
6f6c4c4e62b00c16401a09e2fd151595.ppt
- Количество слайдов: 57
Nature Reviews Genetics
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 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, 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 unknown
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.
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 into DNA molecules, – can carry other genes. Inserts someplace else
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 Sequence Genetically Link Development Physiology Cell Biology
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
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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, – 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 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 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
PCR Strategy Big Primers
…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 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 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 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 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 Complexes, Protein Chips (not required), Allosteric Interactions, Mass Spectroscopy. etc.
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 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.
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 not associate with bait, or transcription machinery.
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 matings per day , » • Automatic sample loading, reading and image analysis.
Two Hybrid Analysis
Yeast Interactome >1, 200 Proteins
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) 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 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. 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. • 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 rat brains. Cell death and Huntington’s Disease (HD) morphology resulted.
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 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 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 (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.
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 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 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 the database, + total peptide mass info = TQLYEYLQR
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, • Other experimental techniques are used to confirms interactions (including Y 2 H). DNA Damage Repair Network
Yeast Protein Interactome
Questions Review
Next Finish Up, Review Lectures online at my Course Materials Page. Read through pp. 579 of the Strategies Paper.