Cen

Shuttle vector jcut with BamHI

Shuttle vector jcut with BamHI

Yeast genomic DNA

Partially digest with Sau3A

Partially digest with Sau3A

Ligate

Ligate

Transform E. coli Screen for ampicillin resistance

Transform E. coli Screen for ampicillin resistance

Isolate and pool recombinant plasmids from 105 transformed ,, E. coli colonies

Assay yeast genomic library by functional complementation plasmids can be introduced into mutant yeast cells to identify the wild-type gene that is defective in the mutant strain.

Libraries constructed for the purpose of screening among yeast gene sequences usually are constructed from genomic DNA rather than cDNA. Because Saccharomyces genes do not contain multiple introns, they are sufficiently compact so that the entire sequence of a gene can be included in a ge-nomic 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 9-19a (see page 369). This vector contains the basic elements that permit cloning of DNA fragments in E. coli. In addition, the shuttle vector contains an autonomously replicating sequence (ARS), which functions as an origin for DNA replication in yeast; a yeast centromere (called CEN), which allows faithful segregation of the plasmid during yeast cell division; and a yeast gene encoding an enzyme for uracil synthesis (URA3), which serves as a selectable marker in an appropriate yeast mutant.

To increase the probability that all regions of the yeast genome are successfully cloned and represented in the plas-mid library, the genomic DNA usually is only partially digested to yield overlapping restriction fragments of = 10 kb. These fragments are then ligated into the shuttle vector in

▲ EXPERIMENTAL FIGURE 9-20 Screening of a yeast genomic library by functional complementation can identify clones carrying the normal form of mutant yeast gene. In this example, a wild-type CDC gene is isolated by complementation of a cdc yeast mutant. The Saccharomyces strain used for screening the yeast library carries ura3~ and a temperature-sensitive cdc mutation. This mutant strain is grown and maintained at a permissive temperature (23 °C). Pooled recombinant plasmids prepared as shown in Figure 9-19

which the polylinker has been cleaved with a restriction enzyme that produces sticky ends complementary to those on the yeast DNA fragments (Figure 9-19b). 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 assure that each region of yeast DNA has a high probability of being represented in the library at least once.

Figure 9-20 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 9-6). 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 placed at 36 °C, a nonpermissive temperature for are incubated with the mutant yeast cells under conditions that promote transformation. The relatively few transformed yeast cells, which contain recombinant plasmid DNA, can grow in the absence of uracil at 23 °C. When transformed yeast colonies are replica-plated and placed 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.

Library of yeast genomic DNA carrying URA3 selective marker

Library of yeast genomic DNA carrying URA3 selective marker

Temperature-sensitive cdc-mutant yeast; ura3~ (requires uracil)

Transform yeast by treatment with LiOAC, PEG, and heat shock

Temperature-sensitive cdc-mutant yeast; ura3~ (requires uracil)

Only colonies carrying a URA3 marker are able to grow

Only colonies carrying a URA3 marker are able to grow

Transform yeast by treatment with LiOAC, PEG, and heat shock

Plate and incubate at permissive temperature on medium lacking uracil

Replica-plate and incubate at nonpermissive temperature

Only colonies carrying a wild-type CDC gene are able to grow

Only colonies carrying a wild-type CDC gene are able to grow

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 subcloning and DNA sequencing, topics we take up in the next section.

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