Introduction to C. elegans

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Introduction to C. elegans and RNA interference
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Why study model organisms?
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The problem

• In order to understand biology, we need to learn
  about the function of the underlying genes
• How can we find out what genes do?
• We need a way to uncover these functions

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How do geneticists study gene function?
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How do geneticists study gene function?
Disrupt the gene and analyze the resulting
phenotype

• Forward genetics
• Classical approach
• A gene is identified by studying mutant
• phenotype and mutant alleles
• The gene must be cloned for further
• functional analysis

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How do geneticists study gene function?
Disrupt the gene and analyze the resulting
phenotype

• Reverse genetics
• Start with gene sequence information
• Engineer a loss of function phenotype to
• evaluate gene to function

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Forward Genetics
Starting point A mutant animal End point
Determine gene function

• Have a mutant phenotype and wish to determine
  what gene sequence is associated with it
• Allows identification of many genes involved in a
  given biological process
• Mutations in essential genes are difficult to
  find
• Works great in model organisms

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What makes a good model organism?
Ease of cultivation Rapid reproduction Small
size
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Why are mutants in model organisms useful?
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Lets see how similar our genes are to model
organisms
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A comparison of genomes
Model organism Haploid genome size (Mb) Estimated of genes
S. cerevisiae 13 6,022
C. elegans 100 14,000
A. thaliana 120 (estimated) 13,000-60,000
D. melanogaster 170 15,000
M. musculus 3,000 100,000
Homo sapien (not a model) 3,000 100,000
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Many genes are conserved in model organisms
http//www.ncbi.nlm.nih.gov/entrez/query.fcgi?dbh
omologene
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The model organism Caenorhabditis elegans
Electron micrograph of a C. elegans hermaphrodite
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Caenorhabditis elegans
Profile Soil nematode Genome size 100
Mb Number of chromosomes 6 Generation time
about 2 days Female reproductive capacity 250
to 1000 progeny
Special characteristics Strains Can Be
Frozen Hermaphrodite Known cell lineage pattern
for all 959 somatic cells Only 302
neurons Transparent body Can be characterized
genetically About 70 of Human Genes have related
genes in C. elegans
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C. elegans cell division can be studied in the
transparent egg
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C. elegans cell lineage is known
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Nuclei and DNA can be visualized
Kelly, W. G. et al. Development 2002129479-492
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How do geneticists identify genes?
Answer They perform a mutagenesis screen. 1.
Mutagenize the organism to increase the
likelihood of finding mutants 2. Identify
mutants 3. Map the mutation 4. Determine the
molecular function of the gene product 5. Figure
out how the gene product interacts with other
gene products in a pathway
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Sort through the mutations identified
Linkage mapping and complementation analysis.
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What are the limitations of Forward Genetics?

• 1. Some genes cannot be studied by finding
• mutations
• Genes performing an essential function
• Genes with redundant functions
• 2. Finding mutants and mapping is time-consuming
• 3. Mutagenesis is random
• Cannot start with a known gene and make a
• mutant

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Genome sequencing has identified many genes
Model organism Haploid genome size (Mb) Estimated of genes
S. cerevisiae 13 6,022
C. elegans 100 14,000
A. thaliana 120 (estimated) 13,000-60,000
D. melanogaster 170 15,000
M. musculus 3,000 100,000
Homo sapien (not a model) 3,000 100,000
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Can the function of a gene be studied when all we
have is the DNA sequence?
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Reverse Genetics
Starting point Gene sequence End point
Determine gene function

• Have a gene in hand (genome sequence, for
  example), and want to know what it does.
• Can be used to correlate a predicted gene
  sequence to a biological function
• Goal is to use the sequence information to
  disrupt the function of the gene

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Some approaches to Reverse Genetics

• Targeted deletion by homologous recombination
• Specific mutational changes can be made
• Time consuming and limited to certain organisms
• Mutagenesis and screening for deletions by PCR
• Likely to completely abolish gene function
• Time consuming and potentially expensive
• Antisense RNA
• Variable effects and mechanism not understood

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A new, fast, generally applicable technique was
needed
And the winner is..
RNAi
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How did we come to understand how RNAi works?
Examining the antisense RNA technique revealed
that the model for how it worked was wrong.
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The old model Antisense RNA leads to
translational inhibition
mRNA is considered the sense strand antisense RNA
is complementary to the sense strand
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The old model Antisense RNA leads to
translational inhibition
This can give the same phenotype as a mutant
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An experiment showed that the antisense model
didnt make sense

• The antisense technology was used in worms...
• Puzzling results were produced both sense and
  antisense RNA preparations were sufficient to
  cause interference.
• What could be going on?

1995 Guo S, and Kemphues KJ. First noticed that sense RNA was as effective as antisense RNA for suppressing gene expression in worm
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When researchers looked closely, they found that
double-stranded RNA caused the silencing!
Negative control
uninjected
Potent and specific genetic interference
by double-strandedRNA in Caenorhabditis
elegans Andrew Fire, SiQun Xu, Mary K.
Montgomery, Steven A. Kostas, Samuel E.
Driver Craig C. Mello
mex-3B antisense RNA
mex-3B dsRNA
Double-stranded RNA injection reduces the levels
of mRNA
1998 Fire et al. First described RNAi phenomenon in C. elegans by injecting dsRNA into C. elegans which led to an efficient sequence-specific silencing and coined the term "RNA Interference".
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dsRNA Hypothesis explains the white petunias

• Purple plants should become purpler...
• Instead, they became whiter.
• How could this be happening?
• The multiple inserted copies of chalcone synthase
  were producing double stranded RNA

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RNAi in worms easier than baking pie!
We are going to inactivate genes by RNAi by
feeding

• Feeding worms bacteria that express dsRNAs or
  soaking worms in dsRNA sufficient to induce
  silencing (Gene 263103, 2001 Science 282430,
  1998).