Chapter 18: Structure of DNA

• Replication is only for DNA

• DNA is transcribed into RNA
• RNA is translated into protein
• Due to protein varying so much, it was thought in the 1800’s to be genetic code
• Timeline of DNA
○ Late 20’s: Griffith Transformation Experiment


 Transformation is uptake of naked DNA into cell
 Conclusion: the bacteria can pass something on
 Griffith’s most intriguing disc:-pneumonia can be injuced by injecting animals with a mixture of
life R-strain bacteria and dead S-strain bacteria.
 Genetic transformation- is referred to the active (though still unknown) substance in the S
cells as the “transforming principle”

○ 40’s: Hershey and Chase Experiment
 Used a bacteriophage
• Combined with radioisotopes P and 35S
32

○ 32
P will show up in DNA
○ 35
S will show up protein
• Put mixture of each in a blender (Isotope, phage, and host)
○ In P, supernatant is hot
32

○ In S, pellet is hot
35

○ Chargoff
 Analyzed DNA from different species
 Percentages differ with base pairs but stay the same within species
 Also discovered that G amount = C amount and T amount = A amount

January 15, 2009

• A worldwide frenzy started to figure all of this out

• Watson and Crick

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 ○ Didn’t do experiments, just interpreted data
• Wilkins and Franklin
○ X-ray crystallographers
○ Wilkins showed Watson and Crick Franklin’s data
• Watson and Crick figured out the mystery in 1953
• Watson’s next involvement was with the human genome project by lobbying congress
• Bottom Line: DNA Structure
○ Double helix with sugar-phosphate backbone
 Sugars have Nitrogen containing bases
○ Right hand helix with minor and major grooves
○ If a big protein (or whatever) pushes in the major groove, the structure can bend
○ 1 helix twist in 3.4 nm
○ 10 base pairs/turn
○ Bacterial genome: 3 million base pairs
○ Human genome: 3 x 109 base pairs
○ Β-DNA is the most common DNA form
○ Z-DNA is left-handed caused by some base pair combos
○ A-DNA is usually seen only in lab situations from a lack of salt
• How to build DNA
○ Building blocks are nucleotides
 Nucleotide has ribose, 5C sugar, N-containing ‘base’ and a phosphate group

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  A and G are purines (2 rings) and C and T are primidines (1 ring)
○ ATP is ribonucleotide, denoted dATP for the difference
 For the three phosphates, they are named α, β, γ (α is closest to the structure)
○ RNA has uracil instead of thymine
○ Dehydration reaction adds nucleotides (remove water to connect monomers)
○ dATP, dCTP, dGTP, dTTP are substrates for replication
○ γ and β leave as pyrophosphate
○ add to 3’ end
○ makes phosphodiester bond (C-O-P-O-C)
○ Nucleic acid strain is highly negatively charged
○ 5’ end has free phosphate
○ 3’ end has free hydroxyl group
○ Backbone is held together by the phosphodiester bonds
○ Strands must be aligned with an inverted strand
○ H bonding (bonding with partial charges) is responsible for holding nucleotides together (A-T and G-C)
○ C-G has 3 bonds while A-T has 2
• RNA is typically single stranded
• DNA supercoils to relieve extratorsional strain
○ Topoisomer: how the DNA physically is (supercoiled or normal)
○ Topoisomerases can convert 1 topoisomer to another

January 20, 2009

• Hydrogen bonds hold the bases together

• Double-stranded DNA becomes single stranded by melting or denaturation
○ They can re-pair by renaturation
○ Done by temp changes, pH, salt, strong polar solvents
• Can measure DNA with spectrophotometer
○ Necleic acids absorb at 260nm
○ There is an absorbance shift as double stranded becomes single stranded
○ Tm: melting temperature, temp to which half the molecules are melted
○ Can use melting to introduce mutation (very small ones, albeit)
• Hybridization: single stranded going back to double stranded
• G to C content is not only important but also where the G-C is
• Humans have LOTS of repeats, making pairing faster
• Cor curve: concentration of DNA
○ Temp is constant
• Repeated DNA
○ Tandem repeats (same chunk over and over)
 Size and repeat numbers vary
○ Repeats of certain genes is associated with disease
○ Number of repeats varies heavily from person to person
○ Can occur not in tandem as well
 Due to mobile ‘jumping DNA’
• Over 90% of human DNA codes for shit
• DNA packaging
○ Bacterial DNA
 Most bacterial chromosomes are circular
 Have (+) proteins holding structure
 Also have plasmids (circular extra-chromosomal DNA)

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 ○ Mammal DNA Packaging
 DNA is wrapped around (+) charged proteins
 Chromatin: DNA and associated proteins
 Histone: basic proteins that associate with DNA
• 8 histones pair up to form a nucleosome
• H1 sits inbetween
 30nm fiver, DNA chilling around nucleosome in a solenoid-ish form (a bit kinked)
 These further compress to form looped domains
• Held in loops by scaffold proteins

Lab January 20, 2009

STOP: Plasmid Time!

• Copy number of plasmid varies

• Ori: origin of replication
• pUC 19 (2685 bp)-our plasmid
• Plasmids can contribute
○ F plasmid
○ Antibiotic resistance
○ Virulence factors
• pUC has ampicillin-ase (β-lactamase)
○ Sometimes called βla or ampr to denote resistance
○ Allows you to select for only transformed cells
• Transformation
○ Bacteria
 E. coli strain DH5α recA1 is our choice
○ Make competent cells
 Method 1) Wash in H2O and electroporate
 Method 2) Wash in Ca2= or some other dication
○ Transformation efficiency
 106-108 transformations/µg DNA
• Procedure
○ Thaw out competent cells
○ Add pUC (.5µg/µl)
○ 10 minutes on ice
○ Heat shock-5 min
○ Add broth, dilute, and plate (make sure you have some antibiotic!)

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