Chapter 7: Linkage, Recombination, and Eukaryotic Gene Mapping PDF

Chapter 7: Linkage, Recombination, and Eukaryotic Gene Mapping 7.1 Linked Genes Do Not Assort Independently Recall: Principle of segregation: Alleles separate during meiosis. Independent assortment: Alleles at one locus sort independently from alleles at another...

Chapter 7: Linkage, Recombination, and Eukaryotic Gene Mapping 7.1 Linked Genes Do Not Assort Independently Recall: Principle of segregation: Alleles separate during meiosis. Independent assortment: Alleles at one locus sort independently from alleles at another locus. Recombination: Alleles sort into new combinations. Recall independent assortment: Mendel’s Dihybrid Crosses Non- Recombinant recombinant  gametes gametes  Recombination is the sorting of alleles into new combinations. F1 gametes can be non-recombinant gametes (same as the parent) or recombinant gametes (new combination)  Bateson, Saunders and Punnett (1905) reported Non-independent Assortment of Flower Color & Pollen Shape in Sweet Peas: expected 9:3:3:1 ratio was not obtained 381 progeny, expected 214:71:71:24 = 238 non-recombinant progeny & 142 recombinant progeny; instead had 339 non-recombinant progeny & 42 recombinant progeny 7.2 Linked Genes Segregate Together and Crossing Over Produces Recombination Between Them Notation for crosses with linkage: Complete linkage leads to nonrecombinant gametes and nonrecombinant progeny Crossing over between linked genes leads to recombinant gametes and recombinant progeny Crossing over leads to recombination; if crossing over occurs between two normally linked loci, they will sort independently.  Crossing Over Between Linked Genes Crossing over occurs between non-sister chromatids on homologous pairs of chromosomes Chromatids participating in the cross over are recombinant (50% of gametes) Those not participating in the cross over are non-recombinant (50% of gametes)  Complete Linkage Compared with Independent Assortment can be observed with testcrosses Note the appearance of nonrecombinant and recombinant progeny Figure 7.4  Standard notation for genes showing independent assortment: (P) AA BB x aa bb  (F1) Aa Bb  (F2) 9 A_B_ : 3 A_bb : 3 aaB_ : 1 aabb  If genes are linked on the same chromosome, we need to indicate which alleles are linked together: Coupling: WT genes linked on the same chromosome 1 : 2 : 1 A B a b A B A B A B a b (P) X  (F1)  (F2) A B a b a b A B a b a b 3 : 1 AB/AB x ab/ab  (F1) AB/ab  (F2) 1 AB/AB : 2 AB/ab : 1 ab/ab Repulsion: WT and mutant genes are linked on the same chromosome 1 : 2 : 1 A b a B A b A b A b a B (P) X  (F1)  (F2) A b a B a B A b a B a B 1 : 2 : 1 Ab/Ab x aB/aB  (F1) Ab/Ab  (F2) 1 Ab/Ab : 2 Ab/aB : 1 aB/aB  Crossing Over Between Linked Genes Calculating recombination frequency: Number of recombinant progeny Recombination frequency = X 100% Total number of progeny Meiosis with and without crossing over together results in more than 50% non- recombinant gametes (15 recombinants/123 total) x 100% = 12.2% recombination frequency  Coupling and repulsion configuration of linked genes  Coupling (cis configuration): One chromosome contains both wild-type alleles and one chromosome contains both mutant alleles. A B a b  Repulsion (trans configuration): Wild-type allele and mutant allele are found on the same chromosome. A b a B The coupling or repulsion arrangement of linked genes on a chromosome affects the results of a testcross (recall a testcross is an unknown x homozygous recessive  Results of a testcross (Aa Bb X aa bb) with complete linkage, independent assortment & linkage with some crossing over Situation Progeny of Testcross Independent assortment Aa Bb (nonrecombinant) 25% Independent assortment aa bb (nonrecombinant) 25% Independent assortment Aa bb (recombinant) 25% Independent assortment aa Bb (recombinant) 25% Complete linkage (genes in coupling) AB / ab (nonrecombinant) 50% Complete linkage (genes in coupling) ab / ab (nonrecombinant) 50% Linkage with some crossing over Together equal (genes in coupling) AB / ab (nonrecombinant) more than Linkage with some crossing over 50% of (genes in coupling) ab / ab (nonrecombinant) offspring Linkage with some crossing over (genes in coupling) Ab / ab (recombinant) Together equal less than 50% Linkage with some crossing over of offspring (genes in coupling) aB / bb (recombinant)  Concept Check For single crossovers, the frequency of recombinant gametes is half the frequency of crossing over because a. a testcross between a homozygote and heterozygote produces ½ heterozygous and ½ homozygous progeny. b. the frequency of recombination is always 50%. c. each crossover takes place between only two of the four chromatids of a homologous pair. d. crossovers occur in about 50% of meiosis. The following testcross produces the progeny shown: AaBb x aabb  10 AaBb, 40 Aabb, 40 aaBb, and 10 aabb. Which statement is most true regarding the AaBb parent? a. The genes are in repulsion b. The genes are in coupling c. The genes are assorting independently d. Not enough information is provided  Linked Genes Segregate Together and Crossing Over Produces Recombination Between Them: Evidence for the Physical Basis of Recombination Walter Sutton’s chromosome theory of inheritance – Genes are physically located on chromosomes. Nettie Stevens and Edmund Wilson’s – Sex was associated with a specific chromosome in insects. Calvin Bridges’s – Nondisjunction of X chromosomes was related to the inheritance of eye color in Drosophila. Harriet Creighton and Barbara McClintock (1931) – Intrachromosomal recombination was the result of physical exchange between chromosomes. – Experiment diagrammed on the next slide  Barbara McClintock (left) & Harriet Creighton (right) provided evidence that genes are located on chromosomes. Note that the other sister chromatids are not represented here Identified a curious chromosome: densely staining knob on one end and an extra piece (a translocation) on the other; Colored and Waxy kernel alleles in repulsion  Predicting the Outcomes of Crosses with Linked Genes when the recombination frequency is known Recombination frequency allows us to predict expected offspring for a cross with linked genes Recall: the recombination frequency is (recombinant progeny / total progeny) x 100%  Gene mapping with recombination frequencies Genetic maps are determined by recombinant frequency; (Physical maps are based on physical [nucleotide] distances.)  the map units of genetic maps are “map units” or “centiMorgans (cM)”  Constructing a genetic map with two-point testcrosses:  Genetic distances measured with recombination rates are approximately additive Gene pair Recombination Frequency (%) A and B 5% (5 cM) B and C 10% (10 cM) A and C 15% (15 cM) A and D 8% B and D 13% C and D 23%  OR Add more crosses to determine orientation Double crossovers can occur between genes, giving the illusion of reduced recombination frequency (and thus cM distances) But, we can also use double crossovers to our advantage  7.3 A Three-Point Testcross Can Be Used to Map Three Linked Genes Constructing a genetic map with the three-point testcross  More efficient mapping technique than two point crosses  The order of the three genes can be established in a single set of progeny  Some double crossovers can usually be detected  Provides more accurate map distances 1. Identify the nonrecombinant progeny (two most numerous phenotypes). 2. Identify the double-crossover progeny (two least numerous phenotypes). 3. Compare the phenotypes of double-crossover progeny with the phenotypes of nonrecombinant progeny. They should be alike in two characteristics and differ in one. 4. The characteristic that differs between the double crossover and the nonrecombinant progeny is encoded by the middle gene. Three Types of Crossovers with Three Linked Loci Gametes  Constructing a genetic map with a three point testcross in Drosophila 1. *Note that genes are named after the mutant phenotypes: scarlet eyes (st), ebony body color (e), spineless bristles (ss) 2. *At this stage of the problem, we do not know the order or the recombination frequency (i.e., map units) 3. *In Drosophila, crossing over does not occur in males, so heterozygous females are always used to mapped genes P: F1: Testcross: Determining Gene Order in a 3-Point Test Cross The results of a 3-point test-cross can be used to map linked genes. 1. Identify the non- recombinant offspring (most prevalent). 2. Identify the dble cross-over offspring (least prevalent). Writing the results of a three-point testcross with the loci in the correct order allows the crossover locations to be determined. With the genes in the correct order, the recombination frequencies (= map units or cM) can be calculated 100%x(5+3)/755=1.06% (used to find the middle gene but does not directly contribute to cM in the map 100%x(50+52+5+3)/755 =14.6% (st and ss are 14.6 cM apart 100%x(43+41+5+3)/755 =12.2% (ss and e are 12.2 cM apart) Determining Mapping Distance in a 3-Point Test Cross  Concept Check A three-point testcross is carried out between three linked genes. The most abundant progeny are s+r+c+ and s r c. The least abundant progeny are s r c+ and s+r+c. Four other progeny, s+r c, s r+c, s r+c+, and s+r c+ are present in intermediate abundance. Which is the middle locus? A) S locus B) R locus C) C locus D) Not enough information is provided  Drosophila melanogaster has four linkage groups corresponding to its four pairs of chromosomes. Note that any two genes more than 50 cM apart will assort independently  Double crossover frequencies tend to be less frequent than expected based on multiplying single crossover frequencies Crossovers are thought to occur at points of physical stress along chiasmas One crossover may relieve stress and reduce the probability of a second crossover Coefficient of Coincidence and Interference calculate suppression (~interference) of crossovers – Coefficient of Coincidence = (Number of observed double crossovers) (Number of expected double crossovers) – Interference = (1) – (coefficient of coincidence)  Concept Check In analyzing the results of a three- point testcross, a student determines that the interference is −0.23. What does this negative interference value indicate? a. Fewer double crossovers took place than expected on the basis of single-crossover frequencies. b. More double crossovers took place than expected on the basis of single-crossover frequencies. c. Fewer single crossovers took place than expected. d. There was a crossover in one region.  Concept Check In analyzing the results of a three- point testcross, a student determines that the interference is −0.23. What does this negative interference value indicate? a. Fewer double crossovers took place than expected on the basis of single-crossover frequencies. b. More double crossovers took place than expected on the basis of single-crossover frequencies. c. Fewer single crossovers took place than expected. d. There was a crossover in one region. Effects of two-, three-, and four-strand double crossovers on recombination between two genes; Double crossovers give the appearance of reduced recombination  Recombination rates exhibit extensive variation & underestimate the true physical (i.e., sequence) distance between genes Levels of recombination vary widely – Among species – Among chromosomes of a single species – Along chromosome regions (centromeres / telomeres) – Between males & females (i.e., male flies have none) Recombination hotspots (more than espected) and regions where recombination is repressed Consequently, genetics maps and physical maps only approximate each other cM distance between genes is not a reliable estimate of nucleotide distance between genes 7.4 Physical-Mapping Methods Are Used to Determine the Physical Positions of Genes on Particular Chromosomes Somatic-cell hybridization Deletion mapping Physical mapping through molecular analysis – In situ hybridization Genetics technologies advance rapidly, e.g., whole- genome sequencing makes some earlier techniques obsolete.  Somatic-cell hybridization can be used to determine which chromosome contains a gene of interest Somatic-cell hybridization is used to assign a gene to a particular human chromosome. Deletion mapping has revealed the chromosomal locations of a number of human genes. Recessive genes display the phenotype in the hemizygous condition  Physical Chromosome Mapping Through Molecular Analysis Fluorescence In Situ Hybridization (FISH) uses a single-stranded complementary DNA probe for the gene. Where the fluorescent probe binds to the chromosomes in a karyotype indicates the chromosome and location of the corresponding gene  Chapter 7 Linkage, Recombination, & Eukaryotic Gene Mapping Suggested problems to help your comprehension & test your knowledge (7th edition): Worked Problems #2 and #3 on pages 212 and 214 *Application Questions and Problems (starting on page 215) #15, 16, 17, 18, 22, 28, 31, 32, 38, 40, 41 *Questions marked with an asterisk have answers provided in the back of the book.