Mary Rose Lamb
Since the summer of 1991, I have been studying the genetics and molecular biology of the unicellular green alga, Chlamydomonas reinhardtii. This work is being done in collaboration with Carol Dieckmann, Telsa Mittlelmeier, and Mike Rice at the University of Arizona.
Chlamydomonas is a photosynthetic organism, so it is advantageous for the cell to be able to swim toward light at an appropriate level. At moderate light intensities, the cell swims toward the light (positive phototaxis). At high light intensities, the cell stops swimming briefly (photophobic behavior) and then swims away from the light (negative phototaxis). We are studying positive phototaxis using the tools of genetics and molecular biology. This problem can be broken down into three major areas of study: How does the cell perceive the direction and intensity of the light? How does the cell transmit the information about the light to the flagella? How do the flagella respond to that information?
What is currently known about light signal reception in Chlamydomonas?
The cell organelle that is responsible for light reception is the eyespot. This structure is located at a precise place in the cell and is thought to be composed of structures in the chloroplast and structures in the portion of the plasma membrane directly above chloroplast portion of the eyespot. The plasma membrane is thought to contain the pigment responsible for light reception, rhodopsin. This is the same pigment involved in vertebrate vision. The chloroplast portion of the eyespot is made up of 2-4 layers of lipid-carotenoid granules each separated by a layer of thylakoid membrane. This is what gives the eyespot its characteristic orange appearance in the light microscope. We are interested in how the eyespot is assembled because it is assembled in a precise place in the cell after each cell division and because it is assembled in two different cellular compartments: the chloroplast and the plasma membrane. We are taking a genetic approach to the problem. In the fall of 1991, we assembled a collection of 170 mutants that failed to swim toward light. Each of those mutants was examined by light microscopy to look for potential eyespot assembly mutants. We found 4 mutants with reduced (mini) eyespots, 5 mutants with multiple eyespots, and 16 mutants with no eyespots. We've done the basic genetics on these mutants and we know that all the mini-eyespot mutants are in a single complementation group and all the multi-eye mutants are in a second complementation group. The eyeless mutants fall into two complementation groups. One of these complementation groups is represented by a single mutant. The mini- and multi-eye mutants are located on the same linkage group (chromosome) as is the eyeless mutant with a single representative. The other eyeless complementation group is located on linkage group #10 and, until we got around to doing the genetics, we had assumed that our mutants were identical with the previously identified gene ey-1 (which also has an eyeless phenotype). Our new eyeless mutant complements ey-1 and its alleles ey-550 and ey-627. We're still trying to figure that one out because an ey-1 mutant crossed with our eyeless strains failed to produce wild type recombinants.
Where is the project headed? What could you be a part of if this interests you?
- We are always in the process of making mutants using insertional mutagenesis so that we can actually clone and sequence the genes that are mutated, something that was extremely difficult with the mutants made using UV. We have yet to isolate an eyeless mutant for any of the known genes by insertional mutagenesis, a surprising result, given the fact that eyeless mutants were 2/3 of the UV-produced eyespot mutants. If you would like to begin the project by isolating your own mutants and carrying through to sequencing, you could do that.
- Clearly assembly of the eyespot involves interactions among polypeptides. What proteins do the polypeptides we've mutated interact with? That question can be studied by isolating suppressor mutants of the eyespot mutants. Extra-genic suppressor mutants should be located in the gene or genes of interacting polypeptides. As we clone and sequence some of the eyespot genes, this can also be approached using a two-hybrid assay, a way of fishing interacting proteins out of a pool. Once we have sequences available for genes, this can also be done using a two-hybrid approach to look for direct interaction between proteins.
- In earlier versions of this document, I suggested a hunt for mutations in the chloroplast DNA that affect phototaxis and eyespot formation. Leeann Mirous used 5-fluorodeoxyuridine, a mutagen that affects both the nuclear and chloroplast genomes, to try to isolate chloroplast mutants. She got five eyespot mutants: two eyeless, one mini-eye, one multi-eye, and one we call "mixed up mess". We've done the genetics on two, the mini and multi. They are not chloroplast mutants (a disappointment, but not a complete surprise). The real surprise is that neither is a new allele of the previously identified genes for mini and multi. These are the first new genes with those phenotypes in ten years of intermittent mutagenesis. Where are these genes? What are these genes? We need to map the mutations to a particular linkage group and get the DNA sequence. This starts out as a classical genetics project and then moves to a molecular project.
- Last year, Nichole Beddes tried to get to the gene sequence for eye-1, the first eyespot mutation identified. Because there is no insertional mutant for it, we hoped to use the Chlamy genome sequence to get to the gene. Unfortunately, it took much longer to get the sequence than the sequencers expected. The sequence is now available and can be used to search for candidate genes on Linkage Group X. This is a molecular project that would involve some bioinformatics, some cloning, and a lot of hopeful transformation.
Roberts DGW, Lamb MR, and Dieckmann CL. 2001. Characterization of the EYE2 gene required for eyespot assembly in Chlamydomonas reinhardtii. Genetics 158: 1037-1049.
Lamb MR, Dutcher SK, Worley CK, and Dieckmann CL. 1999. Eyespot-assembly mutants in Chlamydomonas reinhardtii. Genetics 153: 721-9.