Molecular - Microarray and expression Flashcards

1
Q

What is a microarray?

A

DNA probes fixed to a glass or silicon surface (chip)

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2
Q

What does fixed mean?

A

Anchored via covalent bonds

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3
Q

What is used as probes?

A

Portions of genes are often created by exon-directed oligo probes. Can also use synthesized oligos.

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4
Q

How big are microarrays?

A

100-1000 wells. Affymetrix = 250,000 SNPs

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5
Q

What do we target with microarrays?

A

mRNA is labelled such as by RT transformation and labeling. These are hybridized to chip and expression is assessed.

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6
Q

Colouring

A

Many options. Two colours for 2 different genes; red for upregulated and blue for down. Intermediates also possible.

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7
Q

Aribidopsis thaliana microarray example

A

Can compare genes in different tissues, exposed to different chemicals, or even diseased states. Blue is underexpressed, red is over.

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8
Q

What questions can we analyze with microarray?

A

Detect SNPs, disease alleles or cancer, housekeeping vs tissue specific, development changes, harmless vs pathogenic, (simple arrays can even screen pathogens)

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9
Q

Next generation?

A

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

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10
Q

Problems with the hybridization system

A

Need to be optimized for each (pH, temp). Can be tricky

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11
Q

Three general patterns of generating localized or specific expression

A

Direct localization, contact-dependent, diffusable signals. These can lead to “asymmetry” of a cell

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12
Q

Overview of expression

A

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

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13
Q

mRNA localization

A

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.

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14
Q

UTR means

A

Untranslated region (found at 3’ end of mRNA after stop codon)

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15
Q

bicoid and oskar example

A

In drosophila embryo. Direct localization, bicoid at the future anterior end, and oskar at posterior (detected with in situ)

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16
Q

Hunchback expression in drosophila embryo

A

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.

17
Q

Expression of Caudal in drosophila embryo

A

Opposite of hunchback: nanos turns it on and bicoid causes it to be degraded, so caudal is expressed mostly at the post. end

18
Q

Cell to cell contact or signaling

A

Signaling cascade alters gene expression via TF activation. Integrin acts as a receptor and binds ECM (fibronectin, collagen, sometimes proteoglycans)

19
Q

Integrin signaling pathway

A

When bound, integrin activates Src (Tyr. kinase, dimer autophosphorylation), activates FAK - creates phosphotyrosine motif, binds grb, sos, activates Ras, Raf, then MAPK pathway

20
Q

MAPK role

A

mitogen-activated protein kinase: phosphorylates (activates) TFs (jun, fos), turn on genes for S phase - committed to mitosis

21
Q

MAPK family

A

MAPK activated by MAPKK, which is activated by MAPKKK, which is activated by MAP4K. Many pathways! Activated by ECM contact, stress, GF

22
Q

Culture growth

A

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.

23
Q

What diffusable signals do cells respond to?

A

Paracrine/Endocrine/Gap junction

24
Q

Repairs to vascular system. Step 1.

A

Contact induced expression. A cut exposes ECM, platelets bind to it and release thrombin. This converts fibrinogen to fibrin, which polymerizes and makes clots.

25
Q

Repairs to vascular system. Step 2.

A

Paracrine signaling. Platelets also release EGF, stimulates MAPK path, stimulates endothelial cell division.

26
Q

Repairs to vascular system. Step 3.

A

Endothelial cells secrete matrix and release plasminogen activator; plasmin degrades fibrin, clot is degraded.

27
Q

EGF

A

Endothelial growth factor - need to rebuild what was lost during damage - activate MAPK pathway

28
Q

tPA (tissue plasminogen activator)

A

Given to stroke and heart attack victims. tPA will activate plasmin and it will degrade blood clots.