nature reviews genetics. yeast eukaryote model organism eukaryote; –mitochondria, –organelles,...
Post on 20-Jan-2016
227 Views
Preview:
TRANSCRIPT
Nature Reviews Genetics
Yeasteukaryote model organism
Eukaryote;
– mitochondria,
– organelles,
– cell cycle, etc.
Eukaryote Plus;
– haploid, diploid,
– extra-chromosomal DNA.
Saccharomyces cerevisiaeBaker’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 Projectvs. human genome
12.1 Mb Genomic DNA sequence (Human, 3,000 Mb)
70% coding sequence (Human, 1.8%)
Few Introns (Humans many)
6012 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,
Before
After
foreign DNAhomologous regionhomologous region
Transposons
Transposons: whole units of DNA that have the ability to insert themselves into DNA molecules,
– can carry other genes.
Someplace
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 GeneticsFunctional Genomics
Gene DNASequence
Gene Disruption PhenotypeAnalysis
Function
MutateDNA Sequence
DevelopmentPhysiology
Cell BiologyGenetically Link
…no start codon, no promoter.
lox Haemaglutinin(HA)
transp.transp.
…+ returns functional transcript, or at least, an HA tagged peptide that has been targeted.
...inserted randomly into a genomic library.
yeast genomic library
Fig. 1
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 Tn3 and Tn7 insert non-randomly into DNA.
Up Sides?
Site Directed Mutagenesis
• Systematic deletion of each ORF in the genome,
– homologous recombination replaces the gene with a selectable marker, and a DNA “barcode”,
• UPTAG,• DOWNTAG.
Whole set available:$1,500Fig. 2
uptag downtag
• “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.”
Fig. 1. Chemical genomic screening by using a high-density cell array
• 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.
DNA Microarray
http://www.bio.davidson.edu/courses/genomics/chip/chip.html
Homologous RecombinationUPTAG / DOWNTAG
Fig. 2a
PCR Strategy
Big Primers
Conditional Mutants
Conditional Mutants: mutants that have observable phenotypes under a given set of
growth conditions.
Hybridize/Measure signal,
- absent/altered signal indicates that the cell with that particular barcode has low fitness.
Grow deletion strains under restrictive conditions, PCR U/D-
tags, label DNA,
…each strain has one gene KO’d.
…one strain each for >5,900 genes.
Fig. 2b
DNA Protein InteractionsInteractome #1
Epitope tag a transcription factor of interest.
Formaldehyde cross-link TFs to the DNA...
Shear (cut) genomic DNA into small fragments.
cont. next pageFig 3.
DNA Protein InteractionsInteractome #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.
DNA Protein InteractionsInteractome #1
Antibodies to the HA protein are used to collect
the target TF/DNA fragments.
The microarray has the promoters for known genes
arrayed.
SBF, SPO11, etc.
ProteomicsProtein-Protein Interactions
Yeast Two Hybrid (Y2H),
Protein Chips (not required),
Mass Spectroscopy.
Signal Transduction Pathways,
Heteromeric Protein Complexes,
Allosteric Interactions,
etc.
GAL4 Transcription Activatornative yeast transcription factor
One Protein, Two Functional Domains
BD: Binding Domain,
AD: Activation Domain.
GAL4
Yeast Life Cycle
Yeast Two Hybrid Vectors
...separate GAL4 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.
...mate haploid cells, each expressing the recombinant proteins, – one with bait,
– the other(s) with prey (target).
protein of interest
i.e. constructed from a cDNA library.
cDNAs are derived from mRNA sequences.
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.
Yeast Interactome>1,200 Proteins
Two Hybrid Analysis
Single Bait StrategyWhat 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 (polyQ) 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.
...mate bait and prey cells, each expressing recombinant proteins,
– diploids that have restored GAL4 activated gene expression contain peptides that interact.
htt
Expressing C. elegans proteins.
Set-Up
Bait/Prey Interaction
...found a “C. elegans” protein (K08E3.3b) that interacts with N-terminal htt in two-hybrid tests.
CIP4 in Human Brains
• A: Normal, B ---> D increasing Huntinton’s symptoms.
• Red Arrows represent CIP4 protein localization. Blue arrow points to brain lesions.
• A human homolog of the C. elegans K08E3.3b protein is the Cdc42-interacting protein 4 (CIP4).
• Neuronal CIP4 immunoreactivity increased with neuropathological severity in the neostriatum of HD patients.
CIP4 is Sufficient for HD Symptoms
CIP4 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 cdc42 interacting protein (CIP4),
– CIP4 found at high levels in HD patient’s brains,
• CIP4 sufficient to cause HD-like symptoms in rats.
Y2H 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: Y2H identified interactions require subsequent confirmation.
Protein Arrays
Proteomics II
Proteomics III
Mass Spectrometry
ProteomeProtein - Protein Interactions
Protein ComplexesPeptide 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
ProteomeProtein - ProteinProtein ComplexesPeptide Sequencingetc.
Peptide Mass Mapping“Mass Fingerprinting”
...proteins are cleaved by proteolytic enzymes in a sequence specific manner,
– 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.
1.457 kD
10.003 kD
13.457 kD
8.222 kD
22.655 kD
=One, and only one, 55.792
kD protein in the data base w/ specific fragment
pattern.
Tandem Mass Spectroscopy(MS-MS)
...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.
Often provides enough information to unambiguously identify the entire protein when MS data is compared to online databases.
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.
693.37(EYL)1098.55
+
total peptide mass info
=
TQLYEYLQR
...single entry in the database,
Mass Difference = Amino Acid Weight
Protein-Protein Interactions
• Interacting proteins are co-precipitated, and excised from 2-D Page gels
– gel slices are run through MS-MS,
– 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 Y2H).
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.
top related