Lecture 2

• Lab2

• Allele classification

• Genetic screens

• Epistasis

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

Sunday heat shock times

Arrival time

Heat shock

Groups

2:55 3:20 1-1 1-2

Monday heat shock times

Arrival time

Heat shock

Groups

1:55 2:20 2-1 2-3

2:35 3:00 2-2

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

Functional allelewild-type allele

Allele classification

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)

Partial expression of a gene product

Regulatory mutant resulting in partial expression

weak alleles-hypomorphic alleles

Gut

Brain

Hypomorphic regulatory mutant

gene something

embryo of something

ORF

Gut enhancer

Gut

Brain

gene something

ORF

Gut enhancer

Gut

Brain

Wild-type mutant

Hypomorphic regulatory 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 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.

Antennapedia can be misexpressed either by spontaneous mutation or by genetic engineering.

Antenna to second leg transformation

hsp Antp ry+

Summary of experiments on Antennapedia

Antennaprimordia

Legprimordia

wild type

Antplf

Antpgf

antenna

antenna antenna

leg

leg

leg

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.

homodimer heterodimer

Complex inactive Complex inactive

Mutation must not affect the ability of the complex to form.

X X

X

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

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

Genetic screens

Genetic analysis requires genetic variants.

Saturation screens

An attempt to identify as many genes whose productscontribute to the process that you are studying as isstatistically and technically possible.

Genetic screen for leucine auxotrophic yeast

Yeast cells

mutagenesis

Random pool of DNAsequence changes

Replica plate tominimal media

+leu -leu

Complementation analysis

leu1 X leu2

leu1

leu2

leu1

leu2leu+

leu+

leu- leu+

Non-complementationin same complementation group=same gene.

Complementation

XXX X

#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

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

Real example

A B C D E

A Biochemical Pathway

Epistasis

Epistatic interactions are assayed by comparingthe phenotype of a double mutant organismwith that of the singly mutant organisms.

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

Control of sporulation by sporulation inducingfactor (sif) in a hypothetical fungus

No sporulation Sporulation

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

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.

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.

Tollgf ventralized

Tollgf spz ventralizedpelle Tollgf dorsalizedtube Tollgf dorsalizeddl Tollgf dorsalized

What does this additional information tell you aboutthe order of function?