Hollingsworth, R.R., R.M Ostroff, M.B. Klein, L.A. Niswander, & R.A. Sclafani (1992)
Molecular genetic studies of the Ccd7 protein kinase and induced mutagenesis in yeast.
Genetics 132: 53-62.
This article is an example of mutation analysis, the detailed characterization of several mutant alleles of a gene. It is an important first sep in determining the specific function or functions of the gene product of a gene and the molecular mechanism by which it carries out those functions. Article 4 reported that Cdc7p exhibits sequence homology to Cdc28p, a known protein kinase. Hollingsworth & Sclafani (1990) and Yoon & Campbell (1991) purified Cdc7 protein and demonstrated that it has protein kinase activity in vitro. While these findings are suggestive, none proves that the essential function of Cdc7p is associated with its protein kinase activity. Penguins and chickens have wings but neither species flies. Just because an animal has wings one cannot assume it uses them to fly. Similarly, if a protein contains a particular sequence motif one cannot assume that this is functionally significant for this particular protein. It must be demonstrated by mutation analysis.
Moreover, different alleles of a gene may have subtly different phenotypes that could indicate that the encoded product has more than one cellular function. Multiple functions are often associated with different structural regions of the protein. Analysis of several alleles having different phenotypes can be useful for assigning specific functions to particular regions of a protein. This type of analysis is referred to as a structure-function analysis and is used to identify functional domains of a protein. These domains could represent regions responsible for interaction with other proteins (such as in a heterodimer), or with enzymatic substrates or cofactors (such as ATP or heme), or DNA-binding domains (such as is found in some activator and repressor proteins). A single protein with multiple cellular functions is likely to have a complex multidomain structure and exhibit a pleiotropic phenotype.
This article reports on the sequencing of several cdc7-ts alleles. Other mutations in essential domains associated with protein kinase activity, such as the ATP-binding site, were introduced by in vitro mutagenesis, and these alleles are characterized as well. The analysis demonstrates that the protein kinase activity of Cdc7p is essential for its role in DNA replication and repair.
1. This article describes the cloning of several cdc7 temperature-sensitive alleles using a method based on gene conversion of a plasmid-borne copy of CDC7.
(a) Describe the method used, which must include the role of the herpes virus TK gene. Use a diagram to illustrate your answer that shows the chromosomal cdc7-ts gene, the plasmid construct, and the number and location of the recombination events needed for gene conversion of the plasmid.
(b) What is the phenotype of the transformant before and after the conversion event described above with regard to 5-fluorodeoxyuridine resistance and temperature-sensitivity?
2. Table 2 and Figure 1 summarize the results of sequence analysis of the nine cdc7 mutant alleles. Seven of these cdc7 alleles were isolated using procedures that ensure that independent, and presumably different alterations would be obtained. Despite this, alleles 1, 2, and 5 and alleles 7 and 90 are identical alterations. Hypothesize why particular changes were isolated multiple times. Remember that the mutagens used to induce the sequence alterations do not preferentially attack one guanine residue over another.
3. Define missense suppressor. In your own words, describe why SOE1, a tRNA mutation, suppresses the cdc7-7 allele. (You will need to use information from the genetic code for your answer.) Would you expect the soel suppressor mutation to be dominant or recessive and why?
4. Mutant allele cdc7-10 was constructed by in vitro mutagenesis of codons 76 and 77 that code for the two essential lysines of the putative ATP-binding domain of Cdc7 kinase.
(a) Describe the method used by the authors to demonstrate whether cdc7-10 is a lethal mutation, that is, a loss of Cdc7p kinase activity causes a defect in cell division.
(b) Describe a method that could be used to demonstrate that cdc7-10 does not complement a cdc7A null allele.
(c) Are you surprised that cdc7-10 is not a conditional mutation? Explain.
5. The authors conclude that the results obtained with cdc7-10 provide strong evidence that the kinase activity of Cdc7 protein is essential for its function in cell division. Why is it necessary to demonstrate that sufficient Cdc7-10 protein is present? In other words, could they have come to the same conclusion if they had found that Cdc7-10 protein levels were undetectable?
6. Table 3 shows the results of a study to determine if cdc7 mutants exhibit a different rate of mutation than wild-type.
(a) How was the mutation rate measured?
(b) Do cdc7 mutations exhibit different rates of spontaneous mutation compared with wild-type and how was this determined?
(c) Do the different cdc7 mutant alleles exhibit differences in mutation rate? Discuss.
(d) Do the different cdc7 mutant alleles exhibit differences in mutation rate when UV light is used as the mutagenic agent? What might be the significance of this finding?
7. Define 'segregation lag' according to the authors' usage. What do the results in Table 5 indicate with regard to the functional activity of these temperature-sensitive alleles at the permissive temperature? Are they truly normal at the permissive temperature or are there indications of the mutational defect?
8. The mutant allele cdc7-23 was made by in vitro mutagenesis.
(a) What is linker scanning insertional mutagenesis?
(b) List the phenotypes of cdc7-23 mutant strains.
9. Is there a structure-function relationship between the hyper- versus hypo-mutability phenotype?
10. What is the functional implication of the finding that the hypo- or hyper-mutability phenotype of cdc7 mutations is recessive to wild-type?
11. What conclusions, if any, do the authors come to regarding the role of Cdc7 protein kinase in mutation and repair?
Hollingsworth, R.E. & R.A. Sclafani (1990) DNA metabolism gene CDC7 from yeast encodes a serine (threonine) protein kinase. Proc. Natl Acad. Sci. USA 87: 6272-6276. Yoon, H.Y. & J.L. Campbell (1991) The CDC7 protein of Saccharomyces cerevisiae is a phosphoprotein that contains protein kinase activity. Biochemistry 88: 3574-3578.
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