Article 4

Patterson, M., R.A. Sclafani, W.L. Fangman, & J. Rosamond (1986) Molecular characterization of cell cycle gene CDC7 from Saccharomyces cerevisiae. Mol. Cell. Biol. 6: 15901598.

1. The Introduction to this article summarizes the phenotype of strains carrying temperature-sensitive mutations of CDC7 as follows. At the nonpermissive temperature cdc7 mutants arrest prior to the initiation of DNA replication as budded cells with a single nucleus lacking an elongated spindle but having a divided spindle pole body. Several laboratories investigated other aspects of the cdc7 phenotype and found a number of interesting roles for Cdc7p outside of its role in mitotic cell division. List these.

2. The authors cloned CDC7 by complementation of the cdc7-l allele.

(a) Explain in general terms (that is, do not specifically refer to cdc7 or the cdc7 phenotype) the concept behind this approach, namely cloning by complementation.

(b) What changes would you have to make if you only had dominant mutant alleles available?

3. The libraries were made from partial digests using enzymes that cut frequently in the Saccharomyces genome. Moreover, different enzymes were used to make different libraries. Propose reasons why.

4. How does the isolation of several complementing clones help in localizing the gene of interest?

5. Why were URA+ colonies selected after strain 158 was transformed with the YEp24 library?

6. YIp5 is a pBR322-based vector containing the URA3 gene and no ARS element is present on this vector. Plasmid pMP104 was constructed by inserting a DNA fragment containing the putative CDC7 gene into this vector, (a) Diagram the integration of pMP104 into the cdc7-l gene of strain SB158.

Use the diagram below to help you.

(b) Diagram the cross between a strain carrying an integrated copy of plasmid pMP104 and another strain of opposite mating type with the genotype cdc7-l ura3 TRP1 in transformants with the following two possible sites of integration. For each cross state whether PD, NPD, or TT tetrads would be expected and give the phenotype (TRP, CDC, URA) of the spores in each tetrad type obtained.

(i) Integration occurred at the cdc7-l gene. The vertical line indicates the position of the mutant alteration in cdc7-l. Show the integration event at a site to the right of the mutation.

(ii) Integration occurred at ura3-52 instead. (URA3 is unlinked to CDC7.)

trpl

cdc7-l

7. Diagram the crossover(s) that are required for the 'rescue by recombination' method used to localize the alteration in cdc7-l to the left end of the map. Remember, the plasmid does not integrate.

8. How might you improve the vector used for the construction of the library so that you did not have to worry about multicopy suppression?

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