Intra- and lnterspecific Complementation of Membrane-inexcitable Mutants of Paramecium

Membrane excitation was the basis for backward swimming of Paramecium facing stimulus. According to standard genetic tests, inexcitable mutants fell into three complementation groups for both Paramecium tetraurelia (pwA, pwB, and pwC) and Paramecium caudatum (cnrA, cnrB, and cnrC). Cytoplasm from a wild type transferred to a mutant through microinjection restored the excitability. Transfusions between genetically defined complementation groups of the same species effected curing, whereas transfusions between different mutants (alleles) of the same group or between sister cells of the same mutant clone did not. Cytoplasmic transfers of all combinations among the six groups of mutants of the two species showed that any cytoplasm, except those from the same group, was able to cure. Since the pawns and the caudatum nonreversals complement one another through transfusion, they appeared to belong to six different complementation groups. The extent of curing, the amount of transfer needed to cure, and the time course of curing were characteristic of the group that received the transfusion. Variations in these parameters further suggested that the six groups represented six different genes. Because the donor cytoplasms from either species.were equally effective quantitatively in curing a given mutant, the curing factors were not species specific. These factors are discussed. Ciliated protozoa such as Paramecium perform avoiding reactions when stimulated by chemicals, heat, or touch at the anterior (12). An avoiding reaction consists of transient backward swimming that results from the reversal of ciliary-beat direction. Ciliary reversal is due to an increase in intraciliary Ca ++ , which flows in through the opened calcium channel (21). The voltage-dependent opening of the calcium channel, the essence of membrane excitation, has been investigated in great detail recently using voltage clamps (5, 6, 19). Some ciliates can be genetically manipulated (22, 31). Mutants defective in their membranes or axonemes and, therefore, in their behavior, have been isolated and characterized ( 16, 17, 34). Members of one class of behavioral mutants have no avoiding reactions although they are perfectly motile. Such mutants are called pawns (after the chess piece) in Paramecium tetraurelia (14, 15), caudatum nonreversal (CNR) ~ in Paramecium caudatum (32, 34), and tetrahymena nonreversal in Tetrahymena pyriformis (33). Extensive electrophysiological studies show that these mutants fail in generating the calcium-action potential (18, 33, 34). Upon an abrupt depo~Abbreviations used in this paper. CNR, caudatum nonreversal. larization with a voltage clamp, the Ca ++ inward current in the wild type, which peaks within several ms, is greatly reduced or absent in pawns (24, 25, 28). Complementation in F~ and phenotypic segregation in F2 have led to the conclusion that the more than 200 lines of pawns (representing more than 100 independent mutations) belong to pwA, pwB, and pwC (4, 15, 16), and the seven lines of CNR belong to cnrA, cnrB, and cnrC (32). Diffusible substances in the cytoplasm of the wild type can restore excitability in the mutants. This can be achieved naturally through the cytoplasmic bridge during conjugation (l) or artificially through microinjection (7-9, I 1). Transferring the cytoplasm of a live wild-type donor or various fractions of a wild-type homogenate to a mutant recipient effects the "cure." Curing occurs hours after injection, lasts 3-4 d, and corresponds to the recovery of Ca ++ current upon step depolarization under voltage clamp (7, 8). Such cytoplasmic "transfusion" among many pawns shows that cytoplasmic complementation strictly parallels the genetic complementation so that microinjection can be used to classify the pawns (8). The question of whether any pawns are genetically equivTHE JOURNAL OF CELL BIOLOGY • VOLUME 97 AUGUST 1983 378-382 378 © The Rockefeller University Press 0021-9525/83/08/0378/05 $1.00 on A ril 8, 2017 D ow nladed fom Published August 1, 1983