In most eukaryotic organisms, the products of different meiotic cells are released together into one organ, so it is not possible to identify which gametes arose from a particular meiotic cell, or meiocyte, at the beginning of prophase I. However, for fungi known as ascomycetes, the four haploid products of meiosis are all found together in a common sac, called an ascus. Our ability to recover all of the meiotic products together and know that they occur as a result of the same events in meiosis has greatly
furthered our understanding of meiosis. The topics in this Tool Box have a close connection to Chapter 9 when we will discuss how recombination is used to determine the location of genes on chromosomes. We introduce them here to re-enforce your understanding of the behavior of homologous chromosomes and sister chromatids at MI and MII, without focusing on how these events are used to create genetic maps.
The four products of meiosis correspond to the four chromatids—two sister chromatids from a pair of homologous chromosomes—as shown in Figure A. These four chromatids form a tetrad. Each chromatid in ascomycetes will become one ascospore; the analysis of these ascospores is known as tetrad analysis. Since each ascospore corresponds to one chromatid, each ascospore is haploid. (The ascospore has one chromatid for each of the chromosomes in the haploid set, but typically only one or a few chromosomes are considered in tetrad analysis, which is how Figure A shows it.) Most eukaryotes can only be grown as diploids, with the haploid phase limited to the gametes. By contrast, ascospores grow into colonies or filaments as haploids, so their genotype can be directly scored from the phenotypes.
Figure A: Tetrads and ascospores. In fungi known as ascomycetes, each of the four chromatids of a tetrad at meiosis becomes one ascospore, which can then grow into haploid colonies or filaments, revealing their genotypes. While this drawing has the chromatids in the tetrad and the ascospores in the ascus in the same order (chromatid 1 gives rise to the ascospore at the top, for example), this only occurs in fungi with ordered tetrads and is presented in this way for clarity.
While many different types of genetic variants are known, the most widely used are nutritional variants or auxotrophs. These are mutants that have lost the ability to produce certain nutrients, and thus they cannot grow unless that nutrient is added to their growth media. For example, ade mutants (called ad in the ascomycete Neurospora) cannot grow, unless adenine is added to the media, since the mutation eliminates the ability of the cell to make its own adenine. This concept was introduced in Chapter 2 with the experiments of Beadle and Tatum who used auxotrophic mutants of Neurospora that could not synthesize arginine.
Ditypes and tetratypes
Let’s consider an example using the budding yeast Saccharomyces cerevisiae, one of the best studied eukaryotes and the most familiar ascomycete. A haploid strain that cannot grow without added folic acid (fol2) and without additional adenine (ade3) is crossed to a haploid strain that grows without any additional nutrients; the two genes are on the same chromosome. The resulting diploid cell is allowed to undergo meiosis, called sporulation in yeast and other fungi. The ascus containing the ascospores is collected; the ascus is cut open, and the ascospores are grown on media with different added nutrients to determine their genotypes.
The fol2 and ade3 genes are located near each other on the same chromosome, as illustrated in Figure B. Note that one of the parents lacked both the ability to make folic acid and the ability to make adenine, so one of its chromosomes is labeled fol2 ade3 (on both chromatids), while the other parental strain could make both chemicals and is labeled fol2+ ade3+. Each homologue is shown in Figure B(i). This diploid cell then goes through meiosis, and a crossover forms at some location between these homologues. If the crossover is not between these two genes but occurs somewhere else on the chromosome, as shown in Figure B(i), two types of spores will be found in the ascus—ones that can make neither folic acid or adenine, and ones that can make them both. Because there are only two types of ascospores, this is known as a ditype ascus. Because the two types have the same genotypes as the two original parental strains, this is known as a parental ditype.
Restriction enzymes can also be used as tools for analyzing natural variation in DNA sequences simply because their recognition sites are specific. An example is shown in Figure C. One individual may have the sequence GAATTC at a specific site while a different individual may have AAATTC at the same site. The change in the nucleotide sequence can be detected because the site in the first individual can be cut in vitro with EcoRI while the site in the second individual cannot but cut. This will result in a size difference in the length of the restriction fragment between the two individuals that can be readily detected by agarose gel electrophoresis. This is known as a restriction fragment length polymorphism or RFLP. RFLPs provide yet another phenotype, one that could be correlated with one observed from another assay but that does not need to be; it is a direct look at the underlying DNA sequence. Because many hundreds of restriction enzymes are known, each with a specific recognition site, tens of thousands of RFLPs have been found as natural variation among genomes.
Figure C: Ordered tetrads. This figure show ascospores and tetrads as they would occur in Neurospora, in which the diploid cell is heterozygous for the ad-6 gene. The colors represent the genotype at the ad-6 locus, which is also written out. (i) The crossover has occurred between the ad-6 gene and the centromere. Two important features are shown. First, the ascospores always form in the same order as the chromatids in the tetrad. To see this, the chromatids can be traced, beginning at the centromere on the right. This results in ordered tetrads. Second, the haploid product of meiosis undergoes a mitotic division, as indicated by the red circle and the branching arrows, so there are eight ascospores. The pattern of the ascospores is 2:2:2:2 when the crossover occurs between the gene and the centromere. (ii) An ascus in which the pattern of the ascospores is 4:4 is shown. This arises when the crossover does not occur in the region between the centromere and the gene, shown here to the left of ad-6.
In Figure C(i), the crossover occurred between the centromere and the gene ad-6. The ascospores have the arrangement 2:2:2:2—two with ad-6, two with +, two with ad-6, and two with +. This is known as a second division segregation because the ad-6 alleles (on chromatids 1 and 2) separated with their centromere
at the second meiotic division; in other words, when the centromeres divided at MII, the ad-6 alleles divided as well. In Figure C(ii), the crossover did not occur between the centromere and ad-6, so the ascospores have the arrangement 4:4. This is called a first division segregation, because the ad-6 allele and the + allele segregated from each other at the first meiotic division.
The actual process of tetrad analysis is, in fact, done in the reverse order of how we have just described it, that is, the genotypes of the ascospores are observed and the location of the crossover is inferred from the pattern in the ascus. When the molecular composition of centromeres was not known, it was sometimes difficult to locate them on the chromosome with respect to any of the genes. Crosses in these organisms helped to demonstrate the behavior of the centromere at MI and MII and ultimately led to an understanding of their molecular composition.
Tetrads and gene conversion
Let’s return to a single gene and the centromere to illustrate another important point about meiosis that was found from tetrad analysis. With ordered tetrads, a crossover between the gene and the centromere results in the 2:2:2:2 ascus, shown in Figure C(i).
However, when such experiments were performed in the 1950s, a different type of ascus was also often found. This ascus had a 6:2 arrangement of the ascospores; rather than having four ascospores of each type, there were six of one type and only two of the other type, as shown in Figure D. This was unexpected when it was first encountered because it requires the crossover to be non-reciprocal between the two chromatids, so that more of one type were found than the other type. The terminology is that the ad-6 allele was “converted” to an ad-6+ allele, so a 6:2 ascus is called a gene conversion. The frequency of gene conversion varies with the organism, but it is found in all eukaryotes and is not rare; it is often as common as reciprocal exchange.
Figure D: Gene conversion. While 4:4 and 2:2:2:2 asci are the expected types, as shown in Figure C, another frequent type is an ascus with a 6:2 (or 2:6) pattern of ascospores. From this pattern, it is inferred that one of the ad-6 alleles (dark blue) was “converted” to an ad-6+ allele (light blue) by some process. This process is called gene conversion; the presence of gene conversion provided important insights into the mechanism by which crossing over occurs. While this example shows that ad-6 is converted to ad-6+, the opposite also occurs, whereby ad-6+ is converted to ad-6. The rest of the chromatid involved in conversion is shown dotted and in a different shade because we cannot infer what has occurred, except at the centromere (from the order of the ascospores) and at the ad-6 locus (from the growth on medium lacking adenine).
Gene conversion, which can occur from the + allele to the mutant allele, or vice versa, was important in constructing models by which recombination occurs. The Holliday junction model for recombination that is described in Box 6.2 was designed specifically to account for the high frequency of gene conversion. If the Holliday junction diagrammed in Box 6.2 Figure A is resolved by “cutting” the strands horizontally, rather than vertically, gene conversion occurs. Thus, gene conversion, like crossing over itself, is not an unusual feature that occurs only occasionally during meiosis. Rather, it is an inevitable outcome of the process by which reciprocal exchange occurs and was important evidence for understanding how recombination itself happens. While we usually focus on reciprocal exchanges, unless the specifics of meiosis are being investigated, gene conversion is an equally important biological process.
Most examples of gene conversion arise from mismatch repair, a process discussed in Chapter 4. During the process of recombination, discussed in Box 6.2, the DNA molecule has mismatched bases. Imagine that the ad-6 allele has an A:T base pair, while the ad-6+ allele has G:C at the same site. During recombination, the A can end up paired with the C, a mismatch. This could be repaired to an A:T base pair, which is the ad-6 allele. Alternatively, it could be repaired to a G:C base pair, which is the ad-6+ allele. Thus, conversion will occur because
recombination creates mismatches, which can be repaired to either base or allele.
The term “gene conversion” is often used more generally now to refer to many types of exchange in eukaryotes that are directional and not reciprocal, resulting in more of one type of haploid product than the other. However, the original (and still primary) use of the term came from an analysis of tetrads and the unexpected occurrence of asci with a 6:2 arrangement of ascospores.