Download The Big Picture: an outline of the concepts covered to date

Recombination and Mapping (cont’d)
1
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
2
Sex specific Differences
-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
3
Sex specific differences: human chromosome 12.
4
geneB
geneC
geneD
geneE
geneA
Recombination hot spots
A
B
C
d
e
a
b
c
D
E
-hotspots of recombination and recombination deserts*****
5
Chromosome Position specific effects on recombination
Drosophila X
Recomb
Freq
Cen
2MU
Fw
2MU
Wy
Fa
Tel
Pa
Fa and Pa are 2MU apart -low recombination freq
Fw and Wy are 2MU apart- high recombination freq
Real distance
Fa-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
6
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?
One major reason for this is that the recombination rates are
not equal through the length of the chromosome.
7
Recombination hot spots
Recombination rate along chromosome 12
Sequencing the whole genome of a family:
On average ~100 crossovers in maternal genome
On average ~60 crossovers in paternal genome
Recombination is 1.7x more frequent in females than males
~100 out of ~160 Crossovers were at hotspots of
recombination
dad
mom
J. C. Roach et al., Science 328, 636-639 (2010)
9
xxxxxxxxx
10
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 function
Parts of a gene cannot function
2
Genes are fundamental units of mutation
The gene changes as a whole from one form to
another
3
Genes are fundamental units of structure
The gene is indivisible by recombination
We will go through experiments that show that
recombination does occur within a gene
11
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 bristles
f=forked bristles
carnation is a recessive mutation that alters the
normal bright Red-eyes to a dull red color
carnation
Forked bristles
C=normal red eyes
c=dull carnation eyes
?
12
Recombination
How do you detect recombination between these genes?
f
c
F C
f c
x
F C
Y
F
C
f
c
F
recom
C
Y
FC
parental
(X-linked)
Y
FC
WT
WT
400
fc
WT
Forked carnation 400
Fc
WT
carnation
fC
WT
forked
50
50
100/900
The presence of individuals with recombinant phenotypes
indicates that recombination has occurred between these two
genes
Recombination frequency is ~10%
13
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
C
F
f1
f2
14
Recombination frequency
Looking for recombination between two mutations within the
same gene, we are dealing with extremely small rates of
Recombination
For example
The Drosophila X chromosome is approximately 10 megabases
(10,000,000 bps)
The white gene (w) is about 1000 bp
The 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
15
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
16
Intragenic recombination cross
w1
w2
w1
w2
To detect rare recombinants between w1 and w2
We perform the following cross:
w1/w2
x
W/Y
w1
W
Y
w2
W
w1
w2
w1
w2
W
Y
W/w1 Red
w1/Y White
W/w2 Red
w2/Y White
W/w1,2 Red
w1,2/Y White
W/W Red
W/Y Red
parental
recom
17
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)
8/10,000
0.08 MU or cM
The results demonstrate that the gene is divisible by
recombination
Mutations in a gene occur in different positions within
that gene
18
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
19
IC
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
CCCCCCCCCCCCCCCCCCCCTCCCC
GGGGGGGGGGGGGGGGGGGGAGGGG
CCACCCCCCCCCCCCCCCCCCCCCC
GGTGGGGGGGGGGGGGGGGGGGGGG
Analysis in the bacteriophage T4 reveals that
recombination can occurs between single nucleotides
20
21
Mitotic recombination
Although not as frequent, recombination between homologous
chromosomes occasionally occurs in mitosis as well as meiosis.
This was first discovered in Drosophila by Kurt Stern and now
has important implications for the origins of some human
cancers
Stern made the following cross:
ｷ
y = yellow body
ｷ
sn = singed bristles
ｷ
y+ = normal body
ｷ
sn+ = normal bristles
The F1 females
These should be phenotypically normal
22
Mitotic recombination
Although not as frequent, recombination between homologous
chromosomes occurs occasionally in mitosis as well as meiosis.
This was first discovered in Drosophila by Kurt Stern and now
has important implications for the origins of some human
cancers
Stern made the following cross:
ｷ
y = yellow body color
ｷ
sn = singed bristles
ｷ
y+ = normal body color
ｷ
sn+ = normal bristles
y+ sn
y+ sn
The F1 females
x
y sn+
y sn+
y+ sn
y sn+
These should be phenotypically normal
23
Mitotic recombination
However, some females in a background of normal bristle
and normal body color had sectors of singed bristle,
normal colored tissue next to sectors of yellow body color
normal bristle length.
y+ sn
y sn+
Because these sectors were adjacent to one another, Stern
thought that the two spots must be reciprocal products of the
same event.
That event may be a rare crossover between homologs during
the mitotic divisions.
24
A normal mitotic division would occur as follows:
y+ sn
y sn+
y+ sn
y sn+
Replication
y+ sn
y sn+
y+ sn
y sn+
Segregation
y+ sn
y sn+
Genotypically identical
Daughter cells
y+ sn
y sn+
25
If you have recombination in mitosis
Replication
y+ sn
y sn+
y+ sn
y sn+
Segregation
Genotypically different
Daughter cells
26
If you have recombination in mitosis
Replication
y+ sn
y sn+
y+ sn
y sn+
y+ sn
y+ sn
y sn+
y sn+
y+ sn
y+ sn
y sn+
y sn+
Segregation
y+ sn
y+ sn
Genotypically different
Daughter cells
y sn+
y sn+
27
Mitotic recombination
This only happens in somatic cells in your body
It is therefore not passed on to your progeny.
So Why is this important?
28
Rb
While mitotic recombination is rare, it does occur.
It can convert a heterozygous cell into a homozygous mutant
cell and a wild-type.
Formation of homozygosity in cells can be carcinogenic if a
mutated gene becomes homozygous in somatic cells!!!
Retinoblastoma (Rb) is a human tumor that sometimes results
from a mitotic recombination event.
Rb is a childhood cancer of the eye
It occurs from birth to 4 years of age
If discovered early enough, treatment is 90% effective
29
Rb
There are two forms of Rb:
Hereditary and sporadic
Hereditary Rb: patients typically develop multiple eye tumors
involving both eyes. These tumors develop at an early age.
Siblings often develop the same sort of tumors.
Sporadic Rb (60% of the cases): The development of the eye
tumor is a spontaneous event in the patient with no history
of the disease. Tumors develop only in one eye. Occurs later
than hereditary Rb
30
Hereditary Retinoblastoma
Rb+/Rb(phenotypically normal)
Mutation
Rb-/Rb- cell
(eye tumor)
Sporadic Retinoblastoma
Rb+/Rb+
(phenotypically normal)
First point mutation
Rb+/Rb- cell
(phenotypically normal)
Second mutation
Rb-/Rb- cell
(eye tumor)
31
Knudson proposed the following model to explain both forms of
Rb:
In Knudson's model of hereditary Rb, instead of a mutation
producing cells homozygous for the Rb gene, a mitotic
recombination event could also result in homozygosity for the
Rb gene
Rb+/-
Rb+/+
Inherited
Rb+/+
Non-hereditary
Second
Point mutation
-
Chromosome loss
Mitotic
recombination
-
32
33
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
A
a
A
a
B
OR
b
b
B
34
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
No exceptions to Mendellian laws
at the level of the gene, but
Phenotype ratios are modified
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.
35
a
10
b
20
c
20
d
15
e
15
f
g
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%
36
a
10
b
20
c
20
a
f
a
f
A
F
A
F
d
15
e
f
15
af
aF
Af
AF
5
g
P
R
R
P
Recombinant/total = 2/4=50%
Maximum freq is 50%
37
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
38
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
Seed color
chr1
Flower color
chr1
Pod shape
chr4
Flower position
chr4
Stem length
chr4
Pod color
chr5
Seed shape
chr7
False
39
Ratios
A
a
A
a
A
a
b
B
A
a
b
B
x
A
a
3:1
x
a
a
1:1
x
A
a
b
B
9:3:3:1
x
a
a
b
b
1:1:1:1
40
Chromosomes, genes, alleles, proteins, phenotypes
Each chromosome in G1 has one DNA molecule
Each 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
blanco
Forked
bristle
Shaven body
white
yellow
chromosome
Many genes
Genes on
DNA
white1
white2
41