Microinjection of cytoplasm as a test of complementation in Paramecium.

Abstract

Mutants in P. tetraurelia, unable to generate action potentials, were isolated as cells which show no backward swimming in response to ionic stimulation. These pawn mutants belong to at least 3 complementation groups designated pwA, pwB and pwC. Microinjection of cytoplasm from a wild-type donor into a pawn recipient of any of the 3 complementation groups restores the ability of the pawn to generate action potentials and hence swim backward. The cytoplasm from a pawn cannot restore a recipient of the same complementation group but that from a pawn of a different group can. Electrophysiological analysis demonstrated that the restoration of backward swimming is not due to a simple addition of ions but represents a profound change in the excitable membrane of the recipient pawn cells. Using known pawn mutants and those which had previously been unclassified, a perfect concordance of genetic complementation and complementation was established by cytoplasic transfer through microinjection. This method has been used to classify pawn mutants that are sterile or hard-to-mate and to examine the ability of cytoplasms from different species of ciliated protozoa to restore the ability to swim backward in the pawn mutants of P. tetraurelia. A cell homogenate was also fractionated to further purify the active components. Transfer of cytoplasm between cells by microinjection can be a valid and systematic method to classify mutants. This test is simpler to perform than the genetic complementation test and can be used under favorable conditions in mutants that are sterile and in cells of different species.