C. elegans lecture

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C. elegans lecture Kaveh Ashrafi kaveh.ashrafi_at_
ucsf.edu N412C Genentech Hall 415.514.4102
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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
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Sydney Brenner
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Goldstein lab movie (http//www.bio.unc.edu/facult
y/goldstein/lab/movies.html)
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• OVERVIEW
• C. elegans as an experimental system

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• Life cycle
• Short reproductive maturation time large number
  of progeny

From wormatlas www.wormatlas.org
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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) -repro
ductive system
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Sex
ADULT MALE
autosomes (pairs) sex chromosome(s)
5 XX
5 XO
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body plan of an adult hermaphrodite
Hermaphrodites are self fertilizing because they
contain both oocytes and sperm
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Attractions for developmental biology
neurobiology invariant somatic cell lineage
Cell divisions give rise to 1090 cells. 959
survive, 131 die gtdiscovery of genetic basis of
programmed cell death.
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Genetics of Development, Physiology, behavior
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
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II. GENETIC BASICS
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Self progeny vs. cross progeny
X
I, II, III IV, V, X
I, II, III IV, V, X
I, II, III IV, V, X
I, II, III IV, V
I, II, III IV, V, X
I, II, III IV, V, X
50 XX 50 XO F1 hermaphrodites are
heterozygous at all loci F1 males are
heterozygous at all autosomal loci, hemizygous on
X
100 XX F1 have genotype of parent (clonal)
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Example of a genetic cross in C. elegans
UNCuncoordinated movement unc-40(e271) I a
recessive mutation
unc-40 (e271)/unc-40 (e271)
X
/
F1 self progeny 100 Unc, 100
hermaphrodite unc-40/unc-40 cross
progeny 100 non-Unc (WT),
unc-40/
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Example of a genetic cross in C. elegans
Take F1 that is cross progeny, single onto a new
plate, allow to self F2 (from self fertilization
of cross progeny)
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III. GENETIC SCREENS Point of entry into a
biological process. A simple screen that
can produce informative, tractable mutations with
strong and specific phenotypes.
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A simple forward genetics screening strategy
Po
/

m1


m2


m5
m6



m3
m7
m4
F1 m/
can identify dominant mutations
F2 / m/ m/m can identify
dominant recessive mutations
F3 can identify maternal effect mutations
shows of mutations identified in F2 breed true
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From screen to gene identity Determine if the
mutants breed true Backcross Determine nature
of the mutation (e.g. dominant/recessive) Determi
ne of complementation groups Determine
molecular identity mapping
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Positional mapping using SNPs
Po F1 F2
X
Select F2 progeny with desired phenotype
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Rescue Transgenics
Inject DNA fragments from wild type into mutant
animals to identify Rescuing region. Sequence
DNA region from mutant to identify mutation.
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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/specificit
  y of desired phenotype/pathway