CH7.3 - Functional Genomics Techniques

Lecture Scope & Learning Goals

• Techniques covered aim to infer gene function by studying their protein products.

  • Localize proteins inside cells and track their dynamics.

  • Quantify expression levels of individual proteins (single-gene scale).

  • Identify protein–protein interactions (PPIs).

  • Monitor global changes in gene expression (transcriptome-wide scale).

• Practical importance

  • Pinpointing a protein’s sub-cellular address often hints at its biological role.

  • Expression profiles reveal inducible, cell-cycle, developmental, or stress-response genes.

  • Mapping interactions erects functional networks/pathways.

  • Systems-wide assays (microarrays, RNA-seq in later chapters) capture holistic physiology.

Protein Localization – Concepts & Methods

• Why localization matters

  • Example shown (image dated $02/2009$): red puncta sit where a chromosome (blue) meets the mitotic spindle (green).

  • Hypothesis: "mystery" protein could mediate kinetochore–microtubule attachment.

• Two mainstream localization strategies:

1. Fluorescent Protein Fusions (e.g., GFP)

• GFP = Green Fluorescent Protein

  • Wild-type isolated from jellyfish Aequorea victoria.

  • Barrel-shaped β-can structure encapsulates a self-forming chromophore that auto-fluoresces green.

  • Mutational tweaking shifts emission spectra → palette of blues, cyans, yellows, reds.

• Experimental pipeline

  • Clone open reading frame (ORF) of target gene in-frame with ORF encoding GFP (or other FP).

  • Express fusion protein in cells/organism.

  • Fluorescence microscope reveals every location the fusion visits.

• Example

  • In E. coli, a GFP-tagged plasmid-binding protein lights up discrete foci that correspond to circular plasmids floating within the rod-shaped cell.

  • Diagram interpretation: central nucleoid (bulk chromosome) is diffuse; bright green dots mark multiple plasmids.

• Pros / Cons

  • Live-cell compatible, dynamic.

  • Tag may hinder folding/targeting; requires genetic manipulation.

2. Immunofluorescence (IF)

• Relies on antibody specificity + fluorescent dyes.

• Reagents

  • Primary antibody: binds epitope on protein of interest.

  • Secondary antibody: binds Fc portion of primary; covalently linked to fluorophore (FITC, Alexa, Cy dyes, etc.).

• Protocol outline

  1. Fix cells (paraformaldehyde, methanol) → immobilize proteins.

  2. Permeabilize membranes so antibodies can enter.

  3. Incubate with primary Ab.

  4. Wash; incubate with species-specific fluorescent secondary Ab.

  5. Image individual color channels; optionally merge to assess co-localization.

• Demonstration slide (human fibroblast)

  • DNA polymerase visualized in red.

  • PCNA (sliding clamp) in green.

  • BrdU (thymidine analog incorporated into newly synthesized DNA) in blue.

  • Merged image shows strong overlap → confirms biochemical partnership during S-phase.

• Advantages vs GFP

  • Works with endogenous (untagged) proteins.

  • Multiplexing: different fluorophores track several proteins simultaneously.

  • Drawbacks: requires fixation (static snapshots); antibody quality crucial.

Protein Detection in Extracts – Western (Immuno) Blot

• Purpose: detect and quantify a specific protein from a complex lysate.

• Workflow

  1. Lyse cells → total protein.

  2. Treat with SDS + β-mercaptoethanol.

  • SDS coats proteins with negative charge ∝ length.

  1. SDS-PAGE: polyacrylamide gel electrophoresis separates by molecular mass.

  • Small proteins migrate further toward positive cathode.

  1. Blot transfer: electro- or capillary-blot proteins onto nitrocellulose/PVDF membrane ("protein paper").

  2. Block nonspecific sites (milk/BSA).

  3. Incubate with primary Ab → wash.

  4. Incubate with enzyme- or fluor-conjugated secondary Ab.

  5. Visualize bands (chemiluminescence, colorimetric, fluorescence).

• Key distinctions

  • Gel shows many bands; final blot ideally displays one band = protein/epitope recognized.

• Case study: RecA induction in Deinococcus radiodurans

  • Cells irradiated; samples collected hourly (lanes $1,3,4,5,6,7$ h post-exposure).

  • Western blot probed with anti-RecA Ab.

  • Band intensity rises after irradiation → suggests DNA-damage-inducible repair response.

Identifying Protein–Protein Interactions

A. Co-Immunoprecipitation (Co-IP)

• Principle: antibody “pull-down” of bait protein drags along any bound partners.

• Steps

  1. Express bait protein (± epitope tag; e.g., HA, FLAG, Myc).

  2. Lyse cells → whole-protein extract.

  3. Incubate with immobilized antibody (agarose, magnetic beads).

  4. Wash away non-specific proteins.

  5. Elute precipitate → SDS-PAGE.

  6. Detect prey proteins by staining, western, or mass spectrometry for ID.

• Interpretation

  • Extra bands beyond bait band = potential interactors (protein $X$, protein $Y$).

  • Controls required (beads-only, non-specific Ab) to weed out false positives.

B. Yeast Two-Hybrid (Y2H) Assay

• Exploits modular nature of yeast GAL4 transcription factor.

  • DBD (DNA-binding domain) binds upstream activating sequences (UAS).

  • AD (activation domain) recruits RNA polymerase II.

• Strategy

  • Physically separate GAL4 domains; fuse DBD to protein X (bait) and AD to protein Y (prey).

  • Transform fusion plasmids into yeast strain carrying UAS-linked reporter gene (HIS3, LacZ, GFP, etc.).

  • Interaction between X and Y re-assembles functional transcription factor → reporter expression.

• Read-outs

  • Growth on histidine-deficient medium, blue color on X-gal, fluorescent output, etc.

• Pros/Cons

  • Screens vast libraries for interactors.

  • False positives (promiscuous AD) and false negatives (membrane proteins) exist; often validated by Co-IP.

Global Gene-Expression Profiling – DNA Microarrays

• Purpose: simultaneously assess mRNA levels of thousands of genes.

• Construction

  • Glass slide/"chip" spotted with single-stranded DNA (ssDNA) oligos representing each gene (one spot per gene).

• Comparative hybridization protocol

  1. Collect total RNA from sample 1 (condition A) and sample 2 (condition B).

  2. Use reverse transcriptase to synthesize complementary DNA (cDNA).

  • Incorporate fluorescent nucleotides: sample 1 labeled green, sample 2 labeled red.

  1. Denature → ss-cDNA; mix and hybridize to microarray.

  2. Wash away unbound cDNA; scan slide with laser.

• Color logic & interpretation

  • $\text{Green spot}$ → gene active only in sample 1.

  • $\text{Red spot}$ → gene active only in sample 2.

  • $\text{Yellow/orange} = \text{Red} + \text{Green}$ → gene expressed in both; hue intensity indicates relative expression.

  • Darker/brighter shades denote expression magnitude (fluor intensity ∝ mRNA abundance).

• Applications

  • Environmental responses (cold vs heat shock).

  • Developmental time-courses.

  • Cancer vs normal tissue diagnostics.

• Limitations

  • Relies on prior knowledge of gene sequences (cannot detect novel transcripts).

  • Dynamic range narrower than RNA-seq; subject to background noise.

Integration & Contextual Notes

• Many concepts connect to prior chapters on DNA replication and repair:

  • PCNA’s co-localization with DNA polymerase echoes its role as sliding clamp (Chapter 6).

  • RecA up-regulation post-irradiation ties into SOS DNA-damage response mechanisms.

• Ethical & practical implications

  • GFP imagery revolutionized live-cell imaging but raises GMO regulatory questions.

  • Microarray-based diagnostics guide therapeutic decisions yet must protect patient genomic privacy.

• Quantitative underpinnings (informal)

  • Electrophoretic mobility $\mu = \frac{q}{6\pi\eta r}$ illustrates why SDS uniform charge allows size-based separation (constant $q/m$).

  • Hybridization thermodynamics (melting temperature $T_m$) influences microarray specificity.

Study Tips & Potential Exam Prompts

• Diagram a western blot workflow; annotate where antibodies act.

• Predict localization pattern if GFP tag disrupts nuclear localization signal.

• Design controls for co-immunoprecipitation experiment.

• Interpret a sample microarray heat-map: identify up- vs down-regulated genes.

• Contrast advantages of Y2H vs Co-IP for membrane-spanning proteins.