C. elegans lecture

Kaveh Ashrafi

kaveh.ashrafi@ucsf.edu

N412C Genentech Hall415.514.4102

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

Sydney Brenner

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

I. OVERVIEWC. elegans as an experimental system

Life cycleShort reproductive maturation time & large

number of progeny

From wormatlas: www.wormatlas.org

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

ADULT MALE

Sex

autosomes (pairs) sex chromosome(s)

5 XX

5 XO

body plan of an adult hermaphrodite

Hermaphrodites are self fertilizing because they contain bothoocytes and sperm

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.

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

II. GENETIC BASICS

Self progeny vs. cross progeny

~100% XX

F1 have genotype of parent (clonal)

X

50% XX 50% XO

F1 hermaphrodites are heterozygous at all loci; F1 males are heterozygous at all autosomal loci, hemizygous on X

I, II, IIIIV, V, X

I, II, IIIIV, V, X

I, II, IIIIV, V, X

I, II, IIIIV, V, X

I, II, IIIIV, V, X

I, II, IIIIV, V

Example of a genetic cross in C. elegans

UNC=uncoordinated movementunc-40(e271) I a recessive mutation

X unc-40 (e271)/unc-40 (e271) +/+

F1 :

self progeny: 100% Unc, ~100% hermaphrodite unc-40/unc-40 cross progeny: 100% non-Unc (WT), unc-40/+

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 F1 that is cross progeny, single onto a new plate, allow to self

F2 (from self fertilization of cross progeny)

III. GENETIC SCREENS

Point of entry into a biological process.

A simple screen that can produce informative, tractable mutations with strong

and specific phenotypes.

F1: m/+ can identify dominant mutations

F2: +/+; m/+; m/m can identify dominant & recessive mutations

Po

A simple forward genetics screening strategy

+/+

m1 ++

+ ++

m2

m3+

+

m5m4

+m6 m7

F3: can identify maternal effect mutations; shows of mutations identified in F2 breed true

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

Po

F1

F2

Positional mapping using SNPs

X

Select F2 progeny with desired phenotype

Rescue & Transgenics

*Inject DNA fragments from wild type into mutant animals to identifyRescuing region.

*Sequence DNA region from mutant to identify mutation.

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