Gene Expression

• What is differentiation?


  Process by which cell changes into an overtly specialised cell

  Large number of genes expressed giving specialised characteristics
• What are the 3 basic types of cell?


  Somatic, germ & stem
• How many different cell types are there in the body?


  Around 200
• What makes cells different from eachother?

  Gene expression (all cells contain same genetic info)

  (Express different RNAs & proteins)
• What happens during determination?


  Commitment of undifferentiated cell to particular pathway of specialisation.

  Can occur before overt differentiation

  Few transcription factors activated
• What is Dedifferentiation?


  Reverses differentiation (cell becomes less specialised)

  Can cause cancer as cell can proliferate
• What can you use 2D gel electrophoresis for?


  Compare proteins expressed in different tissues
• What can you do when there's a tumour of unknown origin?

  Use array analyst technique to give it's signature (showing mRNA produced) and match this to one of a known tumour type
• What is gene expression?


  Mechanisms through which genes 'switched on' & transcribed into mRNA
• What is a transcription factor?


  Gene that encodes a protein that activates or represses other genes.

  Often share a common DNA binding domain but have variety of effects.

  Many families of transcription factors: SOX, FOS, CREB
• SOX


  Involved in differentiation of gonads in early development
• FOS


  Responsible for earliest activation of neurones in response to stimulus

  (evolved in brain)
• CREB


  evolved in brain
• How do cells remember what they have specialised as? (cytoplasmic)


  • Transcription factors influence nucleus by modulating gene expression
  • Positive feedback cell-signalling mechanisms ensure stability
• How do cells remember what they have specialised as? (nuclear)


  • heritable changes in gene expression

  e.g, DNA methylation or histone modification = epigenetic
• How do cells remember what they have specialised as? (autocrine)


  • Secreted factors can influence small groups of cells to reinforce differentiated state 'community effects'
• Epigenetics


  Modification of gene expression
• Why might gene expression change functionally?


  To respond to environment

  Up/down regulated
• When might liver cells change their gene expression?

  • alcohol consumption can affect rate of enzyme production
  • adjust enzyme synthesis in response to circulating steroid hormone levels
• When might stratified squamous epithelia change gene expression?


  Keratinise & become thicker in response to injury/damage

  (e.g, in skin cells)
• When might mammary gland epithelial cells change gene expression?


  Pregnancy- increase milk protein production
• Gene expression across life-span:


  • Developmental gene expression

  (maintenance of differentiated state)

  • Gene x environment interactions
  • epigenetic effects
• Typical human cell expresses how much of its genes?


  20%- highly differentiated cells express less
• Replicative cell senescence:


  (Finite) Number of times cells can divide before they enter G0
• What do promotors do?

  Regulate transcription of gene

  Promotor sequence unique for a gene

  RNA polymerase/transcription factors binds to promotor region in gene
• Where does post-translational modification happen?


  Golgi/vesicle.

  Forms final protein
• Types of control levels of gene expression:

  • Transcriptional control
  • RNA processing control
  • RNA transport & localisation control (which mRNA transported)
  • Translational control (which mRNA to be translated)
  • Post-translational control (protein activity regulation)
• What is a TATA box?


  DNA sequence in promotor

  Contains A & T

  Transcription factors bind to promotor proximal elements: activators, enhancer or repressor regions
• What can enhancer regions do?


  Regulate expression of multiple genes

  Confer cell/tissue specificity
• What do we need cortisol activation for?


  Converts noradrenaline to adrenaline
• What does pre-mRNA (heteronuclear RNA) contain? 


  Introns (non-coding) & exons (coding)
• What happens during capping?


  Modified guanosine 5' cap added to mRNA to protect it from degradation by nucleases & aid binding to ribosome.
• Splicing:

  Introns removed & exons joined together- introduces mRNA splice variants (1 is selected) to produce different forms of proteins
• Why is a poly A tail added to mRNA at 3' end?

  Protect it from degradation & stabilise- done before export to cytoplasm
• How can mRNA be silenced?

  Short non-coding RNAs (microRNAs) (miRNAs)

  Blocks expression

  Cancer can develop as certain microRNAs could cause cells to become metastatic
• What is PCR?


  Polymerase chain reaction- minute amount DNA amplified from sample for visualisation, cloning, sequencing or manipulation.
• What is RT-PCR?

  Reverse transcription PCR: mRNA converted to DNA using reverse transcriptase.

  Has improved sensitivity of RNA analysis (Northern blotting, RNAase protection assay)
• Northern blotting:


  Identify RNA in sample- problematic as RNA unstable & degrades quickly so hard to measure
• In situ hybridisation histochemistry (ISHH):


  Detect nucleic acid in fixed tissue using a labelled nucleic acid probe to bind to target through nucleobase pairing

  Target= chromosomal DNA, RNA or infective viral DNA/RNA

  Primer concentrates on localisation of RNA

  Examine spatial & temporal resolution of nucleic acid localisation

  Determine when, where, how much RNA expressed

  E.g, mRNA- monitor developmental expression of specific gene products, cell/tissue types in which it's found
• Spatial resolution:


  Where RNA produced