C' elegans Molecular Genetics

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C. elegans Molecular Genetics

• Dewey C. Royal, Ph.D.

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C. elegans Molecular Genetics

• Transgenic Nematodes
• The physical map the Genome
• Reverse Genetics
• Deletion
• RNAi
• Mapping with Molecular Markers
• Tc1 polymorphisms
• SNPs

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Introducing Transgenes into the Germline

• Gene of interest
• Cosmid of genomic sequence to test for rescue of
  a mutant.
• Transcriptional reporter (GFP or lacz fused to 5
  regulatory sequences) to determine tissue of
  expression.
• Translational reporter (GFP fused in frame to ORF
  of a worm protein) to examine subcellular
  localization.
• Marker to follow the transgene
• lin-15 DNA injected into lin-15(n765ts) mutants
  (score complementation of the mutation).
• rol-6(su1006dm) DNA injected into any strain
  gives dominant Rol phenotype.
• GFP reporter causes worms to glow green under
  fluorescent light a dominant marker.

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Exogenous DNA forms concatemers
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The Physical Map

• About 80of the genome is in cosmids typical
  insert size of 30kb.
• The remaining 20 of the genome was in gaps that
  have been bridged by YACs typical insert size of
  250 kb.
• Placing the clones in order
• Digest cosmids with HindIII results in 10
  fragments.
• Fill in fragments with 32P labeled nucleotides.
• Digest fragments further with a frequent cutter,
  Sau3A.
• Run fragments on an acrylamide gel.
• Match overlapping fingerprints to identify
  adjacent, overlapping clones.

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http//www.wormbase.org
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• Forward Genetics Randomly isolating mutations in
  genes that affect a biological process, then
  cloning the underlying genes by genetic mapping.
• Reverse Genetics Using this sequence of a cloned
  gene to generate a mutation in that gene, then
  characterizing the resulting phenotype.

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Reverse Genetics with Transposons

• Tc1 is a 1.6 kb transposon with 54 bp inverted
  repeats at its termini. About 30 are found in the
  Bristol strain of C. elegans
• Transpositions occur in mut-2 strains.
• Can randomly hopinto a gene and inactive it.
• Not all transpositions cause a phenotype.
• Imprecise excision of Tc1 can generate a small
  deletion in a gene of interest.

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Reverse Genetics with RNAi

• Make sense and antisense transcripts
  corresponding to ORF of the gene that you are
  knocking out.
• Anneal transcripts to make double stranded RNA.
• OR
• Generate sense and antisense transcripts in E.
  coli.
• Feed E. coli to nematodes.
• Inject dsRNA into P0 adult nematodes.
• Observe F1 progeny for phenotype.

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STS Mapping by PCR

• Tc1 transposons create sequence-tagged sites
  (STS).
• The Bristol strain contains about 30 Tc1
  insertions.
• The Bergerac strain contains about 500 Tc1
  insertions.

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