Relevant bibliographies by topics / Genome cloning

Contents

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

Academic literature on the topic 'Genome cloning'

Author: Grafiati
Published: 23 September 2022
Last updated: 28 September 2022

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Journal articles on the topic "Genome cloning"

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Potgieter, A. C., A. D. Steele, and A. A. van Dijk. "Cloning of complete genome sets of six dsRNA viruses using an improved cloning method for large dsRNA genes." Journal of General Virology 83, no. 9 (September 1, 2002): 2215–23. http://dx.doi.org/10.1099/0022-1317-83-9-2215.

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Cloning full-length large (>3 kb) dsRNA genome segments from small amounts of dsRNA has thus far remained problematic. Here, a single-primer amplification sequence-independent dsRNA cloning procedure was perfected for large genes and tailored for routine use to clone complete genome sets or individual genes. Nine complete viral genome sets were amplified by PCR, namely those of two human rotaviruses, two African horsesickness viruses (AHSV), two equine encephalosis viruses (EEV), one bluetongue virus (BTV), one reovirus and bacteriophage Φ12. Of these amplified genomes, six complete genome sets were cloned for viruses with genes ranging in size from 0·8 to 6·8 kb. Rotavirus dsRNA was extracted directly from stool samples. Co-expressed EEV VP3 and VP7 assembled into core-like particles that have typical orbivirus capsomeres. This work presents the first EEV sequence data and establishes that EEV genes have the same conserved termini (5′ GUU and UAC 3′) and coding assignment as AHSV and BTV. To clone complete genome sets, one-tube reactions were developed for oligo-ligation, cDNA synthesis and PCR amplification. The method is simple and efficient compared to other methods. Complete genomes can be cloned from as little as 1 ng dsRNA and a considerably reduced number of PCR cycles (22–30 cycles compared to 30–35 of other methods). This progress with cloning large dsRNA genes is important for recombinant vaccine development and determination of the role of terminal sequences for replication and gene expression.
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Cochrane, Ryan R., Stephanie L. Brumwell, Arina Shrestha, Daniel J. Giguere, Samir Hamadache, Gregory B. Gloor, David R. Edgell, and Bogumil J. Karas. "Cloning of Thalassiosira pseudonana’s Mitochondrial Genome in Saccharomyces cerevisiae and Escherichia coli." Biology 9, no. 11 (October 26, 2020): 358. http://dx.doi.org/10.3390/biology9110358.

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Algae are attractive organisms for biotechnology applications such as the production of biofuels, medicines, and other high-value compounds due to their genetic diversity, varied physical characteristics, and metabolic processes. As new species are being domesticated, rapid nuclear and organelle genome engineering methods need to be developed or optimized. To that end, we have previously demonstrated that the mitochondrial genome of microalgae Phaeodactylum tricornutum can be cloned and engineered in Saccharomyces cerevisiae and Escherichia coli. Here, we show that the same approach can be used to clone mitochondrial genomes of another microalga, Thalassiosira pseudonana. We have demonstrated that these genomes can be cloned in S. cerevisiae as easily as those of P. tricornutum, but they are less stable when propagated in E. coli. Specifically, after approximately 60 generations of propagation in E. coli, 17% of cloned T. pseudonana mitochondrial genomes contained deletions compared to 0% of previously cloned P. tricornutum mitochondrial genomes. This genome instability is potentially due to the lower G+C DNA content of T. pseudonana (30%) compared to P. tricornutum (35%). Consequently, the previously established method can be applied to clone T. pseudonana’s mitochondrial genome, however, more frequent analyses of genome integrity will be required following propagation in E. coli prior to use in downstream applications.
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Takeuchi, T., Q. V. Neri, M. Cheng, Z. Rosenwaks, and G. D. Palermo. "Successful cloning of the male genome." Fertility and Sterility 88 (September 2007): S75. http://dx.doi.org/10.1016/j.fertnstert.2007.07.250.

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Xi, J., D. Graham, K. Wang, and M. Estes. "Norwalk virus genome cloning and characterization." Science 250, no. 4987 (December 14, 1990): 1580–83. http://dx.doi.org/10.1126/science.2177224.

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Takahashi, Seiya. "Animal Cloning: Reprogramming the Donor Genome." Journal of Mammalian Ova Research 21, no. 3 (2004): 74–81. http://dx.doi.org/10.1274/jmor.21.74.

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Zhao, Xinping, Rod A. Wing, and Andrew H. Paterson. "Cloning and characterization of the majority of repetitive DNA in cotton (Gossypium L.)." Genome 38, no. 6 (December 1, 1995): 1177–88. http://dx.doi.org/10.1139/g95-156.

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Repetitive DNA elements representing 60–70% of the total repetitive DNA in tetraploid cotton (Gossypium barbadense L.) and comprising 30–36% of the tetraploid cotton genome were isolated from a genomic library of DNA digested with a mixture of four blunt-end cutting restriction enzymes. A total of 313 clones putatively containing nuclear repetitive sequences were classified into 103 families, based on cross hybridization and Southern blot analysis. The 103 families were characterized in terms of genome organization, methylation pattern, abundance, and DNA variation. As in many other eukaryotic genomes, interspersed repetitive elements are the most abundant class of repetitive DNA in the cotton genome. Paucity of tandem repeat families with high copy numbers (>104) may be a unique feature of the cotton genome as compared with other higher plant genomes. Interspersed repeats tend to be methylated, while tandem repeats seem to be largely unmethylated in the cotton genome. Minimal variation in repertoire and overall copy number of repetitive DNA elements among different tetraploid cotton species is consistent with the hypothesis of a relatively recent origin of tetraploid cottons.Key words: genome analysis, genome evolution, tandemly repetitive DNA sequences, interspersed repetitive DNA sequences, polyploid.
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Aswidinnoor, H., R. J. Nelson, J. F. Dallas, C. L. McIntyre, H. Leung, and J. P. Gustafson. "Cloning and characterization of repetitive DNA sequences from genomes of Oryza minuta and Oryza australiensis." Genome 34, no. 5 (October 1, 1991): 790–98. http://dx.doi.org/10.1139/g91-123.

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The value of genome-specific repetitive DNA sequences for use as molecular markers in studying genome differentiation was investigated. Five repetitive DNA sequences from wild species of rice were cloned. Four of the clones, pOm1, pOm4, pOmA536, and pOmPB10, were isolated from Oryza minuta accession 101141 (BBCC genomes), and one clone, pOa237, was isolated from Oryza australiensis accession 100882 (EE genome). Southern blot hybridization to different rice genomes showed strong hybridization of all five clones to O. minuta genomic DNA and no cross hybridization to genomic DNA from Oryza sativa (AA genome). The pOm1 and pOmA536 sequences showed cross hybridization only to all of the wild rice species containing the C genome. However, the pOm4, pOmPB10, and pOa237 sequences showed cross hybridization to O. australiensis genomic DNA in addition to showing hybridization to the O. minuta genomic DNA.Key words: rice, genome-specific repetitive sequences, Oryza.
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Devor, Eric J., Lingyan Huang, Abdusattor Abdukarimov, and Ibrokhim Y. Abdurakhmonov. "Methodologies for In Vitro Cloning of Small RNAs and Application for Plant Genome(s)." International Journal of Plant Genomics 2009 (June 15, 2009): 1–13. http://dx.doi.org/10.1155/2009/915061.

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The “RNA revolution” that started at the end of the 20th century with the discovery of post-transcriptional gene silencing and its mechanism via RNA interference (RNAi) placed tiny 21-24 nucleotide long noncoding RNAs (ncRNAs) in the forefront of biology as one of the most important regulatory elements in a host of physiologic processes. The discovery of new classes of ncRNAs including endogenous small interfering RNAs, microRNAs, and PIWI-interacting RNAs is a hallmark in the understanding of RNA-dependent gene regulation. New generation high-throughput sequencing technologies further accelerated the studies of this “tiny world” and provided their global characterization and validation in many biological systems with sequenced genomes. Nevertheless, for the many “yet-unsequenced” plant genomes, the discovery of small RNA world requires in vitro cloning from purified cellular RNAs. Thus, reproducible methods for in vitro small RNA cloning are of paramount importance and will remain so into the foreseeable future. In this paper, we present a description of existing small RNA cloning methods as well as next-generation sequencing methods that have accelerated this research along with a description of the application of one in vitro cloning method in an initial small RNA survey in the “still unsequenced” allotetraploid cotton genome.
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Suzuki, Tetsuya, Tomohito Tsukamoto, Eiko Sakai, Hiroyuki Mizuguchi, and Hiroyuki Mizuguchi. "Sequence search for cloning and genome editing." Drug Delivery System 35, no. 3 (July 25, 2020): 255–59. http://dx.doi.org/10.2745/dds.35.255.

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Cockburn, Andrew F., Sharon E. Mitchell, and Jack A. Seawright. "Cloning of the mitochondrial genome ofAnopheles quadrimaculatus." Archives of Insect Biochemistry and Physiology 14, no. 1 (1990): 31–36. http://dx.doi.org/10.1002/arch.940140104.

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Dissertations / Theses on the topic "Genome cloning"

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Wain, Hester Mary. "Targeted mapping of the chicken genome." Thesis, University of Hertfordshire, 1997. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.338594.

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Jones, C. Peter. "Application of large insert cloning technology to genome analysis." Thesis, Open University, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.306253.

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Swan, Daniel. "Cloning and characterisation of arkadia, a recessive, lethal, gene trap mutation." Thesis, Imperial College London, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.324521.

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Moore, Catherine Samantha. "The use of repetitive DNA sequences, in particular retrotransposons, in the genetic analysis of oil palm (Elaeis guineensis)." Thesis, University of Reading, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.340012.

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Shull, Bruce Colin. "Molecular cloning and analysis of the genome of bovine parvovirus." Diss., Virginia Polytechnic Institute and State University, 1987. http://hdl.handle.net/10919/49895.

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The genome of bovine parvovirus (BPV) has been cloned by blunt end ligation of double-stranded virion DNA into the plasmid pUC8. The resulting genomic clones were infectious after transfection into bovine fetal lung (BFL) cells. Sequencing of the plasmids demonstrated that deletions were common at both ends of the cloned BPV genome. Deletions of up to 34 bases at the 3’ end lowered but did not abolish infectivity, while a deletion of 52 bases eliminated infectivity, End label analysis demonstrated the repair of deletions of up to 34 bases at the 3’ end or 35 bases at the 5’ end to the wild type length. Mutually inverted sequence orientations of the palindromic termini, known as the flip and flop forms, can occur during replication of parvovirus DNA. Cloning of BPV terminal sequences permitted the identification of the 3’ flop sequence inversion as a natural component of BPV DNA. This is the first report of sequence inversions within the 3’ end of an autonomous parvovirus. Clones with the 3’ flop or flip conformations were equally infectious. Wild type virion DNA was shown to have predominantly the 3’ flip conformation but a significant amount of 3’ flop was also detected. At the 5’ end, both the flip and flop sequence conformations were identified in nearly equal amounts. The progeny virion DNA from transfection of genomic clones had the same ratio of flip to flop as did wild type at both the 3’ and 5’ ends, regardless of the starting terminal conformations of the genomic clone. These data suggest that, while sequence inversion occurs at both termini during BPV DNA replication, some mechanism exists for the preferential replication of the 3’ flip conformation. Replicative form DNA from BPV infected cells had the same ratio of flip and flop at each end and the same termini as virion DNA. A set of deletion and frameshift mutants affecting each of the coding regions of BPV was constructed using one of the genomic clones. None of these mutants was infectious when transfected into BFL cells, which demonstrates that all three of the major open reading frames are essential for the production of infectious virus.
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Moore, Karen Anne. "Cloning and expression of MCM3 genes in plants." Thesis, University of Exeter, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.312072.

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Bernard, Emmanuelle Alexa. "Cloning and characterisation of the Xenopus laevis bloom's protein." Thesis, University of Sussex, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.367351.

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Bigger, Brian William. "Adaptation of the mitochondrial genome as a vehicle for gene delivery." Thesis, Imperial College London, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.325568.

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Curran, Martin David. "Cloning and characterization of the 5' end of the measles virus genome." Thesis, Queen's University Belfast, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.336037.

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Deng, Ruitang. "Molecular cloning, nucleotide sequencing and genome replication of bovine viral diarrhea virus /." The Ohio State University, 1992. http://rave.ohiolink.edu/etdc/view?acc_num=osu1487779914825349.

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Books on the topic "Genome cloning"

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Flavin, Nora. Cloning and characterisation of the bovine activin receptor type II gene (ActRII): Its localisation to chromosome 2 (BTA2) by somatic cell genetic analysis and the genotyping of an associated microsatelltie UCD2. Dublin: University College Dublin, 1996.

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Dale, Jeremy. From genes to genomes: Concepts and applications of DNA technology. 2nd ed. Chichester, West Sussex: John Wiley & Sons, 2007.

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Goncharov, V. P. Genom i klonirovanie cheloveka: Filosofskiĭ aspekt. Moskva: Sovremennye tetradi, 2003.

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Genetically yours: Bioinforming, biopharming, biofarming. River Edge, NJ: World Scientific, 2002.

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Xu, Huimin. Molecular cloning of the genome of potato virus x for the development of transgenic potato plants resistant to infection by this virus. Ottawa: National Library of Canada, 1990.

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Korochkin, L. I. Genom, klonirovanie i proiskhozhdenie cheloveka. Fri͡azino: Vek 2, 2004.

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

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Morgan, Rose M. The genetics revolution: History, fears, and future of a life-altering science. Westport, Conn: Greenwood Press, 2006.

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von, Schantz Malcolm, and Plant Nick, eds. From genes to genomes: Concepts and applications of DNA technology. 3rd ed. Chichester, West Sussex: John Wiley & Sons, 2011.

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Canada. Library of Parliament, Science and Technology Division. Gene therapy, genetic alteration and cloning. Ottawa: Library of Parliament, 2000.

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Book chapters on the topic "Genome cloning"

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Wong, Dominic W. S. "Gene Targeting and Genome Editing." In The ABCs of Gene Cloning, 187–97. Cham: Springer International Publishing, 2018. http://dx.doi.org/10.1007/978-3-319-77982-9_20.

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Wong, Dominic W. S. "Whole Genome and Next Generation Sequencing." In The ABCs of Gene Cloning, 219–30. Cham: Springer International Publishing, 2018. http://dx.doi.org/10.1007/978-3-319-77982-9_24.

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Kole, C., and P. K. Gupta. "Genome Mapping and Map Based Cloning." In Plant Breeding, 257–99. Dordrecht: Springer Netherlands, 2004. http://dx.doi.org/10.1007/978-94-007-1040-5_11.

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Humphreys-Pereira, Danny A., Taeho Kim, and Joong-Ki Park. "Characterization of nematode mitochondrial genomes." In Techniques for work with plant and soil nematodes, 250–64. Wallingford: CABI, 2021. http://dx.doi.org/10.1079/9781786391759.0250.

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Abstract This chapter presents procedures on polymerase chain reaction (PCR) amplification, protocols for PCR, cloning and sequencing, and mitochondrial genome annotation and gene identification for the characterization of nematodes.
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Humphreys-Pereira, Danny A., Taeho Kim, and Joong-Ki Park. "Characterization of nematode mitochondrial genomes." In Techniques for work with plant and soil nematodes, 250–64. Wallingford: CABI, 2021. http://dx.doi.org/10.1079/9781786391759.0014.

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Abstract This chapter presents procedures on polymerase chain reaction (PCR) amplification, protocols for PCR, cloning and sequencing, and mitochondrial genome annotation and gene identification for the characterization of nematodes.
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Zabarovsky, E. R., M. K. Nurbekov, O. V. Turina, and L. L. Kisselev. "Gene Cloning: Some Methodological Improvements." In Organization and Function of the Eucaryotic Genome, 20. Berlin, Heidelberg: Springer Berlin Heidelberg, 1987. http://dx.doi.org/10.1007/978-3-642-46611-3_24.

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Gresshoff, Peter M. "Positional Cloning of Plant Developmental Genes." In The Handbook of Plant Genome Mapping, 233–56. Weinheim, FRG: Wiley-VCH Verlag GmbH & Co. KGaA, 2005. http://dx.doi.org/10.1002/3527603514.ch10.

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Delgado, Javier A., Steven Meinhardt, Samuel G. Markell, and Rubella S. Goswami. "Gene Cloning Using Degenerate Primers and Genome Walking." In Plant Fungal Pathogens, 611–22. Totowa, NJ: Humana Press, 2011. http://dx.doi.org/10.1007/978-1-61779-501-5_39.

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Gill, Bikram S. "A Century of Cytogenetic and Genome Analysis: Impact on Wheat Crop Improvement." In Wheat Improvement, 277–97. Cham: Springer International Publishing, 2022. http://dx.doi.org/10.1007/978-3-030-90673-3_16.

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AbstractBeginning in the first decade of 1900, pioneering research in disease resistance and seed color inheritance established the scientific basis of Mendelian inheritance in wheat breeding. A series of breakthroughs in chromosome and genome analysis beginning in the 1920s and continuing into the twenty-first century have impacted wheat improvement. The application of meiotic chromosome pairing in the 1920s and plasmon analysis in the 1950s elucidated phylogeny of the Triticum-Aegilops complex of species and defined the wheat gene pools. The aneuploid stocks in the 1950s opened floodgates for chromosome and arm mapping of first phenotypic and later protein and DNA probes. The aneuploid stocks, coupled with advances in chromosome banding and in situ hybridization in the 1970s, allowed precise chromosome engineering of traits in wide hybrids. The deletion stocks in the 1990s were pivotal in mapping expressed genes to specific chromosome bins revealing structural and functional differentiation of chromosomes along their length and facilitating map-based cloning of genes. Advances in whole-genome sequencing, chromosome genomics, RH mapping and functional tools led to the assembly of reference sequence of Chinese Spring and multiple wheat genomes. Chromosome and genomic analysis must be integrated into wheat breeding and wide-hybridizaton pipeline for sustainable crop improvement.
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Hahn, Florian, Laura Sanjurjo Loures, and Vladimir Nekrasov. "A Modular Cloning Toolkit for Genome Editing in Cereals." In Springer Protocols Handbooks, 209–24. New York, NY: Springer US, 2021. http://dx.doi.org/10.1007/978-1-0716-1526-3_10.

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Conference papers on the topic "Genome cloning"

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Collins, Corolyn J., Richard B. Levene, Christina P. Ravera, Marker J. Dombalagian, David M. Livingston, and Dennis C. Lynch. "MOLECULAR CLONING OF THE HUMAN GENE FOR VON WILLEBRAND FACTOR AND IDENTIFICATION OF THE TRANSCRIPTION INITIATION SITE." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642830.

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Most patients with von Willebrand's disease appear to have a defect affecting the level of expression of the von Willebrand factor (vWf) gene. Thus, an understanding of the pathogenesis of von Willebrand's disease will require an analysis of the structure and function of the vWf gene in normals and in patients. To begin such analyses, we have screened a human genomic cosmid library with probes obtained from vWf cDNA and isolated a colinear segment spanning ≈175 kb in five overlapping clones. This segment extends ≈25 kb upstream and ≈5 kb downstream of the transcription start and stop sites for vWf mRNA, implying the vWf gene has a length of ≈150 kb. Within one of these clones, the vWf transcription initiation sites have been mapped. A portion of the promoter region has been sequenced, revealing a typical TATA box, a downstream CCAAT box, and a perfect downstream repeat of the 8 base pairs containing the major transcription start site. Primer extension analysis suggests that sequences contained within the downstream repeat of the transcription start site may be used as minor initiation sites in endothelial cells. Transfection studies are underway to evaluate the role of sequences within this promoter region in gene regulatory activity. Comparative restriction analyses of cloned and chromosomal DNA segments strongly suggests that no major alterations ocurred during cloning and that there is only one complete copy of the vWf gene in the human haploid genome. Similar analyses of DNA from vWf-expressing endothelial cells and non-expressing white blood cells suggests that no major rearrangements are associated with vWf gene expression. Finally, cross hybridization patterns among seven mammalian species suggests a strong conservation of genomic sequences encoding the plasma portion of vWf, but a lower degree of conservation of sequences encoding the N terminal region of provWf.
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"Technological aspects of the search, cloning and production of enzymes for industrial use." In Bioinformatics of Genome Regulation and Structure/Systems Biology (BGRS/SB-2022) :. Institute of Cytology and Genetics, the Siberian Branch of the Russian Academy of Sciences, 2022. http://dx.doi.org/10.18699/sbb-2022-275.

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"GeneCut – a software tool for oligonucleotide design, assembly and cloning of gene constructs." In Bioinformatics of Genome Regulation and Structure/Systems Biology (BGRS/SB-2022) :. Institute of Cytology and Genetics, the Siberian Branch of the Russian Academy of Sciences, 2022. http://dx.doi.org/10.18699/sbb-2022-671.

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Li, Jingjing, Yuan Wang, Bin Sun, Jinfeng Ouyang, Hu Han, Qinqin Huang, Yongbo Yang, and Yi Li. "Notice of Retraction: Genome Cloning of Human Bocavirus (HBoV1) and Prospective Study of NP1 Gene Transcription." In 2011 5th International Conference on Bioinformatics and Biomedical Engineering. IEEE, 2011. http://dx.doi.org/10.1109/icbbe.2011.5781646.

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O'hara, Patrick J., Frank A. Grant, A. Betty, J. Haldmen, and Mark J. Murray. "Structure of the Human Factor VII Gene." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643786.

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Factor VII is a member of a family of vitamin K-dependent, gamma-carboxylated plasma protein which includes factor IX, factor X, protein C, protein S and prothrombin. Activated factor VII (factor Vila) is a plasma serine protease which participates in a cascade of reactions leading to the coagulation of blood. Two overlapping genomic clones containing sequences encoding human factor VII were isolated and characterized. The complete sequence of the gene was determined and found to span 12.8 kilobases. The mRNA for factor VII as demonstrated by cDNA cloning is polyadenylated at multiple sites but contains only one AAUAAA poly-A signal sequence. The mRNA can undergo alternative splicing forming one transcript containing eight segments as exons and another with an additional exon which encodes a larger pre-pro leader sequence. The portion of the pre-pro leader coded for by the additional exon has no known counterpart in the other vitamin K-dependent proteins. The positions of the introns with respect to the amino acid sequence encoded by the eight essential exons of factor VII are the same as those present in factor IX, factor X, protein C and the first three exons of prothrombin. These exons code for domains generally conserved among members of this gene family, including a pre-pro leader (the essential exon la and alternative exon lb), a gamma-carboxylated domain (exons 2 and 3) a growth factor domain (exons 4 and 5) an activation region (exon 6) and a serine protease (exon 8). The corresponding introns in these genes are dissimilar with respect to size and sequence, with the exception of the third intron in factor VII and protein C. Four introns and a portion of exon 8 in factor VII contain regions made up of tandem repeats of oligonucleotide monomer elements. More than a quarter of the intron sequences and more than a third of the 3' untranslated portion of the mRNA transcript consist of these minisatellite tandem repeats. This type of structure is responsible for polymorphisms due to allelic variation in repeat copy number in other areas of the human genome. Tandem repeats can evolve as a result of random crossover in DNA whose sequence is not maintained by selection. This suggests that much of the sequence information present in the introns and untranslated portion of the message is dispensable.
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Yu, Yongkun, Na Li, and Qingpeng Sun. "Cloning and Analysis of LeWRKY2 Genomic DNA." In 2011 5th International Conference on Bioinformatics and Biomedical Engineering (iCBBE). IEEE, 2011. http://dx.doi.org/10.1109/icbbe.2011.5780144.

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Ichinose, A., R. E. Bottenus, K. R. Loeb, and E. W. Davie. "ISOLATION AND CHARACTERIZATION OF THE GENES FOR THE a AND b SUBUNITS OF HUMAN COAGULATION FACTOR XIII." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644652.

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Factor XIII (plasma transglutaminase, fibrin stabilizing factor) is a plasma protein that plays an important role in the final stages of blood coagulation and fibrinolysis. The molecule occurs in blood as a tetramer (a2b2) consisting of two a. subunits and two b subunits. Recently, we have determined the amino acid sequences for both the a. and b subunits of human factor XIII by a combination of cDNA cloning and amino acid sequence analysis. cDNAs coding for the a (3.8 Kb) and b (2.2 Kb) subunits were used for the screening of human genomic DNA libraries. Among 12 × 106 recombinant phage, ∼30 have been shown to contain the sequences for the a subunit and ∼10 have been shown to contain the gene for the b subunit of factor XIII. The clones coding for the a. subunit span ∼90 Kb and have been characterized by restriction mapping. Southern blotting, and DNA sequencing. Both 5’ and 3’ ends of the genomic clones correspond to the 5’ and 3’portions of the cDNA for the a.subunit of factor XIII. The DNA sequence revealed that the activation peptide released ^thrombin (amino acid residues 137), the first putative Ca2+ binding region (around residue 251), the active Site Cys (amino acid residue 314), and the second putative Ca2+ binding region (around residue 473) are encoded by separate exons. Accordingly, the intervening sequences may separate the a subunit into functional and structural domains. The gene organization for the b subunit will also be presented. (Supported by NIH Grant HL 16919.)
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Song, Bo, Su-rong Shuai, Wan-ru Hou, Jun Yang, and Yi-ling Hou. "CDNA, genomic sequence cloning and overexpression of ribosomal protein S9 gene (RPS9) from Ailuropoda." In 2012 International Conference on Computer Science and Information Processing (CSIP). IEEE, 2012. http://dx.doi.org/10.1109/csip.2012.6308914.

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Youssoufiän, H., A. Patel, D. Phillips, H. H. Kazazian, and S. E. Antonarakis. "RECURRENT MUTATIONS AND AN UNUSUAL DELETION IN HEMOPHILIA A." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644014.

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We have identified 15 mutations of the factor VIII (F8) gene from a panel of 107 patients with hemophilia A and have characterized these gene defects byrestriction analysis, oligonucleotide hybridization, cloning and DNA sequencing. Recurrent point mutations that involve CG to TG transitions were identified in exon 18, exon 22, and exon 24; a single CG to TG transition was identified in exon 23; and a CG to CA transition was identified in exon 24. In addition, a Taq I site alteration in intron 4 was identified in a patient with mild hemophilia, which arose dg. S23&in a grandpaternal germ cell. Cloning and sequencing of this region suggests the generation of a newsplice donor site. These data suggest that CG to TG transition is a prominent mechanism of mutation in hemophilia A. Six different deletions were also characterized. In one family, the deletion involved exon 26. However, the deletion endpoints in the male proband were different from those in his carrier mother, suggesting either gonadal mosaicism or a second deletion event in maternal meiosis.Of the 15 mutations, 6 occurred de novo within 2 generations: 4 in males and 2 in females. In these djg.novo mutations paternal age at conception was 35 (range = 32-38) and maternal age was 24 and 27. The ability to discover a sizable number of mutations in the F8 gene producing hemophilia A enables us to determine the frequency and nature of de novo mutations in man.
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Jian Zou, Yi-Ling Hou, Xiang Ding, Wan-Ru Hou, Jun Yang, and Zheng-Song Peng. "CDNA, genomic sequence cloning and sequence analysis of ribosomal protein S3A (RPS3A) gene from giant panda." In 2012 International Conference on Computer Science and Information Processing (CSIP). IEEE, 2012. http://dx.doi.org/10.1109/csip.2012.6308908.

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Reports on the topic "Genome cloning"

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Zhang, Hongbin B., David J. Bonfil, and Shahal Abbo. Genomics Tools for Legume Agronomic Gene Mapping and Cloning, and Genome Analysis: Chickpea as a Model. United States Department of Agriculture, March 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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Dubcovsky, Jorge, Tzion Fahima, and 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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Sherman, Amir, Rebecca Grumet, Ron Ophir, Nurit Katzir, and Yiqun Weng. Whole genome approach for genetic analysis in cucumber: Fruit size as a test case. United States Department of Agriculture, December 2013. http://dx.doi.org/10.32747/2013.7594399.bard.

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The Cucurbitaceae family includes a broad array of economically and nutritionally important crop species that are consumed as vegetables, staple starches and desserts. Fruit of these species, and types within species, exhibit extensive diversity as evidenced by variation in size, shape, color, flavor, and others. Fruit size and shape are critical quality determinants that delineate uses and market classes and are key traits under selection in breeding programs. However, the underlying genetic bases for variation in fruit size remain to be determined. A few species the Cucurbitaceae family were sequenced during the time of this project (cucumber was already sequenced when the project started watermelon and melon sequence became available during the project) but functional genomic tools are still missing. This research program had three major goals: 1. Develop whole genome cucumber and melon SNP arrays. 2. Develop and characterize cucumber populations segregating for fruit size. 3. Combine genomic tools, segregating populations, and phenotypic characterization to identify loci associated with fruit size. As suggested by the reviewers the work concentrated mostly in cucumber and not both in cucumber and melon. In order to develop a SNP (single nucleotide polymorphism) array for cucumber, available and newly generated sequence from two cucumber cultivars with extreme differences in shape and size, pickling GY14 and Chinese long 9930, were analyzed for variation (SNPs). A large set of high quality SNPs was discovered between the two parents of the RILs population (GY14 and 9930) and used to design a custom SNP array with 35000 SNPs using Agilent technology. The array was validated using 9930, Gy14 and F1 progeny of the two parents. Several mapping populations were developed for linkage mapping of quantitative trait loci (QTL) for fruit size These includes 145 F3 families and 150 recombinant inbred line (RILs F7 or F8 (Gy14 X 9930) and third population contained 450 F2 plants from a cross between Gy14 and a wild plant from India. The main population that was used in this study is the RILs population of Gy14 X 9930. Phenotypic and morphological analyses of 9930, Gy14, and their segregating F2 and RIL progeny indicated that several, likely independent, factors influence cucumber fruit size and shape, including factors that act both pre-anthesis and post-pollination. These include: amount, rate, duration, and plane of cell division pre- and post-anthesis and orientation of cell expansion. Analysis of F2 and RIL progeny indicated that factors influencing fruit length were largely determined pre-anthesis, while fruit diameter was more strongly influenced by environment and growth factors post-anthesis. These results suggest involvement of multiple genetically segregating factors expected to map independently onto the cucumber genome. Using the SNP array and the phenotypic data two major QTLs for fruit size of cucumber were mapped in very high accuracy (around 300 Kb) with large set of markers that should facilitate identification and cloning of major genes that contribute to fruit size in cucumber. In addition, a highly accurate haplotype map of all RILS was created to allow fine mapping of other traits segregating in this population. A detailed cucumber genetic map with 6000 markers was also established (currently the most detailed genetic map of cucumber). The integration of genetics physiology and genomic approaches in this project yielded new major infrastructure tools that can be used for understanding fruit size and many other traits of importance in cucumber. The SNP array and genetic population with an ultra-fine map can be used for future breeding efforts, high resolution mapping and cloning of traits of interest that segregate in this population. The genetic map that was developed can be used for other breeding efforts in other populations. The study of fruit development that was done during this project will be important in dissecting function of genes that that contribute to the fruit size QTLs. The SNP array can be used as tool for mapping different traits in cucumber. The development of the tools and knowledge will thus promote genetic improvement of cucumber and related cucurbits.
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Katzir, Nurit, Rafael Perl-Treves, and Jack E. Staub. Map Merging and Homology Studies in Cucumis Species. United States Department of Agriculture, September 2000. http://dx.doi.org/10.32747/2000.7575276.bard.

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List of original objectives (1) Construct a saturated map of melon, using RFLP, SSR, RAPD and Inter-SSR genetic markers. (2) Study the homology between the genomes of cucumber and melon. (3) Add to the Cucumis map, biologically important genes that had been cloned in other plant systems. Background Cucumber and melon are important vegetable crops in Israel and the US. Genome analysis of these crops has lagged behind the major plant crops, but in the last few years genetic maps with molecular markers have been developed. The groups that participated in this program were all involved in initial mapping of cucurbit crops. This grant was meant to contribute to this trend and promote some of the more advanced applications of genome analysis, i.e., map saturation and comparative mapping between cucurbit species. Major achievements The main achievements of the research were (a) the construction of melon maps that include important horticultural traits and Resistance Gene Homologues, (b) the development of approximately 200 SSR markers of melon and cucumber, (c) the preliminary map merging of melon maps and of comparative mapping between melon and cucumber. Implications As a result of this program, we have a good estimate of the applicability of different types or markers developed in one cucurbit species to genetic mapping in other species. Since the linkage groups of melon and cucumber can now be related to each other, future identification of important genes in the two crops will be facilitated. Moreover, the further saturation of the maps with additional markers will now allow us to target several disease resistance loci, horticultural traits for marker-assisted selection, fine mapping and positional cloning.
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Mawassi, Munir, Baozhong Meng, and Lorne Stobbs. Development of Virus Induced Gene Silencing Tools for Functional Genomics in Grapevine. United States Department of Agriculture, July 2013. http://dx.doi.org/10.32747/2013.7613887.bard.

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Grapevine is perhaps the most widely grown fruit crop. To understand the genetic make-up so as to improve the yield and quality of grapes and grape products, researchers in Europe have recently sequenced the genomes of Pinot noir and its inbred. As expected, function of many grape genes is unknown. Functional genomics studies have become the major focus of grape researchers and breeders. Current genetic approaches for gene function studies include mutagenesis, crossing and genetic transformation. However, these approaches are difficult to apply to grapes and takes long periods of time to accomplish. It is thus imperative to seek new ways for grape functional genomics studies. Virus-induced gene silencing (VIGS) offers an attractive alternative for this purpose and has proven highly effective in several herbaceous plant species including tomato, tobacco and barley. VIGS offers several advantages over existing functional genomics approaches. First, it does not require transformation to silence a plant gene target. Instead, it induces silencing of a plant gene through infection with a virus that contains the target gene sequence, which can be accomplished within a few weeks. Second, different plant genes can be readily inserted into the viral genome via molecular cloning and functions of a large number of genes can be identified within a short period of time. Our long-term goal of this research is to develop VIGS-based tools for grapevine functional genomics, made of the genomes of Grapevine virus A (GVA) from Israel and Grapevine rupestris stem pitting-associated virus (GRSPaV) from Canada. GVA and GRSPaV are members of the Flexiviridae. Both viruses have single-stranded, positive sense RNA genomes, which makes them easy to manipulate genetically and excellent candidates as VIGS vectors. In our three years research, several major breakthroughs have been made by the research groups involved in this project. We have engineered a cDNA clone of GVA into a binary vector that is infectious upon delivery into plantlets of micropropagated Vitis viniferacv. Prime. We further developed the GVA into an expression vector that successfully capable to silence endogenous genes. We also were able to assemble an infectious full-length cDNA clones of GRSPaV. In the following sections Achievements and Detailed description of the research activities, we are presenting the outcome and results of this research in details.
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Levin, Ilan, John W. Scott, Moshe Lapidot, and Moshe Reuveni. Fine mapping, functional analysis and pyramiding of genes controlling begomovirus resistance in tomato. United States Department of Agriculture, November 2014. http://dx.doi.org/10.32747/2014.7594406.bard.

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Abstract. Tomato yellow leaf curl virus (TYLCV), a monopartitebegomovirus, is one of the most devastating viruses of cultivated tomatoes and poses increasing threat to tomato production worldwide. Because all accessions of the cultivated tomato are susceptible to these viruses, wild tomato species have become a valuable resource of resistance genes. QTL controlling resistance to TYLCV and other begomoviruses (Ty loci) were introgressed from several wild tomato species and mapped to the tomato genome. Additionally, a non-isogenic F₁diallel study demonstrated that several of these resistance sources may interact with each other, and in some cases generate hybrid plants displaying lower symptoms and higher fruit yield compared to their parental lines, while their respective resistance genes are not necessarily allelic. This suggests that pyramiding genes originating from different resistance sources can be effective in obtaining lines and cultivars which are highly resistant to begomoviruses. Molecular tools needed to test this hypothesis have been developed by our labs and can thus significantly improve our understanding of the mechanisms of begomovirus resistance and how to efficiently exploit them to develop wider and more durable resistance. Five non-allelic Ty loci with relatively major effects have been mapped to the tomato genome using molecular DNA markers, thereby establishing tools for efficient marker assisted selection, pyramiding of multiple genes, and map based gene cloning: Ty-1, Ty-2, Ty-3, Ty-4, and ty-5. This research focused on Ty-3 and Ty-4 due to their broad range of resistance to different begomoviruses, including ToMoV, and on ty-5 due to its exceptionally high level of resistance to TYLCV and other begomoviruses. Our aims were: (1) clone Ty-3, and fine map Ty-4 and Ty-5 genes, (2)introgress each gene into two backgroundsand develop semi isogenic lines harboring all possible combinations of the three genes while minimizing linkage-drag, (3) test the resulting lines, and F₁ hybrids made with them, for symptom severity and yield components, and (4) identify and functionally characterize candidate genes that map to chromosomal segments which harbor the resistance loci. During the course of this research we have: (1) found that the allelic Ty-1 and Ty-3 represent two alternative alleles of the gene coding DFDGD-RDRP; (2) found that ty-5is highly likely encoded by the messenger RNA surveillance factor PELOTA (validation is at progress with positive results); (3) continued the map-based cloning of Ty-4; (4) generated all possible gene combinations among Ty-1, Ty-3 and ty-5, including their F₁ counterparts, and tested them for TYLCV and ToMoV resistance; (5) found that the symptomless line TY172, carrying ty-5, also carries a novel allele of Ty-1 (termed Ty-1ⱽ). The main scientific and agricultural implications of this research are as follows: (1) We have developed recombination free DNA markers that will substantially facilitate the introgression of Ty-1, Ty-3 and ty-5 as well as their combinations; (2) We have identified the genes controlling TYLCV resistance at the Ty-1/Ty-3 and ty-5 loci, thus enabling an in-depth analyses of the mechanisms that facilitate begomovirus resistance; (3) Pyramiding of Ty resistance loci is highly effective in providing significantly higher TYLCV resistance.
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Bertani, Giuseppe. Cloning and Gene Fusion for a Metalloprotein. Fort Belvoir, VA: Defense Technical Information Center, September 1987. http://dx.doi.org/10.21236/ada190914.

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Azem, Abdussalam, George Lorimer, and Adina Breiman. Molecular and in vivo Functions of the Chloroplast Chaperonins. United States Department of Agriculture, June 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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Hulata, Gideon, Thomas D. Kocher, Micha Ron, and Eyal Seroussi. Molecular Mechanisms of Sex Determination in Cultured Tilapias. United States Department of Agriculture, October 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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Mawassi, Munir, Adib Rowhani, Deborah A. Golino, Avichai Perl, and Edna Tanne. Rugose Wood Disease of Grapevine, Etiology and Virus Resistance in Transgenic Vines. United States Department of Agriculture, November 2003. http://dx.doi.org/10.32747/2003.7586477.bard.

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Rugose wood is a complex disease of grapevines, which occurs in all growing areas. The disease is spread in the field by vector transmission (mealybugs). At least five elongated-phloem- limited viruses are implicated in the various rugose wood disorders. The most fully characterized of these are Grapevine virus A (GV A) and GVB, members of a newly established genus, the vitivirus. GVC, a putative vitivirus, is much less well characterized than GV A or GVB. The information regarding the role of GVC in the etiology and epidemiology of rugose wood is fragmentary and no sequence data for GVC are available. The proposed research is aimed to study the etiology and epidemiology of rugose wood disease, and to construct genetically engineered virus-resistant grapevines. The objectives of our proposed research were to construct transgenic plants with coat protein gene sequences designed to induce post-transcriptional gene silencing (pTGS); to study the epidemiology and etiology of rugose wood disease by cloning and sequencing of GVC; and surveying of rugose wood- associated viruses in Californian and Israeli vineyards. In an attempt to experimentally define the role of the various genes of GV A, we utilized the infectious clone, inserted mutations in every ORF, and studied the effect on viral replication, gene expression, symptoms and viral movement. We explored the production of viral RNAs in a GV A-infected Nicotiana benthamiana herbaceous host, and characterized one nested set of three 5'-terminal sgRNAs of 5.1, 5.5 and 6.0 kb, and another, of three 3'-terminal sgRNAs of 2.2, 1.8 and 1.0 kb that could serve for expression of ORFs 2-3, respectively. Several GV A constructs have been assembled into pCAMBIA 230 I, a binary vector which is used for Angrobacterium mediated transformation: GV A CP gene; two copies of the GV A CP gene arranged in the same antisense orientation; two copies of the GV A CP gene in which the downstream copy is in an antigens orientation; GV A replicase gene; GV A replicase gene plus the 3' UTR sequence; and the full genome of GV A. Experiments for transformation of N. benthamiana and grapevine cell suspension with these constructs have been initiated. Transgenic N. benthamiana plants that contained the CP gene, the replicase gene and the entire genome of GV A were obtained. For grapevine transformation, we have developed efficient protocols for transformation and successfully grapevine plantlets that contained the CP gene and the replicase genes of GV A were obtained. These plants are still under examination for expression of the trans genes. The construction of transgenic plants with GV A sequences will provide, in the long run, a means to control one of the most prevalent viruses associated with grapevines. Our many attempts to produce a cDNA library from the genome of GVC failed. For surveying of rugose wood associated viruses in California vineyards, samples were collected from different grape growing areas and tested by RT-PCR for GV A, GVB and GVD. The results indicated that some of the samples were infected with multiple viruses, but overall, we found higher incidence of GVB and GV A infection in California vineyards and new introduction varieties, respectively. In this research we also conducted studies to increase our understanding of virus - induced rootstock decline and its importance in vineyard productivity. Our results provided supporting evidence that the rootstock response to virus infection depends on the rootstock genotype and the virus type. In general, rootstocks are differ widely in virus susceptibility. Our data indicated that a virus type or its combination with other viruses was responsible in virus-induced rootstock decline. As the results showed, the growth of the rootstocks were severely affected when the combination of more than one virus was present.
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