“Coupling” and “repulsion”
Back to Bateson and Punnett
Sample
Observed
Expected
284
215
Purple, long (P_L_)
Purple, round (P_ll)
21
71
Red, long (ppL_)
21
71
Red, round (ppll)
55
24
Total
381
381
Purple, long (P_L_ )
Purple, round (P_ll )
Red, long (ppL_ )
Red, round (ppll )
Observed
Expected (if
SMI)
284
21
21
55
215
71
71
24
(O-E)^2
(O-E)^2
div by E
4761.0
2500.0
2500.0
961.0
Null hypothesis: the genes exhibit SMI.
What is the likelihood that the observed difference is due solely to chance?
Well below 0.1%.
The null hypothesis is rejected.
What is going on? What can explain this “repulsion and coupling”?
Why are these two genes disobeying Mendel’s second law?
http://www.ndsu.edu/instruct/mcclean/plsc431/linkage/linkage1.htm
MCB140 2-2-07 1
MCB140 2-2-07 2
How to screen for a polymorphism
linked to, or directly causing, AMD?
Macular degeneration is a group of
diseases characterized by a breakdown of
the macula. The macula is the center
portion of the retina that makes central
vision and visual acuity possible.
First, what’s the evidence that there is a genetic component
to AMD?
“Numerous studies have attempted to establish dietary and
environmental risk factors that contribute to ARM;
however, heredity has emerged as the primary
determinant of ARM susceptibility (for a review see ref.
16). First-degree relatives of ARM patients are at
between two and four times greater risk of developing
ARM than the first-degree relatives of controls (17,18).
Twin studies have consistently shown high levels of
concordance of the disease among monozygous sibs
and strongly support a genetic etiology (19–22).”
“Age-related maculopathy (ARM), also
known as age-related macular
degeneration (AMD), is the leading cause
of irreversible vision loss in the elderly
population in the USA and the Western
world and a major public health issue.
Affecting nearly 9% of the population over
the age of 65, ARM becomes increasingly
prevalent with age such that by age 75
and older nearly 28% of individuals are
affected (1–6). As the proportion of the
elderly in our population increases, the
public health impact of ARM will become
even more severe. Currently there is little
that can be done to prevent or slow the
progression of ARM (7).”
http://hmg.oupjournals.org/cgi/content/full/9/9/1329#DDD140TB1
22.1
35.2
35.2
40.0
Chi
square
value
132.61
http://hmg.oupjournals.org/cgi/content/full/9/9/1329#DDD140TB1
MCB140 2-2-07 3
How to screen for a polymorphism
linked to, or directly causing, AMD?
MCB140 2-2-07 4
This effort led to chromosome 1q32
Second, how to find the locus? (aka
“candidate region”)
“We collected a set of 364 families (2129
individuals) containing relative pairs
affected with ARM.”
http://hmg.oupjournals.org/cgi/content/full/9/9/1329#DDD14
0TB1
MCB140 2-2-07 5
MCB140 2-2-07 6
1
Now what?
Two SNPs showed the greatest linkage, and they lie in a
260 kb region. This stretch contains the complement H
gene – CFH is a component of the innate immune
system which regulates inflammation, which, in turn, is
consistently implicated in AMD.
“Resequencing revealed a polymorphism in linkage
disequilibrium with the risk allele representing a tyrosinehistidine change at amino acid 402. This polymorphism
is in a region of CFH that binds heparin and C-reactive
protein. Individuals homozygous for the risk alleles have
a 7.4-fold increased likelihood of AMD (95% CI 2.9 to
19).”
Haines et al. Science 308: 419.
MCB140 2-2-07 7
Morgan Science 1911
MCB140 2-2-07 9
Daiger Science 308: 362.
Fig. 5.2
Morgan Science 1911
MCB140 2-2-07 8
MCB140 2-2-07 10
Batrachoseps attenuatus
California Slender Salamander
F.A. Janssens
MCB140 2-2-07 11
MCB140 2-2-07 12
2
Morgan’s observation of linkage
The data
One of these genes affects eye color (pr,
purple, and pr+, red), and the other affects
wing length (vg, vestigial, and vg+,
normal). The wild-type alleles of both
genes are dominant. Morgan crossed pr/pr
· vg/vg flies with pr+/pr+ · vg+/vg+ and then
testcrossed the doubly heterozygous F1
females: pr+/pr · vg+/vg × pr/pr · vg/vg .
1:1:1:1?! ☺
MCB140 2-2-07 13
Sample
AA
ab
Ab
aB
Observed
1339
1195
151
154
Expected (if
drug is
harmless)
710
710
710
710
(O-E)^2
395641
235225
312481
309136
(O-E)^2
div by E
557.2
331.3
440.1
435.4
MCB140 2-2-07 14
These two loci do not follow Mendel’s
second law because they are linked
Chi
square
1764.1
Null hypothesis: genes not linked.
What is the likelihood that the observed difference is due solely to chance?
Ummmmm. Yeah ….
--> null hypothesis, shmull hypothesis.
MCB140 2-2-07 15
MCB140 2-2-07 16
The data
F.A. Janssens
?
MCB140 2-2-07 17
MCB140 2-2-07 18
3
MCB140 2-2-07 19
MCB140 2-2-07 20
MCB140 2-2-07 21
MCB140 2-2-07 22
Recombination Frequency
(Morgan’s data)
1339 red, normal
1195 vermillion, vestigial
151 red, vestigial
154 vermillion, normal
2839 total progeny.
305 recombinant individuals.
305 / 2839 = 0.107
Recombination frequency is 10%.
MCB140 2-2-07 23
MCB140 2-2-07 24
4
Recombination frequency "
a genetic map (Sturtevant’s data)
Unit definition
1% recombinant progeny =
1 map unit =
1 centimorgan (cM) ~ 1 Mb
(note: the latter applies to humans)
MCB140 2-2-07 25
MCB140 2-2-07 26
The met protooncogene and the CFTR gene are 0.7 Mb
apart on chr. 7. In 1000 meiotic events, how many
gametes would you expect to find that have had a
recombination event between the two genes?
Mapping By Recombination
Frequency (Morgan’s data)
1339 red, normal
1195 vermillion, vestigial
151 red, vestigial
154 vermillion, normal
2839 total progeny.
305 recombinant individuals.
305 / 2839 = 0.107
Recombination frequency is 10%.
Map distance between the two loci is 10 m.u.
MCB140 2-2-07 27
MCB140 2-2-07 28
Problem 5.9
If the a and b loci are 20 m.u. apart in
humans, and an AB/ab woman has a
child with an ab/ab man, what is the
probability that this child will be Ab/ab?
MCB140 2-2-07 29
MCB140 2-2-07 30
5
If two genes, A and B, are 80 Mb apart on a
human chromosome, what percentage
recombinant gametes would you expect to be
produced in an individual who is AB/ab?
MCB140 2-2-07 31
MCB140 2-2-07 32
The chromosome as a “linkage group”
If genes are more than 50 map units apart, they behave as if they were unlinked.
MCB140 2-2-07 33
MCB140 2-2-07 34
Bridges (left) and Sturtevant in 1920.
G. Rubin and E. Lewis Science 287: 2216.
MCB140 2-2-07 35
Sturtevant 1961
MCB140 2-2-07 36
6
The three-point testcross
From my perspective, the single most
majestic epistemological
accomplishment of “classical” genetics
MCB140 2-2-07 37
How to Map Genes Using a ThreePoint Testcross
Reading
Two chapters from Morgan’s book (III, on
linkage, and V, on chromosomes).
A short chapter from Sturtevant’s History of
Genetics.
Chapter 5, section 2.
MCB140 2-2-07 39
P
v+/v+ · cv/cv · ct/ct
MCB140 2-2-07 38
×
1. Cross two pure lines.
2. Obtain large number of progeny from F1.
3. Testcross to homozygous recessive
tester.
4. Analyze large number of progeny from
F2.
MCB140 2-2-07 40
v/v · cv+/cv+ · ct+/ct+.
↓
F1
v/v+ · cv/cv+ · ct/ct+ ×
v/v · cv/cv · ct/ct.
↓
Two Drosophila were mated: a
red-eyed fly that lacked a crossvein on the wings and had snipped
wing edges to a vermilion-eyed,
normally veined fly with regular
wings. All the progeny were wild
type. These were testcrossed to a
fly with vermilion eyes, no crossvein and snipped wings. 1448
progeny in 8 phenotypic classes
were observed.
Map the genes.
MCB140 2-2-07 41
MCB140 2-2-07 42
7
1. Rename and rewrite cross
2. Rewrite data
Arrange in descending order, by frequency.
For data like these, no need to calculate χ2. Begin (you don’t have to, but it helps) by designating the
genes with letters that look different in UPPER and lowercase (e.g., not “W/w” but “Q/q” or “I/i”):
eye color: v+/v = E/e
vein on wings: cv+/cv = N/n
shape of wing: ct+/ct = F/f (you fly using wings)
P:
EE nn ff
x
ee NN FF
test-cross:
Ee Nn Ff
x
ee nn ff
NCOs
DCOs
e
E
e
E
e
E
E
e
N
n
n
N
n
N
n
N
F
f
F
f
f
F
F
f
MCB140 2-2-07 43
580
592
45
40
89
94
5
3
MCB140 2-2-07 44
3. Determine gene order
3b. Determine gene order
e
N
F
580
E
n
f
592
With the confusion cleared away, determine gene order by
e
n
F
45
comparing most abundant classes (non-recombinant, NCO) with
E
N (least abundant,
f
40
double-recombinant
DCO), and figuring out,
which one allele pair needs to be swapped between the parental
e
n
f
89
chromosomes in order to get the DCO configuration. This one
E
N
F
94
allele pair will be of the gene that is in the middle.
E
n
F
5
e
N
f
3
NCOs:
DCOs:
Enf
EnF
eNF
eNf
Gene order: E F N (or N F E).
MCB140 2-2-07 45
MCB140 2-2-07 46
4. E and F
4b. F and N
Next, map distance between genes E and F by comparing the
number of single recombinants (COs) for those two genes
with the number of NCOs.
e N F
580
E n f
592
e n F
45
E N f
40
e n f
89
E N F
94
e N f
3
E n F
5
RF=(89+94+3+5)/1448=0.132
Now, map distance between genes F and N by comparing
the number of single recombinants (COs) for those two genes
with the number of NCOs.
e
E
e
E
e
E
e
E
N
n
n
N
n
N
N
n
F
f
F
f
f
F
f
F
580
592
45
40
89
94
3
5
RF=(45+40+3+5)/1448=0.064
The F and N genes are separated by 6.4 m.u.
The E and F genes are separated by 13.2 m.u.
MCB140 2-2-07 47
MCB140 2-2-07 48
8
4c. E and N
5. The map (ta-daaa!)
Finally, map distance between genes E and N by
comparing the number of single recombinants (COs)
for those two genes and the number of DCOs for those
two genes with the number of NCOs. Count DCOs
twice because they represent two recombination
events, and to calculate the correct RF we must, by
definition, count every recombination event that
occurred between those two genes (even if it doesn’t
result in a recombinant genotype for those two genes!).
e
E
e
E
e
E
e
E
N
n
n
N
n
N
N
n
F
f
F
f
f
F
f
F
580
592
45
40
89
94
3
5
RF=(45+40+89+94+3+5+3+5)/1448=0.196
The E and N genes are separated by 19.6 m.u.
MCB140 2-2-07 49
6. Interference
MCB140 2-2-07 50
Final map:
A crossover event decreases the likelihood
of another crossover event occurring
nearby.
E FN
13.2 m.u.
6.4 m.u.
|-------------- 19.6 m.u.----------|
For dessert, do not forget to calculate interference for these loci.
The mathematical probability of seeing a DCO in this area is
equal to the product of probabilities of seeing a CO between E-F and seeing a CO between F--N:
p(expected DCOs)=0.132 x 0.064=0.008448
This means we should have seen 0.008448 x 1448= 12 DCOs. We
only saw 3 + 5 = 8, i.e. the observed frequency of DCOs is
8/1448 = 0.005524.
Interference is equal to 1 minus the “coefficient of coincidence”
= 1 - p(O)/p(E) = 35% " 35% of the double-recombination
events that were expected to have occurred based on
probabilistic considerations didn’t because of interference.
MCB140 2-2-07 51
MCB140 2-2-07 52
MCB140 2-2-07 53
MCB140 2-2-07 54
9
Ensuring you learn
A path
Office hours:
Monday, Feb. 12
Monday, Feb. 19
(both in Barker 101, noon – 1 pm)
Quiz: week of Feb. 12
Review session for midterm:
Monday, Feb. 12 (location TBA, 6.30 pm – 10 pm)
Note: postponement of study until review session
is a recipe for guaranteed failure.
MCB140 2-2-07 55
MCB 140
↓
Intern at Sangamo (" PubMed)
~2
- 3months
↓
RA at Sangamo (~1.5 years)
↓
Life
(grad. school / med. school / law school / etc)
MCB140 2-2-07 56
1. Sangamo " PubMed
2. urnov эт berkeley дот edu
MCB140 2-2-07 57
10