Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.
David M. Bedwell, Ph.D.
Department of Microbiology
E-mail: [email protected]
Learning Objective: Understand the features of the unicellular eukaryote Saccharomyces cerevisiae (baker’s yeast) that make it an ideal model genetic system to study how many highly conserved eukaryotic cellular processes are carried out.
Haploid cellDiploid cell
Volume (mm3) 70 120
Diameter (mm) 4 5-6
Mating Type (MAT) Locus
Transposition mediated by HO recombinase
Mating Type Switching in Yeast
The nuclear genome in a diploid yeast cell represents only ~85% of the total genetic material. Other genetic elements include self-replicating plasmids, mitochondrial DNA, and dsRNA genomes.
Each of the yeast DNA sequences identified in this way was called an autonomously replicating sequence (ARS).
The presence of ARS, CEN, and TEL elements allow the stable maintenance of extremely large linear DNA fragments in yeast. Such constructs are called Yeast Artificial Chromosomes (YACs). DNA fragments as large as 1 million base pairs can be stably maintained in this manner.
Maximum Capacity of Vectors
Vector TypeLength of DNA (kb)
P1 vector 100
Construction of YACs in vitro
In vivo Construction of YACs
(as of the year 2000)
Genome-Wide Approaches to the Analysis of Yeast Protein Function (cont.)
Genome-wide GFP fusions and epitope tags Function (cont.)
Transposon mutagenesis approach for genome-wide GFP fusions (to monitor protein localization) and epitope tags (to detect proteins immunologically)
Transposon hopping carried out into yeast genomic library in E. coli
GFP = Green fluorescent protein
Genome-wide Function (cont.)LacZ fusions and epitope tags
Transposon mutagenesis approach for genome-wide LacZ fusions (to monitor gene expression) and epitope tags (to detect proteins)
Use similar approach as shown in previous slide to insert transposons randomly into every gene in the yeast genome.
Competitive growth assay for phenotypic analysis Function (cont.)
using the yeast deletion collection
Large pools of heterozygous or homozygous yeast deletion strains are grown in the presence or absence of a drug (pink squares). In the collection, each gene deletion (red and blue) is flanked by two sequences that contain unique barcodes (up-tag and down-tag). After DNA extraction, the barcodes are amplified using primers from conserved sequences in the tags and hybridized to barcode microarrays. Relative abundance of each bar-coded PCR product is compared between arrays from treated or untreated samples. Absence of a hybridization signal in the treated sample reveals sensitivity of the corresponding deletion strain (red deletion) to the drug. Note that this scheme depicts the hybridization of treated and untreated samples to different arrays.
Haploinsufficiency occurs when a diploid organism only has a single functional copy of a gene (with the other copy inactivated by mutation) and the single functional copy of the gene does not produce enough of a gene product (typically a protein) to bring about a wild-type condition, leading to an abnormal or diseased state.
Drug-induced haploinsufficiency occurs when lowering the dosage of a gene encoding a potential drug target from two copies to one copy confers hypersensitivity to the drug.
(A) A copy number reduction of a drug target gene (red circles) from two to one limits the amount of a potential drug target and confers hypersensitivity to the drug (pink squares), whereas the drug has no effect in the presence of two functional gene copies.
(B) Haploinsufficiency cannot be observed when the potential drug target is in excess even when one gene copy is deleted.
(a) A bifurcated cellular pathway will have distinct networks on genetic- and physical interaction maps. Proteins A, B, C and D (blue) and proteins 1, 2, 3, and 4 (green) are members of two functionally redundant pathways required to perform an essential function. Proteins A, B and C interact with each other physically, so do proteins 1, 2 and 3.
(b) A protein interaction map, or physical interaction map, identifies interactors based on protein-protein interactions.
(c) A genetic interaction map identifies interactors based on functions without the requirement that the proteins must interact. The combination of these two complementary approaches can be used to deduce a cellular pathway and, in principle, enable the construction of a wiring diagram of the yeast cell.
One way to reveal the functions of the remaining nonessential genes and to identify essential processes is to perform a systematic genome-wide synthetic-lethality analysis.
Synthetic lethality describes any combination of two separately non-lethal mutations that leads to inviability, whereas synthetic sickness indicates a combination of two separate non-lethal mutations that confers a growth defect more severe than that of either single mutation.
The interpretation is that synthetic sickness reflects an important genetic interaction, whereas synthetic lethality reflects an essential interaction.
A haploid strain containing the query mutation (red) is combined with the arrayed deletion collection of the opposite mating type (blue). After mating, diploids are selected and sporulated to yield the haploid progeny. For the crucial selection step against diploids, the query strain contains a special construct that allows for the selection of haploids from one mating type.
In the case of a synthetic sick or lethal interaction, the double mutant is compromised or cannot be recovered.
(i) A MAT strain carrying a query mutation (bni1) linked to a dominant selectable marker, such as the nourseothricin-resistance marker natMX that confers resistance to the antibiotic nourseothricin, and an MFA1promoter-HIS3 reporter is crossed to an ordered array of MATa viable yeast deletion mutants, each carrying a gene deletion mutation linked to a kanamycin-resistance marker (kanMX). Growth of resultant heterozygous diploids is selected for on medium containing nourseothricin and kanamycin.
(ii) The heterozygous diploids are transferred to medium with reduced levels of carbon and nitrogen to induce sporulation and the formation of haploid meiotic spore progeny.
(iii) Spores are transferred to synthetic medium lacking histidine, which allows for selective germination of MATa meiotic progeny because these cells express the MFA1pr-HIS3 reporter specifically.
(iv) The MATa meiotic progeny are transferred to medium that contains both nourseothricin and kanamycin, which then selects for growth of double-mutant meiotic progeny.
(A) bni1::natR cells were crossed to a test array containing 96 deletion mutants, each arrayed in quadruplicate in a square pattern. bnr1 was duplicated within the array. The final array that selects for growth of the bni1 double mutants is shown. Synthetic lethal/sick interactions lead to the formation of residual colonies (yellow circles) that were relatively smaller than the equivalent colony on the wild-type control plate. Synthetic lethal/sick interactions were scored with bnr1, cla4, and bud6. When the query mutation was identical to one of the gene deletions within the array, double mutants could not form because haploids carry a single copy of each allele; therefore, bni1 appeared synthetic lethal with itself.
(B) Tetrad analysis of meiotic progeny derived from diploid cells heterozygous for bni1 and either bnr1, cla4, or bud6. Tetratypes (T) contain one double-mutant spore; nonparental ditypes (NPD) contain two double-mutant spores; and parental ditypes (PD) lack double-mutant spores. The spores were micromanipulated onto distinct positions on the surface of agar medium and then allowed to germinate to form a colony. bni1 bnr1 and bni1 cla4 double mutants are inviable and therefore fail to form a colony, whereas bni1 bud6 double mutants showed a synthetic slow growth (sick) phenotype (yellow arrows). The genetic make-up of the double mutants was inferred by replica plating the colonies to medium containing nourseothricin, which selects for growth of bni1::natR cells, and kanamycin, which selects for growth of the bnr1, cla4, and bud6 gene-deletion mutants.
Genetic interaction network representing the synthetic lethal/sick interactions determined by SGA analysis. Genes are represented as nodes, and interactions are represented as edges that connect the nodes; 291 interactions and 204 genes are shown [Science 294: 2364 (2001)].
(A) To construct abait in the yeast two-hybrid system, a protein of interest X is fused to theDNA binding domain (DBD) of a transcription factor. When expressed on its own in yeast, the bait will not activate transcription since it lacks a transcriptional activation domain (AD).
(B) Likewise, a prey is constructed by fusing a second protein of interest Y to the AD of a transcription factor. The AD-Y fusion is unable to activate transcription on its own, since it is not situated near a promoter.
(C) Co-expression of the interacting DBD-X and AD-Y fusion proteins reconstitutes a functional transcription factor situated at a promoter. Consequently, the reporter gene located downstream of the reporter is activated, and the protein-protein interaction between the proteins X and Y is measured using the product of the reporter gene. Common reporter genes in yeast two-hybrid systems include auxotrophic growth markers, such as the HIS3 or ADE2 genes, or a color marker, such as lacZ.
~6000 activation domain hybrids in MAT (SGA)a strain arrayed in microtiter format (16 plates)
Replica to lawns in MAT strain of DNA-binding domain hybrid 1 and plate diploids on selective media.
Score positives by position and repeat for additional 5999 DNA-binding domain hybrids.
Genome-Wide Screen for Protein-Protein Interactions using Yeast 2-hybrid Assay
(A) A scaffold bait is constructed by fusing a DNA binding domain (DBD) to a small molecule binding protein such as dihyrofolate reductase (DHFR). Simultaneously, each yeast cell expresses a particular activation domain (AD)-fused prey from a cDNA library.
(B) A hybrid compound consisting of a small molecule covalently linked to methotrexate is added, which crosses the yeast cell membrane and binds to the DBD-DHFR bait via its methotrexate part. In this way, the other part of the small molecule is displayed by the scaffold bait.
(C) If the AD-prey binds to the small molecule displayed from the scaffold bait, a functional transcription factor is reconstituted via the small molecule-protein interaction, resulting in activation of the downstream reporter gene.
(A) An interacting protein pair is expressed as the fusion of DBD-X [a protein of interest X fused to the DNA binding domain (DBD)] and AD-Y [a protein of interest Y fused to the activation domain (AD)]. The reconstitution of an artificial transcription factor in the yeast two-hybrid system activates the downstream reporter gene, which converts a compound added to the medium into a toxic end product, resulting in yeast cell death.
(B) Addition of a compound, which interferes with the protein-protein interaction, prevents reconstitution of the hybrid transcription factor. Consequently, the reporter gene is not activated, and the yeast grows on selective medium.
5-FOA Selection for Ura3 interaction- cells
Cdc25-2 encodes a temperature-sensitive form of a membrane bound guanine nucleotide exchange factor (GEF or GDP-release factor). It normally indirectly regulates adenylate cyclase through the activation of Ras1p and Ras2p by stimulating the exchange of GDP for GTP. The CDC25 gene product is required for progression through the G1 phase of the cell cycle. The membrane localization of a constitutively activated ras protein (mRas) complements the mutation.
(A) An integral membrane protein or a membrane-associated protein is expressed as a bait in a yeast strain carrying a mutation in the cdc25 gene. The cdc25-2 mutation prevents growth of the yeast strain at the non-permissive temperature of 36°C.
(B) Co-expression of an interacting protein fused to a constitutively active ras mutant (mRas) targets the mRas-Y fusion to the membrane, where it acts to complement the cdc25-2 mutation, leading to yeast growth at 36°C.
The membrane yeast two-hybrid (MbYTH) system interaction
(A) A bait is constructed by expressing an integral membrane protein or a membrane-associated protein X as a fusion to the C-terminal half of ubiquitin (C), followed by a transcription factor (L). Fusion of the transcription factor to the integral membrane protein prevents its translocation to the nucleus and activation of the reporter genes. A prey is constructed by expressing an interacting protein Y as a fusion to the N-terminal half of ubiquitin (N).
(B) If bait and prey interact, the N- and C-terminal halves of ubiquitin are forced into very close proximity and reassociate to form split-ubiquitin, which is recognized and cleaved by ubiquitin-specific proteases (UBPs; also known as de-ubiquitinating enzymes, or DUBs) in the cytosol. The cleavage liberates the transcription factor from the membrane, followed by its translocation to the nucleus and activation of reporter genes.
The TAPtagconsists of a protein A domain and a calmodulin binding protein (CBP) that are separated by a recognition sequence for the tobacco etch virus (TEV) protease. The TAP sequence is integrated in-frame at the C terminus (or N terminus) of a gene.
Protein extracts are made from large cultures of cells expressing the TAP-tagged proteins. In a first purification step, the fusion proteins bind to immunoglobulin G (IgG) beads by their protein A domain. Cleavage by the TEV protease then releases the fusion proteins and the associated proteins or protein complexes from the IgG beads.
In a second purification step, the fusion proteins bind to calmodulin-coated beads by their CBP domain in the presence of Ca2+. After additional washing, the tagged proteins are eluted by the addition of the chelator EGTA that binds to Ca2+. Purified protein complexes are then separated by denaturing gel electrophoresis.
Distinct protein bands are excised and digested into small peptides with trypsin. The peptides are then identified by mass spectrometry, and the identity of the proteins is determined by database searches.
Part III: interactionFinal Points
(if time allows)
Study of Non-Mendelian Inherited Determinants interaction
Isolating temperature-sensitive (ts) mutants allows essential genes to be studied
A beautiful example of the power of yeast genetic analysis: Understanding the Secretory Pathway
Screens for synthetic genetic and chemical genetic interactions
(B) The principle of synthetic lethality. Inactivation (deletion) of two genes (red and blue) in redundant pathways leads to loss of viability, whereas inactivation of either one gene has no effect.
(C) Drug-induced inhibition of growth in haploid or homozygous diploid deletion strains (chemical-genetic interaction). A protein product of a gene (red circles) is inactivated by treatment with a drug (pink squares). The protein product of a second gene (blue circles) is by itself not essential but prevents loss of viability in the presence of the drug. Deletion of the second gene leads therefore to hypersensitivity to a dose of the drug that that is not lethal in a wild-type cell.