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 ␣-complementation of E. coli ␤-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.

Bacteriophage PM2 presently is the only member of the Corticoviridae family. The virion consists of a protein-rich lipid vesicle, which is surrounded by an icosahedral protein capsid. The lipid vesicle encloses a supercoiled circular double-stranded DNA genome of 10,079 bp. PM2 belongs to the marine phage community and is known to infect two gram-negative Pseudoalteromonas species. In this study, we present a characterization of the PM2 genome made using the in vitro transposon insertion mutagenesis approach. Analysis of 101 insertion mutants yielded information on the essential and dispensable regions of the PM2 genome and led to the identification of several new genes. A number of lysis-deficient mutants as well as mutants displaying delayed-and/or incomplete-lysis phenotypes were identified. This enabled us to identify novel lysis-associated genes with no resemblance to those previously described from other bacteriophage systems. Nonessential genome regions are discussed in the context of PM2 genome evolution.

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Background Pharmacogenetic testing has become increasingly important in drug development and pharmaco‐vigilance processes. To understand the inter‐individual differences in active pathways, tests that target multiple drug metabolizing and drug interacting proteins are required. Method A DNA microarray was developed that consists of an array‐of arrays for genotyping 16 samples simultaneously (the DrugMEt™ Test). Genotyping is accomplished in a single reaction with allele‐specific primers for 27 SNPs of eight highly polymorphic genes: CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A5, TPMT, NAT2, and MDR1. The accuracy and reproducibility were evaluated, and a method comparison with the TaqMan™ assay was performed. The applicability of the method was investigated with samples from different ethnic backgrounds. Results For the various SNPs, an accuracy >99%, and reproducibility of >95%, was observed. The method comparison indicated >95% concordance between the DrugMEt™ Test and TaqMan™ assay. The genotype frequencies of the Caucasian, African American, and Asian populations matched those published in the literature. Conclusions The multigene DrugMEt™ microarray provides a straight‐forward basis for the selection of a drug compound for future development, and identification of the potential responder population. Moreover, drug‐interacting pathways may be identified. Clinical Pharmacology & Therapeutics (2005) 77, P64–P64; doi: