Secondary Level Screening of Chlamydomonas Reinhardtii Mutants Defective in Circadian Gene Expression

• Main Author: Huang, Mingya
• Format: Others
• Published: TopSCHOLAR® 2001
• Subjects: Biology
• Online Access: http://digitalcommons.wku.edu/theses/667; http://digitalcommons.wku.edu/cgi/viewcontent.cgi?article=1670&context=theses

To elucidate the signal transduction chain mediating circadian clock control, this work focuses on the isolation of Chlamydomonas reinhardtii mutants which are defective in circadian gene expression. In a previous study, the reporter gene ARS2 encoding the arylsulfatase enzyme was fused to the promoter of the circadian-regulated CABII-1 gene and transformed into the Chlamydomonas nucleus. The ble marker was introduced into the genome of this transformant via insertional mutagenesis to generate mutants defective in circadian CABII-1 expression. Potential mutants were selected based on aberrant single-point accumulative arylsulfatase activity. In this study, the arylsulfatase activity over the entire growth cycle was further investigated in these mutants and the reliability of the single-point screen was assessed. Of the 16 strains whose accumulative arylsulfatase activity did not differ from the nonmutagenized control in the single-point screen, 12 still showed no significant difference in a multiple-point screen. Of the 9 potential mutants with significant difference to the control in the single-point screen, 3 showed no significant difference in the multiple-point screen. Subsequently, 8 of the candidate mutants with aberrant reporter enzyme activity in the multiple-point screen were characterized by the abundance of their mRNA. The peak-to-trough ratio of CABII-1 and ARS2 transcript abundance was significantly reduced in 4 of these mutants.