PARENT SESSION
Posters P3D Genomic, proteomic and related technologies. Abstracts (720-730)

Random insertional mutagenesis in Chlamydomonas reinhardtii: an attempt to saturate for sulfate acclimation mutants. Steve Pollock^*,1, Wirulda Pootakham¹, Daniel Zimardi¹, Arthur Grossman¹, ¹ Department of Plant Biology, Stanford, California, USA

ABSTRACT- When Chlamydomonas reinhardtii is deprived of exogenous sulfur it derepresses expression of several genes required for the active acquisition and assimilation of environmental sulfate and down-regulates metabolic processes such as photosynthesis. One gene that is derepressed during sulfur deprivation is the cell wall-associated protein arylsulfatase (ARS), which cleaves sulfate from organic compounds making it available for import into the cell. In sulfate sufficient medium, ARS mRNA and protein are undetectable. When the cells are transferred to medium devoid of sulfur, the abundance of ARS increases within 2 h and reaches a maximum at 8 h. We are using a random insertional mutagenesis strategy, involving the insertion of a gene conferring paromomycin resistance, to disrupt components of the signal transduction pathway that sense sulfur levels and stimulate the transcription of ARS, and other genes in C. reinhardtii during sulfur deprivation. Since ARS activity is easily assayed on solid medium, it is an excellent reporter for detecting mutants that are unable to properly sense a low sulfur environment. An adaptor-mediated PCR method has been used to obtain DNA sequences that flank the site of insertion of the paromomycin resistance cassette in strains exhibiting aberrant ARS activity. Using the recently available draft of the C. reinhardtii genome sequence, the locus of the insertion can be readily identified. Those genes altered in the ARS-deficient mutants will be discussed.

KEY WORDS: sulfur, genome