Chapter 5

CHAPTER 5
CHROMOSOME MAPPING IN
EUKARYOTES
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Chapter 5 Outline - Learning

Objectives
1. Genes Linked on the Same Chromosome Segregate Together
2. Crossing Over Serves as the Basis for Determining the Distance between Genes
in Chromosome Mapping
3. Determining the Gene Sequence during Mapping Requires the Analysis of
Multiple Crossovers
4. As the Distance between Two Genes Increases, Mapping Estimates Become
More Inaccurate
5. Drosophila Genes Have Been Extensively Mapped
6. Lod Score Analysis and Somatic Cell Hybridization Were Historically Important in
Creating Human Chromosome Maps
7. Chromosome Mapping Is Currently Performed Using D N A Markers and
Annotated Computer Databases
8. Crossing Over Involves a Physical Exchange between Chromatids
9. Exchanges Also Occur between Sister Chromatids During Mitosis
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Maya Hobeika Kahwagi © USEK 2023-2024

 1903: Sutton pointed out the likelihood of many more “unit factors” than
chromosomes
 Chromosomes are the unit of transmission in meiosis, not genes

 Linked genes can not undergo independent assortment
 Frequency of crossing over on a single chromosome is proportional to distance

between them
 Crossing over results in recombination
 Chromosome maps: indicate relative location of genes on chromosome
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I- Genes Linked on the Same

Chromosome Segregate Together
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I.1- Meiotic Consequences
Depending on the relative position of the genes on the chromosomes, there could be:
 Independent assortment
 No linkage exhibited
 Linkage without crossing over
 Complete linkage
 Linkage with crossing over
 Generates recombinant (crossover) gametes
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I.2- Linkage Ratio and Groups
 Linkage Ratio
 Unique F2 phenotypics ratios in matings involving genes with complete linkage.
 E.g. mutations heavy veins (hv) and brown eyes (bw)
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NB: 1:2:1 in cases of trans-linkage (dominant/recessive vs recessive/dominant)

And 3:1 (3/4, 1/4) in cases of cis-linkage (dominant/dominant vs recessive/recessive)
𝐴𝐵 𝑎𝑏
P: 𝑥
𝐴𝐵 𝑎𝑏
𝐴𝐵
F1: AB ab
𝑎𝑏
AB AB AB
AB ab
ab AB ab
ab ab
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 Linkage group
 Genes on the same chromosome are part of a linkage group
 Number of linkage groups should correspond to haploid number of chromosomes
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II- Crossing Over Serves as the Basis for
Determining the Distance between
Genes in Chromosome Mapping
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 Very low probability of complete linkage for two randomly selected genes on the
same chromosomes
 Crosses will almost always produce a percentage of offspring resulting from
recombinant gametes.
 Percentage depending on the distance between the two genes along the chromosome.
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II.1- Morgan and Crossing Over
 Morgan was the first geneticist to discover the phenomenon of X-linkage (in
Drosophila)
 Investigation of numerous mutations on the X chromosome
 Some puzzling results when 2 genes studied simultaneously
Females [yellow body, white eyes] x wt males
Females [miniature wings, white eyes] x wt males
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II.1- Morgan and Crossing Over
Morgan’s Questions /answers

 What was the source of gene separation?
 Previous knowledge of cytological observations (F. A. Janssens and others): Synapsed
chromosomes in meiosis wrap around each other = chiasmata
 X-shaped intersections with points of overlap
 Points of genetic exchange
 Why did the frequency of the apparent separation vary depending on the genes being
studied?
 Percentage of offspring resulting from recombinant gametes depends on distance
between two genes on same chromosome
 Two genes located close to each other along a chromosome are less likely to have
chiasma
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II.2- Sturtevant and Mapping
 Sturtevant (Morgan’s student)

 First to realize that these results can be used to create maps
 Compiled data from crosses
 Recombination frequencies between linked genes are additive
 Frequency of exchange is estimate of relative distance between two genes
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II.2- Sturtevant and Mapping
 Map unit (mu)

 1 percent recombination between two genes on chromosome
 Also called centi-Morgans (cM)
 Relative distances, not exact ones
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II.3- Single Crossovers
 During meiosis, a limited number of crossover events occur RANDOMLY in each

tetrad
 the closer the two loci along the axis of the chromosome, the less likely that any single
crossover event will occur between them.
 The farther apart two linked loci, the more likely a random crossover event will occur in
between them.
 Single crossover (SCO)
 Occurs between two nonsister chromatids
 Recombination is observed in MAX 50 percent of gametes
 In genes 50 map units apart, crossing over can be expected between 100
percent of tetrads
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II.3- Single Crossovers
 After a single exchange between two nonsister chromatids in tetrad stage

 Two noncrossover (parental) gametes produced
 Two crossover (recombinant) gametes produced
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III- Determining the Gene Sequence
during Mapping Requires the Analysis
of Multiple Crossovers
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III.1- Multiple Exchanges
 Single crossover
 Used to determine distance between two linked genes
 Double crossover
 Double exchanges of genetic material
 Used to determine order AND distance between three linked genes
 Genes must be heterozygous for two alleles
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 3 couples of alleles to analyze DCO

 Review of simple probability calculations:
 Exchange probability between genes (A and B) or (B and C) is directly correlated to
distance between loci
 p(DCO) = p(CO1)x p(CO2) [product law]
 if p(CO between A and B)= 20% and p(CO between B and C)= 30%
then p(DCO)= 20% x 30% = 0,2x0,3=0,06=6%
 expected frequency of double-crossover gametes is extremely low

 very large numbers of offspring are required to detect double-crossover events
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III.2- Three-Point Mapping
 Three criteria of three-point mapping

 Parent must be heterozygous for all three genes under consideration
 Phenotypic class must reflect genotype of gametes of parents
 Sufficient number of offspring must be produced for representative sample
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III.2- Three-Point Mapping in Drosophila
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1- determine order
2- determine distances
d(y-w): p(SCO1) + p(DCO) = 1,5% + 0,06%
so 1,56cM
d(w-ec) : p(SCO2) + p(DCO) = 4% + 0,06%
so 4,06cM
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 Noncrossover F2 phenotypes
 Occur in greatest proportion of offspring
 Double-crossover (DCO) phenotypes
 Occur in the smallest proportion
 Reciprocal classes of phenotypes
 F2 phenotypes complement each other
 Derived from heterozygote
 Have wild type and mutant for all three genes
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Determining Gene Sequence

 Method 1 based on three possible arrangements of genes
1. 3 possible sequences
2. Determine phenotypes of DCO for any given sequence
3. Compare to given DCO sequence
4. If not OK, try another one
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Determining Gene Sequence

 Method 2 uses three possible arrangements
 Also considers that following double-crossover event, the switched allele is in the middle
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III.3- An Autosomal Mapping Problem in Maize
 Differences with previous example:

 autosomal genes and not X-linked
 Other symbols used
 recessive mutant genes bm (brown midrib), v (virescent seedling), and pr (purple aleurone) are
linked on chromosome 5
 female plant known to be heterozygous for all three traits
 male homozygous for all three recessive mutant alleles (equivalent to performing a testcross)
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Parental
Switched gene in the middle

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IV- As the Distance between Two
Genes Increases, Mapping
Estimates Become More
Inaccurate
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 Expected frequency of multiple exchanges between two genes predicted from

distance between them
 Genes farther apart increase the probability of undetected crossovers
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IV.1- Interference and the Coefficient of
Coincidence
 Product law ➔ expected frequency of multiple exchanges calculated from
distance between genes
 example, in the previous maize cross: distance between v and pr=22.3 mu, and
distance between pr and bm=43.4 mu
 DCOexp=(0.223)×(0.434)=0.097=9.7%
 Often in mapping experiments, the observed DCO frequency is less than the
expected number of DCOs
7,8% instead of 9,7%
 Interference : Inhibition of further crossover events another crossover event nearby
 Reduces expected number of multiple crossovers
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IV.1- Interference and the Coefficient of

Coincidence
 Coefficient of coincidence to quantify the disparities that result from interference
C = Observed DCO / expected DCO
Example in maize: C = 0,078/0,097 = 0,804
 Interference I = 1 – C
Example in maize: 1 – 0,804 = 0,196
 Interference is:
 Complete when no double crossovers occur
 Positive: Fewer double-crossover events than expected occur
 I is a positive number
 Negative: More double-crossover events than expected occur
 I is a negative number
 19.6 % fewer double crossovers occurred than expected.

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IV.2- Interference and distance
 Positive interference most often observed

 In C. elegans
 only one crossover event per chromosome
 Interference along each chromosome is complete (C=1.0)
 In other organisms, the closer genes are, the more positive interference occurs.
 Interference in Drosophila often complete within 10 mu
 Physical constraints preventing the formation of closely spaced chiasmata

contribute to interference
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IV.2- Interference and distance
 Two genes close together

 Positive interference occurs
 Accuracy of mapping is high
 Distance between genes increases
 Interference decreases
 Accuracy of mapping decreases
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V- Drosophila Genes Have Been
Extensively Mapped
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 Large number of mutants in organisms such as

 Drosophila
 Maize
 Mice
 Allows for construction of extensive chromosome mapping
 Virtually every morphological feature of the fruit fly subjected to mutation.

 Each locus affected by mutation first localized to one of the four chromosomes (or linkage
groups) then mapped in relation to other genes in the same group
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X < II and III
IV miniscule
Correlation between physical and genetic maps
confirmed by cytological observations
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VI- Lod Score Analysis and Somatic Cell

Hybridization Were Historically Important
in Creating Human Chromosome Maps
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VI.1- Lod Score Method
 In humans, genetic experiments involving carefully planned crosses and large

numbers of offspring are neither ethical nor feasible
 Earliest linkage studies based on pedigree analysis
 lod score (= log of the odds)

 Relies on probability calculations
 Demonstrates linkage between two genes when linkage analysis relies primarily on
pedigrees
 Assesses probability that pedigree with two traits reflects genetic linkage between them
 Lod score accuracy is limited by the extent of the pedigree
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VI.2- Somatic Cell Hybridization
 Made possible the assigning of human genes to their respective chromosomes

 Involves fusing two cells into a single hybrid cell: heterokaryon
 Possible to fuse cells of different origins (e.g. human and mouse)
 As the heterokaryons are cultured in vitro:
 Nuclei fuse together, creating a synkaryon.
 After many generations, chromosomes from one of the two parental species are gradually
lost
 In the case of human–mouse hybrids, preferential loss of human chromosomes (rather than mouse
chromosomes)
 Possible to assign human genes to the chromosomes on which they reside
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VI.2- Somatic Cell Hybridization
 Synteny testing: correlation of the presence or absence of each chromosome with

the presence or absence of each gene product
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VII- Chromosome Mapping is Now

Possible Using DNA Markers and
Annotated Computer Databases
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VII.1- DNA Markers
 DNA markers
 Short segments of DNA with known sequence and location
 Useful landmarks for mapping
 Earliest examples of DNA markers:
 RFLPs and microsatellites
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VII.2- RFLP and Microsatellites
 RFLP
 Restriction fragment length polymorphism
 Polymorphic sites
 Generated when specific DNA sequences are recognized and cut by restriction enzyme
 Microsatellites
 Short repetitive sequences
 Found throughout genome
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VII.3- SNP
 Single Nucleotide Polymorphism (pronounced snip)

 Found throughout genome
 Used by geneticists to identify and locate related genes
 Used to screen for diseases
 Example: Cystic fibrosis
 Gene located by using DNA markers
 Life-shortening autosomal recessive exocrine disorder
 Gene causing disorder found on chromosome 7
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VIII- Crossing Over Involves a Physical

Exchange between Chromatids
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Chiasmata and Crossing Over
 Genetic mapping techniques used to study relationship between chiasmata and

crossing over
 Mapping in maize
 Used cytological markers
 Established crossing over involves a physical exchange of chromosome regions
 2 linked genes on chromosome 9 of the maize plant

 Alleles colorless (c) and colored (C) control endosperm coloration
 Alleles starchy (Wx) and waxy (wx) control the carbohydrate characteristics of the
endosperm.
 The maize plant studied was heterozygous at both loci.
 One of the homologs contained two unique cytological markers.
 a densely stained knob at one end of the chromosome
 a translocated piece of another chromosome (8) at the other end. 53
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IX- Exchanges Also Occur between
Sister Chromatids During Mitosis
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IX.1- Sister Chromatid Exchanges
 Sister chromatid exchanges (SCEs)

 occur during mitosis
 do not produce new allelic combinations
 Reciprocal exchanges similar to crossing over
 Between sister chromatids (crossing over is between NONsisters)
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IX.2- Harlequin Chromosomes
 Sister chromatids involved in mitotic exchanges

 Identification and study of SCEs by several unique staining
techniques
 Patch-like appearance of chromosomes = Harlequins
 cells replicate for two generations in the presence of a base analog (e.g.
bromodeoxyuridine BrdU, an analog of thymidine).
 For each chromosome, one chromatid with one strand of DNA “labeled” and the other with both
strands labeled.
 Differential stain to visualize chromatids with the analog in both strands stain less brightly than
chromatids with BrdU in only one strand.
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IX.3- Relevance
 The significance of SCEs is still uncertain but great interest in this phenomenon
 Increase in SCE frequency:
 By agents that induce chromosome damage (e.g., viruses, X rays, ultraviolet light, and
certain chemical mutagens).
 In Bloom Syndrome (mutation of BLM gene on chromosome 15)
 Rare human disorder (autosomal recessive)
 Prenatal and postnatal retardation of growth
 great sensitivity of the facial skin to the sun
 immune deficiency
 predisposition to malignant and benign tumors
 Etc.
 Excessive SCEs + chromosomal breaks and rearrangements
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X- Did Mendel Encounter Linkage?
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 Often said that Mendel was lucky enough not to encounter linkage
 7 genes / 7 chromosomes in pea
 Modern genetics allowed to link several genes on the same chromosome, but:
 Either too far apart (i.e. behaved as independent)
 Either their combination not studied/unpublished by Mendel.
 If he had:
 Might not have recognized basic patterns of inheritance
 Might not have interpreted them correctly
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Chapter 5 - Chromosome Mapping in Eukaryotes

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Chapter 5 - Chromosome Mapping in Eukaryotes

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Chapter 5 Outline - Learning

Maya Hobeika Kahwagi © USEK 2023-2024

 Chromosomes are the unit of transmission in meiosis, not genes

 Frequency of crossing over on a single chromosome is proportional to distance

I- Genes Linked on the Same

Maya Hobeika Kahwagi © USEK 2023-2024

Maya Hobeika Kahwagi © USEK 2023-2024

Maya Hobeika Kahwagi © USEK 2023-2024

NB: 1:2:1 in cases of trans-linkage (dominant/recessive vs recessive/dominant)

I.2- Linkage Ratio and Groups

Maya Hobeika Kahwagi © USEK 2023-2024

Maya Hobeika Kahwagi © USEK 2023-2024

Maya Hobeika Kahwagi © USEK 2023-2024

Morgan’s Questions /answers

II.2- Sturtevant and Mapping

 Sturtevant (Morgan’s student)

Maya Hobeika Kahwagi © USEK 2023-2024

 Map unit (mu)

II.3- Single Crossovers

 During meiosis, a limited number of crossover events occur RANDOMLY in each

Maya Hobeika Kahwagi © USEK 2023-2024

II.3- Single Crossovers

 After a single exchange between two nonsister chromatids in tetrad stage

Maya Hobeika Kahwagi © USEK 2023-2024

III.1- Multiple Exchanges

Maya Hobeika Kahwagi © USEK 2023-2024

III.1- Multiple Exchanges

 3 couples of alleles to analyze DCO

 expected frequency of double-crossover gametes is extremely low

Maya Hobeika Kahwagi © USEK 2023-2024

 Three criteria of three-point mapping

III.2- Three-Point Mapping in Drosophila

Maya Hobeika Kahwagi © USEK 2023-2024

Maya Hobeika Kahwagi © USEK 2023-2024

III.2- Three-Point Mapping in Drosophila

Determining Gene Sequence

Maya Hobeika Kahwagi © USEK 2023-2024

Determining Gene Sequence

III.3- An Autosomal Mapping Problem in Maize

 Differences with previous example:

Maya Hobeika Kahwagi © USEK 2023-2024

Switched gene in the middle

Maya Hobeika Kahwagi © USEK 2023-2024

 Expected frequency of multiple exchanges between two genes predicted from

Maya Hobeika Kahwagi © USEK 2023-2024

IV.1- Interference and the Coefficient of

 19.6 % fewer double crossovers occurred than expected.

Maya Hobeika Kahwagi © USEK 2023-2024

 Positive interference most often observed

 Physical constraints preventing the formation of closely spaced chiasmata

IV.2- Interference and distance

 Two genes close together

Maya Hobeika Kahwagi © USEK 2023-2024

 Large number of mutants in organisms such as

 Virtually every morphological feature of the fruit fly subjected to mutation.

Maya Hobeika Kahwagi © USEK 2023-2024

VI- Lod Score Analysis and Somatic Cell

Maya Hobeika Kahwagi © USEK 2023-2024

 In humans, genetic experiments involving carefully planned crosses and large

 lod score (= log of the odds)

VI.2- Somatic Cell Hybridization

 Made possible the assigning of human genes to their respective chromosomes