Transcript The Genetics of C elegans (Brenner)

The Genetics of
Caenorthabditis Elegans
Brenner, 1974
Lecture by Assaf Tal
Talk Outline
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Background for Paper
Concept of a Genetic Map
Present Experimental System
Present & Discuss Results
Further Work
From Genes to Structure
“How do genes specify the structure of an
organism?”
- Brenner, 1974
Sydney Brenner (1927 - )
 Discoverer of frameshift mutations (1962)
 Believed “classical” molecular biology was
“solved”
Letter to Max Perutz (1963): “ ... Attempt to
define the unitary steps of development in
terms of genetic analysis … “
1963 Research Proposal: “The New Major
problem in molecular biology is the genetics
… of control mechanisms”
The Goal:
To study the connection between micro
(genes) and macro (development) on a
multi-cellular “simple” organism
A Genetic Map
Genetics Refresher
Homologous
Chromosomes
Gene
Alleles
(recessive/dominant)
Gene:
 Set of bases in DNA
Genotype: Aa
 Paradigm: 1 Gene per
Protein
Phenotype: “what we see”
Allele: A particular Gene
Realization
Mutant: “non-natural” phenotype
Wild Type: “common” phenotype
How to Make a Genetic Map
Step I
are
Find out how many genes
responsible for each phenotype
( “Complementary Analysis”)
Step II
Place them on Chromosomes
( “Linkage Mapping”)
Step III
Determine their distances &
refine (“Linkage, 3-point
Mapping”)
Complementary Groups
Gene A
Protein A
Gene B
Protein B
Blue Eyes
Same
Complementary
Group
How to Find
Complementary
Groups?
Induce Mutations in
Population
Some disrupt
Gene A
Some disrupt
Gene B
No blue eyes
No blue eyes
Breed & Observe
Phenotypes
Complementation Analysis
Recessive Mutations
Case I: Allelic Mutations
Mutation I
Mutation II
Mutant
Phenotype
Case II: Non-Allelic Mutations
Mutation I
Mutation II
Wild Type
Mapping “Linkage Groups”
“Unlinked” Reproduction
A
a
a
B
b
a
b
25%
AB
25%
aB
25%
Ab
25%
ab
b
AB ab

a b ab
Mapping “Linkage Groups”
“Linked” Reproduction with Recombination (w/ probability p)
Recombination
Cis
w/
A
a
a
a
B
b
b
b
Prob. p
A
a
a
a
b
B
b
b
AB
aB
1 p
2
p/2
ab
Ab
1 p
2
p/2
Mapping “Linkage Groups”
Placing Genes on Chromosomes (to 0th order):
Recombination Frequency (p) ~ Distance of Genes
Part of X-chromosome
of Drosophila
C. Elegans – “Nature’s Gift”
Small ( ~ 1 mm).
Fast life cycle ~ adulthood in
less than 24 hrs, live 3 weeks
959 somatic cells.
Most adults are
hermaphrodites.
Genetic composition:
5AA + XX
Rare males:
5AA + XO
Easy to handle in large
quantities.
Popularity of C. Elegans
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First organism to have its DNA sequenced
(1998).
Only organism to survive Columbia shuttle
crash (2003)
Exhibit same symptoms as humans when
quit smoking.
www.wormbase.org
Popularity of C. Elegans
Search TermGoogle Entries
C. Elegans
2,550,000
Drosophila
1,040,000
Depeche Mode
4,120,000
God
421,000,000
Sex
460,000,000
Isolating Mutants
For recessive mutations:
The F2 generation of an
infected parent will be 25%
homozygote mutants
Continue for another
generation to ensure
mutants can reproduce
Classifying Mutants
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Recessive vs. Dominant
Need to maintain wild-type male population
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Male population 0.02% in nature
Keep male population by crossing w/
hermaphrodites
In real life, we need to take into account codominant mutations, sex-linked mutations,
etc …
Some Mutants . . .
Gene Mapping in C. Elegans
Example: Complementation test for Recessive Mutations
Allelic Mutations
Mutant 1 Mutant 2
Mutant
Non-Allelic Mutations
Mutant 1 Mutant 2
Allelic Mutations
Non- Allelic Mutations
Mutant
WT
Mutant
WT
Hermaphrodite
Male
Hermaphrodite
Male
WT Male
Mut. Herm,
50% Male
50% Male
WT
Mutants
WT Male
Mut. Herm,
Wild Type
Phenotype
100% Male WT
Brenner’s Map
Brenner’s Map
Conclusions (cont.)
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Map 258 Autosomal Mutations into 77
Complementation Groups
Accompanying paper: ~ 6.7x107 base pairs
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Naïve reasoning: ~ 6.7x104 proteins (genes)
EMS induces mutations at rate 5x10-4/gene
Mutations per worm: ~ 34
In practice: induced lethal frequency is 0.15 per X
chromosome
Conclusion: 300 “essential” X-Chrom. Genes
Scaled estimate: 2000 “essential” genes
Further Work
John White
John Sulston
Bob Horvitz
Mapping the
Nervous system
Tracing cell
lineage,
apoptosis
Connection
between worms
& humans
Tracing the Cell Lineage
• Somatic cells vs. Germ cells
• John Sulston: the first to observe cell
differentiation in a multicellular
organism in real time
• Always the same
• Cells die of themselves
• C. Elegans is ideal:
• Simple (<1000 somatic cells)
• Transparent
Complete Lineage Map:
Cell Differentiation is “Rigid”
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Nature or nurture?
Development of
Reproduction
Organs
It seems that
nature!
Do genes really
specify the
development?
Genes dictate development
• 302 Neurons for adult hermaphrodite (382 for male)
• To map: 20,000 slices, 0.05μm thick
• Lineage of neurons can be traced
• Re-enter: Brenner
Induce
movementrelated Mutation
Examine
Nervous
System
Check if it
is
Hereditary
Genes indeed dictate development!!!
Cell Death (Apoptosis)
• Cell death:
• Injury, infection, . . .
• By their own volition:
• By external signals
• By internal “pre-programming”
• Purposes: structure formation, removing aged cells, …
• In C. Elegans, 131 cells are “programmed to die”
• Internal preprogramming ↔ genetic origin?
Evidence of Cell “Fate”
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J. E. Sulston, Philos. Trans. R. Soc. London Ser.B 1976, 275, 287-298: The
nuc-1, controlling the activity of DNA endonuclease
E.M.Hedgecock,J.E.Sulston,J.N.Thomson, Science 1983, 220, 1277-1279:
ced-1, ced-2 genes responsible for cell engulfment (phagocytosis)
“Jackpot”: ced-3
 H. R. Horvitz et al. ,Neurosci. Comment. 1982, 1,56-65.
 H. M. Ellis, H. R. Horvitz, Cell 1986, 44, 817-829.
Visual Evidence of Cell Death
Programmed cell death does
not occur in a ced-3 mutant.
Taken from Horvitz (1986)
(bar = 10 micron)
Mapping the Genetic Pathway
From Worms to Humans
Biological Universality
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C. Elegans shares about 35% of its genome
with humans
Horvitz Nobel lecture, 2002:
“One point that emerges . . . is the striking similarity of genes . . .
among organisms . . . I like to refer to this theme as “the principle of
biological universality” . . . and it underlies my conviction strong
conviction that the . . . study of the biology of any organism is likely
to lead to findings of importance in the understanding of other
organisms, including ourselves.”
The End
Thank you!
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