TOPIC 8

• CAUSES OF MUTATION?

  1. High energy ionising radiation

  • alpha/ beta particles
  • X-rays/ UV light
  • disrupt DNA structure

  1. Chemicals

  • NO2 directly alters DNA structure/ interferes with transcription
  • benzopyrene inactivates tumour-suppressant genes
  • mustard gas
• OESTROGEN ON GENE TRANSCRIPTION?

  1. Lipid-soluble
  2. Simple diffusion through phospholipid bilayer
  3. Oestrogen binds with complementary receptor molecule of transcription factor in cytoplasm
  4. Changes shape of DNA binding site on transcription factor (can bind to DNA so activated)
  5. Transcription factor enters nucleus through nuclear pore
  6. Binds to specific base sequences on DNA
  7. Stimulates RNA polymerase to bind to gene
  8. Catalyses transcription = mRNA
  9. Translation of mRNA produces protein
  10. Gene expressed
• EPIGENETICS DEFINITION?

  heritable changes in gene function with no change in DNA base sequence/ that is not due to mutation but due to environment

  • diet/ stress/ pollution
  • eukaryotes only
• ACTIVE VS INACTIVE GENES?

  INACTIVE

  • tightly packed
  • cannot be read
  • epigenetic silencing
  • heterochromatin
  • decreased acetylation (deacetylation) of histones
  • increased methylation of DNA

  ACTIVE GENES

  • unwraps
  • DNA exposed
  • easily transcribed
  • euchromatin
• DEACETYLATION?

  • acetyl groups have negative charges
  • increased positive charges on histones
  • increased attraction to phosphate groups on DNA
  • stronger association
  • DNA not accessible to transcription factors
• METHYLATION?

  • adding CH3 to cytosine bases (CpG sites) of DNA
  • prevents binding of transcription factors to DNA
  • attracts protein that condense DNA-histone complex
• RNA INTERFERENCE?

  prevents translation by breaking down mRNA in eukaryotes and some prokaryotes

  1. Enzyme cuts large, double-stranded molecules of RNA
  2. Into smaller, double-stranded small interfering RNA
  3. Strands separate = siRNA
  4. siRNA has specific base sequence complementary to target gene
  5. One strand of siRNA combines with enzyme
  6. Guides enzyme to mRNA molecule
  7. Pairs bases with complementary ones on section of mRNA of target gene
  8. Enzyme cuts mRNA into smaller sections
  9. Polypeptide cannot be produced via translation (mRNA cannot bind to ribosome)
  10. No gene expression
• BENIGN VS MALIGNANT?

  • B normal nucleus, M larger and darker nucleus
  • B specialised cells, M unspecialised cells
  • B adhesion molecules (cells stick together) surrounded by capsule, M metastasis (no adhesion molecules) and no capsule (finger-like projections into tissues)
  • B localised effects, M systemic effects
• ONCOGENE?

  • mutations of proto-oncogenes
  • permanently activated
  • receptor protein permanently activated (no growth factor)
  • or code for growth factor so produced in excessive amounts
  • few inherited, most acquired
• ABNORMAL METHYLATION OF GENES?

  • hypermethylation of tumour suppressor genes (BRCA1)
  • hypomethylation of oncogenes
• OESTROGEN AND CANCER?

  • after menopause, risk of breast cancer increases
  • fat cells produce more oestrogen
  • tumours
  • further increases oestrogen concentration
  • WBCs drawn to tumour
  • further increases oestrogen concentration
  • tumour develops
• WHY IS DETERMINING GENOME/ PROTEOME EASIER FOR SIMPLE ORGANISMS?

  • one, circular piece of DNA not associated with histones
  • no non-coding sections of DNA
• IN VIVO GENE CLONING?

  1. DNA fragment isolated
  2. mRNA to cDNA using reverse transcriptase
  3. restriction endonucleases cut fragments
  4. gene machine+ PCR (oligonucleotides - smaller, overlapping single strands of nucleotides - assembled)
  5. Add promoter and terminator regions (ensure replication)
  6. Inserted into vectors (e.g. plasmids) using restriction endonucleases and DNA ligase
  7. Transformation into bacteria (ice cold CaCl2 and heat shock)
  8. Bacteria multiply
  9. Identify successful using marker genes
  10. antibiotic resistance, replica plating
  11. GFP
  12. enzyme
• WHY TRANSFORMATION NOT ALWAYS EFFECTIVE?

  • only few bacterial cells accept plasmid
  • some plasmids close again
  • DNA fragment ends join together
• IN VITRO CLONING?

  (DNA fragments isolated then polymerase chain reaction)

  REQUIREMENTS

  • DNA fragment
  • DNA polymerase
  • primers
  • nucleotides
  • thermocycler

  STEPS

  1. Heat to 95

  • denatures DNA
  • breaks hydrogen bonds between bases
  • two template strands

  1. Cool to 55

  • primers anneal to start of template strand
  • complementary base pairing
  • hydrogen bonds

  1. Heat to 72

  • DNA nucleotides pair with complementary bases
  • hydrogen bonds form (A/T, C/G)
  • new strand synthesised faster

  1. DNA polymerase joins nucleotides and makes sugar-phosphate backbone (condensation reaction/ phosphodiester bonds)
  2. Repeat for other template = two DNA molecules
• PURPOSE OF DNA POLYMERASE?

  catalyses formation of phosphodiester bonds between nucleotides to create sugar-phosphate backbone
• PRIMERS?

  • short nucleotide sequence
  • base set complementary to those at ends of DNA fragments
  • make DNA double stranded so DNA polymerase binds
  • prevents template strands from rejoining
• IMPORTANCE OF KNOWING BASE SEQUENCE IN PCR?

  for primers
• DNA PROBE?

  short, single-stranded length of DNA with bases complementary to target base sequence (gene) with marker gene attached
• LOCATING SPECIFIC ALLELES?

  1. Fragment of DNA with complementary base sequence to allele produced
  2. Polymerase chain reaction
  3. DNA probe synthesised by attaching marker to DNA fragment
  4. Heat DNA of sample to separate strands
  5. Cooled in mixture of DNA probes
  6. Hybridisation (bind)
  7. Wash off unattached probes
  8. Hybridised DNA labelled
• GENETIC FINGERPRINTING?

  • non-coding regions of DNA contain short, repeating sequences (VNTRs)

  1. Extraction of DNA fragment
  2. Amplification using PCR
  3. Digestion using specific restriction endonucleases (VNTRs intact)
  4. Separation of fragment using gel electrophoresis
  5. Hybridisation of VNTRs at specific complementary base sequences with DNA probes
  6. Location depending on probes
• HOW DO TOTIPOTENT CELLS LEAD TO CELL SPECIALISATION?

  translate only part of their DNA
• KEY FACT ABOUT ONLY STEM CELLS?

  can divide by mitosis