Biochem Ch. 19-22 LGs

• Metabolic pathways


  Expressed in different areas for the greatest efficiency
• Cori cycle


  1. Muscles produce lactate under anaerobic conditions

  2. Lactate is exported to the liver for gluconeogenesis

  3. Liver exports glucose back to the muscles
• Hormones


  Substances produced by one tissue that affect the function of other tissues throughout the body
• Insulin


  • Released by B-cells of the pancreatic islets in response to high blood glucose
  • Signals fuel abundance promoting fuel storage while limiting release of stored fuels
• Glucagon


  Small peptide hormone
• Insulin signaling


  Activates phosphatases that dephosphorylate glycogen synthase (activating it) and glycogen phosphorylase (inhibiting it)
• Type-1 diabetes


  • Juvenile onset, no insulin made (5-10% of diabetics)
  • Autoimmune disease that kills pancreatic B-cells
  • Treated with insulin injections
• Type 2 diabetes


  • Adult onset, insulin resistance (90-95% of diabetics)
  • Underlying cause unknown, but closely linked to obesity and inactivity
  • Treated with lifestyle changes, drugs that lower blood glucose, or bariatric surgery
• Hyperglycemia leads to long term effects such as cataracts, kidney failure, and cardiovascular damage
• Acute effects of diabetes include hypoglycemia (if too much insulin is injected) and ketoacidosis
• DNA replication


  1. Helicase unwinds the DNA helix

  2. SSB binds single-stranded regions

  3. DNA polymerase adds deoxynucleotides

  4. Primase introduces RNA primer

  5. RNase H removes RNA primer

  6. Ligase joins fragments
• Leading strand


  The one strand that is continuously synthesized
• Lagging strand


  The other strand that is synthesized in segments (Okazaki fragments)
• Telomeres


  • Repeating sequence of DNA folded into a loop and binds protective proteins
  • Solves the end-shortening problem by telomerase adding repeats to 3' ends
• Cells that replicate indefinitely have telomerase activity, most cells stop expressing telomerase to protect against cancer
• Linear DNA is susceptible to end-joining by DNA repair machinery or degradation by exonucleases
• Nucleosome


  Linear DNA is negatively supercoiled by wrapping the double helix around a core
• Proofreading function of polymerase


  Polymerase slows down due to bulge caused by mispairing, 3'-5' exonuclease removes misincorporated nucleotide
• Euchromatin


  Lightly packed DNA that is heavily transcribed
• Heterochromatin


  Tightly packed DNA (30nm fiber)
• Cancer


  • Characterized by uncontrolled cell division
  • Results from accumulated mutations or modifications of DNA
• Mutations may be induced by environmental factors, genetic predisposition, viral infection, or spontaneous mutations
• Transversion


  Mutation leading to a switch between purine and pyrimidine
• Transition


  Purine to purine or pyrimidine to pyrimidine mutation
• Abasic sites


  Thousands of bases are spontaneously hydrolyzed from ribose each day
• Deamination


  Cytosine undergoes a transamination reaction with water
• DNA uses thymine instead of uracil so that if deamination occurs, uracil is recognized as an error and can be removed
• DNA repair mechanisms


  1. Base excision repair

  2. Nucleotide excision repair

  3. Mismatch repair

  4. Nonhomologous end-joining

  5. Homologous recombination

  6. Alkyltransferases
• Restriction enzymes


  • Isolated from bacteria, cut double-stranded DNA within a defined recognition sequence that is usually palindromic
  • Bacteria methylate their own genomic DNA to prevent cleavage, but will degrade viral DNA
• Restriction enzyme recognition sites are directional (5'-3') and complementary sticky ends allow reannealing and ligation of DNA fragments
• Plasmids


  • Small circular DNAs that replicate independently in bacteria
  • Engineered as cloning vectors with an origin of replication, antibiotic resistance gene, and polylinker region
• PCR (Polymerase Chain Reaction)


  1. Amplifies any DNA sequence provided some sequence info is known on either side of the target DNA

  2. Involves repeated cycles of DNA denaturation, primer annealing, and DNA extension
• Illumina sequencing


  Simultaneously sequences thousands of DNA fragments by detecting fluorescent signals as complementary deoxynucleotides are added
• CRISPR-Cas9


  Bacterial system that uses guide RNAs to direct the Cas9 enzyme to cleave specific DNA sequences, can be used for gene editing
• Sequencing can be applied to determine the sequence of entire genomes
• RNAP


  RNA polymerase in E. coli
• CRISPR-Cas9 system


  1. Cas9 protein uses guide RNAs to locate and destroy complementary DNA sequences in invading viruses

  2. Plasmids expressing Cas9 and guide RNAs against specific genes may be used in research applications
• Inactivation of targeted genes by CRISPR-Cas9


  Caused by non-homologous end-joining (NHEJ), resulting in a non-functional protein product
• Editing of genes by CRISPR-Cas9


  Achieved through homology-directed repair (HDR) after Cas9 cleavage, if replacement DNA is introduced
• RNA polymerase (RNAP)


  Synthesizes new RNA strands from a DNA template