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

Overview

Journal Mol Gen Genet

Specialties Genetics
Molecular Biology

Date 1993 Oct 1

PMID 8232215

Citations 4

Authors

L Jaquet

M Lollier

J L Souciet

S Potier

Affiliations

Soon will be listed here.

Abstract

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.

Citing Articles

Large-Scale Survey of Intraspecific Fitness and Cell Morphology Variation in a Protoploid Yeast Species.

Jung P, Sigwalt A, Ohnuki S, de Montigny J, Ohya Y, Schacherer J G3 (Bethesda). 2016; 6(4):1063-71.

PMID: 26888866 PMC: 4825641. DOI: 10.1534/g3.115.026682.

Integrated allosteric regulation in the S. cerevisiae carbamylphosphate synthetase - aspartate transcarbamylase multifunctional protein.

Serre V, Penverne B, Souciet J, Potier S, Guy H, Evans D BMC Biochem. 2004; 5:6.

PMID: 15128434 PMC: 434488. DOI: 10.1186/1471-2091-5-6.

Uracil-induced down-regulation of the yeast uracil permease.

Seron K, Blondel M, Haguenauer-Tsapis R, Volland C J Bacteriol. 1999; 181(6):1793-800.

PMID: 10074071 PMC: 93577. DOI: 10.1128/JB.181.6.1793-1800.1999.

As in Saccharomyces cerevisiae, aspartate transcarbamoylase is assembled on a multifunctional protein including a dihydroorotase-like cryptic domain in Schizosaccharomyces pombe.

Lollier M, Jaquet L, Nedeva T, Lacroute F, Potier S, Souciet J Curr Genet. 1995; 28(2):138-49.

PMID: 8590465 DOI: 10.1007/BF00315780.

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