Chapter 10 

 HYBRIDIZATION 



Winge and Laustsen (1939) hybridized yeasts by placing a hap- 

 loid ascospore from one strain in close proximity to an ascospore 

 of a second strain by use of the micromanipulator. When all con- 

 ditions are favorable, the two spores fuse to produce a diploid hy- 

 brid cell. We (1943) have used this method, but obtained relatively 

 few hybrids by ascospore to ascospore matings. The method has 

 the peculiar disadvantage that the characteristics of the parents 

 cannot be determined since the single haploid spore used as a par- 

 ent is consumed in the mating process. This is particularly im- 

 portant in genetical analysis. 



We developed a new procedure based on the fact that single as- 

 cospores from Saccharomyces cerevisiae often produce persistently 

 haploid cultures. It is possible to hybridize these with other simi- 

 larly derived haploid cultures simply by mixing the cells together 

 in an appropriate medium. These mixtures result in copulation if 

 each culture is paired with a complementary type. One parent 

 culture can be mated to a large number of other clones. The par- 

 ents can be classified as to biochemical and other characteristics 

 previous to the matings and the progeny can be classified subse- 

 quently. 



We make hybrids between clones of different mating types by 

 mixing the haploid cells from the different cultures together en 

 masse and after the diploid cells have been produced and have sporu- 

 lated, we select 4-spored asci from the mixture, dissect them and 

 grow each haploid ascospore separately in pure culture. This tech- 

 nique has been condemned by Winge and Roberts because they hold 

 that illegitimate hybridizations occur too frequently to make the 

 method reliable. We have done two things which validate our tech- 

 nique. 



(1) Our stocks have been selected for types which practically 

 never produce asci from single ascospore cultures. 



(2) Our hybrids are currently heterozygous for from 9 to 12 

 alleles and the detection of illegitimacy in such well marked matings 

 is direct and unambiguous. Among more than a thousand tetrads 



so marked, we have not found any illegitimate ones. 



The special advantage of this method over the technique of spore - 

 to-epore matings is that both parents can be fully characterized be- 

 fore the matings are made and parents can be selected which will 

 insure that the hybrid be heterozygous for all the known loci. This 



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