The Genetics of Caenorthabditis Elegans Brenner, 1974

1. The Genetics of Caenorthabditis ElegansBrenner, 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 Homologous Chromosomes Gene Alleles (recessive/dominant) • Gene: • Set of bases in DNA • Paradigm: 1 Gene per Protein • Allele: A particular Gene Realization 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 IIPlace them on Chromosomes ( “Linkage Mapping”) Step IIIDetermine their distances & refine (“Linkage, 3-point Mapping”)

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

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

10. Mapping “Linkage Groups” “Unlinked” Reproduction A a a a B b b b 25% AB 25% aB 25% Ab 25% ab

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

12. Mapping “Linkage Groups” Placing Genes on Chromosomes (to 0th 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 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

16. 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

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 Example: Complementation test for Recessive Mutations Non- Allelic Mutations Allelic Mutations Allelic Mutations Non-Allelic Mutations Mutant Hermaphrodite WT Male Mutant Hermaphrodite WT Male Mutant 1 Mutant 2 Mutant 1 Mutant 2 Mut. Herm, Mut. Herm, WT Male WT Male Mutant Phenotype Wild Type 50% Male WT 50% Male Mutants 100% Male WT

20. Brenner’s Map

21. Brenner’s Map

22. 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

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

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 Programmed cell death does not occur in a ced-3 mutant. Taken from Horvitz (1986) (bar = 10 micron)

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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