TOWARD CLONING GENES BY COMPLEMENTATION IN PARAMECIUM

Conventional methods of gene cloning by complementing mutant defects i s made difficult by the 800 ploidy of the Paramecium macronucleus. How ever, this nucleus is some 30 mu m in diameter and readily propagates exogenous DNA fragments as cells divide. These attributes allow for ma ssive injection of engineered DNA fragments and their maintenance in t he transformed descendant. If a genomic DNA fraction injected into a m utant macronucleus effects complementation, it should be possible to s ort a fractional library to isolate the complementing gene. Here, we i nvestigated four aspects of establishing this method for general use. First, using the cloned CAA gene as a test case, we further investigat ed transformation by macronuclear injection and showed that phenotypic reversion is directly correlated with the copy number of the transgen e, even when it is of a recessive allele, cam(2), which has a missense mutation but produces a partially functional protein. Second, we exam ined the copy number of the transgene established in cells of older cl onal age and discussed the likely dilution of the transgene in younger descendants of the injected cell. Third, we showed that the degree of phenotypic reversion is correlated with the transgene product, the ca m(2) calmodulin protein in the cell. Fourth, we extended the investiga tion to very recessive mutants whose genes are to be cloned. We showed that size fractions of wild-type genomic DNA digests effect strong ph enotypic reversions in several pawn mutants, setting the stage for clo ning these Ca2+-channel related genes. The general usefulness of this method in cloning genes that complement recessive alleles and current limitations of this method in dealing with dominant alleles are assess ed and discussed.