GENE CONVERSION 26-34 



Situations such as these could obscure Mendelian segregations 

 and lead to the incorrect conclusion that gene transformation had 

 occurred. Originally G G g g spore tetrads (where either allele 

 is highly mutable) might be finally diagnosed as G G G G, G G G g, 

 G g g g, or g g g g. 



The decisive criterion for determining whether the non-Mende- 

 lian segregations involve gene conversions or mutations during 

 growth on glucose would be, of course, the abilities of the four seg- 

 regants to ferment at the earliest stage of growth, viz., the spore 

 stage. In the absence of a test sufficiently sensitive to detect fer- 

 mentation by a single spore, other means must be employed to in- 

 vestigate the problem. 



Mundkur (1949) has investigated this problem. He reported 

 experiments in which the fermentative abilities of tetrads derived 

 from "converting" pedigrees were tested under conditions in which 

 their multiplication was restricted and, therefore, before muta- 

 tions could have obscured the results. 



Previous pedigrees in which irregular segregations of galactose 

 and melibiose markers were analyzed (Tables 26-1 and 26-2) were 

 derived from crosses in which culture CIA was used as one of the 

 parents. CIA is an a mating type capable of fermenting sucrose, 

 galactose, melibiose, alpha methyl glucoside and maltose; it is an 

 adenine -independent white culture and is thiamin-, inositol-, and 

 pantothenate -independent, but is pyridoxine -dependent. An ade- 

 nine -dependent pink haplophase culture was used as a mate for CIA 

 and its pink descendants afforded an added advantage. Two crosses 

 involving CIA and one of its direct descendants (609) were analyz- 

 ed. 



After all the four (or three) spores in a'tetrad had germinated 

 to produce microcolonies ranging in population from 100,000 to 

 500,000 cells per colony, each entire microcolony was transplant- 

 ed and streamed over a small area on a glucose agar slant. This 

 procedure insures a thorough mixing of mutant fermenter sectors 

 that might have arisen during growth of the microcolony. A small 

 number of cells was then immediately scraped off the slant surface 

 and inoculated into molten 1.75% nutrient agar at 40°C. containing 

 2% galactose as the carbon source. This molten agar was poured 

 over a layer of solidified non-nutrient 3% agar in a sterile petri 

 plate. A third layer of non-nutrient agar was poured over the 

 galactose agar after the latter had solidified. This method pre- 

 vents the fermenter colonies in close proximity from being con- 

 fluent in growth and also minimizes contaminations. The same 

 procedure was employed in making melibiose pour plates. Fig, 

 26-1 describes the method diagramatically. 



The residual haplophase yeast on the slant was allowed to in- 

 cubate 24 hours and was then streaked over the entire slant sur- 



