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Journal of Virology, January 2003, p. 123-134, Vol. 77, No. 1
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.1.123-134.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

A Direct Transposon Insertion Tool for Modification and Functional Analysis of Viral Genomes

Heikki Vilen,1 Juha-Matti Aalto,1 Anna Kassinen,1 Lars Paulin,2 and Harri Savilahti1*

Program in Cellular Biotechnology,1 DNA Synthesis and Sequencing Laboratory, Institute of Biotechnology, Viikki Biocenter, University of Helsinki, Helsinki, Finland2

Received 8 July 2002/ Accepted 18 September 2002

Advances in DNA transposition technology have recently generated efficient tools for various types of functional genetic analyses. We demonstrate here the power of the bacteriophage Mu-derived in vitro DNA transposition system for modification and functional characterization of a complete bacterial virus genome. The linear double-stranded DNA genome of Escherichia coli bacteriophage PRD1 was studied by insertion mutagenesis with reporter mini-Mu transposons that were integrated in vitro into isolated genomic DNA. After introduction into bacterial cells by electroporation, recombinant transposon-containing virus clones were identified by autoradiography or visual blue-white screening employing {alpha}-complementation of E. coli ß-galactosidase. Additionally, a modified transposon with engineered NotI sites at both ends was used to introduce novel restriction sites into the phage genome. Analysis of the transposon integration sites in the genomes of viable recombinant phage generated a functional map, collectively indicating genes and genomic regions essential and nonessential for virus propagation. Moreover, promoterless transposons defined the direction of transcription within several insert-tolerant genomic regions. These strategies for the analysis of viral genomes are of a general nature and therefore may be applied to functional genomics studies in all prokaryotic and eukaryotic cell viruses.

* Corresponding author. Mailing address: Institute of Biotechnology, Viikki Biocenter, P.O. Box 56, Viikinkaari 9, 00014 University of Helsinki, Finland. Phone: 358-9-191 59516. Fax: 358-9-191 59366. E-mail: harri.savilahti{at}helsinki.fi.

Journal of Virology, January 2003, p. 123-134, Vol. 77, No. 1
0022-538X/03/$08.00+0     DOI: 10.1128/JVI.77.1.123-134.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.





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