Genetic complementation is carried out by crossing the Yfg^- MATa mutant to each of the tester strains MATa yfg1, MATa yfg2, etc., as well as the normal control strain MATa. These yfg1, yfg2, etc., are previously defined mutations causing the same phenotype. The diploid crosses are isolated and the Yfg trait is scored. The Yfg⁺ phenotype in the heterozygous control cross establishes that the Yfg^- mutation is recessive. The Yfg^- phenotype in MATa yfg1 cross, and the Yfg⁺ phenotype in the MATa yfg2, MATa yfg3, etc., crosses reveals that the original Yfg^- mutant contains a yfg1 mutation.

Meiotic analysis can be used to determine if a mutation is an alteration at a single genetic locus and to determine genetic linkage of the mutation both to its centromere and to other markers in the cross. As illustrated in Figure 7.1, the MATa yfg1 mutant is crossed to a normal MATa strain. The diploid is isolated and sporulated. Typically, sporulated cultures contain the desired asci with four spores, as well as unsporulated diploid cells and rare asci with less than four spores. The sporulated culture is treated with snail extract which contains an enzyme that dissolves the ascus sac, but leaves the four spores of each tetrad adhering to each other. A portion of the treated sporulated culture is gently transferred to the surface of a petri plate or an agar slab. The four spores of each cluster are separated with a microneedle controlled by a micromanipulator. After separation of the desired number of tetrads, the ascospores are allowed to germinate and form colonies on complete medium. The haploid segregants can then be scored for the Yfg⁺ and Yfg^- phenotypes. Because the four spores from each tetrad are the product of a single meiotic event, a 2:2 segregation of the Yfg⁺:Yfg^- phenotypes is indicative of a single gene. If other markers are present in the cross, genetic linkage of the yfg1 mutation to the other markers or to the centromere of its chromosome could be revealed from the segregation patterns.

The molecular characterization of the yfg1 mutation can be carried out by cloning the wild-type YFG1⁺ gene by complementation, as illustrated in Figure 7.1 and described below (Section 11.1 Cloning by Complementation).

 

Figure 7.1. General approaches for genetic analysis. As an example, a MATa strain is mutagenized and a hypothetical trait, Yfg^- (Your Favorite Gene) is detected. The Yfg^- mutant is analyzed by three methods, complementation, meiotic analysis and molecular cloning (see the text).

7.2 Tetrad analysis

Meiotic analysis is the traditional method for genetically determining the order and distances between genes of organisms having well-defined genetics systems. Yeast is especially suited for meiotic mapping because the four spores in an ascus are the products of a single meiotic event, and the genetic analysis of these tetrads provides a sensitive means for determining linkage relationships of genes present in the heterozygous condition. It is also possible to map a gene relative to its centromere if known centromere-linked genes are present in the cross. Although the isolation of the four spores from an ascus is one of the more difficult techniques in yeast genetics, requiring a micromanipulator and practice, tetrad analysis is routinely carried out in most laboratories working primarily with yeast. Even though linkage relationships are no longer required for most studies, tetrad analysis is necessary for determining a mutation corresponds to an alteration at a single locus, for constructing strains with new arrays of markers, and for investigating the interaction of genes.

Figure 7.2. Origin of different tetrad types. Different tetrad types (left) are produced with genes on homologous (center) or nonhomologous (right) chromosomes from the cross AB x ab. When PD > NPD, then the genes are on homologous chromosomes, because of the rarity of NPD, which arise from four strand double crossovers. The tetratype (T) tetrads arise from single crossovers. See the text for the method of converting the %T and %NPD tetrads to map distances when genes are on homologous chromosomes. If gene are on nonhomologous chromosomes, or if they greatly separated on the same chromosome, then PD = NPD, because of independent assortment, or multiple crossovers. Tetratype tetrads of genes on nonhomologous chromosomes arise by crossovers between either of the genes and their centromere, as shown in the lower right of the figure. The %T can be used to determine centromere distances if it is known for one of the genes (see the text).

There are three classes of tetrads from a hybrid which is heterozygous for two markers, AB x ab: PD (parental ditype), NPD (non-parental ditype) and T (tetratype) as shown in Figure 7.2. The following ratios of these tetrads can be used to deduce gene and centromere linkage:

	PD	NPD	T
	AB	aB	AB
	AB	aB	Ab
	ab	Ab	ab
	ab	Ab	aB
Random assortment	1 :	1 :	4
Linkage	>1 :	<1
Centromere linkage	1 :	1 :	<4

There is an excess of PD to NPD asci if two genes are linked. If two genes are on different chromosomes and are linked to their respective centromeres, there is a reduction of the proportion of T asci. If two genes are on different chromosomes and at least one gene is not centromere-linked, or if two genes are widely separated on the same chromosome, there is independent assortment and the PD : NPD : T ratio is 1 : 1 : 4. The origin of different tetrad types are illustrated in Figure 7.2.

The frequencies of PD, NPD, and T tetrads can be used to determine the map distance in cM (centimorgans) between two genes if there are two or lesser exchanges within the interval:

The equation for deducing map distances, cM, is accurate for distances up to approximately 35 cM. For larger distances up to approximately 75 cM, the value can be corrected by the following empirically-derived equation:

Similarly, the distance between a marker and its centromere cM', can be approximated from the percentage of T tetrads with a tightly-linked centromere marker, such as trp1:

The inheritance of non-Mendelian elements can be revealed by tetrad analysis. For example, a cross of r⁺ MATa and r^- MATa haploid strains would result in r⁺ MATa/MATa and r^- MATa/MATa diploid strains, the proportion of which would depend on the particular r^- strain. Each ascus from a r⁺ diploid strain contains four r⁺ segregants or a ratio of 4:0 for r⁺:r^-. In contrast, a cross of pet1 MATa and PET1⁺ MATa strains would result in a PET1⁺/pet1 MATa/MATa diploid, which would yield a 2:2 segregation of PET1⁺/pet1. Similar, the other non-Mendelian determinants also produce primarily 4:0 or 0:4 segregations after meiosis.

Another means for analyzing non-Mendelian elements is cytoduction, which is based on the segregation of haploid cells, either MATa or MATa, from zygotes. Haploid cells arise from zygotes at frequencies of approximately 10^-3 with normal strains, and nearly 80% with kar1 crosses, such as, for example, kar1 MATa x KAR1+ MATa. While the haploid segregants from a kar1 cross generally retains all of the chromosomal markers from either the MATa or MATa parental strain, the non-Mendelian elements can be reassorted. For example, a MATa canR1 kar1 [r^- y^- kil-o] x MATa CAN^S1 [r⁺ y⁺ kil-k] cross can yield MATa can^R1 kar1 haploid segregants that are [r⁺ y⁺ kil-k], [r^- y⁺ kil-k], etc. In addition, high frequencies of 2 mm plasmids and low frequencies of chromosome can leak from one nucleus to another.