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Nature Reviews Genetics Nature Reviews Genetics

Yeast eukaryote model organism Eukaryote; – mitochondria, – organelles, – cell cycle, etc. Eukaryote Yeast eukaryote model organism Eukaryote; – mitochondria, – organelles, – cell cycle, etc. Eukaryote Plus; – haploid, diploid, – extra-chromosomal DNA. Saccharomyces cerevisiae Baker’s Yeast

Yeast Genome Project • Yeast Genome Project finished in 1996, – 1. 2 x Yeast Genome Project • Yeast Genome Project finished in 1996, – 1. 2 x 107 DNA base pairs, • 16 chromosomes, 230 kb - 2, 352 kb, • ~6, 000 Open Reading Frames (ORFs), – Only ~4% of the genes have introns, • > 70% of the genome is coding.

Yeast Genome Project vs. human genome 12. 1 Mb Genomic DNA sequence (Human, 3, Yeast Genome Project vs. human genome 12. 1 Mb Genomic DNA sequence (Human, 3, 000 Mb) 70% coding sequence (Human, 1. 8%) Few Introns (Humans many) 6022 Genes (Human, 20 -25, 000) About 70% of the genes found in humans, are found in yeast.

Known/Unknown (2001) 3, 780 genes with some characterization 560 homologous with other organisms ~1900 Known/Unknown (2001) 3, 780 genes with some characterization 560 homologous with other organisms ~1900 unknown

Assigning Gene Function Geneticist: gene sequence, expression, etc. Biochemist: enzymatic function, etc. Cell Biologist: Assigning Gene Function Geneticist: gene sequence, expression, etc. Biochemist: enzymatic function, etc. Cell Biologist: cellular location, etc. - especially Protein/DNA Interactions Protein/Protein Interactions Protein/Membrane Interactions etc.

The Awesome Power of Yeast Genetics Homologous Recombination Transposons Life Cycle etc. The Awesome Power of Yeast Genetics Homologous Recombination Transposons Life Cycle etc.

Homologous Recombination • the replacement of a gene with an exogenous gene through equal Homologous Recombination • the replacement of a gene with an exogenous gene through equal crossing over, homologous region foreign DNA homologous region Before After

Transposons Someplace Transposons: whole units of DNA that have the ability to insert themselves Transposons Someplace Transposons: whole units of DNA that have the ability to insert themselves into DNA molecules, – can carry other genes. Inserts someplace else

Hologous Recombination and Transposons • Serve as shuttles to carry experimental DNA sequences into Hologous Recombination and Transposons • Serve as shuttles to carry experimental DNA sequences into yeast, – Regulatory sequences (promoters) drive the expression of, • Mutant Genes: for structure function analysis, • Reporter Genes: code for enzymes that signal their presence in specific cells, • Epitope Tags: proteins tagged with a foreign peptide sequence that binds to a specific antibody, – etc.

Reverse Genetics Functional Genomics Gene DNA Sequence Gene Disruption Phenotype Analysis Function Mutate DNA Reverse Genetics Functional Genomics Gene DNA Sequence Gene Disruption Phenotype Analysis Function Mutate DNA Sequence Genetically Link Development Physiology Cell Biology

transp. lox …no start codon, no promoter. transp. . inserted randomly into a genomic transp. lox …no start codon, no promoter. transp. . inserted randomly into a genomic library. Haemaglutinin (HA) yeast genomic library …+ returns functional transcript, or at least, an HA tagged peptide that has been targeted. Fig. 1

? Biology 470 WEB Page ? Biology 470 WEB Page

Up Sides? Transposon Down Sides • Insertions are essentially generated at random; – it Up Sides? Transposon Down Sides • Insertions are essentially generated at random; – it is very difficult to mutagenize all genes within a genome by transposon mutagenesis alone, • but really, transposon-specific biases in target-site selection, – for reasons not fully understood, transposons such as Tn 3 and Tn 7 insert non-randomly into DNA.

Site Directed Mutagenesis uptag downtag • Systematic deletion of each ORF in the genome, Site Directed Mutagenesis uptag downtag • Systematic deletion of each ORF in the genome, – homologous recombination replaces the gene with a selectable marker, and a DNA “barcode”, • UPTAG, • DOWNTAG. Fig. 2 Whole set available: $1, 500

Fig. 1. Chemical genomic screening by using a high-density cell array • “Of the Fig. 1. Chemical genomic screening by using a high-density cell array • “Of the 14 gene deletions that produce the rapamycin-enhanced phenotype, 13 genes have human homologs that showed >30% identity (highly significant) at the protein level, and most of them encode mitochondrial proteins. ” • “Because mitochondrial dysfunction is known to underlie the pathogenesis of a wide range of neurodegenerative disorders…our result suggests that rapamycin may be useful in preventing the progression of these diseases, including Alzheimer's, Parkinson's, and Huntington's diseases and brain aging. ”

DNA Microarray • DNA arrayed at high density on a solid substrate, • In DNA Microarray • DNA arrayed at high density on a solid substrate, • In this experiment, DNA complementary to each ORF’s UPTAG and DOWNTAG is arrayed in an ordered fashion. http: //www. bio. davidson. edu/courses/genomics/chip. html

Homologous Recombination UPTAG / DOWNTAG Fig. 2 a Homologous Recombination UPTAG / DOWNTAG Fig. 2 a

PCR Strategy Big Primers PCR Strategy Big Primers

…each strain has one gene KO’d. Conditional Mutants …one strain each for >5, 900 …each strain has one gene KO’d. Conditional Mutants …one strain each for >5, 900 genes. Grow deletion strains under restrictive conditions, PCR U/Dtags, label DNA, Hybridize/Measure signal, - absent/altered signal indicates that the cell with that particular barcode has low fitness. Fig. 2 b Conditional Mutants: mutants that have observable phenotypes under a given set of growth conditions.

Fig. 1. Chemical genomic screening by using a high-density cell array • “Of the Fig. 1. Chemical genomic screening by using a high-density cell array • “Of the 14 gene deletions that produce the rapamycin-enhanced phenotype, 13 genes have human homologs that showed >30% identity (highly significant) at the protein level, and most of them encode mitochondrial proteins. ” • “Because mitochondrial dysfunction is known to underlie the pathogenesis of a wide range of neurodegenerative disorders…our result suggests that rapamycin may be useful in preventing the progression of these diseases, including Alzheimer's, Parkinson's, and Huntington's diseases and brain aging. ”

Formaldehyde cross-link TFs to the DNA. . . DNA Protein Interactions Interactome #1 Epitope Formaldehyde cross-link TFs to the DNA. . . DNA Protein Interactions Interactome #1 Epitope tag a transcription factor of interest. Shear (cut) genomic DNA into small fragments. Fig 3. cont. next page

DNA Protein Interactions Interactome #1 Antibodies to the HA protein are used to collect DNA Protein Interactions Interactome #1 Antibodies to the HA protein are used to collect the target TF/DNA fragments. …target probes from genome with HA-tagged TF, …reference probes from genome with TF deleted.

SBF, SPO 11, etc. DNA Protein Interactions Interactome #1 Antibodies to the HA protein SBF, SPO 11, etc. DNA Protein Interactions Interactome #1 Antibodies to the HA protein are used to collect the target TF/DNA fragments. The microarray has the promoters for known genes arrayed.

Proteomics Protein-Protein Interactions Signal Transduction Pathways, Yeast Two Hybrid (Y 2 H), Heteromeric Protein Proteomics Protein-Protein Interactions Signal Transduction Pathways, Yeast Two Hybrid (Y 2 H), Heteromeric Protein Complexes, Protein Chips (not required), Allosteric Interactions, Mass Spectroscopy. etc.

GAL 4 Transcription Activator native yeast transcription factor 4 L GA One Protein, Two GAL 4 Transcription Activator native yeast transcription factor 4 L GA One Protein, Two Functional Domains BD: Binding Domain, AD: Activation Domain.

Yeast Life Cycle Yeast Life Cycle

Yeast Two Hybrid Vectors . . . separate GAL 4 Binding Domain and Activation Yeast Two Hybrid Vectors . . . separate GAL 4 Binding Domain and Activation Domain, . . . create chimeric proteins, on expression vectors, – Bait Gene fused to the Binding Domain Gene, – Target (prey) Gene fused to the Activation Domain Gene.

Yeast Two Hybrid Vectors …in a diploid cell. Yeast Two Hybrid Vectors …in a diploid cell.

c. DNAs are derived from m. RNA sequences. protein of interest i. e. constructed c. DNAs are derived from m. RNA sequences. protein of interest i. e. constructed from a c. DNA library. . mate haploid cells, each expressing the recombinant proteins, – one with bait, – the other(s) with prey (target).

No Interaction Bait/Prey . . . bait binds DNA, . . . prey does No Interaction Bait/Prey . . . bait binds DNA, . . . prey does not associate with bait, or transcription machinery.

Bait/Prey Interact . . . bait binds DNA, . . . prey associates with Bait/Prey Interact . . . bait binds DNA, . . . prey associates with bait, . . . activation domain is then in proximity to transcriptonal machinery, . . . reporter gene turned on.

Lot’s of Love; Genetix • High throughput screening, • As many as 100, 000 Lot’s of Love; Genetix • High throughput screening, • As many as 100, 000 matings per day , » • Automatic sample loading, reading and image analysis.

Two Hybrid Analysis Two Hybrid Analysis

Yeast Interactome >1, 200 Proteins Yeast Interactome >1, 200 Proteins

Single Bait Strategy What interacts with the protein implicated in Huntington’s Disease? PNAS 100(5): Single Bait Strategy What interacts with the protein implicated in Huntington’s Disease? PNAS 100(5): 2712 -2717

Abstract • Huntington’s disease (HD) is a neurodegenerative disease caused by polyglutamine (poly. Q) Abstract • Huntington’s disease (HD) is a neurodegenerative disease caused by polyglutamine (poly. Q) expansion in the protein huntingtin (htt). • Pathogenesis in HD seems to involve the formation of neuronal intranuclear inclusions and the abnormal regulation of transcription and signal transduction. • To identify previously uncharacterized htt-interacting proteins in a simple model system, a yeast two-hybrid screen was used with a Caenorhabditis elegans “protein expression” library.

Set-Up htt Expressing C. elegans proteins. . mate bait and prey cells, each expressing Set-Up htt Expressing C. elegans proteins. . mate bait and prey cells, each expressing recombinant proteins, – diploids that have restored GAL 4 activated gene expression contain peptides that interact.

Bait/Prey Interaction . . . found a “C. elegans” protein (K 08 E 3. Bait/Prey Interaction . . . found a “C. elegans” protein (K 08 E 3. 3 b) that interacts with N-terminal htt in two-hybrid tests.

CIP 4 in Human Brains • A: Normal, B ---> D increasing Huntinton’s symptoms. CIP 4 in Human Brains • A: Normal, B ---> D increasing Huntinton’s symptoms. • Red Arrows represent CIP 4 protein localization. Blue arrow points to brain lesions. • A human homolog of the C. elegans K 08 E 3. 3 b protein is the Cdc 42 interacting protein 4 (CIP 4). • Neuronal CIP 4 immunoreactivity increased with neuropathological severity in the neostriatum of HD patients.

CIP 4 is Sufficient for HD Symptoms CIP 4 protein was over expressed in CIP 4 is Sufficient for HD Symptoms CIP 4 protein was over expressed in rat brains. Cell death and Huntington’s Disease (HD) morphology resulted.

The Skinny …and, how many species involved? • Bait: Human, • Target (prey): C. The Skinny …and, how many species involved? • Bait: Human, • Target (prey): C. elegans (roundworm), – bait/target match found. • C. elegans target gene has a human homolog cdc 42 interacting protein (CIP 4), – CIP 4 found at high levels in HD patient’s brains, • CIP 4 sufficient to cause HD-like symptoms in rats.

Y 2 H Weaknesses • False Positives, – some Baits are “sticky”, sticks to Y 2 H Weaknesses • False Positives, – some Baits are “sticky”, sticks to lots of Targets, – some “Targets” are sticky, sticks to lots of “Baits”, – fortuitous activation of marker promoter, • usually assay for multiple markers, • False Negatives, – clone fidelity, – protein conformation (especially membrane bound proteins), – protein modifications (phosphorylation, glycosylation, etc. ), • Artifacts: Y 2 H identified interactions require subsequent confirmation.

Proteomics II Protein Arrays Proteomics II Protein Arrays

Proteomics III Mass Spectrometry Proteome Protein - Protein Interactions Protein Complexes Peptide Sequencing etc. Proteomics III Mass Spectrometry Proteome Protein - Protein Interactions Protein Complexes Peptide Sequencing etc.

Mass Spectrometry • Molecules to be analyzed, referred to as analytes are first ionized Mass Spectrometry • Molecules to be analyzed, referred to as analytes are first ionized (usually in a vacuum), • Newly charged (protonated) molecules are introduced into an electric and/or magnetic field in gas phase, • Their path through the field is a function of the mass to charge ratio m/z, • m/z of the ionized species can be used to deduce the mass of the analyte with high precision.

Proteomics and Mass Spec MALDI ESI-MS Proteome Protein - Protein Complexes Peptide Sequencing etc. Proteomics and Mass Spec MALDI ESI-MS Proteome Protein - Protein Complexes Peptide Sequencing etc.

Peptide Mass Mapping “Mass Fingerprinting”. . . proteins are cleaved by proteolytic enzymes in Peptide Mass Mapping “Mass Fingerprinting”. . . proteins are cleaved by proteolytic enzymes in a sequence specific manner, 22. 655 k. D 8. 222 k. D 1. 457 k. D 10. 003 k. D 13. 457 k. D = One, and only one, 55. 792 k. D protein in the data base w/ specific fragment pattern. – thus, each protein in a proteome has a unique peptide mass subset, • these subsets can be computationally derived from protein databases, i. e via translated genomic DNA sequences, • experimentally determined unknowns can be compared, via computers, to online databases for identification, . . scalable, multiple samples can be deposited at once, computers sort out the constituents.

Tandem Mass Spectroscopy (MS-MS) Often provides enough information to unambiguously identify the entire protein Tandem Mass Spectroscopy (MS-MS) Often provides enough information to unambiguously identify the entire protein when MS data is compared to online databases. . mass spectrometry can also be used to obtain sequence to identify peptides, – treatment with sequence specific protelytic enzymes provides information on the terminal residues, – the mass of the peptide fragment is determined, – a short amino acid sequence from the peptide is obtained.

MS-MS MS #1: peptide fingerprinting is performed, – peptides that have an appropriate mass MS-MS MS #1: peptide fingerprinting is performed, – peptides that have an appropriate mass for further study are isolated, MS #2: selected peptides are bombarded with argon gas, making random fractures in the peptide backbone, and mass spec is repeated, - the mass of each of these fragments is measured.

Mass Difference = Amino Acid Weight 693. 37(EYL)1098. 55. . . single entry in Mass Difference = Amino Acid Weight 693. 37(EYL)1098. 55. . . single entry in the database, + total peptide mass info = TQLYEYLQR

Protein-Protein Interactions • Interacting proteins are coprecipitated, and excised from 2 -D Page gels Protein-Protein Interactions • Interacting proteins are coprecipitated, and excised from 2 -D Page gels – gel slices are run through MSMS, – computers de-convolute slices with multiple proteins. 2 -D Page

Interaction Mapping • Multiple proteins isolated in single gel slices are candidate interactors, • Interaction Mapping • Multiple proteins isolated in single gel slices are candidate interactors, • Other experimental techniques are used to confirms interactions (including Y 2 H). DNA Damage Repair Network

Yeast Protein Interactome Yeast Protein Interactome

Questions Review Questions Review

Next Finish Up, Review Lectures online at my Course Materials Page. Read through pp. Next Finish Up, Review Lectures online at my Course Materials Page. Read through pp. 579 of the Strategies Paper.