The Genetics of Caenorthabditis Elegans

Brenner, 1974

Lecture by Assaf Tal

Talk Outline

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

Gene:

Set of bases in DNA

Paradigm: 1 Gene per Protein

Allele: A particular Gene Realization

Homologous Chromosomes

Gene

Alleles(recessive/dominant)

Genotype: Aa

Phenotype: “what we see”

Wild Type: “common” phenotype

Mutant: “non-natural” phenotype

How to Make a Genetic Map

Step I Find out how many genes are 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

Blue Eyes

Induce Mutations in Population

No blue eyes

Gene B

Protein A Protein B

How to Find Complementary

Groups?

Same Complementary

Group

Some disrupt Gene A

Some disrupt Gene B

No blue eyes

Breed & Observe Phenotypes

Complementation Analysis

Mutation I Mutation II

Case I: Allelic Mutations Case II: Non-Allelic Mutations

Mutation I Mutation II

Recessive Mutations

Mutant

Phenotype

Wild Type

Mapping “Linkage Groups”“Unlinked” Reproduction

A B a b

a b a bA a

B b bb

a a

25% aB

25% ab

25% AB

25% Ab

Mapping “Linkage Groups”“Linked” Reproduction with Recombination (w/ probability p)

A a

B b bb

a a

ab

AB

A a

Bb bb

a a

aB

p/2

Cis

1

2

p

Recombination

w/

Prob. p

1

2

p

Ab

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

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 Term Google 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

Recessive vs. Dominant Need to maintain wild-type male population

Male population 0.02% in nature Keep male population by crossing w/

hermaphrodites In real life, we need to take into account co-

dominant mutations, sex-linked mutations, etc …

Some Mutants . . .

Gene Mapping in C. Elegans

Mutant 1 Mutant 2

Allelic Mutations Non-Allelic Mutations

Example: Complementation test for Recessive Mutations

Mutant

Phenotype

Wild Type

Mutant 1 Mutant 2

Allelic Mutations

Mutant

Hermaphrodite

WT

Male

WT Male

Mutant

Hermaphrodite

WT

Male

WT MaleMut. Herm, Mut. Herm,

Non- Allelic Mutations

50% Male

WT

50% Male

Mutants

100% Male WT

Brenner’s Map

Brenner’s Map

Conclusions (cont.)

Map 258 Autosomal Mutations into 77 Complementation Groups

Accompanying paper: ~ 6.7x107 base pairs 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

Mapping the Nervous system

John Sulston

Tracing cell lineage, apoptosis

Bob Horvitz

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”

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 movement-

related 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” 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

Taken from Horvitz (1986)

(bar = 10 micron)

Programmed cell death does not occur in a ced-3 mutant.

Mapping the Genetic Pathway

From Worms to Humans

Biological Universality

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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