The Isolation and Characterization of Saccharomyces cerevisiae Mutants That Constitutively Express Purine Biosynthetic Genes

Abstract

In response to an external source of adenine, yeast cells repress the expression of purine biosynthesis pathway genes. To identify necessary components of this signalling mechanism, we have isolated mutants that are constitutively active for expression. These mutants were named bra (for bypass of repression by adenine). BRA7 is allelic to FCY2, the gene encoding the purine cytosine permease and BRA9 is ADE12, the gene encoding adenylosuccinate synthetase. BRA6 and BRA1 are new genes encoding, respectively, hypoxanthine guanine phosphoribosyl transferase and adenylosuccinate lyase. These results indicate that uptake and salvage of adenine are important steps in regulating expression of purine biosynthetic genes. We have also shown that two other salvage enzymes, adenine phosphoribosyl transferase and adenine deaminase, are involved in activating the pathway. Finally, using mutant strains affected in AMP kinase or ribonucleotide reductase activities, we have shown that AMP needs to be phosphorylated to ADP to exert its regulatory role while reduction of ADP into dADP by ribonucleotide reductase is not required for adenine repression. Together these data suggest that ADP or a derivative of ADP is the effector molecule in the signal transduction pathway.

CTP sensing and Mec1ATR-Rad53CHK1/CHK2 mediate a two-layered response to inhibition of glutamine metabolism

“…Hence, while the relative growth-inhibiting effect of DON is weaker in fui1Δ mutants, the fui1Δ mutation does not confer an absolute growth advantage on DON. The genes deleted in the strongly sensitive strains were linked to glutamine-related processes: URA8 encodes one of two CTP synthases (human: CTPS1 , CTPS2 ), which catalyze the glutamine-dependent conversion of UTP to CTP and are directly and irreversibly inhibited by DON [ 27 ]; HPT1 (human: HPRT1 ), encodes the hypoxanthine-guanine phosphoribosyltransferase which facilitates purine salvage, a parallel pathway to the DON-sensitive purine de novo synthesis [ 28 ]; RTG1 encodes a TOR-inhibited transcription factor which is activated by glutamine starvation and promotes the expression of glutamine synthesis genes [ 29 ]. The weaker chemogenomic DON interactors, Rhb1 (a putative Rheb-like GTPase involved in trans-membrane transport), Par32 (an adapter protein that links TORC1 activity to amino acid transporter localization), and Fui1 (a uridine permease), control uptake processes and may affect DON or glutamine uptake.…”

Enriching for direct regulatory targets in perturbed gene-expression profiles

“…This gene set is believed to be controlled in part by the transcriptional repressors Rox1 and Mot3, which act by recruiting the co-repressor complex Ssn6/Tup1 [ 21 - 23 ]; this may explain the participation of TUP1 in the component. ADE2 and HPT1 are both involved in purine biosynthesis, and deletion of HPT1 is known to activate ADE2 transcription via a feedback pathway dependent on an adenine metabolite [ 24 , 25 ].…”

Yeast Double Transporter Gene Deletion Library for Identification of Xenobiotic Carriers in Low or High Throughput

“…We selected commercial xenobiotics, including agrochemicals and drugs for both human and animal use, and prepared 10 mM stock solutions (20 mM for artesunate; 40 mM for dl -4-hydroxy-3-methoxymandelic acid and tamoxifen) in 100% DMSO of the compounds purchased from Sigma-Aldrich (Merck Group) (see Table S1 in the supplemental material). We selected 5-fluorocytosine (catalog number F7129; Sigma-Aldrich, Merck Group) as a positive control for the assays, as the deletion of FCY2 (YER056C; encoding the purine-cytosine permease) is well characterized and provides a resistant phenotype to this compound ( 8 , 52 ).…”