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The molecular analysis of two high copy number suppressors of a yeast cell cycle protein kinase mutant

Donovan, Joseph; (1994) The molecular analysis of two high copy number suppressors of a yeast cell cycle protein kinase mutant. Doctoral thesis (Ph.D), UCL (University College London). Green open access

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Abstract

The S. cerevisiae protein kinase Dbf2 carries out an essential function in late mitosis, and its kinase activity is cell cycle regulated around anaphase/telophase. Originally it was identified as a temperature-sensitive cell cycle mutation, dbf2-ts, that is defective for S phase, the dbf2 mutation causing a 40-minute delay in the initiation of DNA synthesis. However, subsequent analysis showed that the dbf2 execution point is later in the cell cycle during nuclear division. Consistent with a late mitotic role for Dbf2, dbf2 cells accumulate as large, swollen "dumbbells" with divided chromatin and fully extended spindles, when incubated at the restrictive temperature. To further explore the physiological role of Dbf2, a S. cerevisiae high copy genomic library was screened for sequences that could rescue the dbf2 temperature- sensitive phenotype. Five different suppressor genes were isolated, that were named pSDB21-25. Both pSDB24 and 25 are allele-specific, rescuing only one of the three dbf2 alleles. The characterization of these two molecular suppressors forms the basis of the work described in this report. SDB25 was sequenced and found to encode p40, a previously characterized substrate and inhibitor of Cdc28 kinase activity. Antibodies were raised against Sdb25 and used to show that Sdb25 is a phosphoprotein, and that Sdb25 has an associated kinase activity that is CDC28-dependent. Remarkably, Sdb25 transcript levels, protein levels and associated kinase activity are precisely cell cycle regulated, sharing a common peak in late mitosis. Moreover, Sdb25 protein levels and associated kinase activity are sharply upregulated at the peak of Dbf2 kinase activity in cells released from a dbf2-ts block. The Sdb25 protein then disappears around START in the next cell cycle. This indicates that SDB25 function is confined to M/G1, and morphological analysis of sdb25∆ cells supports this conclusion. The second allele-specific suppressor of DBF2, SDB24, is 2463 nucleotides long and is a newly identified yeast gene. SDB24 is expressed under cell cycle control, and is maximally abundant in early S phase. Deletion of SDB24 does not affect cell viability, although deletion of both SDB24 and DBF2 is lethal for the cell.

Type: Thesis (Doctoral)
Qualification: Ph.D
Title: The molecular analysis of two high copy number suppressors of a yeast cell cycle protein kinase mutant
Open access status: An open access version is available from UCL Discovery
Language: English
Additional information: Thesis digitised by ProQuest.
Keywords: Biological sciences; Cell cycle regulation
URI: https://discovery.ucl.ac.uk/id/eprint/10106989
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