A1 Refereed original research article in a scientific journal
Functional Genomics on Potato virus A: Virus genome-wide map of sites essential for virus propagation
Authors: Kekarainen T, Savilahti H, Valkonen JPT
Publisher: COLD SPRING HARBOR LAB PRESS, PUBLICATIONS DEPT
Publication year: 2002
Journal name in source: GENOME RESEARCH
Journal acronym: GENOME RES
Volume: 12
Issue: 4
First page : 584
Last page: 594
Number of pages: 11
ISSN: 1088-9051
DOI: https://doi.org/10.1101/gr.220702
Abstract
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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