Download early RNs, crossing over initiates, then synapsis begins Chiasmata

Bouquet stage (transient),
Synapsis complete, Chiasmata
early RNs, crossing over
late RNs
(one per late
initiates, then synapsis begins
RN)
Synaptonemal complex
Most early RNs degrade
Actually two
sister chromatids
Spindle forms
Axial elements
Early RNs
Zygotene
RNs contain
enzymes
involved in
recombination
Pachytene spread in maize, silver stained to show Synaptonemal Complexes, with
each chromosome arm labeled and RNs identified (arrows)
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Table 1. Events in meioticprophase
Stage in meiotic
prophase
Chromosome
morphology
and SC
morphogenesis
Bouquet
formation
DSB repair
Cytological
signs of
recombination
Leptotene
axial elements
begin to
develop
telomeres
begin to
cluster
DSBs appear
early nodules
Zygotene
chromosome
synapsis
initiates
telomeres
tightly
clustered
DSBs
disappear
early nodules
Pachytene
chromosomes
fully synapsed
telomeres
disperse
double
Holliday
junctions
late nodules
Diplotene
SC
disassembled;
chromosomes
condense
mature
recombinants
chiasmata
Diakinesis
further
chromosome
compaction
chiasmata
The precise sequence of events varies somewhat from one organism to another. Meiotic
DSB repair has been studied exclusively in S . cerevisiae, whereas most observations of
recombination nodules and all observations of chiasmata have been made in other
organisms. (SC) Synaptonemal complex; (DSB) double-strand break.
Homolog A
Chromatid 1
3’ ATCGATCGATCG 5’
5’ TAGCTAGCTAGC 3’
Homolog A
Chromatid 2
3’ ATCGATCGATCG 5’
5’ TAGCTAGCTAGC 3’
Homolog B
Chromatid 1
3’ ATCG GCTAATCG 5’
5’ TAGCCGATTAGC 3’
Homolog B Chromatid 1 serves as template for DNA repair of Homolog A Chromatid 2 :
Homolog A
Chromatid 2
3’ A TCGGC CGATCG 5’
Homolog B
Chromatid 1
5’ TAGCCGATTAGC 3’
Homolog B
Chromatid 2
3’ ATCG GCTAATCG 5’
5’ TAGCCGATTAGC 3’
Homolog A
Homolog B
Assume the DSB occurs in Homolog A Chromatid 2:
5’ – 3’ exonuclease does its thing:
3’ A T
5’ TAGCTA
Chromatid 2
Chromatid 1
CGATCG 5’
GC 3’
5’ TAGCTA ATTA G C 3’
3’ ATCG GCTAATCG 5’
Two cuts are made to resolve double Holliday junction:
3’ ATCGAT
5’ TAGCTA
CGATCG 5’
GCTAGC 3’
3’ A T
5’ TAGCTA
CGATCG 5’
GC 3’
Homolog A
Homolog B
Chromatid 2
Chromatid 1
3’ A TC G
5’ TAGCCGATTA
Homolog A
Homolog B
Chromatid 2
Chromatid 1
5’ TAGCTAATTA
3’ ATCG
G CCGATCG 5’
GC 3’
5’ – 3’ exonuclease does its thing:
GC 3’
GCTAATCG 5’
2
The first and second halves of this sequence are swapped between homologs:
Homolog A
Chromatid 2
3’ A TC G GCTAATCG 5’
Homolog A
Chromatid 2
5’ TAGCTAATTA GC 3’
Homolog B
Homolog B
Chromatid 1
Chromatid 1
Then, remembering that there are two other chromatids that were not involved in
the recombination, and considering only the four polymorphic bases, and
considering only the 5’ – 3’ strand sequences, the four meiotic products are:
3’ ATCG GCCGATCG 5’
5’ TAGCCGATTAGC 3’
ATCG
HeteroduplexDNA strands are repaired by DNA mismatch repair enzymes. If the
top strand of chromatid is used as the correct strand and the bottom strand is
repaired, the result is:
Homolog A
Chromatid 2
CGA T
ATCG
OR
CGA T
ATCG
OR
TAAT
ATCG
OR
TAAT
CGGC
CGAT
CGGC
CGAT
CTAG
CTAG
CTAG
CTAG
3’ A TC G GCTAATCG 5’
5’ TAGC CGATTA GC 3’
Or if the bottom strand is used as “correct” and the top strand is repaired:
Homolog A
Chromatid 2
3’ A TC GATTAATCG 5’
5’ TAGCTAATTA GC 3’
1:1:2 ratio!
1:3 ratio
1:1 ratio
You might be able to see this if you could recover all four prod ucts of one meiosis
and if you were looking in exactly the right place at the right time!
Recombination Hotspots
Recombination frequency within and between a1 and sh1 in maize
(Civardi et al., 1994):
Physical Distance
Recomb. Freq.
Bp/ %recomb
Within a1
1 kbp
0.0046
217 bp
DNA sequence structure of two maize inbredsin a1- sh2 interval and cM
per bp in each region
Four expressed genes in this region
Between sh1 and a1
140 kbp
0.09
1,555 bp
To identify where the recombinations were occurring in this region, Yao
et al. (2002) did a similar study and mapped the recombination
breakpoints using a high density of markers. To identify the di fferent
sequence elements in the region, they sequenced the entire 140 kb
region from both parents.
“Interloop region” – no predicted or expressed genes
Genes tend to be more recombinogenic, retrotransposon regions tend to have little
recombination, but not all genes are hotspots and not all hotspots are genes.
Yao, Hong et al. (2002) Proc. Natl. Acad. Sci. USA 99, 6157-6162
Copyright ©2002 by the National Academy of Sciences
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Transposable elements
Using bronze mutants to study recombination
hotspots in homozygous backgrounds
•Ac transposable element is autonomous: it encodes the gene for the transposase
enzyme that allows it to jump. Some maize lines have active Ac.
•Ds element is non -autonomous: it is a derivative of Ac but its transposase gene
does not function. However, when Ac is present in same genome, Ds can jump with
the help of the transposase encoded by Ac
•Retrotransposons – transposons that propagate through RNA intermediate. There
are many sequences derived from retrotransposons in maize genome, but none are
active.
Estimating recombination frequency between bz and Ac sites
bz-Ds
Ac bz-s
bronze
bronze
Non-Recombinant TC
progeny (most)
bz
×
Fu et al., 2002
Recombination between bz and nearby Ac
insertion sites
bz
Ac activates Ds to
jump, restoring bz
gene function:
purple spotted
kernels (TC progeny
segregate 1:1)
0.0006
cM/kb
bronze
Recombinant TC progeny
(extremely rare)
If Ac jumps to unlinked chromosome
BEFORE meiosis it can be inherited
along with bz-Ds allele and will cause
purple spots. TC progeny segr. 1:3
0.02 – 0.05
cM/kb
Triangles are Ac insertion sites, peach color boxes are genes, remainder is
nested retrotransposon mess
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Recombination Hotspots
• Genes tend to be hotspots.
• Genes tended to have higher sequence
conservation, maybe important for crossing over
to occur.
• Large nested retrotransposon blocks (~50% of
maize genome) is recombinationally inactive –
even when in homozygous state. Also highly
methylated.
• Perhaps 2 levels of control – chromatin state
(exposed chromosome regions more easily
accessed by enzymes) plus sequence similarity.
Crossing-over within genes
generates novel alleles
More recombination
• Why might it be important to not permit
crossing over to occur in retrotransposon
sequence blocks?
• Further evidence for importance of DSBs
is that transposase enzymes increase
frequency of recombination locally (and
are known to cause DNA breaks).
One crossover = 50%
recombination
A--------------- B
A--------------- B
a--------------- b
a--------------- b
Deletion
Mismatch pairing in tandemly repeated genes can create new haplotypes:
→
A--------------- B Parental gamete
A--------------- b Recombinant gamete
a --------------- B Recombinant gamete
a --------------- b Parental gamete
What happens if you have two crossovers between the same genes in
the same meiosis?
e.g., creation of novel
Rp1 “alleles” in maize
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Double crossovers – four possibilities:
A--------------- B
A--------------- B
a--------------- b
a--------------- b
→
A--------------- B
A--------------- B
a --------------- b
a --------------- b
Parental gamete
Parental gamete
Parental gamete
Parental gamete
A--------------- B
A--------------- B
a--------------- b
a--------------- b
→
A--------------- b
A--------------- b
a --------------- B
a --------------- B
Recombinant gamete
Recombinant gamete
Recombinant gamete
Recombinant gamete
A--------------- B
A--------------- B
a--------------- b
a--------------- b
→
A--------------- B
A--------------- b
a --------------- b
a --------------- B
Parental gamete
Recombinant gamete
Recombinant gamete
Parental gamete
A--------------- B
A--------------- B
a--------------- b
a--------------- b
→
A--------------- b
A--------------- B
a --------------- B
a --------------- b
Recombinant gamete
Parental gamete
Recombinant gamete
Parental gamete
If all four of these events are equally likely, then we still expect 50% recombinant
gametes to occur! In fact, any number of crossovers greater than zero will
produce 50% recombinant gametes.
Crossover Interference
•
If recombination events (having one or more crossovers in an interval) occur
independently of each other on the chromosome, then the relationship between
recombination frequencies between three loci is as follows:
rAC = rAB(1 - rBC ) + rBC (1 - rAB) = rAB + rBC – 2rABrBC .
•
If a recombination event in one interval hinders a simultaneous recombination even in
an adjacent interval, then interference is said to occur. To account for interference,
the equation is written as:
rAC = rAB + rBC – 2CrABrBC = rAB + rBC – 2(1-i)rABrBC ,
where C = coefficient of coincidence, and i = coefficient of interference = 1 - C.
•
If there is no interference (pairs of recombination events occur in random positions),
then i = 0, and we have the original equation.
•
If a crossover in interval A – B always completely suppresses recombination in
interval B – C and vice versa, then there is complete interference and i = 1, and
rAC = rAB + rBC .
Crossing-over and Recombination
•
•
•
•
Crossing-over is the cause.
Recombination is the observed result.
The relationship is not 1:1.
0 crossovers in an interval = 0
recombination between markers defining
the interval.
• >0 crossovers in an interval = 50%
recombination between markers.
Crossover Interference
• Unless there is complete interference,
recombination frequencies will not be additive
along the chromosome:
r AC < r AB + r BC
• Map functions are used to linearize genetic
maps by converting observed recombination
frequencies to unobserved but expected
numbers of crossovers (based on assumptions
about interference).
• Before discussing map functions, what do we
know about crossover interference?
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Crossover interference can be studied directly by
observing late recombination nodules
Crossover interference can be
studied directly by observing late
recombination nodules
• Sherman and Stack (1995) reported the
following results from a survey of 270
female meioses in tomato:
• There is a 1:1 relationship between late
recombination nodules (RNs) and
chiasmata.
• Every synaptonemal complex (SC) has at
least one RN.
There are no RNs at telomeres or centromeres
No RNs at telomeres
No RNs at centromeres
Genetic map units (based on 1 RN = 50 c M) overlayed on tomato SC maps:
Note that very large physical distances near centromere are genetically
tightly linked!
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Sherman and Stack (1995) summary:
negative interference:
more double
crossovers than
expected
• Fig 17 sherman and stack
positive interference:
fewer double
crossovers than
expected
•
•
•
•
•
RNs are more common in euchromatin than
heterochromatin.
The number of RNs per chromosome pair is mainly
related to the length of euchromatin in the
chromosome pair.
Crossover interference is common: a RN in one
chromosome arm seems to drive the second RN into
the other chromosome arm.
Both positive and negative crossover interference was
observed. Pairs of RNs tend not to occur in closely
adjacent intervals as often as expected, nor did they
tend to occur in very distant intervals, instead, pairs
tended to occur more frequently than expected at
intermediate distances.
Problem for mapping functions is that interference
varies throughout genome!
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