Genes and Chromosomes

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Chapter 24

• Genes and Chromosomes

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One gene, one gene product
Colinearity of nucleic DNA, mRNA, and polypeptide
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DNA molecular weights have been determined by
various methods

• Hydrodynamics (ultracentrifugation)
• Sedimentation rates can characterize particles
• Electron microscopy
• Autoradiography
• Base pair of NaDNA has known mass (660 Da) and
  known width (3.4 Å)

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Viral DNA

• Parasitic particles with DNA (or RNA) surrounded
  by protein coat
• Plant and some bacterial/animal viruses have
  small RNA genomes
• DNA viral genomes vary in size
• Some are circular during certain periods in their
  life cycle

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Bacterial DNA

• Circular, double-stranded
• Contour length ?1.7 mm
• Many also contain plamids
• Self-replicating, circular, small-large
• Some carry advantageous traits (drug-resistance,
  e.g.)

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Eukaryotic DNA

• Smallest eukaryotic genome (S. cerevisiae) is
  almost 3X as large as that of e. coli
• DNAs are arranged into chromosomes
• Haploid number (N) total number of unique
  chromosomes in organism
• Diploid number (2N) total number of chromosomes
  (most somatic cells contain 2 copies/chromosome)
• Haploid number is species-specific
• Mitochondria also contain chromosomes (mtDNA)
• Smaller than nuclear chromosomes
• 2-10 copies/mitochondria
• mtDNA codes for some mitochondrial proteins,
  tRNAs, rRNAs but
• Most mitochondrial proteins are coded for by
  nuclear DNA

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Eukaryotic DNA

• Most genes contain intervening seqments of DNA
  that do not code for amino acids
• These are called intervening sequences (INTRONS)
• Coded sections are called EXONS

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Fractionation of Nucleic Acids

• De-proteination of DNA
• Proteins are precipitated in CHCl3/ROH or phenol
  solution and centrifuged
• Detergents, guanidine chloride, treatment with
  proteases, or high salt dissociates DNA-protein
  complexes
• EtOH then precipitates DNA-RNA mixtures
• Nucleic acids must be protected from ubiquitous
  nucleases
• EDTA
• Autoclave all glassware

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Fractionation of Nucleic Acids

• Chromatography
• Hydroxyapatite (Ca5(PO4)3OH) binds
  double-stranded DNA with high affinity
• Concentration gradients can be used to elute
  single vs. double stranded DNA
• Thermal chromatography elute single-stranded
  DNA, gradually raise temperature of column,
  melting double-stands.
• Elution time ? Tm
• mRNA can be separated by affinity chromatography

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Tm 41.1XGC 16.6 log Na 81.5 where Tm
is DNA melting temperature and XGC is mole
fraction of G-C bp
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Fractionation of Nucleic Acids

• Electophoresis
• Small-medium DNAs can be separated by PAGE
• Agarose must be used for larger molecules
• Good system for separation of plasmids from
  chromosomal DNA
• Large DNAs (gt100 bp) cannot travel through
  agarose
• PFG pulse-field gel electrophoresis

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Fractionation of Nucleic Acids

• Electrophoresis, continued
• DNA is stained by planar aromatic dyes
• Ethidium
• Acridine orange
• Proflavin

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Fractionation of Nucleic Acids

• Electrophoresis, continued
• Southern transfer technique (blotting)
• Electrophorese duplex DNA
• Treat with 0.5 M NaOH to melt duplex
• Tranfer contents of gel to nitrocellulose paper,
  which binds single-strand DNA tightly
• Vacuum dry paper at high temperature (80 C) to
  fix DNA in place
• Treat with small sequence of 32P-labeled
  complimentary probe
• Allow probe and DNA strand to hybidize, wash
  unbound probe, autoradiograph on X-ray film

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Fractionation of Nucleic Acids

• Ultracentrifugation
• Equilibrium density gradient in CsCl solution
  separates DNA by buoyant density (?) according to
  
• 1.660 0.098 XGC
• This can be used to determine G-C/A-T ratio
• Mitochondrial/chloroplast DNA can be separated
• Difference in density of single- vs.
  double-stranded DNA allows separation
• High density of RNA requires Cs2SO4 gradient
• rRNA Sucrose gradient is used rRNA is, in fact,
  classified and described by its sedimentation
  behavior

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Chromosome Organization

• Chromosomes DNA-RNA-protein complex called
  chromatin
• Structural features vary with cell cycle
• Chromatin exists as euchromatin (less dense) and
  heterochromatin (more dense)
• Histones Proteins of chromatin
• Nucleosomes First level of chromosome
  organization

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Histones

• 5 major classes H1, H2A, H2B, H3, H4
• All are rich in Arg and Lys

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Histones, continued

• Often post-translationally modified
• Methylation, acetylation
• All at conserved Arg, His, Lys, Ser, Thr
• All decrease positive charge
• Despite high degree of homology among species,
  degree of modification varies widely
• Weird example Lys 119 of 10 of H2As are bonded
  covalently to ubiquitin

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Nucleosomes

• Observations that led to elucidation of structure
• 4/5 histones are present in equal numbers in
  chromatin Half that number is H1
• Diffraction patterns indicate repeated, regular
  structures every ? 100 Å
• Purified DNA generates identical pattern when
  incubated with equimolar amounts of histones (not
  H1)
• Electron micrographs show 100 Å-diameter beads
  connected by thin strands
• Treatment of chromatin by double-strand-specific
  nuclease cleaved DNA between beads
• Crosslinking experiments indicated the following
  composition
• (H2A)2(H2B)2(H3)2(H4)2 per 200 bp DNA
• Further digestion revealed octamer above was
  associated with 146 bp (bead) and H1 with linker

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