Project description:Knockout mutants of polyketide synthase genes were generated (pks1, pks2, pks3, pks14, pks18, pks37) and compared to wild-type AX2 strain of Dictyostelium discoideum using Agilent custom microarrays. The Dictyostelium cells were harvested at eight developmental stages (Amoeboid, Intiation of starvation, loose aggregate, Streaming, Mound, Slug, early culminant, Fruiting body) and subjected to microarray based expression profiling.
Project description:We identified 3418 genes transcribed at a level of at least two copies each. We identified many transcripts involved in protein translation, cell maintenance and metabolism, as expected for vegetative cells. The most highly expressed cell signaling genes include ubiquitin, smlA, and nucleotide exchange factors RasGEF F and Ras GEF G. Additionally, we identified many genes previously reported to be expressed only during later stages of development including dutA, actin8, thioredoxin3, culmination specific protein 45D, discoidin II and yelA. We isolated total RNA from Dictyostelium amoeba growing in HL5 media with shaking and at a density of 2.4 X 10(7) cells/ml using Ambion RNAqueous isolation kit. We prepared the SAGE library using an Invitrogen kit and being careful to reduce the amount of ditags in the concatemer ligation reaction to 25% of the manufacturer's suggested mass. Concatemers were sequenced at the Molecular Research Core Facility on the campus of Idaho State University using an Applied Biosystems 3100 Genetic Analyzer. We identified SAGE tags by comparison to the Dictyostelium genome sequence available on the dictybase website.
