A GENETIC SCREEN TO ISOLATE GENES REGULATED BY THE YEAST CCAAT-BOX BINDING-PROTEIN HAP2P

We have developed a screening method to isolate yeast genes regulated by a specific transcription activator. The screen is based on the use of expression libraries in which the lacZ reporter gene is placed unde r control of yeast regulatory elements. Two partially representative l ibraries, constructed by different methods, were used to isolate genes regulated by the yeast CCAAT-box binding protein Hap2p. Among 26 fusi ons shown to be regulated by Hap2p only CYT1 was known to be regulated by this activator. Sequence analysis revealed that most of the remain ing regulated fusions are in new yeast genes, while some are in previo usly characterized yeast genes (PTP1, RPM2, SDH1). Optimal expression of these three genes also requires Hap3p and Hap4p and is regulated by carbon source. Hap2p was known to regulate expression of genes involv ed in Krebs cycle, electron transport and heme biosynthesis. Our resul ts suggest that Hap2p could play a more general role by regulating oth er mitochondrial processes such as protein import and phosphate transp ort (PTP1) or maturation of mitochondrial tRNAs (RPrM2). Among the rem aining regulated fusions, two of them correspond to open reading frame s (ORFs) on chromosomes III and XI whose nucleotide sequences have bee n entirely determined. The use of this approach to functionally analys e ORFs of unknown function is discussed.