Hox2.1 locus. No homologous recombinants were identified. One clone, known as a 'pick-up' clone, was identified. It is thought that the targeting vector has found homology with the target sequence, picked up DNA from the locus (in this case 3' to sequence within the construct) and integrated elsewhere in the genome. An integration site, which the promoterless Hox2.1-neo targeting vector has integrated into twice, despite non-homology, has been characterised. It is suggested that this locus may be a site of frequent integration. The sequence flanking the construct was amplified by inverse PCR, cloned and sequenced. The properties which may make it highly targetable are discussed. It was shown to have weak promoter function.
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9

Jiang, Xiaoyan. "Insertional mutagenesis by provirus in retrovirus-induced lymphoid murine tumors." Thesis, McGill University, 1995. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=28790.

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Ahi-1 was initially identified as a common helper provirus integration site on mouse chromosome 10 in 16% of Abelson pre-B-cell lymphomas and shown to be closely linked to Myb proto-oncogene. By using long-range restriction mapping, we have mapped the Ahi-1 locus approximately 35 kbp downstream of the Myb gene. To test whether provirus integration in the Ahi-1 region enhances the expression of Myb by cis-acting mechanism, we have also examined Myb gene expression in A-MuLV-induced pre-B-lymphomas. Our data have revealed that there is no clear evidence for such activation in the tumors we have tested, indicating that provirus insertion in the Ahi-1 region may activate a novel gene, apparently involved in tumor formation. In addition, another provirus integration site which was identified in 14% of immature T-cell thymomas induced by Mo-MuLV infection of MMTV${{ rm D} over C}$-Myc transgenic mice has recently been mapped within the Ahi-1 region. We have used exon amplification to identify genes that may cooperate with v-Ab1 or c-Myc in B- and T-cell transformation. We have indeed identified a novel Ahi-1 gene which encodes two major RNA species of 2 kbp and 5 kbp. The Ahi-1 gene is expressed in several organs of the mouse and rat and is highly expressed in brain and testis. One cluster of the proviruses was found to integrate 3$ sp prime$ of noncoding region of the Ahi-1 in an inverse transcriptional orientation. The Ahi-1 is highly conserved in evolution and encodes a 297 amino acid protein. The predicted Ahi-1 protein contains a SH3 domain and four potential SH3-binding sites, which function to mediate specific protein-protein interactions during signaling. The human homologue of the Ahi-1 gene has been cloned and mapped to chromosome 6q 23-24, and located approximately 330 kbp downstream of the Myb gene. The Ahi-1 is a novel gene which may play important roles in the signal transduction and tumor development.
Mis-2 and Mis-4 loci were identified as two new common provirus integration sites in Moloney MuLV-induced thymomas. Mis-2 has been mapped on mouse chromosome 10 and located 160 and 40 kbp downstream of Myb and Ahi-1 genes. The Mis-4 locus has been located on chromosome 15 at about 60 and 30 kbp downstream of Myc and Mlvi-4 genes. Myc RNA levels are elevated in tumors harboring a Mis-4 rearrangement compared with some of those with no Mis-4 rearrangement, indicating that provirus integration affected Myc expression by long-distance activation. Both loci may contain novel genes involved in tumor formation.
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10

Franz, Marie-Josee. "Analysis of factor-independent mutants induced by retroviral insertional mutagenesis." Thesis, Brunel University, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.262514.

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Книги з теми "Insertion mutagenesi"

1

French, Neil Simon. Insertional mutagenesis cloning of oncogenes. Manchester: University of Manchester, 1995.

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2

Dupuy, Adam J., and David A. Largaespada, eds. Insertional Mutagenesis Strategies in Cancer Genetics. New York, NY: Springer New York, 2011. http://dx.doi.org/10.1007/978-1-4419-7656-7.

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3

Dupuy, Adam J. Insertional mutagenesis strategies in cancer genetics. New York: Springer, 2011.

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4

Dupuy, Adam J. Insertional mutagenesis strategies in cancer genetics. New York: Springer, 2011.

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5

Chen, Hai Shiene. Characterization of the HIV reverse transparencies by insertion mutagenesis. Ottawa: National Library of Canada, 1993.

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6

K, Trower Michael, ed. In vitro mutagenesis protocols. Totowa, N.J: Humana Press, 1996.

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7

In vitro mutagenesis protocols. 3rd ed. Totowa, NJ: Humana Press, 2010.

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8

Wisconsin-Madison), International Symposium on Plant Transposable Elements (1987 University of. Plant transposable elements. New York: Plenum Press, 1988.

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9

E, Lambert Michael, McDonald John F. 1947-, Weinstein I. Bernard, Cold Spring Harbor Laboratory, and Abbott Laboratories, eds. Eukaryotic transposable elements as mutagenic agents. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory, 1988.

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10

Dupuy, Adam J., and David A. Largaespada. Insertional Mutagenesis Strategies in Cancer Genetics. Springer, 2011.

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Частини книг з теми "Insertion mutagenesi"

1

Jankowicz-Cieslak, Joanna, Florian Goessnitzer, Bradley J. Till, and Ivan L. Ingelbrecht. "Induced Mutagenesis and In Vitro Mutant Population Development in Musa spp." In Efficient Screening Techniques to Identify Mutants with TR4 Resistance in Banana, 3–20. Berlin, Heidelberg: Springer Berlin Heidelberg, 2022. http://dx.doi.org/10.1007/978-3-662-64915-2_1.

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AbstractMutagenesis of in vitro propagated bananas is an efficient method to introduce novel alleles and broaden genetic diversity. Mutations can be induced by treatment of plant cells with chemical mutagens or ionizing radiation. The FAO/IAEA Plant Breeding and Genetics Laboratory established efficient methods for mutation induction of in vitro shoot tips in banana using physical and chemical mutagens as well as methods for the efficient discovery of EMS-induced single nucleotide mutations in targeted genes or amplicons and identification of large genomic changes, including deletions and insertions. Mutagenesis of in vitro propagated tissues requires large populations serving as starting material, and a long process to dissolve genetic mosaics (chimeras) resulting from the mutagenesis of multicellular tissues. However, treating shoot apical meristems of tissue cultured bananas with a mutagen is a commonly used practice for banana mutation breeding programmes, and still the most effective. In our previous studies, we showed that chimeras, unique mutations accumulated in different cells of the plant propagule, could be rapidly removed via isolation of shoot apical meristems and subsequent longitudinal bisection. Further, induced mutations were maintained in mutant plants for several generations. We established such systems for inducing and maintaining both point mutations caused via EMS mutagenesis as well as insertions and deletions caused by gamma irradiation and describe hereafter methods for dose selection, gamma irradiation and chimera dissolution.
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Ito, Takuya, Reiko Motohashi, and Kazuo Shinozaki. "Preparation of Transposon Insertion Lines and Determination of Insertion Sites in Arabidopsis Genome." In In Vitro Mutagenesis Protocols, 209–19. Totowa, NJ: Humana Press, 2002. http://dx.doi.org/10.1385/1-59259-194-9:209.

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Slape, Christopher, and Peter D. Aplan. "Retroviral Insertional Mutagenesis." In Encyclopedia of Cancer, 1–4. Berlin, Heidelberg: Springer Berlin Heidelberg, 2015. http://dx.doi.org/10.1007/978-3-642-27841-9_5081-2.

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Slape, Christopher, and Peter D. Aplan. "Retroviral Insertional Mutagenesis." In Encyclopedia of Cancer, 4067–71. Berlin, Heidelberg: Springer Berlin Heidelberg, 2016. http://dx.doi.org/10.1007/978-3-662-46875-3_5081.

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Slape, Christopher, and Peter D. Aplan. "Retroviral Insertional Mutagenesis." In Encyclopedia of Cancer, 3293–96. Berlin, Heidelberg: Springer Berlin Heidelberg, 2011. http://dx.doi.org/10.1007/978-3-642-16483-5_5081.

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6

Amsterdam, Adam, and Nancy Hopkins. "Insertional Mutagenesis in Zebrafish." In Development, 371–87. Berlin, Heidelberg: Springer Berlin Heidelberg, 1999. http://dx.doi.org/10.1007/978-3-642-59828-9_22.

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Wu, Sareina Chiung-Yuan, Kommineni J. Maragathavally, Craig J. Coates, and Joseph M. Kaminski. "Steps Toward Targeted Insertional Mutagenesis With Class II Transposable Elements." In Chromosomal Mutagenesis, 139–51. Totowa, NJ: Humana Press, 2008. http://dx.doi.org/10.1007/978-1-59745-232-8_10.

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8

Takeda, Junji, Zsuzsanna Izsvák, and Zoltán Ivics. "Insertional Mutagenesis of the Mouse Germline With Sleeping Beauty Transposition." In Chromosomal Mutagenesis, 109–25. Totowa, NJ: Humana Press, 2008. http://dx.doi.org/10.1007/978-1-59745-232-8_8.

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Bassett, Kody A., Melanie R. Mormile, and Ronald L. Frank. "Whole-Genome Identification and Characterization of Bacterial Insertion Sequences Using Bioinformatic Tools." In Microbial Transposon Mutagenesis, 171–80. New York, NY: Springer New York, 2019. http://dx.doi.org/10.1007/978-1-4939-9570-7_16.

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Pettitt, Stephen J., E.-Pien Tan, and Kosuke Yusa. "piggyBac Transposon-Based Insertional Mutagenesis in Mouse Haploid Embryonic Stem Cells." In Chromosomal Mutagenesis, 15–28. New York, NY: Springer New York, 2014. http://dx.doi.org/10.1007/978-1-4939-1862-1_2.

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Тези доповідей конференцій з теми "Insertion mutagenesi"

1

DeJesus, Michael A., and Thomas R. Ioerger. "Improving discrimination of essential genes by modeling local insertion frequencies in transposon mutagenesis data." In BCB'13: ACM-BCB2013. New York, NY, USA: ACM, 2013. http://dx.doi.org/10.1145/2506583.2506610.

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Tinghu, Qiu N., Laura Vera Ramirez, Roland Rad, and Jeffrey E. Green. "Abstract 653: Identification of metastasis promoting genes using the PiggyBac insertional mutagenesis system." In Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA. American Association for Cancer Research, 2016. http://dx.doi.org/10.1158/1538-7445.am2016-653.

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3

Ranzani, Marco, Daniela Cesana, Cynthia C. Bartholomä, Francesca Sanvito, Michela Riba, Mauro Pala, Fabrizio Benedicenti, et al. "Abstract 3169: Lentiviral vector-based insertional mutagenesis identifies new clinically relevant liver cancer genes." In Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC. American Association for Cancer Research, 2013. http://dx.doi.org/10.1158/1538-7445.am2013-3169.

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4

Vera Ramirez, Laura, Sven Bilke, Robert Walker, Paul Meltzer, Tinghu Qiu, Roland Rad, and Jeffrey E. Green. "Abstract 1576: Insertional mutagenesis to identify molecular mechanisms of breast cancer dormancy and metastasis." In Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA. American Association for Cancer Research, 2016. http://dx.doi.org/10.1158/1538-7445.am2016-1576.

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Mann, Karen M., Nancy A. Jenkins, and Neal G. Copeland. "Abstract B9: Metastasis-associated loci in pancreatic cancer identified using Sleeping Beauty insertional mutagenesis." In Abstracts: AACR Special Conference on Pancreatic Cancer: Progress and Challenges; June 18-21, 2012; Lake Tahoe, NV. American Association for Cancer Research, 2012. http://dx.doi.org/10.1158/1538-7445.panca2012-b9.

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Datta, Shalini, Jean-Charles Nault, Andrea Franconi, Sandrine Imbeaud, Maxime Mallet, Gabrielle Couchy, Eric Letouze, et al. "Abstract 919: Adeno-associated virus 2 (AAV2) induces recurrent insertional mutagenesis in human hepatocellular carcinomas." In Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA. American Association for Cancer Research, 2015. http://dx.doi.org/10.1158/1538-7445.am2015-919.

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Maurin, Michelle, Devin J. Jones, Mik A. Black, Justin Y. Newberg, Nancy A. Jenkins, Neal G. Copeland, and Karen M. Mann. "Abstract A37: SB insertional mutagenesis identifies new metastasis-promoting tumor suppressor genes in pancreatic cancer." In Abstracts: AACR Special Conference: Advances in Modeling Cancer in Mice: Technology, Biology, and Beyond; September 24-27, 2017; Orlando, Florida. American Association for Cancer Research, 2018. http://dx.doi.org/10.1158/1538-7445.mousemodels17-a37.

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Koudijs, Marco J., Jaap Kool, Daoud Sie, Pramudita Prasetyanti, Edwin Cuppen, Anton Berns, John Hilkens, Maarten van Lohuizen, David Adams, and Jos Jonkers. "Abstract 2208: High-resolution analysis of insertional mutagenesis screens to study genetic interactions in heterogeneous tumors." In Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC. American Association for Cancer Research, 2010. http://dx.doi.org/10.1158/1538-7445.am10-2208.

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9

Powell, Tracy A., Branden S. Moriarity, Aaron L. Sarver, and David A. Largaespada. "Abstract C3: Candidate osteosarcoma metastatic drivers identified in highly clonal metastases using transposon-mediated insertional mutagenesis." In Abstracts: AACR Special Conference on Tumor Invasion and Metastasis - January 20-23, 2013; San Diego, CA. American Association for Cancer Research, 2013. http://dx.doi.org/10.1158/1538-7445.tim2013-c3.

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Holmes, W. E., H. R. Lijnen та D. Collen. "CHARACTERIZATION OFα2-ANTIPLASMIN.REACTIVE SITE VARIANTS PRODUCED BY SITE-DIRECTED MUTAGENESIS". У XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644766.

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α2-Antiplasmin (α2AP) is the primary physiological plasmin inhibitor in human plasma. The inhibition is rapid (second order rate constants (k1) are expressed as M−1 s−1 ) (k1 = 2 × 107) and occurs as the consequence of an irreversible 1:1 stoichiometric complex formation; the exact nature of and the forces involved in complex formation are not fully understood. In fact, what makes α2AP an inhibitor, rather than simply a substrate remains unresolved. Recently, we deduced the primary structure of α2 AP from the sequence of its cDNA. 95%of this sequence was confirmed by amino acid (aa) sequence analysis of naturalα2 AP (α2 AP)? The 452 aa molecule contains 2 disulfide bonds and 4 glycosylated Asn residues, aa sequence alignment confirmed α2AP's membership in the Serpin family. The reactive site sequence as determined by NH2 - and COOH-terminal aa sequence analysis of the plasmin-modified inhibitor and the released M−r ∼ 8000 peptide is Met362-Ser363-Arg364-Met365-Ser366, P3-P2-P1-P'1-P'2, respectively.Natural and engineered P1 residue substitutions in the Serpin α2 -antitrypsin ( α2 AT) have shown altered specificities and efficiencies. To further examine the role of P and P' residues in determining Serpin specificity, in the present study we have by site-directed mutagenesis, deleted (△) the P'l-Met365 residue of a AP thereby producing a recombinant (r) inhibitor (r α2 AP△Met365) whose putative new reactive site mimics that of antithrombin III (ATIII) and a AT-Pittsburgh (Pl-Arg-P'1-Ser). A second variant was constructed (ra2AP△Arg364) in which the Pl-Arg364 residue was deleted, producing the new sequence Met362-Ser363-Met364-Ser365, containing 2 potential sites analogous to the Pl-P'l, Met-Ser reactive site of α2 AT. The variants and r α2 AP were expressed in CH0 cells, purified and compared with n α2 AP, α2AT and ATIII for the ability to inhibit plasmin, thrombin, trypsin and elastase. n α2 AP and r α2 AP had nearly identical inhibition constants and like ATIII did not inhibit neutrophil elastase. Without heparin both α2 APs and ATIII inhibited thrombin moderately (k1 = 2 to 4× 103 ). Bovine trypsin was neutralized by the α2 APs with k1 = 3 × 106 and by ATIII with k1 = 1 × 105. The α2APs inhibited plasmin (k1 = 2 ×107 ) much more efficiently than ATIII (K1 =2 × 103 ). In contrast, was a highly effective antielastase (k1 = 1 × 107 ), a poor plasmin and thrombin inhibitor ancl inhibited bovine trypsin with = 2 × 10. As reported by others, α2 AT-Pittsburg has greatly reduced antielastase activity and greatly enhanced antithrombin activity. Analysis of ra APAMet365 revealed little change in activity toward plasmin, trypsin and elastase. Thus, α2 AP has no absolute requirement for Met .in the P'l position in order to effectively inhibit plasmin and trypsin. The other P^ subsites appear to be spatially flexible as deletion of the natural P'l residue must displace them. Contrary to prediction a 20-fold decrease in antithrombin activity was observed rather than an enhanced activity. Analysis of rα2 AP△Arg364 showed that it is unreactive with plasmin, trypsin and thrombin, but that it has acquired a significant antielastase activity (k1 = 1.5 × 105). The exact PI residue(s) has not been determined but removal of the bulky basic Arg364 may have resulted in accessibility of the predicted reactive site(s) peptide bond(s) Met362-Ser363 or Met364-Ser365 to the active site cleft of elastase. α2AP'Enschede', a natural mutant with deficient antiplasmin activity, was shown to contain an Ala insertion between aa 353 and 357, 7 to 10 positions NH2-terminal to its reactive site (Holmes et al., this meeting). This mutation results in conversion of α2 AP'Enschede' from an inhibitor to a substrate that retains a high affinity for the active site of plasmin.
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Звіти організацій з теми "Insertion mutagenesi"

1

John, Vogel P. Final technical report for: Insertional Mutagenesis of Brachypodium distachyon DE-AI02-07ER64452. Office of Scientific and Technical Information (OSTI), October 2015. http://dx.doi.org/10.2172/1224441.

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2

Barefoot, Susan, Benjamin Juven, Thomas Hughes, Avraham Lalazar, A. B. Bodine, Yitzhak Ittah, and Bonita Glatz. Characterization of Bacteriocins Produced by Food Bioprocessing Propionobacteria. United States Department of Agriculture, August 1992. http://dx.doi.org/10.32747/1992.7561061.bard.

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Objectives were to further characterize activity spectra of dairy propionibacteria bacteriocins, jenseniin G and propionicin PLG-1, purify them, examine the role of cell walls in resistance, examine their interactions with cytoplasmic membrane, explain producer immunity, and clone the responsible genes. Inhibitory spectra of both bacteriocins were further characterized. Propionicin was most effective in controlling Gram-positive, rather than Gram-negative organisms; it controlled growth of sensitive cells both in a culture medium and a model food system. Jenseniin inhibited yogurt cultures and may help prevent yogurt over-acidification. Both were active against botulinal spores; jenseniin was sporostatic; propionicin was sporicidal. Jenseniin was produced in broth culture, was stable to pH and temperature extremes, and was purified. Its molecular mass (3649 Da) and partial amino acid composition (74%) were determined. A blocked jenseniin N-terminus prevented sequencing. Methods to produce propionicin in liquid culture were improved, and large scale culture protocols to yield high titers were developed. Methods to detect and quantify propionicin activity were optimized and standardized. Stability of partially purified propionicin was demonstrated and an improved purification scheme was developed. Purified propionicin had a 9328-Da molecular mass, contained 99 amino acids, and was significantly hydrophobic; ten N-terminal amino acids were identified. Propionicin and Jenseniin interacted with cytoplasmic membranes; resistance of insensitive species was cell wall-related. Propionicin and jenseniin acted similarly; their mode of action appeared to differ from nisin. Spontaneous jenseniin-resistant mutants were resistant to propionicin but nisin-sensitive. The basis for producer immunity was not resolved. Although bacteriocin genes were not cloned, a jenseniin producer DNA clone bank and three possible vectors for cloning genes in propionibacteria were constructed. In addition, transposon Tn916 was conjugatively transferred to the propionicin producer from chromosomal and plasmid locations at transfer frequencies high enough to permit use of Tn916 for insertional mutagenesis or targeting genes in propionibacteria. The results provide information about the bacteriocins that further supports their usefulness as adjuncts to increase food safety and/or quality.
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Shai, Yechiel, Arthur Aronson, Aviah Zilberstein, and Baruch Sneh. Study of the Basis for Toxicity and Specificity of Bacillus thuringiensis d-Endotoxins. United States Department of Agriculture, January 1996. http://dx.doi.org/10.32747/1996.7573995.bard.

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The report contains three parts which summarizes the three years achievements of the three participating research groups; The Weizmann group, Tel-Aviv group and Purdue group. The firs part describes the achievements obtained by Shai's group toward the elucidation of the mechanism of membrane insertion and the structural organization of the pores formed by the Cry3A and Cry1Ac B. thuringiensis d-endotoxins. For that purpose Shai's group synthesized, fluorescently labeled and structurally and functionally characterized peptides corresponding to the seven helices that compose the pore-forming domain of Cry3A toxin, including mutants peptides and the hairpin a4G-a5 of both Cry3A and Cry 1Ac toxins composed of a4, a5 and the loop connecting a4-a5. Among the synthesized peptides were three mutated a4 helices based on site directed mutagenesis done at Aronson's group that decreased or increased Cry 1Ac toxicity. The results of these studies are consistent with a situation in which only helices a4 anda5 insert into the membrane as a helical hairpin in an antiparallel manner, while the other helices lie on the membrane surface like ribs of an umbrella (the "umbrella model"). In order to test this model Shai's group synthesized the helical hairpin a4<-->a5 of both Cry3A and Cry 1 Ac toxins, as well. Initial functional and structural studies showed direct correlation between the properties of the mutated helices and the mutated Cry1Ac. Based on Shai's findings that a4 is the second helix besides a5 that insert into the membrane, Aronson and colleagues performed extensive mutation on this helix in the CrylAc toxin, as well as in the loop connecting helices 4 and 5, and helix 3 (part two of the report). In addition, Aronson performed studies on the effect of mutations or type of insect which influence the oligomerization either the Cry 1Ab or Cry 1Ac toxins with vesicles prepared from BBMV. In the third part of the report Zilberstein's and Sneh's groups describe their studies on the three domains of Cry 1C, Cry 1E and crylAc and their interaction with the epithelial membrane of the larval midgut. In these studies they cloned all three domains and combinations of two domains, as well as cloning of the pore forming domain alone and studying its interaction with BBMV. In addition they investigated binding of Cry1E toxin and Cry1E domains to BBMV prepared from resistant (R) or sensitive larvae. Finally they initiated expression of the loop a4G<-->a5 Cry3A in E. coli to be compared with the synthetic one done by Shai's group as a basis to develop a system to express all possible pairs for structural and functional studies by Shai's group (together with Y. Shai).
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Xu, Jin-Rong, and Amir Sharon. Comparative studies of fungal pathogeneses in two hemibiotrophs: Magnaporthe grisea and Colletotrichum gloeosporioides. United States Department of Agriculture, May 2008. http://dx.doi.org/10.32747/2008.7695585.bard.

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Plant pathogenic fungi have various life styles and different plant infection strategies. Hemibiotrophs like Magnaporthe grisea and Colletotrichum species develop specialized structures during plant infection. The goal of this study was to identify, characterize, and compare genes required for plant infection in M. grisea and C. gloeosporioides. Specific objectives are to: 1) further characterize genes identified in the preliminary studies of C. gloeosporioides and M. grisea;2) identify and characterize additional fungal genes tagged by GFP; and 3) identify in planta growth and appressorium-specific genes by subtractive hybridization and transcript profiling by the LongSAGE method. In this study, the PI and Co-PI collaborated closely on studies in M. grisea and C. gloeosporioides. In M. grisea, REMI and ATMT were used to transform the wildtype with promoter-less EGFP constructs. A total of 28 mutants defective in different plant infection processes or expressing EGFP during plant infection were identified. Genes disrupted in five selected mutants have been identified, including MG03295 that encodes a putative Rho GTPase. In transformant L1320, the transforming vector was inserted in the MIRI gene that encodes a nuclear protein. The expression of MIRI was highly induced during infection. Deletion and site-directed mutagenesis analyses were used to identify the promoter regions and elements that were essential for induced in planta expression of MIRI. This was the first detailed characterization of the promoter of an in planta gene in M. grisea and the MIRI promoter can be used to monitor infectious growth. In addition, the Agilent whole-genome array of M. grisea was used for microarray analyses with RNA samples from appressoria formed by the wild-type shain and the pmkl and mstl2 mutants. Over 200 genes were downregulated in the mst I 2 and pmkl mutants. Some of them are putative transcription factors that may regulate appressorium formation and infectious hyphal growth. In C. gloeosporioides, various REMI mutants showing different pathogenic behavior were identified and characterized. Mutants N3736 had a single insertion and was hyper-virulent. The gene disrupted in mutant3736 (named CgFMOI) encodes a FAD-dependent monooxygenase. Expression analyses linked the expression of the CgFMOI gene with the necrotrophic phase of fungal infection, and also suggest that expression of CgFMOl is unnecessary for the first stages of infection and for biotrophy establishment. All CgFMOl-silenced mutants had reduced virulence. In REMI mutant N159, the tagged gene encodes a putative copper transporter that is homologue of S. cerevisiae CTR2. In yeast, Ctr2 is a vacuolar transporter for moving copper from the vacuole to the cytoplasm. The gene was therefore termed CgCTR2. In addition to characterization of CgCTR2, we also conducted comparative analyses in M. grisea. The M. grisea CgCTR-2 homolog was isolated, knockout strains were generated and characterized and the M. grisea was used to complement the Nl 59 C. gloeosporioides mutant. Overall, we have accomplished most of proposed experiments and are in the process of organizing and publishing other data generated in this project. For objective 3, we used the microarray analysis approach. Several genes identified in this study are novel fungal virulence factors. They have the potential to be used as targets for developing more specific or effective fungicides. In the long run, comparative studies of fungal genes, such as our CgCTR2 work, may lead to better disease control strategies.
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Chamovitz, Daniel, and Albrecht Von Arnim. Translational regulation and light signal transduction in plants: the link between eIF3 and the COP9 signalosome. United States Department of Agriculture, November 2006. http://dx.doi.org/10.32747/2006.7696515.bard.

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The COP9 signalosome (CSN) is an eight-subunit protein complex that is highly conserved among eukaryotes. Genetic analysis of the signalosome in the plant model species Arabidopsis thaliana has shown that the signalosome is a repressor of light dependent seedling development as mutant Arabidopsis seedlings that lack this complex develop in complete darkness as if exposed to light. These mutant plants die following the seedling stage, even when exposed to light, indicating that the COP9 signalosome also has a central role in the regulation of normal photomorphogenic development. The biochemical mode of action of the signalosome and its position in eukaryotic cell signaling pathways is a matter of controversy and ongoing investigation, and recent results place the CSN at the juncture of kinase signaling pathways and ubiquitin-mediated protein degradation. We have shown that one of the many CSN functions may relate to the regulation of translation through the interaction of the CSN with its related complex, eukaryotic initiation factor (eIF3). While we have established a physical connection between eIF3 subunits and CSN subunits, the physiological and developmental significance of this interaction is still unknown. In an effort to understand the biochemical activity of the signalosome, and its role in regulating translation, we originally proposed to dissect the contribution of "h" subunit of eIF3 (eIF3h) along the following specific aims: (i) Isolation and phenotypic characterization of an Arabidopsis loss-of-function allele for eIF3h from insertional mutagenesis libraries; (ii) Creation of designed gain and loss of function alleles for eIF3h on the basis of its nucleocytoplasmic distribution and its yeast-two-hybrid interactions with other eIF3 and signalosome partner proteins; (iii) Determining the contribution of eIF3h and its interaction with the signalosome by expressing specific mutants of eIF3h in the eIF3h- loss-of function background. During the course of the research, these goals were modified to include examining the genetic interaction between csn and eif3h mutations. More importantly, we extended our effort toward the genetic analysis of mutations in the eIF3e subunit, which also interacts with the CSN. Through the course of this research program we have made several critical scientific discoveries, all concerned with the apparent diametrically opposed roles of eIF3h and eIF3e. We showed that: 1) While eIF3e is essential for growth and development, eIF3h is not essential for growth or basal translation; 2) While eIF3e has a negative role in translational regulation, eIF3h is positively required for efficient translation of transcripts with complex 5' UTR sequences; 3) Over-accumulation of eIF3e and loss-of-function of eIF3h both lead to cop phenotypes in dark-grown seedlings. These results were published in one publication (Kim et al., Plant Cell 2004) and in a second manuscript currently in revision for Embo J. Are results have led to a paradigm shift in translation research – eIF3 is now viewed in all systems as a dynamic entity that contains regulatory subuits that affect translational efficiency. In the long-term agronomic outlook, the proposed research has implications that may be far reaching. Many important plant processes, including developmental and physiological responses to light, abiotic stress, photosynthate, and hormones operate in part by modulating protein translation [23, 24, 40, 75]. Translational regulation is slowly coming of age as a mechanism for regulating foreign gene expression in plants, beginning with translational enhancers [84, 85] and more recently, coordinating the expression of multiple transgenes using internal ribosome entry sites. Our contribution to understanding the molecular mode of action of a protein complex as fundamental as eIF3 is likely to lead to advances that will be applicable in the foreseeable future.
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Epel, Bernard, and Roger Beachy. Mechanisms of intra- and intercellular targeting and movement of tobacco mosaic virus. United States Department of Agriculture, November 2005. http://dx.doi.org/10.32747/2005.7695874.bard.

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To cause disease, plant viruses must replicate and spread locally and systemically within the host. Cell-to-cell virus spread is mediated by virus-encoded movement proteins (MPs), which modify the structure and function of plasmodesmata (Pd), trans-wall co-axial membranous tunnels that interconnect the cytoplasm of neighboring cells. Tobacco mosaic virus (TMV) employ a single MP for cell- cell spread and for which CP is not required. The PIs, Beachy (USA) and Epel (Israel) and co-workers, developed new tools and approaches for study of the mechanism of spread of TMV that lead to a partial identification and molecular characterization of the cellular machinery involved in the trafficking process. Original research objectives: Based on our data and those of others, we proposed a working model of plant viral spread. Our model stated that MPᵀᴹⱽ, an integral ER membrane protein with its C-terminus exposed to the cytoplasm (Reichel and Beachy, 1998), alters the Pd SEL, causes the Pd cytoplasmic annulus to dilate (Wolf et al., 1989), allowing ER to glide through Pd and that this gliding is cytoskeleton mediated. The model claimed that in absence of MP, the ER in Pd (the desmotubule) is stationary, i.e. does not move through the Pd. Based on this model we designed a series of experiments to test the following questions: -Does MP potentiate ER movement through the Pd? - In the presence of MP, is there communication between adjacent cells via ER lumen? -Does MP potentiate the movement of cytoskeletal elements cell to cell? -Is MP required for cell-to-cell movement of ER membranes between cells in sink tissue? -Is the binding in situ of MP to RNA specific to vRNA sequences or is it nonspecific as measured in vitro? And if specific: -What sequences of RNA are involved in binding to MP? And finally, what host proteins are associated with MP during intracellular targeting to various subcellular targets and what if any post-translational modifications occur to MP, other than phosphorylation (Kawakami et al., 1999)? Major conclusions, solutions and achievements. A new quantitative tool was developed to measure the "coefficient of conductivity" of Pd to cytoplasmic soluble proteins. Employing this tool, we measured changes in Pd conductivity in epidermal cells of sink and source leaves of wild-type and transgenic Nicotiana benthamiana (N. benthamiana) plants expressing MPᵀᴹⱽ incubated both in dark and light and at 16 and 25 ᵒC (Liarzi and Epel, 2005 (appendix 1). To test our model we measured the effect of the presence of MP on cell-to-cell spread of a cytoplasmic fluorescent probe, of two ER intrinsic membrane protein-probes and two ER lumen protein-probes fused to GFP. The effect of a mutant virus that is incapable of cell-to-cell spread on the spread of these probes was also determined. Our data shows that MP reduces SEL for cytoplasmic molecules, dilates the desmotubule allowing cell-cell diffusion of proteins via the desmotubule lumen and reduces the rate of spread of the ER membrane probes. Replicase was shown to enhance cell-cell spread. The data are not in support of the proposed model and have led us to propose a new model for virus cell-cell spread: this model proposes that MP, an integral ER membrane protein, forms a MP:vRNAER complex and that this ER-membrane complex diffuses in the lipid milieu of the ER into the desmotubule (the ER within the Pd), and spreads cell to cell by simple diffusion in the ER/desmotubule membrane; the driving force for spread is the chemical potential gradient between an infected cell and contingent non-infected neighbors. Our data also suggests that the virus replicase has a function in altering the Pd conductivity. Transgenic plant lines that express the MP gene of the Cg tobamovirus fused to YFP under the control the ecdysone receptor and methoxyfenocide ligand were generated by the Beachy group and the expression pattern and the timing and targeting patterns were determined. A vector expressing this MPs was also developed for use by the Epel lab . The transgenic lines are being used to identify and isolate host genes that are required for cell-to-cell movement of TMV/tobamoviruses. This line is now being grown and to be employed in proteomic studies which will commence November 2005. T-DNA insertion mutagenesis is being developed to identify and isolate host genes required for cell-to-cell movement of TMV.
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