A new technique is described for selection of temperature-sensitive mutants affecting gametogenesis in Chiamydomonas reinhardi. The first mutant found by this technique is characterized. Cells exhibiting the gaim-phenotype are capable of sexual agglutination, but cannot form zygotes at the restrictive temperature. The mutation, however,
The temperature-sensitive gametogenesis-defective mutant, gain-1 is sex-limited, expressed only in mating type minus (rot-), and can sexually agglutinate but not fuse at the restrictive temperature (35~ with gametes of wild type (wt) mt +.Thin-section, freeze-cleave, and scanning electron microscopy reveal that the gain-1 phenotype is dependent on both the temperature at which the cells undergo nitrogen starvation (and therefore gamete formation) and the temperature at which the cells are maintained during the 12 h before mating. Under all conditions of gametogenesis at 35~ each gam-1 cell produces a normal-appearing membrane-associated mating structure that fails to activate in response to flagellar agglutination. Varying with the conditions of gametogenesis, on the other hand, are the agglutination and signaling properties of the gain-1 flagella. The two mutant phenotypes displayed by gain-1 have been denoted gam-l-I and gam-l-II. An agglutination reaction involving gam-I-I cells does not result in activation of the wt mt § mating structure. A more stable agglutination reaction, which can result in activation of the wt mt+ mating structure, is characteristic of gam-l-II cells, but because the gam-1 rot-mating structure still fails to activate, cell fusion is precluded. We conclude that the gain-1 mutation affects flagellar component(s) involved in establishing an effective, signal-generating agglutination reaction.KEY WORDS mutant temperature sensitivity flagellar membrane sexual agglutination signaling mating structureThe sexual cycle of the single-celled eukaryoteChlamydomonas reinhardi represents an easily controlled developmental process. Gametogenesis is initiated when the haploid cells are starved for nitrogen (11). Both mating types then develop the ability to agglutinate by their flagellar tips (6, 11), a reaction that occurs immediately when gametes of opposite mating types are mixed. During gamete formation, mating-type-specific mating structures also develop in association with the plasma membrane (2,6,9,14,17). These mating structures remain unactivated unless stimulated by some sort of sexual agglutination. The activated structure of mating-type plus (mt +) is a long, 74 J. CELL BIOLOGY ~ The Rockefeller University Press
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