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Contents Definition of proteomics Protein profiling Protein-protein interactions Yeast two-hybrid method Protein chips TAP tagging/Mass spectrometry Biochemical genomics Pregenomics biochemical assays Methods used to find genes responsible for specific biochemical activity before the inception of genomics Laboriously purify responsible protein Often expensive and time consuming Expression cloning Introduce cDNA pools into cells Look for biochemical activity in those cells Caveat: Often difficult to detect biochemical activity in cell’s biochemical “background” Biochemical genomics ( “Enzomics”? ) Genome of an organism is already known Approach Construct plasmids for all ORFs Attach ORFs to sequence that will facilitate purification Transform cells Isolate ORF products Test for biochemical activity Biochemical genomics in yeast 6,144 ORF yeast strains made ORFs fused to glutathione S-transferase (GST) for purification purposes Biochemical assay revealed three new biochemical reactions associated with yeast ORFs pre-tRNA Ligated tRNA tRNA halves Abc1 35 64 Microfluidics Proteomics requires greater automation Microfluidics: a “lab on a chip” Microvalves and pumps allow control of nanoliter amounts Can control biochemical reactions A microfluidics chip Microfluidics in action loading mixing 500 mm compartmentalization purging Summary I Goals of proteomics Identify and ascribe function to proteins under all biologically plausible conditions Proteomics methods 2-D gel electrophoresis for separating proteins on the basis of charge and molecular weight Mass spectrometry for identifying proteins by measuring the mass-to-charge ratio of their ionized peptide fragments Protein chips to identify proteins, to detect protein– protein interactions, to perform biochemical assays, and to study drug–target interactions Summary II Proteomics methods (continued) Yeast two-hybrid method for studying protein–protein interactions Biochemical genomics for high-throughput assays Some accomplishments of proteomics Example: yeast Yeast two-hybrid method reveals interactome Transcriptional regulatory networks deduced Biochemical genomics uncovers new ORF functions Subcellular localization of proteins Genomics IV: “Phenomics” High-Throughput Genetics Applications of genomics approaches to genetics Background Genetics is the study of gene function Genomics is changing the way genetics is performed Gene function is inferred from the resulting phenotype when the gene is mutated Global, high-throughput approaches Genomics approaches are being applied to both forward and reverse genetics Forward and reverse genetics Forward genetics starts with identification of interesting mutants Reverse genetics starts with a known gene and alters its function Then aims to discover the function of genes defective in mutants 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 Basics of forward genetics Forward genetics usually starts with mutagenesis of organism Can use chemicals Or can use radiation e.g., ethyl methyl sulfonate (EMS) e.g., X rays Then screen progeny of mutagenized individuals for phenotypes of interest Genomics applied to genetics Genomics characterized by the following: High throughput Global approach Using automation to speed up a process All genes in genome Applied to both forward and reverse genetics Genomics and forward genetics High-throughput genetic screens Candidate-gene approach Insertional mutagenesis To go from phenotype to gene Loss-of-function mutation Activation tagging Enhancer trapping 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 Industrial setting for screens 2002 Para digm Genetics, Inc. All rights reserved. Used with permission. 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: 2002 Para digm Genetics, Inc. All rights reserved. Used with permission. compare to wild type From phenotype to gene Once an interesting mutant is found and chromosome characterized, we want to find the gene in which the mutant occurred Positional cloning First use genetic mapping Then use chromosome walking contig candidate genes mutation 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 Insertional mutagenesis Alternative to chromosome walking Insert piece of DNA that disrupts genes Inserts randomly in chromosomes Make collection of individuals To reduce time and effort required to identify mutant gene Each with insertion in different place Screen collection for phenotypes Use inserted DNA to identify mutated gene Insertional mutagens Transposable elements Mobile elements jump from introduced DNA Or start with a small number of nonautonomous elements Mobilize by introducing active element e.g., P elements in Drosophila e.g., AC/DS elements in plants Single-insertion elements e.g., T-DNA in plants Once insert, can’t move again Basics of reverse genetics Reverse genetics starts with known genes E.g., from genomic sequencing Goal: to determine function through targeted modulation of gene activity Decrease Increase Ways to modulate gene activity Delete gene Homologous recombination Works well in yeast Can be done in mouse and flies Interfere with transcription Antisense RNA Interfering RNA (RNAi) Identify gene affected by mutagenesis Insertional or chemical Reverse-genetics example Gene that encodes muscle-specific transcription factor in mouse neo genome locus myogenin selection Myogenin Homologous recombination used to delete gene Mice born, but can’t make muscle targeting vector Tk neo product of homologous recombination selectable marker disrupts myogenin gene RNAi and antisense RNA Double-stranded RNA able to disrupt gene expression Cells have machinery that destroy doublestranded RNA 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 Finding random mutations in your gene of interest (or every gene in the genome) Random insertion of transposons Random point mutations/indels Screening an insertion library 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 PCR primers insert gene Z PCR amplification insert gene Z + – amplification product on gel indicates presence of insert near gene P element piggyBac Summary of P element Gene Disruption Project Insect transposon vectors Host range of transposons Mosquito Silkmoth Flour Beetle Transformation of Planaria (roundworm) Potential for broad host range transposons in mutagenesis Insertional mutagenesis (random) Transgenic RNAi Homologous recombination? (a la Drosophila) TILLING Method for finding mutations produced by chemical mutagens in specific genes Chemical mutagenesis Usually produces point mutations Very high mutagenic efficiency Generally gives more subtle phenotypes than insertions e.g., hypomorphs, temperature sensitive mutants TILLING in Arabidopsis I EMS mutagenize seed EMS used to mutagenize Arabidopsis Grow individual mutagenized lines Make primers flanking gene of interest Amplify using PCR gene Z WT gene Z mutant PCR amplification from wild type and mutant WT mutant 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 |||||| TACGCC CTG ||| GAC Cel 1 Arabidopsis TILLING Project Mapping mutations by DHPLC Prepare heteroduplexes as for Cel I method Rather than digest sample, separate on denaturing HPLC Cleaner result, but much slower analysis than Cel I digestion Mapping mutations by TGCE 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 Summary II Reverse genetics From gene to function Genomics approaches to reverse genetics RNAi screens Identifying mutations in insertional libraries TILLING