Micro 302 - Yeast Model
"Wild" Wine
"Wild" Wines or BioWines: - Normally, the grape juice is sterilized to kill off the wild yeast. They then introduce a specific yeast. Imparts a lot of flavours to the wine to predictably produce many batches w/ the same flavour. - A lot of young people in France are rebelling against the country's very strict wine restrictions. i.e. there's a social/societal aspect to it. - A bit of a gamble b/c they don't know what flavours these WT yeasts will impart Avril Creek: Half their wines utilize wild yeast now!
Lifecycle of S. cerevisiae? (two forms)
- 2 diff mating types: a-type + α-type --> will conjugate to form a dikaryotic organism, fusion --> a/α diploid Two forms of yeast cells can survive & grow: haploid + diploid. Haploid cells: Undergo a simple lifecycle of mitosis & growth. Under conditions of high stress will die. The asexual form of the fungus. Diploid cells: Preferential 'form' of yeast in nature. Similarly undergo a simple lifecycle of mitosis & growth. Under conditions of stress, diploid cells can undergo sporulation, entering meiosis and producing four haploid spores, which can subsequently mate. This is the sexual form of the fungus. - Rate at which mitotic cell cycle progresses often differs substantially between haploid and diploid cells. Under optimal conditions, yeast cells can double their population every 100 minutes.[13][14] However, growth rates vary enormously both between strains and between environments.
Why Yeast as a model system?
- 20% of human disease gene have homologues in yeast! - Fundamental (euk) processes are highly conserved (transcription, translatioin, cell cycle, chromatin, DNA rep, mitosis, signalling, metabolism, transporters, etc.) - 2001, 2013,2016 Nobel Prizes in Medicine: Cell cycle, Vesicular trafficking, autophagy (involved yeast)!
Systems biology
- A holistic perspective - The study of an organism as an integrated & interacting network of genes, proteins, molecules & biochemical rxns. - Requires systematic data collection for molecules in a cell - e.g. Proteomics, genomics, metabolomics
2. Is the phenotype caused by a single mutation?
- Perform via meiotic analysis - If we have more than one mutation, we shouldn't have this 2 + 2 situation (i.e. 2n + 2n = tetrad), as the original cells are diploid and for one gene. a) Cross mutant with MATa YFG+ (WT), then sporulate b) Dissect resulting 4 spores from tetrad c) Score for Yfg+ & Yfg- (i.e. Discard the colonies that aren't 2+2 (two black, two white)
What are some examples of Laboratory studies of yeast (e.g. what do we study about them that's relevant to humans)? (7)
- Secretory pathway - Cell cycle - Systems biology - Metabolism - G-protein coupled receptors - Transcription factors - MAPK pathways - i.e. We care about yeast b/c it shares many fundamental processes w/ our cells
Tetrad analysis
- The physical manipulation of of a tetrad of yeast spores (products of meiosis) in order separate the 4 spores - First must digest the ascus w/ digestive enzymes --> the spores will flatten out, then are physically separated using a glass needle. Can be spotted onto new agar - Can then test Mendelian genetics
Unique characteristics that Eukaryotes possess (compared to Prokaryotes) (8)
- organelles - lipids of PM - nucleus - chromosomes - chromatin - gene regulation (very sophisticated and complex) - mRNA transport (i.e. translation occurs outside nucleus) - cytoskeleton - Are typically diploid (not all though)
Two forms of yeast cellular division and examples of those Genus spp that utilize them?
1) Budding - e.g. Saccharomyces cerevisae (i.e. bakers yeast; involved in most studies, esp. studying cell cycle) 2) Fission - e.g. Schizosaccharomyces pombe (i.e. divide by binary fission)
What 3 Factors (and their fn's) are induced by Fus3 (in the yeast mating pathway)?
1) Cell fusion (FUS1) 2) Nuclear fusion (KAR1) 3) a CDK inhibitor (FAR1) - i.e. Ensures that all cells involved in mating are in the G1 phase
What is the cell cycle of yeats
1) M-phase = Mitosis + cytokinesis 2) G2 phase 3) S Phase = DNA replication 4) G2 Phase Checkpoints: G1S, SG2, G2M
What are 3 examples of pathogenic yeast? Why are they hard to treat/eradicate?
1. Candida albicans - commonly pathogenic in humans (opportunistic) 2. Cryptococus gatti - killed 8 people btwn 1998 and 2003. Infects resp tract :/ 3. Ustilago maydis (Huitlacoche) - A mexican delicacy; causes corn smut (harmful to corn, but actually edible!) Are difficult ot target in terms of antibiotics b/c their cell structure is very similar to humans'. Hard to find what component of the cell to target w/out harming the host (i.e. human)
HOw to Use yeast as a tool to Identify the ligand for human 'Orphan' GPCR's
1. Create a GPCR chimera (human ligand binding site + yeast Ste2 cytoplasmic domain) --> the yeast 'mating response' will be activated w/ whatever would bind to the human GPCR a) A result of the yeast mating pathway is expression of MSG5. In order to detect this transcription, replace MSG5 with GFP
What is process is undertaken during a Mutant hunt? (4)
1. Create mutations at random; screen for phenotype of interest (want a single mutation though) 2. Is the phenotype caused by a single mutation? 3. How many genes are involved in the process? 4. Find the mutations, and therefore the genes involved
About the Pheromone (Mating) Response Pathway in Yeast (5)
1. Involves GPCR's 2. Involves a MAPK pathway (SteII, Ste7, and Fus3 - physically associated w/ each other on a scaffold) 3. Signalling: a) specificity b) amplification c) attenuation (Must have some way to dampen/shut off the signal)
What are Mendel's Laws?
1. Law of Dominance 2. Law of Segregation 3. Law of Independent Assortment
How then do we rewire the MAPK pathway of the yeast to detect ligands for the human orphan GPCR (steps)
1. Remove the native Ste2 ligand binding component from yeast. The starting lab strain will have the genotype By4741: MATa, ura3, leu3, his3, met15 Then utilize homologous recombination to insert/replace desired DNA seq's: Perform PCR w/ 'fusion' primers to replace Ste2 with Auxotrophic marker LEU2 --> all mutated cells should be capable of growing without Leucine now. Confirm replacement by comparing to a COntrol (WT yeast. If perform SDS-Page, look for presence of Ste2 in control and absence in mutant?
What 3 'roles' can Yeast occupy (Hint: think relationship to its environment/host)
1. Symbiotic - More so mutualistic in this case. Exist w/ another organism. Both organisms tend to benefit 2. Pathogenic - One organism is harmed (host), while the other benefits. 3. Saprotrophic - Feed on 'dead' material --> facilitate decay
When was Saccharomyces cerevisiae's genome sequence completed?
1996
TAP tag
= Tandem Affinity Purification A type of chromatography; we've engineered and modified a select protein to bind to a specific column (a TAP tag) - it has two affinity tags --> allows us to perform very clean purifications. Have Protein A and Calmodulin Binding Protein (CBP) w/ a TEV cleavage site (specific protease site that yeast do not possess), so yeast proteases won't cleave this in the absence of this signal.
G-Protein Coupled Receptors
A 7 transmembrane domain, single polypeptide that has a series of cytoplasmic loops. Is clustered in the membrane. When the receptor binds to a ligand, it will alter the way these helices interact with each other --> change in cytoplasmic loops --> activation of heterotrimeric G-protein (consists of α, β, λ subunits). When the GPCR is activated, will break into α + β/λ --> elicit other biochemical reactions. In this case, its the β/λ subunit that will activated mating - Are popular drug targets (40% of pharmaceuticals!) - Present in ALL eukaryotes (sensory, dz processes, etc.)
Cdc
A CDK = "cell division cycle" Tells us that mutants involved in the following experiment arre involved in the cell cycle. - Doesn't actuall specify the specific proteins involved that elicit the mutaiton though!
Proteome chips/microarrays
A high-throughput method used to track the interactions and activities of proteins, and to determine their function, and determining function on a large scale. Its main advantage lies in the fact that large numbers of proteins can be tracked in parallel. The chip consists of a support surface such as a glass slide, nitrocellulose membrane, bead, or microtitre plate, to which an array of capture proteins is bound. Probe molecules, typically labeled with a fluorescent dye, are added to the array. Any reaction between the probe and the immobilized protein emits a fluorescent signal that is read by a laser scanner. Protein microarrays are rapid, automated, economical, and highly sensitive, consuming small quantities of samples and reagents.
What is an 'orphan' receptor?
A receptor that has been isolated and sequenced, but the ligand to which it binds (what activates it) is unknown.
Saccharomyces cerevisiae
A saprotrophic yeast! A model organism of great scientific interest.
Polymerase Chain Reaction (PCR)
A technique for amplifying specific DNA in vitro by incubating with special primers, DNA polymerase molecules, and nucleotides. Most PCR methods rely on thermal cycling --> Exposes reactants to repeated cycles of heating and cooling to permit different temperature-dependent reactions: a) DNA melting b) Enzyme-driven DNA replication. Employs 2 main reagents: 1) Primers (short, single strand DNA fragments known as oligonucleotides that are a complementary seq to target DNA region) 2) DNA polymerase. 1st Step: Two strands of DNA double helix are physically separated at a high temperature in a process called DNA melting. 2nd Step: the temp is lowered & primers bind to the complementary sequences of DNA. - The 2 DNA strands then become templates for DNA polymerase to enzymatically assemble a new DNA strand from free nucleotides, the building blocks of DNA. - As PCR progresses, the DNA generated is itself used as a template for replication, --> a chain reaction in which original DNA template is exponentially amplified.
Ste12
A transcription factor that activates the transcription & translation of msg5
Homologous recombination
A type of genetic recombination in which nucleotide sequences are exchanged between two similar or identical molecules of DNA After a double-strand break occurs, sections of DNA around the 5' ends of the break are cut away in a process called resection. In the strand invasion step that follows, an overhanging 3' end of the broken DNA molecule then "invades" a similar or identical DNA molecule that is not broken. Homologous recombination that occurs during DNA repair tends to result in non-crossover products, in effect restoring the damaged DNA molecule as it existed before the double-strand break.
auxotrophic markers
A wild-type allele of a gene that encodes a key enzyme for the production of an essential monomer used in biosynthesis There aren't a lot of good antibiotic resistance genes to use. Therefore, instead will use auxotrophic markers (i.e. problem in a catabolic pathway --> a specific metabolite must be provided for in the media (e.g. leucine) in order for growth to occur. Laboratory strains willl have a collection of metabolic genes disrupted for study Allows for the use of plasmid DNA's w/ these auxotrophic markers. These plasmids will thus restore the disrupted metabolic pathway
How are genes or Open Reading Frames of yeast named?
According to genomic location Y = Yeast W = Watson strand
What is another situation (think a virus/disease) in which the TAP tag methodology can be applied to other systems?
Added TAP tag to the different HIV proteins. Once they infected cells, scientists were able to establish what proteins would interact w/ the diff HIV proteins.
What is an example of a GPCR commonly employed in animals (Hint: think survival)?
Adrenaline (epinephrine) receptor Or Adrenergic receptor. --> Fight or flight response
What do: Gain of function, Overexpression, & Inducible (over)expression experiments allow you to do?
Allows you to overproduce a specific gene product & regulate when it is produced
Why do yeast cells involved in mating have to be arrested in the G1 phase?
B/c in order for each cell to mate, they must be in the SAME stage & can't be undergoing growth or replication
S. cerevisiae - Bakers & brewers yeasts
Break down glucose (via fermentation) to produce carbon dioxide & ethanol CO2 --> gives the rise to breads Ethanol --> beer and wine! e.g. Marmite
How can you study 'aging' in yeast?
Can study aging, b/c every time a bud breaks off of the mother cell, it leaves a scar as well as specific proteins - Will usually undergo 18-20 budding events before senescence (death)
What did the scientists do to study the prediction that there'd be two mutant phenotypes and two WT haploids as a result of tetrad dissection?
Compared a plate with WT spores (control) to a plating with the tetrad spores on media with Abx Gentamycin - Also created a way in which to select for those spores w/ altered genes
What are the inhibitors of transcription factor Ste12?
Dig1 and Dig2
Finding & Implications for paper: Brewer's yeasts mate inside the guts of hibernating wasps
Finding: Researchers have found that diff strains of S. cerevisiae mingle & mate like crazy inside the guts of hibernating wasps. Implication: The findings suggest that wasps might help to foster yeast biodiversity, w/ important implications for ecology & industry
What two types of experiments are the major drivers in genetic analysis?
Gain of function Loss of function experiments
Example of an inducible expression experiment
Galactose inducible expression Add Gal promoter in front of a gene of interest (e.g. YFG1) --> expression inhibited in presence of glucose --> expression low in presence of raffinose --> expression high in presence of galactose Therefore, also allows for a way in which we can rapidly shut off transcription of a gene (e.g. by adding glucose)
Yeast nomenclature
Gene: YGF1 = normal WT allele yfg1 = mutant, non-functional allele yfg1-3 = a specific mutant non-fn'l allele (i.e. the 3rd mutant) ∆yfg1 = complete deletion or 'knock-out' yfg1::NAT = NAT resistance gene replacing YFG1 pGAL::YFG1 = gal promoter controlling YFG1 expression Protein: Yfg1, Yfg1p, or YFG1
What are the benefits/pros to using yeast as a model organism
Grow a lot more slowly than bacteria, but don't require extensive resources, media, serum, etc. Less expensive. Have smaller, less-complex genomes compared to humans. - Simple & fast propagation; cheap & safe (1x10^7 cells per mL) - Can have dispersed cells in a suspension; or can be plated - Haploid or diploid forms (Can vary the nutritional qualities of the media to make yeast either haploid or diploid. Diploids good for creating KO or introducing an edit (foreign DNA) - Easy to transform (intro foreign DNA. e.g. via plasmid) - Extremely efficient homologous recombination (if DNA is intro'd that has homologous NT's, it can be incorp'd into yeast genome) - Many reagent resources available (for PCR?)
Saccharomyces cerevisiae size & shape?
Haploid cell: Volume = 70µm³ Diameter = 4µm Diploid cell: Volume = 120µm³ Diameter = 5-6µm - Its larger size (compared to bacteria) allow us to better visualize budding events
Colocalization (in terms of determining where each protein is localized in a cell)
In fluorescence microscopy, colocalization refers to observation of the spatial overlap between two (or more) different fluorescent labels, each having a separate emission wavelength, to see if the different "targets" are located in the same area of the cell or very near to one another. The definition can be split into two different phenomena: co-occurrence, which refers to the presence of two (possibly unrelated) fluorophores in the same pixel, and correlation, a much more significant statistical relationship between the fluorophores indicative of a biological interaction. This technique is important to many cell biological and physiological studies during the demonstration of a relationship between pairs of bio-molecules. Colocalization is used in real-time single-molecule fluorescence microscopy to detect interactions between fluorescently labeled molecular species. In this case, one species (e.g. a DNA molecule) is typically immobilized on the imaging surface, and the other species (e.g. a DNA-binding protein) is supplied to the solution. The two species are labeled with dyes of spectrally resolved. The molecules are detected as spots appearing on the surface in real-time, and their locations are found to within 10-20 nm by fitting of point-spread functions. Since typical sizes of biomolecules are on the order of 10 nm, this precision is usually sufficient for calling of molecular interactions
SGA Summary
Involve Synthetic Lethal/Sick interactions Shows evidence that two genes act on independent but complementary pathways Can compare/contrast to physical interactions
Is it easier or harder to conduct a Loss-of-function experiment?
It often proves to be more problematic. But is relatively straightforward in yeast
What does Msg5 do in terms of Fus3?
It's a protein phosphatase that can inactivate Fus3
Experiment involving Tetrad Dissection and yeast mutant
Mutant possessed a gene segment encoding for gentamycin resistance.
What coordinates mating in yeast?
Pheromones (e.g. α-factor, a-factor Mediated by receptors of group Ste (so called b/c if yeast lack these genes, they're unable to conjugate)
Process of Tandem Affinity Purification (TAP)
Protein of interest fused to TEV site, CBP. Then perform pull-downs (you have your giant bead and you attach your protein of interest - often through an affinity tag. The beads are large and heavy --> can spin them out. Can be used as a method of purification. You can also add in IgG, which will bind all over the surface of Protein A (produced by pathogenic S. aureus). If we then spin the beads out, we will have all of our select protein. Then cleave this off w/ TEV --> CBP to our protein 1 and this will bind only in the presence of calcium to its binding partner. Thus, perform a second purification step CBP will bind to calmodulin on the beads (in the presence of Calcium. Why do we do two pull-downs? In our tube, we have this construct with mutliple proteins still in it. We want to see what our "bait" protein binds to... So if two proteins interact, they become attached. E.g. protein 4 sticks to the 'bait', they come down together (are pulled down). Key to the tandem affinity tag is that you do the first pull-down with, say, protein 1 to the protein A. Then cleave this off with TEV = CBD with protein 1 and this will bind only in the presence of Ca2+ to its binding partner. 2nd purification step: CBD will bind the Calmodulin to perform a second purification (in the presence of Ca2+. At the end of the pull-down, you drain off all the liquid, add SDS-PAGE loading buffer, which will release the proteins from the beads and completely denatures the proteins Then run these proteins on a gel (SDS-PAGE), then use Mass Spec (bait is the control) to establish these networks of interaction. It's pretty quick and easy. And the end product (interaction map) is "clean". Keep in mind that proteins may come off after binding (may have a limited time). Can be used to establish e.g. yeast protein interactions. So it's semi-quantitative (i.e. you can see the RELATIVE amounts of proteins).
Another method of labelling a protein or nucleic acid besides fluorescent tag?
Radio label using ³²P
What is the advantage of sexual reproduction?
Results in a mixing of genes --> genetic variability
Why are yeast useful in terms of Mendelian genetics?
S. cerevisiae make it easy & helpful to directly observe Mendelian allele segregation
Why are the kinases involved in a MAPK cascade physically associated on a scaffolding in the cell?
Scaffold prevents cross-talk between other MAPK pathways i.e. there are many other MAPK pathways taking place at the same time w/in a cell
Final step in SGA experiment
Second to last array of wells is an important first control, as you'd expect the first (half-KO yeast cell) to still growth Last array: Compare to above wells - looks to see where there's no/slow growth.
Genetic interactions (image)
Several genes affect a single trait.
MAPK
Signal transduction/cascade typically goes: GPCR --> activation of heterotrimeric G protein --> phosphorylation cascade (amplification) --> activation of target proteins or activation of transcription for select products
Whats a transcription factor activated by the yeast mating pathway?
Ste12
Procedure for SGA's
Synthetic genetic array analysis is generally conducted using colony arrays on petriplates at standard densities (96, 384, 768, 1536). To perform a SGA analysis in S. cerevisae, the query gene deletion is crossed systematically with a deletion mutant array (DMA) containing every viable knockout ORF of the yeast genome (currently 4786 strains).[9] The resulting diploids are then sporulated by transferring to a media containing reduced nitrogen. The haploid progeny are then put through a series of selection platings and incubations to select for double mutants. The double mutants are screened for SSL interactions visually or using imaging software by assessing the size of the resulting colonies
Why perform two pull-downs with Tandem Affinity Purification?
Tandem = involves two tag processes It makes it cleaner; with pull-downs, sometimes you get non-specific interactions. With two pull-downs, you can be pretty confident that what sticks/associates with your bait interacts directly (or indirectly). e.g. Bait bind directly to grey and pink ones, and is bound indirectly to yellow, blue and green proteins. Con: You can't guarantee that the TAP tag doesn't interfere w/ protein fusion
Interactome
The whole set of molecular interaction in a particular cell. Specifically refers to physical interactions among molecules (such as those among proteins, aka protein-protein interactions/PPIs; or btwn small molecules & proteins), but can also describe sets of indirect interactions among genes (genetic interactions). The interactomes based on PPIs should be associated to the proteome of the corresponding spp in order to provide a global view ("omic") of all the possible molecular interactions that a protein can present
What is an example of a Counter Selectable Marker?
URA3 in the presence of 5-FOA URA3 converts 5-FOA into the toxic compound 5-fluorouracil Therefore, Ura+ cells are kiled by presence of 5-FOA Whereas, Ura- cells are resistance to this compound
4. Find the mutations, and therefore the genes involved (i.e. find the gene in which the mutation has occurred)
Use Molecular (direct) Complementation - You now have minimal # of mutants in the complementation groups... - Find a DNA fragment that rescues each mutant's LOF phenotype --> Will need a library of as many genes as possible
3. How many genes are involved in the process?
Use method of Genetic Complementation (test for determining whether two mutations associated with a specific phenotype represent two different forms of the same gene (alleles) or are variations of two different genes.) - There may be 100s of mutants, so you need to reduce the number to further characterize mutation - Cross each mutant with each other, isolate the diploids, then score the phenotype (see if it grows on galactose) - If you don't get growth, it means that the two mutants that are crossed have the SAME mutation. - Say you combine mut1 with mut2 and it actually grows... This shows that they have mutations at different regions/genes!
How do prevent diploid contamination (after inefficient sporulation) when isolating double mutants? (3)
Use recessive selection drugs: 1) Canavanine: toxic arginine analogue --> will kill yeast with a functional CAN1 transporter (can1 yeast are resistant) 2) Thialysine: toxic lysine analogue --> will kill yeast with a functional LYP1 transporter (lyp1 yeast are resistant) 3) MFA1pro-HIS3 - only MATa cells grow on media lacking histidine
drug resistance markers
Used to 'mark' and select for genetic manipulations e.g. Gene G418 - confers resistance to genetecin (G418) Gene NAT - confers resistance to nourseothricin (cloNAT)
IF YFG1 is an essential gene, how can you study alleles or mutants of this gene?
Via Plasmid shuffling: If you have an essential gene, you can do a TEMPORARY KO. a) Intro YFG on a plasmid with URA3 (converts to toxic product); Can then create a KO of YFG (yfg1:NAT)no b/c will still survive due to plasmid w/ YFG1 b) Introduce plasmid with mutated YFG1* with HIS3 auxotrophic marker --> makes yeast diploid. c) Also make another strain w/ a homozygous diploid WT form of YFG d) Remove selection fo rthe WT plasmid (i.e. grow with uracil) --> URA3 plasmid is lost b/c uracil is provided. e) Plate the resulting cells w/ 5-FOA --> will extinguish any cells that sitll have a fn'l URA3
How do we understand/ID Where a protien is localized?
Via high throughput microscopy... 1) Use localization 2) Then colocalization w/ 'RFP-tagged' reference proteins that rep 22 distinct categories of localization ( to determine the localization of all of these ORF's)
SGA study performed by Boone et al, in Nature Reviews
WT alleles are drawn as open circles and the Deletion mutations are drawn as closed circles. SO we have these sets that are grown (every single one of these has one gene KO'd; i.e. every single spot is a haploid w/ one gene knocked out) 4,874 spots! Each one of these psots will have a DIFFERENT deletion. We are interested in mfg, so that's our query - what's my gene involved in? What role dose it play? So, we take this gene of interest & knock it out in a haploid, then cross them, to make double KO's. IF you have a double KO, and one gene requires interaction with another, you would expect no proliferation. At what point or does it even, does your gene - when it's added - is it going to cause a lack of proliferation or cell death? So you're essentially making synthetic genetic mutations.
1. Create mutations at random; screen for phenotype of interest (usually an LOF in specific conditions) - Example
We are looking for the ability of cells to grow on galactose. - When plating with glucose (non-selective media), everything should grow. - When we grow on galactose, those lacking capability to metabolize galactose will die (becomes a selective media)
When are temperature-sensitive mutants actually necessary for genetic experiments?
When the experiment involves an essential gene - i.e. you can't KO an essential gene, so instead you create a temperature-sensitive mutant.
What are examples of Wild, commercial and laboratory forms of Saccharomyces cerevisiae?term-4
Wild: On wheat, the dustiness on grapes Commercial: Food production (e.g. fermentation of food preservation and production of beverages/breads); Biopharmaceuticals Laboratory: Studies on cell cycle, etc.
Coding region of S. cerevisiae vs. human's and the implications?
With S. cerevisiae, most of the genetic material is coded --> genes that will actually be transcribed into proteins, for example. - Regulation is highly simplified (e.g. single promoter); only 1 intron --> not a good model for alternative splicing. Whereas, humans have ~5 introns. With humans, A very small amount of our genetic material is actually transcribed. But these other regions are responsible for regulation (very important) --> are believed to give rise to the complexity of our genome/organism
Example in which GST-collection enabled protein purification & assays in high throughput: Global ID of protein kinase substrates
With this study, they've taken every ORF and have modified each an every one with an affinity tag called GST, which will bind to Glutathione. Each one is in an individual well. Can therefore, do high-throughput... in which you harvest the cells, isolate the proteins based on their interaction w/ glutathione (small-scale protein purification). You can then take those proteins and you use a microarray printer... The only proteins that we end up with are the GST-bound proteins... Now we have this plate that has a "pure" form of every protein in the yeast genome. Print these in duplicates (two dots) on specialized glass slide. Also did anti-GST to see where each of the dots are. You also have to leave certain gaps as reference points to know where you are. In this study, they isolated a kinase in which they wanted to know what its target protein/cellular target was. Therefore took this slide, incubated it with the kinase, radiolabelled p32, then developed it on an autorad --> ID proteins that were phosphor'd by that kinase (then referred to reference which indicated where each protein was). Limitations: How do you ensure that the proteins on the plate are correctly folded (or folded at all), the orientation of the proteins, etc. Possible uses: Look for protein-protein interactions, phospholipid interactions, Ab specificity, etc.
Mutant hunts for non-essential processes - Example?
Would like to find all genes that are needed for growth on Galactose
What makes yeast an excellent model organism for studying cell cycle?
You can actually determine where a yeast is in the cell cycle based on budding size!
DNA microarray
a collection of microscopic DNA spots attached to a solid surface. Scientists use DNA microarrays to measure the expression levels of large numbers of genes simultaneously or to genotype multiple regions of a genome. Each DNA spot contains picomoles (10−12 moles) of a specific DNA sequence, known as probes (or reporters or oligos). These can be a short section of a gene or other DNA element that are used to hybridize a cDNA or cRNA (also called anti-sense RNA) sample (called target) under high-stringency conditions. Probe-target hybridization is usually detected and quantified by detection of fluorophore-, silver-, or chemiluminescence-labeled targets to determine relative abundance of nucleic acid sequences in the target.
Synthetic Genetic Array
a high-throughput technique for exploring synthetic lethal and synthetic sick genetic interactions (SSL).[1] SGA allows for the systematic construction of double mutants using a combination of recombinant genetic techniques, mating and selection steps. Using SGA methodology a query gene deletion mutant can be crossed to an entire genome deletion set to identify any SSL interactions, yielding functional information of the query gene and the genes it interacts with. The results of this study showed that genes with similar function tend to interact with one another and genes with similar patterns of genetic interactions often encode products that tend to work in the same pathway or complex
What was Lee Hartwells genetic approach to answer: How many genetic factors? How to ID them?
a) CHemically intro mutations into the genome at random b) Isolate conditional mutants (i.e. are temperature sensitive) c) Collect the temp-sensitive mutants, grow at reg 23 deg Celsius d) Then Shift to 36 deg Celsius (non-permissable temp) --> see what's happening to the yeast cells microscopically. Findings: FOund that for certain mutated cells (e.g. cdc 15), all the cells were 'ocked' at the SAME position in the cell cycle - i.e. they have synchronous cessation If this was a mutation involving metabolism, we wouldn't see this synchronous culture - i.e. they'd be dying at many different points at random b/c they are 'sick'
Describe mating in yeast
a- or α-factor binds to a Ste receptor (a to Ste3 on an α cell, and vice versa) --> Shmoo formation --> Membrane fusion --> Nuclear migration & fusion (aka Karyogamete)
Transcriptome
all the mRNA molecules transcribed from a genome - A comprehensive study of the mRNA's
Pulldown assay
an in vitro technique used to detect physical interactions between two or more proteins and an invaluable tool for confirming a predicted protein-protein interaction or identifying novel interacting partners. --> purification of protein complexes! Uses a purified & tagged protein as a "bait" to bind any interacting proteins. The method consists of first immobilizing the tagged protein (bait) on an affinity ligand specific to the tag, creating an affinity support to capture & purify other proteins (prey) that interact with the bait. Once the prey proteins have been incubated with an immobilized bait protein, interacting complexes are eluted using an eluting buffer depending on the affinity ligand. Each experiment needs proper controls to demonstrate that characterized interactions are not an artifact. A positive control: Consists of an immobilized bait protein alone - is necessary to verify proper attachment of tagged bait protein to the affinity support. To ID and eliminate false positives caused by nonspecific binding of prey proteins to the affinity support, cell lysates or purified proteins can be analyzed after being passed through a minus bait support. Following a pull-down experiment, protein fractions are resolved by SDS-PAGE & then visualized by gel staining or western-blotting detection.
CDK's
cyclin dependent kinases that bind to & activate cyclins (ALWAYS PRESENT) --> i.e regulate the cell cycle
How to make a Knock-Out
e.g. Total replacement of Gene X with natMX (Abx resistance) Utilize homologous recombination: Design primers (complementary to 5' and 3' ends) & carry out PCR for natMX on a plasmid. Additionally on those primers, we have ~45 NT of homology upstream and downstream of the gene of interest (Gene X) Can then select for these homologous recombination events by plating on the Abx to which this mutated cell is now resistant
Counter Selectable Markers
i.e. If the cell has a particular, functioning gene, it will kill the cell. - The opposite would be forward selection markers (yeast cell growth requires expression of the gene. e.g. HIS3) - Can be used to select agains (i.e. kill) a cell that expresses a counter selectable marker gene
Why is it that the lichen Bryoria tortuosa is toxic, while B. fremontii is edible?
lichen = algal cell + fungi growing together as a single, functioning organism - Found that the algae and fungi were identical... BUT: B. tortuosa had a third organism (a yeast) that was producing the toxin.
