Cloning by functional complementation of a mouse cDNA encoding a homologue of CDC25, a Saccharomyces cerevisiae RAS activator.
In the yeast Saccharomyces cerevisiae genetic and biochemical evidence indicates that the product of the CDC25 gene activates the RAS/adenylyl cyclase/protein kinase A pathway by acting as a guanine nucleotide protein. Here we report the isolation of a mouse brain cDNA homologous to CDC25. The mouse cDNA, called CDC25Mm, complements specifically point mutations and deletion/disruptions of the CDC25 gene. In addition, it restores the cAMP levels and CDC25-dependent glucose-induced cAMP signalling in a yeast strain bearing a disruption of the CDC25 gene. The CDC25Mm-encoded protein is 34% identical with the catalytic carboxy terminal part of the CDC25 protein and shares significant homology with other proteins belonging to the same family. The protein encoded by CDC25Mm, prepared as a glutathione S-transferase fusion in Escherichia coli cells, activates adenylyl cyclase in yeast membranes in a RAS2-dependent manner. Northern blot analysis of mouse brain poly(A)+ RNA reveals two major transcripts of approximately 1700 and 5200 nucleotides. Transcripts were found also in mouse heart and at a lower level in liver and spleen.
Martegani
E
E
Dipartimento di Fisiologia e Biochimica Generali, Università degli Studi di Milano, Italy.
Vanoni
M
M
Zippel
R
R
Coccetti
P
P
Brambilla
R
R
Ferrari
C
C
Sturani
E
E
Alberghina
L
L
D000262:Adenylyl Cyclases / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D018797:Cell Cycle Proteins; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D004247:DNA / Q000235:genetics*; D004271:DNA, Fungal / Q000235:genetics; D004789:Enzyme Activation; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000235:genetics; D015966:Gene Expression Regulation, Fungal; D005816:Genetic Complementation Test; D005838:Genotype; D005947:Glucose / Q000494:pharmacology; D005982:Glutathione Transferase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010957:Plasmids; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D012689:Sequence Homology, Nucleic Acid; D018631:ras Proteins; D020666:ras-GRF1
C542351:CDC25 protein, S cerevisiae; D018797:Cell Cycle Proteins; D004271:DNA, Fungal; D005656:Fungal Proteins; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D020666:ras-GRF1; D011061:Poly A; D012313:RNA; D004247:DNA; D000242:Cyclic AMP; D005982:Glutathione Transferase; D019204:GTP-Binding Proteins; C489015:RAS2 protein, S cerevisiae; D018631:ras Proteins; D000262:Adenylyl Cyclases; D005947:Glucose