Yeast gene cloning experiment

Summary

A general method for cloning yeast genes by complementation using a putative mutation. The mutation is cdc101-1, which is both recessive and temperature sensitive for yeast growth.

Operation method

complementarities

Materials and Instruments

yeasts

Move

1. Transform lea 2 cdc 101-1 yeast strain with a Saccharomyces cerevisiae DNA library containing LEU 2 as a selection marker, incubate at 23°C, and screen 2,000-20,000 ^Leu+ transformants based on the size of the inserted fragments.2. Photocopy plate transformants, transfer them to pre-warmed selective plates and incubate at 37°C to screen for complementary temperature-sensitive mutant phenotypes. After overnight incubation, colonies containing plasmids inserted with complementary cdc 101-1 mutant gene fragments as well as potential CDC ¹⁰¹⁺ revertant mutants containing random non-complementary plasmids may grow. Each single colony that emerged on the plate was re-stratified and saved for further analysis.3. Obtain ^{plasmid-less Leu-} strains from yeast candidates, observe their ability to grow at 37°C, and retain those transformants that can grow at 37°C if they contain the plasmid.4. isolate plasmid DNA by extracting total DNA from the transformed yeast identified in step 3 and transforming E. coli separately. re-transform the plasmid DNA into the mutated yeast strain and confirm that the correct plasmid has been isolated.

5. analyze the plasmids by restriction mapping to determine if the genomic DNA insert fragments that appear in the different plasmids overlap. This analysis may help to determine the boundaries of this complementary region. Construct different insertion and deletion mutations and examine their ability to complementarily mutate in yeast mutant strains. This will provide more precise information about gene localization within the cloned insertion fragments.6. Introduce a yeast selective marker into a site in the center of the complementary region (based on the information obtained in step 5).7. Re-transform the disrupted plasmid into the original mutant strain and screen the mutant phenotype to check whether complementation has been completely disrupted. Construct diploid strains containing one copy of the wild-type gene and one copy of the disrupted gene.

8. after spore formation and dissection, cross the haploid spore product containing the disrupted gene copy (usually identified by the selective marker introduced in step 6) with a strain containing the original mutation; if this diploid is phenotypically identical to the original mutant strain, this indicates that complementation is not present, suggesting that the genes associated with the mutation have been cloned, and also make a control experiment in which the haploid containing the disrupted gene (which may itself A control experiment in which a haploid containing the disruption gene (which may itself exhibit a non-wild-type phenotype) is crossed with a wild-type spore goes to show that this diploid phenotype is wild-type (indicating that disruption is a recessive mutation).

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