Good afternoon!

Welcome to weeks 5 – 8 !
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 https://participant.turningtechnologies.eu/en/join

Session ID: bio411p

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 Which of the following does droplet
single cell sequencing NOT require?
A. Microfluidics
B. Bar coded oligonucleotides
C. Biotinylation
D. Reverse Transcription
E. NGS sequencing
Which of the follow does the yeast two hybrid
system NOT utilize?
A. DNA binding domain fusion
proteins
B. Activation domain fusion
proteins
C. ‘Bait’ and ‘Prey’ proteins
D. Epitope tagged proteins
 Phage display is useful for
A. Generating polyclonal antibodies
B. Screening hybridoma cell lines
C. Identifying antigen binding
sequences
D. ‘Humanizing’ mAbs
 Focus of weeks 5 – 8

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Deciphering genome function
Topics for weeks 5 – 8

• Week 5: forward genetic screens

• Week 6: functional genomic screens

• Week 7: chemical genomics and

synthetic lethal screens

• Week 8: interfering with proteins and

analysing non-model organisms

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Deciphering genome function
Contents and organisation: reminder
• Thursday, first hour: general notions about the week’s topic
(some of these notions already known to some of you)
• Thursday, second hour: two ”case studies” exemplifying the application
of the week’s approaches to given scientific questions
• Final version of the lecture on Moodle by Thursday evening
• Exercises weeks 5 and 6: read one paper ahead of time -given on
Thursday; exercise questions related to that paper and to the lecture
• Exercises weeks 7 and 8: graded group project assigned in week 7
(Monday November 6th, 4:15PM), to be handed in by midnight on Friday
November 17th)
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 Forward genetic screens

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How can mutations reveal function?

?
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Forward genetic screens
Today’s menu

• Forward genetic screens: general notions

• Two case studies: deciphering cell cycle control

in S. cerevisiae and embryogenesis in Drosophila

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Introductory notions
Some definitions
• Locus: site of a given gene in the genome

• Mutation: change from the wild-type sequence in a given gene

• Allele: alternative form of a given gene (e.g. mutant allele)

• Genotype: DNA sequence of an individual (at a given gene -

sometimes understood at the genome level)

• Phenotype: detectable traits of an individual

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Introductory notions
Haploid (N) Diploid (2N)
Reminder

gametes somatic cells

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Introductory notions
Haploid (N) Diploid (2N)
Heterozygous versus homozygous

• Heterozygous (at a given locus): m/+

• Homozygous (at a given locus): m/m

gametes somatic cells
Heterozygous Homozygous

m m m

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Recessive vs dominant see
also
week 2

• If only homozygous has a phenotype typically recessive

> recessive allele

• If heterozygous has a phenotype

> dominant allele
null
allele
• Special case: haploinsufficiency,
whereby heterozygous loss-of- typically
function has a phenotype (rare) dominant

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Albinism: recessive trait
Parents
Loss-of-function
Normal Normal
Aa ´ Aa mutation in
Tyrosinase, one of
Sperm the genes essential
A a for melanin synthesis
Eggs
AA
Aa Fully penetrant
A Normal
Normal
(carrier)
(100% of the
individuals affected)
Aa
Normal aa
a Albino
(carrier)

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 Achondroplasia: dominant trait
Gain-of-function
Parents
mutation in FGR3, a
Dwarf Normal
gene that negatively
Dd ´ dd regulates bone growth
Sperm Fully penetrant
D d (100% of the
Eggs individuals affected)
Dd dd
d Dwarf Normal

Dd dd
d Dwarf Normal
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Steps for a successful genetic screen
Step 1: choosing the right model organism

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What criteria are important?
• Appropriate for given biological question
• Ease of scoring a given phenotype
• Ease of growth (including short generation time)
• Ease of genetic manipulation (including mating)
• Ethical considerations
• Sustainability considerations (environment + laboratory)
• Generality of conclusions drawn
• Eventual importance for human health and society

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Isogenic vs non-isogenic see
also
week 2
• In an isogenic model organism, all individuals have the
same genetic background (at the least initially…).

Isogenic Non-isogenic (but inbred) Non-isogenic

S. cerevisiae D. melanogaster

D. rerio
C. elegans

M. musculus H. sapiens

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 Phenotypic variability
• Same mutation, different outcome –can be due to differences
in the genetic background
Normal With tumor

Retinoblastoma (Rb): tumor suppressor

(some types of Rb mutations)
24 Daruma: leptin receptor
Steps for a successful genetic screen
Step 2: conducting the actual screen

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Steps for a successful genetic screen
Step 2: conducting the actual screen

Forward Reverse genetics

genetics (functional genomics)
targeted

random
next
week
this
week
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Screen vs selection
random mutagenesis

screen

selection

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How does one generate mutations?
• Wait and see (spontaneous mutations)…

Thomas H. Morgan Drosophila eye

wild-type white

Science 32: 120 (1910)

28 Nobel prize in 1933

How does one generate mutations?
• Wait and see (spontaneous mutations)…

Thomas H. Morgan Drosophila eye

wild-type white

Science 32: 120 (1910)

29 Nobel prize in 1933

How does one generate mutations?
• Too slow for most organisms (and Hermann J. Muller
scientists...) -unless there is a selection
step

• In 1926, Müller used X-rays to induce

mutations experimentally, increasing
their frequency >10’000 fold

Nobel prize in 1946

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Some commonly used mutagens see
also
week 2

• Ionizing radiations (e.g. X-rays, gamma rays) typically generate

large changes (deletions, inversions, translocations, …)
• Chemical mutagens (e.g. alkylating agents such as EMS or
ENU) typically generate point mutations (silent, missense or
nonsense), but also small deletions
• EMS: ethyl methanesulfonate leads to hydrolysis of purine
deoxyribose bond; as a result, any of the four bases can be
introduced upon DNA replication
• ENU: N-ethyl-N-nitrosourea leads to random alkylation of
nucleic acids; as a result, DNA replication is inaccurate and
random bases are introduced

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Conditional mutations
• Strong loss-of-function mutations in
essential genes are difficult -and
sometimes impossible- to isolate

• Alternative: conditional mutations

(e.g. temperature sensitive mutations)

• More generally: acute inactivation

can be advantageous vs chronic
inactivation (e.g. UAS-GAL4 system in
Drosophila, Lox-Cre system in mice,
chemical genetics) see
also
week 2
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 https://participant.turningtechnologies.eu/en/join

Session ID: bio411p

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Which geneticist said the following?
Two years work wasted, I have been breeding those
flies for all that time and I’ve got nothing out of it.

A. Gregor Mendel
B. Thomas Morgan
C. Hermann Muller
D. Seymour Benzer
E. Brian McCabe

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Simplifying: complementation analysis
• Allows one to determine whether two recessive mutant alleles
with a similar homozygous phenotype affect the same gene
wild-type C. elegans unc-32(e189) (Unc phenotype)

complements (wild-type)
> different genes
fails to complements (Unc)
> recessive mutations > same gene
doi: 10.1895/wormbook.1.24.1 (2005) trans-heterozygous

• Although complementation analysis allows one to reach the

36 correct conclusion in most cases, there are rare exceptions
Ordering the action of genes
• The analysis of double-mutants (in two different genes) can
reveal their order of action in a linear pathway
• Works best when nature of pathway is known
• This is referred to as epistasis analysis

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 Epistasis analysis exemplified
+/+; m1/m1; +/+; m1/m1;
+/+ +/+ m2/m2 m2/m2

Gene 1: Gene 2:
TYRP2 Tyrosinase
Dopachrome tautomerase
(tyrosine-related protein 2) 2 epistatic to 1
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Mosaic analysis to test autonomy
• Reveals in which cell the function of a given gene is required
wild-type (A+B) mutant (A+B)

phenotype
A B A B
configurations

A B A B cell autonomous
mosaic

A B A B cell non-autonomous
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Steps for a successful genetic screen
Step 4: identifying the mutated gene

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Identifying the gene of interest
• Linkage analysis (> chromosome identified)

• Recombination mapping (> region on chromosome identified)

• Deficiency mapping (> region on chromosome identified)

• Before: positional cloning (can take a long time… e.g. 13 years for per)

• Nowadays: whole genome sequencing (aided by recombination mapping)

• Key experiment in any case: rescue by wild-type copy

(or else: induce the same mutation using CRISPR/Cas9)
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Forward genetic screens: key points
• Let the organism tell you what is important

• Different mutagens can be used

• Loss-of-function, gain-of-function and conditional alleles

• Complementation analysis

• Epistasis analysis, mosaic analysis

• Gene identification

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Forward genetic screens
Today’s menu

• Forward genetic screens: general notions

• Two case studies: deciphering cell cycle control

in S. cerevisiae and embryogenesis in Drosophila

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Cell cycle control in S. cerevisiae

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Key role of Cdk/Cyclin complexes
Nobel Prize in Physiology or Medicine 2001

Leland Hartwell Tim Hunt Paul Nurse

S. cerevisiae sea urchin S. pombe
(budding yeast) (fission yeast)

45 https://www.youtube.com/watch?v=mJNyKHRAp0k
What drives the cell cycle?

?
bud (daughter)

parent (mother)

G1 S

M G2

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Duration of typical human cell cycle?

A. 4 hours
B. 12 hours
C. 24 hours
D. 48 hours
Cell cycle at work in human cells
mAzami Green-Geminin (fragment)
(from coral Galaxea fascicularis)

mKusabira Orange-Cdt1 (fragment)

(from coral Fungia concinna)

49 Sakaue-Sawano et al.; 2008

What drives the cell cycle?

?
bud (daughter)

parent (mother)

G1 S

M G2

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A series of revealing experiments
• Fusion of cells at different cell cycle stages
bud (daughter)

parent (mother)

Rao and Johnson, 1970

Conclusion: S et M phases contain activities

that bring G1 cells into the S or M “state”
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What is the nature of these activities?
bud (daughter)

parent (mother)

G1 S ?
?
G2
M

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S. cerevisiae cell cycle see
also
week 2
• Factoids: ~10 µm-long, ~90 min cell cycle
bud (daughter)

parent (mother)

bud (daughter)

parent (mother)

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Principle of Cdc genetic screen
• Hypothesis: components of the cell cycle engines must be essential,
hence the use of temperature-sensitive mutants

• Haploid cells grown at permissive temperature of 23oC before being

shifted to the restrictive temperature of 36oC

• Key advance: use of photo-microscopy to analyze phenotypes of

Cell-division cycle (Cdc) mutants

• Relative size of bud and mother cell reveals phenotype

• Focused on mutants with reproducible phenotypes, distinguishing

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execution point and termination point
Illustration: first three Cdc mutants
wild-type cdc-1
cdc-2

cdc-1

White : execution point

cdc-2 cdc-3 Black: termination point

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Cdc28: evolutionarily conserved Cdk
• Cdk/Cyclin complexes:
engines of the cell cycle
From basic
• Function by phosphorylating research to the
key substrates to enable clinic: FDA-
transitions between cell cycle approved CDK
phases (e.g. G1/S and G2/M) inhibitors

ribociclib

Genetics 74: 267 (1973)

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Embryogenesis in Drosophila

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Genetic control of embryogenesis
Nobel Prize in Physiology or Medicine 1995

Edward Lewis Christiane Eric Wieschaus

Nüsslein-Volhard

https://www.ibiology.org/development-
58 and-stem-cells/developmental-genes/
Drosophila life cycle see
also
week 2

• Factoids: ~10 mm long,

~ 12 days life cycle,
embryogenesis lasts
~ 12 hours

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A powerful EMS-based genetic screen
• Analyze larval epidermis (cuticle), which harbors landmarks for
position and polarity

• Block agar method to increase throughput

• Dominant temperature-sensitive mutant to kill unwanted progeny

during screen, thereby also significantly increasing throughput

• Critical importance of team work

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Larval cuticle reflects embryo pattern
bud (daughter)

parent (mother)

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First instar larva
Drosophila embryonic fate map
bud (daughter)

wild-type cuticle (ventral)

parent (mother)

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 Creative use of genetics
DTS91: isogenize chromosome II
dominant
temperature-
bud (daughter)

sensitive lethal
(at 29o) parent (mother)

CyO: balancer, kill unwanted progeny

prevents
recombination
(homozygous
lethal)
focus on defects in larval cuticle

white-eyed

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An interesting cast of characters

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An impressive cast of characters
• Evolutionarily conserved
components, important
for development and From basic
physiology across research to the
metazoan evolution, clinic: FDA-
approved
including in human beings
Hedgehog
pathway
inhibitors

Sonidegib
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Two case studies: key points
• The awesome power of genetic screens

• Proper choice of model organism

• Design of genetic screen to address biological question

• Cdc screen in S. cerevisiae

• Embryonic lethal screen in Drosophila

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How can mutations reveal function?

?
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Forward genetic screens can help!

√
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Monday session
• In preparation for Monday: read “Clock Mutants of Drosophila
melanogaster” (on Moodle)

• On Monday: exercises regarding this paper and the lecture

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Primer on attached X chromosomes
• Compound or attached X chromosomes (denoted X∧X) consist of
two X chromosomes sharing one centromere, so that they are
always inherited together
• Note that X to autosome ratio determines sex in Drosophila
X mutation
EMS

X
X

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Do not forget to express your views

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Questions?

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