Yeast Genomic Libraries Can Be Constructed with Shuttle Vectors and Screened by Functional Complementation

A collection of DNA molecules each cloned into a vector molecule is known as a DNA library. When genomic DNA from a particular organism is the source of the starting DNA, the set of clones that collectively represent all the DNA sequences in the genome is known as a genomic library. In some cases, a DNA library can be screened for the ability to express a functional protein that complements a recessive mutation. Such a screening strategy would be an efficient way to isolate a cloned gene that corresponds to an interesting recessive mutation identified in an experimental organism. To illustrate this method, referred to as functional complementation, we describe how yeast genes cloned in special E. coli plasmids can be introduced into mutant yeast cells to identify the wild-type gene that is defective in the mutant strain. Because Saccharomyces genes do not contain multiple introns, they are sufficiently compact that the entire sequence of as many as 10 genes can be included in a genomic DNA fragment inserted into a plasmid vector.

To construct a plasmid genomic library that is to be screened by functional complementation in yeast cells, the plasmid vector must be capable of replication in both E. coli cells and yeast cells. This type of vector, capable of propagation in two different hosts, is called a shuttle vector. The structure of a typical yeast shuttle vector is shown in Figure 6-15a. This vector contains the basic elements that permit cloning of DNA fragments in E. coli as well as sequences required for its propagation in yeast.

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EXPERIMENTAL FIGURE 6-15 A yeast genomic library can be constructed in a plasmid shuttle vector that can replicate in yeast and in E. coli. (a) Components of a typical plasmid shuttle vector for cloning Saccharomyces genes. The presence of a yeast replication origin (ARS) and a yeast centromere (CEN) allows stable replication and segregation in yeast. Also included is a yeast selectable marker such as URA3, which allows a ura3 mutant to grow on medium lacking uracil. Finally, the vector contains sequences for replication and selection in E. coli (ORI and ampr) and a polylinker for easy insertion of yeast DNA fragments. (b) Typical protocol for constructing a yeast genomic library. Partial digestion of total yeast genomic DNA with Sau3A is adjusted to generate fragments with an average size of ∼10 kb. The vector is prepared to accept the genomic fragments by digestion with BamHI, which produces the same sticky ends as Sau3A. Each transformed clone of E. coli that grows after selection for ampicillin resistance contains a single type of yeast DNA fragment.

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To increase the probability that all regions of the yeast genome will be successfully cloned and represented in the plasmid library, the genomic DNA is usually only partially digested to yield overlapping restriction fragments of ∼10 kb. These fragments are then ligated into a shuttle vector in which the polylinker has been cleaved with a restriction enzyme that produces sticky ends complementary to those on the yeast DNA fragments (Figure 6-15b). Because the 10-kb restriction fragments of yeast DNA are incorporated into the shuttle vectors randomly, at least 105 E. coli colonies, each containing a particular recombinant shuttle vector, are necessary to ensure that each region of yeast DNA has a high probability of being represented in the library at least once.

Figure 6-16 outlines how such a yeast genomic library can be screened to isolate the wild-type gene corresponding to one of the temperature-sensitive cdc mutations mentioned earlier in this chapter. The starting yeast strain is a double mutant that requires uracil for growth due to a ura3 mutation and is temperature sensitive due to a cdc28 mutation identified by its phenotype (see Figure 6-6b). Recombinant plasmids isolated from the yeast genomic library are mixed with yeast cells under conditions that promote transformation of the cells with foreign DNA. Since transformed yeast cells carry a plasmid-borne copy of the wild-type URA3 gene, they can be selected by their ability to grow in the absence of uracil. Typically, about 20 petri dishes, each containing about 500 yeast transformants, are sufficient to represent the entire yeast genome. This collection of yeast transformants can be maintained at 23 °C, a temperature permissive for growth of the cdc28 mutant. The entire collection on 20 plates is then transferred to replica plates, which are maintained at 36 °C, a nonpermissive temperature for cdc mutants. Yeast colonies that carry recombinant plasmids expressing a wild-type copy of the CDC28 gene will be able to grow at 36 °C. Once temperature-resistant yeast colonies have been identified, plasmid DNA can be extracted from the cultured yeast cells and analyzed by DNA sequencing, a topic we take up shortly.

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EXPERIMENTAL FIGURE 6-16 Screening of a yeast genomic library by functional complementation can identify clones carrying the normal form of a mutant yeast gene. In this example, a wild-type CDC gene is isolated by complementation of a cdc yeast mutant. The yeast genomic library prepared as shown in Figure 6-15 is transformed into a ura3, temperature-sensitive cdc mutant strain. The relatively few transformed yeast cells, which contain recombinant plasmid DNA, can grow in the absence of uracil at 23 °C. When these colonies are replica-plated and incubated at 36 °C (a nonpermissive temperature), only clones carrying a library plasmid that contains the wild-type copy of the CDC gene will survive. LiOAC = lithium acetate; PEG = polyethylene glycol.