Saccharomyces cerevisiae cdc15 mutants arrested at a late stage in anaphase are rescued by Xenopus cDNAs encoding N-ras or a protein with beta-transducin repeats.
We have constructed a Xenopus oocyte cDNA library in a Saccharomyces cerevisiae expression vector and used this library to isolate genes that can function in yeast cells to suppress the temperature sensitive [corrected] defect of the cdc15 mutation. Two maternally expressed Xenopus cDNAs which fulfill these conditions have been isolated. One of these clones encodes Xenopus N-ras. In contrast to the yeast RAS genes, Xenopus N-ras rescues the cdc15 mutation. Moreover, overexpression of Xenopus N-ras in S. cerevisiae does not activate the RAS-cyclic AMP (cAMP) pathway; rather, it results in decreased levels of intracellular cAMP in both mutant cdc15 and wild-type cells. Furthermore, we show that lowering cAMP levels is sufficient to allow cells with a nonfunctional Cdc15 protein to complete the mitotic cycle. These results suggest that a key step of the cell cycle is dependent upon a phosphorylation event catalyzed by cAMP-dependent protein kinase. The second clone, beta TrCP (beta-transducin repeat-containing protein), encodes a protein of 518 amino acids that shows significant homology to the beta subunits of G proteins in its C-terminal half. In this region, beta Trcp is composed of seven beta-transducin repeats. beta TrCP is not a functional homolog of S. cerevisiae CDC20, a cell cycle gene that also contains beta-transducin repeats and suppresses the cdc15 mutation.
Ernst Boehringer Institute, Vienna, Austria.
W
W
Spevak
B D
BD
Keiper
C
C
Stratowa
M J
MJ
Castañón
D000705:Anaphase; D000818:Animals; D001483:Base Sequence; D002453:Cell Cycle; D018797:Cell Cycle Proteins; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D004247:DNA / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics; D019204:GTP-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D015870:Gene Expression; D005796:Genes; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D009841:Oligonucleotides / Q000737:chemistry; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000235:genetics; D016415:Sequence Alignment; D015293:Transducin / Q000378:metabolism*; D029867:Xenopus Proteins; D014982:Xenopus laevis / Q000235:genetics; D044844:beta-Transducin Repeat-Containing Proteins
C471646:BTRC protein, Xenopus; C082279:CDC15 protein; D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D009841:Oligonucleotides; D012333:RNA, Messenger; D029867:Xenopus Proteins; D044844:beta-Transducin Repeat-Containing Proteins; D004247:DNA; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; D015293:Transducin; D016283:Proto-Oncogene Proteins p21(ras)