Download unc-40 - UCSF Biochemistry

C. elegans lecture
Kaveh Ashrafi
[email protected]
N412C Genentech Hall
415.514.4102
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. OVERVIEW
C. elegans as an experimental system
Life cycle
Short 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
Sex
ADULT MALE
autosomes (pairs)
5
5
sex chromosome(s)
XX
XO
body plan of an adult hermaphrodite
Hermaphrodites are self fertilizing because they contain both
oocytes 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
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
II.
GENETIC BASICS
Self progeny vs. cross progeny
X
I, II, III
IV, V, X
I, II, III
IV, V, X
~100% XX
F1 have genotype of
parent (clonal)
I, II, III
IV, V, X
I, II, III
IV, V, X
50% XX
I, II, III
IV, V
I, II, III
IV, V, X
50% XO
F1 hermaphrodites are heterozygous at
all loci; F1 males are heterozygous at
all autosomal loci, hemizygous on X
Example of a genetic cross in C. elegans
UNC=uncoordinated movement
unc-40(e271) I a recessive mutation
unc-40 (e271)/unc-40 (e271)
X
+/+
F1 :
self progeny:
100% Unc, ~100% hermaphrodite unc40/unc-40
cross progeny: 100% non-Unc (WT),
unc-40/+
Example of a genetic cross in C. elegans
Take F1 that is cross progeny, single onto a new plate, allow to se
F2 (from self fertilization of cross progeny)
unc-40
+
unc-40
+
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?
III.
GENETIC SCREENS
Point of entry into a biological process.
A simple screen that can produce informative,
tractable mutations with strong and specific phenotypes.
A simple forward genetics screening strategy
Po
+/+
m1 + m2 +
+ + + m3
F1:
F2:
m/+
+/+; m/+; m/m
m6
+
m4
+
+
m5 m7
can identify dominant mutations
can identify dominant & recessive mutations
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
Positional mapping using SNPs
Po
X
F1
Select F2 progeny with desired phenotype
F2
Rescue & Transgenics
*Inject DNA fragments from wild type into mutant animals to iden
Rescuing 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