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Nucleic Acids Research, 2003, Vol. 31, No. 18 e107
© 2003 Oxford University Press

Rapid generation of gene disruption constructs by in vitro transposition and identification of a Dictyostelium protein kinase that regulates its rate of growth and development

Tomoaki Abe, Judith Langenick and Jeffrey G. Williams*

School of Life Sciences, University of Dundee, Wellcome Trust Biocentre, Dow Street, Dundee DD1 5EH, UK

*To whom correspondence should be addressed. Tel: +44 1382 345823; Fax: +44 1382 345386; Email: j.g.williams{at}dundee.ac.uk

We describe a rapid method for creating Dictyo stelium gene disruption constructs, whereby the target gene is interrupted by a drug resistance cassette using in vitro transposition. A fragment of genomic DNA containing the gene to be disrupted is amplified by PCR, cloned into a plasmid vector using topoisomerase and then employed as the substrate in an in vitro Tn5 transposition reaction. The transposing species is a fragment of DNA containing a Dictyostelium blasticidin S resistance (bsr) cassette linked to a bacterial tetracycline resistance (tetr) cassette. After transposition the plasmid DNA is transformed into Escherichia coli and clones in which the bsr-tetr cassette is inserted into the Dictyostelium target DNA are identified. To demonstrate its utility we have employed the method to disrupt the gene encoding QkgA, a novel protein kinase identified from the Dictyostelium genome sequencing project. QkgA is structurally homologous to two previously identified Dictyostelium kinases, GbpC and pats1. Like them it contains a leucine-rich repeat domain, a small GTP-binding (ras) domain and a MEKK domain. Disruption of the qkgA gene causes a marked increase in growth rate and, during development, aggregation occurs relatively slowly to form abnormally large multicellular structures.


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