Frequencies of sectored colonies of various phenotypes reflecting mitotic recombination in a wild-type strain and in strains with high and low levels of DNA polymerase alpha.

Following growth of cells in medium containing glucose (AMC45), or raffinose with 0.05% or 0.005% galactose (AMC20), cells were plated on glucose-containing medium (AMC45) or raffinose with 0.05% galactose (AMC20) and allowed to form colonies. These colonies were then replica-plated to medium lacking uracil or tryptophan, or containing hygromycin. The first number in each column is the number of colonies of each sectoring class. The number in parentheses represent the rate of the event. Since we can detect only one half of the reciprocal crossover (RCO) events, the frequency of sectored colonies was doubled to estimate the rate of RCOs (see text for details). The “Total” column shows the total rate of all recombination events occurring between homologues within the rDNA. Some patterns of sectoring are unambiguous in defining the type and position of the exchange. Other patterns are ambiguous unless further tests are performed. Sectored colonies in which one sector is HygR Trp+ Ura− and the other sector is is HygS Trp− Ura+ indicate reciprocal crossovers (RCOs) between the HPH marker and CEN12 (Figure 2C). An RCO between the HPH and TRP1 markers results in Trp+ Ura− and Trp− Ura+ sectors (Figure 2A). A RCO between TRP1 and URA3 results in Ura+/Ura− sectors (Figure 2B); tetrad dissection of the Ura+ sector results in four Ura+ spores. A break-induced replication (BIR) event initiated on the URA3-containing chromatid between HPH and TRP1 results in Ura+/Ura− sectors (Figure 2D); tetrad dissection of the Ura+ sector results in 2∶2 segregation for both the TRP1 and URA3 markers, whereas dissection of the Ura− sector results in 4+∶0− segregation for the TRP1 marker. In the colonies in the “Gene conversion” class, the colony has Ura+/Ura− sectors. By tetrad dissection, the Ura+ sector yield two Ura+ and two Ura− spores. The conversion event could be local (deleting only the URA3 insertion) or represent a BIR event that occurs distal to the TRP1 insertion. A BIR event initiated by a break on the HPH-containing chromatid results in a Trp+/Trp− sectored colony; dissection of the Trp+ results in 2∶2 segregation of the TRP1 marker. The category of sister-chromatid recombination includes formation of extrachromosomal circles (Figure 2E), unequal sister-chromatid exchange (Figure 2F), and gene conversion of the TRP1 marker. We performed a physical analysis (described in Text S1) that indicated that, in six of nine colonies with Trp+/Trp− sectors in the AMY20 strain, the Trp+ cells had two copies of the TRP1 insertion, as expected from unequal sister-strand exchange.