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Download early RNs, crossing over initiates, then synapsis begins Chiasmata
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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) 1 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 3 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 4 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 5 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? 6 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! 7 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! 8