1. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Genomics Book (20 chapters)  Chapter 7: High throughput genetics  Chapter 8: Proteomics  Chapter 13: Genome Structure  Chapter 14: Human origin  Chapter 15: Genomics and Medicine  Chapter 18: Pharmacogenomics  Chapter 19: Genomics and Agriculture  Chapter 20: Ethical issues of genomics

2. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Chapter 7 High-Throughput Genetics Applications of genomics approaches to genetics

3. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Contents  Basics of forward genetics  Genomics approaches to forward genetics  Basics of reverse genetics  Genomics approaches to reverse genetics

4. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Background  Genetics is the study of gene function  Gene function is inferred from the resulting phenotype when the gene is mutated  Genomics is changing the way genetics is performed  Global, high-throughput approaches  Genomics approaches are being applied to both forward and reverse genetics

5. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Forward and reverse genetics  Forward genetics starts with identification of interesting mutant phenotype  Then aims to discover the function of genes defective in mutants by chromosome walking  Reverse genetics starts with a known gene and alters its function by transgenic technology  Then aims to determine the role of the gene from the effects on the organism  This chapter focuses on applications of genomics to genetics in model organisms

6. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Basics of forward genetics  Forward genetics usually starts with mutagenesis of organism  Can use chemicals  e.g., ethyl methyl sulfonate (EMS)  Or can use radiation  e.g., X rays  Then screen progeny of mutagenized individuals for phenotypes of interest

7. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Phenotype  Empirical definition: features that are different from those of the wild type (normal)  Can be something visible  e.g., change in hair color or anatomy  Or may require invasive analysis  e.g., different mobility of enzyme during electrophoresis

8. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Mouse hair-color mutant Beige

9. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Drosophila anatomical mutant Mutant with legs instead of antennae wild

10. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Genetic analysis  First step in analysis of mutants: precisely describe phenotype  Basis of predicting gene function: identify precisely what has malfunctioned (wrong) in the mutant  That is what the gene product does in the wild type

11. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Phenotype to gene function  Example: mouse  Small-eye mutant  Gene codes for Pax6 transcription factor  Required for normal eye development  Same transcription factor required for eye development in humans and fruit flies Poor rat

12. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Genetic screens  For diploid organisms  Two chromosomes  Mutagenesis performed  Then mutagenized individuals are mated or self-crossed  Screen progeny for mutant phenotypes

13. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Recessive traits  Recessive trait  Normally loss-of- function mutation  When heterozygous parents are crossed, the mutant phenotype appears in 1/4 of the progeny heterozygous no phenotype homozygous wildtype no phenotype homozygous mutant phenotype

14. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Dominant traits  Dominant trait  Normally gain-of- function mutation  When heterozygous parents are crossed, the mutant phenotype appears in 3/4 of the progeny heterozygous mutant phenotype homozygous wildtype no phenotype homozygous mutant phenotype

15. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Genomics applied to genetics  Genomics characterized by the following:  High throughput  Using automation to speed up a process  Global approach  All genes in genome  Applied to both forward and reverse genetics

16. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Genomics and forward genetics  High-throughput genetic screens  Candidate-gene approach  To go from phenotype to gene  Insertional mutagenesis  Loss-of-function mutation  Activation tagging  Enhancer trapping

17. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 High-throughput genetic screens  Some genetic screens are relatively straightforward  e.g., For a visible phenotype like eye color  If phenotype is subtle or needs to be measured, the screen is more time consuming  Examples  Seed weight  Behavioral traits

18. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Industrial setting for screens

19. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 High-throughput genetic screen  Paradigm Genetics, Inc. performs “phenotypic profiling”  Take measurements of mutants’ physical and chemical parameters  e.g., plant height, leaf size, root density, and nutrient utilization  Different developmental times: compare to wild type

20. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 From phenotype to gene  Once an interesting mutant is found and characterized, we want to find the gene in which the mutant occurred  Positional cloning  First use genetic mapping  Then use chromosome walking  Needle in hay stack chromosomecontigcandidate genesmutation

21. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Candidate-gene approach  If the mutated gene is localized to a sequenced region of the chromosome, then look for genes that could be involved in the process under study  Last step: confirm gene identification  Rescue of phenotype  Mutations in same gene in different alleles Tau mutation in circadian rhythm

22. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Insertional mutagenesis  Alternative to chromosome walking  To reduce time and effort required to identify mutant gene  Insert piece of DNA that disrupts genes  Inserts randomly in chromosomes  Make collection of individuals  Each with insertion in different place  Screen collection for phenotypes  Use inserted DNA to identify mutated gene

23. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Insertional mutagens  Transposable elements  Mobile elements jump from introduced DNA  e.g., P elements in Drosophila  Or start with a small number of nonautonomous elements  Mobilize by introducing active element  e.g., AC/DS elements in plants  Single-insertion elements  e.g., T-DNA in plants  Once insert, can’t move again

24. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Insertional mutagenesis in Arabidopsis  T-DNA inserts into plant chromosome  Screen for mutations that affect flower formation  Make genomic library from mutant DNA  Probe with T-DNA  Identify mutant gene T-DNA inserts into gene probe library made from T-DNA tagged mutant with T-DNA sequence DNA flanking T-DNA to identify gene AGAMOUS T-DNA probe

25. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Activation tagging  A variation on insertional mutagenesis  Makes gain-of-function mutations instead of loss-of-function mutations  Potential to identify gene function not detectable through loss-of-function screens  Useful for the following cases:  Functionally redundant genes  Genes required for viability

26. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Activation tagging in Arabidopsis I  Strong constitutive viral promoter  CaMV 35S  Inserted randomly  With T-DNA  When inserts are near a gene, the following results occur:  Activation  Constitutive expression  Can result in abnormal phenotype gene X constitutive expression of gene X 35S enhancer T-DNA vector

27. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Activation tagging in Arabidopsis II  Examples of mutant phenotypes found in activation-tagging screen  In an activation-tagging experiment carried out by Detlef Weigel’s laboratory at the Salk Institute, many different abnormal phenotypes were observed for Arabidopsis. Among the genes that were activated were Flowering Locus T (FT), which controls flowering time, and genes that control plant growth and leaf shape.

28. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Enhancer trapping  Type of insertional mutagenesis  Used to find genes with interesting expression patterns  Insert carries a reporter gene  Expresses foreign protein  No effect on organism  Enhancer trap  Has minimal promoter in front of reporter gene  Enhancer near point of insertion acts on it

29. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 By chance it will go to right place Enhancer Need very large population to increase your chances Not good for all genes! Randon=m event. Need targeted approach!

30. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Enhancer trapping in Drosophila I  Use transposon P element  Carries reporter gene  -galactosidase  Hops into genome  When lands near enhancer, activates gene expression  Expression similar to that of neighboring gene gene Y enhancer P element vector gene Y enhancer  -galactosidase TATA P element recognition sites

31. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Enhancer trapping in Drosophila II  Reporter gene: Green Fluorescent Protein (GFP)  The enhancer trap has inserted into a gene expressed in part of the fly eye

32. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Gene trapping  Similar to enhancer trapping  Instead of minimal promoter has splice acceptor site (AG) before reporter gene  Expressed only when correctly spliced  Usually causes gene disruption as well GT AG

33. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Basics of reverse genetics To find out the function of a known gene  Reverse genetics starts with known genes  e.g., from genomic sequencing  Goal: to determine function through targeted modulation of gene activity  Decrease  Increase

34. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Ways to modulate gene activity  Delete gene  Homologous recombination  Works well in yeast  Can be done in mouse and flies not plants  Interfere with transcription  Antisense RNA  Interfering RNA (RNAi)  Identify gene affected by mutagenesis  Insertional or chemical

35. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Reverse-genetics example  Gene that encodes muscle-specific transcription factor in mouse  Myogenin required  Homologous recombination used to delete gene  Mice born, but can’t make muscle targeting vector neo genome locus product of homologous recombination selectable marker disrupts myogenin gene Tk myogenin neo selection

36. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 RNAi and antisense RNA  Double-stranded RNA able to disrupt gene expression  Cells have machinery that destroy double- stranded RNA: viruses/ cDNA  Appears to be basis for the following:  Interfering RNA (RNAi)  Double-stranded RNA introduced into cells  Antisense RNA  Introduce complementary RNA  Forms double-stranded RNA in cells

37. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 RNAi mechanism I  RNAi probably depends on a system used to detect the following:  Double-stranded (ds) RNA viruses  Other abnormal gene expression  Initially characterized in the following:  Fungus  Quelling in Neurospora  Plants  Resistance to spread of virus

38. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 RNAi mechanism II  Cellular RNase recognizes dsRNA  Cleaves to small (23 bp) fragments  Fragments hybridize to transcripts  RNA-dependent RNA polymerase forms dsRNA  RISC nuclease chews up dsRNA RNApol RNase RISC (RNA induced silencing complex)

39. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Systematic RNAi screens  In the roundworm, C. elegans, RNAi is easy to do  Soak in solution of dsRNA  Feed bacteria expressing dsRNA  Or inject dsRNA  Makes systematic RNAi screens possible  ~ 75% of time, RNAi gives a reduction in RNA levels  (Not 100% silencing but leaky)  Has been used to reduce expression of genes  On particular chromosomes, or  Expressed at a particular developmental time

40. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 RNAi on all genes on chromosome  Goal: In C. elegans, determine function of all 2,300 genes on chromosome III  RNAi constructs made for each gene  Worms microinjected with double-stranded RNA T7T3 add T3 + T7 polymerase predicted ORF construct with promoter sequence dsRNA Dr. A. Hyman

41. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Phenotypes found in RNAi screen Cytokinesis (phenotypic class C6) Pronuclear migration (phenotypic class C1) Pronuclear/nuclear appearance (phenotypic class B1) Wild typeRNAi embryos

42. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 RNAi screen of embryonic genes  Microarray analysis identified genes active in early embryogenesis  Used RNAi to target each gene  Worms injected with RNAi construct  Progeny tested for phenotypes

43. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Wild-type C. elegans embryogenesis

44. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Effects of RNAi on actin

45. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Finding genes in libraries of mutants cDNA  Goal: Find known genes disrupted in collections of mutants  Screening of functional-genomics libraries  Can be used for mutations that affect lethality or fertility  Screen heterozygotes  Can either use PCR to identify mutations in a particular gene or sequence flanking sequences of all inserts

46. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Screening an insertion library (Insertion in promoter region)  PCR used to find insertion  One primer complementary to insert  Other primer complementary to gene  If get an amplification product then you have insertion  Sequence product for exact location gene Z insert PCR primers gene Z insert PCR amplification +– amplification product on gel indicates presence of insert near gene

47. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 TILLING  Method for finding mutations produced by chemical mutagens in specific genes  Chemical mutagenesis  Usually produces point mutations  Generally gives more subtle phenotypes than insertions  e.g., hypomorphs, temperature sensitive mutants

48. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 TILLING in Arabidopsis I  EMS used to mutagenize Arabidopsis  Grow individual mutagenized lines  Make primers flanking gene of interest  Amplify using PCR WT mutant gene Z WT mutant PCR amplification from wild type and mutant EMS mutagenize seed

49. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 TILLING in Arabidopsis II  Denature DNA from pools of mutant lines  Allow to hybridize to wild-type DNA  Detect mismatches in hybridized DNA  Denaturing HPLC  Cel I enzyme cuts at mismatches  Sequence to identify site of mutation ATGCGGACTG |||||| ||| TACGCCGGAC ATGCGG CTG |||||| ||| TACGCC GAC Cel 1 +

50. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Summary I  Forward genetics  Mutation to gene function  Genetic screens  Cloning genes identified in screens  Genomics approaches to forward genetics  High-throughput genetic screens  Insertional mutagenesis  Activation tagging  Enhancer trapping and gene trapping

51. © 2005 Prentice Hall Inc. / A Pearson Education Company / Upper Saddle River, New Jersey 07458 Summary II  Reverse genetics  From gene to function  Genomics approaches to reverse genetics  RNAi screens  Identifying mutations in insertional libraries  TILLING