Selection of functional cDNAs by complementation in yeast.
Yeast cDNA was prepared in a yeast expression plasmid to generate a cDNA plasmid pool composed of approximately 40,000 members. Several yeast mutants were transformed with the cDNA plasmid pool, and the cDNAs for ADC1, HIS3, URA3, and ASP5 were isolated by functional complementation. Restriction enzyme analysis confirmed the genetic identity of the ADC1, HIS3, and URA3 cDNAs and demonstrated that the URA3 cDNA contains 5' noncoding sequences. The relative abundance of the various cDNAs in the cDNA plasmid pool paralleled the abundance of the mRNAs in total poly(A)+ RNA, which ranged from approximately 0.01% to 1%. The utility of this approach to isolate rare cDNAs from higher eukaryotes is discussed.
D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis*; D004262:DNA Restriction Enzymes; D004251:DNA Transposable Elements; D004926:Escherichia coli / Q000235:genetics; D005816:Genetic Complementation Test; D010957:Plasmids; D011061:Poly A / Q000235:genetics; D012313:RNA / Q000235:genetics; D012333:RNA, Messenger; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D004251:DNA Transposable Elements; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D004247:DNA; D004262:DNA Restriction Enzymes