CDC14 of Saccharomyces cerevisiae. Cloning, sequence analysis, and transcription during the cell cycle.

J Wan,H Xu,M Grunstein

doi:10.1016/s0021-9258(19)49907-9

J Wan, H Xu + Show 1 more

Open Access

https://doi.org/10.1016/s0021-9258(19)49907-9

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Abstract

We have cloned, mapped and sequenced the complete CDC14 gene of Saccharomyces cerevisiae and characterized its transcription during the cell cycle. CDC14 was found within a 3.5-kilobase pair XhoI-XbaI fragment of chromosome VI. The DNA sequence reveals an open reading frame capable of encoding a 423-amino acid polypeptide. Protein sequence comparisons through the Prosite, GenBank and EMBL databases allowed us to identify a conserved protein tyrosine phosphatase active site in the encoded CDC14 protein beginning at amino acid 153. Disruption demonstrates that CDC14 is an essential gene. The level of the CDC14 transcript appears to be weakly cell cycle-regulated and has a periodicity which lags approximately 15 min behind histone HTB1 mRNA accumulation levels. DNA sequence analysis has identified a region within the CDC14 promoter which bears a striking resemblance (15 out of 21 base pairs identity) to the cell cycle regulation region of the promoter of the histone H2A1-H2B1 (HTA1-HTB1) gene pair. The cell cycle regulation sequence is responsible for the periodic accumulation and hydroxyurea sensitivity of the histone HTA1-HTB1 message. However, unlike histone mRNA, which is repressed upon hydroxyurea arrest, CDC14 mRNA appears to be unaffected. This suggests that CDC14 and histone genes are regulated by different mechanisms during the cell cycle. Furthermore, superhelical density measurements suggest that CDC14 is not involved in nucleosome assembly.

Highlights

CDC14 gene of Saccharomyces cerevisiae and char- cdcl4t”strains aregrown at themaximum permissive temperacterizedits transcription during tcheell cycle
CDCl4 ature, and it has been suggested that CDC14 functions in was found within a 3.5-kilobase pair XhoI-XbaI frag- some aspect of DNA metabolism
(Sharon and Simchen1,990a, 199Ob).To shed light on CDC14 function, we have cloned and sequenced the gene, mapped its transcript, shown that it is essential by a disruption, and examined CDCl4 mRNA synthesis duringthe cell cycle

Summary

THEJOURNAOLF BIOLOGICACHLEMISTRY

8 1992 by The American Society for Biochemistry and Molecular Biology, Inc. Vol 267, No 16. CDCl4 ature, and it has been suggested that CDC14 functions in was found within a 3.5-kilobase pair XhoI-XbaI frag- some aspect of DNA metabolism (Sharon and Simchen1,990a, 199Ob).To shed light on CDC14 function, we have cloned and sequenced the gene, mapped its transcript, shown that it is essential by a disruption, and examined CDCl4 mRNA synthesis duringthe cell cycle. Arrest, CDCl4 mRNA appears to be unaffected This l(ts), u r d , his, adel, ade, tyrl, lys, gal. YC29a: a, cdcll-l(ts), suggests that CDCl4 and histone genes are regulated urd-52, his, gal- (product of a cross between cdc14(7-41) and by different mechanisms during the cell cycle. Nested Deletions and Sequencing-The plasmids pJWc2OO and pJWc300 were used to sequence the 3.5-kb XhoI-XbaI fragment from the 5’ to 3’ direction relative to CDCl4 ORF.

RESULTS

Characterizianngd Cloning

DISCUSSION