Nullisomic Tetrahymena. II. a Set of Nullisomics Define the Germinal Chromosomes.

Crosses of a diploid Tetrahymena thermophila to a strain with a haploid germinal nucleus result in chromosome loss during meiosis in the haploid. The resulting monosomics can be made nullisomic by a special cross that induces homozygosis of a meiotic product of the germinal nucleus, but retention of the parental somatic nucleus. The creation and testing of single nullisomics for three of the five chromosome pairs and a triple nullisomic missing another pair is presented. Taken together, these strains make possible a series of crosses in which all but one of the chromosomes is missing in one parent. This set of nullisomics can, therefore, be used to map any mutation in Tetrahymena to a specific chromosome.

Induced resistance to 6-methylpurine and cycloheximide in tetrahymena. I. Germ line mutants of T. thermophila.

Two new mutant lines of Tetrahymena thermophila (T. pyriformis, syngen 1), each conferring resistance to a different agent, are described. Resistance to cycloheximide and 6-methylpurine are each determined by dominant genes, ChxA2 and Mpr; the traits show phenotypic assortment. The method used to select these mutations, the critical importance of backcrossing to wild type following mutagenesis, and the utility of these marker genes in further mutagenic selection schemes and studies of the sexual cycle of Tetrahymena are noted.

Isolation of homozygous mutants after induced self-fertilization in Tetrahymena.

Genomic exclusion, an unusual cytogenetic sequence during mating in Tetrahymena pyriformis, results in the production of homozygous germinal nuclei by the diploidization of haploid nuclei following meiosis. A method is presented for selecting cells that have made new somatic nuclei from these homozygous germinal nuclei, a step necessary for phenotypic expression of new mutations; this variation of the normal set of events is termed short-circuit genomic exclusion. The utility of thisapproach for obtaining induced mutations is demonstrated by the isolation and analysis of a strain homozygous for a recessive mutation conferring resistance to 2-fluoroadenosine. Occurring in about 5% of the unmutagenized pairs in specific crosses, short-circuit genomic exclusion should be of general use for the isolation of dominant or recessive induced mutations in this protozoan.

Positive selection for mating with functional heterokaryons in Tetrahymena pyriformis.

Allelic assortment of Tetrahymena pyriformis makes possible the isolation of cells which contain both a heterozygous germ line nucleus and a somatic nucleus which expresses phenotypes of only one member of allelic pairs. Cycloheximide-sensitive segregants have been isolated from the vegetative progeny of cells heterozygous for a dominant mutation conferring resistance to cycloheximide; such segregants are called functional heterokaryons. Since progeny from crosses of these cells are cycloheximide-resistant, addition of the drug provides positive selection for successful exconjugants. The timing for expression of the new phenotype during conjugation is presented and used to identify what appear to be immature progeny of round one mating in genomic exclusion. Usefulness of functional heterokaryons in a number of genetic and developmental studies is discussed.