The mutants gam-10 and gam-11 have provided the first solid evidence that adhesion between mating structures is a necessary step preceding gamete fusion.  Gametes of these temperature sensitive, mt-, sex-limited mutants are able to recognize mt+ gametes, agglutinate, signal, remove their walls and activate their mating structures.  The activated mating structures of opposite mating type cells are then able to recognize each other and adhere via their mating structures.  The adhering mating structures are, however, unable to fuse.

             The adhesive interactions between wild type gametes must be initiated by recognition between the membranes of activated mating structures of opposite mating type cells.  Fusion occurs so rapidly that early mating structure interactions (adhesion) have not been visualized in matings between wild type gametes.  These fusion-defective mutants, however, when mated with wild type, accumulate at the stage where interacting mating structures are attached, because the membranes cannot fuse.

              A scanning electron micrograph of such adhering mating structures is shown above, and a thin section transmission electron micrograph is shown below.  In the thin section, a fuzzy material, probably glycoprotein, is seen between the two mating structures.

            A number of mutants expressed only in mt+ (imp-1, imp-11, fus+ and bs-37) have been shown to be defective in mating structure adhesion (there is, as yet, no genetic evidence for mt- involvement).  The sex-limited mt- mutants gam-10, gam-11 and gam-1-II have defects in mating structure fusion (there is, as yet no evidence for mt+ involvement in gamete fusion).  Therefore, at present, it appears that mt+ gametes control mating structure recognition and adhesion, while mt- gametes control fusion (fertilization).

            Mutational blocks appear to be the best way to study individual steps in the mating reaction, since appropriate mutants can stop these intercellular interactions at intermediate stages.   In addition, the use of conditional mutants allows one to "turn on" or "turn off" mating ability at will, observe changes occurring during recovery from restrictive conditions, and study the loss of mating ability when cells are placed at the restrictive temperature. 

            We have produced additional fusion-defective mutants using insertional mutagenesis.  Mutants were produced by transforming cells with the ble plasmid, that confers resistance to the antibiotic Zeocin^TM.   We are presently producing additional mutants using the pHyg3 plasmid as the mutagen.   Insertions of the plasmid within genes required for fusion have tagged two region in the genome.  We are now attempting to complement these candidate genes using BACs (bacterial artificial chromosomes) which contain genes found near the site of the insertions.  Once we have determined which genes are mutated in these fusion-defective mutants, we will be able to analyze how they function.