Lorencz, A.T. & S.I. Reed (1986) Sequence analysis of temperature-sensitive mutations in the Saccharomyces cerevisiae gene CDC28. Mol. Cell. Biol. 6: 4099-4103.
In this article the authors sequence several independent mutant alleles of CDC28. They find that these mutations cluster in two distinct regions of the Cdc28 protein.
In Article 12 we will see that two of these mutations, cdc28-4 and cdc28-lN, that map to one of these two regions define different cell cycle functions of Cdc28 protein kinase.
1. Figure la shows how the authors synthesized the double-stranded DNA fragments of CDC28 that they used to localize the sites of the alterations in the 11 cdc28-ts mutations. These laborious methods are no longer necessary given the advent of polymerase chain reaction (PCR). Describe how you would use PCR-based methods to synthesize a series of five overlapping DNA fragments spanning the CDC28 open reading frame (ORF). Use the diagram below to illustrate primer pair location. Draw an arrow at the 3' end of each primer and number both members of each pair (1, 2, 3, 4, and 5).
2. Explain why it is probable that the cdc28-ts alterations are in the ORF and not likely to be in the promoter region.
3. Draw a diagram illustrating the gene conversion event between the DNA fragment of CDC28 and the genomic cdc28-ts mutation in each of the following cases. Show the exchange sites and the chromosomal product.
(a) The alteration lies within the fragment sequence.
(b) The alteration lies outside of the fragment sequence.
4. Explain in your own words what the authors mean when they say that 'fragments spanning the mutation are able to rescue the temperature-sensitive mutant phenotype'.
5. Illustrate the gap repair method used to recover the mutation in the genome onto a gapped plasmid as follows.
(a) Diagram the YRp7 plasmid construct used to create the 'gapped' plasmids.
(b) How are the gaps created in the construct shown in (a)?
(c) Diagram a gene conversion event between a gapped plasmid and the chromosome. Include the plasmid product of this conversion event.
(d) Is the chromosomal copy altered as a result of this gene conversion event?
(e) What is the Cdc phenotype of the transformed strain after the gene conversion event?
(f) What happens to host cells if gene conversion of the gapped region does not occur?
6. The sequence alterations in 11 cdc28-ts mutants were determined but only 6 mutant alterations are reported in Figure 2. Explain.
7. The ts mutations clustered in two regions of the Cdc28 protein, codons 120130 and the C-terminal 50 residues. Moreover, the same alteration was isolated independently many times. The authors suggest that this implies that these regions of the Cdc28 protein are uniquely important for the cell cycle function of Cdc28 protein kinase. Discuss their reasons for excluding the following explanations.
(a) EMS mutagenesis is not random.
(b) Only alterations to these restricted regions of the Cdc28 protein can cause temperature sensitivity.
(c) These regions of the Cdc28 protein are involved in functions required of all kinases.
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