Techniques to study input and output pathways of the circadian clock in Chlamydomonas

Matthew Young, Daniel Byrd, and Sigrid Jacobshagen

Department of Biology, Western Kentucky University, Bowling Green, KY, USA

The plant-like cryptochrome (CPH1) in Chlamydomonas represents one of the candidate photoreceptors for entrainment of the circadian clock to environmental light/dark cycles and therefore a potential component of the circadian clock input pathway. To investigate this possibility, we have cloned an RNA interference construct for plant-like cryptochrome in an effort to reduce the expression level of this protein. Strains with reduced cryptochrome can then be tested for reduced abilities to entrain to environmental light/dark cycles. In a two-step strategy, we first cloned a fragment of the genomic DNA for CPH1 into the pCB740 plasmid in place of the HSP70B gene. pCB740 was kindly provided by Christoph Beck and contains the HSP70A/RBCS2 fusion promoter in front of the HSP70B gene. pCB740 also contains the Chlamydomonas ARG7 gene as a marker. In a second step, we cloned the respective cDNA fragment of CPH1 behind the genomic fragment in reverse orientation. We are currently verifying our construct by DNA sequencing. In our study of the output pathway of the circadian clock in Chlamydomonas , we screened for mutants defective in circadian transcription of the LHCB-1 gene. Mutants were created through insertional mutagenesis using the ble marker (pSP124S). To verify the insertions, we designed a PCR assay with primers hybridizing to the ble gene. We are currently testing our PCR assay with genomic DNA from our mutants and control strains. The assay thus far does not reproducibly amplify the ble gene, and therefore cannot yet function as a true test of successful insertion.

e-mail address of presenting author: sigrid.jacobshagen@wku.edu