Toward cloning genes by complementation in Paramecium

Abstract

Conventional methods of gene cloning by complementing mutant defects is made difficult by the 800 ploidy of the Paramecium macronucleus. However, this nucleus is some 30 microns in diameter and readily propagates exogenous DNA fragments as cells divide. These attributes allow for massive injection of engineered DNA fragments and their maintenance in the transformed descendant. If a genomic DNA fraction injected into a mutant macronucleus effects complementation, it should be possible to sort a fractional library to isolate the complementing gene. Here, we investigated four aspects of establishing this method for general use. First, using the cloned CAM gene as a test case, we further investigated transformation by macronuclear injection and showed that phenotypic reversion is directly correlated with the copy number of the transgene, even when it is of a recessive allele, cam2, which has a missense mutation but produces a partially functional protein. Second, we examined the copy number of the transgene established in cells of older clonal 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 cam2 calmodulin protein in the cell. Fourth, we extended the investigation to very recessive mutants whose genes are to be cloned. We showed that size fractions of wild-type genomic DNA digests effect strong phenotypic reversions in several pawn mutants, setting the stage for cloning these Ca(2+)-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 assessed and discussed.