Relevant bibliographies by topics / Site-Directed Mutagenesi

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  1. Journal articles
  2. Dissertations / Theses
  3. Books
  4. Book chapters
  5. Conference papers
  6. Reports

Academic literature on the topic 'Site-Directed Mutagenesi'

Author: Grafiati
Published: 9 March 2023
Last updated: 10 March 2023

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Journal articles on the topic "Site-Directed Mutagenesi"

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Loke, Paxton, and Tiow-Suan Sim. "A Comparison of Three Site-Directed Mutagenesis Kits." Zeitschrift für Naturforschung C 56, no. 9-10 (October 1, 2001): 810–13. http://dx.doi.org/10.1515/znc-2001-9-1021.

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Abstract In this comparative study, three different mutagenesis kits, namely the MutaGene phagemid in vitro mutagenesis kit (Bio-Rad), the Transformerä Site-Directed mutagenesis kit (Clontech) and the Quik-change site-directed mutagenesis kit (Stratagene) were used for the mutagenesis of IPNS genes. However, a large difference in mutation efficiencies among these kits was encountered. Furthermore, these kits employ different strategies with its own individual strengths and weaknesses. Thus, a comparison among these three kits to evaluate their usefulness and improvements on the strategy adopted by the Quik-change site-directed mutagenesis kit, which was the kit of choice for our work, are presented for the benefit of research work.
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Carter, P. "Site-directed mutagenesis." Biochemical Journal 237, no. 1 (July 1, 1986): 1–7. http://dx.doi.org/10.1042/bj2370001.

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de Vries, G. E. "Site-directed mutagenesis." Trends in Plant Science 5, no. 7 (July 2000): 276. http://dx.doi.org/10.1016/s1360-1385(00)01699-x.

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Nybo, Kristie. "Site-directed mutagenesis: colony growth." BioTechniques 50, no. 2 (February 2011): 87–89. http://dx.doi.org/10.2144/000113609.

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Erb, Laurie, Richard Garrad, Yanjun Wang, Tom Quinn, John T. Turner, and Gary A. Weisman. "Site-directed Mutagenesis of P2UPurinoceptors." Journal of Biological Chemistry 270, no. 9 (March 3, 1995): 4185–88. http://dx.doi.org/10.1074/jbc.270.9.4185.

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Weiner, M. P., and G. L. Costa. "Rapid PCR site-directed mutagenesis." Genome Research 4, no. 3 (December 1, 1994): S131—S136. http://dx.doi.org/10.1101/gr.4.3.s131.

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MacDonald, Nicholas J., José M. P. Freije, Mary L. Stracke, Richard E. Manrow, and Patricia S. Steeg. "Site-directed Mutagenesis ofnm23-H1." Journal of Biological Chemistry 271, no. 41 (October 11, 1996): 25107–16. http://dx.doi.org/10.1074/jbc.271.41.25107.

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Carey, Michael F., Craig L. Peterson, and Stephen T. Smale. "PCR-Mediated Site-Directed Mutagenesis." Cold Spring Harbor Protocols 2013, no. 8 (August 2013): pdb.prot076505. http://dx.doi.org/10.1101/pdb.prot076505.

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Costa, Gina L., and Michael P. Weiner. "Rapid PCR Site-Directed Mutagenesis." Cold Spring Harbor Protocols 2006, no. 1 (January 1, 2006): pdb.prot4144. http://dx.doi.org/10.1101/pdb.prot4144.

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Imai, Kiyohiro, Kenzo Fushitani, Gentaro Miyazaki, Koichiro Ishimori, Teizo Kitagawa, Yoshinao Wada, Hideki Morimoto, Isao Morishima, Daniel T. b. Shih, and Jeremy Tame. "Site-directed mutagenesis in haemoglobin." Journal of Molecular Biology 218, no. 4 (April 1991): 769–78. http://dx.doi.org/10.1016/0022-2836(91)90265-8.

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Dissertations / Theses on the topic "Site-Directed Mutagenesi"

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Rúnarsdóttir, Saga. "Site-Directed Mutagenesis Studies of FucO." Thesis, Uppsala universitet, Institutionen för kemi - BMC, 2009. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-235136.

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Al-Khatib, Haifa Yousef. "Site Directed Mutagenesis of Dienelactone Hydrolase." Thesis, University of North Texas, 1995. https://digital.library.unt.edu/ark:/67531/metadc277940/.

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The clcD gene encoding dienelactone hydrolase (DLH) is part of the clc gene cluster for the utilization of the B-ketoadipate pathway intermediate chlorocatechol. The roles that individual amino acids residues play in catalysis and binding of the enzyme were investigated. Using PCR a 1.9 kbp clcD fragment was amplified and subcloned yielding a 821 bp BamHi to ZscoRI subclone in the plasmid pUC19.
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Chen, Wei 1965. "Site Directed Mutagenesis Of Dienelactone Hydrolase." Thesis, University of North Texas, 1992. https://digital.library.unt.edu/ark:/67531/metadc500900/.

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The role of individual amino acid residues of the enzyme dienelactone hydrolase was investigated. Using the polymerase chain reaction (PCR), a 1.9 kbp clcD fragment was amplified and subcloned yielding a 821 bp BamHI to EcoRI clcD subclone in the plasmid pUC19. Site-specific mutants of dienelactone hydrolase were created using mismatched oligonucleotides to prime DNA synthesis. Specifically modified proteins from mutated clcD genes (Arg 81 to alanine, Tyr 85 to phenylalanine and Arg 206 to alanine), were encoded by the mutant clones. Enzyme assays showed that dienelactone hydrolase activity of the mutants Arg 81 and Arg 206 was totally abolished. The DLHase enzyme activity of mutant Tyr 85 is greatly decreased by approximately two thirds.
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Marc, Daniel. "La myristylation de la proteine de capside vp4 du poliovirus; son role dans le cycle viral." Paris 7, 1991. http://www.theses.fr/1991PA077059.

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La proteine de capside vp4 du poliovirus, ainsi que des precurseurs vp0 et p1, est myristlee: sa glycine n-terminale est liee de maniere covalente a un acide gras tetra-decanoique. Cette modification co-traductionnelle est determinee par la sequence n-terminale de la proteine (gly#1ala#2gln#3val#4ser#5ser#6). Par mutagenese dirigee a l'aide d'oligonucleotides, nous avons modifie dans le cadn viral la sequence codant pour le signal de myristylation de la proteine. Des transcrits genomiques portant les differentes mutations ont ete synthetises in vitro, et leurs proprietes analysees in vitro par traduction en systeme acellulaire, et in vivo apres transfection de cellules de primates. La transfection du transcrit portant la mutation ser#5thr conduit a une production de virus possedant une proteine normalement myristylee. Toutes les autres mutations qui empechent totalement (gly#1arg et gly#1ala) ou partiellement (ser#5pro et ala#2pro) la myristylation de la proteine vp0 in vivo, abolissent l'infectivite des transcrits. Ces dernieres mutations n'empechent pas la replication des transcrits in vivo, mais affectent la maturation proteolytique du precurseur p1 in vitro. En outre, dans le cas des mutations de la glycine no 1, l'assemblage viral n'a pas lieu, le defaut se situant au niveau de l'assemblage des pentameres 14s. Dans le cas des deux autres mutations (ser#5pro et ala#2pro), des particules virales matures s'assemblent en quantite reduite, mais ces virons ne sont pas infectieux et semblent defectueux dans les etapes precoces de l'infection. La myristylation de la proteine vp4 et de ses precurseurs est donc indispensable au cycle viral. Elle joue role important au niveau de l'assemblage viral, mais semble egalement impliquee dans les etapes precoces de la decapsidation
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White, Malcolm F. "Yeast phosphoglycerate mutase studied by site-directed mutagenesis." Thesis, University of Edinburgh, 1989. http://hdl.handle.net/1842/24419.

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Wang, Xiaoshan. "Site-Directed Mutagenesis in Francisella Tularensis by Allelic." Thesis, Virginia Tech, 2007. http://hdl.handle.net/10919/36440.

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Francisella tularensis is a Gram-negative, facultative intracellular coccobacillus and the etiologic agent of tularemia for a wide variety of vertebrate and invertebrate animal species. Several species and subspecies of Francisella are currently recognized. However, the majority of infections are caused by F. tularensis subspecies tularensis (type A) and subspecies holarctica (type B). Given the low infectious dose, multiple transmission routes, severity of illness, and lack of licensed vaccines, F. tularensis has long been considered a potential biological weapon and is now classified as a category A select agent by the National Institutes of Health and the Centers for Disease Control and Prevention.

The investigation of the mechanisms of pathogenesis by F. tularensis type A and B strains is hindered by the difficulty and lack of methods to mutate the putative genes that encode for virulence factors. New genetic tools have been developed that have enabled mutagenesis of F. tularensis type A and type B stains. However, site-specific mutations remain difficult to execute or these methods generate random mutations. In this study a novel method was developed to create site-directed mutations in a putative capsule biosynthesis locus to knock out encapsulation of the attenuated F. tularensis live vaccine strain. Two suicide vectors for mutagenesis of F. tularensis were constructed based on the commercial PCR cloning vector pSC-A. These vectors were created by inserting into the cloning site a kanamycin resistance gene boarded upstream by 1.3 kb of N-terminal DNA and downstream by 1.3 kb of C-terminal DNA that flanks the target gene. Cryotransformation was used to introduce the vectors into F. tularensis. Open reading frame (ORF) FTT0793, which may encode for an ABC transporter involved in capsule export, was initially selected for mutagenesis in order to generate a mutant that was nonencapsulated, but could still synthesize capsule and induce a host immune response. Mutagenesis of this gene was successful. However, phenotypic assays could not confirm that the mutant was nonencapsulated compared to the parent. Therefore, adjacent ORFs FTT0798 and FTT0799, which may encode for a galactosyl transferase and mannosyl transferase, respectively, were also deleted to completely knock out capsule synthesis. The resulting mutant appeared to be nonencapsulated as determined by negative staining transmission electron microscopy.

In this study, a plasmid and method for generating allelic exchange mutants is reported, which should be useful for generating additional mutants of F. tularensis for use in clarifing the roles of specific genes. This vector is currently being used to make a nonencapsulated mutant of a virulent type A strain to determine the role of capsule in virulence.


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Bowker-Kinley, Melissa M. "Pyruvate dehydrogenase kinase Kinetics, site-directed mutagenesis, and regulation /." [Bloomington, Ind.] : Indiana University, 2005. http://wwwlib.umi.com/dissertations/fullcit/3183930.

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Thesis (Ph.D.)--Indiana University, 2005.
Source: Dissertation Abstracts International, Volume: 66-07, Section: B, page: 3690. Chair: Robert A. Harris. Title from dissertation home page (viewed Oct. 5, 2006).
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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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Schmidt, William Richard. "Site-directed mutagenesis of the ncd microtubule motor protein." Thesis, This resource online, 1996. http://scholar.lib.vt.edu/theses/available/etd-12302008-063348/.

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Books on the topic "Site-Directed Mutagenesi"

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

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

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Lloyd, John S. Heterologous expression and site-directed mutagenesis of soluable methane monooxygenase. [s.l.]: typescript, 1997.

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Provart, Nicholas J. A structure-function analysis of pea carbonic anhydrase by site directed mutagenesis. Ottawa: National Library of Canada = Bibliothèque nationale du Canada, 1993.

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Horst, Bluethmann, and Ohashi Pamela S, eds. Transgenesis and targeted mutagenesis in immunology. San Diego: Academic Press, 1994.

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Yang, Winnie Yongchen. Site-directed mutagenesis and purification of gpNu1, the small subunit of bacteriophage lambda terminase. Ottawa: National Library of Canada, 1993.

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Book chapters on the topic "Site-Directed Mutagenesi"

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Carrigan, Patricia E., Petek Ballar, and Sukru Tuzmen. "Site-Directed Mutagenesis." In Methods in Molecular Biology, 107–24. Totowa, NJ: Humana Press, 2010. http://dx.doi.org/10.1007/978-1-61737-954-3_8.

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Adair, John R., and T. Paul Wallace. "Site-Directed Mutagenesis." In Springer Protocols Handbooks, 347–60. Totowa, NJ: Humana Press, 1998. http://dx.doi.org/10.1007/978-1-59259-642-3_28.

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James, Roberta M., and Paul Dickinson. "Site-Directed Mutagenesis." In Springer Protocols Handbooks, 361–81. Totowa, NJ: Humana Press, 1998. http://dx.doi.org/10.1007/978-1-59259-642-3_29.

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Bhattacharya, Animesh. "Site-directed Mutagenesis." In Encyclopedia of Systems Biology, 1954–55. New York, NY: Springer New York, 2013. http://dx.doi.org/10.1007/978-1-4419-9863-7_1474.

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Gooch, Jan W. "Site-Directed Mutagenesis." In Encyclopedic Dictionary of Polymers, 924. New York, NY: Springer New York, 2011. http://dx.doi.org/10.1007/978-1-4419-6247-8_14811.

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Johnson, Alfred C., and Marvin Reitz. "Site-Directed Mutagenesis." In Recombinant DNA Principles and Methodologies, 699–719. Boca Raton: CRC Press, 2021. http://dx.doi.org/10.1201/9781003067658-19.

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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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Strain-Damerell, Claire, and Nicola A. Burgess-Brown. "High-Throughput Site-Directed Mutagenesis." In Methods in Molecular Biology, 281–96. New York, NY: Springer New York, 2019. http://dx.doi.org/10.1007/978-1-4939-9624-7_13.

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McClellan, Michael J. "In Vitro Site Directed Mutagenesis." In Methods in Molecular Biology, 87–95. New York, NY: Springer US, 2023. http://dx.doi.org/10.1007/978-1-0716-3004-4_8.

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Kim, Yang-Gyun, and Stefan Maas. "Multiple Site-Directed Mutagenesis In Vitro." In In Vitro Mutagenesis Protocols, 29–36. Totowa, NJ: Humana Press, 2002. http://dx.doi.org/10.1385/1-59259-194-9:029.

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Conference papers on the topic "Site-Directed Mutagenesi"

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Holmes, W. E., H. R. Lijnen, and D. Collen. "CHARACTERIZATION OFα2-ANTIPLASMIN.REACTIVE SITE VARIANTS PRODUCED BY SITE-DIRECTED MUTAGENESIS." In 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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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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ISPAS, GABRIELA, I. FAMELAER, FATIMA ELMALKI, and M. JACOBS. "LUCIFERASE KNOCK – OUT MUTANTS BY SITE DIRECTED MUTAGENESIS OF THE AMP BINDING SITE." In Proceedings of the 11th International Symposium. WORLD SCIENTIFIC, 2001. http://dx.doi.org/10.1142/9789812811158_0042.

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Yu, Yao-Chuan. "Site-directed mutagenesis studies support postulated MtNPF1.7 structure and transport mechanism." In ASPB PLANT BIOLOGY 2020. USA: ASPB, 2020. http://dx.doi.org/10.46678/pb.20.1332481.

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Li, Mingtao, Xiaoyu You, and Kunrong Mei. "Site-directed mutagenesis of Saccharomyces cerevisiae genome using mismatch PCR product." In International Conference on Biomedical and Intelligent Systems (IC-BIS 2022), edited by Ahmed El-Hashash. SPIE, 2022. http://dx.doi.org/10.1117/12.2660375.

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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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Wiedenmann, Joerg, Beatrice Vallone, Fabrizio Renzi, Karin Nienhaus, Sergey V. Ivanchenko, Carlheinz Roecker, and Gerd U. Nienhaus. "Dimeric variants of the red fluorescent protein eqFP611 generated by site-directed mutagenesis." In Biomedical Optics 2004, edited by Alexander P. Savitsky, Lubov Y. Brovko, Darryl J. Bornhop, Ramesh Raghavachari, and Samuel I. Achilefu. SPIE, 2004. http://dx.doi.org/10.1117/12.529370.

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Ren, Qiang, Fei Luo, Weichao Ding, and Haifeng Lu. "An Improved NSGAII Algorithm Based on Site-Directed Mutagenesis Method for Multi-Objective Optimization." In 2019 IEEE Symposium Series on Computational Intelligence (SSCI). IEEE, 2019. http://dx.doi.org/10.1109/ssci44817.2019.9002847.

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He, Yi-Wu, Mark P. Krebs, Judy Herzfeld, H. G. Khorana, and Kenneth J. Rothschild. "FTIR spectroscopy, site-directed mutagenesis, and isotope labeling: a new approach for studying membrane proteins." In Luebeck - DL tentative, edited by Herbert M. Heise, Ernst H. Korte, and Heinz W. Siesler. SPIE, 1992. http://dx.doi.org/10.1117/12.56283.

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NEU, GEORGIA, JOACHIM BENTROP, KARIN SCHWAB, and REINHARD PAULSEN. "SITE DIRECTED MUTAGENESIS OF PHOSPHORYLATION SITES IN THE C-TERMINAL REGION OF DROSOPHILA RH1 OPSIN." In Proceedings of the International School of Biophysics. WORLD SCIENTIFIC, 2001. http://dx.doi.org/10.1142/9789812799975_0008.

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Reports on the topic "Site-Directed Mutagenesi"

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Needleman, R. Site-directed mutagenesis of an energy transducing protein: Bacteriorhodopsin. Final report, July 15, 1992--July 14, 1996. Office of Scientific and Technical Information (OSTI), May 1998. http://dx.doi.org/10.2172/661520.

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2

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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Stern, David B., and Gadi Schuster. Manipulation of Gene Expression in the Chloroplast: Control of mRNA Stability and Transcription Termination. United States Department of Agriculture, December 1993. http://dx.doi.org/10.32747/1993.7568750.bard.

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Chloroplasts are the site of photosynthesis and of other essential biosynthetic activities in plant cells. Chloroplasts are semi-autonomous organelles, since they contain their own genomes and protein biosynthetic machinery, but depend on the coordinate expression of nuclear genes to assemble macromolecular complexes. The bioeingineering of plants requires manipulation of chloroplast gene expression, and thus a knowledge of the molecular mechanisms that modulate mRNA and protein production. In this proposal the heterotrophic green alga Chlamydomonas reinhardtii has been used as a model system to understand the control and interrelationships between transcription termination, mRNA 3' end processing and mRNA stability in chloroplasts. Chlamydomonas is a unique and ideal system in which to address these issues, because the chloroplast can be easily manipulated by genetic transformation techniques. This research uncovered new and important information on chloroplast mRNA 3' end formation and mRNA stability. In particular, the 3' untranslated regions of chloroplast mRNAs were shown not to be efficient transcription terminators. The endonucleolytic site in the 3' untranslated region was characterized by site directed mutagensis and the role of several 3' untranslated regions in modulating RNA stability and translation has been studied. This information will allow us to experimentally manipulate the expression of chloroplast genes in vivo by post-transcriptional mechanisms, and should be widely applicable to other higher plant systems.
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Spiers, Donald, Arieh Gertler, Harold Johnson, and James Spain. An In Vitro and In Vivo Investigation of the Diverse Biological Activities of Bovine Placental Lactogen. United States Department of Agriculture, August 1993. http://dx.doi.org/10.32747/1993.7568087.bard.

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In order to understand the structure-function relationship of bovine placental lactogen (bPL) and initiate production of material for in vivo testing, 28 different bPL analogues were prepared by either truncation or site-directed mutagenesis. The effect of these mutations was determined by measuring binding capacity, ability to homodimerize extracellular domains (ECDs) of several lactogenic and somatogenic receptors, and by in vitro bioassays. Two analogues were prepared in large amounts for in vivo studies. These studies (a) identified the residues responsible for the somatogenic activity of bPL (K73, G133, T188) and for both lactogenic and somatogenic activity (N-terminus, K185, Y190); (b) allowed preparation of bPL analogues with selectively abolished or reduced somatogenic activity; and (c) provided a tool to understand the kinetic difference between lactogenic and somatogenic receptors. In vivo studies using rodent and dairy models showed that bovine growth hormone (bGH) is superior to bPL in stimulating growth and lactation. Likewise, bGH has greater somatogenic activity in different age groups and thermal environments. Initial studies of bPL analog T188 suggest that its lactogenic potential is superior to bGH. Effective experimental models have now been developed and tested for analysis of new bPL analogs.
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Gutnick, David, and David L. Coplin. Role of Exopolysaccharides in the Survival and Pathogenesis of the Fire Blight Bacterium, Erwinia amylovora. United States Department of Agriculture, September 1994. http://dx.doi.org/10.32747/1994.7568788.bard.

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Fireblight, a disease of apples and pears, is caused by Erwinia amylovora. Mutants of E. amylovora that do not produce the extreacellular polysaccharide (EPS), amylovoran, are avirulent. A similar EPS, stewartan, is produced by E. stewartii, which caused Stewart's wilt of corn, and which has also been implicated in the virulence of this strain. Both stewartan and amylovoran are type 1 capsular polysaccharides, typified by the colanic acid slime produced by Escherichia coli. Extracellular polysaccharide slime and capsules are important for the virulence of bacterial pathogens of plants and animals and to enhance their survival and dissemination outside of the host. The goals of this project were to examine the importance of polysaccharide structure on the pathogenicity and survival properties of three pathogenic bacteria: Erwinia amylovora, Erwinia stewartii and Escherichia coli. The project was a collaboration between the laboratories of Dr. Gutnick (PI, E. coli genetics and biochemistry), Dr. Coplin (co-PI, E. stewartii genetics) and Dr. Geider (unfunded collaborator, E. amylovora genetics and EPS analysis). Structural analysis of the EPSs, sequence analysis of the biosynthetic gene clusters and site-directed mutagenesis of individual cps and ams genes revealed that the three gene clusters shared common features for polysaccharide polymerization, translocation, and precursor synthesis as well as in the modes of transcriptional regulation. Early EPS production resulted in decreased virulence, indicating that EPS, although required for pathogenicity, is anot always advantageous and pathogens must regulate its production carefully.
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Stern, David, and Gadi Schuster. Manipulation of Gene Expression in the Chloroplast. United States Department of Agriculture, September 2000. http://dx.doi.org/10.32747/2000.7575289.bard.

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The steady-state level of a given mRNA is determined by its rates of transcription and degradation. The stabilities of chloroplast mRNAs vary during plant development, in part regulating gene expression. Furthermore, the fitness of the organelle depends on its ability to destroy non-functional transcripts. In addition, there is a resurgent interest by the biotechnology community in chloroplast transformation due to the public concerns over pollen transmission of introduced traits or foreign proteins. Therefore, studies into basic gene expression mechanisms in the chloroplast will open the door to take advantage of these opportunities. This project was aimed at gaining mechanistic insights into mRNA processing and degradation in the chloroplast and to engineer transcripts of varying stability in Chlamydomonas reinhardtii cells. This research uncovered new and important information on chloroplast mRNA stability, processing, degradation and translation. In particular, the processing of the 3' untranslated regions of chloroplast mRNAs was shown to be important determinants in translation. The endonucleolytic site in the 3' untranslated region was characterized by site directed mutagensis. RNA polyadenylation has been characterized in the chloroplast of Chlamydomonas reinhardtii and chloroplast transformants carrying polyadenylated sequences were constructed and analyzed. Data obtained to date suggest that chloroplasts have gene regulatory mechanisms which are uniquely adapted to their post-endosymbiotic environment, including those that regulate RNA stability. An exciting point has been reached, because molecular genetic studies have defined critical RNA-protein interactions that participate in these processes. However, much remains to be learned about these multiple pathways, how they interact with each other, and how many nuclear genes are consecrated to overseeing them. Chlamydomonas is an ideal model system to extend our understanding of these areas, given its ease of manipulation and the existing knowledge base, some of which we have generated.
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Nelson, Nathan, and Charles F. Yocum. Structure, Function and Utilization of Plant Photosynthetic Reaction Centers. United States Department of Agriculture, September 2012. http://dx.doi.org/10.32747/2012.7699846.bard.

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Light capturing and energy conversion by PSI is one of the most fundamental processes in nature. In the heart of these adaptations stand PSI, PSII and their light harvesting antenna complexes. The main goal of this grant proposal was to obtain by X-ray crystallography information on the structure of plant photosystem I (PSI) and photosystem II (PSII) supercomplexes. We achieved several milestones along this line but as yet, like several strong laboratories around the world, we have no crystal structure of plant PSII. We have redesigned the purification and crystallization procedures and recently solved the crystal structure of the PSI supercomplex at 3.3 Å resolution. Even though this advance in resolution appears to be relatively small, we obtained a significantly improved model of the supercomplex. The work was published in J. Biol. Chem. (Amunts et al., 2010). The improved electron density map yielded identification and tracing of the PsaK subunit. The location of an additional 10 ß-carotenes, as well as 5 chlorophylls and several loop regions that were previously uninterruptable have been modeled. This represents the most complete plant PSI structure obtained thus far, revealing the locations of and interactions among 17 protein subunits and 193 non-covalently bound photochemical cofactors. We have continued extensive experimental efforts to improve the structure of plant PSI and to obtain PSII preparation amenable to crystallization. Most of our efforts were devoted to obtain well-defined subcomplexes of plant PSII preparations that are amenable to crystallization. We studied the apparent paradox of the high sensitivity of oxygen evolution of isolated thylakoids while BBY particles exhibit remarkable resilience to the same treatment. The integrity of the photosystem II (PSII) extrinsic protein complement as well as calcium effects arise from the Ca2+ atom associated with the site of photosynthetic water oxidation were investigated. This work provides deeper insights into the interaction of PsbO with PSII. Sight-directed mutagenesis indicated the location of critical sites involved in the stability of the water oxidation reaction. When combined with previous results, the data lead to a more detailed model for PsbO binding in eukaryotic PSII.
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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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