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A Genetics Laboratory Module Involving Selection and Identification of Lysine Synthesis Mutants in the Yeast

    Authors: JILL B. KEENEY1,*, RUTH REED2
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    Affiliations: 1: Department of Biology and; 2: Department of Chemistry, Juniata College, Huntingdon, Pennsylvania 16652
    AUTHOR AND ARTICLE INFORMATION AUTHOR AND ARTICLE INFORMATION
    • Instructions for media preparation are given at the end of the paper.
    • *Corresponding author. Mailing address: Department of Biology, Juniata College, 1700 Moore St., Huntingdon, PA 16652. Phone: (814) 641-3577. Fax: (814) 641-3685. E-mail: keeney@juniata.edu.
    • Copyright © 2000, American Society for Microbiology. All Rights Reserved.
    Source: J. Microbiol. Biol. Educ. May 2000 vol. 1 no. 1 26-30. doi:10.1128/154288100X14285805552330
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    Abstract:

    We have developed a laboratory exercise, currently being used with college sophomores, which uses the yeast to convey the concepts of amino acid biosynthesis, mutation, and gene complementation. In brief, selective medium is used to isolate yeast cells carrying a mutation in the lysine biosynthesis pathway. A spontaneous mutation in any one of three separate genetic loci will allow for growth on selective media; however, the frequency of mutations isolated from each locus differs. Following isolation of a mutated strain, students use complementation analysis to identify which gene contains the mutation. Since the yeast genome has been mapped and sequenced, students with access to the Internet can then research and develop hypotheses to explain the differences in frequencies of mutant genes obtained.

References & Citations

1. Bhattacharjee JK1985a-Aminoadipate pathway for the biosynthesis of lysine in lower eukaryotesCrit Rev Microbiol1213115110.3109/104084185091044273928261 http://dx.doi.org/10.3109/10408418509104427
2. Chattoo BB, Sherman F, Azubalis DA, Fjellstedt TA, Mehnert D, Ogur M1979Selection of lys2 mutants of the yeast Saccharomyces cerevisiae by the utilization of a-aminoadipateGenetics93516517248969
3. Feller A, Dubois E, Ramos F, Pierard A1994Repression of the genes for lysine biosynthesis in Saccharomyces cerevisiae is caused by limitation of Lys14-dependent transcriptional activationMol Cell Biol14641164187935367
4. Manney TR, Manney ML1992Yeast: a research organism for teaching geneticsAm Biol Teacher5442610.2307/4449533 http://dx.doi.org/10.2307/4449533
5. Montelone BA, Williamson B, Mayo L1995Rapid transformation of a color mutant of yeastAm Biol Teacher5717110.2307/4449955 http://dx.doi.org/10.2307/4449955
6. Ramos F, Dubois E, Pierard A1988Control of enzyme synthesis in the lysine biosynthetic pathway of Saccharomyces cerevisiaeEvidence for a regulatory role of gene LYS14Eur. J. Biochem.17117117610.1111/j.1432-1033.1988.tb13773.x3123231 http://dx.doi.org/10.1111/j.1432-1033.1988.tb13773.x
7. Stanford University25August1999Saccharomyces Genome Database
8. Storts DR, Bhattacharjee JK1989Properties of revertants of lys2 and lys5 mutants as well as alpha-aminoadipatesemialdehyde dehydrogenase from Saccharomyces cerevisiaeBiochem. Biophys. Res. Commun.16118218610.1016/0006-291X(89)91578-72499333 http://dx.doi.org/10.1016/0006-291X(89)91578-7
9. Watson JD, Hopkins NH, Roberts JW, Steitz JA, Weimer AM1987Molecular biology of the gene4th ed550594Benjamin/CummingsMenlo Park, Calif
10. White BT1999The red & white yeast labAm Biol Teacher6160060410.2307/4450775 http://dx.doi.org/10.2307/4450775
11. Zaret KS, Sherman F1985a-Aminoadipate as a primary nitrogen source for Saccharomyces cerevisiae mutantsJ. Bacteriol.1625795833921525
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2000-05-01
2017-11-20

Abstract:

We have developed a laboratory exercise, currently being used with college sophomores, which uses the yeast to convey the concepts of amino acid biosynthesis, mutation, and gene complementation. In brief, selective medium is used to isolate yeast cells carrying a mutation in the lysine biosynthesis pathway. A spontaneous mutation in any one of three separate genetic loci will allow for growth on selective media; however, the frequency of mutations isolated from each locus differs. Following isolation of a mutated strain, students use complementation analysis to identify which gene contains the mutation. Since the yeast genome has been mapped and sequenced, students with access to the Internet can then research and develop hypotheses to explain the differences in frequencies of mutant genes obtained.

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Figures

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FIG. 1

An overview of the a-aminoadipate pathway for lysine biosynthesis. In a cell growing with abundant nitrogen, a-aminoadipate, produced from the transaminase reaction, is converted to lysine via several biochemical steps. Mutations in the and genes (encoding the a-aminoreductase enzyme) result in sufficient levels of the reverse reaction to allow nitrogen assimilation using a-aminoadipate as the nitrogen source.

Source: J. Microbiol. Biol. Educ. May 2000 vol. 1 no. 1 26-30. doi:10.1128/154288100X14285805552330
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FIG. 2

Diagram of the procedure of streaking yeast cells to obtain single colonies.

Source: J. Microbiol. Biol. Educ. May 2000 vol. 1 no. 1 26-30. doi:10.1128/154288100X14285805552330
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FIG. 3

The complementation test. (A) Yeast strains containing mutations in , , and (horizontal lines) were replicated onto YPD medium perpendicular to a-aminoadipate-resistant yeast strains (vertical lines) of opposite mating type. After allowing mating to occur, the plate was replica plated onto medium lacking lysine. (B) The replica plate to medium lacking lysine, showing the results of the complementation test. Unknowns 1, 2, and 4 contain a mutation in , as shown by the lack of growth at the intersection with the strain. Unknown 3 contains a mutation in . Note the leaky growth phenotype of the strains, indicated by the light growth outside the squares of complementing growth.

Source: J. Microbiol. Biol. Educ. May 2000 vol. 1 no. 1 26-30. doi:10.1128/154288100X14285805552330
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