Lecture 4 (Murray) Flashcards
Epigenetics and epitranscriptomics in yeast
What are characteristics of Saccharomyces cerevisiae
(budding yeast)
Basic Traits
- Found on grape skins; used in wine and bread making.
- Size: ~5 micrometers, visible under a light microscope.
- Rapid division (~19 minutes).
- Bud size indicates cell cycle stage.
- Bud scars reveal division history.
Conservation and Domestication
- Long-term storage by freezing or freeze-drying.
- Domesticated for industrial purposes.
- First pure isolation: late 1800s.
Scientific Significance
- Model organism since the 1930s.
- Genetic engineering: shuffle vectors for DNA insertion (since 1978).
- Selectable markers: auxotrophic strains for experiments.
- Gene knock-outs: systematic gene studies and screens.
Genomic Milestones
- First fully sequenced eukaryotic genome.
- “Clean” genetics for studying inheritance and molecular processes.
Saccharomyces cerevisiae
Life cycle
What are the advantages of this life cycle for research?
Yeast Life Cycle
- Haploid cells (“a” and “α”) exist (one set of chromosomes), reproducing asexually via mitosis
- Mating occurs when “a” and “α” cells fuse, requiring mating projections (“shmoo”) to combine genetic material and form a diploid cell.
- Diploid cells (a/α) contain two sets of chromosomes and can reproduce through mitosis, producing more diploid cells.
- Under specific conditions, diploid cells undergo meiosis and sporulation, resulting in four haploid spores (two “a” and two “α”) with genetic diversity from recombination.
- Spores germinate when conditions improve, forming new haploid cells and restarting the cycle.
Advantages
Single-hit phenotypes are visible in haploids, making genetic analysis straightforward. Mutations and their effects are easier to predict and study.
Mating Type Genes in S. cerevisiae
Mating Type Genes in S. cerevisiae
- MAT gene: Determines mating type.
- HML (α) and HMR (a): Silent loci with alternate mating type information.
- Switching: Information from HML or HMR copied into MAT via recombination.
- HO endonuclease: Cleaves MAT, initiating recombination.
- Regulation: HO expression delayed in daughter cells to ensure proper timing.
- Swi genes: Regulate HO expression through chromatin remodeling.
- Outcome: Alternates mating types, promotes genetic diversity.
Silencing at HMR and HML in S. cerevisiae
Silencing at HMR and HML in S. cerevisiae
- Flanking sequences: Silencers E and I repress genes at HML and HMR.
- Recruitment: Silencers contain a ORC, Rep1, and Abf1 Binding site.
- Sir complex: Together they recruit Silent Information Regulators Sir2, Sir3, Sir4 to form heterochromatin
- Sir2: NAD+-dependent histone deacetylase (H4K16).
- Mechanism: Deacetylation compacts chromatin and prevents transcription.
- NAD+: Cleaved and consumed in the reaction.
- Sir3/Sir4: Bind deacetylated histones, recruit Sir proteins, spread silencing.
- Purpose: Keep HML and HMR inactive until recombination.
Characteristics of the S. cerevisiae genome
Genome Characteristics
Genome Size: Approximately 12,000 KB (12 MB) of DNA.
Chromosome Count: Haploid genome contains 16 chromosomes.
Gene Density: 1 gene per 2 KB, very dense genome with minimal non-coding sequence.
Total Genes: Contains approximately 5,076 genes (short ORFs).
Genomic Features
Introns: Only 4% of all genes contain an intron, meaning very few mRNAs require splicing.
Gene Types: Includes both protein-coding genes and non-coding RNAs (e.g., TLC1, the telomerase RNA component, which is part of the polymerase enzyme).
Key DNA Sequences
Origin of Replication: Contains Autonomous Replicative Sequences (ARS), which are defined sequences that initiate DNA replication.
Centromeres: Each chromosome has a centromere with a short, specific centromeric sequence (CEN), essential for chromosome segregation.
Ribosomal DNA (rDNA): Highly repetitive region coding for rRNA, with 100-200 copies per 9 KB unit of DNA.
Ty Elements: Transposons that have moved within the genome over evolutionary time, contributing to genomic variability.
Centromere Structure in S. cerevisiae
Centromere biology in S. cerevisiae
- Size: ~125 base pairs.
- Structure: Single CENP-A nucleosome.
- Function: Kinetochore assembly, spindle attachment.
- Comparison: Simpler than regional centromeres in other organisms.
Kinetochore components in S. cerevisiae
Kinetochore components in S. cerevisiae
- The kinetochore of S. cerevisiae assembles on a 125 bp point centromere, which consists of three regions: CDEI, CDEII, and CDEIII.
- Cbf1binds to CDEI, Cse4 (a histone H3 variant) forms a centromeric nucleosome at CDEII, and the Cbf3 complex binds to CDEIII, which is crucial for kinetochore assembly.
The kinetochore has a three-layered structure
- Inner kinetochore interacts with centromeric DNA.
- Central kinetochor links the inner and outer components.
- Outer kinetochore connects to microtubules for chromosome segregation.
- The Dam1 and Ndc80 complexes anchor the kinetochore to microtubules, enabling proper chromosome movement.
- Compared to higher eukaryotes, the kinetochore in S. cerevisiae is simpler, suited to its small point centromeres while maintaining accurate chromosome segregation.
What is the HiC method?
Hi-C Method for 3D DNA Modeling
Purpose: Technique to determine the 3D proximity of DNA sequences within a cell.
Process:
1. Crosslinking: Use formaldehyde to chemically crosslink DNA strands. If two chromosomes are close, they can form crosslinks.
2. Fill and Tag: Fill staggered DNA ends and ligate with nucleotides tagged with biotin, marking the junction.
3. Cleavage and Enrichment: Perform a second digestion to cleave crosslinks, then pull down on biotin tags to enrich sequences that were ligated together.
4. Sequencing and Analysis: Use high-throughput sequencing to identify sequence positions and analyze frequency of fragment proximity.
Outcome: Provides insights into the 3D organization of chromosomes by identifying DNA regions frequently in close proximity.
What has been done in the Yeast deletion collection?
Yeast deletion collection
- Purpose: Identify genes required for growth under specific conditions using a systematic deletion approach.
Process:
- Gene deletion: Create strains with specific gene deletions tagged with unique barcodes (uptags and downtags).
- Pool deletion strains: Combine strains with tagged deletions.
- Growth condition: Expose pooled strains to chosen condition.
- Purify DNA: Extract genomic DNA from pooled strains.
- PCR amplification: Amplify uptags and downtags (strain-specific barcodes).
- Hybridization: Bind PCR products to a microarray chip.
- Data analysis: Measure barcode intensity, calculate growth rate for each strain.
Concept of Synthetic lethality
Concept of Synthetic Lethality
- Two gene deletions cause cell death; single deletions have no effect.
- Reveals gene functions and interactions by creating genetic interaction networks.
Gene Grouping
- Similar synthetic interactions indicate shared pathways or complexes.
- Example: mfg1d, genY, and genZ in the same pathway show similar phenotypes upon deletion.
Testing
- Systematic deletion of mfg1d with all other genes.
- Identifies related interactions and functional groups.
Synthetic genetic array?
Synthetic Genetic Array (SGA) Analysis
Definition: A technique to study genetic interactions in (S. cerevisiae)
How SGA Works
- Gene Deletion: A collection of yeast strains with individual gene deletions is created.
- Crossing Mutants: Each deletion strain is crossed with another strain containing a target gene deletion to generate double mutants.
- Growth Selection: The double mutants are grown under specific conditions to observe fitness effects.
- Barcode Analysis: Unique DNA barcodes allow tracking of mutant strains, and their growth defects are analyzed.
Significance: Helps identify gene networks, essential genes, and potential drug targets through synthetic lethal interactions.
Yeast Two-Hybrid Assay (Y2H)
Purpose and Applications
- Detect protein-protein interactions to map interaction networks.
- Study gene function and protein roles in cellular pathways.
- Applicable to genes from other organisms.
Method
Protein Components:
- Bait: DNA-binding domain (DBD) fused to Protein X.
- Prey: Activation domain (AD) fused to Protein Y.
Interaction Mechanism:
- No interaction: DBD and AD remain separate, no reporter gene activation (e.g., white colonies with LacZ).
- Interaction: DBD and AD join, activating the reporter gene (e.g., blue colonies with LacZ).
Tandem Affinity Purification (TAP)
Purpose and Applications
- Study protein complexes by isolating and identifying interactions.
- Achieve high specificity through two-step purification with minimal contaminants.
- Map protein networks, identify drug targets, and analyze functional roles in proteomics.
Method
TAP Tag Fusion
- Fuse target protein with a TAP tag (e.g., Protein A + calmodulin-binding peptide, separated by a TEV cleavage site).
Protein Complex Isolation
- First purification: Bind Protein A to IgG column, wash away contaminants.
- TEV protease cleavage: Release bait protein with its complex.
Second Affinity Purification
- Pass eluate through calmodulin-binding column, remove contaminants.
- Elute complex using EGTA.
Complex Analysis
- Use mass spectrometry (MS) to identify proteins in the purified complex.
Sc2.0 Project (Synthetic Yeast 2.0)
The first fully synthetic eukaryotic genome project
Synthetic Yeast 2.0 Project
- First fully synthetic eukaryotic genome project.
- Aim: Create a synthetic genome for industrial applications.
- Redesign: Remove non-essential genes, introduce tags/markers.
Process:
- Synthesize chunks of the redesigned genome.
- Gradually replace the native genome with synthetic chunks.
- Use tags/markers to track genome replacement.
Challenges:
- Potential lethal combinations from compromised chromosomes; project not yet completed.
Karyotype engineering in yeast
Chromosome Fusion Study
Idea
- Progressively fuse chromosomes in S. cerevisiae from 16 to 12, 8, 4, and finally 2.
- Test viability of yeast with fewer, larger chromosomes.
Key Points
- CRISPR Fusion: Used CRISPR-Cas9 to target telomeres and fuse chromosomes step by step.
- Results: Yeast viable with 2 chromosomes; gene expression affected but essential functions intact.
- Significance: Demonstrates chromosome structure flexibility and simplifies genetic engineering with fewer chromosomes.
- Reproductive Isolation: Meiosis fails between strains with 16 vs. 2 chromatids, creating a reproductively isolated strain akin to a new species with the same DNA sequence.
Advantages of S. cerevisiae and S. pombe as model organisms?
End of Lecture Question
Genome Simplicity:
- S. cerevisiae: Small and well-annotated genome makes it easy to study and manipulate genetic pathways.
- S. pombe: Compact genome with introns allows for studies on splicing and more complex gene regulation.
Cell Cycle Studies:
- S. cerevisiae: Simpler cell cycle machinery provides a straightforward model for basic eukaryotic cell division.
- S. pombe: Highly conserved cell cycle machinery makes it an excellent model for studying processes relevant to human cells.
Post-Translational Modifications:
- S. cerevisiae: Limited modifications, but sufficient for understanding basic eukaryotic processes.
- S. pombe: Post-translational modifications closely resemble human systems, enabling insights into human biology.
