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LT20 Chlamydomonas
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UCL BIOL2005 Genetic Systems: Chlamydomonas
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ChlamydomonasGeneral Features
Unicellular eukaryotic green microalgae
of the Chlorophyta
10-20 um in diameter
More than 600 species described – found
in marine, freshwaters, damp soil, snow –
C. reinhardtii most studied
C. reinhardtii – phototrophic and
heterotrophic growth: understanding
photosynthesis, chloroplast biology, cell
motility, phototaxis, circadian rhythms other aspects of eukaryotic cell biology
- Can survive in the dark on acetate – can generate photosynthesis mutants which
are viable
- 2 flagella identical to human cilia – motility
- Can only fit one chloroplast – plants cells fit more as they are larger
The 3 genomes
Nuclear: 120Mb, 17 chromosomes, ~15 000 protein-coding genes
Chloroplast genome: circular, 204kb genome within chloroplast, each cell containing
~80 copies, 99 genes (68 protein coding genes + rRNA and tRNA genes)
Mitochondrial genome: small, linear 15.8kb molecule, each cell contains ~50 copies,
13 genes (8 protein coding genes +2rRNA and 3 tRNA genes)
Life cycle
Vegetative reproduction
Vegetative cells are haploid
Cells grow by multiple fission to
form clonal colonies
Cell division can be synchronised by
12 hr light/12 hr dark cycle
Cultivation is rapid (doubling time ~8
hours) and has simple nutrient
requirements
Inheritance Patterns
, Nuclear mutations – inherited according to
Mendelian rules (ie. 2:2 in tetrad)
Organelle mutations; chloroplast and
mitochondrial are inherited uniparentally (ie. 4:0
in tetrad)
Chloroplast genomes are inherited from mt+
parent
Mitochondrial genomes are inherited from mt-
parent
Random Mutagenesis
Conducted on vegetative cells: cells are haploid, all visible mutations give a
phenotype irrespective of dominant/recessive nature
UV mutagenesis: cells are exposed to UV light and plated on solid medium, select
mutants after 10% survival, usually causing point mutations, surviving cells can be
screened phenotypically
Chemical mutagenesis: cells in liquid culture exposed to random mutagenic agent
such as ethyl methane sulfonate (EMS), cells are transferred to a sold medium and
surviving cells are screened phenotypically
Searching for startch mutants – cells treated with UV, surviving cells exposed to
iodine vapour to stain cellular starch, non-stained cells isolated
Isolating photosystem II mutants: cells grown up in liquid medium, exposed to EMS,
surviving cells plated out on solid medium, surviving cells assayed by fluorescence by
looking for low chlorophyll fluorescence emitting cells
DNA Transformation
All 3 genomes transformable
3 steps: Entry (getting DNA into cell/compartment), Insertion (ensure DNA is
incorporated into the genome), Selection (picking out correctly transformed cells)
Entry
- biolistics (DNA used to coat tungsten or gold particles, lawn bombarded with the
particles)
- glass beads (suspension of cells vortexed with glass beads and target DNA,
abrasive forces cause transient membrane lesions, DNA diffuses into cell)
- Electroporation (high V electrical field applied across a cell suspension, causes
transient membrane pores and charge gradient, negatively charged DNA drawn
into the cells)
Insertion of DNA into the genome
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