Nasmyth, K.A. & S.I. Reed (1980) Isolation of genes by complementation in yeast: molecular cloning of a cell-cycle gene. Proc. Natl Acad. Sci. USA 77: 2119-2123.
1. These questions refer to the construction of the plasmid library.
(a) List the essential features of the vector YRp7.
(b) Which type of library is this (genomic, cDNA, other)?
(c) Describe the preparation of insert DNA fragments. Include the genotype of the strain that was the source of the insert DNA.
(d) The DNA fragments were inserted into the BamHl site of YRp7. Would you expect to be able to release all of these insert fragments using BamHll Explain.
(e) How large is the library? The S. cerevisiae genome is 1.2 x 107 bp (1.2 x 104 kbp or 12 Mbp). Is the authors' estimate that the library contains each gene, in whole or in part, in about ten independent library clones reasonable? Explain.
2. These questions refer to the identification of yeast transformants containing a library plasmid carrying the putative CDC28 gene.
(a) Why were Trp+ transformants selected and how were they selected?
(b) What selection method was used to identify transformants with the Cdc+ phenotype?
(c) Why is this cloning approach called 'cloning by complementation'?
3. Figure 1 shows the results of Southern analysis of several Trp+ Cdc+ transformants probed with pBR322 plasmid DNA.
(a) What is the significance of the finding that some transformants exhibit only a plasmid-sized fragment while others had high molecualar weight forms or both?
(b) Is it surprising that those transformants that exhibit only plasmid-sized fragments lose both the Trp+ and Cdc+ phenotypes when grown under nonselective conditions? Explain.
(c) Describe the method used to recover the plasmid from the unstable transformants in E. coli.
(d) Restriction mapping and Southern analysis were used to demonstrate that three of the recovered plasmids contained overlapping inserts. Describe how you would use Southern analysis to distinguish vector-derived from insert-derived fragments in a Hindlll digest of YRp7-CDC28( 1). Describe how you would use Southern analysis to demonstrate which Hindlll fragments of YRp7-CZ»C2S(3), the plasmid with the smallest insert, are also contained in the YKpl-CDC28(\) insert.
(e) How is it helpful to have several complementing plasmids with overlapping inserts of different sizes for localizing the complementing gene within the insert fragment?
4. Unpublished genetic mapping studies determine the distance between CDC28 and TYR1 to be 7 map units (centamorgans). What is the significance of the finding that YRp7-CZ>C25(l) complements both cdc28-4 and tyrP.
5. Diagram a hypothetical integration event between YRp7-CZ>C2<S(3) and the chromosomal cdc28-4 gene. Use the diagram below of the region of chromosome II of strain SR668-2 containing both cdc28-4 and tyrl mutations. Include a diagram of the circular YRp7-C£)C2#(3) plasmid, the position of the recombination event between plasmid and chromosome, and the map of the chromosomal product of the integration event. (The vertical line marks the position of the mutant sequence alteration in cdc28-4.)
6. How does the product of the integration event described in question 5 differ if the position of the recombination site is to the right versus the left of the mutant alteration?
7. Table 2 presents the results of a cross between strain SR668-2 containing an integrated copy of YRp7-CZ>C2S(3) (refer to this allele as cdc28-4:CDC28-TRP1) with strain KN-86.
(a) Diagram the cross indicating the genotype and phenotype of the parent strains and the resulting diploid. Include the TYR1, CDC28, and TRP1 genes.
(b) Give the genotype of the four ascospores in a PD tetrad.
(c) What is the origin of the small number of tetratype asci that were obtained?
8. The results in Table 2 determined the linkage of the integrated TRP1 gene to tyrl. How would you determine the linkage of the integrated TRP1 to cdc28-4 using these same tetrads? Would you expect the map distance to be smaller, larger, or the same as the map distance to tyrl? Explain.
9. Show the calculation that the authors made to conclude that 1 map unit is equivalent to a maximum of 1.75 kbp in this region of chromosome II.
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