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Flashcards in Gene Regulation And Expression Deck (16):
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Structure of RNA

Long, un branched polymer of ribonucleoside monophosphate moieties joined with phosphodiester linkages

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Levels at which gene expression is regulated

Epi genetic factors
Transcription factors
RNA processing
mRNA stability
Translation
Post-translation modification

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Effect of DNA methylation

Can cause long-term inactivation of genes in cellular differentiation

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Types of histone modifications

- phosphorylation (loosen)
- acetylation (promotes initiation of transcription)
- methylation (condense)

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Definition of transcription factors

Proteins that bind cis-regulatory elements in DNA to either positively or negatively regulate gene expression

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Domains of an activator transcription factor

DNA binding
Activation

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Methods of repressor TFs

- directly through repression domain
- indirectly through influencing chromatin structure
- by preventing activators from binding

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RNA interference

Inhibition of gene expression by RNA molecules

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How miRNA regulates gene expression

Mature miRNA couple with RISC and degrade mRNA by direct cleavage or inhibit protein synthesis

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Why is RNA chemically unstable?

Spontaneous cleavage of phosphodiester backbone

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How to protect against RNases

Wear gloves
Use RNAse free tubes and Pipettes
Use RNase free chemicals
Supplement reactions with RNase inhibitors
Good sterile technique
Pretreat materials with heat
Wash with DEPC-treated water

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Adv and disadv of in situ hybridisation

A = detect expression of mRNA in cells and tissues
D = difficult to quantitate levels

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RNA extraction

- gain access (detergent)
- release RNA (denature proteins)
- protect RNA (chelate endogenous nucleases)
- purify RNA
- concentrate RNA

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Main ingredients for RNA isolation

- guanidium thiocyanate (lyse cells)
- proteinase k, b- mercaptoethanol, SDS (degrade protein)
- DNase 1 (degrades genomic DNA)
- phenol (separates RNA)
- isopropanol (precipitates RNA)

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Advantages of NB

Gold standard of RNA analysis
Detect multiple transcripts

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Disadvantages of NB

Hazardous chemicals
Long process
RNA degradation