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Genetic Model Systems : Yeast. David M. Bedwell, Ph.D. Department of Microbiology BBRB 432 Phone: 934-6593 E-mail: firstname.lastname@example.org Web: www.microbio.uab.edu/Bedwell/.
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Genetic Model Systems: Yeast David M. Bedwell, Ph.D. Department of Microbiology BBRB 432 Phone: 934-6593 E-mail: email@example.com Web: www.microbio.uab.edu/Bedwell/ 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.
References • An Introduction to the Genetics and Molecular Biology of the Yeast Saccharomyces cerevisiae, byFred Sherman. http://dbb.urmc.rochester.edu/labs/sherman_f/yeast/index.html • Saccharomyces Genome Database (SGD): http://www.yeastgenome.org/ • Overview of diverse genome-wide technologies: Yeast-based functional genomics and proteomics technologies: the first 15 years and beyond. B. Suter, D. Auerbach and I. Stagler, Biotechniques 40: 625-642 (2006).
Part I: Nuts and bolts of yeast genetics and molecular biology that make it such a powerful genetic system
Size and Shape of Yeast Haploid cellDiploid cell Volume (mm3) 70 120 Diameter (mm) 4 5-6
Why Use the Yeast Genetic System? • Non-pathogenic, so can be handled with few precautions. • Rapid growth rate. • Grow as dispersed cells that facilitate replica plating and mutant isolation. • Highly versatile DNA transformation system. • Can be maintained in stable haploid and diploid states that facilitate genetic analyses. • Small genome size (1.2 x 107 base pairs) is only ~3.5x larger than E. coli. • Very active homologous recombination machinery. • Novel techniques (2-hybrid, YACs) make yeast valuable for studies of many organisms.
Yeast Life Cycle • Haploid yeast cells exist as one of two mating types: MATa or MAT. “Wild” yeast (found in nature) are homothallic, which means they rapidly change mating type during the haploid phase and then mate to form diploids. Thus, only the diploid phase of the life cycle is stable.
Yeast Life Cycle (cont.) • Laboratory yeast strains are heterothallic due to a mutation in the gene encoding HO recombinase. This allows them to be maintained stably as either haploid or diploid cells. The stable haploid state is essential for the utility of yeast as a genetic model system.
HML HMRa (Silent) (Silent) Mating Type (MAT) Locus Cassette Replaced Transposition mediated by HO recombinase a HML HMRa (Silent) (Silent) Mating Type Switching in Yeast • Rapid mating type switching is possible in homothallic “wild type” yeast, leading to rapid mating and diploid cell formation (this is BAD for genetics!). • In heterothallic cells the HO recombinase gene is defective. This makes mating type switching impossible, so cells are locked in one haploid mating type or the other (this is GOOD for genetics!).
DNA Transformation and Recombination • DNA transformation is extremely efficient in yeast (>104 transformants/g DNA possible). • Both linear and circular DNA can be introduced and recombined into the genome. • Plasmids capable of self-replication can also be introduced. • Several different transformation protocols are available, and include: • Transformation of spheroplasts • Transformation of cells treated with lithium salts • Transformation by electroporation • Mutagenesis can be accomplished in cells transformed with synthetic oligonucleotides (needs a positive selection). • Mitochondrial transformation can also be accomplished using high-velocity microprojectile bombardment devices (Biolistic guns) with tungsten microprojectiles coated with the DNA to be introduced.
Yeast Genetic Information 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.
Strategy To Identify Replication Origins in Yeast Cells Each of the yeast DNA sequences identified in this way was called an autonomously replicating sequence (ARS).
Yeast Artificial Chromosomes (YACs) 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) Plasmid 20 Phage 25 Cosmid 45 P1 vector 100 BAC 200 YAC 1000
cloning sites Construction of YACs in vitro • Important features of YAC vectors • Circular form, ori, and Amp gene allows propagation of plasmid in E. coli. • Digestion with BamHI linearizes plasmid w/ terminal telomeres. • Digestion w/ SfiI, NotI, or SmaI allows insertion of 200-800 kb of DNA by 3-way ligation. • Selection w/ TRP1 and URA3 allow positive selection of both arms of linear YAC. • ARS1, CEN4 sequences allow propagation in yeast. • Counterscoring for loss of SUP4-o allows identification of clones with foreign DNA inserted by colony color.
In vivo Construction of YACs • Foreign DNA Insertion into YAC vectors by in vivo recombination • Provide homology to ends of genomic target insert in the internal ends of YAC arms. • Transform two arms (unligated) and insert DNA into yeast. • Selection w/ TRP1 and URA3 allow positive selection of DNA molecules containing both arms of YAC. • Homologous recombination between insert DNA and YAC arms is only way to provide stable maintenance of YAC during replication in yeast. • ARS1, CEN4, and TEL sequences allow long-term propagation in yeast.
The Yeast Genome Project • Yeast genome sequencing project finished in 1996 (complete genome contains 1.2 x 107 base pairs). • 16 chromosomes (ranging in size from 230k base pairs to 2,352k base pairs. • ~6,000 ORFs originally thought to be in the yeast genome (estimates ranged from 5,700 to 6,300); now appears to be ~6,400. • Only ~4% of yeast genes contain introns (when present, usually one small intron close to the start of the coding sequence). • Compact genome organization, with genes representing ~70% of the total sequence. • The yeast genome contains a limited amount of repetitive DNA. 52 complete copies of the TY retrotransposon were found, as well as 264 solo LTRs (or other remnants) that represent the footprints of previous transposition events.
Categories of Yeast Protein Function (as of the year 2000)
Genome-Wide Approaches to the Analysis of Yeast Protein Function • Yeast deletion analysis: • Knock out every gene in the yeast genome. • Examine the phenotypic consequences of each knockout under different growth conditions. • Complete set of knockouts (4800 for haploid set) now available for $1500. • Protein over-expression: • Examine the effects of over-producing each gene in the yeast genome. • Genome wide analysis of gene expression: • Use microarrays (aka gene chips) to examine the transcriptional regulation of each gene in the genome under different growth conditions. • Use -galactosidase fusions to monitor gene expression.
Genome-Wide Approaches to the Analysis of Yeast Protein Function (cont.) • Protein localization: • Use epitope tags and GFP fusions to every gene in the genome to examine the subcellular location of the protein products. • Yeast protein interactions: • Use 2-hybrid analysis and glutathione S-transferase (GST) fusions to determine the interacting partners of every protein in the yeast genome. • Yeast protein function: • Assay for specific enzyme functions in pools of GST-tagged constructs. • Possible to identify the source of an enzyme activity with no more than 84 assays. Start with 64 assays of pooled samples from individual 96 well trays to screen the entire genome, then narrow down the candidates by assaying the 8 rows from the positive trays, followed by the 12 individual wells in the positive row.
Genome-wide GFP fusions and epitope tags 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 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 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.
Drug-induced haploinsufficiency in heterozygous diploid strains 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.
Genetic versus physical interaction maps (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.
Synthetic lethal screening using synthetic genetic arrays (SGA) 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.
Synthetic Genetic Array (SGA) methodology Procedure (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.
Double-mutant array and tetrad analysis for SGA (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.
Results of Synthetic Genetic Array (SGA) 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)].
The yeast two-hybrid system (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.
Uses of the Yeast 2-Hybrid Assay • Test the association of two specific proteins that are believed to interact on the basis of other criteria. • Define domains or amino acids that are critical for the interactions of two proteins that are known to interact. • Screen libraries for proteins that interact with a specific protein.
~6000 activation domain hybrids in MATa 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
Small molecule yeast two-hybrid screening (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.
Screening for compounds that inhibit a protein-protein interaction (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. URA3 +5-FOA URA3 + 5-FOA
5-FOA Selection for Ura3- cells • The URA3 gene is a common selectable marker for plasmids. • The URA3 gene encodes orotidine 5´- phosphate decarboxylase (required for uracil biosynthesis). • 5-fluoroorotic acid (5-FOA) is converted to the toxic compound 5-fluorouracil by the URA3 gene product. • Due to this conversion, Ura+ cells are killed by 5-FOA while Ura- cells are resistant to this compound. Ura+ Dead :-( 5-FOA Alive :-) Ura-
Two-hybrid with membrane proteins- the reverse Ras system 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 (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.
Overview of the tandem affinity purification (TAP-tagging) 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: Final Points (if time allows)
Study of Non-Mendelian Inherited Determinants • The mitochondrial genome • Mitochondrial DNA is ~76k base pairs • Inherited as a cytoplasmic element • Yeast mitochondrial genome contains ~9 genes • Prions • infectious proteins that function as cytoplasmically-inherited transmissible elements. • Yeast forms of prions include the [PSI+] factor, an altered form of the yeast Sup35 (eRF3) protein. • Mammalian examples of prions include Mad Cow Disease, Crutchfield-Jacob disease, and Kuru.
Examples of how studies using yeast can reveal how human cells work • Cell cycle studies by Lee Hartwell resulted in 2001 Nobel Prize in Physiology and Medicine. • Mitotic spindle assembly depends on the completion of DNA synthesis • The concept of START • Identification of cdc28, the first cyclin-dependent kinase • Other crucial concepts critical for cancer, etc. • Dissection of the secretory pathway by Randy Shekman • Identified dozens of complementation groups involved in protein transport throughout the secretory pathway • Many other studies of conserved processes such as DNA synthesis, transcription, translation, cell signaling, etc., etc.
Examples of things you can’t study using yeast- if its not conserved, forget it! • MicroRNAs are not present in yeast (along with the requisite machinery such as the RISC complex), so you can’t study microRNA function or use silencing RNAs (siRNAs). • Yeast don’t use complex developmental programs to make complex structures like organs. • Etc., etc.
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.