1. C. elegans lecture Kaveh Ashrafi kaveh.ashrafi@ucsf.edu N412C Genentech Hall 415.514.4102

2. Genetics concepts: -diploid genetics: *somatic tissue is diploid all the time *hermaphrodite genetics -multicellular organism: * when & where gene function is required (mosaic analysis, tissue/developmental stage specific promoters, cell ablation) -forward and reverse screens

3. Sydney Brenner

4. Goldstein lab movie ( http://www.bio.unc.edu/faculty/goldstein/lab/movies.html)

5. I.OVERVIEW C. elegans as an experimental system

6. Life cycle Short reproductive maturation time & large number of progeny From wormatlas: www.wormatlas.org

7. Basic anatomy: tube within a tube Outer tube -body wall-cuticle -epithelial system -muscle system -excretory system -nervous system (hermaphrodite: 302 neurons, 5000 synaptic connections) only organism for which complete wiring diagram known Pseudocoelomic cavity -fluid-filled; transport Inner tube -alimentary system (pharynx/intestine) -reproductive system

8. ADULT MALE Sex autosomes (pairs) sex chromosome(s) 5XX 5XO

9. body plan of an adult hermaphrodite Hermaphrodites are self fertilizing because they contain both oocytes and sperm

10. Attractions for developmental biology & neurobiology: invariant somatic cell lineage Cell divisions give rise to 1090 cells. 959 survive, 131 die ==>discovery of genetic basis of programmed cell death.

11. How do cells adopt their fates? (cellular basis of asymmetry, differentiation programs) How do they end up in the right place at the right time? How do cell come together to form organs/tissues? (3D migration, programmed cell death, developmental timing) How do cells communicate with each other? (signaling cascades, neuroendocrine pathways) Molecular genetic analysis of disease processes, physiology, & behavior Genetics of Development, Physiology, & behavior

12. II.GENETIC BASICS

13. Self progeny vs. cross progeny ~100% XX F 1 have genotype of parent (clonal) X 50% XX 50% XO F 1 hermaphrodites are heterozygous at all loci; F 1 males are heterozygous at all autosomal loci, hemizygous on X I, II, III IV, V, X I, II, III IV, V, X I, II, III IV, V, X I, II, III IV, V, X I, II, III IV, V, X I, II, III IV, V

14. Example of a genetic cross in C. elegans UNC=uncoordinated movement unc-40(e271) I a recessive mutation X unc-40 (e271)/unc-40 (e271) +/+ F 1 : self progeny: 100% Unc, ~100% hermaphrodite unc- 40/unc-40 cross progeny: 100% non-Unc (WT), unc-40/+

15. Example of a genetic cross in C. elegans 1/4 unc-40/unc-40 1/2 unc-40/+ 1/4 +/+ Phenotypic ratios for recessive alleles? Dominant alleles? What are the sex ratios? What if mutation is on X? unc-40+ unc-40 + Take F 1 that is cross progeny, single onto a new plate, allow to self F 2 (from self fertilization of cross progeny)

16. III.GENETIC SCREENS Point of entry into a biological process. A simple screen that can produce informative, tractable mutations with strong and specific phenotypes.

17. F 1: m/+ can identify dominant mutations F 2: +/+; m/+; m/m can identify dominant & recessive mutations PoPo A simple forward genetics screening strategy +/+ m1++ +++ m2 m3 + + m5 m4 + m6 m7 F 3: can identify maternal effect mutations; shows of mutations identified in F 2 breed true

18. From screen to gene identity *Determine if the mutants breed true *Backcross *Determine nature of the mutation (e.g. dominant/recessive) *Determine # of complementation groups *Determine molecular identity: mapping

19. PoF1F2PoF1F2 Positional mapping using SNPs X Select F 2 progeny with desired phenotype

20. Rescue & Transgenics *Inject DNA fragments from wild type into mutant animals to identify Rescuing region. *Sequence DNA region from mutant to identify mutation.

21. general considerations regarding screens Specificity of phenotype under study Robustness of phenotype under study You always have to balance the ease of screening scheme/assay with the desired targeting/specificity of desired phenotype/pathway