Reactivation of the ATCase domain of the URA2 gene complex: a positive selection method for Ty insertions and chromosomal rearrangements in Saccharomyces cerevisiae.
Genetic rearrangements such as deletions or duplications of DNA sequences are rarely detected in the yeast Saccharomyces cerevisiae. We have developed a screening system using the URA2 gene coding for the bi-functional CPSase-ATCase (carbamyl phosphate synthetase - aspartate transcarbamylase) to select positively for these kinds of events. Nonsense mutations in the CPSase region cause a complete loss of the ATCase activity because of their strong polar effect. Thirty-seven ATCase+ revertants were isolated from a strain containing three nonsense mutations in the proximal CPSase region. Genetic and structural analysis of the URA2 locus in these strains allowed us to characterize two major classes of revertants. In the first, an entire copy of a Ty transposon was found to be inserted in the CPSase coding domain. This event, which represents a new form of Ty-mediated gene activation was further analysed by mapping the Ty integration site in 26 strains. In a second class of revertants, we observed chromosomal rearrangements and, in particular, duplication of the ATCase region and its integration in a new chromosomal environment in which this sequence becomes active.
Roelants
F
F
Laboratoire de Microbiologie et Génétique URA 1481, Université Louis Pasteur/CNRS, Institut de Botanique, Strasbourg, France.
Potier
S
S
Souciet
J L
JL
de Montigny
J
J
D001221:Aspartate Carbamoyltransferase / Q000235:genetics*; D001483:Base Sequence; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing) / Q000235:genetics*; D002872:Chromosome Deletion; D004251:DNA Transposable Elements; D015321:Gene Rearrangement; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics*; D009154:Mutation; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*
D004251:DNA Transposable Elements; D009097:Multienzyme Complexes; C019820:carbamoyl phosphate synthase-aspartatecarbamoyl transferase complex; D001221:Aspartate Carbamoyltransferase; D002223:Carbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)