Determination of the Frequency of Minichromosome Loss to Assess Chromosome Stability in Fission Yeast

Quantitative assessment of chromosome stability in specific genetic backgrounds or under conditions of environmental stress can be addressed by direct cytological examination of chromosome transmission errors (using live or fixed imaging); however, in many cases, this is impractical, particularly when the rate of loss is low. Model chromosomes that allow simple and convenient assessment of chromosome stability are therefore useful. Ch16 is a 530-kb minichromosome constructed by the deletion of large portions of chromosome 3 termini. Ch16 carries the ade6-M216 allele, which interallelically complements the ade6-M210 mutation. Hence, Ade+ is an indication of the presence of Ch16, and Ade- indicates its loss. Ade+ and Ade- are phenotypically discernible as white and red colonies, respectively, on media containing limiting amounts of adenine. When a single cell bearing Ch16 divides on a plate to give rise to two daughter cells, one of which has lost Ch16, it will result in the formation of a half-sectored colony (half of the colony is red and the other half is white). The frequency of half-sectored colonies provides an accurate estimate of mitotic minichromosome loss per cell division. This protocol describes a method to determine half-sectored colony frequency and potential problems associated with the method.