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Caenorhabditis elegans

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Caenorhabditis elegans. 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 - PowerPoint PPT Presentation

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Page 1: Caenorhabditis elegans

Caenorhabditis elegans

Page 2: Caenorhabditis elegans

Sydney Brenner (1927 - )

•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

The Genetics of Caenorhabditis Elegans, 1973

Page 3: Caenorhabditis elegans

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

Page 4: Caenorhabditis elegans

C. Elegans lifecycle

Page 5: Caenorhabditis elegans

HandlingIsolated from soil(see first picture on the right)

Cultures reside on small platesCovered w/ E.Coli lawn that provides nutrition for the worms

Preparation of monoxenic culturesGerms & worms are killed with a chemical, but eggs survive

Long storage via freezingEarly larvae survive freezing for weeks

Individual worms can be examined Lifted with paper strips and studied under the microscope.

Page 6: Caenorhabditis elegans

A plate with C. Elegans

Page 7: Caenorhabditis elegans

Refresher: diploid cellsC. Elegans is a diploid organism with 6 pairs of chromosomes

I II III IV V X

5 autosomes

1 sex chromosome

A pair of homologous chromosomes

I II III IV V X

zygote

Regular cellGametes

I II III IV V X

One from mommy: ovum

One from daddy: sperm

mitosys

Page 8: Caenorhabditis elegans

XO sex-determination systemSex is determined by the number of X chromosomes:

Ovum Sperm Ovum Sperm

XX XO

Note that the ovum always contains an X chromosome, but the sperm may or may not.

Page 9: Caenorhabditis elegans

A small twist: HermaphroditesAn XX worm produces both ovum and spermThus, it can self-fertilize to produce progeny

In the wild, self-fertilizing hermaphrodites tend to homozygosity: homologous chromosomes contain identical alleles

(A+a)(A+a) = AA + aa + 2Aa2(A+a)(A+a) = 2AA + 2aa + 4Aa

4(A+a)(A+a) = 4AA + 4aa + 8Aa

-a---A--

Consider a pair of chromosomes heterozygous in trait A/a: -a--

-A--

-a---a--

-A---A--

-a---A--

-A---a--

x =

egg ovum

Page 10: Caenorhabditis elegans

Hermaphrodites

Figure A:Arrows point to head, tail, and vulva

Figure B:Anus

Figure D:An egg leaving the vulva

Page 11: Caenorhabditis elegans

MalesIn the progeny of self-fertilizing hermaphrodites, there is an occasional male due to nondisjunction (<0.1%)

Males can mate with hermaphrodites, and their sperm has advantage over hermaphrodite’s own sperm.

This fan-shaped tail is the male’s reproductive organ. It also allows to distinguish males on the plate.

Page 12: Caenorhabditis elegans

Big picture

1. Induce a mutation in one of the chromosomes

2. Create a line homologous in this mutation (aa)

3. Isolate and study several different phenotypes4. Create a genetic map of the worm5. See why C. Elegans is a great model organism

“Dumpy” worm

-----a--Suppose “a” is a recessive mutation:

-a---a--

Page 13: Caenorhabditis elegans

Inducing mutations

Sperms Ovums Zygote

Sexual timeline of a hermaphrodite worm:

Introduce a powerful mutagen:EMS - ethane methyl sulfonate 

Since sperms are already produced, only ovums contribute a mutated chromosome

Page 14: Caenorhabditis elegans

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

Page 15: Caenorhabditis elegans

Mutation phenotypes

Page 16: Caenorhabditis elegans

Blistered phenotype on a plate

Blistered worm

Page 17: Caenorhabditis elegans

Isolating recessive alleles

-a------

--------

-a------

-a------

-----a--

-a------

--------

-a---a--

1. Start with wild type hermaphrodite

2. Induce mutation in the ovum

3. Let the baby self-fertilize

4. Examine the progeny

Site of mutation

P

F1

F2

¼ mutant phenotype

¾ wild phenotype

5. Pick out homologous mutants

Page 18: Caenorhabditis elegans

Autosomal vs sex-linked mutations

-a-- ---a-- --

---- a----- a-

Cross the homologous mutant with wild-type males & examine progeny

CASE I: autosomal mutation CASE II: X-linked mutation

---- ---- --

---- ---- --

Homologous mutant

Wild male (1 X chromosome)

+ +

-a-- ---- --

-a-- ------ --

---- ---- a-

---- a----- --

Progeny male always gets its X chromosome from mother

Progeny female gets one X chromosome from each parent

= =

IV X IV X

+

=

♂ ♂

♀ ♀

Page 19: Caenorhabditis elegans

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?

Page 20: Caenorhabditis elegans

Genetic complementation

• 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

-a---a--

-a---a--

---?---?

10 plates with homologous mutants with the same phenotype:

Page 21: Caenorhabditis elegans

Complementation & cis-trans test

-a---a--

Cross the homologous mutant with wild-type males & examine progeny

CASE I: same gene CASE II: different genes

1st homologous mutant (male)

2nd homologous mutant (female)

+

Progeny has different phenotypes!

=

IV IV

+

=

-a---a--

-a---a--

-a---a--

+

=

---b---b

-a-----b

Still exhibits mutation! Restored wild phenotype!

Cis-trans test allows to group mutants of the same phenotype into complementation groups

Page 22: Caenorhabditis elegans

Cis-trans test in C.Elegans

-a---a--

Generic cis-trans test requires that mutant males mate with hermaphrodites.But: mutated males with many phenotypes (e.g., uncoordinated) can’t mate!

1st homologous mutant (female)

Wild-type male

+

=

+

=

--------

-a------

Progeny males (wild phenotype!)

+

=

-a---a--

-a---a--

2nd homologous mutant (female)

+

-----a--

or

-a---a--

+

=

--------

-a------

+

=

---b---b

-a-----b

-------b

or

=Examine presence of mutated phenotype in progeny males!

mutant wild wild wild

Page 23: Caenorhabditis elegans

Next step: linkage groups

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 seesix linkage groups,mapping to 6 chromosomes:

I II III IV V X

-a---a--

-a---a--

-a---a--

-a---a--

--b---b-

--b---b-

--b---b-

---c---c

--b---b-

---c---c

-a---a--

--b---b-

---c---c

10 plates w/ blistered homologs3 plates w/ blistered homologs, corresponding to 3 different genes

cis-trans

Page 24: Caenorhabditis elegans

Aside: Cis and trans configurationsConsider a worm that has two recessive mutations: “u” and “d”, but is exhibiting wild phenotype. There are two ways this could happen:

----u--d

cis

Chromosome I is mutation-freeChromosome II carried both u & d

u------d

trans

Chromosome I is carries uChromosome II carries 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.

Page 25: Caenorhabditis elegans

Constructing a trans heterozygoteu---u---

---d---d

Start with two homozygous mutants:

u---u---

--------

Wild ♂Mutant1 ♀

+ = u-------

Baby ♂

+ ---d---d

Mutant2 ♀

=

u------d

-------d

u------d-------d

Two types of progeny:

(a)

(b)

To filter out (b), let the progeny self-fertilize:

-------d

u------d

+

+

- ¼ uu, ¼ dd, ½ wild

- ¼ dd, 1½ wild Discard

Use

Page 26: Caenorhabditis elegans

Meiosis in trans heterozygote

u------d

---du--- ----u--d

Consider a pair of homologous chromosomes:

What are the possible gamete configurations?

These are regular gametes, when one chromosome in the pair entirely goes to the gamete

These gametes resulted from chromosomal crossover, when the pair of parental chromosomes got mixed during meiosis:

Page 27: Caenorhabditis elegans

Progeny in trans heterozygote (I)u------d

---du--- ----u--d

u------d

u---

---d

u--d

----

Sperms:

Ovums: u---u---

---du---

u--du---

----u---

u------d

---d---d

u--d---d

-------d

u---u--d

---du--d

u--du--d

----u--d

u-------

---d----

u--d----

--------

1-P

1-P

P

P

P = probability of crossover

Page 28: Caenorhabditis elegans

Progeny in trans heterozygote (II)u------d

---du--- ----u--d

u------d

u---

---d

u--d

----

Sperms:

Ovums: Unc

Wild

Unc

Wild

Wild

Dpy

Dpy

Wild

Unc

Dpy

UncDpy

Wild

Wild

Wild

Wild

Wild

1-P

1-P

P

P

We observe Mendelian ratio 9:3:3:1

Page 29: Caenorhabditis elegans

Locating linkage groups

Suppose genes “u” and “d” are on different chromosomes:Then, they segregate independently, with P=0.5

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!

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 = 1P(unc+dpy) = P2

Ratio = P(unc+dpy)/P(dpy) = P2

u------d

Consider self-progeny of trans heterosyzote:

u- ---- -d

Page 30: Caenorhabditis elegans

Results: classification of linkage groups

1st chromosome

2nd chromosome

Etc…

Page 31: Caenorhabditis elegans

Results: mapping of mutantsWe can guess the order of genes on each chromosome by using P, the recombination probability, as the yard stick:

Page 32: Caenorhabditis elegans

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

Page 33: Caenorhabditis elegans

Ease of observation

The worm is transparent, and we can see all of its ~1000 cells in a microscope

Page 34: Caenorhabditis elegans

Developmental biology

It is possible to trace the fate of each cell in the growing worm

Page 35: Caenorhabditis elegans

Complete cell lineage

Constant number of cells: 959 in hermaphrodite, and 1031 in male

Page 36: Caenorhabditis elegans

Programmed cell death (apoptosis)

131 cells in the developing worm embryo die by apoptosis in a predetermined way

Page 37: Caenorhabditis elegans

First complete genetic map

100 million base pairs~20,000 genes

Page 38: Caenorhabditis elegans

One of the simplest nervous systems

Nervous system consists of 302 neurons that form a small-world networkTheir interconnections have been completely mapped out

Page 39: Caenorhabditis elegans

Gene silencing via RNA interferenceInject 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

Page 40: Caenorhabditis elegans

Some of the sources

A couple of intro genetics textbookshttp://www.wormbook.org/chapters/www_nematodeisolation/nematodeisolation.html http://www.wooster.edu/biology/wmorgan/bio306/C.elegans_Week3_Directions.htmlhttp://www.sanger.ac.uk/Projects/C_elegans/ http://www.wormbook.org/chapters/www_dominantmutations/dominantmutations.htmlhttp://www.wormatlas.org/handbook/anatomyintro/anatomyintro.htm http://www.wormclassroom.org/ge.htmlhttp://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.htmlhttp://www.nematodes.org/teaching/devbio3/index.shtml http://www.translational-medicine.com/content/2/1/39/figure/F1