Date of Award
Doctor of Philosophy
Plant Pathology and Microbiology
Martin H. Spalding
Chlamydomonas reinhardtii shows adaptive changes in order to survive in limiting CO2 conditions because they live in quite variable conditions of CO2 availability. We are trying to identify mutants defective in the acclimation response to limiting CO 2. Nuclear transformation to complement an Arg- phenotype was used to generate insertional mutants, and Cah1 expression and having high CO2-requiring (HCR) phenotype were used as reporters for induction of genes involved in acclimation to limiting CO 2.;Among a collection of insertionally generated mutants, a mutant has been isolated that showed no pCA1 protein and no Cah1 mRNA. This mutant strain, designated cah1-1, has been confirmed to have a disruption in the Cah1 gene caused by a single Arg7 insert. cah1-1 is its lack of any significant growth phenotype, indicating pCA1 is not essential for function of the CO 2-concentrating mechanism or for growth of C. reinhardtii at limiting CO2 concentrations.;Eight independently isolated mutants of C. reinhardtii that require high CO2 for photoautotrophic growth were tested by complementation group analysis. Two of the new mutants formed a single complementation group along with the previously described mutant cia-5, which appears to be defective in the signal transduction pathway for low CO 2 acclimation. The other mutations represent six additional, independent complementation groups.;From approximately 7000 transformants screened for lack of Cah1 expression, HCR90 was identified. This mutant shows a recessive HCR phenotype that co-segregates with the Arg+ phenotype and presence of 1 Arg insert. HCR90 does not show any allelism to the known HCR mutants. Km (Ci) of HCR90 was found to be higher than that of wild type, suggesting HCR90 lacks a functional CCM. After 5' and 3' sequences flanking the Arg insert were obtained, genomic clones from a C. reinhardtii BAC library were identified by using both flanking DNA as a probe. The 5' and 3' flanking sequences identified non-overlapping sets of BACs, indicating a rather large disruption. Since HCR90 grows poorly in low CO2, complementation of the mutation should be possible under direct selection in low CO2 . Transformations with a cDNA expression plasmid library for complementing of HCR90 phenotype are in progress.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu
Van, Kyujung, "Insertional mutagenesis of the signal transduction pathway for low CO2 acclimation in Chlamydomonas reinhardtii " (2001). Retrospective Theses and Dissertations. 458.