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Caenorhabditis elegans
Anton Kapliy
February 17, 2009
Sydney Brenner (1927 - )
The Genetics of Caenorhabditis Elegans, 1973
•South African biologist (originally chemist)
•D.Phil from Oxford
•Extensive work in molecular biology
•Nobel Prize in 2002
Established C. Elegans as a model organism to study
genetics and cell development.
In his honor, another worm was named C. Brenneri
Meet C. Elegans
Small nematode worm (roundworm)
Natural habitat: soil
Length: ~1 mm
Food: E.Coli
Life cycle: ~3 days
Cellular structure: ~1000 eukaryotic cells; ~300 neurons
First multi-cellular organism to have its genome sequenced
C. Elegans lifecycle
Handling
Isolated from soil
(see first picture on the right)
Cultures reside on small plates
Covered w/ E.Coli lawn that provides nutrition for the worms
Preparation of monoxenic cultures
Germs & worms are killed with a chemical, but eggs survive
Long storage via freezing
Early larvae survive freezing for weeks
Individual worms can be examined
Lifted with paper strips and studied under the microscope.
A plate with C. Elegans
Refresher: diploid cells
C. Elegans is a diploid organism with 6 pairs of chromosomes
Gametes
Regular cell
One from
mommy:
ovum
A pair of homologous
chromosomes
I II III IV V X
zygote
mitosys
I II III IV V X
I II III IV V X
One from
daddy:
sperm
5 autosomes
1 sex chromosome
XO sex-determination system
Sex is determined by the number of X chromosomes:
Ovum
Sperm
Ovum
Sperm
Note that the ovum always
contains an X chromosome, but
the sperm may or may not.
XX
XO
A small twist: Hermaphrodites
An XX worm produces both ovum and sperm
Thus, it can self-fertilize to produce progeny
In the wild, self-fertilizing hermaphrodites tend to homozygosity:
homologous chromosomes contain identical alleles
egg
Consider a pair of chromosomes
heterozygous in trait A/a:
-a--A--
ovum
x
-a--A--
=
-a--a--
-a--A--
-A--A--
-A--a--
(A+a)(A+a) = AA + aa + 2Aa
2(A+a)(A+a) = 2AA + 2aa + 4Aa
4(A+a)(A+a) = 4AA + 4aa + 8Aa
Hermaphrodites
Figure A:
Arrows point to head,
tail, and vulva
Figure B:
Anus
Figure D:
An egg leaving the vulva
Males
In the progeny of self-fertilizing hermaphrodites, there is an
occasional male due to nondisjunction (<0.1%)
This fan-shaped tail is the
male’s reproductive organ.
It also allows to
distinguish males on the
plate.
Males can mate with hermaphrodites, and
their sperm has advantage over
hermaphrodite’s own sperm.
Big picture
1. Induce a mutation in one of the chromosomes
Suppose “a” is a recessive mutation:
----a--
2. Create a line homologous in this mutation (aa)
“Dumpy” worm
-a--a--
3. Isolate and study several different phenotypes
4. Create a genetic map of the worm
5. See why C. Elegans is a great model organism
Inducing mutations
Sexual timeline of a hermaphrodite worm:
Sperms
Ovums
Introduce a powerful mutagen:
EMS - ethane methyl sulfonate
Zygote
Since sperms are already
produced, only ovums
contribute a mutated
chromosome
Properties of mutations
•EMS works at DNA level by producing point mutations:
G/C > A/T
•EMS is a very powerful mutagen:
mutation rate = ~5x10-4 per gene per generation
With ~100 identifiable genes, this means 1 in 20 worms mutate
•Most mutations in C. Elegans are recessive
In this discussion, I will ignore dominant/semidominant
Mutation phenotypes
Blistered phenotype on a plate
Blistered worm
Isolating recessive alleles
P
1. Start with wild type hermaphrodite
Site of mutation
-------
-a-----
2. Induce mutation in the ovum
-a-----
-a-----
3. Let the baby self-fertilize
F1
4. Examine the progeny
-a-- -a-- ---- ----a-- ---- -a-- ----
5. Pick out homologous mutants
F2
¼ mutant
phenotype
¾ wild
phenotype
Autosomal vs sex-linked mutations
Cross the homologous mutant with wild-type males & examine progeny
CASE II: X-linked mutation
CASE I: autosomal mutation
IV
X
IV
-a--a--
---
-------
Homologous mutant
+
-------
+
Wild male (1 X chromosome)
--
=
-------
=
-a-----
--
-a-----
---
X
aa+
-=
♂
Progeny male always gets its X
chromosome from mother
---♂
----
♀
Progeny female gets one X
chromosome from each parent
---♀
----
aa--
Experimental results: phenotypes
As mentioned before, virtually all mutations are recessive.
Located means that the mutation was mapped on one of the chromosomes
Note that there are several autosomal blistered mutants. What are they?
Genetic complementation
10 plates with homologous mutants
with the same phenotype:
-a--a--
-a--a----?
---?
• Given 10 independent mutants with blistered
phenotype:
– Do we know that the same gene is responsible in each case?
– Or could multiple genes cause the same phenotype?
• If different genes cause the same phenotype in two
mutants, they are said to show genetic complementation
• To find out: use Cis-Trans test
Complementation & cis-trans test
Cross the homologous mutant with wild-type males & examine progeny
CASE II: different genes
CASE I: same gene
IV
IV
-a--a--
1st homologous mutant (male)
-a--a--
+
+
+
-a--a--
2nd homologous mutant (female)
=
-a--a--
=
=
Progeny has different phenotypes!
Still exhibits mutation!
---b
---b
-a----b
Restored wild phenotype!
Cis-trans test allows to group mutants of the same phenotype into complementation groups
Cis-trans test in C.Elegans
Generic cis-trans test requires that mutant males mate with hermaphrodites.
But: mutated males with many phenotypes (e.g., uncoordinated) can’t mate!
-a--a-+
+
-------
-a--a-mutant
=
---or
-a-wild
------=
=
-a-----
Progeny males (wild phenotype!)
+
-a--a--
+
Wild-type male
=
-a-----
-a--a--
1st homologous mutant (female)
+
+
2nd
---b
---b
homologous mutant (female)
=
Examine presence of mutated
phenotype in progeny males!
-a----b
wild
=
---or
---b
wild
Next step: linkage groups
3 plates w/ blistered homologs,
corresponding to 3 different genes
10 plates w/ blistered homologs
-a--a--a--a--
-a--a--
--b--b-
--b--b-
---c
---c
-a--a--
--b--b-
--b--b-
---c
---c
cis-trans
-a--a--
--b--b-
---c
---c
The cis-trans test performed on 10 independent mutations tell us how many
are truly independent – that is, caused by different genes. In the example
above, we reduced the problem to 3 independent mutations (genes).
Next step is to determine the linkage groups of these genes. Genes in
different linkage groups segregate independently (acc. to Mendel)
In hindsight, we expect to see
six linkage groups,
mapping to 6 chromosomes:
I II III IV V X
Aside: Cis and trans configurations
Consider a worm that has two recessive mutations: “u” and “d”, but is
exhibiting wild phenotype. There are two ways this could happen:
trans
cis
u-----d
---u--d
Chromosome I is carries u
Chromosome II carries d
Chromosome I is mutation-free
Chromosome II carried both u & d
Both are wild type, since u and d are recessive!
These configurations are called double heterozygotes
Next slide shows how we can construct one.
Constructing a trans heterozygote
Start with two homozygous mutants:
u--u---
---d
---d
Two types of progeny:
Mutant1 ♀
u--u---
+
Baby ♂ Mutant2 ♀
Wild ♂
-------
=
u------
+
---d
---d
To filter out (b), let the progeny self-fertilize:
u--- u--+
---d ---d
---- ---+
---d ---d
- ¼ uu, ¼ dd, ½ wild
- ¼ dd, 1½ wild
=
u-----d
(a)
------d
(b)
Use
Discard
Meiosis in trans heterozygote
Consider a pair of homologous chromosomes:
u-----d
What are the possible gamete configurations?
u---
---d
These are regular gametes, when
one chromosome in the pair
entirely goes to the gamete
u--d
----
These gametes resulted from
chromosomal crossover, when the
pair of parental chromosomes got
mixed during meiosis:
Progeny in trans heterozygote (I)
u-----d
P = probability of
crossover
1-P
Sperms:
Ovums:
u--1-P
---d
u-----d
u--d
P
----
P
u---
---d
u--u-----d
u--u--d
u-----u---
u-----d
---d
---d
u--d
---d
------d
u--d
u--u--d
---d
u--d
u--d
u--d
---u--d
----
u--------d
---u--d
----------
Progeny in trans heterozygote (II)
We observe Mendelian ratio 9:3:3:1
u-----d
1-P
Sperms:
Ovums:
u--1-P
---d
u-----d
u--d
P
----
P
u---
---d
u--d
----
Unc
Wild
Unc
Wild
Wild
Dpy
Dpy
Wild
Unc
Dpy
Wild
Wild
Wild
Unc
Dpy
Wild
Wild
Locating linkage groups
Consider self-progeny of
trans heterosyzote:
u-----d
Pick out all dumpy worms and count how many are also uncoordinated:
P(dpy) = (1-P)2 + 2P(1-P) + P2 = 1 – 2P + P2 +2P – 2P2 + P2 = 1
P(unc+dpy) = P2
Ratio = P(unc+dpy)/P(dpy) = P2
Suppose genes “u” and “d” are on different chromosomes:
Then, they segregate independently, with P=0.5
u- --- -d
If genes u and d are on the same chromosome, the measured ratio will
quadratically diverge from 0.52 = 0.25 – making it a very sensitive test!
Results: classification of linkage groups
1st chromosome
2nd chromosome
Etc…
Results: mapping of mutants
We can guess the order of genes on each chromosome by using P, the
recombination probability, as the yard stick:
Intermission
Brenner’s paper establishes C. Elegans as a perfect model organism because:
•Worms are easy to handle and quick to multiply
•Availability of very potent mutagen
•Hermaphrodites can maintain homozygous recessive alleles
•Hermaphrodites can self-fertilize even with mutations that impair movement
•Rare males allow to mix genetic traits
Actually, there is a lot more that can be done with C. Elegans – the final few
slides summarize some of its interesting features & recent developments
Ease of observation
The worm is transparent, and we can see all of its ~1000 cells in a microscope
Developmental biology
It is possible to trace the fate of each cell in the growing worm
Complete cell lineage
Constant number of cells: 959 in hermaphrodite, and 1031 in male
Programmed cell death (apoptosis)
131 cells in the developing worm embryo die by apoptosis in a predetermined way
First complete genetic map
100 million base pairs
~20,000 genes
One of the simplest nervous systems
Nervous system consists of 302 neurons that form a small-world network
Their interconnections have been completely mapped out
Gene silencing via RNA interference
Inject double-stranded RNA
Enzyme dicer breaks dsRNA into a
cascade of small-interfering RNA
siRNA bind to another enzyme:
RNA-induced Silencing Complex
RISCs silence the matching sequence
in the messenger RNA
Some of the sources
A couple of intro genetics textbooks
http://www.wormbook.org/chapters/www_nematodeisolation/nematodeisolation.html
http://www.wooster.edu/biology/wmorgan/bio306/C.elegans_Week3_Directions.html
http://www.sanger.ac.uk/Projects/C_elegans/
http://www.wormbook.org/chapters/www_dominantmutations/dominantmutations.html
http://www.wormatlas.org/handbook/anatomyintro/anatomyintro.htm
http://www.wormclassroom.org/ge.html
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?indexed=google&rid=ce2.section.100
http://fruitfly4.aecom.yu.edu/labmanual/16a.html
http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/C/Caen.elegans.html
http://en.wikipedia.org/wiki/Caenorhabditis_elegans
http://en.wikipedia.org/wiki/Apoptosis
http://www.bio.unc.edu/faculty/goldstein/lab/movies.html
http://www.loci.wisc.edu/outreach/text/celegans.html
http://www.nematodes.org/teaching/devbio3/index.shtml
http://www.translational-medicine.com/content/2/1/39/figure/F1