Molecular - Microarray and expression Flashcards
(28 cards)
What is a microarray?
DNA probes fixed to a glass or silicon surface (chip)
What does fixed mean?
Anchored via covalent bonds
What is used as probes?
Portions of genes are often created by exon-directed oligo probes. Can also use synthesized oligos.
How big are microarrays?
100-1000 wells. Affymetrix = 250,000 SNPs
What do we target with microarrays?
mRNA is labelled such as by RT transformation and labeling. These are hybridized to chip and expression is assessed.
Colouring
Many options. Two colours for 2 different genes; red for upregulated and blue for down. Intermediates also possible.
Aribidopsis thaliana microarray example
Can compare genes in different tissues, exposed to different chemicals, or even diseased states. Blue is underexpressed, red is over.
What questions can we analyze with microarray?
Detect SNPs, disease alleles or cancer, housekeeping vs tissue specific, development changes, harmless vs pathogenic, (simple arrays can even screen pathogens)
Next generation?
Being modified to work with RNA. No need for hybridizing. Short reads but usually enough to identify what is being expressed. Can look at whole transcriptome or short RNA
Problems with the hybridization system
Need to be optimized for each (pH, temp). Can be tricky
Three general patterns of generating localized or specific expression
Direct localization, contact-dependent, diffusable signals. These can lead to “asymmetry” of a cell
Overview of expression
Recruit RNA-Pol-II to promoter: TF-II core factors include TBP (TATA binding protein), D, A and B, F bind. Then comes Pol-II, E, H (turn on), J (fall off). Activators increase the rate of expression
mRNA localization
Microtubules are +/- ends, so cytoskeleton is intrinsically asymmetrical. Adaptor proteins anchor to 3‘UTR of mRNA and motor proteins (dynein/kinesin). This acts like an address for mRNA to be delivered to.
UTR means
Untranslated region (found at 3’ end of mRNA after stop codon)
bicoid and oskar example
In drosophila embryo. Direct localization, bicoid at the future anterior end, and oskar at posterior (detected with in situ)
Hunchback expression in drosophila embryo
mRNA is found throughout, but expressed unevenly. Hunchback gene has bicoid promoter, so strongly expressed in ant. end. Hunchback also has nanos response element, which causes Poly A tail to be shortened and hunchback is degraded. Nanos is found only in post. end, so hunchback is degraded there.
Expression of Caudal in drosophila embryo
Opposite of hunchback: nanos turns it on and bicoid causes it to be degraded, so caudal is expressed mostly at the post. end
Cell to cell contact or signaling
Signaling cascade alters gene expression via TF activation. Integrin acts as a receptor and binds ECM (fibronectin, collagen, sometimes proteoglycans)
Integrin signaling pathway
When bound, integrin activates Src (Tyr. kinase, dimer autophosphorylation), activates FAK - creates phosphotyrosine motif, binds grb, sos, activates Ras, Raf, then MAPK pathway
MAPK role
mitogen-activated protein kinase: phosphorylates (activates) TFs (jun, fos), turn on genes for S phase - committed to mitosis
MAPK family
MAPK activated by MAPKK, which is activated by MAPKKK, which is activated by MAP4K. Many pathways! Activated by ECM contact, stress, GF
Culture growth
Cells depend on ECM binding to grow, You can treat cells with collagen or ECM to stimulate growth. Cells are monolayer inhibited because they lose contact with EMC when they start stacking and then stop dividing.
What diffusable signals do cells respond to?
Paracrine/Endocrine/Gap junction
Repairs to vascular system. Step 1.
Contact induced expression. A cut exposes ECM, platelets bind to it and release thrombin. This converts fibrinogen to fibrin, which polymerizes and makes clots.
