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Published online before print March 20, 2002, 10.1101/gr.220702
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Vol. 12, Issue 4, 584-594, April 2002

LETTER
Functional Genomics on Potato Virus A: Virus Genome-Wide Map of Sites Essential for Virus Propagation

Tuija Kekarainen,1 Harri Savilahti,2 and Jari P.T. Valkonen1,3

1 Department of Plant Biology, Swedish University of Agricultural Sciences (SLU), S-75007 Uppsala, Sweden; 2 Program in Cellular Biotechnology, Institute of Biotechnology, Viikki Biocenter, FIN-00014 University of Helsinki, Finland

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.

3 Corresponding author.

12:584-594 ©2002 by Cold Spring Harbor Laboratory Press  ISSN 1088-9051/02 $5.00
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