introduction to c. elegans and rna interference why study model organisms?
TRANSCRIPT
• 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
The problem:
Forward genetics:• Classical approach• A gene is identified by studying mutant phenotype and mutant alleles• The gene must be cloned for further functional analysis
Disrupt the gene and analyze the resulting phenotype
How do geneticists study gene function?
Reverse genetics:• Start with gene sequence information• Engineer a loss of function phenotype to evaluate gene to function
Disrupt the gene and analyze the resulting phenotype
How do geneticists study gene function?
Forward Genetics
• 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
Starting point: A mutant animalEnd point: Determine gene function
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
A comparison of genomes
Species Number of Genes HomoloGene
Input Grouped groups
H.sapiens 23,516* 19,336 18,480P.troglodytes 21,526 13,009 12,949C.familiaris 19,766 16,761 16,324M.musculus 31,503 21,364 19,421R.norvegicus 22,694 18,707 17,307G.gallus 18,029 12,226 11,400D.melanogaster 14,017 8,093 7,888A.gambiae 13,909 8,417 7,882C.elegans 20,063* 5,137 4,909S.pombe 5,043 3,210 3,174S.cerevisiae 5,863 4,733 4,583K.lactis 5,335 4,454 4,422E.gossypii 4,726 3,944 3,935M.grisea 11,109 6,290 5,884N.crassa 10,079 5,908 5,902A.thaliana 26,659 11,180 10,857O.sativa 33,553 11,022 9,446P.falciparum 5,222 971 950
Many genes are conserved in modelorganisms
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=homologene
Caenorhabditis elegans
Profile
Soil nematodeGenome size: 100 MbNumber of chromosomes: 6Generation time: about 2 daysFemale reproductive capacity: 250 to 1000 progeny
Special characteristicsStrains Can Be FrozenHermaphroditeKnown cell lineage pattern for all 959 somatic cellsOnly 302 neuronsTransparent bodyCan be characterized geneticallyAbout 70% of Human Genes have related genes in C. elegans
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
How do geneticists identify genes?
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
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
Genome sequencing has identified many genes
Reverse Genetics
Starting point: Gene sequenceEnd 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
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
How did we come to understand how RNAi works?
Examining the antisense RNA technique revealed that the model for how it
worked was wrong.
The old model: Antisense RNA leads to translational inhibition
mRNA is considered the sense strand
antisense RNA is complementary to the sense strand
This can give the same phenotype as a mutant
The old model: Antisense RNA leads to translational inhibition
An experiment showed that the antisense model didn’t 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
When researchers looked closely, they found that double-stranded RNA caused the
silencing!
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".
Negative control uninjected
mex-3B antisense RNA mex-3B dsRNA
Double-stranded RNA injection reduces the levels of mRNA
Potent and specificgenetic interference bydouble-strandedRNA inCaenorhabditis elegansAndrew Fire*, SiQun Xu*, Mary K. Montgomery*,Steven A. Kostas*†, Samuel E. Driver‡ & Craig C. Mello‡
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