The main area of interest
in the lab is to isolate the gene controlling sexual fusion. To accomplish this, we have isolated new fusion-defective mutants using insertional mutagenesis. Cells
have been transformed with the ble or pHyg3 plasmid and new fusion-defective mutants have been selected. We have produced 6 fusion-defective
insertional mutants and there are several students in the lab who are selecting additional mutants. One of the goals
of the lab is to do saturation mutagenesis, to determine the total number of genes that are required for sexual cell fusion
in this organism.
We have probed Southern blots of several
of these insertional mutants; all but one of the mutants appears to have a single insertion of the plasmid. We have used TAIL-PCR to clone the DNA flanking the inserted plasmid in
two of the mutants. The DNA isolated was sequenced and blasted at the Chlamydomonas genome site.
Two different genomic regions were identified.
Analysis
of this DNA (including complementation analysis with BACs) is now in progress.
We are
also using bioinformatics to begin to analyze the proteins identified by the insertions. The two putative membrane proteins
identified appear to have interesting properties suggesting roles in the fusion process.
Light and electron micrographic
analysis of these proteins are also in progress.
Confirmation
of the genes/proteins causing the fusion-defective phenotype will lead to a number of important new areas of investigation:
-
We will compare the sequences of wild type and mutant versions of the genes to locate the regions of the protein necessary
for its function.
-
We will tag the genes with green fluorescent protein to follow the timing of synthesis of the protein they code for and
determine the localization of the protein on gametes and zygotes. This should
allow us to understand how fusion proteins control the final stage in fertilization in this organism and perhaps help others
to understand the interactions that occur between sperm and eggs.
- We will also run
RNAi experiments to determine the effects of gene knock-down. We will study both the genes we have identified by the
mutagenesis experiments and other genes we identify using bioinformatics.
Student projects have included mutagenesis and isolation
of new mating mutants and light and scanning electron microscope studies of mutants.
We have studied the mechanism of action of reagents known to effect signaling, adhesion or fusion in other systems
to determine if mating is affected, and if so, at what stage. Reagents used include
calcium channel blockers, various protease inhibitors, plant hormones, progesterone, caffeine and gadolinium, which blocks
stretch-activated channels in the plasma membrane.
Studends
have also studied specialized membrane domains using chemicals that interact with different components of the plasma membrane,
microscopic analysis of the carbohydrates and proteins on the surface of cells, and attempts to force the fusion of fusion
defective mutants using polyethylene glycol and electrofusion.
To study the proteins and glycoproteins involved
in the interactions between opposite mating type gametes, students have labeled the amino acids or the carbohydrates on plasma
membrane (glyco)proteins with biotin. These biotinylated proteins have then been
analyzed using electrophoresis and western (protein) blotting. We are also using
surfactants (detergents) to understand how the Chlamydomonas fusion protein functions.
