In all, 67 genes in the Saccharomyces genome database are annotated as cdc genes, of which 32 genes are directly involved in cell division. The rest regulate cell polarity and bud growth without taking part directly in the process of cell division. Many of these 32 genes have been cloned by complementation (see Chapter 8). For example, cdc28 mutation in S. cerevisiae arrests growth of cells at a point "Start" in G1, when the cell commits to enter the cell cycle. To clone this gene, a cdc28 ts mutant is transformed with a genomic library from wild (CDC28) yeast strain, the cells plated and incubated at non-permissive temperature (37°C) and the rare colonies in which CDC28 gene was incorporated survived. (We shall follow the convention of naming a gene with three letters and a number. For S. cerevisiae, the wild-type alleles are written in uppercase letters in italics and mutant recessive alleles are written in lowercase letters in italics). The plasmid-carrying CDC28 gene is recovered, sequenced and from the DNA sequence the encoded protein is identified as a protein kinase, an enzyme that transfers the terminal phosphate group from ATP onto a serine/threonine residue of another protein. Cloning of several cdc genes revealed that many of them are protein kinases whereas others are phosphatases (enzymes that remove phosphate groups from protein substrates). Paul Nurse showed that the human CDC2 kinase was able to complement the loss of function mutation in yeast (Lee and Nurse, 1987), underscoring the evolutionary conservation of the yeast and the human kinase. In the meantime, work with sea urchin eggs by Tim Hunt led to identification of a 45 kd protein, whose levels in dividing cells fluctuated at a regular interval, that he called cyclin. Biochemical analysis in different organisms reveals that binding of cyclin to the protein kinase regulated the enzyme activity. The first of two important conclusions from these studies was that the cyclical nature of eukaryotic cell cycle is controlled by phosphorylation/dephosphorylation of cyclin-dependent kinases (CDKs) and by the targeted degradation of cyclin protein as shown in Figure 6.4. The second important conclusion was that the mechanism of cell cycle regulation is highly conserved in all organisms from yeast to human. Leland Hartwell, Paul Nurse and Tim Hunt received the Nobel Prize in 2001 for their contributions to understanding the fundamental process of cell division.
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