2. Genome, mutants and transgenic worms 2nd lecture:

3. C.elegans genome Approximately equally sized holocentric chromosomes - 5 autosomes, one X. Clone-based physical map Alan Coulson, John Sulston, Sydney Brenner, and Jonathan Karn (1986) Toward a Physical Map of the Genome of the Nematode Caenorhabditis elegans. PNAS October 15, vol. 83

4. WormBase www.wormbase.org www.wormbase.org Aim to capture, curate and distribute information about C.elegans biology Object oriented, web-based; data base downloadable The central data repository for C.elegans

5. ACeDB - Designed and built originally for the C. elegans database, AceDB (www.acedb.org) is an object oriented database used in genome sequencing projectswww.acedb.org It forms the core of the WormBase site as well. A C.elegans DataBase (Sanger Institute) developed since 1989

6. Genome Projects 1.To establish an integrated Web-based database and research interface. 2.To assemble physical and genetic maps of the genome. 3.To generate and order genomic and expressed gene sequences. 4.To identify and annotate the complete set of genes encoded within a genome. 5.To compile atlases of gene expression. 6.To accumulate functional data, including biochemical and phenotypic properties of genes. 7.To characterize DNA sequence diversity. 8.To provide the resources for comparison with other genomes.

7. Genomes that have already been sequenced Phage ΦX174 1977 Mycoplasma genitalium1995 Haemophilus influenzae 1995 Yeast 1996 E. coli 1997 Caenorhabditis elegans 1998 Drosophila 2000 Arabidopsis thaliana 2001 Rice 2002 Anopheles gambiae 2002 Human genome 2003

8. Science 282, 2012 (1998) The first multicellular organism genome sequenced 97 Mb over 19,000 genes (~ 5000 essential) The 97-Mb sequence is a composite of  2527 cosmids,  257 YACs,  113 fosmids, and  44 PCR products

9. Gene density

10. Science, 282 (1998) C. elegans Genome Project Catalogue

11. Pairwise comparisons of protein C. elegans 47 37 21 27 9 26 16 20 26 51 7436 About 70 % of human genes have a clear C. elegans homolog Human genes can often rescue the worm mutant E. coli S. cerevisiae H. sapiens

13. Genetic nomenclature  Genes defined by mutation dpy-5  Genes defined by sequence similarities mlc-3  Genes with related properties unc-131  Homologous genes wrn-1; sir-2.1  Pseudogenes have suffix ps msp-48ps  tag = temporarily assigned gene name  Alleles have a single or double letter followed by a number dpy-5(e61)  The letter identifies the isolating laboratory  Optional suffixes hc17ts  Wild type (N2) alleles are designated with + dpy-5(+) CGC Nomenclature

14. Genetic nomenclature Gene knockouts  Small deletions (<5 kb) – laboratory prefix, number, sometimes optional suffix te (transposon-excision) or ko (knockout)  Insertion of selectable marker - alleles: double colon, selected marker zhp-3(jf61::unc-119+)  In case deletion affects two genes - gene-1&gene-2(allele) rad-54&tag-157(ok615)  Deletions affecting more than two genes are named as defficiencies (Def)

15. Genetic nomenclature Two or three UPPERCASE letters followed by a number The strain letter prefixes refer to laboratory of origin and are distinct from the allele letter prefixes Multiple mutant alleles carried in one strain are organised by chromosome, and chromosomes separated by semicolons A strain is a set of individuals of a particular genotype with a capacity to produce more individuals of the same genotype. MT688 is a strain of genotype unc-32(e189) / lin-12(n137) III; him-5(e1467) V

16. Natural isolates and SNPs Worms are polymorphic like humans N2 (isolated from Bristol, UK) is the reference strain There are many natural isolates (eg. from Australia, Hawaii and Germany) Natural isolates have single nucleotide polymorphisms (SNPs) SNPs are DNA sequence variations that occur when a single nucleotide (A,T,C,or G) in the genome sequence is changed.

17. Natural isolates StrainSourceSNPs / bp  N2 Bristol, UKancestral  TR403 Madison, WI1:8750  RC301 Freiburg, Germany 1:7740  AB1 Adelaide, Australia 1:2430  CB4857 Claremont, CA1:1445  CB4856 Hawaii1:876      

18. Mutants http://130.15.90.245/photos.htm Bli Muv trp-4 wtUnc Bmd

19. C. elegans as a genetic tool Function Gene Forward Genetics  Random mutagenesis  Transposons Reverse Genetics  PCR identification of rearrangements (site-directed mutagenesis)  RNAi Gene Function (behavior, disease, phenotype) (tyrosine hydroxylase, P-450, 5HT receptor) ??

20. Forward genetics Random mutagenesis  Random mutagen (EMS/TMP-UV) to generate point mutations or small deletions  Analysis of F2 for the selected phenotypes  Mapping using visible markers and polymorphic DNA sequences Gene targeting techniques based on homologous recombination are not available in C.elegans

21. Mutagenesis Mutants resistant to the chemical under investigation are selected on F2 culture plates containing the chemical (in this case 10 -4 M levamisole). These mutants are then picked out individually and isolated on dishes in the continued presence of the active compound. The progeny that breed true will be homozygous and will therefore retain resistance to the compound and can be used to identify by genetic mapping the gene(s) in which mutations caused resistance.

22. Forward Genetics Transposons: discrete segment of DNA moving in the genome,encoding a transposase Normally present in C.elegans in different copies (strain-dependent) Activated by forced expression of transposases Most common:Tc1 (“cut and paste mechanism”) Insertional mutagenesis with Tc1 will generate mutant alleles tagged by the transposon that can be used for identify the mutated gene Problems: 1) Other Tc elements are mobilized in the mutator strain 2) Several copies of the Tc1 (identification the mutagenic insertion) 3) Transposition cannot be controlled Solution: mobilization of Mos-1 element (a Tc1 absent in C.elegans) achieved by conditional expression of the Mos-1 transposase Transposons

23. Transposon-mediated mutagenesis a double-strand cut, made by the transposase protein, on both sides of the transposon (red) liberates the transposon, which can subsequently integrate at a novel genomic position. inflicted genomic damage: (1) insertions into other genes can result in mutations, hence the mutator phenotype, and (2) the occurrence of a DSB that results from transposon excision that is either repaired via the homologous recombination or the NHEJ (non-homologous end joining) repair pathway.

24. Genetic and genome-wide RNAi screens for mut genes Animals that were paralysed because of a transposon insertion in the unc-22 or unc-54 genes were fed on dsRNA-producing bacterial clones each targeting a different C. elegans gene. Temporal alleviation of transposon silencing can restore UNC-22 or UNC-54 function.

25. Reverse genetics  RNA interference  Specific KnockOut KO/RNAi gene Phenotype(s) http://celeganskoconsortium.omrf.org/ http://shigen.lab.nig.ac.jp/c.elegans/index.jsp Specific KO: EMS/TMP mutagenesis (deletions) PCR to identify the mutated gene

26. C. elegans Gene Knockout Consortium ultimate goal is to produce null alleles of all known genes in the C.elegans genome chemical mutagenesis PCR with nested primers identify animals in the mutagenized population with deletions at the targeted locus

28. Gene expression 3 approaches to study gene expression in C.elegans: 1)Reporter-gene fusion with transformation (GFP, LacZ) 2)In situ hybridization using mRNA 3)Immunofluorescence with specific antibody

29. Purposes of transformation: identification of genes by rescuing a mutant phenotype using a WT copy of the gene or the gene homolog from another species expression pattern using the gene of interest with reporter (gene_X_promoter::GFP) interference of a biological process by overexpression of WT or mutated gene

30. Transformation  Introduced in the early 1980s  DNA is injected into the cytoplasm of the gonads (syncytium).  Usually, a marker (e.g. genepromoter::GFP) is co- injected  The DNA can pass through the germline in the form of extrachromosomal array 40X DIC http://130.15.90.245/photos.htm

31. Integration of the transgene into worm genome X- or gamma radiation Expose a plate with ca 50 L4 worms bearing the extrachromosomal array, to 3500–4800 rads of x- or gamma rays You can use the hospital x-ray machine Look for worms positive for marker - in case of GFP fluorescent worms - who give a 100% fluorescent progeny  pick a few hundred F1s to individual plates  pick 4-5 F2s from each F1