Download Lecture 2

Lecture 2
• Lab2
• Allele classification
• Genetic screens
• Epistasis
Groups
Group 1-1
Josh Farhi
Tyler Madden
Group 1-2
Youssef Neema
Christine Schmidt
Group 2-1
Caitlin Carlisle
Kourtney Gordon
Nina Nissan
Group 2-2
Nicole Stabler
Rachel Edgar
Group 2-3
Kathleen Shah
Camille Fong Chih Kai
Sunday heat shock times
Arrival
time
2:55
Heat
shock
3:20
Groups
1-1
1-2
Monday heat shock times
Arrival
time
1:55
Heat
shock
2:20
2-1
2:35
3:00
2-2
Groups
2-3
Where do we do the heat shocks?
Room 361 of the Western Science Center.
How do I get there on Sunday if the front doors of WSC
are locked? Tunnels.
A) Middlesex College front door turn left down to the
staircase in the middle of the hallway. Go down to
tunnel that leads to the WSC.
B) Natural Science Center to Physics and find a tunnel
leading to WSC.
C) Natural Science Center to B&G third floor to WSC
Allele classification
Functional allele
wild-type allele
Active gene product
Loss-of-function allele (lf)
Generally recessive
Two classes important for this course
Loss-of-function allele (lf)
Null alleles-amorphic alleles
Completely inactive
gene product
Loss-of-function allele (lf)
Null alleles-amorphic alleles
No gene product
Regulatory mutant resulting in
no expression
Loss-of-function allele (lf)
weak alleles-hypomorphic alleles
Partially inactive gene
product
Loss-of-function allele (lf)
weak alleles-hypomorphic alleles
Partially inactive gene product:
multifunctional protein with only 1 of
2 functions affected by the change.
Loss-of-function allele (lf)
weak alleles-hypomorphic alleles
Partial expression of
a gene product
Regulatory mutant resulting in
partial expression
Hypomorphic regulatory mutant
Gut enhancer
ORF
gene something
Brain
Gut
embryo of something
Hypomorphic regulatory mutant
Gut enhancer
ORF
gene something
Brain
Brain
Gut
Wild-type
Gut
mutant
Gain-of-function alleles (gf)
Generally associated with misregulation of a gene
product’s activity, and are generally dominant.
Three examples
Many developmentally important genes are
expressed in a spatially restricted pattern.
The pattern of the expression is important
for the phenotype of the organism. Non- or
mis-expression of the gene can result in a
phenotype.
Antennapedia is expressed in
the second thoracic segment
where the second leg will form.
Leg to antenna
transformation.
Antennapedia can be misexpressed either by spontaneous
mutation or by genetic engineering.
+
Antp
ry
hsp
Antenna to second leg transformation
Summary of experiments on Antennapedia
Antenna
primordia
Leg
primordia
antenna
leg
antenna
antenna
leg
leg
wild type
Antplf
Antpgf
Dominant negative (dn)
antimorph
Criteria
The gene product works in a complex, either with itself
or another protein, and the activity of all the proteins
in the complex is essential for the total activity of the
complex.
homodimer
X
Complex inactive
heterodimer
X
Complex inactive
X
Mutation must not affect the ability
of the complex to form.
A dominant negative allele reduces activity to a
greater extent than a null allele when heterozygous.
dn
X
wt
wt
x
x x
x
1 :
1/2 wild type activity
2
:
1
1/4 wild type activity
Multiple copies of the dn allele or overexpression
will strongly inhibit wild type activity.
dn
X
X
X
X
x x
x x Only rarely will a
complex of two
x x wild type proteins
form.
x
x x
wt
Genetic screens
Genetic analysis requires genetic variants.
Saturation screens
An attempt to identify as many genes whose products
contribute to the process that you are studying as is
statistically and technically possible.
Genetic screen for leucine auxotrophic yeast
Yeast cells
mutagenesis
Replica plate to
minimal media
+leu
Random pool of DNA
sequence changes
-leu
Complementation analysis
leu1 X leu2
leu1
X
X
leu2
leuNon-complementation
in same complementation group
=same gene.
leu1
X
leu+
leu+
X
leu2
leu+
Complementation
Example of 100 mutants and 1,000 genes required
for leucine biosynthesis.
901
900
#
Complementation
groups (genes)
100
Poisson
distribution
zero group=e-m
98
1
0 1 2 3
#hits (numbers of independent allele/
complementation group (gene))
Example of 100 mutants and 10 genes required
for leucine biosynthesis.
e-10=4.5X10-5
3
#
Complementation 2
groups (genes)
1
0
10
20
#hits (numbers of independent allele/
complementation group (gene))
Real example
A Biochemical Pathway
A
B
C
D
E
Epistasis
Epistatic interactions are assayed by comparing
the phenotype of a double mutant organism
with that of the singly mutant organisms.
Epistasis: Criteria for the two mutations
A. Have related phenotypes
growth control
sex determination
dorsal ventral axis determination
B. Work on a pathway that makes a distinct decision
growth/nongrowth
male/female
expression/nonexpression
C. The two mutations have distinct/opposite phenotypes
all males versus all females
expression always ON versus always OFF
Ventralized versus Dorsalized
Control of sporulation by sporulation inducing
factor (sif) in a hypothetical fungus
No sporulation
Sporulation
The pathway that controls sporulation
Receptor protein
kinase
Kinase Inhibitor of
sporulation
Inducer of
sporulation Spo genes
-sif
Inactive
OFF
Inactive
OFF
Active Inactive
ON
OFF
No
expression
OFF
+sif
Active
ON
Active
ON
Inactive
OFF
Active
ON
Expression
ON
Epistasis example
Drosophila embryos have a dorsal and ventral side.
Mutations exist where the mother lays eggs where
the embryo develops with only the dorsal side lacking
The ventral side (dorsalized). Mutations exist also that
ventralize the embryo.
Loss-of-function alleles
spz
Toll
pelle
tube
dl
phenotype
dorsalized
dorsalized
dorsalized
dorsalized
dorsalized
cact
ventralized
Double mutants
spz cact
Toll cact
pelle cact
tube cact
dl cact
ventralized
ventralized
ventralized
ventralized
dorsalized
What does the above data tell you about the order of function?
Draw out the pathway indicating positive and negative interactions.
Tollgf
ventralized
Tollgf spz
pelle Tollgf
tube Tollgf
dl Tollgf
ventralized
dorsalized
dorsalized
dorsalized
What does this additional information tell you about
the order of function?