Lecture 2 Lab2 Allele classification Genetic screens Epistasis.
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Transcript of Lecture 2 Lab2 Allele classification Genetic screens Epistasis.
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Lecture 2
• Lab2
• Allele classification
• Genetic screens
• Epistasis
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Group 1-1Josh FarhiTyler Madden
Group 1-2Youssef NeemaChristine Schmidt
Group 2-1Caitlin CarlisleKourtney GordonNina Nissan
Group 2-2Nicole StablerRachel Edgar
Group 2-3Kathleen ShahCamille Fong Chih Kai
Groups
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Sunday heat shock times
Arrival time
Heat shock
Groups
2:55 3:20 1-1 1-2
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Monday heat shock times
Arrival time
Heat shock
Groups
1:55 2:20 2-1 2-3
2:35 3:00 2-2
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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 WSCare locked? Tunnels.A) Middlesex College front door turn left down to the staircase in the middle of the hallway. Go down totunnel that leads to the WSC.B) Natural Science Center to Physics and find a tunnelleading to WSC.C) Natural Science Center to B&G third floor to WSC
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Functional allelewild-type allele
Allele classification
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)
Partial expression of a gene product
Regulatory mutant resulting in partial expression
weak alleles-hypomorphic alleles
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Gut
Brain
Hypomorphic regulatory mutant
gene something
embryo of something
ORF
Gut enhancer
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Gut
Brain
gene something
ORF
Gut enhancer
Gut
Brain
Wild-type mutant
Hypomorphic regulatory mutant
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Gain-of-function alleles (gf)
Generally associated with misregulation of a gene product’s 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 importantfor the phenotype of the organism. Non- ormis-expression of the gene can result in aphenotype.
Antennapedia is expressed inthe second thoracic segmentwhere the second leg will form.
Leg to antennatransformation.
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Antennapedia can be misexpressed either by spontaneous mutation or by genetic engineering.
Antenna to second leg transformation
hsp Antp ry+
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Summary of experiments on Antennapedia
Antennaprimordia
Legprimordia
wild type
Antplf
Antpgf
antenna
antenna antenna
leg
leg
leg
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Dominant negative (dn)antimorph
Criteria
The gene product works in a complex, either with itselfor another protein, and the activity of all the proteinsin the complex is essential for the total activity of thecomplex.
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homodimer heterodimer
Complex inactive Complex inactive
Mutation must not affect the ability of the complex to form.
X X
X
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A dominant negative allele reduces activity to a greater extent than a null allele when heterozygous.
1/2 wild type activity 1/4 wild type activity
X
wt wt
dn
x
xx x
1 : 2 : 1
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Multiple copies of the dn allele or overexpressionwill strongly inhibit wild type activity.
Only rarely will a complex of twowild type proteinsform.
X
wt
dn
XXX
x x
x x
x x
x x
x
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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 productscontribute to the process that you are studying as isstatistically and technically possible.
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Genetic screen for leucine auxotrophic yeast
Yeast cells
mutagenesis
Random pool of DNAsequence changes
Replica plate tominimal media
+leu -leu
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Complementation analysis
leu1 X leu2
leu1
leu2
leu1
leu2leu+
leu+
leu- leu+
Non-complementationin same complementation group=same gene.
Complementation
XXX X
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#hits (numbers of independent allele/complementation group (gene))
#Complementationgroups (genes)
Example of 100 mutants and 1,000 genes requiredfor leucine biosynthesis.
901
98
1
Poisson distributionzero group=e-m
0 1 2 3
100
900
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Example of 100 mutants and 10 genes requiredfor leucine biosynthesis.
e-10=4.5X10-5
#Complementationgroups (genes)
#hits (numbers of independent allele/complementation group (gene))
0 10 20
1
2
3
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Real example
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A B C D E
A Biochemical Pathway
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Epistasis
Epistatic interactions are assayed by comparingthe phenotype of a double mutant organismwith that of the singly mutant organisms.
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Epistasis: Criteria for the two mutations
A. Have related phenotypesgrowth controlsex determinationdorsal ventral axis determination
B. Work on a pathway that makes a distinct decisiongrowth/nongrowthmale/femaleexpression/nonexpression
C. The two mutations have distinct/opposite phenotypesall males versus all femalesexpression always ON versus always OFFVentralized versus Dorsalized
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Control of sporulation by sporulation inducingfactor (sif) in a hypothetical fungus
No sporulation Sporulation
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The pathway that controls sporulation
-sif
+sif
Receptor proteinkinase
Kinase Inhibitor ofsporulation
Inducer of sporulation Spo genes
InactiveOFF
InactiveOFF
InactiveOFF
InactiveOFF
ActiveON
ActiveON
ActiveON
ActiveON
NoexpressionOFF
ExpressionON
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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 lackingThe ventral side (dorsalized). Mutations exist also thatventralize the embryo.
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Loss-of-function alleles phenotypespz dorsalizedToll dorsalizedpelle dorsalizedtube dorsalizeddl dorsalized
cact ventralized
Double mutantsspz cact ventralizedToll cact ventralizedpelle cact ventralizedtube cact ventralizeddl 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 ventralizedpelle Tollgf dorsalizedtube Tollgf dorsalizeddl Tollgf dorsalized
What does this additional information tell you aboutthe order of function?