Gene Regulation And Expression Flashcards
Levels at which gene expression is regulated
Epi genetic factors Transcription factors RNA processing mRNA stability Translation Post-translation modification
Structure of RNA
Long, un branched polymer of ribonucleoside monophosphate moieties joined with phosphodiester linkages
Effect of DNA methylation
Can cause long-term inactivation of genes in cellular differentiation
Types of histone modifications
- phosphorylation (loosen)
- acetylation (promotes initiation of transcription)
- methylation (condense)
Definition of transcription factors
Proteins that bind cis-regulatory elements in DNA to either positively or negatively regulate gene expression
Domains of an activator transcription factor
DNA binding
Activation
Methods of repressor TFs
- directly through repression domain
- indirectly through influencing chromatin structure
- by preventing activators from binding
RNA interference
Inhibition of gene expression by RNA molecules
How miRNA regulates gene expression
Mature miRNA couple with RISC and degrade mRNA by direct cleavage or inhibit protein synthesis
Why is RNA chemically unstable?
Spontaneous cleavage of phosphodiester backbone
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
Adv and disadv of in situ hybridisation
A = detect expression of mRNA in cells and tissues D = difficult to quantitate levels
RNA extraction
- gain access (detergent)
- release RNA (denature proteins)
- protect RNA (chelate endogenous nucleases)
- purify RNA
- concentrate RNA
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)
Advantages of NB
Gold standard of RNA analysis
Detect multiple transcripts
