CLASSICAL GENETICS: TETRAD ANALYSIS and RECOMBINATION References 1. Perkins, D.D. (1962) Crossing-over and interference in a multiply marked chromosome arm of Neurosopora. Genetics 47, 1253-1274. Classic paper describing the analysis of 1262 hand-dissected tetrads 2. Szostak, J.W., Orr-Weaver, T.L., Rothstein, R.J. and Stahl, F.W. (1983) The double-strand-break repair model for recombination. Cell 33, 23-25. The observation that double-stranded breaks stimulate recombination during mitosis prompts a model for meiotic recombination. 3. Sun, H., Treco, D. Schultes, N.P. and Szostak, J.W. (1989) Double-strand breaks at an initiation site for meiotic gene conversion. Nature 338, 87-90. Physical evidence for the model: Identification of double-stranded breaks at a meiotic recombination hotspot. 4. Keeney, S., Giroux, C.N. and Kleckner, N. (1997) Meiosis-specific DNA double-strand breaks are catalyzed by Spo11, a member of a widely conserved protein family. Cell 88, 375-84. Identification of Spo11, a topoisomerase-like protein, covalently coupled to DNA at the site of the break. 5. Allers, T. and Lichten, M. (2001) Differential Timing and Control of Noncrossover and Crossover Recombination during Meiosis. Cell 106, 47-57. Initial evidence that the double-stranded break model is not correct in detail. 6. Lorenz, A. and Whitby (2006) Crossover promotion and prevention. Biochemical Society Transactions 34, 537-540. A clear review describing the known eukaryotic meiotic genetic recombination pathways Learning objectives: Describe Tetrad Types produced by meiosis Understand genetic consequences of linkage of genes to centromeres Undersand how the 1:1:4 ratio of tetrad types occurs for genes unliked to each other and their centromeres Know the Perkins Formula Define gene conversion Understand current models for genetic recombination and their consequences for meiosis The Awesome Power of Tetrad Analysis Review from previous lecture: 1) homologs disjoin during meiosis I, 2) sister chromatids disjoin in meiosis II producing four haploid products. In ascomycete fungi (such as S. cerevisiae and S. pombe), these four products are packaged in an ascus (a sac) and the four spores are termed a tetrad. These spores can be separated and germinated (grown) to produce haploid colonies which can be analyzed for their genotype. We will consider the segregation of two genes, A and B, in a cross. Three types of tetrads can be produced: parental ditype (PD) in which the genotype of each spore is the same as one of the two parents, nonparental ditype (NPD) in which the genotype of each of the spores is different from that of the parents, and tetratype (T) in which all 4 combination of alleles are present. For example in the cross ABxab, a PD tetrad would contain AB AB ab ab; an NPD tetrad would be Ab Ab ab ab; and a T tetrad would correspond to AB Ab ab ab. 1
Consider the cross ABxab. If A(a) and B(b) are on different chromosomes but inseparable from their centromeres: A segregates from a in the first division and B segregates from b in the first division. PD:NPD:T = 1:1:0. Recombination frequeny=recombinant products/total = 50% 2
Consider: A(a) locus is inseparable from its centromere but B(b) locus is close but separable from its centomere: With no crossover between B and its centomere, PD and NPD tetrads are produced as in the previous case because B and b segregate from each other in the first division. With a crossover between B and its centromere, B and b segregate from each other in the second division producing a tetratype (T). T s arise from crossovers between a locus and its centomere. Distance to centromere = 50 x Tetratypes/Total (n.b. formula only works for genes fairly close to the centromere). 3
If A(a) and B(b) are freely separable from their centromeres, what will be PD:NPD:T? a 4
Summary of Tetrad Analysis Genes on separate chromosomes PD:NPD:T 1. Both genes inseparable from their centromeres 1:1:0 2. One inseparable from centromere, but other is linked 1:1:<4 3. One inseparable from centromere, other far 1:1:4 4. Both far 1:1:4 Genes on the same chromosome PD:NPD:T 1. Linked PD>NPD* 2. Far from each other 1:1:4 *Do you see why this is the case? Production of anything other than a PD ascus requires one or more crossovers between the two loci (see Appendix). *A Chi-squared test can be use to determine whether PD>NPD. See Chi-squared analysis handout for some examples. Determining Genetic Distances The Perkins Formula (see Appendix for a derivation) allows the calculation of map distances. What is remarkable is that because tetrad analysis follows all of the products of a single meiosis, distances greater than 50 cm can be determined from a single cross involving two loci! Note that in the examination of progeny from a cross at random (random spore analysis), two unlinked loci produce a recombination frequency of 50%. The formula: Distance in centimorgans= 50(T+6NPD)/(T+NPD+PD) 5
Questions (for 1-3 the question is: where are the two genes located with respect to each other?): 1. pet8 TRP1 x PET8 trp1 PD=NPD, no T 2. pet8 GAL1 x PET8 gal1 PD=NPD, 15% of tetrads are T 3. lys2 GAL1 x LYS2 gal1 PD:NPD:T = 101:23:259 4. What is the definition of a centromere? 5. What might 4:0 segregation of a phenotype mean? 6
Gene Conversion: Nonreciprocal transfer of genetic information Exceptions to the 2:2 segregation pattern are seen. For example, in a cross between ARG4 and arg4, 2% of the tetrads demonstrate 1:3 segregation and 2% a 3:1 segregation. About 1/3 of the time, this pattern is associated with a crossover betweeen the flanking markers PET1 and THR1. The nonreciprocal transfer of information is called gene conversion. This observation together with the observation that that double-stranded breaks stimulate recombination during mitosis led to the double-strand break model for genetic recombination. 7
single-end invasion DNA synthesis second end capture hdna heteroduplex DNA (hdna) 8
Resolution, Cross-overs, Mismatch repair, and Gene Conversion What about gene conversion that occur without crossing over? It turns out that after the single suppressed by unknown mechanism! M vs. m is a difference in the DNA sequences between the parental chromosomes in the cross (e.g. a mutation!) 9
end invasion step of double-strand break repair, repair can occur via a different pathway that does not produce a crossover called synthesis-dependent strand annealing (SDSA). Sounds complicated, but it isn t: In addition to the double-holiday junction and SDSA pathway, there is a third recombination 10
pathway that operates in budding yeast that is mediated by the Mus81 protein. It turns out that different organisms use different pathways: dhj Pathway (crossover) SDSA pathway (non-crossover) Mus81 pathway (crossover) S. cerevisiae X X X S. pombe X X C. elegans X X D. melanogaster X X X Mice/Humans X X X 11
Appendix: The Perkins Formula [Perkins(1949) Genetics 34,607] 12
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