Reed, S.I., J.A. Hadwiger, & A.T. Lorencz (1985) Protein kinase activity associated with the product of the yeast cell division cycle gene CDC28. Proc. Natl Acad. Sci. USA 82: 40554059.
Lorencz and Reed (1984) describe the sequence analysis of CDC28. They found that the encoded protein exhibits sequence homology to several vertebrate oncogenes including src (21% homology), fes (25.5% homology), fps (24.5% homology), yes (25% homology), and v-raf (24% homology). Most of these oncogenes have been shown to encode protein kinases and so the authors of this article attempt to demonstrate that CDC28 also encodes a protein kinase.
1. Polyclonal antibody directed against an in-frame LacZ-Cdc28 fusion protein had been prepared by Reed (1982) and this was used here to precipitate Cdc28 protein from yeast extracts. Figure 1 shows an analysis of the yeast proteins precipitated by this antibody.
(a) Why were the yeast cells grown in the presence of 35S-methionine?
(b) Define preimmune serum.
(c) About 7-10 different proteins are precipitated from the yeast cell extract by the antibody. Give two reasons (other than the molecular weight) why the authors conclude that the 36 kD protein is a Cdc28 protein and not the other precipitated proteins, particularly the 70 kD protein.
2. Briefly describe the kinase assay developed here to determine the protein kinase activity of the immune precipitate.
(a) What is the basis for the authors' conclusion that this phosphorylation of the 40 kD protein is dependent on the presence of the Cdc28 protein in the immune precipitate?
(b) How do the authors know that the phosphorylation of the 40 kD protein is occurring in the immune precipitate and not in vivo prior to preparation of the cell extract?
3. Extracts prepared from cells expressing cdc28-4 lacked kinase activity even at the permissive temperature and with no decrease in Cdc28-4 mutant protein levels. Based on this finding alone the authors cannot conclude that it is the kinase activity of the Cdc28 protein and not some other as yet unidentified function that is required for its role in the cell cycle. Discuss why this finding is inconclusive.
4. The finding that the protein kinase activity of the immune precipitate prepared from cells expressing the cdc28-13 mutant allele is temperature sensitive is the most compelling argument supporting the authors' conclusion that the kinase activity of Cdc28 protein is its essential function in regulating the cell cycle. Discuss.
Lorencz, A.T. & S.I. Reed (1984) Primary structure homology between the product of yeast cell division control gene CDC28 and vertebrate oncogenes. Nature 307: 183-185. Reed, S.I. (1982) Preparation of product-specific antisera by gene fusion: antibodies specific for the product of the yeast cell-division-cycle gene CDC28. Gene 20: 255-265.
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