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2002
LSU-HHMI Summer Undergraduate Research Program |
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 Kristen
R. LeBleu (James V. Moroney, LSU Dept. of Biological Sciences)
Genetic and Molecular Characterization of BleR Insertional
Mutants in Chlamydomonas reinhardtii
Chlamydomonas reinhardtii, a eukaryotic unicellular alga, utilizes
a carbon concentrating mechanism (CCM) to fix carbon more efficiently
in low CO2 environments. To determine the genes involved in
the CCM, insertional mutants were generated by transforming
C. reinhardtii, strain D66, with the BleR gene. The BleR gene
confers resistance to the antibiotic Zeocin. Transformants resistant
to Zeocin were then screened in low CO2, high CO2, and acetate.
Those that appeared sick or died on low CO2 or in the light
on acetate were selected for further analysis. These mutants
were then divided into two categories: HFL (high fluorescent
mutants) and SLC (sick on low CO2 mutants).
In HFL mutants the BleR insert disrupted the DNA coding for
a part of the photosynthetic apparatus. With photosynthesis
interrupted, the HFL mutants grow poorly in light even with
a carbon source and fluoresce under long-wave UV light. SLC
mutants have normal photosynthesis on high CO2 and grow normally
on acetate in the light but become sick on low CO2.
This poster describes how we determined whether the inserted
BleR gene was causing the HFL or SLC phenotype. The methods
utilized were tetrad analysis and the random spore method. This
involved crossing the mutants with the wild type cc-124 and
screening the progeny. When the progeny demonstrated resistance
to Zeocin and had the mutant phenotype, it confirmed that the
BleR insert was genetically linked to the disruption in the
CCM. Our hypothesis is that the genes disrupted by the BleR
insert are important to the functioning of the CCM. The mutants
were further characterized using iPCR.
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