Lab2

1
Lecture 2

• Lab2
• Allele classification
• Genetic screens
• Epistasis

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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
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Sunday heat shock times
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Monday heat shock times
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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 BG third floor to WSC
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Allele classification
Functional allele wild-type allele
Active gene product
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Loss-of-function allele (lf)
Generally recessive
Two classes important for this course
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Loss-of-function allele (lf)
Null alleles-amorphic alleles
Completely inactive gene product
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Loss-of-function allele (lf)
Null alleles-amorphic alleles
No gene product
Regulatory mutant resulting in no expression
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Loss-of-function allele (lf)
weak alleles-hypomorphic alleles
Partially inactive gene product
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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.
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Loss-of-function allele (lf)
weak alleles-hypomorphic alleles
Partial expression of a gene product
Regulatory mutant resulting in partial expression
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Hypomorphic regulatory mutant
Gut enhancer
ORF
gene something
Brain
Gut
embryo of something
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Hypomorphic regulatory mutant
Gut enhancer
ORF
gene something
Brain
Brain
Gut
Gut
mutant
Wild-type
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Gain-of-function alleles (gf)
Generally associated with misregulation of a gene
products activity, and are generally dominant.
Three examples
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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.
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Antennapedia can be misexpressed either by
spontaneous mutation or by genetic engineering.
Antp
ry
hsp
Antenna to second leg transformation
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Summary of experiments on Antennapedia
Leg primordia
Antenna primordia
antenna
leg
wild type
antenna
antenna
Antplf
leg
leg
Antpgf
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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.
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heterodimer
homodimer
X
X
Complex inactive
Complex inactive
X
Mutation must not affect the ability of the
complex to form.
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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 2 1
1/2 wild type activity
1/4 wild type activity
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Multiple copies of the dn allele or
overexpression will strongly inhibit wild type
activity.
dn
x
x
X
Only rarely will a complex of two wild type
proteins form.
X
x
x
X
x
x
X
x
x
x
wt
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Genetic screens
Genetic analysis requires genetic variants.
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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.
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Genetic screen for leucine auxotrophic yeast
Yeast cells
mutagenesis
Replica plate to minimal media
leu
-leu
Random pool of DNA sequence changes
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Complementation analysis
leu1 X leu2
leu1
leu1
leu
X
X
X
X
leu2
leu2
leu
leu-
leu
Non-complementation in same complementation
group same gene.
Complementation
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Example of 100 mutants and 1,000 genes
required for leucine biosynthesis.
901
900
Poisson distribution zero groupe-m
Complementation groups (genes)
98
100
1
0
1
2
3
hits (numbers of independent allele/ complementat
ion group (gene))
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Example of 100 mutants and 10 genes required for
leucine biosynthesis.
e-104.5X10-5
3
Complementation groups (genes)
2
1
0
10
20
hits (numbers of independent allele/ complementat
ion group (gene))
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Real example
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A Biochemical Pathway
A
B
C
D
E
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Epistasis
Epistatic interactions are assayed by
comparing the phenotype of a double mutant
organism with that of the singly mutant organisms.
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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
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Control of sporulation by sporulation
inducing factor (sif) in a hypothetical fungus
No sporulation
Sporulation
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The pathway that controls sporulation
Receptor protein kinase
Inhibitor of sporulation
Inducer of sporulation
Kinase
Spo genes
-sif
No expression OFF
Inactive OFF
Active ON
Inactive OFF
Inactive OFF
sif
Active ON
Inactive OFF
Active ON
Active ON
Expression ON
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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.
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Loss-of-function alleles phenotype spz dorsal
ized Toll dorsalized pelle dorsalized tube
dorsalized dl dorsalized cact ventra
lized Double mutants spz cact ventralized Toll
cact ventralized pelle cact ventralized tub
e cact ventralized dl cact dorsalized
What does the above data tell you about the order
of function? Draw out the pathway indicating
positive and negative interactions.
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Tollgf ventralized Tollgf spz ventralized p
elle Tollgf dorsalized tube Tollgf
dorsalized dl Tollgf dorsalized
What does this additional information tell you
about the order of function?