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1
Recombination and Mapping (cont’d)
2
Factors affecting MU
In most cases the order of genes revealed by mapping techniques correspond to the order of genes determined by sequencing.
In contrast, actual physical distance between genes does not show direct correspondence to map units.
Gene order, but not gene distance, is usually consistent between genetic and physical maps.
-for genes far apart, double, triple etc crossovers affect MU
-Species specific differences
Humans 1MU is ~ 1 million bp; Yeast 1MU is ~ 5000 bp
-Extreme example: In Drosophila males, there is no recombination during meiosis
Sex specific Differences
3
-Sex specific differences
For example markers D12s7 and Pah
males Rf= 9%
females Rf= 22%
Extreme example: In Drosophila males, there is no recombination during meiosis
4
Sex specific differences: human chromosome 12.
Recombination hot spots
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gen
eA
gen
eB
gen
eC
gen
eD
gen
eE
D Ea b c
A B C d e
-hotspots of recombination and recombination deserts*****
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Chromosome Position specific effects on recombination
Fa and Pa are 2MU apart -low recombination freqFw and Wy are 2MU apart- high recombination freq
Real distanceFa-Pa = 1x106 bp
Fw-Wy = 0.5x106 bp
Fw-Wy are closer together along the DNA but because of higher recombination rate they appear to be farther apart
Cen Tel
RecombFreq
Drosophila X
Fw Wy Fa Pa
2MU 2MU
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One major reason for this is that the recombination rates are not equal through the length of the chromosome.
2m.u lies between Pa and Fa- these two genes are located near the telomere
2m.u lie between Fw and Wy- these two genes are located in the middle of the chromosome
What can you conclude about the physical distance between these two sets of genes?
Recombination hot spots
Recombination rate along chromosome 12
xxxxxxxxx
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Intragenic recombination
Up until this point, genes have been viewed as a linear array of indivisible functional units on a chromosome
The tenets of the model are
1 Genes are fundamental units of functionParts of a gene cannot function
2 Genes are fundamental units of mutationThe gene changes as a whole from one form to another
3 Genes are fundamental units of structureThe gene is indivisible by recombination
We will go through experiments that show that recombination does occur within a gene
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Recombination and genes
If recombination occurs within a gene, how do you detect it?
How do we detect recombination between two different genes
Drosophila: Two genes
forked (f) is a recessive mutation that gives rise to forked bristles
F=normal bristlesf=forked bristles
carnation is a recessive mutation that alters the normal bright Red-eyes to a dull red color
C=normal red eyesc=dull carnation eyes?
Fork
ed
bri
stl
es
carn
ati
on
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Recombination
How do you detect recombination between these genes?
f c
F C x F Cf c Y
F C
f c
F C
Y
The presence of individuals with recombinant phenotypesindicates that recombination has occurred between these two genesRecombination frequency is ~10%
(X-linked)
parental
recom
FC Y
FC
fc
Fc
fC
WT
WT
WT
WT
WT
Forked carnation
carnation
forked
400
400
50
50
100/900
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What about recombination within a gene
Does recombination occur within a gene?Is there something special about a gene that makes it indivisible by recombination
If recombination occurs within a gene how would you detect it?
Recombination rates are a function of the distance between genes
Greater the distance between genes, higher the recombination frequency
For forked and carnation, Rf was approximately 10%These two genes are 240,000 bp apart
To find out if recombination occurs within a gene we can look for recombination between two mutations within the same gene
f c
F C
f1 f2
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Recombination frequency
Looking for recombination between two mutations within the same gene, we are dealing with extremely small rates of Recombination
For exampleThe Drosophila X chromosome is approximately 10 megabases (10,000,000 bps)The white gene (w) is about 1000 bpThe white gene represents about 1/10,000th the length of the X-chromosome
To look for recombination within a gene we perform the identical set of crosses used to look for recombination between genes
w1
w2
w1
w2
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Recombination frequency
To look for recombination within a gene we perform the identical set of crosses used to look for recombination between genes
For example we isolate two independent mutations in the white gene
w1 and w2
(How do we know that w1 and w2 are in the same gene?)
w1
w2
w1
w2
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Intragenic recombination cross
To detect rare recombinants between w1 and w2We perform the following cross:
w1/w2 x W/Y
w1
w2
W
Y
Y
W/w1 Red
W
w1
w2
W
w1/Y White
w2/Y White
w1,2/Y White
W/Y Red
w1
w2
w1
w2
parental
recom
W/w2 Red
W/w1,2 Red
W/W Red
w2w1
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The result
The cross produced the following results
10,000 males obtained
9996 were white eyed
4 were red eyed
Map distance = # recombinants/total progeny
4+4/10,000 (why 4+4?) because 4 recombinants were white and have to be added to the 4 red to get total recombinants)
8/10,0000.08 MU or cM
The results demonstrate that the gene is divisible by recombination
Mutations in a gene occur in different positions within that gene
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Normal gene ATG GGG GGG TTT CCC TTT AAA
Mutant1 ATG CGG GGG TTT CCC TTT AAA
Mutant2 ATG GGG GGG TTT CCC ATT AAA
mut1 mut2 x normal/Y
Parental classes
1 ATG CGG GGG TTT CCC TTT AAA
2 ATG GGG GGG TTT CCC ATT AAA
Recombinant classes
3 ATG CGG GGG TTT CCC ATT AAA
4 ATG GGG GGG TTT CCC TTT AAA
IC
19
Definitions
Intragenic recombination: Recombination occurring within a gene
Intergenic recombination: Recombination occurring between genes
Fine structure Analysis: Mapping a large number of mutations within a single gene through recombination
CCCCCCCCCCCCCCCCCCCCTCCCCGGGGGGGGGGGGGGGGGGGGAGGGG
CCACCCCCCCCCCCCCCCCCCCCCCGGTGGGGGGGGGGGGGGGGGGGGGG
Analysis in the bacteriophage T4 reveals that recombination can occurs between single nucleotides
20
21
The Big Picture: an outline of the concepts covered to date
1. Genes are physical units of hereditary that carry information from one generation to the next
2. Mendel elucidated the following principles regarding the inheritance patterns of genes
A. Each diploid individual contains two copies of a given gene
B. Each Gene can have different forms called alleles. There are two alleles in a diploid individual
The form that is expressed phenotypically in the heterozygote is known as the dominant allele. It is an operational definition
C. These copies (alleles) segregate from one another to form gametes. There is a single copy of each gene in a gamete (one allele of a gene in a gamete)
D. Different genes assort independently from one another during gamete formation (unless they are on the same chromosome and are linked)
3. The inheritance pattern of genes parallels the behavior of chromosomes at meiosis. This generated the hypothesis that genes reside on chromosomes
OR
A
aA
a
B
bb
B
22
The Big Picture
4. Exceptional patterns of chromosome segregation
The X/X and X/Y sex chromosomal system produces exceptional segregation patterns because males contain only one copy of X-linked genes
Non-disjunction: homologous chromosomes migrate to the same pole during meiosis
5. Exceptional expression (phenotype) patterns:
Incomplete dominance,
Co-dominance,
Lethal alleles
6. Genes that reside close to one another on the same chromosome do not assort independently- linkage +++++exceptions to Mendellian laws at the level of independent assortment of two genes+++++
7. Occasionally recombination occurs between these linked genes. The higher the frequency of recombination between any two genes, the greater the distance between them. Recombination frequencies serve as a useful method of mapping genes along a chromosome.
No exceptions to Mendellian lawsat the level of the gene, but Phenotype ratios are modified
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a b c d e f g10 20 20 15 15 5
This map means that there is a 20% recombination frequency between the genes b and c and a 5% recombination frequency between the genes f and g
Genes very far apart on the same chromosome will appear to assort independently
How many map units between a and f?
a-f = 80 cM
What is the recombination freq between a and f?
Is it 80%, less than 80%, more than 80%
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a b c d e f g10 20 20 15
15 5
a f
a f
A F
A F
af PaF RAf RAF P
Recombinant/total = 2/4=50%
Maximum freq is 50%
25
The largest distance that can be measured by this technique is 50MU.
50% also indicates NO LINKAGE
If two genes are very far apart on the same chromosome, use markers between these genes to more accurately map the genes
Therefore when you obtain a recombination frequency of 50% this means that either:
the genes are on two different chromosomes
OR very far apart on the same chromosome
26
Mendel studied 7 traits that assorted independently.
The only explanation for this behavior is that the genes controlling these traits are located on different chromosomes.
True False
Seed color chr1
Flower color chr1
Pod shape chr4
Flower position chr4
Stem length chr4
Pod color chr5
Seed shape chr7
27
Ratios
A Aa x a 3:1
A aa x a 1:1
A b A ba B x a B 9:3:3:1
A b a ba B x a b 1:1:1:1
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Chromosomes, genes, alleles, proteins, phenotypes
Each chromosome in G1 has one DNA moleculeEach chromosome in G2 has two DNA molecules (2 sister chromatids)
Each chromosome has many genes
A gene has many forms- alleles - two alleles in a diploid
Each allele produces a protein that give rise to a phenotype
Different alleles are caused by different changes in the same gene
wh
iteyellow
Sh
aven
bod
y
Fork
ed
b
ristl
e
bla
nco
white1
Many genes
Genes on DNA
white2
chromosome