Germ-line knockout heterokaryons of an essential alpha-tubulin gene enable high-frequency gene replacement and a test of gene transfer from somatic to germ-line nuclei in Tetrahymena thermophila.
The haploid Tetrahymena thermophila genome contains a single alpha-tubulin (ATU) gene. Using biolistic transformation, we disrupted one of the two copies of the ATU gene in the diploid germ-line micronucleus. The heterozygous germ-line transformants were made homozygous in the micronucleus by mating to a star strain containing a defective micronucleus. This mating, known as round 1 genomic exclusion, resulted in two heterokaryon clones of different mating types which have both copies of the ATU gene knocked out in the micronucleus but only wild-type genes in the polycopy somatic macronucleus. When these heterokaryons were mated, the exconjugant progeny cells did not grow because the new somatic macronuclei do not have any alpha-tubulin genes. However, when these conjugants were transformed with a functional marked ATU gene, viable transformants were obtained that contained the transforming ATU gene at the homologous locus in the new macronucleus. The exconjugant progeny could be rescued at a high efficiency (900 transformants per microg of DNA) with a wild-type ATU gene. Unlike previous macronuclear transformation protocols, this strategy should allow introduction of highly disadvantageous (but viable) mutations into Tetrahymena, providing a powerful tool for molecular and functional studies of essential genes. These knockout heterokaryons were used to demonstrate that gene transfer from somatic macronuclei to germ-line micronuclei occurs rarely if at all.
Hai
B
B
Department of Biology, University of Rochester, NY 14627, USA.
Gorovsky
M A
MA
D000818:Animals; D002459:Cell Fusion; D002467:Cell Nucleus / Q000235:genetics*; D003433:Crosses, Genetic; D018628:Gene Dosage; D018014:Gene Transfer Techniques; D005804:Genes, Lethal; D017125:Genes, Protozoan; D005822:Genetic Vectors; D018503:Genome, Protozoan; D006579:Heterozygote; D006720:Homozygote; D048631:Micronucleus, Germline / Q000235:genetics*; D009154:Mutation; D011003:Ploidies; D016808:Tetrahymena thermophila / Q000235:genetics*; D014170:Transformation, Genetic; D014404:Tubulin / Q000235:genetics*
D014404:Tubulin