Thematische Bibliographien / Molecular gene cloning

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Auswahl der wissenschaftlichen Literatur zum Thema „Molecular gene cloning“

Autor: Grafiati

Veröffentlicht am 18. Mai 2024

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Zeitschriftenartikel zum Thema "Molecular gene cloning"

Kawai, Shuji, Hiroyuki Honda, Takaaki Tanase, Masahito Taya, Shinji Iijima und Takeshi Kobayashi. „Molecular Cloning ofRuminococcus albusCellulase Gene“. Agricultural and Biological Chemistry 51, Nr. 1 (Januar 1987): 59–63. http://dx.doi.org/10.1080/00021369.1987.10867974.

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He, Xiao-Ping, Zhao-Shen Li, Zhen-Xing Tu, Xue Pan, Yan-Fang Gong, Jun Gao und Jing Jin. „Molecular cloning of human canstatin gene“. World Chinese Journal of Digestology 12, Nr. 10 (2004): 2329. http://dx.doi.org/10.11569/wcjd.v12.i10.2329.

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Iijima, Shinji, Takeshi Uozumi und Teruhiko Beppu. „Molecular Cloning ofThermus flavusMalate Dehydrogenase Gene“. Agricultural and Biological Chemistry 50, Nr. 3 (März 1986): 589–92. http://dx.doi.org/10.1080/00021369.1986.10867438.

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IKEDA, Akemi, Masuhiro TAKATA, Takahide TANIGUCHI und Kenji SEKIKAWA. „Molecular Cloning of Bovine CD14 gene.“ Journal of Veterinary Medical Science 59, Nr. 8 (1997): 715–19. http://dx.doi.org/10.1292/jvms.59.715.

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Kazemi, B., F. Fallahian ., N. Seyed ., M. Bandepour ., A. Shabani . und P. Ghadam . „Molecular Cloning of the Streptokinase Mutant Gene“. Pakistan Journal of Biological Sciences 9, Nr. 3 (15.01.2006): 546–48. http://dx.doi.org/10.3923/pjbs.2006.546.548.

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KAWAI, Shuji, Hiroyuki HONDA, Takaaki TANASE, Masahito TAYA, Shinji IIJIMA und Takeshi KOBAYASHI. „Molecular cloning of Ruminococcus albus cellulase gene.“ Agricultural and Biological Chemistry 51, Nr. 1 (1987): 59–63. http://dx.doi.org/10.1271/bbb1961.51.59.

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Zhou, C., S. H. Huang und A. Y. Jong. „Molecular Cloning of Saccharomyces cerevisiae CDC6 Gene“. Journal of Biological Chemistry 264, Nr. 15 (Mai 1989): 9022–29. http://dx.doi.org/10.1016/s0021-9258(18)81897-x.

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Robert Klein, Jürgen, Bernhard Henrich und Roland Plapp. „Molecular cloning of theEnvC gene ofEscherichia coli“. Current Microbiology 21, Nr. 6 (Dezember 1990): 341–47. http://dx.doi.org/10.1007/bf02199435.

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Clouston, W. M., B. A. Evans, J. Haralambidis und R. I. Richards. „Molecular cloning of the mouse angiotensinogen gene“. Genomics 2, Nr. 3 (April 1988): 240–48. http://dx.doi.org/10.1016/0888-7543(88)90008-0.

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Russell, Mark W., Paul Kemp, Lihong Wang, Lawrence C. Brody und Seigo Izumo. „Molecular cloning of the human HAND2 gene“. Biochimica et Biophysica Acta (BBA) - Gene Structure and Expression 1443, Nr. 3 (Dezember 1998): 393–99. http://dx.doi.org/10.1016/s0167-4781(98)00237-1.

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Dissertationen zum Thema "Molecular gene cloning"

Seto, Nina Oi Ling. „The copper-zinc superoxide dismutase gene from Drosophila melanogaster : attempts to clone the gene using two mixed sequence oligonucleotide probes“. Thesis, University of British Columbia, 1987. http://hdl.handle.net/2429/26534.

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Superoxide dismutase is an enzyme which scavenges superoxide radicals and is thought to be a longevity determinant, as there exists a positive correlation between superoxide dismutase concentration and maximum life span potential. The cytosolic CuZn superoxide dismutase in D. melanogaster has been purified and sequenced, but the gene has not been cloned. However, when it is available the CuZn SOD gene may be reintroduced into the Drosophila genome via the P-element transformation system so its effects on the life span potential of Drosophila may be studied. This study describes attempts to clone the CuZn SOD gene from D. melanogaster using two mixed sequence oligonucleotide probes. The SI probe corresponds to amino acids 43-48 of the protein sequence and contains 128 different oligonucleotide sequences representing all possible codon combinations predicted from the amino acid sequence. The GT3 probe is targeted to amino acids 90-95 of the protein. In this probe, deoxyguanosine was placed in positions where all four nucleotides may occur to decrease probe heterogeneity. The probes were used to screen D. melanogaster Canton-S and Oregon-R genomic lambda libraries. Three positive clones isolated from the Canton-S library had identical nucleotide sequence in the GT3 probe binding region, and sequencing of the probe binding site revealed that one member of the GT3 probe had formed a 15 bp duplex with the phage DNA. Screening of the Oregon-R library produced four clones which hybridized with both GT3 and S1 probes. When these phage DNA were hybridized to polytene chromosomes by in situ hybridization, none mapped to 68AB on the third chromosome, the location of the CuZn SOD gene. These results suggest that modification of the classical strategy used in this study is necessary, and implications on probe design are discussed.
Medicine, Faculty of
Biochemistry and Molecular Biology, Department of
Graduate

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Bates, Nancy Carol. „Characterization of cbg : a cloned gene encoding an extracellular [beta]-glucosidase from Cellulomonas fimi“. Thesis, University of British Columbia, 1987. http://hdl.handle.net/2429/26163.

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A group of Escherichia coli clones harbouring recombinant pBR322 plasmid, containing Cellulomonas fimi DNA inserts, that reacted with antiserum to C.fimi culture supernatant, was screened for their ability to hydrolyze carboxymethyl cellulose (CMC) and 4-methylumbeliferyll-β-D-cellobioside (MUC). A clone, pEC62, hydrolyzed MUC but did not hydrolyze CMC. The recombinant enzyme encoded by pEC62 was shown to be a β-glucosidase (cellobiase). C.fimii itself was shown to encode an extracellular β-glucosidase in C.fimi. This is the first report of an extracellular β-glucosidase from a bacterium. Deletion analysis localized the cloned gene (cbg)to the tet promoter proximal region of the 7.0 kilobase insert of pEC62. Further analysis and sequence data showed a highly active derivative of pEC62 contained a translational gene fusion between lacZ of pUC13 and cbg. From this data, a location for the cbg start site was proposed.
Science, Faculty of
Microbiology and Immunology, Department of
Graduate

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Moser, Bernhard. „Molecular cloning, characterization and expression of the endoglucanase C gene of Cellulomonas fimi and properties of the native and recombinant gene products“. Thesis, University of British Columbia, 1988. http://hdl.handle.net/2429/29036.

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In addition to substrate-associated cellulases, Cellulomonas fimi secretes endoglucanases ( endo-1, 4-β-D-glucan glucanohydrolases, EC 3.2.1.4. ) which are recovered from the cellulose-free culture supernatant of cells grown on microcrystalline cellulose. Two such enzymes, C3.1 and C3.2 with Mrs of 130'000 and 120'000, respectively, were purified to homogeneity. The two endoglucanases were shown to share the same N-terminal amino acid sequence and to hydrolyze carboxymethylcellulose ( CMC ) with similar efficiencies ( 236u/mg protein for C3.1 and 367u/mg protein for C3.2 ). The recombinant lambda vector L47.1-169 was identified from a C.fimi DNA-lambda library on the basis of hybridization with C3.1/2-specific oligonucleotide probes. The subclone pTZ18R-8 only moderately expressed CMCase activity. The 5'-terminus of cenC ( the gene coding for C3.1/2 ) was localized in the insert by Southern transfer experiments and nucleotide sequence analysis. Results from total C.fimi RNA-DNA hybrid protection analyses defined the boundaries of cenC in pTZ18R-8 and led to the tentative identification of -10 and -35 promoter sequences. To improve the expression of cenC, its entire coding sequence, except for the start codon GTG, was fused in frame to the ATG codon of a synthetic ribosomal binding site ( PTIS ) and placed under the transcriptional control of the lac p/o system. Induction of the resulting clone ( JM101[pTZP-cenC] ) led to impaired growth in liquid cultures because overproduction of CenC inhibited cell division'" and eventually led to cell death. Analysis of cell fractions by SDS-PAGE revealed a dominant ( >10% of total cell extract proteins ), clone-specific protein with a Mr of approximately 140'000 which was found exclusively in the cytoplasmic fraction. Conversely, 60% of the total CMC-hydrolyzing activity was localized in the periplasmic fraction indicating that the export of CenC is required for maximal expression of endoglucanase activity. Isolation of the cellulolytic activities from an osmotic shockate led to the purification to homogeneity of two recombinant cellulases, CenC1 and CenC2, with Mr of 130'000 and 120'000, respectively. Both enzymes hydrolyzed CMC with similar efficiencies ( 278u/mg protein for CenC1 and 390u/mg protein for CenC2 ). In addition, amino acid sequence analyses showed the two enzymes to have the same N-termini as the native enzymes and proved furthermore that the CenC leader peptide was functional in Escherichia coli.
Science, Faculty of
Microbiology and Immunology, Department of
Graduate

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Woodruff, Wendy Anne. „Cloning and characterization of the oprF gene for protein F from Pseudomonas aeruginosa“. Thesis, University of British Columbia, 1988. http://hdl.handle.net/2429/29218.

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The oprF gene encoding porin protein F from Pseudomonas aeruginosa was cloned onto a cosmid vector into Escherichia coli. Protein F was expressed in large amounts in E. coli and retained its heat- and reduction-modifiable and immunological characteristics. The cloned oprF gene product was purified from E. coli and characterized with respect to pore-forming ability in black lipid bilayers. Small channels, with an average single channel conductance of approximately 0.4 nS, were observed. A similar small channel size was observed for native protein F. The oprF sequences were used as a DNA-DNA hybridization probe with chromosomal DNA from the 17 IATS (International Antigen Typing Scheme) strains of P. aeruginosa, 52 clinical isolates and the non-aeruginosa Pseudomonads. Conservation of oprF sequences was observed among all the P. aeruginosa strains and to a lesser extent among the non-aeruginosa strains of the P. fluorescens rRNA homology group. Insertion mutations in the oprF gene were created in vivo by Tn1mutagenesis of the cloned gene in E. coli and in vitro by insertion of the streptomycin-encoding Ω fragment into the cloned gene, followed by transfer of the mutated protein F gene back into P. aeruginosa and homologous recombination with the chromosome. The oprF mutants were characterized by gel electrophoresis and immunoblotting, and it was shown that the mutants had lost protein F. The P. aeruginosa oprF mutants were characterized with respect to growth rates, antibiotic permeability and cell surface hydrophobicity. The results of these studies indicated that major alterations in the cell surface had occurred and that the cells were unable to grow in a non-defined liquid medium without added electrolytes. Marginal differences were observed in MICs (minimum inhibitory concentrations) of hydrophilic antibiotics for the oprF mutants compared with their protein F-sufficient parents. The putative roles of protein F in antibiotic permeability and general outer membrane permeability are discussed. Evidence for extensive homologies between protein F, the OmpA protein of E. coli and PHIII of Neisseria gonorrhoeae are presented. A role for protein F in prophylactic anti-Pseudomonas therapy, as a target for vaccine development, is proposed.
Science, Faculty of
Microbiology and Immunology, Department of
Graduate

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Wakarchuk, Warren William. „The molecular cloning and characterization of a Beta-glucosidase gene from an Agrobacterium“. Thesis, University of British Columbia, 1987. http://hdl.handle.net/2429/27559.

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The β-glucosidase (Abg) from ATCC 21400, an Agrobacterium species, was purified to homogeneity. The protein was cleaved with cyanogen bromide and the peptides were purified by reversed phase high pressure liquid chromatography. The partial amino-acid sequences for three CNBr peptides, CNBr1, CNBr2 and CNBr3, were determined by automated Edman degradation. A sequence from CNBr2 was used to synthesize a mixture of oligonucleotides which was used as a hybridization probe to identify a recombinant DNA clone carrying the gene for β-glucosidase. A single clone was isolated which expressed an enzymatic activity that hydrolyzed several β-glucosides. The enzymatic activity produced by this clone could be adsorbed by rabbit antiserum raised against the Agrobacterium enzyme. The direction of transcription of the β-glucosidase gene was determined by verifying the DNA sequence 3' to the oligonucleotide probe binding site. After subcloning the gene a high level of expression was obtained in the plasmid vector pUC18 using the lacZ gene promoter. The nucleotide sequence of the 1599 bp insert in pABG5 was determined using the chain terminator method. The start of the protein coding region was determined by aligning the amino terminal sequence of the protein with the predicted amino acid sequence of the cloned gene. The open reading frame was 1387 nucleotides and contained 458 codons. The molecular weight calculated from the deduced amino acid sequence agreed with that observed from both the native and recombinant enzymes. The predicted amino acid composition from the open reading frame matched with that determined for the native β-glucosidase. The stop codon of this coding region was followed by a potential stem loop structure which may be the transcriptional terminator. There was a region of the deduced Abg sequence which had homology to a region from two other β-glucosidase sequences. This region of homology contained a putative active site by analogy with the active site of hen egg white lysozyme.
Science, Faculty of
Microbiology and Immunology, Department of
Graduate

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Zhang, Ling 1962. „Molecular cloning and characterization of the chicken ornithine decarboxylase gene“. Thesis, McGill University, 1994. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=22831.

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Ornithine decarboxylase (ODC) is the rate determining enzyme in the biosynthesis of polyamines which are essential for cell growth. In chickens, significantly higher bioactivity is reported in broiler than in egg layer strains of chickens (Bulfield et al., 1988). To characterize the genetic differences in growth rates and ODC levels in chickens, an ODC cDNA and genomic gene were cloned and sequenced. Sequencing of ODC cDNA revealed that this clone (pODZ3: 2,052 bp) was not a full length of ODC cDNA and contained 2 putative introns. The open reading frame (introns deleted) coded for a protein of 404 amino acids which had about 85% amino acid identity with human ODC. Sequencing of genomic ODC clone (pODG2-8: 5098 bp) represented the 3$ prime$ end of ODC gene from downstream of intron 7. Northern blotting of chicken RNA probed with the insert of pODZ3 revealed 2 hybridizable messages of 1.6 and 2.1 kb, respectively. In addition, analysis of MspI restriction fragment length polymorphism (RFLP) using the 3$ prime$ end of ODC gene as a probe suggested that two MspI RFLPs present in the lean line of broiler chickens was related to selection of high lean body mass.

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Fisher, Simon E. „Positional cloning of the gene responsible for Dent's disease“. Thesis, University of Oxford, 1995. http://ora.ox.ac.uk/objects/uuid:22f6e7a5-4f00-41c9-a1d3-1b05899f22c0.

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The hypervariable locus DXS255 in human Xp11.22 has a heterozygosity exceeding 90% and has therefore facilitated the localization of several disease genes which map to the proximal short arm of the X chromosome, including the immune deficiency Wiskott-Aldrich syndrome and the eye disorders retinitis pigmentosa, congenital stationary night blindness and Aland Island eye disease. In addition, a microdeletion involving DXS255 has been identified in patients suffering from Dent's disease, a familial X-linked renal tubular disorder which is characterized by low molecular weight proteinuria, hypercalciuria, nephrocalcinosis, nephrolithiasis (kidney stones) and eventual renal failure. Two YAC contigs were constructed in Xp11.23-p11.22 in order to aid transcript mapping; the first centred on the DXS255 locus, the second mapping distal to the first and linking the genes GATA, TFE3 and SYP to the OATL1 cluster. Eleven novel markers were generated, one of which contains an exon from a novel calcium channel gene. Four putative CpG islands were detected in the region. Analysis of the microdeletion associated with Dent's disease using markers from the DXS255 contig demonstrated that it is confined to a 370kb interval. A YAC overlapping this deletion was hybridized to a kidney-specific cDNA library to isolate coding sequences that might be implicated in the disease aetiology. The clones thus identified detect a 9.5kb transcript which is expressed predominantly in kidney, and originate from a novel gene (CLCN5) falling within the deleted region. Sequence analysis indicates that the 746 residue protein encoded by this gene is a new member of the C1C family of voltage-gated chloride channels. The coding region of CLCN5 is organized into twelve exons, spanning 25-30kb of genomic DNA. Using the information presented in this thesis, other studies have identified deletions and point mutations which disrupt CLCN5 activity in further patients affected with X-linked hypercalciuric nephrolithiasis, confirming the role of this locus in renal tubular dysfunction.

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Pauliny, Angela. „Cloning and molecular characterisation of the zebrafish colourless gene“. Thesis, University of Bath, 2002. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.393804.

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Baker, N. E. „Wingless : A gene required for segmentation in Drosophila“. Thesis, University of Cambridge, 1985. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.377244.

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Oza, Kalpesh. „Cloning of a DNA repair gene (uvsF) from Aspergillus“. Thesis, McGill University, 1989. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=59577.

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In order to clone the DNA repair gene of Aspergillus nidulans, uvsF$ sp-$ pyrG$ sp-$ strains were transformed with a genomic library in a plasmid vector that carried the pyr-4 gene of Neurospora which complements pyrG mutants of Aspergillus. Out of the several transformants obtained, four were like wild type. For rescuing plasmids, transformant DNA was digested with Bg/II and self ligated, and used for transformation of E. coli. Two types of plasmids were obtained; these two had a region in common ($<$1.0 kb) that was not a simple overlap and gave evidence for rearrangements. Surprisingly, only the plasmids with the larger insert of Aspergillus DNA were able to complement uvsF$ sp-$ in the secondary transformation. Northerns of polyA$ sp+$-enriched mRNA, probed with this plasmid, showed three bands. However, its subclone which spans the shared region hybridized to only one of them (1.0 kb). Two cDNA and five genomic clones were identified. The two cDNA clones though not identical, cross-hybridized. Three out of five genomic clones were identical. The cDNA hybridized to a short segment (2.2 kb) of one of the three types of genomic clones, locating the putative uvsF gene sequence.

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Bücher zum Thema "Molecular gene cloning"

A, Lund Peter, und Minchin Steve, Hrsg. Gene cloning. New York: Taylor & Francis Group, 2007.

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Howe, Christopher. Gene Cloning and Manipulation. 2. Aufl. Leiden: Cambridge University Press, 2007.

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Gene cloning and manipulation. Cambridge [England]: Cambridge University Press, 1995.

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Brown, T. A. Gene cloning: An introduction. 3. Aufl. London: Chapman and Hall, 1995.

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Gene cloning: An introduction. 2. Aufl. London: Chapman and Hall, University and Professional Division, 1990.

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The ABCs of gene cloning. New York: Chapman & Hall, 1997.

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Winnacker, Ernst-L. From genes to clones: Introduction to gene technology. Weinheim: VCH, 1987.

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Winnacker, Ernst-L. From genes to clones: Introduction to gene technology. Weinheim: VCH Verlagsgesellschaft, 1987.

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J, Boulnois G., und University of Leicester, Hrsg. Gene cloning and analysis: A laboratory guide. Oxford: Blackwell Scientific Publications, 1987.

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Winnacker, Ernst L. From genes to clones: Introduction to gene technology. Weinheim, Federal Republic of Germany: VCH, 1987.

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Buchteile zum Thema "Molecular gene cloning"

Cortes, Mauricio, James R. Mensch, Miriam Domowicz und Nancy B. Schwartz. „Proteoglycans: Gene Cloning“. In Methods in Molecular Biology, 3–21. Totowa, NJ: Humana Press, 2011. http://dx.doi.org/10.1007/978-1-61779-498-8_1.

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Habener, Joel F. „Approaches to Gene Cloning“. In Molecular Cloning of Hormone Genes, 1–9. Totowa, NJ: Humana Press, 1987. http://dx.doi.org/10.1007/978-1-4612-4824-8_1.

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Rougeon, François, Jean-Jacques Panthier, Inge Holm, Florent Soubrier und Pierre Corvol. „The Renin Gene“. In Molecular Cloning of Hormone Genes, 321–42. Totowa, NJ: Humana Press, 1987. http://dx.doi.org/10.1007/978-1-4612-4824-8_13.

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Habener, Joel F. „Gene Structure and Regulation“. In Molecular Cloning of Hormone Genes, 11–51. Totowa, NJ: Humana Press, 1987. http://dx.doi.org/10.1007/978-1-4612-4824-8_2.

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Moore, David D., Richard F. Selden, Edouard Prost, Daniel S. Ory und Howard M. Goodman. „The Human Growth-Hormone Gene Family“. In Molecular Cloning of Hormone Genes, 121–35. Totowa, NJ: Humana Press, 1987. http://dx.doi.org/10.1007/978-1-4612-4824-8_6.

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Jagodic, Maja, und Pernilla Stridh. „Positional Gene Cloning in Experimental Populations“. In Methods in Molecular Biology, 3–24. New York, NY: Springer New York, 2014. http://dx.doi.org/10.1007/7651_2014_108.

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Running, Katherine L. D., und Justin D. Faris. „Rapid Cloning of Disease Resistance Genes in Wheat“. In Compendium of Plant Genomes, 187–212. Cham: Springer International Publishing, 2023. http://dx.doi.org/10.1007/978-3-031-38294-9_10.

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AbstractWheat is challenged by rapidly evolving pathogen populations, resulting in yield losses. Plants use innate immune systems involving the recognition of pathogen effectors and subsequent activation of defense responses to respond to pathogen infections. Understanding the genes, genetic networks, and mechanisms governing plant-pathogen interactions is key to the development of varieties with robust resistance whether through conventional breeding techniques coupled with marker selection, gene editing, or other novel strategies. With regards to plant-pathogen interactions, the most useful targets for crop improvement are the plant genes responsible for pathogen effector recognition, referred to as resistance (R) or susceptibility (S) genes, because they govern the plant’s defense response. Historically, the molecular identification of R/S genes in wheat has been extremely difficult due to the large and repetitive nature of the wheat genome. However, recent advances in gene cloning methods that exploit reduced representation sequencing methods to reduce genome complexity have greatly expedited R/S gene cloning in wheat. Such rapid cloning methods referred to as MutRenSeq, AgRenSeq, k-mer GWAS, and MutChromSeq allow the identification of candidate genes without the development and screening of high-resolution mapping populations, which is a highly laborious step often required in traditional positional cloning methods. These new cloning methods can now be coupled with a wide range of wheat genome assemblies, additional genomic resources such as TILLING populations, and advances in bioinformatics and data analysis, to revolutionize the gene cloning landscape for wheat. Today, 58 R/S genes have been identified with 42 of them having been identified in the past six years alone. Thus, wheat researchers now have the means to enhance global food security through the discovery of R/S genes, paving the way for rapid R gene deployment or S gene elimination, manipulation through gene editing, and understanding wheat-pathogen interactions at the molecular level to guard against crop losses due to pathogens.

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Carter, Glen P., Dena Lyras, Rachael Poon, Pauline M. Howarth und Julian I. Rood. „Methods for Gene Cloning and Targeted Mutagenesis“. In Methods in Molecular Biology, 183–201. Totowa, NJ: Humana Press, 2010. http://dx.doi.org/10.1007/978-1-60327-365-7_12.

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Malcolm, Colin A., und Lucinda M. C. Hall. „Cloning and Characterisation of a Mosquito Acetylcholinesterase Gene“. In Molecular Insect Science, 57–65. Boston, MA: Springer US, 1990. http://dx.doi.org/10.1007/978-1-4899-3668-4_7.

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Margolis, Frank L. „Molecular Cloning of Olfactory-Specific Gene Products“. In Molecular Neurobiology of the Olfactory System, 237–65. Boston, MA: Springer US, 1988. http://dx.doi.org/10.1007/978-1-4613-0989-5_11.

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Konferenzberichte zum Thema "Molecular gene cloning"

Fang, GuiJie, und XianFeng Qiao. „Molecular Cloning and Analysis of Hubei White Swine Myostatin Gene“. In 2010 2nd International Conference on Information Engineering and Computer Science (ICIECS). IEEE, 2010. http://dx.doi.org/10.1109/iciecs.2010.5678159.

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Yin, Heng, Xiaoming Zhao und Yuguang Du. „Cloning and Molecular Characterization of a SKP1 Gene from Brassica napus“. In 2009 3rd International Conference on Bioinformatics and Biomedical Engineering (iCBBE). IEEE, 2009. http://dx.doi.org/10.1109/icbbe.2009.5162512.

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Moheer, Reyad Qaed Al, Farah Diba Abu Bakar und Abdul Munir Abdul Murad. „Molecular cloning and characterization of alpha - galactosidase gene from Glaciozyma antarctica“. In THE 2015 UKM FST POSTGRADUATE COLLOQUIUM: Proceedings of the Universiti Kebangsaan Malaysia, Faculty of Science and Technology 2015 Postgraduate Colloquium. AIP Publishing LLC, 2015. http://dx.doi.org/10.1063/1.4931247.

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Zhou, Tao, Anchun Cheng, Mingshu Wang, Dekang Zhu, Xiaoyue Chen, An-chun Cheng, Ming-shu Wang et al. „Molecular Cloning and Characterization of the UL10 Gene from Duck Enteritis Virus“. In 2010 4th International Conference on Bioinformatics and Biomedical Engineering (iCBBE). IEEE, 2010. http://dx.doi.org/10.1109/icbbe.2010.5515899.

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Li, Lijuan, Anchun Cheng, Mingshu Wang, Dekang Zhu, Renyong Jia, Qihui Luo, Hengmin Cui et al. „Molecular Cloning and Sequence Analysis of the Duck Plague Virus gI Gene“. In 2010 4th International Conference on Bioinformatics and Biomedical Engineering (iCBBE). IEEE, 2010. http://dx.doi.org/10.1109/icbbe.2010.5516186.

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Zhu, Qing-Hua. „Molecular Cloning and Sequence Analysis of psbA Gene Fragment from Cladosiphon okamuranus“. In 2010 4th International Conference on Bioinformatics and Biomedical Engineering (iCBBE). IEEE, 2010. http://dx.doi.org/10.1109/icbbe.2010.5516813.

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Odeph, Margaret, Qian Yang und Jackson Phiri. „Molecular cloning of ERG 2 gene to effect knockdown in Chaetomium cupreum“. In 2011 6th International Conference on Broadband and Biomedical Communications (IB2Com). IEEE, 2011. http://dx.doi.org/10.1109/ib2com.2011.6217938.

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Fang, GuiJie, und XianFeng Qiao. „Molecular Cloning and Analysis of Glutathione S-Transferase Gene of Schistosoma japonicum“. In 2010 2nd International Conference on Information Engineering and Computer Science (ICIECS). IEEE, 2010. http://dx.doi.org/10.1109/iciecs.2010.5678431.

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Odeph, Margaret, Qian Yang und Jackson Phiri. „Molecular cloning of ERG 2 gene to effect knockdown in chaetomium cupreum“. In 2012 IEEE International Conference on Information Science and Technology (ICIST). IEEE, 2012. http://dx.doi.org/10.1109/icist.2012.6221602.

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Yang, Xiaoyuan, Anchun Cheng, Mingshu Wang, Dekang Zhu, Xiaoyue Chen, Renyong Jia, Qihui Luo, Yi Zhou und Zhengli Chen. „Molecular cloning and sequence analysis of the duck enteritis virus Us4 gene“. In 2010 3rd International Conference on Biomedical Engineering and Informatics (BMEI). IEEE, 2010. http://dx.doi.org/10.1109/bmei.2010.5639851.

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Berichte der Organisationen zum Thema "Molecular gene cloning"

Dubcovsky, Jorge, Tzion Fahima und Ann Blechl. Positional cloning of a gene responsible for high grain protein content in tetraploid wheat. United States Department of Agriculture, September 2003. http://dx.doi.org/10.32747/2003.7695875.bard.

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High Grain Protein Content (GPC) is a desirable trait in breadmaking and pasta wheat varieties because of its positive effects on quality and nutritional value. However, selection for GPC is limited by our poor understanding of the genes involved in the accumulation of protein in the grain. The long-term goal of this project is to provide a better understanding of the genes controlling GPC in wheat. The specific objectives of this project were: a) to develop a high-density genetic map of the GPC gene in tetraploid wheat, b) to construct a T. turgidum Bacterial Artificial Chromosome (BAC) library, c) to construct a physical map of the GPC gene and identify a candidate for the GPC gene. A gene with a large effect on GPC was detected in Triticum turgidum var. dicoccoides and was previously mapped in the short arm of chromosome 6B. To define better the position of the Gpc-B1 locus we developed homozygous recombinant lines with recombination events within the QTL region. Except for the 30-cM region of the QTL these RSLs were isogenic for the rest of the genome minimizing the genetic variability. To minimize the environmental variability the RSLs were characterized using 10 replications in field experiments organized in a Randomized Complete Block Design, which were repeated three times. Using this strategy, we were able to map this QTL as a single Mendelian locus (Gpc-B1) on a 2.6-cM region flanked by RFLP markers Xcdo365 and Xucw67. All three experiments showed that the lines carrying the DIC allele had an average absolute increase in GPC of 14 g/kg. Using the RFLP flanking markers, we established the microcolinearity between a 2.l-cM region including the Gpc-B1 gene in wheat chromosome 6BS and a 350-kb region on rice chromosome 2. Rice genes from this region were used to screen the Triticeae EST collection, and these ESTs were used to saturate the Gpc-B1 region with molecular markers. With these new markers we were able to map the Gpc-B1 locus within a 0.3-cM region flanked by PCR markers Xucw83 and Xucw71. These flanking markers defined a 36-kb colinear region with rice, including one gene that is a potential candidate for the Gpc-B1 gene. To develop a physical map of the Gpc-B1 region in wheat we first constructed a BAC library of tetraploid wheat, from RSL#65 including the high Gpc-B1 allele. We generated half- million clones with an average size of l3l-kb (5.1 X genome equivalents for each of the two genomes). This coverage provides a 99.4% probability of recovering any gene from durum wheat. We used the Gpc-BI flanking markers to screen this BAC library and then completed the physical map by chromosome walking. The physical map included two overlapping BACs covering a region of approximately 250-kb, including two flanking markers and the Gpc-B1 gene. Efforts are underway to sequence these two BACs to determine if additional wheat genes are present in this region. Weare also developing new RSLs to further dissect this region. We developed PCR markers for flanking loci Xucw79andXucw71 to facilitate the introgression of this gene in commercial varieties by marker assisted selection (httQ://maswheat.ucdavis.edu/ orotocols/HGPC/index.hlm). Using these markers we introgressed the Gpc-B1 gene in numerous pasta and common wheat breeding lines.

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Zhang, Hongbin B., David J. Bonfil und Shahal Abbo. Genomics Tools for Legume Agronomic Gene Mapping and Cloning, and Genome Analysis: Chickpea as a Model. United States Department of Agriculture, März 2003. http://dx.doi.org/10.32747/2003.7586464.bard.

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The goals of this project were to develop essential genomic tools for modern chickpea genetics and genomics research, map the genes and quantitative traits of importance to chickpea production and generate DNA markers that are well-suited for enhanced chickpea germplasm analysis and breeding. To achieve these research goals, we proposed the following research objectives in this period of the project: 1) Develop an ordered BAC library with an average insert size of 150 - 200 kb (USA); 2) Develop 300 simple sequence repeat (SSR) markers with an aid of the BAC library (USA); 3) Develop SSR marker tags for Ascochyta response, flowering date and grain weight (USA); 4) Develop a molecular genetic map consisting of at least 200 SSR markers (Israel and USA); 5) Map genes and QTLs most important to chickpea production in the U.S. and Israel: Ascochyta response, flowering and seed set date, grain weight, and grain yield under extreme dryland conditions (Israel); and 6) Determine the genetic correlation between the above four traits (Israel). Chickpea is the third most important pulse crop in the world and ranks the first in the Middle East. Chickpea seeds are a good source of plant protein (12.4-31.5%) and carbohydrates (52.4-70.9%). Although it has been demonstrated in other major crops that the modern genetics and genomics research is essential to enhance our capacity for crop genetic improvement and breeding, little work was pursued in these research areas for chickpea. It was absent in resources, tools and infrastructure that are essential for chickpea genomics and modern genetics research. For instance, there were no large-insert BAC and BIBAC libraries, no sufficient and user- friendly DNA markers, and no intraspecific genetic map. Grain sizes, flowering time and Ascochyta response are three main constraints to chickpea production in drylands. Combination of large seeds, early flowering time and Ascochyta blight resistance is desirable and of significance for further genetic improvement of chickpea. However, it was unknown how many genes and/or loci contribute to each of the traits and what correlations occur among them, making breeders difficult to combine these desirable traits. In this period of the project, we developed the resources, tools and infrastructure that are essential for chickpea genomics and modern genetics research. In particular, we constructed the proposed large-insert BAC library and an additional plant-transformation-competent BIBAC library from an Israeli advanced chickpea cultivar, Hadas. The BAC library contains 30,720 clones and has an average insert size of 151 kb, equivalent to 6.3 x chickpea haploid genomes. The BIBAC library contains 18,432 clones and has an average insert size of 135 kb, equivalent to 3.4 x chickpea haploid genomes. The combined libraries contain 49,152 clones, equivalent to 10.7 x chickpea haploid genomes. We identified all SSR loci-containing clones from the chickpea BAC library, generated sequences for 536 SSR loci from a part of the SSR-containing BACs and developed 310 new SSR markers. From the new SSR markers and selected existing SSR markers, we developed a SSR marker-based molecular genetic map of the chickpea genome. The BAC and BIBAC libraries, SSR markers and the molecular genetic map have provided essential resources and tools for modern genetic and genomic analyses of the chickpea genome. Using the SSR markers and genetic map, we mapped the genes and loci for flowering time and Ascochyta responses; one major QTL and a few minor QTLs have been identified for Ascochyta response and one major QTL has been identified for flowering time. The genetic correlations between flowering time, grain weight and Ascochyta response have been established. These results have provided essential tools and knowledge for effective manipulation and enhanced breeding of the traits in chickpea.

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Ozias-Akins, P., und R. Hovav. molecular dissection of the crop maturation trait in peanut. Israel: United States-Israel Binational Agricultural Research and Development Fund, 2020. http://dx.doi.org/10.32747/2020.8134157.bard.

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Crop maturation is one of the most recognized characteristics of peanut, and it is crucial for adaptability and yield. However, not much is known regarding its genetic and molecular control. The goals of this project were to study the molecular-genetic components that control crop maturation in peanut and identify candidate genes. Crop maturation was studied directly by phenotyping the maturity level or through other component traits such as flowering pattern and branching habit. Six different RIL populations (HH, RR, CC, FNC, TGT and FLIC) were used for the genetic analysis. In total, 14 QTLs were found for maturity level. The phenotypic explanation values ranged in 5.3%-18.6%. Common QTL were found between maturity level and harvest index (in RR and CC populations), branching habit (in HH population), flowering pattern/branching rate (in CC and TGT populations) and pod size (in CC population). Further investigations were done to define genes that control maturity level and the component traits. A map-based cloning approach was used to identify a major candidate gene for branching habit - a novel AhMADS-box gene (AhMADS). AhMADS was mainly expressed in the lateral shoot, the organ in which the difference between branching habit occurs. Sequence alignment analysis found SNPs in AhMADS that cause to exon/intron splicing alterations. Overexpression study of AhMADs-box in tobacco under 35S control revealed one line with a spreading-like lateral shoot indicating that AhMADS may be the causing effect of BH and therefore indirectly controls maturity level. In addition, several candidate genes were defined that may control flowering pattern. An RNA expression study was performed on two parental lines, Tifrunner and GT-C20, identifying four candidate genes in the flowering regulatory pathway that were down-regulated at the mainstem (non-flowering) compared to the first (flowering) shoot, indicating their influence on flowering pattern. Also, another candidate gene was identified, Terminal Flowering 1-like (AhTFL1), which was located within a small segment in chromosome B02. A 1492 bp deletion was found in AhTFL1 that completely co-segregates with the flowering pattern phenotype in the CC population and two independent EMS-mutagenized M2 families. AhTFL1 was significantly less expressed in flowering than non-flowering branches. Finally, a field trial showed that an EMS line (B78) mutagenized in AhTFL1 is ~18% days earlier than the control (Hanoch). In conclusion, our study revealed new insights into the molecular basis for the fundamentally important crop maturity trait in peanut. The results generated new information and materials that will promote informed targeting of peanut idiotypes by indirect selection and genomic breeding approaches.

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Prusky, Dov, und Jeffrey Rollins. Modulation of pathogenicity of postharvest pathogens by environmental pH. United States Department of Agriculture, Dezember 2006. http://dx.doi.org/10.32747/2006.7587237.bard.

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Until recently, environmental pH was not considered a factor in determining pathogen compatibility. Our hypothesis was that the environmental pH at the infection site, which is dynamically controlled by activities of both the host and the pathogen, regulates the expression of genes necessary for disease development in Colletotrichum gloeosporioides and Sclerotinia sclerotiorum. This form of regulation ensures that genes are expressed at optimal conditions for their encoded activities.Pectate lyase encoded by pelB, has been demonstrated to play a key role in virulence of C. gloeosporioides in avocado fruit. Polyglacturonase synergism with oxalic acid production is considered to be an essential pathogenicity determinant in the interactions of S. sclerotiorum with its numerous hosts. A common regulatory feature of these virulence and pathogenicity factors is their dependence upon environmental pH conditions within the host niche to create optimal conditions for expression and secretion. In this proposal we have examined, 1) the mechanisms employed by these fungi to establish a suitable pH environment, 2) the molecular levels at which genes and gene products are regulated in response to environmental pH, and 3) the molecular basis and functional importance of pH-responsive gene regulation during pathogenicity. The specific objectives of the proposal were: 1. Characterize the mechanism of local pH modulation and the effect of ambient pH on the expression and secretion of virulence factors. 2. Provide evidence that a conserved molecular pathway for pH-responsive gene expression exists in C. gloeosporioides by cloning a pacC gene homologue. 3. Determine the role of pacC in pathogenicity by gene disruption and activating mutations. Major conclusions 1. We determined the importance of nitrogen source and external pH in the secretion of the virulence factor pectate lyase with respect to the ambient pH transcriptional regulator pacC. It was concluded that nitrogen source availability and ambient pH are two independent signals for the transcriptional regulation of genes required for the disease process of C. gloeosporioides and possibly of other pathogens. 2. We also determined that availability of ammonia regulate independently the alkalinization process and pelB expression, pecate lyase secretion and virulence of C. gloeosporioides. 3. Gene disruption of pacC reduced virulence of C. gloeosporioides however did not reduced fully pelB expression. It was concluded that pelB expression is regulated by several factors including pH, nitrogen and carbon sources. 4. Gene disruption of pacC reduced virulence of S. slcerotiourum Creation of a dominant activating

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Hulata, Gideon, Thomas D. Kocher, Micha Ron und Eyal Seroussi. Molecular Mechanisms of Sex Determination in Cultured Tilapias. United States Department of Agriculture, Oktober 2010. http://dx.doi.org/10.32747/2010.7697106.bard.

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Tilapias are among the most important aquaculture commodities worldwide. Commercial production of tilapia is based on monosex culture of males. Current methods for producing all-male fingerlings, including hormone treatments and genetic manipulations, are not entirely reliable, in part because of the genetic complexity of sex determination and sexual differentiation in tilapias. The goals of this project are to map QTL and identify genes regulating sex determination in commonly cultured tilapia species, in order to provide a rational basis for designing reliable genetic approaches for producing all-male fingerlings. The original objectives for this research were: 1) to identify the gene underlying the QTL on LG1 through positional cloning and gene expression analysis; 2) to fine map the QTL on LG 3 and 23; and 3) to characterize the patterns of dominance and epistasis among QTL alleles influencing sex determination. The brain aromatase gene Cyp19b, a possible candidate for the genetic or environmental SD, was mapped to LG7 using our F2 mapping population. This region has not been identified before as affecting SD in tilapias. The QTL affecting SD on LG 1 and 23 have been fine-mapped down to 1 and 4 cM, respectively, but the key regulators for SD have not been found yet. Nevertheless, a very strong association with gender was found on LG23 for marker UNH898. Allele 276 was found almost exclusively in males, and we hypothesized that this allele is a male-associated allele (MAA). Mating of males homozygous for MAA with normal females is underway for production of all-male populations. The first progeny reaching size allowing accurate sexing had 43 males and no females. During the course of the project it became apparent that in order to achieve those objectives there is a need to develop genomic infrastructures that were lacking. Efforts have been devoted to the development of genomic resources: a database consisting of nearly 117k ESTs representing 16 tissues from tilapia were obtained; a web tool based on the RepeatMasker software was designed to assist tilapia genomics; collaboration has been established with a sequencing company to sequence the tilapia genome; steps have been taken toward constructing a microarray to enable comparative analysis of the entire transcriptome that is required in order to detect genes that are differentially expressed between genders in early developmental stages. Genomic resources developed will be invaluable for studies of cichlid physiology, evolution and development, and will hopefully lead to identification of the key regulators of SD. Thus, they will have both scientific and agricultural implications in the coming years.

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Droby, Samir, Michael Wisniewski, Martin Goldway, Wojciech Janisiewicz und Charles Wilson. Enhancement of Postharvest Biocontrol Activity of the Yeast Candida oleophila by Overexpression of Lytic Enzymes. United States Department of Agriculture, November 2003. http://dx.doi.org/10.32747/2003.7586481.bard.

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Enhancing the activity of biocontrol agents could be the most important factor in their success in controlling fruit disease and their ultimate acceptance in commercial disease management. Direct manipulation of a biocontrol agent resulting in enhancement of diseases control could be achieved by using recent advances in molecular biology techniques. The objectives of this project were to isolate genes from yeast species that were used as postharvest biocontrol agents against postharvest diseases and to determine their role in biocontrol efficacy. The emphasis was to be placed on the yeast, Candida oleophila, which was jointly discovered and developed in our laboratories, and commercialized as the product, Aspire. The general plan was to develop a transformation system for C . oleophila and either knockout or overexpress particular genes of interest. Additionally, biochemical characterization of the lytic peptides was conducted in the wild-type and transgenic isolates. In addition to developing a better understanding of the mode of action of the yeast biocontrol agents, it was also our intent to demonstrate the feasibility of enhancing biocontrol activity via genetic enhancement of yeast with genes known to code for proteins with antimicrobial activity. Major achievements are: 1) Characterization of extracellular lytic enzymes produced by the yeast biocontrol agent Candida oleophila; 2) Development of a transformation system for Candida oleophila; 3) Cloning and analysis of C.oleophila glucanase gene; 4) Overexpression of and knockout of C. oleophila glucanase gene and evaluating its role in the biocontrol activity of C. oleophila; 5) Characterization of defensin gene and its expression in the yeast Pichiapastoris; 6) Cloning and Analysis of Chitinase and Adhesin Genes; 7) Characterization of the rnase secreted by C . oleophila and its inhibitory activity against P. digitatum. This project has resulted in information that enhanced our understanding of the mode of action of the yeast C . oleophila. This was important step towards enhancing the biocontrol activity of the yeast. Fungal cell wall enzymes produced by the yeast antagonist were characterized. Different substrates were identified to enhance there production in vitro. Exo-b-1, 3 glucanase, chitinase and protease production was stimulated by the presence of cell-wall fragments of Penicillium digitatum in the growing medium, in addition to glucose. A transformation system developed was used to study the role of lytic enzymes in the biocontrol activity of the yeast antagonist and was essential for genetic manipulation of C . oleqphila. After cloning and characterization of the exo-glucanase gene from the yeast, the transformation system was efficiently used to study the role of the enzyme in the biocontrol activity by over-expressing or knocking out the activity of the enzyme. At the last phase of the research (still ongoing) the transformation system is being used to study the role of chitinase gene in the mode of action. Knockout and over expression experiments are underway.

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Dubcovsky, Jorge, Tzion Fahima, Ann Blechl und Phillip San Miguel. Validation of a candidate gene for increased grain protein content in wheat. United States Department of Agriculture, Januar 2007. http://dx.doi.org/10.32747/2007.7695857.bard.

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High Grain Protein Content (GPC) of wheat is important for improved nutritional value and industrial quality. However, selection for this trait is limited by our poor understanding of the genes involved in the accumulation of protein in the grain. A gene with a large effect on GPC was detected on the short arm of chromosome 6B in a Triticum turgidum ssp. dicoccoides accession from Israel (DIC, hereafter). During the previous BARD project we constructed a half-million clones Bacterial Artificial Chromosome (BAC) library of tetraploid wheat including the high GPC allele from DIC and mapped the GPC-B1 locus within a 0.3-cM interval. Our long-term goal is to provide a better understanding of the genes controlling grain protein content in wheat. The specific objectives of the current project were to: (1) complete the positional cloning of the GPC-B1 candidate gene; (2) characterize the allelic variation and (3) expression profile of the candidate gene; and (4) validate this gene by using a transgenic RNAi approach to reduce the GPC transcript levels. To achieve these goals we constructed a 245-kb physical map of the GPC-B1 region. Tetraploid and hexaploid wheat lines carrying this 245-kb DIC segment showed delayed senescence and increased GPC and grain micronutrients. The complete sequencing of this region revealed five genes. A high-resolution genetic map, based on approximately 9,000 gametes and new molecular markers enabled us to delimit the GPC-B1 locus to a 7.4-kb region. Complete linkage of the 7.4-kb region with earlier senescence and increase in GPC, Zn, and Fe concentrations in the grain suggested that GPC-B1 is a single gene with multiple pleiotropic effects. The annotation of this 7.4-kb region identified a single gene, encoding a NAC transcription factor, designated as NAM-B1. Allelic variation studies demonstrated that the ancestral wild wheat allele encodes a functional NAC transcription factor whereas modern wheat varieties carry a non-functional NAM-B1 allele. Quantitative PCR showed that transcript levels for the multiple NAMhomologues were low in flag leaves prior to anthesis, after which their levels increased significantly towards grain maturity. Reduction in RNA levels of the multiple NAMhomologues by RNA interference delayed senescence by over three weeks and reduced wheat grain protein, Zn, and Fe content by over 30%. In the transgenic RNAi plants, residual N, Zn and Fe in the dry leaves was significantly higher than in the control plants, confirming a more efficient nutrient remobilization in the presence of higher levels of GPC. The multiple pleiotropic effects of NAM genes suggest a central role for these genes as transcriptional regulators of multiple processes during leaf senescence, including nutrient remobilization to the developing grain. The cloning of GPC-B1 provides a direct link between the regulation of senescence and nutrient remobilization and an entry point to characterize the genes regulating these two processes. This may contribute to their more efficient manipulation in crops and translate into food with enhanced nutritional value. The characterization of the GPC-B1 gene will have a significant impact on wheat production in many regions of the world and will open the door for the identification of additional genes involved in the accumulation of protein in the grain.

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Azem, Abdussalam, George Lorimer und Adina Breiman. Molecular and in vivo Functions of the Chloroplast Chaperonins. United States Department of Agriculture, Juni 2011. http://dx.doi.org/10.32747/2011.7697111.bard.

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We present here the final report for our research project entitled "The molecular and in vivo functions of the chloroplast chaperonins”. Over the past few decades, intensive investigation of the bacterial GroELS system has led to a basic understanding of how chaperonins refold denatured proteins. However, the parallel is limited in its relevance to plant chaperonins, since the plant system differs from GroEL in genetic complexity, physiological roles of the chaperonins and precise molecular structure. Due to the importance of plant chaperonins for chloroplast biogenesis and Rubisco assembly, research on this topic will have implications for many vital applicative fields such as crop hardiness and efficiency of plant growth as well as the production of alternative energy sources. In this study, we set out to investigate the structure and function of chloroplast chaperonins from A. thaliana. Most plants harbor multiple genes for chaperonin proteins, making analysis of plant chaperonin systems more complicated than the GroEL-GroES system. We decided to focus on the chaperonins from A. thaliana since the genome of this plant has been well defined and many materials are available which can help facilitate studies using this system. Our proposal put forward a number of goals including cloning, purification, and characterization of the chloroplast cpn60 subunits, antibody preparation, gene expression patterns, in vivo analysis of oligomer composition, preparation and characterization of plant deletion mutants, identification of substrate proteins and biophysical studies. In this report, we describe the progress we have made in understanding the structure and function of chloroplast chaperonins in each of these categories.

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Dubcovsky, Jorge, Tzion Fahima und Ann Blechl. Molecular characterization and deployment of the high-temperature adult plant stripe rust resistance gene Yr36 from wheat. United States Department of Agriculture, November 2013. http://dx.doi.org/10.32747/2013.7699860.bard.

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Stripe rust, caused by Puccinia striiformis f. sp. tritici is one of the most destructive fungal diseases of wheat. Virulent races that appeared within the last decade caused drastic cuts in yields. The incorporation of genetic resistance against this pathogen is the most cost-effective and environmentally friendly solution to this problem. However, race specific seedling resistance genes provide only a temporary solution because fungal populations rapidly evolve to overcome this type of resistance. In contrast, high temperature adult plant (HTAP) resistance genes provide a broad spectrum resistance that is partial and more durable. The cloning of the first wheat HTAP stripe rust resistance gene Yr36 (Science 2009, 323:1357), funded by our previous (2007-2010) BARD grant, provided us for the first time with an entry point for understanding the mechanism of broad spectrum resistance. Two paralogous copies of this gene are tightly linked at the Yr36 locus (WKS1 and WKS2). The main objectives of the current study were to characterize the Yr36 (WKS) resistance mechanism and to identify and characterize alternative WKSgenes in wheat and wild relatives. We report here that the protein coded by Yr36, designated WKS1, that has a novel architecture with a functional kinase and a lipid binding START domain, is localized to chloroplast. Our results suggest that the presence of the START domain may affect the kinase activity. We have found that the WKS1 was over-expressed on leaf necrosis in wheat transgenic plants. When the isolated WKS1.1 splice variant transcript was transformed into susceptible wheat it conferred resistance to stripe rust, but the truncated variant WKS1.2 did not confer resistance. WKS1.1 and WKS1.2 showed different lipid binding profiling. WKS1.1 enters the chloroplast membrane, while WKS1.2 is only attached outside of the chloroplast membrane. The ascorbate peroxidase (APX) activity of the recombinant protein of TmtAPXwas found to be reduced by WKS1.1 protein in vitro. The WKS1.1 mature protein in the chloroplast is able to phosphorylate TmtAPXprotein in vivo. WKS1.1 induced cell death by suppressing APX activity and reducing the ability of the cell to detoxify reactive oxygen. The decrease of APX activity reduces the ability of the plant to detoxify the reactive H2O2 and is the possible mechanism underlying the accelerated cell death observed in the transgenic plants overexpressing WKS1.1 and in the regions surrounding a stripe rust infection in the wheat plants carrying the natural WKS1.1 gene. WKS2 is a nonfunctional paralog of WKS1 in wild emmer wheat, probably due to a retrotransposon insertion close to the alternative splicing site. In some other wild relatives of wheat, such as Aegilops comosa, there is only one copy of this gene, highly similar to WKS2, which is lucking the retrotransposon insertion. WKS2 gene present in wheat and WKS2-Ae from A. showed a different pattern of alternative splice variants, regardless of the presence of the retrotransposon insertion. Susceptible Bobwhite transformed with WKS2-Ae (without retrotansposon insertion in intron10), which derived from Aegilops comosaconferred resistance to stripe rust in wheat. The expression of WKS2-Ae in transgenic plants is up-regulated by temperature and pathogen infection. Combination of WKS1 and WKS2-Ae shows improved stripe rust resistance in WKS1×WKS2-Ae F1 hybrid plants. The obtained results show that WKS1 protein is accelerating programmed cell death observed in the regions surrounding a stripe rust infection in the wheat plants carrying the natural or transgenic WKS1 gene. Furthermore, characterization of the epistatic interactions of Yr36 and Yr18 demonstrated that these two genes have additive effects and can therefore be combined to increase partial resistance to this devastating pathogen of wheat. These achievements may have a broad impact on wheat breeding efforts attempting to protect wheat yields against one of the most devastating wheat pathogen.

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Wagner, D. Ry, Eliezer Lifschitz und Steve A. Kay. Molecular Genetic Analysis of Flowering in Arabidopsis and Tomato. United States Department of Agriculture, Mai 2002. http://dx.doi.org/10.32747/2002.7585198.bard.

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The primary objectives for the US lab included: the characterization of ELF3 transcription and translation; the creation and characterization of various transgenic lines that misexpress ELF3; defining genetic pathways related to ELF3 function regulating floral initiation in Arabidopsis; and the identification of genes that either interact with or are regulated by ELF3. Light quality, photoperiod, and temperature often act as important and, for some species, essential environmental cues for the initiation of flowering. However, there is relatively little information on the molecular mechanisms that directly regulate the developmental pathway from the reception of the inductive light signals to the onset of flowering and the initiation of floral meristems. The ELF3 gene was identified as possibly having a role in light-mediated floral regulation since elj3 mutants not only flower early, but exhibit light-dependent circadian defects. We began investigating ELF3's role in light signalling and flowering by cloning the ELF3 gene. ELF3 is a novel gene only present in plant species; however, there is an ELF3 homolog within Arabidopsis. The Arabidopsis elj3 mutation causes arrhythmic circadian output in continuous light; however, we show conclusively normal circadian function with no alteration of period length in elj3 mutants in dark conditions and that the light-dependent arrhythmia observed in elj3 mutants is pleiotropic on multiple outputs regardless of phase. Plants overexpressing ELF3 have an increased period length in constant light and flower late in long-days; furthermore, etiolated ELF3-overexpressing seedlings exhibit a decreased acute CAB2 response after a red light pulse, whereas the null mutant is hypersensitive to acute induction. This finding suggests that ELF3 negatively regulates light input to both the clock and its outputs. To determine whether ELF3's action is phase dependent, we examined clock resetting by light pulses and constructed phase response curves. Absence of ELF3 activity causes a significant alteration of the phase response curve during the subjective night, and overexpression of ELF3 results in decreased sensitivity to the resetting stimulus, suggesting that ELF3 antagonizes light input to the clock during the night. Indeed, the ELF3 protein interacts with the photoreceptor PHYB in the yeast two-hybrid assay and in vitro. The phase ofELF3 function correlates with its peak expression levels of transcript and protein in the subjective night. ELF3 action, therefore, represents a mechanism by which the oscillator modulates light resetting. Furthermore, flowering time is dependent upon proper expression ofELF3. Scientifically, we've made a big leap in the understanding of the circadian system and how it is coupled so tightly with light reception in terms of period length and clock resetting. Agriculturally, understanding more about the way in which the clock perceives and relays temporal information to pathways such as those involved in the floral transition can lead to increased crop yields by enabling plants to be grown in suboptimal conditions.

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