Chapter 4 Gene Linkage and Genetic Mapping 1
Important Definitions Locus = physical location of a gene on a chromosome Homologous pairs of chromosomes often contain alternative forms of a given gene = alleles Different alleles of the same gene segregate at Meiosis I Alleles of different genes assort independently in gametes Genes on the same chromosome exhibit linkage: inherited together 2
Genetic Mapping Gene mapping determines the order of genes and the relative distances between them in map units 1 map unit = 1 cm (centimorgan) In double heterozyote: Cis configuration = mutant alleles of both genes are on the same chromosome = ab/ab Trans configuration = mutant alleles are on different homologues of the same chromosome = Ab/aB 3
Genetic Mapping Gene mapping methods use recombination frequencies between alleles in order to determine the relative distances between them Recombination frequencies between genes are inversely proportional to their distance apart Distance measurement: 1 map unit = 1 percent recombination (true for short distances) 4
Genetic Mapping Recombination results from crossing-over between linked alleles The 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. 5
Genetic Map Figure 04.05: The frequency of recombination is used to construct a genetic map. 6
Genetic Mapping Genes with recombination frequencies less than 50 percent are on the same chromosome = linked Linkage group = all known genes on a chromosome Two 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 7
Recombination Recombination between linked genes occurs at the same frequency whether alleles are in cis or trans configuration Recombination frequency is specific for a particular pair of genes Recombination frequency increases with increasing distances between genes No matter how far apart two genes may be, the maximum frequency of recombination between any two genes is 50 percent. 8
Genetic Mapping Recombination changes the allelic arrangement on homologous chromosomes Figure 04.04: Crossing-over between two genes. 9
Genetic Mapping The map distance (cm) between two genes equals one half the average number of crossovers in that region per meiotic cell The recombination frequency between two genes indicates how much recombination is actually observed in a particular experiment; it is a measure of recombination 10
Figure 04.06: Diagram of chromosomal configurations in 50 meiotic cells. 11
Genetic Mapping Over 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 12
Genetic Mapping The frequency of recombination between two mutant alleles is independent of whether they are present in the same chromosome or in homologous chromosomes. 13
Figure 04.02: The frequency of recombination between two mutant alleles. 14
Gene Mapping: Double Crossing Over Two exchanges taking place between genes, and both involving the same pair of chromatids, result in nonrecombinant chromosomes Figure 04.08: Crossovers between marker genes A and B. 15
Figure 04.12: The result of two crossovers in the interval between two genes is indistinguishable from independent assortment of the genes. 16
Genetic vs. Physical Distance Map distances based on recombination frequencies are not a direct measurement of physical distance along a chromosome Recombination hot spots overestimate physical length Low rates in heterochromatin and centromeres underestimate actual physical length 17
Figure 04.11: Chromosome 2 in Drosophila as it appears in metaphase of mitosis and in the genetic map. 18
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 doublerecombinant gametes. The effect of double crossing-over is exchange of members of the middle pair of alleles between the chromosomes 19
Table 04.01 Interpreting in a Three-Point Cross. 20
Table 04.T02: Table 4.2 Comparing Reciprocal Products in a Three-Point Cross. 21
Genetic Mapping Mapping function: the relation between genetic map distance and the frequency of recombination Chromosome interference: crossovers in one region decrease the probability of a second crossover close by Figure 04.17: Mapping functions. 22
Genetic Mapping Coefficient of coincidence = observed number of double recombinants divided by the expected number Interference = 1-coefficient of coincidence 23
Mapping Genes in Human Pedigrees Methods of recombinant DNA technology are used to map human chromosomes and locate genes Genes can then be cloned to determine structure and function Human pedigrees and DNA mapping are used to identify dominant and recessive disease genes Polymorphic DNA sequences are used in human genetic mapping. 24
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. 25
Genetic Polymorphisms The presence in a population of two or more relatively common forms of a gene or a chromosome is called polymorphism A 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) 26
SNPs SNPs are abundant in the human genome. The density of SNPs in the human genome averages about one per 1300 bp Identifying 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. 27
Figure 04.18: SNP genotype of an individual. 28
RFLPs Restriction endonucleases are used to map genes as they produce a unique set of fragments for a gene There are more than 200 restriction endonucleases in use, and each recognizes a specific sequence of DNA bases EcoR1 cuts double-stranded DNA at the sequence 5'-GAATTC-3' wherever it occurs 29
Figure 04.19: The restriction enzyme EcoRI cleaves double-stranded DNA wherever the sequence 5-GAATTC-3 is present. 30
RFLPs Differences in DNA sequence generate different DNA cleavage sites for specific restriction enzymes Two different alleles will produce different fragment patterns when cut with the same restriction enzyme due to differences in DNA sequence Figure 04.20: A minor difference in the DNA sequence of two molecules can be detected if the difference eliminates a restriction site. 31
SSRs A 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 chromosome When 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 molecule There is an average of one SSR per 2 kb of human DNA 32
Figure 04.21B: A type of genetic variation that is widespread in most natural populations of animals and plants. 33
Table 04.T03: Data from International Human Genome Sequencing Consortium, Nature 409 (2001): 860-921 34
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 SNPs Restriction enzyme cleavage of polymorphic alleles that are different in RFLP pattern produces different size fragments by gel electrophoresis 35
Figure 04.22: Human pedigree showing segregation of SSR alleles. 36
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. 37
European American Yakut Japanese BiAka Mbuti Datog Hadza Figure 04.F23: Amylase copy number varies with the amount of starch in human diets. Data from G.H. Perry, et al. Nat. Genet. 39 (2007): 1256-1260. 38
Figure 2 - Diet and AMY1 copy number variation. (a) Comparison of qpcr-estimated AMY1 diploid copy number frequency distributions for populations with traditional diets that incorporate many starch-rich foods (high-starch) and populations with traditional diets that include little or no starch (low-starch). (b) Cumulative distribution plot of diploid AMY1 copy number for each of the seven populations in the study. Perry et al. Nature Genetics 39, 1256-1260 (2007) 39
Figure 3 - High-resolution fiber FISH validation of AMY1 copy number estimates. Red (approx10 kb) and green (approx8 kb) probes encompass the entire AMY1 gene and a retrotransposon directly upstream of (and unique to) AMY1, respectively. (a) Japanese individual GM18972 was estimated by qpcr to have 14 (13.73 plusminus 0.93) diploid AMY1 gene copies, consistent with fiber FISH results showing one allele with ten copies and the other with four copies. (b) Biaka individual GM10472 was estimated by qpcr to have six (6.11 plusminus 0.17) diploid AMY1 gene copies, consistent with fiber FISH results. (c) The reference chimpanzee (Clint; S006006) was confirmed to have two diploid AMY1 gene copies.perry et al. Nature Genetics 39, 1256-1260 (2007) 40
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Tetrad Analysis In some species of fungi, each meiotic tetrad is contained in a saclike structure, called an ascus Each product of meiosis is an ascospore, and all of the ascospores formed from one meiotic cell remain together in the ascus Figure 04.24: Formation of an ascus containing all of the four products of a single meiosis. 45
Tetrad Analysis Several features of ascus-producing organisms are especially useful for genetic analysis: They are haploid, so the genotype is expressed directly in the phenotype They produce very large numbers of progeny Their life cycles tend to be short 46
Ordered and Unordered Tetrads Organisms like Saccharomyces cerevisiae, produce unordered tetrads: the meiotic products are not arranged in any particular order in the ascus Unordered 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 tetrads The geometry of meiosis is revealed in ordered tetrads 47
Tetrad Analysis: Unordered Tetrads In tetrads when two pairs of alleles are segregating, three patterns of segregation are possible Parental ditype (PD) = two parental genotypes Nonparental ditype (NPD) = only recombinant combinations Tetratype (TT) = all four genotypes observed Figure 04.26: Types of unordered asci produced with two genes in different chromosomes. 48
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 duplicated The exchange process consists of the breaking and rejoining of the two chromatids, resulting in the reciprocal exchange of equal and corresponding segments 49
Tetrad Analysis When genes are unlinked, the parental ditype tetrads and the nonparental ditype tetrads are expected in equal frequencies: PD = NPD Linkage is indicated when nonparental ditype tetrads appear with a much lower frequency than parental ditype tetrads: PD» NPD Map distance between two genes that are sufficiently close that double and higher levels of crossing-over can be neglected, equals 1/2 x (number TT / total number of tetrads) x 100 50
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 genes The fact that the arrangement of meiotic products is ordered also makes it possible to determine the recombination frequency between any particular gene and its centromere 51
Figure 04.28: The life cycle of Neurospora crassa. 52
Tetrad Analysis: Ordered Tetrads Homologous centromeres of parental chromosomes separate at the first meiotic division The centromeres of sister chromatids separate at the second meiotic division When there is no crossover between the gene and centromere, the alleles segregate in meiosis I A crossover between the gene and the centromere delays segregation alleles until meiosis II 53
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 100 This formula is valid when the gene is close enough to the centromere and there are no multiple crossovers 54
Figure 04.29 (top): First- and second-division segregation in Neurospora. 55
Figure 04.28 (bottom): First- and second-division segregation in Neurospora. 56
Gene Conversion Most asci from heterozygous Aa diploids demonstrate normal Mendelian segregation and contain ratios of 2A : 2a in four-spored asci, or 4A : 4a in eight-spored asci. Occasionally, aberrant ratios are also found, such as 3A : 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 57
Gene Conversion Gene conversion is frequently accompanied by recombination between genetic markers on either side of the conversion event, even when the flanking markers are tightly linked Gene conversion results from a normal DNA repair process in the cell known as mismatch repair Gene conversion suggests a molecular mechanism of recombination 58
Figure 04.31: Mismatch repair resulting in gene conversion. 59
Recombination Recombination is initiated by a double-stranded break in DNA The size of the gap is increased by nuclease digestion of the broken ends These gaps are repaired using the unbroken homologous DNA molecule as a template The repair process can result in crossovers that yield chiasmata between nonsister chromatids 60
Figure 04.32: Double-strand break in a duplex DNA molecule. Adapted from D. K. Bishop and D. Zickler, Cell 117 (2004): 9-15 61
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 loop The juxtaposed free ends are joined together, further unwinding and exchange of pairing partners increase the length of heteroduplex region -- process of branch migration 62
Recombination: Holliday Model One of two ways to resolve the resulting structure, known as a Holliday junction, leads to recombination, the other does not The breakage and rejoining is an enzymatic function carried out by an enzyme called the Holliday junction-resolving enzyme 63
Figure 04.33: EM of a Holliday structure. Illustration modified from B. Alberts. Essential Cell Biology. Garland Science, 1997. Illustration reproduced with permission of Huntington Potter, Johnnie B. Byrd Sr., Alzheimer s Center & Research Institute 64