DNA History and Experiments

• 1869: Friedrich Miescher discovered nuclein, which contains carbon, hydrogen, oxygen, nitrogen, and phosphorus, and is found in the nucleus.
• Early 1900's: Chromosomes were found to contain genetic information, including protein, DNA, and RNA.

Griffith's Transformation Experiment

• R-Strain: Benign, rough, lacks polysaccharide coat.
• S-Strain: Has a shiny coat, virulent, and will cause pneumonia and death.
• Living R strain was transformed into S strain.
• The mechanism was unclear to Griffith.

Avery's Experiments

• Sought to identify the factor responsible for genetic transformation.
• By extracting fractions of bacteria, it was found that DNA is the macromolecule responsible for transformation.

Hershey-Chase Experiments

• Determined what the carrier of genetic information is.
• Bacteriophage T2, composed of 50% DNA and 50% protein, was used.
• Phosphorous is a component of DNA, so labeled DNA gets progeny phages with labeled DNA.
• Sulfur is only found in protein, so it's exclusive to it.
• The bacterial cell is transformed into a phage factory until it lyses.
• The hypothesis was that the injected part was the genetic information used in organisms.
• Radioactive-labeled phages
• Blender: Used to remove phage ghosts from the bacterial cell.
• Centrifugation: Radioactivity was in the pellet with P32, and in the supernatant with S35 because protein was not injected into the cell.

Nucleotides

• Composed of 5-carbon pentose sugars.
• RNA has a 2' OH group.

Nitrogenous Bases

• Purines: Consist of 2 rings.

• Pyrimidine: Consists of a single ring.

• Phosphate group is present.

• Nucleotides can contain 1, 2, or 3 phosphates.

• dNMP's, dNDP's, dNTP's

Polynucleotide Chains

• Have polarity.
• Phosphodiester linkages exist between the 3' OH group and the 5' phosphate group.
• Built in the 5' to 3' direction.

Erwin Chargaff's Base Composition Studies

• Approximately 50% of bases are purines (A, G).
• Approximately 50% are pyrimidines (C, T).

Franklin & Wilkins: X-Ray Diffraction Studies

• DNA is helical
• There are 0.34 nm and 3.4 nm regularities.

DNA structure

• Complementary base pairs are A-T and C-G.
• More C-G pairs result in increased difficulty to denature due to 3 hydrogen bonds.

Watson and Crick's Model

• Two antiparallel polynucleotide chains wound around each other in a right-handed helix.
• Sugar-phosphate backbones are on the outside of the double helix.
• Bases are 0.34 nm apart, connected by hydrogen bonds.
• 10 bases make 1 full turn.
• Grooves of unequal size form between backbones.
• Helix has a diameter of 2 nm.

RNA Structure

• Includes rRNA, mRNA, tRNA, etc.
• Single-stranded and can bind to itself when possible.

DNA application

• 1c of DNA could hypothetically store 100 million hours of HD video.
• The entire internet could fit in a 4L jug.

Experimentally Manipulating DNA

• Separation is done kinetically
• Denaturation can be studied experimentally.

Melting Temperatures

• Can be observed in the lab by spectrometer.
• Labs measured at 260 nm.
• Labs increase when strands separate.
• Tm is the temperature at which 1/2 of absorbance change is reached.
• Hyperchromatic shift: UV light is shielded by bases when the double helix is present, hence why it's absorbed.
• Bases are less shielded when single-stranded.

Factors Affecting Tm

• GC content: Harder to break because of hydrogen bonds.
• Base stacking: van der Waals forces between a single strand.
• Degree of complementarity: Strands aren't always fully bonded together; more bonds mean it's harder to break.

Bioinformatics

• Combination of computer science and biology.
• Aims to determine regions of the genome that correspond to genes, what proteins genes encode, how genes influence each other, and how they function.
• Genome: all the genetic material of an organism.
• Transcriptome: what mRNA is being made
• Proteome: all the proteins produced or modified by an organism or system
• Microbiome: bacteria with us
• Myobiome: fungi with us

Genome Information

• Approximately 20,000 protein-coding genes.
• Protein-coding genes make up about 2% of the genome.
• The sequencing project was completed in 2003.

Comparing Genomes

• Similarity between two human genomes is about 99.7%.
• Single Nucleotide Polymorphisms (SNPs).
• Copy number variation (CNVs).
• Most phenotypic differences are due to SNPs.

Repeat DNA

• 1960s: Britten & Kohne discovered.
• Experiment involved lysing DNA samples, denaturing them, and measuring the rate of denaturation, repeating sections come together more quickly.
• Compared bacteria (diverse, not a lot of repetitive DNA) to bovine.

Tandem Repeated DNA

• Satellite DNA / Simple Sequence Repeats: ~10-15% of the genome.
• Regular satellite: 10^5 - 10^7 bp.
• Minisatellite: 10^2 - 10^5.
• Microsatellite: 10^1 - 10^2.
• Usually in non-coding regions and doesn't cause phenotypic change.
• Exceptions include Huntington's disease, fragile X syndrome, and muscular dystrophy.
• Present in centromeres and telomeres; given by repeats.

DNA fingerprinting

• Restriction Fragment Length Polymorphisms (RFLP).
• Minisatellite flanked by restriction enzyme cut sites.
• Analysis of short repeated sequences (STR's) is now done instead of RFLPs.
• Criminal cases test 13 different STR sites.
• Odds of two of the same: 1 in 10^5

Interspersed Repeated DNA

• ~25-50% of the genome.
• Scattered throughout, up to 10,000 bp long.
• Consists of families of transposons, which move around and leave copies of themselves behind.
• LINES (long) 6000-8000bp; contain the genes required for their own mobilization.
• SINES: <500bp; doesn't contain the genes required for their own mobilization.

Bacterial Chromosomes

• Usually circular
• Located in the nucleoid region of the cell
• Localized by non-DNA components (proteins + RNA molecules)
• Negatively supercoiled
• One loop is approximately 20,000 bp.

Supercoiling

• Twisting the double helix into a more compact form.
• Originally found in circular plasmids and viral DNA.
• Linear DNA can supercoil in regions where DNA is anchored, e.g., scaffold protein.
• Twisted in the opposite direction as the DNA helix -> LEFT HANDED
• Same direction as the DNA helix -> RIGHT HANDED

Topoisomerases (TI's)

• Convert DNA between relaxed and supercoiled states
• Type 1: makes nicks in one strand, causing relaxation of supercoiled form.
• Type II: Uses ATP to introduce OR relax supercoiling, requires a double-strand break.
• DNA gyrase is an example (bacteria).

Bacterial Plasmids

• Small, circular DNA
• Replicate autonomously, made on their own inside a bacterial cell.
• Types: F (fertility), R (resistance), virulence, metabolic, colicinogenic, cryptic.

Eukaryotic Chromatin

• Eukaryotic DNA is bound to a complex series of proteins
• Forms fibers of 10-30 nm in diameter: chromatin
• During cell division, chromatin fibers condense into chromosomes.

Histones

• Proteins that help organize DNA into chromosomes (structural support).
• Bound to DNA by ionic bonds
• The 5 proteins: H1, H2A, H2B, H3, H4

Nucleosomes

• Repeating structural units on chromatin = beads on a string
• Each contains a histone protein core, an octamer of 8 histones made of dimers of H2A, H2B, H3, H4
• ~146 bp wrapped around a ~10nm diameter

H1 + Chromatin

• Nucleosome formation is the first step in DNA packaging.
• Second step is when linker DNA binds to linker histone (H1), increasing diameter from 10 to 30nm, by attaching to a nudeosome
• Nucleosomes undergo further packaging.

Further Packaging of DNA

• 30nm fibre is packed into a zigzag.
• then fold into looped domains of 50,000-100,000bp (300nm)
• euchromatin
• looped domains attach to insoluble protein network

Histone + Chromatin Modification

• Remodeling alters level of gene expression

Heterochromatin

• Regions of DNA not actively transcribed, more compacted.
• Looped domains fold onto each other
• Low accessibility to DNA.
• ~700nm diameter

Euchromatin

• Actively being transcribed, so more loosely packed

Nuclear structure

• Mems are separated by ~30nm perinuclear space
• Outer mem is continuous with endomem system + studded in places with ribosomes
• Also contains proteins that bind actin + IF with cytoskeleton
• Nuclear pores allow passage in + out of nucleus
• Nuclear pore complex, multi subunit complex with diameter of ~120mm (30nucleoporins)
• Centre of pore is filled with large transporter protein

Nucleus

• Site of chrom storage, transcription + ribosome synthesis
• Boundary defined by a nuclear envelope
• IN: proteins for transcription or chrom replication, ribosomal proteins
• OUT: tRNA, mRNA, rRNA, ribosomal subunits for protein synthesis

Nuclear import

• Proteins that are syn in cytoplasm + function in nucleus contains a nuclear localization signal (NLS)
• 8-30 AA's, like a fake ID
• Proline, Lysline, Arginine
• A protein called importin in the cytoplasm recognizes the NLS + binds to the protein
• Complex then moves to the nucleus via transporter protein at center of nuclear pore complex
• Inside the nucleus
• RAN activated by RCC1
• RAN GTP binds to importin
• importin releases cargo
• RAN GTP importin transported out of nucleus through NPC
• In cytoplasm
• GTP hydrolyzed by RAN GAP (turns off RAN)
• Importin is released
• RAN GDP taken back into nucleus by NTF2

Nuclear export

• Nuclear export signal on adaptor proteins that bind to RNA cargo "NES"
• Exportin proteins bind RAN-GTP

Nucleoskeletons

• Help maintain shape + provide a skeleton for chromatin fibers
• nuclear lamins localize to inner surface of inner mem
• intermediate filaments
• If defected, e.g. Hutchinson-Gilford progeria syndrome

Chromosome territories

• The chromatin of each chromosome has its own location
• Observed using Fluorescence in situ hybridization (FISH)
• Influences gene expression
• Territory is quite vague, some closer to boundary, some closer to the center; migrates to center if it is being used more, more peripheral if not
• Can vary during lifetime of cell + between cells

Nucleolus

• Where ribosomal subunits are assembled
• Size varies with activity (liver vs. fat cell)
• Two major regions:
• Fibrils: DNA being transcribed into rRNA
• Granules: rRNA's being packaged with proteins.

Nucleolus organizer region (NOR)

• Stretch of DNA containing many copies of rRNA genes

Bacterial DNA Replication

• Watson-Crick model describes DNA as a double-stranded helix that unwinds and acts as a template.
• Three potential mechanisms:
  • Semiconservative replication
  • Conservative replication
  • Dispersive replication
• Meselson + Stahl:
  • Grew bacteria in ^15N.
    • Gets incorporated into DNA.
    • Centrifuging gives distinct band (heavy).
  • Grew bacteria for 1 gen in ^14N.
    • Gets incorporated into DNA.
    • Centrifuging gives distinct intermediate band.
  • Grew for 2nd gen in ^14N.
    • Centrifuging gives 2 bands: intermediate and light.
• Begins at a replication origin (Ori).
• Replication forks.
• Proceeds bidirectionally.
• Theta (θ) rep.
• Once finished, linked circles are separated by Topoisomerase.

Bacterial origin of rep

• Consensus seq's high in AT base pairs because it takes less energy to break apart 2 H bonds of A-T than the 3 between G-C.