Genetic analysis of yeast strains lacking negative feedback control: a one-step method for positive selection and cloning of carbamoylphosphate synthetase-aspartate transcarbamoylase mutants unable to respond to UTP.
We have undertaken an in vivo genetic approach to the analysis of negative feedback control by uridine triphosphate (UTP) of the yeast carbamoylphosphate synthetase-aspartate transcarbamoylase multifunctional protein (CPSase-ATCase). Using an analog of uracil, 5-fluorouracil, we have constructed a screening system leading, in one step, to selection and cloning of a functional aspartate transcarbamoylase that is defective in negative feedback control by UTP. Due to the nature of the screen, spontaneous or UV-induced mutants could be recovered. Well-characterized cloned mutants have been sequenced and reveal one or two modifications in single codons leading to single amino acid replacements. These amino acid changes occurred either in the CPSase or ATCase domains, abolishing their sensitivity to regulation but not their catalytic activities. Hence the regulatory and catalytic sites are distinct. With the same screening system, it may also be possible to enlarge the scope of the molecular study of the feedback processes to include equivalent proteins in fungi as well as higher eukaryotes.
Jaquet
L
L
Laboratoire de Microbiologie et Génétique, URA-GEM 1481, Université Louis Pasteur/CNRS, Institut de Biologie Moléculaire et Cellulaire, Strasbourg, France.
Lollier
M
M
Souciet
J L
JL
Potier
S
S
D000494:Allosteric Regulation; D000596:Amino Acids / Q000235:genetics; D001221:Aspartate Carbamoyltransferase / Q000037:antagonists & inhibitors* / Q000235:genetics; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000037:antagonists & inhibitors* / Q000235:genetics; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D004795:Enzyme Stability; D005246:Feedback; D005472:Fluorouracil / Q000494:pharmacology; D005799:Genes, Dominant; D006358:Hot Temperature; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors* / Q000235:genetics; D009154:Mutation; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D014544:Uridine Triphosphate / Q000378:metabolism*
D000596:Amino Acids; D009097:Multienzyme Complexes; C019820:carbamoyl phosphate synthase-aspartatecarbamoyl transferase complex; D001221:Aspartate Carbamoyltransferase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing); D005472:Fluorouracil; D014544:Uridine Triphosphate