Genetics 200 A
Fall 2009
Hiten Madhani
Handout 2
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
Genetics 200 A
Fall 2009
Hiten Madhani
Handout 2
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
Genetics 200 A
Fall 2009
Hiten Madhani
Handout 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
Genetics 200 A
Fall 2009
Hiten Madhani
Handout 2
If A(a) and B(b) are freely separable from their centromeres, what will be PD:NPD:T?
a
4
Genetics 200 A
Fall 2009
Hiten Madhani
Handout 2
Summary of Tetrad Analysis
Genes on separate chromosomes
1. Both genes inseparable from their centromeres 2. One inseparable from centromere, but other is linked
3. One inseparable from centromere, other far
4. Both far
PD:NPD:T
1:1:0
1:1:<4
1:1:4
1:1:4
Genes on the same chromosome
1. Linked
2. Far from each other
PD:NPD:T
PD>NPD*
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
Genetics 200 A
Fall 2009
Hiten Madhani
Handout 2
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
Genetics 200 A
Fall 2009
Hiten Madhani
Handout 2
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
Genetics 200 A
Fall 2009
Hiten Madhani
Handout 2
single-end invasion
DNA synthesis
second end capture
hDNA
heteroduplex DNA
(hDNA)
8
Genetics 200 A
Fall 2009
Hiten Madhani
Handout 2
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
Genetics 200 A
Fall 2009
Hiten Madhani
Handout 2
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
Genetics 200 A
Fall 2009
Hiten Madhani
Handout 2
pathway that operates in budding yeast that is mediated by the Mus81 protein. It turns out that
different organisms use different pathways:
S. cerevisiae
dHJ Pathway
(crossover)
X
SDSA pathway
(non-crossover)
X
X
S. pombe
Mus81 pathway
(crossover)
X
X
C. elegans
X
X
D. melanogaster
X
X
X
Mice/Humans
X
X
X
11
Genetics 200 A
Fall 2009
Hiten Madhani
Handout 2
Appendix: The Perkins Formula [Perkins(1949) Genetics 34,607]
12
Genetics 200 A
Fall 2009
Hiten Madhani
Handout 2
13