Textbook assignment: Chapter 5, pages 132-135, Chapter 7, pages 194 - 200 (plus chapter end questions that also apply to lecture 14).
Major concepts
Introduction: The basic concept of tetrad analysis is briefly introduced by the textbook in Chapter 5, pages 132-135, and then analyzed in greater detail in chapter 7, pages 194-200. The textbook uses the mold Neurospora to illustrate the analysis of ordered spores and the green alga, Chlamydomonas, to illustrate the analysis of unordered spores. Because the widely studied budding yeast, Saccharomyces cerevisiae, produces unordered spores similar to those of Chlamydomonas, I have also included material on yeast spores that is not covered in the thextbook. .
Tetrad analysis: In the diploid multicellular organisms that have until now received most of our attention, we have only been able to sample single products of meiosis (randomly selected haploid genomes contained in egg, sperm, or pollen cells). For a more detailed analysis of meiosis, it is desirable to sample all four products from the same meiotic event. This is possible in a number of "simple" organisms in which all four products of meiosis form viable spores that can be recovered individually and analyzed genetically.
Ordered spores: The mold Neurospora crassa produces an ordered set of spores that provide highly detailed information about the products of meiosis. Haploid cells of two different mating types fuse to form a diploid cell (Fig. 7.10), which immediately undergoes meiosis. The developing haploid spores are enclosed in a membranous structure called an ascus, which has an elongated shape and is narrow enough so that nuclei do not move past each other. A single mitotic division occurs after meiosis is compleded such that of each of the four initial products of meiosis forms two identical spores. This permits the meiotic products to be sampled twice as neighboring spores. Because the spores remain physically ordered as they are generated by the meiotic (and mitotic) divisions, it is possible to determine precisely at which meiotic division genetic segregation is occurring (Figures 5.16 and 7.11). The products of first meiotic division are arranged 4:4 along the length of the ascus, and the products of second division are arranged in a 2:2:2:2 relationship.
Using centromeres as markers: Because of the ordered positions of the spores in the ascus, it is possible to use the centromere of each chromosome as a genetic marker. The centromeres from the homologous parental chromosomes separate at the first division and thus exhibit a 4:4 distribution in the ascus. If a crossover occurs between the centromere and a heterozygous genetic marker, the recombinant chromatids will now have an allele from one of the parental chromosomes associated with the centromere from the other. When the recombinant sister chromatid separates from the parental sister chromatid at the second meiotic division, the two products of that division will remain side by side in one half of the ascus, and will divide once mitotically to generate two parental and two recombinant spores. The same thing will also happen in the other half of the ascus, as shown in parts c and d of Figure 5.16, and parts b and c of Figure 7.11.
2:2:2:2 distributions: The formation of two parental and two recombinant spores in each half of the ascus will result in a 2:2:2:2 distribution of possible combinations of centromeres and genetic markers. Although the two centromeres cannot be distinguished in the progeny, the descendants of the two original centromeres remain in the two halves of the ascus in a 4:4 distribution. Thus if a crossover has occurred between non-sister chromatids in the tetrad, there will be a 2:2 distribution of the heterozygous genetic marker in each half of the ascus. If there is no crossover between the marker and the centromere, each of the markers will stay with its original centromere to generate a 4:4 distribution (Fig. 5.16, part a, and Fig. 7.11, part a).
2:4:2 distributions" Because the orientation of the second meiotic division is random, the order of the pairs of parental and recombinant spores in each half of the ascus is also random, generating four possible sequences (the two shown in figure 7.11 plus two others with the position of the orange and blue dots and their accompanying alleles reversed). These four possibilities can be visualized as follows:
where + is wild type at the locus under study and a represents a mutation at that locus. Note that two of these four configurations are seen as a 2:4:2 distribution of ascospores. The critical requirement to verify that recombination has occurred is a 2:2 distribution in each half of the ascus.
Advantages of ordered ascospore analysis: Full analysis of ordered ascospores requires dissecting out the individual spores in order and keeping precise records of the cultures derived from individual spores obtained from specific positions in the ascus. This is a tedious process, but it is essential for three types of study:
Map units in Neurospora: Since only half of the spores in each ascus are recombinant, a map unit is 100 times one half of the asci that contain recombinant spores, divided by the total number of asci. It is also possible to do three point crosses involving the centromere and two markers as well as conventional two point or three point crosses with genetic markers.
Unordered spores in Chlamydomonas: Our textbook uses the sexual cycle of Chalymdomonas, a eukaryotic green alga, to illustrate unordered tetrad anlaysis. Chlamydomonas grows in the vegetative state as a single-celled haploid alga, with two flagella on each cell. There are two genetically determined mating types, designated + and -. Under adverse conditions, such as nitrogen starvation, cells of the two types fuse to form a zygote, which then undergoes meiosis to yield four haploid cells (two of each mating type), which are temporarily enclosed in an ascus. Each of these four cells can be dissected out and grown into a new culture, permitting all four of the products of a single meiosis to be examined. However, the four meiotic products are randomly oriented in the ascus, such that the sequence of meiotic events cannot be analyzed.
Effects of recombination: The first meiotic division separates centromeres of homologous chromosomes and the second division separates sister chromatids. Thus, if there is no crossing over in a dihybrid test cross, two of the spores will be of one parental genotype and the other two of the other parental genotype (Fig. 7.13a). This is referred to as a parental ditype (PD) because it contains two types of spores that are both the same as the parental genotypes. If there is a single crossover event, there will be one spore with each of the parental genotypes and the other two will exhibit reciprical recombination (Fig. 7.13b). This is referred to as a tetratype (TT) because each ascus contains four different types of spores. It is also possible to obtain a non-parental ditype (NPD) if a second crossover involving the other two strands of the meiotic tetrad occurs (Fig 7.13 c,d). This is a relatively less frequent event, and is scored as a double crossover. A similar system of nomenclature can also be used to identify the possible products of independent assortment. However, in cases where independent assortment occurs, all four possible combinations of elleles would be expected to occur with equal frequency (Fig. 7.12).
Map units: A map unit is defined as a recombination frequency that yields 1% recombinant progeny. Because only half of the spores in a TT ascus are recombinant, whereas all spores in an NPD ascus are recombinant, map units are scored as one-half of the number of TT asci plus the number of NPD asci, divided by the total number of asci examined and multiplied by 100 to convert to percentage.
Yeast: The following information about the budding yeast, Saccharomyces cerevissiae, is taken from a lecture given in previous years before we used the current textbnook. This material formed the starting point for writing the description of unordered tetrad analysis in Chlamydomonas, with so much similarity between the two that many parts required little more than changing the name of the organism.
Types of yeast: Two very different types of yeast are widely used in genetic studies. Although they have retained certain shared properties, such as alcoholic fermentation, they have been evolutionarily separated from each other for as long as they have been separated from our own ancestors. They are budding yeast, Saccharomyces cerevisiae, which is widely used as a baking and brewing yeast, and fission yeast, Schizosaccharomyces pombe, which is used in the brewing of an African beer. Because references to "yeast" are often made rather casually as if all yeasts were similar, it is important to look for species identification and to be aware that the two types are very different in many aspects of their life cycles.
Yeast life cycles: In brief summary, S. cerevisiae, multiplies by budding, grows preferentially in the diploid state, and is triggered by starvation to undergo meiosis and form spores. When conditions improve, the spores germinate and become haploid vegetative cells, which normally undergo conjugation at the first opportunity, thus regenerating the diploid state. However, they can also be maintained as haploids. S. pombe, on the other hand, multiplies by a fission process that is very similar to mitosis in higher organisms, lives preferentially as a haploid, and is triggered by starvation to undergo conjugation to form a diploid, which then usually immediately undergoes meiosis and forms haploid spores. The spores germinate when conditions improve and proliferate as haploid vegetative cells. Some strains can be maintained in the diploid state through the use of selective media. S. pombe produces asci containing ordered spores, much like those described above for Neurospora. However, they are small and difficult to work with, and thus seldom studied.
Unordered spores: When a diploid S. cerevisiae (budding yeast) cell undergoes meiosis, all four of the resultant haploid nuclei form viable spores contained in a membranous structure called an ascus (plural = asci). The individual spores can be dissected out and cultured separately to give rise to haploid strains representing each of the four products of meiosis.
Effects of recombination: The first meiotic division separates centromeres of homologous chromosomes and the second division separates sister chromatids. Thus, if there is no crossing over in a dihybrid test cross, two of the spores will be of one parental genotype and the other two of the other parental genotype. This is referred to as a parental ditype (PD) because it contains two types of spores that are both the same as the parental genotypes. If there is a single crossover event, there will be one spore with each of the parental genotypes and the other two will exhibit reciprical recombination. This is referred to as a tetratype (TT) because each ascus contains four different types of spores. It is also possible to obtain a non-parental ditype (NPD) if a second crossover involving the other two strands of the meiotic tetrad occurs. This is a relatively less frequent event, and is scored as a double crossover.
Map units in yeast: A map unit is defined as a recombination
frequency that yields 1% recombinant progeny. Because only half
of the spores in a TT ascus are recombinant, whereas all spores
in an NPD ascus are recombinant, map units are scored as one-half
of the number of TT asci plus the number of NPD asci, divided
by the total number of asci examined and multiplied by 100 to
convert to percentage.