A1 Vertaisarvioitu alkuperäisartikkeli tieteellisessä lehdessä
Functional Genomics on Potato virus A: Virus genome-wide map of sites essential for virus propagation
Tekijät: Kekarainen T, Savilahti H, Valkonen JPT
Kustantaja: COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT
Julkaisuvuosi: 2002
Tietokannassa oleva lehden nimi: GENOME RESEARCH
Lehden akronyymi: GENOME RES
Vuosikerta: 12
Numero: 4
Aloitussivu: 584
Lopetussivu: 594
Sivujen määrä: 11
ISSN: 1088-9051
DOI: https://doi.org/10.1101/gr.220702
Tiivistelmä
Transposition-based in vitro insertional mutagenesis strategies provide promising new approaches for functional characterization of any cloned gene or genome region. We have extended the methodology and scope of such analysis to a complete viral genome. To map genome regions both essential and nonessential for Potato virus A propagation, we generated a genomic 15-bp insertion mutant library utilizing the efficient In vitro DNA transposition reaction of phage Mu. We then determined the proficiency of 1125 mutants to propagate In tobacco protoplasts by using a genetic footprinting strategy that simultaneously mapped the genomic Insertion sites. Over 300 sites critical for virus propagation were identified, and many of them were located in positions previously not assigned to any viral functions. Many genome regions tolerated insertions Indicating less important sites for virus propagation and thus pinpointed potential locations for further genome manipulation. The methodology described is applicable to a detailed functional analysis of any viral nucleic acid cloned as DNA and can be used to address many different processes during viral infection cycles.
Transposition-based in vitro insertional mutagenesis strategies provide promising new approaches for functional characterization of any cloned gene or genome region. We have extended the methodology and scope of such analysis to a complete viral genome. To map genome regions both essential and nonessential for Potato virus A propagation, we generated a genomic 15-bp insertion mutant library utilizing the efficient In vitro DNA transposition reaction of phage Mu. We then determined the proficiency of 1125 mutants to propagate In tobacco protoplasts by using a genetic footprinting strategy that simultaneously mapped the genomic Insertion sites. Over 300 sites critical for virus propagation were identified, and many of them were located in positions previously not assigned to any viral functions. Many genome regions tolerated insertions Indicating less important sites for virus propagation and thus pinpointed potential locations for further genome manipulation. The methodology described is applicable to a detailed functional analysis of any viral nucleic acid cloned as DNA and can be used to address many different processes during viral infection cycles.
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