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Academic literature on the topic 'Mutagenesi sito specifica'

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Published: 11 March 2023

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Journal articles on the topic "Mutagenesi sito specifica"

Cupples, C. G., M. Cabrera, C. Cruz, and J. H. Miller. "A set of lacZ mutations in Escherichia coli that allow rapid detection of specific frameshift mutations." Genetics 125, no. 2 (June 1, 1990): 275–80. http://dx.doi.org/10.1093/genetics/125.2.275.

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Abstract We have used site-directed mutagenesis to alter bases in lacZ near the region encoding essential residues in the active site of beta-galactosidase. The altered sequences generate runs of six or seven identical base pairs which create a frameshift, resulting in a Lac- phenotype. Reversion to Lac+ in each strain can occur only by a specific frameshift at these sequences. Monotonous runs of A's (or of T's on the opposite strand) and G's (or C's) have been constructed, as has an alternating -C-G- sequence. These specific frameshift indicator strains complement a set of six previously described strains which detect each of the base substitutions. We have examined a variety of mutagens and mutators for their ability to cause reversion to Lac+. Surprisingly, frameshifts are well stimulated at many of these runs by ethyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine and 2-amino-purine, mutagens not widely known to induce frameshifts. A comparison of ethyl methanesulfonate, N-methyl-N'-nitro-N-nitrosoguanidine and 2-aminopurine frameshift specificity with that found with a mutH strain suggests that these mutagens partially or fully saturate or inactivate the methylation-directed mismatch repair system and allow replication errors leading to frameshifts to escape repair. This results in a form of indirect mutagenesis, which can be detected at certain sites.

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KURAMITSU, Seiki, and Hiroyuki KAGAMIYAMA. "Site-specific mutagenesis of aspartate aminotransferase." Seibutsu Butsuri 28, no. 1 (1988): 7–11. http://dx.doi.org/10.2142/biophys.28.7.

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Baldrich, Marcus, and Werner Goebel. "Rapid and efficient site-specific mutagenesis." "Protein Engineering, Design and Selection" 3, no. 6 (1990): 563. http://dx.doi.org/10.1093/protein/3.6.563.

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Inouye, Satoshi, Yili Guo, Nicholas Ling, and Shunichi Shimasaki. "Site-specific mutagenesis of human follistatin." Biochemical and Biophysical Research Communications 179, no. 1 (August 1991): 352–58. http://dx.doi.org/10.1016/0006-291x(91)91377-o.

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SOYFER, VALERY N. "Genetic Engineering and Site-Specific Mutagenesis." Annals of the New York Academy of Sciences 452, no. 1 (October 1985): 305–11. http://dx.doi.org/10.1111/j.1749-6632.1985.tb30017.x.

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Sambrook, Joseph, and David W. Russell. "Site-specific Mutagenesis by Overlap Extension." Cold Spring Harbor Protocols 2006, no. 1 (June 2006): pdb.prot3468. http://dx.doi.org/10.1101/pdb.prot3468.

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Foss, K., and William H. McClain. "Rapid site-specific mutagenesis in plasmids." Gene 59, no. 2-3 (January 1987): 285–90. http://dx.doi.org/10.1016/0378-1119(87)90336-2.

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Fujii, J., K. Maruyama, M. Tada, and D. H. MacLennan. "Expression and Site-specific Mutagenesis of Phospholamban." Journal of Biological Chemistry 264, no. 22 (August 1989): 12950–55. http://dx.doi.org/10.1016/s0021-9258(18)51579-9.

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Caffrey, Michael S., and Michael A. Cusanovich. "Site-specific mutagenesis studies of cytochromes c." Biochimica et Biophysica Acta (BBA) - Bioenergetics 1187, no. 3 (September 1994): 277–88. http://dx.doi.org/10.1016/0005-2728(94)90001-9.

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Anthony-Cahill, Spencer J., Michael C. Griffith, Christopher J. Noren, Daniel J. Suich, and Peter G. Schultz. "Site-specific mutagenesis with unnatural amino acids." Trends in Biochemical Sciences 14, no. 10 (October 1989): 400–403. http://dx.doi.org/10.1016/0968-0004(89)90287-9.

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Dissertations / Theses on the topic "Mutagenesi sito specifica"

Mancuso, Rossella <1982&gt. "Immunoconiugati contenenti tossine vegetali o siRNA per la deplezione selettiva di cellule leucemiche. Preparazione, citotossicità e studi di mutagenesi sito-specifica." Doctoral thesis, Alma Mater Studiorum - Università di Bologna, 2012. http://amsdottorato.unibo.it/4507/1/Mancuso_Rossella_Tesi.pdf.

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CD33 is a myeloid cell surface marker absent on normal hematopoietic stem cells and normal tissues but present on leukemic blasts in 90% of adult and paediatric acute myeloid leukaemia (AML) cases. By virtue of its expression pattern and its ability to be rapidly internalized after antibody binding, CD33 has become an attractive target for new immunotherapeutic approaches to treat AML. In this study two immunoconjugates were constructed to contain a humanised single-chain fragment variable antibody (scFv) against CD33 in order to create new antibody-derived therapeutics for AML. The first immunoconjugate was developed to provide targeted delivery of siRNAs as death effectors into leukemic cells. To this purpose, a CD33-specific scFv, modified to include a Cys residue at its C-terminal end (scFvCD33-Cys), was coupled through a disulphide bridge to a nona-d-arginine (9R) peptide carrying a free Cys to the N-terminal. The scFvCD33-9R was able to completely bind siRNAs at a protein to nucleic acid ratio of about 10:1, as confirmed by electrophoretic gel mobility-shift assay. The conjugate was unable to efficiently transduce cytotoxic siRNA (siTox) into the human myeloid cell line U937. We observed slight reductions in cell viability, with a reduction of 25% in comparison to the control group only at high concentration of siTox (300 nM). The second immunoconjugate was constructed by coupling the scFvCD33-Cys to the type 1 ribosome inactivating protein Dianthin 30 (DIA30) through a chemical linking The resulting immunotoxin scFvCD33-DIA30 caused the rapid arrest of protein synthesis, inducing apoptosis and leading ultimately to cell death. In vitro dose-dependent cytotoxicity assays demonstrated that scFvCD33-DIA30 was specifically toxic to CD33-positive cell U937. The concentration needed to reach 50 % of maximum killing efficiency (EC50) was approximately 0.3 nM. The pronounced antigen-restricted cytotoxicity of this novel agent makes it a candidate for further evaluation of its therapeutic potential.

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Tito, Donald. "Site-directed mutagenesis of hydrogenase genes in Azotobacter chroococcum." Thesis, McGill University, 1992. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=56889.

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Accessory hydrogen uptake genes have been identified in a region of the Azotobacter chroococcum genome about 5 kb downstream of the hydrogenase structural genes (hupSL). DNA sequencing has revealed six genes (hupABYCDE) in this region. These genes are probably transcribed in the same direction as hupSL but are probably in a different operon. Mutational analysis had shown that disruption of the hupB, hupY, hupD and hupE genes gives a Hup$ sp-$ phenotype. In the present work additional mutational analysis, using Tn5, a Tn5 -derivative containing a promoterless lacZ gene, and a kanamycin resistance gene, confirms the direction of transcription and the separate nature of the hupABYCDE operon, and extends the region known to be necessary for Hup activity to hupA and possibly to 1.6 kb upstream of hupA.

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Owegi, Margaret. "Site-directed mutagenesis of yeast V-ATPase subunit d." Virtual Press, 2005. http://liblink.bsu.edu/uhtbin/catkey/1319550.

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V-ATPases are enzymes found in all eukaryotic cells. They are organized into a peripheral membrane complex (V1) and an integral membrane complex (V0). VI is responsible for ATP hydrolysis and generates the energy used by Vo to pump protons from the cytosol into the vacuole. Subunit d is a component of Vo possibly located at the interface between V 1 and V. in the V-ATPase complex. We hypothesize that subunit d could be involved in the structural and functional coupling of VI and Vo. This was tested by generating point mutations along the open reading frame of subunit d from yeast. The mutations F94A, H128A, D173A, D217A, D261A, E317A, W325A, E328A and C329A, all in conserved regions of the protein sequence, were characterized by examining their growth phenotype and by assessing their ATPase specific activity, proton transport and V1Vo assembly in purified vacuolar membranes. The mutations E317A, W325A, E328A and C329A had reduced ATPase and proton transport activities. In addition, V1Vo assembly was compromised by the mutation W325A. Our results suggest that residues at the carboxyl-end of subunit d are important for ATPase activity, proton pumping and V1Vo assembly at the membrane.
Department of Chemistry

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Blom, Lillemor. "Investigation of the interactions between the bacterial homologue to actin, and the chaperone GroEL/ES through a combination of protein engineering and spectroscopy." Thesis, Linköping University, Department of Physics, Chemistry and Biology, 2008. http://urn.kb.se/resolve?urn=urn:nbn:se:liu:diva-15818.

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Molecular chaperones help many proteins in the cell reach their native conformation. The mechanism with which they do this has been studied extensively, but has not been entirely elucidated. This work is a continuation of the study done by Laila Villebeck et al. (2007) on the conformational rearrangements in the eukaryotic protein actin in interaction with the eukaryotic chaperone TRiC. In this study the intentions were to analyze the protein MreB, a prokaryotic homologue to actin, when interacting with the prokaryotic chaperone GroEL. The purpose was to investigate if the mechanisms of GroEL and TRiC are similar. The analysis of the conformation of MreB was to be made through calculations of fluorescence resonance energy transfer (FRET) between two positions in MreB labeled with fluorescein. A MreB mutant was made through site-specific mutagenesis to enable labeling at a specific position. Another single mutant and a corresponding double mutant needed for these measurements were avaliable from earlier studies. The results from fluorescence measurements on these mutants indicated that the degree of labeling was insufficient for accurate determination of FRET. Suggestions are made on improvements of the experimental approach for future studies.

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Khaja, Sara. "Site-Directed Mutagenesis in Citrus paradisi Flavonol-Specific 3-O-Glucosyltransferase." Digital Commons @ East Tennessee State University, 2014. https://dc.etsu.edu/etd/2453.

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Flavonoids are plant secondary metabolites that have significant biochemical and physiological roles. Biosynthesis of these compounds involves several modifications, most predominantly glucosylation, which is catalyzed by glucosyltransferases (GTs). A signature amino acid sequence, the PSPG box, is used to identify putative clones and has been shown to be involved in UDP-glucose binding. Site-directed mutagenesis is used to answer questions regarding the structure and function of this family of enzymes, particularly what allows some GTs to be more selective towards some substrates than others. The grapefruit (Citrus paradisi) flavonol-3-O-glucosyltransferase (CpF3GT) is specific for flavonol substrates and will not glucosylate anthocyanidins. Comparison of the CpF3GT sequence with that of Vitis vinifera GT, which glucosylates both flavonols and anthocyanidins, provided the basis for the amino acid substitution of proline 145, alanine 374, and alanine 375 in CpF3GT to threonine, aspartate, and glycine, respectively, to test the affect on GT’s affinity for flavonoid substrates.

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Sheikh, Qaiser Iftikhar. "Exploring the structure and function of bacterial cytosine specific DNA methyltransferases using site-directed mutagenesis." Thesis, University of Sheffield, 2001. http://etheses.whiterose.ac.uk/10258/.

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Point mutations were engineered into the sequence of the multispecific DNA methyltransferase (Mtase) M. SPRI in motif IX, in order to mimic the corresponding motif IX of mono-specific Mtase. A similar approach was adopted to modify the sequence of the monospecific enzyme M. HhaI in motifs IX and X based on the available structure and as a consequence the enzyme regained methylation potential. It was thought that these changes might be sufficient to enable functional exchange of the target recognition domains (TRDs) between a mono- and a multispecific enzyme. However, insertion of various segments of TRD region from M. SPRI into the M. HhaI was not successful (Chapter 4). To establish whether mono- and multispecific Mtases are incompatible in terms of sequence exchanges, a systematic "swapping" of motifs was carried out (Chapter 5). These experiments suggested that there are some enzyme-specific structural interactions between different subunits within each class of Mtases. In second half of this thesis a bacterial two-hybrid system based on the reversible assembly of an engineered form of M. SPRI was developed (Chapter 6). However the Mtase protein does not assemble into an active species until a DNA segment encoding a leucine zipper motif is fused to each of the two halves. Co-transformation of E. coli with the plasmids expressing the C-terminal and N-terminal domains respectively resulted in the abolition of colonies on double antibiotic plates, when an mcr strain was used as host. High performance liquid chromatography was used to estimate the extent of modification of plasmids indirectly. The extent of methylation at specific sequences within a plasmid molecule was readily detected by the corresponding differential susceptibility to digestion by specific restriction enzymes. Using this approach it proved possible to detect different levels of activity produced by wild type and mutant recombinant DNA Methyltransferases with sensitivity and in a semi quantitative manner. In order to analyse the biochemical properties of Mtase, I have developed an in vitro translation-modification assay. Binary studies with the mutants (from Chapter 3 and 5) showed that there were no detectable sequence-specific recognition differences between these enzymes. Taken together, these results suggest that motif IX plays a role in general stabilisation of the enzyme core structure and has a less significant role in DNA recognition.

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Ma, On Ki. "Association of the N-methyl-D-aspartate receptor subunit NR3A with protein phosphatase 2A : structural analysis by site-directed mutagenesis /." View Abstract or Full-Text, 2003. http://library.ust.hk/cgi/db/thesis.pl?BIOL%202003%20MA.

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Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2003.
Includes bibliographical references (leaves 82-99). Also available in electronic version. Access restricted to campus users.

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Chandrasekar, Sowmya. "Probing metal and substrate binding to metallo-[beta]-lactamase ImiS from Aeromonas sobria using site-directed mutagenesis." Oxford, Ohio : Miami University, 2004. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=miami1098134311.

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Liapis, Evagelos. "The supF assay for understanding DNA adduct-induced mutagenesis : traditional application and development of a site-specific version." Thesis, University of Leicester, 2007. http://hdl.handle.net/2381/29719.

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A primary aim of this study was to establish and utilise the supF assay to investigate the mutagenicity of the cancer drug tamoxifen in target endometrial cells. In particular, the supF assay was employed to ascertain the mutations caused by two model reactive intermediates of tamoxifen, alpha-acetoxytamoxifen and 4-hydroxytamoxifen quinone methide (4-OHtamQM) in methylated pSP189 plasmid. The plasmid was methylated in vitro in order to allow application of the LwPy53 algorithm which enables in silico prediction of the Gâ†’T mutation distribution along the human p53 gene, using alpha-acetoxytamoxifen and 4-OHtamQM-induced mutation data from the supF assay.;Relative mutation frequencies increased proportionally with the adduct level for alpha-acetoxytamoxifen, up to âˆ¼15 times the background frequency, while 4-OHtamQM failed to raise the mutation frequency above that of the untreated control. The majority of mutations in alpha-acetoxytamoxifen-treated plasmid were GC â†’TA transversions, while GCâ†’AT transitions predominated in both 4-OHtamQM-treated plasmid and the untreated control. Based on the p53 Gâ†’T transversion predictions, alpha-acetoxytamoxifen induced damage resulted in two hotspots at positions 244 and 273; these mutations might be expected to occur in tamoxifen associated endometrial tumours if tamoxifen adducts play a role in cancer development.;The second part of the thesis was devoted to the development and validation of a novel site-specific assay. The assay, which is a site-specific version of the supF assay, was validated and utilized to investigate the mutagenic potential and types of mutations caused by individual O 6-MeG adducts situated in intact double stranded or gapped plasmids, within two different sequence contexts, in both E. coli and human cells.

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Books on the topic "Mutagenesi sito specifica"

McPherson, M.J., (Ed.), ed. Directed Mutagenesis: A Practical Approach. Oxford: I.R.L. P., 1991.

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J, McPherson M., ed. Directed mutagenesis: A practical approach. Oxford [England]: IRL Press, 1991.

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International Symposium on Site-Directed Mutagenesis and Protein Engineering (1990 Tromsø, Norway). Site-directed mutagenesis and protein engineering: Proceedings of the International Symposium on Site-Directed Mutagensis and Protein Engineering, Tromsø, 27-30 August 1990. Edited by el-Gewely M. Rafaat. Amsterdam: Elsevier Science Publishers., 1991.

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International Symposium on Site-Directed Mutagenesis and Protein Engineering (1990 Tromsø, Norway). Site-directed mutagenesis and protein engineering: Proceedings of the International Symposium on Site-Directed Mutagenesis and Protein Engineering, Tromsø, 27-30 August 1990. Edited by El-Gewely M. Rafaat. Amsterdam: Elsevier Science Publishers, 1991.

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Bolotin, Shelly. Structure function studies of SLAM: Use of site specific mutagenesis to map regions of SLAM which interact iwth measles virus. Ottawa: National Library of Canada, 2003.

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McPherson, M. J. Directed Mutagenesis: A Practical Approach (The Practical Approach Series). Oxford University Press, USA, 1991.

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McPherson, M. J. Directed Mutagenesis: A Practical Approach (Practical Approach Series). Oxford University Press, USA, 1991.

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Vogel, Walter Kevin. Site-directed mutagenesis of the m2 muscarinic acetylcholine receptor. 1997.

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Vogel, Walter Kevin. Site-directed mutagenesis of the m2 muscarinic acetylcholine receptor. 1997.

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Haining, Robert Luddy. Structure/function studies at the active-site region of cyanobacterial ribulose bisphosphate carboxylase/oxygenase by site-directed mutagenesis. 1993.

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Book chapters on the topic "Mutagenesi sito specifica"

Guengerich, F. Peter. "Site-Specific Mutagenesis." In Molecular Life Sciences, 1–3. New York, NY: Springer New York, 2014. http://dx.doi.org/10.1007/978-1-4614-6436-5_159-1.

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Guengerich, Frederick Peter. "Site-Specific Mutagenesis." In Molecular Life Sciences, 1142–44. New York, NY: Springer New York, 2018. http://dx.doi.org/10.1007/978-1-4614-1531-2_159.

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Keravala, Annahita, and Michele P. Calos. "Site-Specific Chromosomal Integration Mediated by ϕC31 Integrase." In Chromosomal Mutagenesis, 165–73. Totowa, NJ: Humana Press, 2008. http://dx.doi.org/10.1007/978-1-59745-232-8_12.

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DeSantis, Grace, and J. Bryan Jones. "Combining Site-Specific Chemical Modification with Site-Directed Mutagenesis." In In Vitro Mutagenesis Protocols, 55–65. Totowa, NJ: Humana Press, 2002. http://dx.doi.org/10.1385/1-59259-194-9:055.

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Klovins, Janis, and Valdis Berzins. "PCR-Based Site-Specific Mutagenesis." In Basic Cloning Procedures, 53–67. Berlin, Heidelberg: Springer Berlin Heidelberg, 1998. http://dx.doi.org/10.1007/978-3-642-71965-3_4.

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Chiba, Kunitoshi, and Dirk Hockemeyer. "Genome Editing in Human Pluripotent Stem Cells Using Site-Specific Nucleases." In Chromosomal Mutagenesis, 267–80. New York, NY: Springer New York, 2014. http://dx.doi.org/10.1007/978-1-4939-1862-1_15.

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Geisinger, Jonathan M., and Michele P. Calos. "Using Phage Integrases in a Site-Specific Dual Integrase Cassette Exchange Strategy." In Chromosomal Mutagenesis, 29–38. New York, NY: Springer New York, 2014. http://dx.doi.org/10.1007/978-1-4939-1862-1_3.

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Meetei, Amom Ruhikanta, and M. R. S. Rao. "Generation of Multiple Site-Specific Mutations by Polymerase Chain Reaction." In In Vitro Mutagenesis Protocols, 95–102. Totowa, NJ: Humana Press, 2002. http://dx.doi.org/10.1385/1-59259-194-9:095.

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Stewart, Charles R. "Site-Specific Mutagenesis of Bacillus subtilis Phage SPO1." In Methods in Molecular Biology, 57–67. New York, NY: Springer New York, 2018. http://dx.doi.org/10.1007/978-1-4939-8940-9_5.

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Swingle, Bryan M. "Oligonucleotide Recombination Enabled Site-Specific Mutagenesis in Bacteria." In Methods in Molecular Biology, 127–32. Totowa, NJ: Humana Press, 2013. http://dx.doi.org/10.1007/978-1-62703-293-3_9.

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Conference papers on the topic "Mutagenesi sito specifica"

Li, Ming. "Highly efficient site-specific mutagenesis in malaria mosquitoes." In 2016 International Congress of Entomology. Entomological Society of America, 2016. http://dx.doi.org/10.1603/ice.2016.115516.

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Ashok Kumar, A., Margaret Insley, Jay Gambee, Sharon J. Busby, and Kathleen L. Berkner. "SITE SPECIFIC MUTAGENESIS WITHIN THE GLA-DOMAIN OF HUMAN FACTOR IX." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644079.

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Factor IX, a plasma protein, plays a critical role in blood coagulation. The biological activity of factor IX as well as several other plasma proteins depends on the presence of gamma-carboxy glutamic acid (Gla) residues in their amino terminal region. In vitro mutagenesis has been used to selectively replace Gla residues of factor IX with aspartic acid (Asp) residues in order to establish the contribution of individual as well as paired Gla residues to the normal functioning of the protein. These substitutions were made at positions 7, 15, 20 and 26 in human factor IX. In addition, residue number 18, a cysteine has been changed to serine in an attempt to disrupt the highly conserved disulfide bond in the gla-domain. The gla-domain mutants will be produced in mammalian cells and compared with native recombinant factor IX. A rapid immunoaffinity purification procedure, which has been used to obtain recombinant factor IX produced in the presence or absence of vitamin K, is being used to purify the mutants. Protein sequence analysis has been used to confirm complete processing and proper gamma-carboxylation of recombinant factor IX. The properties of these mutants as compared to human factor IX will be discussed.

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Yu, Hansong, Shuang Qu, Yuhua Wang, Chunhong Piao, Junmei Liu, and Yaohui Hu. "Cloning and Site-specific Mutagenesis of DS Enzyme Gene of Corynebacterium glutamicum." In 2015 International Conference on Materials, Environmental and Biological Engineering. Paris, France: Atlantis Press, 2015. http://dx.doi.org/10.2991/mebe-15.2015.26.

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Haigwood, N., E.-P. Pâques, G. Mullenbach, G. Moore, L. DesJardin, and A. Tabrizi. "IMPROVEMENT OF T-PA PROPERTIES BY MEANS OF SITE DIRECTED MUTAGENESIS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643841.

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The clinical relevance of tissue-plasminogen-activator (t-PA) as a potent thrombolytic agent has recently been established. It has however been recognized that t-PA does not fulfill all conditions required for an ideal thrombolytic pharmaceutical agent; for example, its physiological stability and its short half life in vivo necessitate the use of very large clinical doses. We have therefore attempted to develop novel mutant t-PA proteins with improved properties by creating mutants by site-directed mutagenesis in M13 bacteriophage. Seventeen mutants were designed, cloned, and expressed in CHO cells. Modifications were of three types: alterations to glycosylation sites, truncations of the N- or C-termini, and amino acids changes at the cleavage site utilized to generate the two chain form of t-PA. The mutant proteins were analyzed in vitro for specific activity, fibrin dependence of the plasminogen activation, fibrin affinity, and susceptibility to inhibition by PAI.In brief, the results are: 1) some unglycosylated and partially glycosylated molecules obtained by mutagenesis are characterized by several-fold higher specific activity than wild type t-PA; 2) truncation at the C-terminus by three amino acids yields a molecule with increased fibrin specificity; 3) mutations at the cleavage site lead zo a decreased inhibition by PAI; and 4) recombinants of these genes have been constructed and the proteins were shown to possess multiple improved properties. The use of site directed mutagenesis has proved to be a powerful instrument to modulate the biological properties of t-PA.

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Nelles, L., H. R. Linjnen, E. Demarsin, D. Collen, and W. E. Holmes. "CHARACTERIZATION OF SINGLE CHAIN UROKINASE-TYPE PLASMINOGEN ACTIVATOR MUTANTS PRODUCED BY SITE-SPECIFIC MUTAGENESIS OF LYS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642909.

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A cDNA encoding full length single chain urokinase-type plasminogen activator (scu-PA) was cloned and sequenced. The recombinant scu-PA (rscu-PA) cDNA as well as the cDNA of two mutants constructed by deoxyoligonucleotide directed mutagenesis of Lys158 in rscu-PA to Gly158 (rscu-PA-Gly158 ) or to Glu158 (rscu-PA-Glu158 ) were inserted into SV40 early promoter/enhancer based expression vectors, which were used to transfect Chinese Hamster Ovary (CHO) cells. The expression products were purified from serum-free conditioned media by immunoadsorption on an insolubilized monoclonal antibody raised against natural scu-PA (nscu-PA), followed by gel filtration.The amidolytic activity of the three rscu-PAs was low (< 500 IU/mg). The mutant rscu-PAs, in contrast to the rscu-PA and nscu-PA, could not be converted into an amidolytically active two-chain form (tcu-PA) by plasmin. The mutant scu-PAs had a very low specific activity (< 1,000 IU/mg) on fibrin plates, whereas wild type rscu-PA had a specific activity < 1000 IU/mg. The mutant scu-PAs did not cause lysis of a I-fibrin labeled plasma clot immersed in citrated human plasma. Serum-free medium from a control transfected CHO cell line showed no significant plasminogen activating activity.In a purified system, both rscu-PA-Gly and rscu-PA-Glu activate plasminogen following Michaelis-Menten kinetics, with a much lower affinity (K = 60-80 yM) but with a higher catalytic rate constant (k2 = B.01 s-1) as compared to the wild type rscu-PA (K =1.0 yM, k = 0.002 s-1).It is concluded thaz conversion of scu-PA to tcu-PA is prerequisite for the activation of plasminogen. However, Lys158 seems to be important for the stability of the Michaelis complex between scu-PA and plasminogen.

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Pittman, Debra D., Louise C. Wasley, Beth L. Murray, Jack H. Wang, and Randal J. Kaufman. "ANALYSIS OF STRUCTURAL REQUIREMENTS FOR FACTOR VIII FUNCTION USING SITE-DIRECTED MUTAGENESIS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644044.

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Factor VIII (fVIII) functions in the intrinsic pathway of coagulation as the cofactor for Factor IXa proteolytic activation of Factor X. fVIII contains multiple sites which are susceptible to cleavage by thrombin, Factor Xa, and activate) protein C. Proteolytic cleavage is required for cofactor activity and may be responsible for inactivation of cofactor activity. In order to identify the role ofthe individual cleavages of fVIII in its activation and inactivation, site-directed DNA mediated mutagenesis of fVIII was performed and the altered forms of fVIII produced and characterized. Conversionof Arg residues to lie residues at amino acid positions 740, 1648, and 1721 resulted in resistance to thrombin cleavage at those siteswith no alteration of in vitro procoagulant activity. Modification of the thrombin cleavage sites at either positions 372 or 1689 resulted in loss of cofactor activity suggesting that these sites are important for activation. Modification of the postulated activated protein C cleavage site at position 336 resulted in fVIII with a higher specific activity than wild type, possibly due to resistance toproteolytic inactivation.DNA mediated mutagenesis was also used to study the role of post-translational biosynthetic modifications of fVIII. Structural characterization of recombinant fVIII suggested the presence of sulfated tyrosine residues within two acidic regions located between amino acid residues 336-372 and 1648-1689. Individual modification of theseTyr residues to Phe had negligible effect on synthesis and in vitrocofactor activity. The effect of combinations of these mutations onsecretion, cofactor activity, and vWF interaction will be presented.

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Jorgensen, M. J., MJ Rabiet, A. B. Cantor, B. Furie, C. L. Brown, C. B. Shoemaker, and B. C. Furie. "VITAMIN K-DEPENDENT γ-CARBOXYLATION OF FACTOR IX REQUIRES A RECOGNITION SITE CONTAINED WITHIN THE PROPEPTIDE." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643564.

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The vitamin K-dependent proteins, including Factor IX (FIX), are calcium-binding proteins that undergo vitamin K-dependent post-translational modification to convert amino terminal glutamic aoid residues to Gla residues. Sequence homology among the propeptides of these proteins suggests a role for this region in designating the adjacent glutamic acid-rich domain for γ-carboxylation during intraoellular processing. Mutations vere made in the propeptide (residues -1 to -18) of FIX, and the effects on γ-carboxylation were assessed. The human FIX cDNA coding sequenoe was modified using oligonucleotide-directed site-specific mutagenesis and was expressed in Chinese hamster ovary cells. The extent of γ-carboxylation of secreted FIX was determined by (1) ability to interact with conformation-specific antibodies directed against the Gla-dependent, metal-stabilized, native structure of FIX, and (2) direct Gla analysis of the alkaline hydrolysate. Using the unmodified coding sequence, 64 ± 17 % of recombinant Factor IX bound to the conformation-specific antibodies, and 9.4 ± 0.7 Gla residues were found (compared with 12 Gla in plasma FIX). When the 18-residue propeptide was deleted, secreted FIX contained no detectable native FIX antigen and no detectable Gla. Similarly, point mutations leading to substitution of Ala for Phe at residue -16 or Glu for Ala at residue -10 led to secretion of FIX containing 2% and 6% native antigen, respectively, and approximately 1-2 Gla residues. The molecular weight of each of the reoombinant FIX species, as estimated by SDS-PAGE, was identical to that of plasma FIX. NH2-terminal sequence analysis of the mutant FIX speoies yielded the NH2-terminal sequence of plasma FIX. These data indicate that the mutations made in the propeptide did not interfere with intracellular proteolytic prooessing of FIX. We conolude that the FIX propeptide participates in defining a recognition site that designates an adjacent glutamic acid-rich domain for γ-carboxylation. The association of the propeptide with the γ-carboxylation recognition site provides the first demonstration of a specific function served by a propeptide in post-translational protein processing.

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Rosenberg, Steven, Karin Hartog, Ole Nordfand, Mirella Ezban, and Rae Lyn Burke. "THE FUNCTIONAL DOMAINS OF COAGULATION FACTOR VIII." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643614.

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The functional domains of Factor VIII have been investigated using site-directed mutagenesis to probe the effect of thrombin cleavage on pro-cofactor/cofactor activity. We have previously shown that clotting activity is obtained upon coexpression of the amino terminal (92 kDa) heavy chain and carboxyl terminal (80 kDa) light chain proteolytic cleavage products as individual, secreted proteins without the909 amino acid central region (Burke et.al., J. Biol. Chem. 261, 12574, 1986). In the present work the thrombin cleavage sites in the heavy and light chains previously characterized by others (D. Eaton et.al., Biochemistry 25, 505, 1986) have been modified to remove these sites and the mutagenized gene reassembled into separate expression vectors for the two chains. Coexpression of wild type and mutant proteins in COS-7 cells has been characterized by coagulant activity, immunological assays specific for each of the two chains, and radioimmuno-precipitations. Alteration of the thrombin cleavage site in the heavy chain (Arg-372→ΔArg-372) leads to loss of coagulant activity, whereas another mutant Arg-372→Lys-372 shows 20-fold reduced activity. Radioimmunoprecipitations and RIA data show that this is not a reflection of reduced synthesis or increased degradation of the mutant polypeptides. These results suggest that Arg-372 is required for the efficient folding, assembly, or proteolytic activation of Factor VIII.

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Schapira, M., B. Waeber, H. R. Brunner, R. Crystal, and M. Courtney. "PROTECTION BY α1-ANTITRYPSIN ALA-357 ARG-358 AGAINST ARTERIAL HYPOTENSION INDUCED BY FACTOR XII FRAGMENT." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642801.

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The specificity of serine protease inhibitors belonging to the serpin superfamily depends on the nature of the reactive center amino acid residues. For example, Met→Arg mutation at the reactive center P1 residue (position 358) alters the specificity of α1-antitrypsin (AT) from the Met-specific enzyme neutrophil elastase to the Arg-specific proteases thrombin, plasma kallikrein (K) and activated Factor XII fragment (XIIf). To obtain an inhibitor species which would inhibit K and XIIf but not thrombin, we now have produced by site-directed mutagenesis of cloned AT cDNA an AT variant having Arg at P1 and Ala at P2. This modification at P2 was made because C1-inhibitor, the major inhibitor of K and XIIf, also has Ala at P2. In purified systems, AT Ala-357 Arg-358 inactivated thrombin, K and XIIf with 2nd-order rate constants of 1.1, 21.8 and 0.6 μM-1 min-1 whereas values of 8.5, 4.2 and 2.1 μM-1 min-1 were found with AT Arg-358. Thus, when compared to AT Arg-358, AT Ala-357 Arg-358 was 5.2 times more efficient for inhibiting K but 7.7 times less efficient for inhibiting thrombin. In vivo, AT Ala-357 Arg-358 (0.7 mg i.v.) did not modify the thrombin time of male Wistar rats while a 2-fold prolongation was seen with 0.7 mg AT Arg-358. However, AT Ala-357 Arg-358 (0.7 mg i.v.) partially prevented the kinin-mediated circulatory collapse induced by XIIf (0.1 μg i.v.) since rats (n=4) treated with this double mutant had a blood pressure fall of 14 ±3 (meaniSD) mmHg while control animals (n = 8) receiving saline or AT Val-358 (0.7 mg i.v.) had a decrease of 27 ± 3 mmHg (p<0.01 by t test). AT Ala-357 Arg-358 has therapeutic potential for disease states with activation of the plasma kinin-forming system such as angioedema attacks or septic shock.

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Pannekok, H., A. J. Van Zonneveid, C. J. M. de vries, M. E. MacDonald, H. Veerman, and F. Blasi. "FUNCTIONAL PROPERTIES OF DELETION-MUTANTS OF TISSUE-TYPE PLASMINOGEN ACTIVATOR." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643724.

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Over the past twenty-five years, genetic methods have generated a wealth of information on the regulation and the structure-function relationship of bacterial genes.These methods are based on the introduction of random mutations in a gene to alter its function. Subsequently, genetic techniques cure applied to localize the mutation, while the nature of the impairedfunction could be determined using biochemical methods. Classic examples of this approach is now considered to be the elucidation of the structure and function of genes, constituting the Escherichia coli lactose (lac) and tryptophan (trp) operons,and the detailed establishment of the structure and function of the repressor (lacl) of the lac operon. Recombinant DNA techniques and the development of appropriate expression systems have provided the means both to study structure and functionof eukaryotic (glyco-) proteins and to create defined mutations with a predestinedposition. The rationale for the construction of mutant genes should preferentiallyrely on detailed knowledge of the three-dimensional structure of the gene product.Elegant examples are the application of in vitro mutagenesis techniques to substitute amino-acid residues near the catalytic centre of subtilisin, a serine proteasefrom Bacillus species and to substituteanamino acid in the reactive site (i.e. Pi residue; methionine) of α-antitrypsin, a serine protease inhibitor. Such substitutions have resulted into mutant proteins which are less susceptible to oxidation and, in some cases, into mutant proteins with a higher specific activity than the wild-type protein.If no data are available on the ternary structure of a protein, other strategies have to be developed to construct intelligent mutants to study the relation between the structure and the function of a eukaryotic protein. At least for a number of gene families, the gene structure is thought to be created by "exon shuffling", an evolutionary recombinational process to insert an exon or a set of exons which specify an additional structural and/or functional domain into a pre-existing gene. Both the structure of the tissue-type plasminogen activator protein(t-PA) and the t-PA gene suggest that this gene has evolved as a result of exon shuffling. As put forward by Gilbert (Science 228 (1985) 823), the "acid test"to prove the validity of the exon shuffling theory is either to delete, insert or to substitute exon(s) (i.e. in the corresponding cDNA) and toassay the properties of the mutant proteins to demonstrate that an exon or a set of adjacent exons encode (s) an autonomousfunction. Indeed, by the construction of specific deletions in full-length t-PA cDNA and expression of mutant proteins intissue-culture cells, we have shown by this approach that exon 2 of thet-PA gene encodes the function required forsecretion, exon 4 encodes the "finger" domain involved in fibrin binding(presumably on undegraded fibrin) and the set of exons 8 and 9 specifies kringle 2, containing a lysine-binding sit(LBS) which interacts with carboxy-terminal lysines, generated in fibrin after plasmic digestion. Exons 10 through 14 encode the carboxy-ter-minal light chain of t-PA and harbor the catalytic centre of the molecule and represents the predominant "target site" for the fast-acting endothelial plasminogen activator inhibitor (PAI-1).As a follow-up of this genetic approach to construct deletion mutants of t-PA, we also created substitution mutants of t-PA. Different mutants were constructed to substitute cDNA encoding thelight chain of t-PA by cDNA encoding the B-chain of urokinase (u-PA), in order to demonstrate that autonomous structural and functional domains of eitherone of the separate molecules are able toexert their intrinsic properties in a different context (C.J.M. de Vries et al., this volume). The possibilities and the limitations of this approach to study the structure and the function of t-PA and of other components of the fibrinolytic process will be outlined.

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Reports on the topic "Mutagenesi sito specifica"

Benkovic, Stephen J. The Use of Combinatorial Heavy and Light Chain Libraries and Site Specific Mutagenesis to Create Antibody Biosensors for Metal Ions. Fort Belvoir, VA: Defense Technical Information Center, April 1997. http://dx.doi.org/10.21236/ada325575.

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Gurevitz, Michael, Michael Adams, and Eliahu Zlotkin. Insect Specific Alpha Neurotoxins from Scorpion Venoms: Mode of Action and Structure-Function Relationships. United States Department of Agriculture, June 1996. http://dx.doi.org/10.32747/1996.7613029.bard.

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This study was motivated by the need to develop new means and approaches to the design of future, environmentally-safe, insecticides. Utilization of anti-insect selective toxins from scorpion venoms and clarification of the molecular basis for their specificity, are a major focus in this project and may have an applicative value. Our study concentrated on the highly insecticidal toxin, LqhaIT, and was devoted to: (I) Characterization of the neuropharmacological and electrophysiological features of this toxin. (II) Establishment of a genetic system for studying structure/activity relationships of the toxin. (III) Analysis of the insecticidal efficacy of an entomopathogenic baculovirus engineered and expressing LqhaIT. The results obtained in this project suggest that: 1) The receptor binding site of LqhaIT on insect sodium channels differs most likely from its analogous receptor site 3 on vertebrate sodium channels. 2) The effects of LqhaIT are presynaptic. Hyperexcitation at the neuromuscular results from dramatic slowing of sodium channel inactivation and enhanced peak sodium currents causes by LqhaIT. 3) The putative toxic surface of LqhaIT involves aromatic and charged amino acid residues located around the C-terminal region and five-residue-turn of the toxin (unpublished). 4) The anti-insect/anti-mammalian toxicity ratio can be altered by site-directed mutagenesis (publication 8). This effect was partly shown at the level of sodium channel function. 5) The insecticidal efficacy of AcNPV baculovirus increased to a great extent when infection was accompanied by expression of LqhaIT (publication 5).

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Gurevitz, Michael, William A. Catterall, and Dalia Gordon. face of interaction of anti-insect selective toxins with receptor site-3 on voltage-gated sodium channels as a platform for design of novel selective insecticides. United States Department of Agriculture, December 2013. http://dx.doi.org/10.32747/2013.7699857.bard.

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Voltage-gated sodium channels (Navs) play a pivotal role in excitability and are a prime target of insecticides like pyrethroids. Yet, these insecticides are non-specific due to conservation of Navs in animals, raising risks to the environment and humans. Moreover, insecticide overuse leads to resistance buildup among insect pests, which increases misuse and risks. This sad reality demands novel, more selective, insect killers whose alternative use would avoid or reduce this pressure. As highly selective insect toxins exist in venomous animals, why not exploit this gift of nature and harness them in insect pest control? Many of these peptide toxins target Navs, and since their direct use via transformed crop plants or mediator microorganisms is problematic in public opinion, we focus on the elucidation of their receptor binding sites with the incentive of raising knowledge for design of toxin peptide mimetics. This approach is preferred nowadays by agro-industries in terms of future production expenses and public concern. However, characterization of a non-continuous epitope, that is the channel receptor binding site for such toxins, requires a suitable experimental system. We have established such a system within more than a decade and reached the stage where we employ a number of different insect-selective toxins for the identification of their receptor sites on Navs. Among these toxins we wish to focus on those that bind at receptor site-3 and inhibit Nav inactivation because: (1) We established efficient experimental systems for production and manipulation of site-3 toxins from scorpions and sea anemones. These peptides vary in size and structure but compete for site-3 on insect Navs. Moreover, these toxins exhibit synergism with pyrethroids and with other channel ligands; (2) We determined their bioactive surfaces towards insect and mammalian receptors (see list of publications); (3) We found that despite the similar mode of action on channel inactivation, the preference of the toxins for insect and mammalian channel subtypes varies greatly, which can direct us to structural features in the basis of selectivity; (4) We have identified by channel loop swapping and point mutagenesis extracellular segments of the Navinvolved with receptor site-3. On this basis and using channel scanning mutagenesis, neurotoxin binding, electrophysiological analyses, and structural data we offer: (i) To identify the residues that form receptor site-3 at insect and mammalian Navs; (ii) To identify by comparative analysis differences at site-3 that dictate selectivity toward various Navs; (iii) To exploit the known toxin structures and bioactive surfaces for modeling their docking at the insect and mammalian channel receptors. The results of this study will enable rational design of novel anti-insect peptide mimetics with minimized risks to human health and to the environment. We anticipate that the release of receptor site-3 molecular details would initiate a worldwide effort to design peptide mimetics for that site. This will establish new strategies in insect pest control using alternative insecticides and the combined use of compounds that interact allosterically leading to increased efficiency and reduced risks to humans or resistance buildup among insect pests.

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Gordon, Dalia, Ke Dong, and Michael Gurevitz. Unexpected Specificity of a Sea Anemone Small Toxin for Insect Na-channels and its Synergic Effects with Various Insecticidal Ligands: A New Model to Mimic. United States Department of Agriculture, November 2010. http://dx.doi.org/10.32747/2010.7697114.bard.

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Motivated by the high risks to the environment and human health imposed by the current overuse of chemical insecticides we offer an alternative approach for the design of highly active insect-selective compounds that will be based on the ability of natural toxins to differentiate between insect and mammalian targets. We wish to unravel the interacting surfaces of insect selective toxins with their receptor sites on voltage-gated sodium channels. In this proposal we put forward two recent observations that may expedite the development of a new generation of insect killers that mimic the highly selective insecticidal toxins: (i) A small (27aa) highly insecticidal sea anemone toxin, Av3, whose toxicity to mammals is negligible; (ii) The prominent positive cooperativity between distinct channel ligands, such as the strong enhancement of pyrethroids effects by anti-insect selective scorpion depressant toxins. We possess a repertoire of insecticidal toxins and sodium channel subtypes all available in recombinant form for mutagenesis followed by analysis of various pharmacological, electrophysiological, and structural methods. Our recent success to express Av3 provides for the first time a selective toxin for receptor site-3 on insect sodium channels. In parallel, our recent success to determine the structures and bioactive surfaces of insecticidal site-3 and site-4 toxins establishes a suitable system for elucidation of toxin-receptor interacting faces. This is corroborated by our recent identification of channel residues involved with these two receptor sites. Our specific aims in this proposal are to (i) Determine the bioactive surface of Av3 toward insect Na-channels; (ii) Identify channel residues involved in binding or activity of the insecticidal toxins Av3 and LqhaIT, which differ substantially in their potency on mammals; (iii) Illuminate channel residues involved in recognition by the anti-insect depressant toxins; (iv) Determine the face of interaction of both site-3 (Av3) and site-4 (LqhIT2) toxins with insect sodium channels using thermodynamic mutant cycle analysis; and, (v) Examine whether Av3, LqhIT2, pyrethroids, and indoxacarb (belongs to a new generation of insecticides), enhance allosterically the action of one another on the fruit fly and cockroach paraNa-channels and on their kdr and super-kdr mutants. This research establishes the grounds for rational design of novel anti-insect peptidomimetics with minimal impact on human health, and offers a new approach in insect pest control, whereby a combination of allosterically interacting compounds increases insecticidal action and reduces risks of resistance buildup.

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Levisohn, Sharon, Maricarmen Garcia, David Yogev, and Stanley Kleven. Targeted Molecular Typing of Pathogenic Avian Mycoplasmas. United States Department of Agriculture, January 2006. http://dx.doi.org/10.32747/2006.7695853.bard.

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Intraspecies identification (DNA "fingerprinting") of pathogenic avian mycoplasmas is a powerful tool for epidemiological studies and monitoring strain identity. However the only widely method available for Mycoplasma gallisepticum (MG) and M. synoviae (MS)wasrandom amplified polymorphic DNA (RAPD). This project aimed to develop alternative and supplementary typing methods that will overcome the major constraints of RAPD, such as the need for isolation of the organism in pure culture and the lack of reproducibility intrinsic in the method. Our strategy focussed on recognition of molecular markers enabling identification of MG and MS vaccine strains and, by extension, pathogenic potential of field isolates. Our first aim was to develop PCR-based systems which will allow amplification of specific targeted genes directly from clinical material. For this purpose we evaluated the degree of intraspecies heterogeneity in genes encoding variable surface antigens uniquely found in MG all of which are putative pathogenicity factors. Phylogenic analysis of targeted sequences of selected genes (pvpA, gapA, mgc2, and lp) was employed to determine the relationship among MG strains.. This method, designated gene targeted sequencing (GTS), was successfully employed to identify strains and to establish epidemiologically-linked strain clusters. Diagnostic PCR tests were designed and validated for each of the target genes, allowing amplification of specific nucleotide sequences from clinical samples. An mgc2-PCR-RFLP test was designed for rapid differential diagnosis of MG vaccine strains in Israel. Addressing other project goals, we used transposon mutagenesis and in vivo and in vitro models for pathogenicity to correlated specific changes in target genes with biological properties that may impact the course of infection. An innovative method for specific detection and typing of MS strains was based on the hemagglutinin-encoding gene vlhA, uniquely found in this species. In parallel, we evaluated the application of amplified fragment length polymorphism (AFLP) in avian mycoplasmas. AFLP is a highly discriminatory method that scans the entire genome using infrequent restriction site PCR. As a first step the method was found to be highly correlated with other DNA typing methods for MG species and strain differentiation. The method is highly reproducible and relatively rapid, although it is necessary to isolate the strain to be tested. Both AFLP and GTS are readily to amenable to computer-assisted analysis of similarity and construction of a data-base resource. The availability of improved and diverse tools will help realize the full potential of molecular typing of avian mycoplasmas as an integral and essential part of mycoplasma control programs.

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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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Gurevitz, Michael, Michael E. Adams, Boaz Shaanan, Oren Froy, Dalia Gordon, Daewoo Lee, and Yong Zhao. Interacting Domains of Anti-Insect Scorpion Toxins and their Sodium Channel Binding Sites: Structure, Cooperative Interactions with Agrochemicals, and Application. United States Department of Agriculture, December 2001. http://dx.doi.org/10.32747/2001.7585190.bard.

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Integrated pest management in modern crop protection may combine chemical and biological insecticides, particularly due to the risks to the environment and livestock arising from the massive use of non-selective chemicals. Thus, there is a need for safer alternatives, which target insects more specifically. Scorpions produce anti-insect selective polypeptide toxins that are biodegradable and non-toxic to warm-blooded animals. Therefore, integration of these substances into insect pest control strategies is of major importance. Moreover, clarification of the molecular basis of this selectivity may provide valuable information pertinent to their receptor sites and to the future design of peptidomimetic anti-insect specific substances. These toxins may also be important for reducing the current overuse of chemical insecticides if they produce a synergistic effect with conventional pesticides. Based on these considerations, our major objectives were: 1) To elucidate the three-dimensional structure and toxic-site of scorpion excitatory, "depressant, and anti-insect alpha toxins. 2) To obtain an initial view to the sodium channel recognition sites of the above toxins by generating peptide decoys through a phage display system. 3) To investigate the synergism between toxins and chemical insecticides. Our approach was to develop a suitable expression system for toxin production in a recombinant form and for elucidation of toxin bioactive sites via mutagenesis. In parallel, the mode of action and synergistic effects of scorpion insecticidal toxins with pyrethroids were studied at the sodium channel level using electrophysiological methods. Objective 1 was achieved for the alpha toxin, LqhaIT Zilberberg et al., 1996, 1997; Tugarinov et al., 1997; Froy et al., 2002), and the excitatory toxin, Bj-xtrIT (Oren et al., 1998; Froy et al., 1999; unpublished data). The bioactive surface of the depressant toxin, LqhIT2, has been clarified and a crystal of the toxin is now being analyzed (unpublished). Objective 2 was not successful thus far as no phages that recognize the toxins were obtained. We therefore initiated recently an alternative approach, which is introduction of mutations into recombinant channels and creation of channel chimeras. Objective 3 was undertaken at Riverside and the results demonstrated synergism between LqhaIT or AaIT and pyrethroids (Lee et al., 2002). Furthermore, negative cross-resistance between pyrethroids and scorpion toxins (LqhaIT and AaIT) was demonstrated at the molecular level. Although our study did not yield a product, it paves the way for future design of selective pesticides by capitalizing on the natural competence of scorpion toxins to distinguish between sodium channels of insects and vertebrates. We also show that future application of anti-insect toxins may enable to decrease the amounts of chemical pesticides due to their synergism.

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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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Fahima, Tzion, and Jorge Dubcovsky. Map-based cloning of the novel stripe rust resistance gene YrG303 and its use to engineer 1B chromosome with multiple beneficial traits. United States Department of Agriculture, January 2013. http://dx.doi.org/10.32747/2013.7598147.bard.

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Research problem: Bread wheat (Triticumaestivum) provides approximately 20% of the calories and proteins consumed by humankind. As the world population continues to increase, it is necessary to improve wheat yields, increase grain quality, and minimize the losses produced by biotic and abiotic stresses. Stripe rust, caused by Pucciniastriiformisf. sp. tritici(Pst), is one of the most destructive diseases of wheat. The new pathogen races are more virulent and aggressive than previous ones and have produced large economic losses. A rich source for stripe-rust resistance genes (Yr) was found in wild emmer wheat populations from Israel. Original Project goals: Our long term goal is to identify, map, clone, characterize and deploy in breeding, novel wild emmer Yr genes, and combine them with multiple beneficial traits. The current study was aiming to map and clone YrG303 and Yr15, located on chromosome 1BS and combine them with drought resistance and grain quality genes. Positional cloning of YrG303/Yr15: Fine mapping of these genes revealed that YrG303 is actually allelic to Yr15. Fine genetic mapping using large segregating populations resulted in reduction of the genetic interval spanning Yr15 to less than 0.1 cM. Physical mapping of the YrG303/Yr15 locus was based on the complete chromosome 1BS physical map of wheat constructed by our group. Screening of 1BS BAC library with Yr15 markers revealed a long BAC scaffold covering the target region. The screening of T. dicoccoidesaccession-specific BAC library with Yr15 markers resulted in direct landing on the target site. Sequencing of T. dicoccoidesBAC clones that cover the YrG303/Yr15 locus revealed a single candidate gene (CG) with conserved domains that may indicate a role in disease resistance response. Validation of the CG was carried out using EMS mutagenesis (loss-of- function approach). Sequencing of the CG in susceptible yr15/yrG303 plants revealed three independent mutants that harbour non-functional yr15/yrG303 alleles within the CG conserved domains, and therefore validated its function as a Pstresistance gene. Evaluation of marker-assisted-selection (MAS) for Yr15. Introgressions of Yr15 into cultivated wheat are widely used now. Recently, we have shown that DNA markers linked to Yr15 can be used as efficient tools for introgression of Yr15 into cultivated wheat via MAS. The developed markers were consistent and polymorphic in all 34 tested introgressions and are the most recommended markers for the introgression of Yr15. These markers will facilitate simultaneous selection for multiple Yr genes and help to avoid escapees during the selection process. Engineering of improved chromosome 1BS that harbors multiple beneficial traits. We have implemented the knowledge and genetic resources accumulated in this project for the engineering of 1B "super-chromosome" that harbors multiple beneficial traits. We completed the generation of a chromosome including the rye 1RS distal segment associated with improved drought tolerance with the Yr gene, Yr15, and the strong gluten allele 7Bx-over-expressor (7Bxᴼᴱ). We have completed the introgression of this improved chromosome into our recently released variety Patwin-515HP and our rain fed variety Kern, as well as to our top breeding lines UC1767 and UC1745. Elucidating the mechanism of resistance exhibited by Yr36 (WKS1). The WHEAT KINASE START1 (WKS1) resistance gene (Yr36) confers partial resistance to Pst. We have shown that wheat plants transformed with WKS1 transcript are resistant to Pst. WKS1 is targeted to the chloroplast where it phosphorylates the thylakoid-associatedascorbateperoxidase (tAPX) and reduces its ability to detoxify peroxides. Based on these results, we propose that the phosphorylation of tAPX by WKS1 reduces the ability of the cells to detoxify ROS and contributes to cell death. Distribution and diversity of WKS in wild emmer populations. We have shown that WKS1 is present only in the southern distribution range of wild emmer in the Fertile Crescent. Sequence analysis revealed a high level of WKS1 conservation among wild emmer populations, in contrast to the high level of diversity observed in NB-LRR genes. This phenomenon shed some light on the evolution of genes that confer partial resistance to Pst. Three new WKS1 haplotypes displayed a resistance response, suggesting that they can be useful to improve wheat resistance to Pst. In summary, we have improved our understanding of cereals’ resistance mechanisms to rusts and we have used that knowledge to develop improved wheat varieties.

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