Introduction to C. elegans and RNA interference
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1 Introduction to C. elegans and RNA interference
2 Why study model organisms?
3 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
4 How do geneticists study gene function?
5 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
6 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
7 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
8 What makes a good model organism? Ease of cultivation Rapid reproduction Small size
9 Why are mutants in model organisms useful?
10 Let s see how similar our genes are to model organisms
11 A comparison of genomes Model organism S. cerevisiae C. elegans A. thaliana D. melanogaster M. musculus Homo sapien (not a model) Haploid genome size (Mb) (estimated) 170 3,000 3,000 Estimated # of genes 6,022 14,000 13,000-60,000 15, , ,000
12 Many genes are conserved in model organisms Species Number of Genes HomoloGene Input Grouped groups H.sapiens 23,516 * 19,336 18,480 P.troglodytes 21,526 13,009 12,949 C.familiaris 19,766 16,761 16,324 M.musculus 31,503 21,364 19,421 R.norvegicus 22,694 18,707 17,307 G.gallus 18,029 12,226 11,400 D.melanogaster 14,017 8,093 7,888 A.gambiae 13,909 8,417 7,882 C.elegans 20,063 * 5,137 4,909 S.pombe 5,043 3,210 3,174 S.cerevisiae 5,863 4,733 4,583 K.lactis 5,335 4,454 4,422 E.gossypii 4,726 3,944 3,935 M.grisea 11,109 6,290 5,884 N.crassa 10,079 5,908 5,902 A.thaliana 26,659 11,180 10,857 O.sativa 33,553 11,022 9,446 P.falciparum 5,
13 The model organism: Caenorhabditis elegans Electron micrograph of a C. elegans hermaphrodite
14 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
15 C. elegans cell division can be studied in the transparent egg
16 C. elegans cell lineage is known
17 Nuclei and DNA can be visualized Kelly, W. G. et al. Development 2002;129:
18 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
19 Sort through the mutations identified Linkage mapping and complementation analysis.
20 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
21 Genome sequencing has identified many genes Model organism S. cerevisiae C. elegans A. thaliana D. melanogaster M. musculus Homo sapien (not a model) Haploid genome size (Mb) (estimated) 170 3,000 3,000 Estimated # of genes 6,022 14,000 13,000-60,000 15, , ,000
22 Can the function of a gene be studied when all we have is the DNA sequence?
23 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
24 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
25 A new, fast, generally applicable technique was needed And the winner is.. RNAi
26
27 How did we come to understand how RNAi works? Examining the antisense RNA technique revealed that the model for how it worked was wrong.
28 The old model: Antisense RNA leads to translational inhibition mrna is considered the sense strand antisense RNA is complementary to the sense strand
29 The old model: Antisense RNA leads to translational inhibition This can give the same phenotype as a mutant
30 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
31 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".
32 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
33 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 263:103, 2001; Science 282:430, 1998).
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