2 Important Definitions Locus = physical location of a gene on a chromosomeHomologous pairs of chromosomes often contain alternative forms of a given gene = allelesDifferent alleles of the same gene segregate at Meiosis IAlleles of different genes assort independently in gametesGenes on the same chromosome exhibit linkage: inherited together
3 Genetic MappingGene mapping determines the order of genes and the relative distances between them in map units1 map unit = 1 cM (centimorgan)In double heterozyote:Cis configuration = mutant alleles of both genes are on the same chromosome = ab/ABTrans configuration = mutant alleles are on different homologues of the same chromosome = Ab/aB
4 Genetic Mapping Gene mapping methods use recombination frequencies between alleles in order to determine the relative distances between themRecombination frequencies between genes are inversely proportional to their distance apartDistance measurement: 1 map unit = 1 percent recombination (true for short distances)
6 Figure 4.2 The frequency of recombination between two mutant alleles
7 Genetic MappingThe frequency of recombination between two mutant alleles is independent of whether they are present in the same chromosome or in homologous chromosomes.
8 Figure 4.3 The frequency of recombination between two genes depends on the genes
9 Genetic MapFigure 04.05: The frequency of recombination is used to construct a genetic map.
10 Genetic MappingRecombination results from crossing-over between linked allelesThe linkage of the genes can be represented as a genetic map, which shows the linear order of the genes along the chromosome spaced so that the distances between genes is proportional to the frequency of recombination between them.
11 Figure 04.04: Crossing-over between two genes. Genetic MappingRecombination changes the allelic arrangement on homologous chromosomesFigure 04.04: Crossing-over between two genes.
12 Genetic MappingGenes with recombination frequencies less than 50 percent are on the same chromosome = linkedLinkage group = all known genes on a chromosomeTwo genes that undergo independent assortment have recombination frequency of 50 percent and are located on nonhomologous chromosomes or far apart on the same chromosome = unlinked
13 Genetic MappingThe map distance (cM) between two genes equals one half the average number of crossovers in that region per meiotic cellThe recombination frequency between two genes indicates how much recombination is actually observed in a particular experiment; it is a measure of recombination
14 Figure 04.06: Diagram of chromosomal configurations in 50 meiotic cells.
15 Figure 4.7 Crossing-over outside the region between two genes is not detectable through recombination
16 Genetic MappingOver an interval so short that multiple crossovers are precluded (~ 10 percent recombination or less), the map distance equals the recombination frequency because all crossovers result in recombinant gametes.Over the short interval genetic map = linkage map = chromosome map
17 Gene Mapping: Double Crossing Over Two exchanges taking place between genes, and both involving the same pair of chromatids, result in nonrecombinant chromosomesFigure 04.08: Crossovers between marker genes A and B.
19 Figure 4.10 Genetic map of chromosome 10 of corn, Zea mays Adapted from an illustration by E.H. Coe.
20 Figure 04.12: The result of two crossovers in the interval between two genes is indistinguishable from independent assortment of the genes.
21 Genetic vs. Physical Distance Map distances based on recombination frequencies are not a direct measurement of physical distance along a chromosomeRecombination “hot spots” overestimate physical lengthLow rates in heterochromatin and centromeres underestimate actual physical length
22 Figure 04.11: Chromosome 2 in Drosophila as it appears in metaphase of mitosis and in the genetic map.
23 Genetic Mapping: Three-Point Cross In any genetic cross, the two most frequent types of gametes are nonrecombinant; these provide the linkage phase (cis versus trans) of the alleles in the multiply heterozygous parent.The two rarest classes identify the double-recombinant gametes.The effect of double crossing-over is exchange of members of the middle pair of alleles between the chromosomes
24 Table 04.01 Interpreting in a Three-Point Cross.
25 Figure 4.14 The order of genes in a three-point testcross
26 Figure 4.15 Result of single crossovers in a triple heterozygote
27 Figure 4.16 Result of double crossovers in a triple heterozygote
28 Table 4.2 Comparing Reciprocal Products in a Three-Point Cross
29 Figure 04.17: Mapping functions. Genetic MappingMapping function: the relation between genetic map distance and the frequency of recombinationChromosome interference: crossovers in one region decrease the probability of a second crossover close byFigure 04.17: Mapping functions.
30 Genetic MappingCoefficient of coincidence = observed number of double recombinants divided by the expected numberInterference = 1-coefficient of coincidence
31 Mapping Genes in Human Pedigrees Methods of recombinant DNA technology are used to map human chromosomes and locate genesGenes can then be cloned to determine structure and functionHuman pedigrees and DNA mapping are used to identify dominant and recessive disease genesPolymorphic DNA sequences are used in human genetic mapping.
32 Mapping Genes in Human Pedigrees Most genes that cause genetic diseases are rare, so they are observed in only a small number of families.Many mutant genes of interest in human genetics are recessive, so they are not detected in heterozygous genotypes.The number of offspring per human family is relatively small, so segregation cannot usually be detected in single sibships.The human geneticist cannot perform testcrosses or backcrosses, because human matings are not dictated by an experimenter.
33 Genetic Polymorphisms The presence in a population of two or more relatively common forms of a gene or a chromosome is called polymorphismA prevalent type of polymorphism is a single base pair difference, simple-nucleotide polymorphism (SNP)SNPs in restriction sites yield restriction fragment length polymorphism (RFLP)Polymorphism resulting from a tandemly repeated short DNA sequence is called a simple sequence repeat (SSR)
34 SNPs SNPs are abundant in the human genome. The density of SNPs in the human genome averages about one per 1300 bpIdentifying the particular nucleotide present at each of a million SNPs is made possible through the use of DNA microarrays composed of about 20 million infinitesimal spots on a glass slide the size of a postage stamp.
36 RFLPsRestriction endonucleases are used to map genes as they produce a unique set of fragments for a geneThere are more than 200 restriction endonucleases in use, and each recognizes a specific sequence of DNA basesEcoR1 cuts double-stranded DNA at the sequence5'-GAATTC-3' wherever it occurs
37 Figure 04.19: The restriction enzyme EcoRI cleaves double-stranded DNA wherever the sequence 5-GAATTC-3 is present.
38 RFLPsDifferences in DNA sequence generate different DNA cleavage sites for specific restriction enzymesTwo different alleles will produce different fragment patterns when cut with the same restriction enzyme due to differences in DNA sequenceFigure 04.20: A minor difference in the DNA sequence of two molecules can be detected if the difference eliminates a restriction site.
39 SSRsA third type of DNA polymorphism results from differences in the number of copies of a short DNA sequence that may be repeated many times in tandem at a particular site in a chromosomeWhen a DNA molecule is cleaved with a restriction endonuclease that cleaves at sites flanking the tandem repeat, the size of the DNA fragment produced is determined by the number of repeats present in the moleculeThere is an average of one SSR per 2 kb of human DNA
40 Figure 04.21B: A type of genetic variation that is widespread in most natural populations of animals and plants.
41 Mapping Genes in Human Pedigrees Human pedigrees can be analyzed for the inheritance pattern of different alleles of a gene based on differences in SSRs and SNPsRestriction enzyme cleavage of polymorphic alleles that are different in RFLP pattern produces different size fragments by gel electrophoresis
42 Figure 04.22: Human pedigree showing segregation of SSR alleles.
43 Copy-number variations (CNVs) A substantial portion of the human genome can be duplicated or deleted in much larger but still submicroscopic chunks ranging from 1 kb to 1 Mb.This type of variation is known as copy-number variation (CNV).The extra or missing copies of the genome in CNVs can be detected by means of hybridization with oligonucleotides in DNA microarrays.
44 Tetrad AnalysisIn some species of fungi, each meiotic tetrad is contained in a sac-like structure, called an ascusEach product of meiosis is an ascospore, and all of the ascospores formed from one meiotic cell remain together in the ascusFigure 04.24: Formation of an ascus containing all of the four products of a single meiosis.
45 Tetrad AnalysisSeveral features of ascus-producing organisms are especially useful for genetic analysis:They are haploid, so the genotype is expressed directly in the phenotypeThey produce very large numbers of progenyTheir life cycles tend to be short
46 Figure 4.24 Life cycle of the yeast Saccharomyces cerevisiae
47 Ordered and Unordered Tetrads Organisms like Saccharomyces cerevisiae, produce unordered tetrads: the meiotic products are not arranged in any particular order in the ascusUnordered tetrads have no relation to the geometry of meiosis.Bread molds of the genus Neurospora have the meiotic products arranged in a definite order directly related to the planes of the meiotic divisions—ordered tetradsThe geometry of meiosis is revealed in ordered tetrads
48 Tetrad Analysis: Unordered Tetrads In tetrads when two pairs of alleles are segregating, three patterns of segregation are possibleParental ditype (PD) = two parental genotypesNonparental ditype (NPD) = only recombinant combinationsTetratype (TT) = all four genotypes observedFigure 04.26: Types of unordered asci produced with two genes in different chromosomes.
49 Tetrad Analysis: Unordered Tetrads The existence of TT for linked genes demonstrates two important features of crossing-over:The exchange of segments between parental chromatids takes place in prophase I, after the chromosomes have duplicatedThe exchange process consists of the breaking and rejoining of the two chromatids, resulting in the reciprocal exchange of equal and corresponding segments
50 Tetrad AnalysisWhen genes are unlinked, the parental ditype tetrads and the nonparental ditype tetrads are expected in equal frequencies: PD = NPDLinkage is indicated when nonparental ditype tetrads appear with a much lower frequency than parental ditype tetrads: PD » NPDMap distance between two genes that are sufficiently close that double and higher levels of crossing-over can be neglected, equals1/2 x (number TT / total number of tetrads) x 100
51 Tetrad Analysis: Ordered Tetrads Ordered asci also can be classified as PD, NPD, or TT with respect to two pairs of alleles, which makes it possible to assess the degree of linkage between the genesThe fact that the arrangement of meiotic products is ordered also makes it possible to determinethe recombination frequency between any particular gene and its centromere
52 Figure 04.28: The life cycle of Neurospora crassa.
53 Tetrad Analysis: Ordered Tetrads Homologous centromeres of parental chromosomes separate at the first meiotic divisionThe centromeres of sister chromatids separate at the second meiotic divisionWhen there is no crossover between the gene and centromere, the alleles segregate in meiosis IA crossover between the gene and the centromere delays segregation alleles until meiosis II
54 Tetrad Analysis: Ordered Tetrads The map distance between the gene and its centromere equals:1/2 x (number of asci with second division segregation/total number of asci) x 100This formula is valid when the gene is close enough to the centromere and there are no multiple crossovers
55 Figure 04.29 (top): First- and second-division segregation in Neurospora.
56 Figure 04.28 (bottom): First- and second-division segregation in Neurospora.
57 Gene ConversionMost asci from heterozygous Aa diploids demonstrate normal Mendelian segregation and contain ratios of2A : 2a in four-spored asci, or 4A : 4a in eight-spored asci.Occasionally, aberrant ratios are also found, such as3A : 1a or 1A : 3a and 5A : 3a or 3A : 5a.The aberrant asci are said to result from gene conversion because it appears as if one allele has “converted” the other allele into a form like itself
58 Gene ConversionGene conversion is frequently accompanied by recombination between genetic markers on either side of the conversion event, even when the flanking markers are tightly linkedGene conversion results from a normal DNA repair process in the cell known as mismatch repairGene conversion suggests a molecular mechanism of recombination
60 Figure 04.31: Mismatch repair resulting in gene conversion.
61 RecombinationRecombination is initiated by a double-stranded break in DNAThe size of the gap is increased by nuclease digestion of the broken endsThese gaps are repaired using the unbroken homologous DNA molecule as a templateThe repair process can result in crossovers that yield chiasmata between nonsister chromatids
62 Figure 04.32: Double-strand break in a duplex DNA molecule. Adapted from D. K. Bishop and D. Zickler,Cell 117 (2004): 9-15
63 Recombination: Holliday Model The nicked strands unwind, switch partners, forming a short heteroduplex region with one strand and a looped-out region of the other strand called a D loopThe juxtaposed free ends are joined together, further unwinding and exchange of pairing partners increase the length of heteroduplex region—process of branch migration
64 Recombination: Holliday Model One of two ways to resolve the resulting structure, known as a Holliday junction, leads to recombination, the other does notThe breakage and rejoining is an enzymatic function carried out by an enzyme called the Holliday junction-resolving enzyme
65 Figure 04.33: EM of a Holliday structure. Illustration modified from B. Alberts. Essential Cell Biology. Garland Science, Illustration reproduced with permission of Huntington Potter,Johnnie B. Byrd Sr., Alzheimer’s Center & Research Institute