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.
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 (bs(r)) cassette linked to a bacterial tetracycline resistance (tet(r)) cassette. After transposition the plasmid DNA is transformed into Escherichia coli and clones in which the bs(r)-tet(r) 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.
Abe
Tomoaki
T
School of Life Sciences, University of Dundee, Wellcome Trust Biocentre, Dow Street, Dundee DD1 5EH, UK.
Langenick
Judith
J
Williams
Jeffrey G
JG
D000595:Amino Acid Sequence; D000818:Animals; D004251:DNA Transposable Elements / Q000235:genetics*; D004023:Dictyostelium / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D018507:Gene Expression Regulation, Developmental; D015971:Gene Expression Regulation, Enzymologic; D017125:Genes, Protozoan / Q000235:genetics; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional / Q000235:genetics / Q000379:methods*; D009154:Mutation; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D011494:Protein Kinases / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
D004251:DNA Transposable Elements; D011494:Protein Kinases