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Amino acid synthesis is the set of biochemical ..
We have developed a laboratory exercise, currently being used with college sophomores, which uses the yeast Saccharomyces cerevisiae 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. The yeast Saccharomyces cerevisiae is a commonly used organism in cell and molecular biology research (reviewed in reference 9). The simple growth requirements and rapid division time of yeast cells make them convenient for laboratory exercises in microbiology, cell biology, and genetics (4). The laboratory exercise described here uses the lysine synthesis pathway to explore the concepts of amino acid biosynthesis,
N2 - In replicating yeast, lysine 63-linked polyubiquitin (polyUb) chains are extended from the ubiquitin moiety of monoubiquitinated proliferating cell nuclear antigen (monoUb-PCNA) by the E2-E3 complex of (Ubc13-Mms2)-Rad5. This promotes error-free bypass of DNA damage lesions. The unusual ability of Ubc13-Mms2 to synthesize unanchored Lys63-linked polyUb chains in vitro allowed us to resolve the individual roles that it and Rad5 play in the catalysis and specificity of PCNA polyubiquitination. We found that Rad5 stimulates the synthesis of free polyUb chains by Ubc13-Mms2 in part by enhancing the reactivity of the Ubc13-Ub thiolester bond. Polyubiquitination of monoUb-PCNA was further enhanced by interactions between the N-terminal domain of Rad5 and PCNA. Thus, Rad5 acts both to align monoUb-PCNA with Ub-charged Ubc13 and to stimulate Ub transfer onto Lys63 of a Ub acceptor. We also found that Rad5 interacts with PCNA independently of the number of monoubiquitinated subunits in the trimer and that it binds to both unmodified and monoUb-PCNA with similar affinities. These findings indicate that Rad5-mediated recognition of monoUb-PCNA in vivo is likely to depend upon interactions with additional factors at stalled replication forks.
All standard amino acids except for lysine.
Laboratory strains of yeast, in the haploid state, are either a or mating type, as determined by the MATa or MAT locus, respectively. A cross between haploids of different mating types forms a stable diploid. Wild-type (e.g., prototrophic) yeasts have no essential amino acids. Thus, yeasts are able to form colonies on minimal media containing a carbon source (usually glucose), a nitrogen source (usually ammonium sulfate), and a minimal medium base containing inorganic salts and vitamins.
Many of the yeast amino acid biosynthetic pathways have been studied in detail, and it has been found that some amino acids (or synthesis intermediates) can be utilized by the yeast cell as a nitrogen source. In yeast and higher fungi, lysine biosynthesis is achieved via the -aminoadipate pathway () (, ). The -aminoadipate, a precursor in this pathway, can be used in place of ammonium sulfate as a nitrogen source by S. cerevisiae, but only in strains carrying a mutation in either the LYS2, LYS5, or LYS14 gene (, ). (Note that by standard yeast genetic nomenclature, all genes are given a three-letter designation followed by a number. Wild-type genes are written in uppercase, e.g., LYS2. Mutant genes are written in lowercase and often referred to by a specific mutation number, e.g., lys2-801.) Thus, -aminoadipate medium can be used to select for spontaneous or induced mutations in these genes, allowing for a mechanistically simple but cognitively informative laboratory exercise on mutation, selection, and complementation.
Lysine biosynthesis in the yeast Candida maltosa: Properties …
The biochemical basis for selection on -aminoadipate can be explained in the context of the lysine biosynthesis pathway. The LYS2 and LYS5 genes each code for a separate subunit of the enzyme -aminoadipate reductase, which converts -aminoadipate into -aminoadipate-semialdehyde (). The inactivation of -aminoadipate reductase (via a mutation in LYS2 or LYS5) allows -aminoadipate in the medium to serve as a nitrogen donor to -ketoglutarate. This occurs by a reversal of the transamination step shown in . The glutamate formed in this reaction then serves as a nitrogen source for the synthesis of other amino acids. In a wild-type (LYS2 LYS5) strain, the active reductase enzyme readily converts -aminoadipate to -aminoadipate-semialdehyde, thus greatly reducing the reverse transaminase reaction ().
LYS14 mutations are also selected on -aminoadipate medium. The LYS14 gene encodes a DNA regulatory protein that increases the expression of several genes involved in lysine biosynthesis, including LYS2 and LYS5 (, ). Thus, in the absence of functional LYS14 gene product, the level of -aminoadipate reductase is sufficiently reduced for -aminoadipate to be the primary nitrogen source for the cell. Additionally, the lysine present in the selective medium represses activation by the LYS14 product.
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lysine biosynthetic process via aminoadipic acid | SGD
Once -aminoadipate-resistant mutants are isolated, the gene locus containing the mutation that confers resistance is identified by complementation analysis. “Tester” yeast strains containing a known mutation in lys2, lys5, or lys14 are each mated with the -aminoadipate-resistant mutant strains, thus forming stable diploids. The diploids are then replica plated onto medium lacking lysine. Growth of the diploid on this medium indicates a functional lysine synthesis pathway. Thus, the haploid parents contain mutations at different genetic loci, and the wild-type loci from each parent are able to “complement” each other, generating a functional pathway. The lack of diploid growth on medium lacking lysine indicates that the parent haploid strains harbor a mutation at the same genetic locus, and neither is able to provide a functional gene product to the synthesis pathway.
Lysine, an amino acid, and zinc, ..
At the conclusion of the exercise, students are required to write an individual report, 4 to 6 pages in length, which includes Introduction, Materials and Methods, Results, Discussion, and Literature Cited sections. The report must include an explanation of the biochemical basis of the -aminoadipate selection. For the discussion, students are expected to conduct a literature search and use the articles they find to explain their results. Basic goals are to have the students gain an understanding of how mutations in different genes in a biochemical synthesis pathway can give rise to similar phenotypes (in this case, -aminoadipate resistance) and to understand how variations in phenotype relate to gene function. To this end, we give the students two basic questions to be answered in the discussion: (i) why is the frequency of lys2 mutations obtained so much higher than that of the other mutations? and (ii) why is the lys14 mutation leaky? If Internet access is available, students can be directed to the Saccharomyces genome website (). A search by gene name will bring up the general gene information, the gene sequence, and links to references about the gene. Students may notice that LYS2 (4,179 bases) is much larger than the LYS5 gene (819 bases) and thus is simply a bigger target for genetic mutation. Students may also theorize how the function and nature of the protein affect what kind of mutations will abolish protein function. For example, the LYS2 structural gene may be much more sensitive to mutational changes than LYS5 or LYS14. Perhaps this subunit contains the active site or a critical structural domain. Students often come up with theories proposing hot spots for mutation, which may also be true. The LYS14 gene product activates several different genes and thus by nature is likely to have a less rigid structure that is tolerant of mutations. We encourage the students to research and think about their own theories to explain the results. Beyond answering the questions listed above, the students are encouraged to discuss relevant information they find concerning the pathway. We expect students to find references relating to the biochemical function of -aminoadipate reductase and the regulatory function of lys14 and to be able to summarize the information in the context of this experiment. Additionally, some students discuss other genes in the pathway and/or the medical significance of lysine.
Effects of Yeast Extract on Poly-ε-Lysine Production in …
The yeast Saccharomyces cerevisiae is a commonly used organism in cell and molecular biology research (reviewed in reference ). The simple growth requirements and rapid division time of yeast cells make them convenient for laboratory exercises in microbiology, cell biology, and genetics (). The laboratory exercise described here uses the lysine synthesis pathway to explore the concepts of amino acid biosynthesis, mutation, and complementation analysis. Laboratory exercises involving yeast adenine mutants have previously been published (, ). The system we describe requires a more in-depth study of a biochemical pathway, as the selection is based on the use of a specific nitrogen source. Additionally, mutations at several genetic loci are selected so that complementation testing can be used to identify the mutant locus. Finally, the resulting mutation may be in either an enzyme or a regulatory protein, so that a full understanding of the exercise requires a student to comprehend how protein function relates to phenotype.
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