Genetics 146, 135-147. Hours indicate time after mixing of starved cells to initiate conjugation. Selection for complete gene replacement by phenotypic assortment enables the distinction between essential and non-essential genes. The initial transformant contains one transformed gene which has been disrupted with an expression cassette containing a drug-selectable marker. Its clonal progeny are serially transferred to higher drug concentrations thus killing cells with fewer copies of the selectable markers.
All arrows indicate multiple generations.
The genotype of the Mpr locus in Round I exconjugants was not determined. The single haploid nucleus in each conjugant then diploidizes and most cells separate without forming a new macronucleus. This process, referred to as Round 1 of genomic exclusion, greatly facilitates complementation testing and mutant rescue by cytoplasmic exchange during conjugation. Round 1 genomic exclusion or phenotypic assortment of heterozygotes each allow production of heterokaryons, cells with different macro- and micronuclear genotypes. Heterokaryons homozygous or heterozygous for a drug resistance gene in the transcriptionally inert micronucleus but having only drug-sensitive alleles in the macronucleus are drug sensitive.