Degree Type

Dissertation

Date of Award

2016

Degree Name

Doctor of Philosophy

Department

Genetics, Development and Cell Biology

Major

Genetics

First Advisor

Martin H. Spalding

Abstract

We demonstrate that CIA5, the master CCM transcription regulator of the Chlamydomonas CCM, has transcription activation activity either in yeast or in Chlamydomonas, and has an acidic activation domain that also is responsible for abnormal SDS-PAGE migration. We are also the first successful overexpression and purification of CIA5 protein from E. coli., which was used to demonstrate DNA binding ability of CIA5/CCM1 in vitro using random binding site selection and gel mobility shift assays, and the development of a mini-CIA5 construct, which only contains a partial CIA5 CDS (nt 1 - 330 plus nt 1282 - 1674). This mini-CIA5, when transformed into cia5, was able to complement the cia5 lethal phenotype in very low CO2. However, detailed assessment of gene expression in the mini-CIA5 complemented cia5 suggested that other CIA5 sequences missing from mini-CIA5 may also be important for a fully functional CIA5. Screening or selection for cia5 suppressors by insertional mutagenesis, UV mutagenesis or EMS mutagenesis identified no candidate colonies other than cia5 revertants that converted the point mutation back to its wild-type form. The inability to identify second-site suppressors further supports and strengthens the significant role of CIA5 in the Chlamydomonas CCM.

Copyright Owner

Bo Chen

Language

en

File Format

application/pdf

File Size

140 pages

Included in

Genetics Commons

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