1. The Genetics of Caenorthabditis Elegans Brenner, 1974 Lecture by Assaf Tal

2. Talk Outline Background for Paper Concept of a Genetic Map Present Experimental System Present & Discuss Results Further Work

3. From Genes to Structure “How do genes specify the structure of an organism?” - Brenner, 1974

4. 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”

5. The Goal: To study the connection between micro (genes) and macro (development) on a multi-cellular “simple” organism A Genetic Map

6. 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

7. 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”)

8. Complementary Groups Gene A Blue Eyes Induce Mutations in Population No blue eyes Gene B Protein AProtein B How to Find Complementary Groups? Same Complementary Group Some disrupt Gene A Some disrupt Gene B No blue eyes Breed & Observe Phenotypes

9. Complementation Analysis Mutation IMutation II Case I: Allelic Mutations Case II: Non-Allelic Mutations Mutation IMutation II Recessive Mutations Mutant Phenotype Wild Type

10. Mapping “Linkage Groups” “Unlinked” Reproduction Aa Bbbb aa 25% aB 25% ab 25% AB 25% Ab

11. Mapping “Linkage Groups” “Linked” Reproduction with Recombination (w/ probability p) Aa Bbbb aa ab AB Aa B b bb aa aB p/2 Cis Recombination w/ Prob. p Ab p/2

12. Mapping “Linkage Groups” Placing Genes on Chromosomes (to 0 th order): Recombination Frequency (p) ~ Distance of Genes Part of X-chromosome of Drosophila

13. 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.

14. 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

15. Popularity of C. Elegans Search TermGoogle Entries C. Elegans2,550,000 Drosophila1,040,000 Depeche Mode4,120,000 God421,000,000 Sex460,000,000

16. Isolating Mutants For recessive mutations: The F 2 generation of an infected parent will be 25% homozygote mutants Continue for another generation to ensure mutants can reproduce

17. 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 …

18. Some Mutants...

19. Gene Mapping in C. Elegans Mutant 1Mutant 2 Allelic MutationsNon-Allelic Mutations Example: Complementation test for Recessive Mutations Mutant Phenotype Wild Type Mutant 1Mutant 2 Allelic Mutations Mutant Hermaphrodite WT Male WT Male Mutant Hermaphrodite WT Male WT Male Mut. Herm, Non- Allelic Mutations 50% Male WT 50% Male Mutants 100% Male WT

20. Brenner’s Map

22. Conclusions (cont.) Map 258 Autosomal Mutations into 77 Complementation Groups Accompanying paper: ~ 6.7x10 7 base pairs Naïve reasoning: ~ 6.7x10 4 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

23. Further Work John White Mapping the Nervous system John Sulston Tracing cell lineage, apoptosis Bob Horvitz Connection between worms & humans

24. 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

25. Complete Lineage Map:

26. Cell Differentiation is “Rigid” Nature or nurture? Development of Reproduction Organs It seems that nature! Do genes really specify the development?

27. 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!!!

28. 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?

29. 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.

30. Visual Evidence of Cell Death Taken from Horvitz (1986) (bar = 10 micron) Programmed cell death does not occur in a ced-3 mutant.

31. Mapping the Genetic Pathway

32. From Worms to Humans

33. 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.”

34. The End Thank you!

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