Molecular characterization of Saccharomyces cerevisiae URA6 gene. DNA sequence, mutagenesis analysis, and cell cycle regulation relevant to its suppression mechanism to cdc8 mutation.
Yeast SOC8 DNA fragment was isolated as a wild type dominant suppressor of cdc8 mutation. We have used Bal31 deletion analysis to define the minimal 1 kilobase HpaI-NcoI DNA element required for complementing the cdc8 mutation. The complementing sequence harbored a multicopy plasmid also enhanced by uridine monophosphate kinase in crude extracts. DNA sequence analysis revealed an open reading frame encoding a protein with a molecular weight of 24,949. Since our SOC8 sequence was identical to that of URA6 gene, which encodes uridine monophosphate kinase, we conclude that SOC8 is allelic with URA6, and we use the term URA6 hereafter. Northern blotting experiments showed that the size of mRNA is about 0.9 kibobases. Primer extension experiments showed multiple transcriptional starting sites primarily located at -160 The size and the deduced amino acid composition are consistent with information obtained from purified uridine monophosphate kinase. Thus, both molecular genetic and biochemical evidence supports a notion that the URA6 is SOC8 encoding a yeast uridine monophosphate kinase. Mutagenesis analysis of its putative nucleotide-binding site, altering essential lysine to glutamic acid, resulted in loss of its uridine monophosphate kinase activity. Complementation analysis studies indicated that the mutated ura6 gene abolished its ability to complement ura6 mutant cells; nor could it suppress cdc8 mutation. Unlike CDC8, the mRNA level of the URA6 gene did not fluctuate throughout the cell cycle; presumably, the temporal order of these two enzymatic activities might be different during cell cycle progression. These data may explain an incomplete suppression of cdc8 by URA6, as previously observed. Taken together, the results support our previous speculation that the suppression of the cdc8 mutation mechanisms by URA6 is due to the provision of the trans-acting dTMP kinase activity to complement the cdc8 defect.
Jiang
Z R
ZR
Department of Pediatrics and Microbiology, University of Southern California School of Medicine, Los Angeles 90027.
Abaigar
L T
LT
Huang
S H
SH
Cai
B
B
Jong
A Y
AY
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D002453:Cell Cycle; D005800:Genes, Fungal; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation; D009703:Nucleoside-Phosphate Kinase / Q000378:metabolism; D010957:Plasmids; D012333:RNA, Messenger / Q000235:genetics; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013379:Substrate Specificity
D012333:RNA, Messenger; C023276:uridine monophosphate kinase; D009703:Nucleoside-Phosphate Kinase; C020093:dTMP kinase