6.4: Genetic analyses of methionine biosynthesis

Looking at the pathway for Met biosynthesis later in this chapter, you may wonder how the gene numbers became associated with specific genes, since the numbers do not correspond to the positions of the reactions encoded by the MET gene products in the pathway. The numbering system reflects the discovery process for the MET genes. The first studies of Met biosynthesis
in yeast were done by geneticists, who used classical genetic screens to isolate met mutants. Genetic screens are important tools for identifying new genes because they are unbiased by prior knowledge of the pathway. In addition, mutation is a random process that should affect all genes involved in producing the phenotype under study. The geneticist begins by treating a parent strain with a chemical or radiation to induce mutations in DNA. The spontaneous mutation rate in
yeast is ~10-8/base/generation, which is much too low for a practical genetic screen. Investigators therefore use mutagen doses that kill up to ~50% of the cells. Cells that survive the mutagenesis typically harbor a large number of mutations, many of which have no effect on the phenotype
that is being screened. Consequently, large numbers of cells are required to uncover all the genes involved in the phenotype. For example, the yeast genome contains ~6000 genes, so a useful genetic screen might involve 20,000 or more cells.

Selective media provide important tools for identifying mutant phenotypes in genetic screens. Depending on the phenotype being studied, investigators may select for mutants using either a positive or negative selection scheme, as shown on the opposite page. The easiest kinds of screens employ positive selection, because only mutant cells grow on selective media. If investigators are analyzing pathways that are important for cell growth, such as Met synthesis, they would probably use a negative selection scheme. In a negative scheme, cells are first cultured

Selection strategies used to isolate yeast mutants.

After the initial mutagenesis, yeast are grown on a plate containing rich (or complete synthetic) media. In thisfigure, the mutagenesis has generated three different mutants in the gene of interest. The mutant colonies
are surrounded by an empty circle. Replicas of the master plate are copied to selective media. In a negative selection scheme, the selective plate lacks a component that is normally present in rich media. In a positive selection scheme, the media contains a selective agent, which is toxic to normal cells, but tolerated bymutant cells. The selective agent is sometimes a toxic analog of a normal cellular metabolite.

on media, such as YPD or YC, that allow all cells to grow. Replicas of these master plates are then made on defined media lacking Met. (Replica plating is outlined in Chapter 12.) Since only wild-type cells grow on the selective media lacking Met, researchers look for colonies on the rich media whose counterparts are missing on the selective media.

The number and spectrum of mutants obtained in a genetic screen are unpredictable, because of the random nature of mutation. As you might expect, a screen might produce
multiple mutants in one gene and no mutations in other genes involved in the phenotype.
After completing a screen, investigators must next determine if the mutations are in the
same or different genes. For this, geneticists rely on genetic mapping (Chapter 5) and/or complementation. Complementation is a functional test of gene activity. In a complementation experiment, introduction of a functional gene from another source rescues a mutant phenotype caused by the defective gene. Classic genetic complementation in yeast takes advantage of the
two yeast mating types and the ability of yeast to survive as both haploid and diploid strains. In a complementation experiment with met mutants, researchers mate a haploid met mutant in either the a or a mating type (MATa or MATa) with a haploid met mutant of the opposite mating type. If the diploid is able to grow in the absence of Met, complementation has occurred, and the metmutations in the two haploid strains must be in different genes. If the diploid is not able to survive on the selective plate, the two haploid strains carry mutations in the same gene (although they are almost certain to be different mutant alleles). A genetic screen can yield multiple mutant alleles of the same gene, which together form a complementation group.

By 1975, yeast labs had isolated collections of met mutants and mapped nine of the metmutations to chromosomes. In a landmark study, Masselot and DeRobichon-Szulmajster (1975) collected 100 met strains from labs around the world and did systematic complementation experiments with all the mutants. Twenty-one complementation groups, representing potential genes, were identified, and the genes were assigned names MET1 through MET25. Many of theMET genes encode enzymes in the Met biosynthetic pathway, which is outlined on the opposite page. Some gene products are involved in the synthesis of cofactors and methyl donors used
in the pathway, while other MET gene products (not shown) are involved in regulation of the pathway (reviewed in Thomas & Surdin-Kerjan, 1992). For the most part, the names assigned in the 1975 study are still used today. A few genes identified in the 1975 study were subsequently shown not to be involved in Met biosynthesis, and others (e.g. MET15, MET17 and MET25) were later shown to represent different alleles of the same gene (D’Andrea et al., 1987).

At the time of the 1975 study, the biochemical reactions in the pathway were largely known, and scientists faced the challenge of associating genes with enzymatic activities. You can see from the pathway that mutations in 11 different MET genes would produce a phenotype in which strains would grow in the presence of methionine, but not in its absence. The scientists narrowed down possible gene-enzyme relationships by analyzing the ability of met strains to
use alternative sulfur sources in the place of methionine (Masselot & DeRobichon-Szulmajster, 1975). Yeast are very versatile in their use of both inorganic and organic sulfur sources. Sulfate is efficiently transported into cells by the Sul1p and Sul2p transporters in the membrane. Sulfite and sulfide are also transported into the cells with a reduced efficiency. Yeast are also able to transport and use Met, Cys, homocysteine and S-adenosylmethionine (AdoMet or SAM) as sulfur sources (reviewed in Thomas and Surdin-Kerjan, 1992). In this lab, you will use selective media in which sulfite or cysteine replaces methionine to distinguish between 3 met mutants. You will also use a differential medium, BiGGY agar, that distinguishes yeast strains by their production of hydrogen sulfide. Differential media allows all mutants to grow, but the mutants produce colonies that can be distinguished from one another by their color or morphology.

Note

The met mutants used in this course were NOT generated by traditional mutagenesis. Instead, the mutants were constructed by a newer molecular approach that requires detailed knowledge of the yeast genome sequence. After the yeast genome project was complete, researchers were interested in obtaining a genome-wide collection of deletion strains, each of which differed from the parental BY4742 strain at a single gene locus. Their approach, which
is discussed in more detail in Chapter 7, takes advantage of the high frequency with which S. cerevisiae undergoes homologous recombination. Each ORF in the S. cerevisiae genome was systematically replaced with a bacterial KAN^R gene (Winzeler et al., 1999). A major advantage of this strategy, sometimes referred to as “reverse genetics,” over the traditional genetic approach is that positive selection can be used to isolate mutants. Only strains with disrupted MET genes are able to grow on media containing analogs of kanamycin. Strains with KAN^R-disrupted genes have other advantages over mutant strains generated with chemical mutagens or radiation treatment.

The strains do not harbor secondary mutations induced by the mutagen treatment and spontaneous reversion to a wild type phenotype is not possible.

Methionine biosynthesis in yeast.

The proteins catalyzing individual steps in Met and Cys biosynthesis are listed next to each step in the pathway. The names of the genes encoding the activities are shown in italicized capital letters, following
S. cerevisiae conventions. The MET1 and MET8 genes encode proteins that are involved in synthesizing siroheme, an essential cofactor for sulfite reductase. The MET7 and MET13 gene products catalyze the last two steps in the synthesis of the methyl donor used by Met6p, homocysteine methyltransferase, to synthesize methionine. (Adapted from Thomas et al.,1992)