Chapter 4 - DNA

• DNA Nucleotide structure

  Phosphate group, Deoxyribose Sugar (2' H), Nitrogenous base
• Pyrimidine - 1 ring, cytosine and thymine, T is uracil in RNA
• Purine - 2 rings, adenine, guanine
• DNA Double Helix

  Anti-parallel strands, Phosphodiester bonds, Hydrogen bonds
• Phosphodiester bonds connect nucleotide to nucleotide in single DNA strand
• Hydrogen bonds connect complementary base pairs on double helix
• Packaging DNA in Eukaryotic cells?

  2m of DNA in nucleus that is 6um in diameter, equal protein mass
• Histones

  positive charge, 102 amino acids (small), highly conserved
• Histone Fold Motifs

  3 alpha helices, 2 loops
• Beads on a String - 10 nm fiber - Level 1


  Nucleosome, Linker DNA and DNA wrapped around histones
• DNA must be free of nucleosomes for transcription to occur
• How to get rid of nucleosomes?

  Chromatin Remodeling Complexes and Histone Acetylation/Deacetylation
• Chromatin Remodeling Complexes

  Use of histone acetylation to loosen DNA for transcription to occur and histone deacetylation for nucleosome restoration, repeats cycle
• Histone Acetylation/Deacetylation

  HATs (histone acetyltransferases), Acetylate histones (adds acetyl group), positive charge neutralized, Lysines don't bind to negative charged DNA (DNA looser), Transcriptional machinery has access to DNA, HDACs reverse process
• 30 nm Fiber - Level 2


  Histone H1, Nucleosomes in solenoid structure, 6 nucleosomes per turn, Histones deacetylated, Inactive genes
• Euchromatin

  Beads on a String and 30 nm Fiber, Genes can switch from active and inactive, Differs in cell type
• Heterochromatin

  Loop Regions and Chromosomes, DNA more condensed, Non-histone proteins used, can't revert to euchromatin
• Looped Regions

  DNA (SARS/MARS) attached to Scaffold/Matrix, DNA attached won't contain active gene
• Chromosomes

  Coiling of scaffold into helix, found in mitosis and meiosis, tightly packed
• DNA Protocols

  Genomic Library, cDNA Library, DNA Sequencing, Sodium Bisulfite Method
• Genomic Library - identification of DNA fragment from entire genome
• Characteristics of a vector

  ORI, Ab resistance gene, Multiple cloning site
• cDNA library - identify particular DNA fragment from set of mRNA being transcribed
• DNA polymerase lays down nucleotides in 5' to 3' direction
• Reading DNA sequence from bottom to top is the strand copied from 5' to 3'
• DNA methylation - mechanism used by cell to turn off gene expression
• Sodium Bisulfite can be used to determine methylation status of a gene
• Any cytosine residues not methylated will converted to uracil in presence of Sodium Bisulfite
• DNA methylated?

  Cytosine protected and remain Cs, recruit HDACs, Lysines on histones retain positive charge, Histone bind DNA more tightly, Transcriptional machinery doesn't have DNA access, Gene is off
• DNA not methylated?

  Cytosine not protected and convert to thymine, Histones become acetylated, Lysines lose positive charges, Histones bind DNA less tightly, Transcriptional machinery has DNA access, Gene is on