How do you clone a gene in C. elegans?

mutant phenotype

?
gene

classical genetic analysis

Linkage analysis
On which chromosome is the mutation (and affected gene) located? Based on Mendels 2nd Law (Principle of independent assortment)
genes or alleles for different traits assort independently during gamete formation

Unlinked genes
(different chromosomes)

Linked genes
(same chromosome)

Linkage analysis

mutant strain

dumpy strain (Dpy)

dpy-5 mut Chromosome ? Chromosome I

Dpy = dumpy phenotype

Linkage analysis
P0

Case 1: mutation is not linked

F2
or or + + or Dpy

Mut

Dpy

Mut

F1

F3
25% Dpy

F3
100% Dpy

assuming the mutation of interest is recessive

Linkage analysis
P0

Case 2: mutation is linked

F2
The gene with the mut allele is linked to the the dpy-5 gene

+ , Mut

Dpy , +

+ , Mut

F1

+/Dpy , Mut/+

assuming the mutation of interest is recessive

Linkage analysis
Perform with marker strains for all six chromosomes

1 Mb

Chromosome

II

III

IV

Tells you to which chromosome your mutation is linked

Mapping experiments
The three-factor mapping experiment
determine where your mutation is located with respect to two known sites

Loci on one chromosome do not assort independently

non-independent assortment
site A site B

Can only be separated through recombination

Recombination
During meiosis, homologous chromosomes pair-up

Creates genetic diversity

mother

father

Recombination
1 map unit (centimorgan)
1% chance of a recombination event between two defined loci

5 map units

dpy-5 unc-29

Dpy Unc phenotype: both dumpy and uncoordinated

Mapping experiments
P0 F2
Here we are looking at Case 3
Case 1

Dpy Unc

site A
Mut Dpy Unc
Case 2

F1

+
Case 3

site B

+ +

Mut

Mapping experiments
P0 F2

Mut

Dpy Unc

Dpy only

F1

+ Unc only

Mapping experiments
F2 F3

Here, the recombination event picked-up the mutation of interest

Dpy only Dpy , Mut

Unc only

Unc only

Mapping experiments
Analysis of all recombinants allows you to determine the order
Mut dpy-5 unc-29 or dpy-5 Mut unc-29 or dpy-5 unc-29 Mut
site A site B

Frequency of recombination events will tell you the distance (map units)

Mapping experiments

site A site B

site A site B

site C site D

Transformation rescue experiment

rescue successful

Gene defined by mutation successfully identified and cloned

Mut

collection of hundreds of overlapping cosmids.

A B C D E F G H I

collection of hundreds of overlapping cosmids.

G H

rescue successful

Use cosmids for rescue experiments

collection of hundreds of overlapping cosmids.

collection of hundreds of overlapping cosmids.

H1
H

H2

H3

H4

H5

H6

Minimal rescuing fragment

cloning

1990s now
months!

years!

mapping

linkage analysis

rescue experiments

Important advances
1998 C. elegans genome sequence published
using the overlapping cosmids

RNAi (RNA-mediated interference)

method to inactivate gene function in a targeted manner

Hawaii strain found to be polymorphic to Bristol strain

1 out of every 1500 base-pairs differs

Important advances
SNP mapping Single-nucleotide polymorphism (SNP)
the variation of a single nucleotide between DNA sequences of biological species
e.g., TGTACGC vs. TGTTCGC

SNP mapping
map your mutation to any of the thousands of SNP sites
Hawaii

Bristol Site A Site B

Important advances
Genome sequencing Sequencing reactions have become cheaper
less than $1,000 to sequence the genome of a C. elegans strain

Cross your mutant with the Hawaii strain

isolate homozygous mutants in the F2 generation pool animals and sequence genomic DNA
Hawaii P0 mutant of interest F1 F2

chromosome region of chromosome transcription unit (gene) exact mutation

it is critical to understand the concept of linkage and mapping.

once you understand it, you can apply it to any process and any genetic model.

ced-9

Hengartner, M.O. and Horvitz H.R. (1994). C. elegans cell survival gene ced-9 encodes a functional homolog of the mammalian proto-oncogene bcl-2. Cell, 76(4), 665-676. http://www.cell.com/retrieve/pii/0092867494905061

1994

shares sequence similarity with bcl-2

egl-1

not only ced-9 but also the entire pathway in which ced-9 acts has been conserved through evolution.

pro-apoptotic acts upstream of ced-9 small, novel protein BH3 domain functionally homologous to the sensors

23% C. elegans CED-9 protein is h o m o l identical o g o u s to human Bcl-2 protein