2. Mutant screens and characterization |
3. Transformation of the chloroplast and nuclear genomes |
6. Cell biology of chlamydomonas |
7. Chlamydomonas reinhardtii |
Besides the practical work several lectures will be given by experts in the field
1.Genetic
analysis: gametogenesis, tetrad analysis and vegetative diploid analysis.
Haploid cells of Chlamydomonas reinhardtii exist either as mating-type
(+) or mating-type
(-) and can be propagated vegetatively. Upon starvation
for nitrogen and blue light illumination, vegetative cells develop into
gametes. Mixing of gametes of opposite mating-type triggers a series
of complex reactions that ultimately lead to the fusion of gametes and,
subsequently, of their nuclei and chloroplasts. The resulting zygote
can be induced to undergo meiosis and to produce a tetrad consisting
of four haploid progeny cells. In such crosses the chloroplast and mitochondrial
genomes are transmitted uniparentally from the (+) and (-) mating-types,
respectively. The students will learn the basic methods for crossing
Chlamydomonas cells and for tetrad analysis. They will also learn how
to produce vegetative diploids.
2. Mutant screens and characterization
Photosynthetic function is dispensable in C. reinhardtii in the presence of a suitable carbon source. Thus, mutants deficient in photosynthetic function can be isolated readily. A useful feature of this alga is that its fluorescence properties are highly sensitive to alterations in the photosynthetic electron transfer chain. Fluorescence measurements will be used as non-invasive diagnostic tools to identify photosynthetic lesions in mutants. Fluorescence video-imaging will also be used to identify mutations that affect the capacity of the algae to adapt to changes in light quantity and quality and that affect non-photochemical quenching or state transitions.
Moreover newly developed sensitive spectroscopic methods will be used for following photosynthetic reactions in the nanosecond to millisecond time range.
-Photosynthetic mutants will be examined using immunoblot analysis and pulse labeling techniques. The latter technique is especially powerful for the analysis
of mutants deficient in the translation of chloroplast-encoded thylakoid polypeptides.
-Mutants deficient in flagellar assembly or function will be isolated. The protein composition of the flagella of known mutants will be examined by PAGE. Deflagellation followed by regeneration of the flagella will be examined.
3. Transformation of the chloroplast and nuclear genomes
Biolistic transformation of the chloroplast can be achieved with a particle gun using appropriate
selectable markers. The most used marker is aadA, a bacterial gene conferring
spectinomycin and streptomycin resistance to the chloroplast. Specific
chloroplast gene disruptions will be performed using the homologous
plastid recombination system. Transformants which will be tested by
PCR analysis for homoplasmicity.
The high yield of nuclear transformation makes it possible to isolate genes that are defective in mutants by genomic complementation with a wild-type cosmid library. This approach will be discussed, together with mapping techniques. Nuclear transformation occurs through random insertion in the nuclear genome. It will be used to produce tagged mutants deficient in photosynthetic electron transport (fluorescence screen).
4. Reverse nuclear genetics
Newly developed methods for the inactivation of specific nuclear genes of Chlamydomonas by RNAi wil be presented.
5. Cell fractionation
Cell wall-deficient strains of Chlamydomonas are very useful for cell fractionation studies, in particular for isolating chloroplasts which can subsequently be separated into stroma, envelope and thylakoid membranes. The various photosynthetic complexes, photosystem II, photosystem I and the cytochrome b6f complex will be purified from detergent-solubilized thylakoid membranes by blue native gel electrophoresis and sucrose gradient centrifugation and their subunits revealed by PAGE. Photochemical activity of the complexes will be measured.
Methods for isolating flagella will also be used and protein analysis will be performed on several different mutants deficient in specific flagellar polypeptides.
6. Cell biology of Chlamydomonas
Nuclear transformation allows one to tag specific proteins and to study their cellular location through immunofluorescence. Students will have the opportunity to test this on specific proteins of the flagellar apparatus.
7. is an ideal organism for studying cellular movement and hydrodynamics as well as flagellar beating and photomovement responses under various environmental conditions or stimuli. In general, movement of individual cells that live under conditions of low Reynold numbers is dominated by viscosity of the medium and this is critical for all cell separation techniques (cell sorting). Beyond this, flagellar beating is crucial for all kinds of epithelial flagella and sperms, and may be nicely studied using C.reinhardtii. (see flagellar section). Motion and responses of individual cells to external stimuli such as light and chemicals will be studied and quantified under the microscope using a computer supported motion analysis system whereas overall responses of cell populations (phototaxis, chemotaxis or phobic responses) will be demonstrated by dynamic light scattering.
Two setups of each: motion analysis and light scattering may be available on demand.
8. Bioinformatics
The sequences of the chloroplast and mitochondrial genomes of C. reinhardtii have been determined and a draft sequence of the nuclear genome of this alga comprising ca. 90% of the genome is available together with 280,000 ESTs. The current bioinformatics tools for analyzing these sequence