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The Genetics of Caenorthabditis Elegans Brenner, 1974

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?”.

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The Genetics of Caenorthabditis Elegans Brenner, 1974

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  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!

  35. Merchandise (only 8.99$)

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