The overexpression of the 3' terminal region of the CDC25 gene of Saccharomyces cerevisiae causes growth inhibition and alteration of purine nucleotides pools.
The CDC25 gene is transcribed at a very low level in S. cerevisiae cells. We have studied the effects of an overexpression of this regulatory gene by cloning either the whole CDC25 open reading frame (pIND25-2 plasmid) or its 3' terminal portion (pIND25-1 plasmid) under the control of the inducible strong GAL promoter. The strain transformed with pIND25-2 produced high levels of CDC25 specific mRNA, induced by galactose. This strain does not show any apparent alteration of growth, both in glucose and in galactose. Instead the yeast cells transformed with pIND25-1, that overexpress the 3' terminal part of CDC25 gene, grow very slowly in galactose medium, while they grow normally in glucose medium. The nucleotides were extracted from transformed cells, separated by HPLC and quantitated. The ATP/ADP and GTP/GDP ratios were almost identical in control and in pIND25-2 transformed strains growing in glucose and in galactose, while the strain that overexpresses the 3' terminal portion of CDC25 gene showed a decrease of ATP/ADP ratio and a partial depletion of the GTP pool. The disruption of RAS genes was only partially able to 'cure' this phenotype. A ras2-ts1, ras1::URA3 strain, transformed with pIND25-1 plasmid, was able to grow in galactose at 36 degrees C. These results suggest that the carboxy-terminal domain of the CDC25 protein could stimulate an highly unregulated GTPase activity in yeast cells by interacting not only with RAS gene products but also with some other yeast G-proteins.
Frascotti
G
G
Dipartimento di Fisiologia e Biochimica Generali, Università di Milano, Italy.
Coccetti
P
P
Vanoni
M A
MA
Alberghina
L
L
Martegani
E
E
D018797:Cell Cycle Proteins; D002851:Chromatography, High Pressure Liquid; D000242:Cyclic AMP / Q000032:analysis; D005656:Fungal Proteins / Q000235:genetics*; D015966:Gene Expression Regulation, Fungal; D005800:Genes, Fungal; D005809:Genes, Regulator; D011905:Genes, ras; D016366:Open Reading Frames; D010641:Phenotype; D010957:Plasmids; D011685:Purine Nucleotides / Q000378:metabolism*; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014158:Transcription, Genetic; D014170:Transformation, Genetic; D020666:ras-GRF1
D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D011685:Purine Nucleotides; D020666:ras-GRF1; D000242:Cyclic AMP