1 Linkage, Recombination, and Crossing Over
2 Mendel said..
3 Heterozygous for two traits GgRr Independent segregation Gametes: GR; Gr; gr; gr; 25% each Phenotypes: YeRo YeWr GrRo GrWr
4 But sometimes Genes Do Not Assort Independently
5 THOMAS HUNT MORGAN Traits in Morgan s cross The yellow body allele (y) is recessive to the gray body allele (y + ) The white eyed allele (w) is recessive to the red eyed allele (w + ) The miniature wing allele (m) is recessive to the normal winged allele (m + ) 5
6 MORGAN s EXPERIMENTS Both the white eye gene (w) (recessive) and a gene for miniature wings (m) (recessive) are on the X chromosome. Morgan (1911) crossed a female white miniature (ww/ m m) with a wild-type male (w^ m^/ Y). In the F1, all males were white-eyed with miniature wings (w m/y), and all females were wild-type for eye color and wing size (w^ m^/w m).
8 MORGAN s EXPERIMENTS F1 female have genotype w w^/ m m^ red/normal possible gametes (w m) (w m^) (w^ m) (w^ m^) 25% each if independent segregation is true males have genotype (w m) white/miniature thus F2 (w m) (w m) 25% white/miniature (w m^) (w m) 25% white/normal (w^ m) (w m) 25% red/miniature (w^ m^) (w m) 25% red/normal
10 F2, the most frequent phenotypes for both sexes were the phenotypes of the parents in the original cross (white eyes with miniature wings, and red eyes with normal wings). Non-parental phenotypes (white eyes with normal wings or red eyes with miniature wings) occurred in about 37% of the F2 flies. Well below the 50% predicted for independent assortment
11 MORGAN S PROPOSAL During meiosis alleles of some genes assort together because they are near each other on the same chromosome. Recombination occurs when genes are exchanged between X chromosomes of the F1 females
12 BATESON & PUNNETT, 1905 Traits of sweet peas Flower color can be either purple or red Purple is dominant to red Pollen shape can be either oblong or round Oblong is dominant to round 12
13 BATESON & PUNNETT, 1905 Purple, long (PPLL) x red, round (ppll) F 1 offspring are purple, long (PpLl)
14 F 2 (crossing PpLl x PpLl; gametes PL Pl pl pl) possess four different phenotypes 14
15 F 2 generation The observed ratio is 15.6 : 1.0 : 1.4 : 4.5 The expected ratio 9 : 3 : 3 : 1 >>> is NOT seen! Parental phenotypes were overrepresented Non parental phenotypes were underrepresented evidence that the two genes do not assort independently The nature of their physical linkage was not apparent to Bateson and Punnett at this time The genes are physically linked on the same chromosome 15
17 Linkage, Recombination, and Crossing Over Genes that are on the same chromosome travel through meiosis together; however, alleles of chromosomally linked genes can be recombined by crossing over.
18 Linkage Phases: Coupling and Repulsion
20 Key Points Linkage between genes is detected as a deviation from expectations based on Mendel s Principle of Independent Assortment. The frequency of recombination measures the intensity of linkage. In the absence of linkage, this frequency is 50 percent; for very tight linkage, it is close to zero.
22 Crossing Over Occurs in Prophase of Meiosis I
24 Key Points Recombination is caused by a physical exchange between paired homologous chromosomes early in prophase of the first meiotic division after chromosomes have duplicated. At any one point along a chromosome, the process of exchange (crossing over) involves only two of the four chromatids in a meiotic tetrad. Late in prophase I, crossovers become visible as chiasmata.
25 Crossing Over MAX 50% recombination
26 Multiple Crossovers
27 Chromosome Mapping Linked genes can be mapped on a chromosome by studying how often their alleles recombine.
28 A Chromosome Map
29 Genetic Map Distances The distance between two points on the genetic map of a chromosome is the average number of crossovers between them.
30 Recombination Mapping with a Two Point Testcross
31 The Recombination Frequency between vg and b is 18% This is equal to 18 map units, or 18 centimorgans (cm) on the genetic map.
32 Recombination Mapping with a Three Point Testcross
33 Calculation of Map Distances
34 Determining the Gene Order There are 3 possible gene orders 1. sc ec cv 2. ec sc cv 3. ec cv sc The two most common classes are the parentals. Among the recombinant classes, the 2 rare classes represent the double crossovers. The gene that is switched in the double crossover classes compared to the parental is the middle gene (in this case, ec).
35 Map unit = centimorgan gene A and B 30% RF gene A and C 10% RF gene B and C 20% RF A C B 35
36 Calculation of Map Distances
37 Interference and the Coefficient of Coincidence Assuming independence, the expected frequency of double crossovers is = The observed frequency of double crossovers was 2/3248 = A crossover in one region inhibited a crossover nearby.
39 Recombination Frequency and Genetic Map Distance
40 Recombination Frequency and Genetic Map Distance
41 Chiasma Frequency and Genetic Map Distance
42 Key Points The genetic maps of chromosomes are based on the average number of crossovers that occur during meiosis. Genetic map distances are estimated by calculating the frequency of recombination between genes in experimental crosses.
43 Key Points Recombination frequencies less than 20 percent estimate map distance directly; however, recombination frequencies greater than 20 percent underestimate map distance because multiple crossover events do not always produce recombinant chromosomes. An average of one chiasma during meiosis is equivalent to 50 centimorgans of genetic map distance.
44 Genetic Distance and Physical Distance
45 Key Points In Drosophila, genes can be localized on maps of the polytene chromosomes by combining recessive mutations with cytologically defined deletions and duplications. A deletion will reveal the phenotype of a recessive mutation located between its endpoints, whereas a duplication will conceal the mutant phenotype. Genetic and cytological maps are colinear; however, genetic distances are not proportional to cytological distances.
46 Linkage Analysis in Humans Pedigree analysis provides ways for localizing genes on human chromosomes.
47 The relation between physical and genetic distances is not constant across the genome. Chiasma counts in human male meiosis show an average of 49 crossovers per cell (Morton et al., 1982). Since each crossover gives 50% recombinants, the chiasma count implies a total male genetic map length of 2450 cm. Chiasmata are more frequent in female meiosis and the total female map length is 4296 cm (excluding the X). Thus over the 3000 Mb autosomal genome, 1 male cm averages 1.05 Mb and 1 female cm averages 0.70 Mb; the sexaveraged figure is 1 cm = 0.88 Mb.
48 Genes on human X chromosome 48
49 Key Points Linkage between human genes can be detected by analyzing pedigrees. Pedigree analysis also provides estimates of recombination frequencies to map genes on human chromosomes.
50 Key Points Pedigree analysis also provides information of linkage between genetic markers and diseases.
51 I:2 alleles a;b I;1 alleles c;d II;1 alleles a;c II;2 alleles a;d II;3 alleles b;d II;4/II;5/II;6 alleles e;f I;1/II;1/III;1 All share the disease and the c allele ; thus the c allele is in linkage with the disease. III;1 alleles c;e III;2 alleles a;f III;3 >> III;6 allele c not present (absent in parents)
52 I:2 alleles a;b I;1 alleles c;d II;1 alleles a;c II;2 alleles a;d II;3 alleles d;b II;4/II;5/II;6 alleles e;f I;1/II;1/III;1/ II;3/ III;6 All share the disease but not the c allele; thus the c allele is not in linkage with the disease. III;1 alleles c;e III;2 alleles a;f III;6 alleles b;f
53 Nail Patella syndrome: (NPS) is a rare genetic disorder with abnormalities of bone, joints, fingernails and kidneys. NPS is characterized by absent or underdeveloped kneecaps and thumbnails. It is estimated to occur in 1/50,000 newborns.
54 Linkage Between the ABO and Nail Patella Loci
56 Calculation of the Recombination Frequency 4/13 offspring (31%) in this pedigree are recombinant. Combining data from many pedigrees, the genetic distance between the ABO and NPS1 loci is 10 cm. Molecular markers can also be mapped.
57 LOD score:in genetics, a statistical estimate of whether two loci are likely to lie near each other on a chromosome and are therefore likely to be inherited together as a package. LOD stands for Logarithm of the odds (to the base 10). A LOD score of three or more is generally taken to indicate that two gene loci are close to each other on the chromosome. A LOD score of three means the odds are a thousand to one in favor of genetic linkage.
58 HHT Genes ENG (HHT1, OMIM #187300) 9q34 [McAllister KA et al., 1994] ACVRL1 (HHT2, OMIM #600376) 12q13 [Johnson DW et al., 1996] SMAD4 (JPHT, OMIM #175050) 18q21 [Gallione CJ et al., 2004]
59 Results II DAU DEF θ=0 θ=0.1 θ=0 θ=0.1 D9S ENG D9S D12S D12S D12S IAC DIT θ=0 θ=0.1 θ=0 θ=0.1 D9S ENG D9S D12S D12S D12S Università di Pavia Laboratorio di Genetica Medica
63 .and if no linkage is observed Still an informative result: The gene is not there!!
64 Genetic markers The number of physically observable genes in humans is very small. Consequently, human genetic maps based on these were not useful. The development of genetic loci that could be observed at the level of DNA was essential to modern human genetics. Two alleles (ie, D vs d) needed to be detected, at the DNA level, to define a DNA marker locus. There are several technical solutions to the observation of DNA differences.
65 Genetic markers In a DNA marker, somewhere in the bp amplified region there must be a DNA sequence difference (polymorphism) between individuals. The most common DNA marker systems examine the number of repeated units in a simple sequence repeat motif, such as CACACACACACACAC. Individuals can vary considerably in the number of CA blocks, making these types of DNA sequences very useful population markers.
66 Genetic markers Single basepair differences, however, are much more common in the genome and so have great potential. Single basepair differences are often called SNPs (Single Nucleotide Polymorphisms). However, the frequency of individuals being different at a single base is much less than CACACACA repeat motifs. Genetic markers are simply signposts along the chromosomes that are readily detected and comparable between laboratory experiments.
67 Genetic markers Ideally, genetic markers should be readily available at a high density across the genome (>100,000). The markers should be easily communicated between lab groups and easily quality controlled. And, the work to obtain the marker information should be low error and inexpensive.
68 distance from centromere, cm Genetic marker map of the mouse chr X N centromere chromosome markers used (84) markers in process (11) available markers (55)
69 SNP genetic marker data
72 Recombination and Evolution Recombination or the lack of it plays a key role in evolution.
73 Key Points Recombination can bring favorable mutations together. Chromosome rearrangements, especially inversions, can suppress recombination. Recombination is under genetic control.
74 LINKED or NON LINKED? A recombination frequency of 50% means that genes are unlinked. There are two ways in which genes maybe unlinked: They may be on separate chromosomes. They may be far apart on the same chromosome.
75 MULTIPLE CROSSOVERS If the genes are on the same chromosome, multiple crossovers can occur. The further apart two loci are, the more likely they are to have crossover events take place between them. The chromatid pairing is not always the same in crossover, so that 2,3, or 4 chromatids may participate in multiple crossover.
76 Mitotic recombination Crossing over during mitosis was first observed by Stern (1936) in Drosophila. The alleles involved are sex linked and recessive to the wild type: Y produces yellow body color instead of wild type grey. Sn produces short, twisty bristles ( signed ) rather than the wild type long, curved ones. Bristles follow body color (y+/ are black, and y/y are yellow.
77 Mitotic recombination Female progeny from the cross y+ sn / y+ sn x y sn+ /Y Generally have wild type phenotype of grey bodies and normal bristles, corresponding to their genetoype (y+ sn / y sn+). But exceptions: Some flies had patches of yellow and/or signed bristles. This could be explained by nondisjunction or chromosomal loss. Other flies had twin spots, adjacent regions of bristles, one yellow and the other signed, a mosaic phenotype. The spots are reciprocal products of the same genetic event, a mitotic crossing over. Mitotic crossover occurred either between the centromere and the sn locus or between the sn and the y locus.
78 Fig Body surface phenotype segregation in a Drosophila strain Peter J. Russell, igenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
79 Fig Production of the twin spot and single yellow spot shown in Figure by mitotic crossing-over Peter J. Russell, igenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
80 Mechanism of Mitotic Crossing over A rare event occurring only in diploid cells, mitotic crossover can result when replicated chromatids come together to form a structure similar to the four strand stage in meiosis. If the starting genotype is d+ e / d e+, the two possible orientations of the resulting chromatids are: One cell with d+ e+ / d+ e+, and one with the d e/ d e. These are the ones that are useful for mapping, because the recessive phenotype can be observed in progeny of the d e / d e cells. Reversal of the alleles, d e+ / d+ e. Phenotypically indistinguishable from non recombinant cells, there are not useful for mapping, but are nonetheless derived from a crossover event.
81 Retinoblastoma Most common childhood eye cancer. Non hereditary (sporadic) form occurs in an individual with no family history of the disease, and affects only one eye (unilateral). Heteditary form affects both eyes (bilateral) and usually occurs at an earlier age than sporadic. A single gene (Rb) on chromosome 13q14 involved. In hereditary retinoblastoma, tumor cells have mutations in both copies of this gene, while other cells in the same individual are heterozygous. The disease is caused by a second mutation that affects the normal RB allele. The second mutation is often identical to the one on the other chromosome, strong circumstantial evidence that the wild type copy of the gene is somehow replaced by the inherited mutated allele. One possible explanation is mitotic recombination.
82 Fig Normal mitotic segregation of genes in a theoretical diploid cell with one homologous pair of chromosomes Peter J. Russell, igenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.
83 Fig Result of a mitosis of the same cell type as the cell in Figure but in which a rare mitotic crossing-over occurs Peter J. Russell, igenetics: Copyright Pearson Education, Inc., publishing as Benjamin Cummings.