Molecular Genetics

Frederick Miescher (1869): The First Discovery of DNA

  • Extracted a substance from the nuclei of white blood cells found in pus.

  • Called it “nuclein”(DNA) because it came from the nucleus.

  • Later renamed nucleic acid due to its acidic properties.

  • Discovery : DNA exists and is located in the nucleus, but its role in inheritance was still unclear

For many years, scientists believed proteins, not DNA, were the molecule of heredity because

  • Chromosomes are ~60% protein and ~40% DNA so proteins seemed more important.

  • Proteins are made of 20 different amino acids while DNA is made up of 4 nucleotides, making proteins seem complex enough to store genetic information and DNA not

Griffith’s Streptococcus pneumoniae Experiment

Griffith worked with Streptococcus pneumoniae, which causes pneumonia. He tested on mice. There are two strains (versions):

S Strain (Smooth):

  • Has a smooth outer capsule made of polysaccharides

  • Capsule protects the bacteria from the mouse’s immune system

  • Immune cells cannot recognize or destroy it

  • Result: mice die

R Strain (Rough, the bacteria looked rough under microscope):

  • No capsule

  • Easily recognized and destroyed by the immune system

  • Result: mice live

He then heat-killed the bacteria to see what effect it would have on the mice, and injected it as per usual, and if the bacteria were dead, they wouldnt be able to have any effect on the mouse. However, when he injected the heat-killed S Strain,

  • Something from the heat-killed S bacteria entered into the live R bacteria

  • The live R bacteria transformed into live S bacteria

  • It now had a capsule (as live S Strain does)

  • Killed the mice

He called it “Transforming Principle”

  • The ability to cause disease moved from one cell to another

  • The trait changed permanently

  • The new S bacteria passed virulence to future generations

  • Did not need to be a living cell to do so

  • Meant it had to be a molecule

  • All this highly hinted at DNA

Avery, MacLeod, and McCarty (1944): DNA Identified

At the time, scientists argued that it was RNA, or proteins, or something else.

They did an experiment and they used the same bacteria as Griffith:

  • S strain

  • R strain

What they did:

  • Killed S strain bacteria

  • Broke them open

  • Extracted all the cellular components

So now they had:

  • DNA

  • Proteins

  • RNA

  • Other molecules

They separated the components into different samples:

  • One sample with proteins

  • One sample with RNA

  • One sample with DNA

They did three tests:

  • Live R strain + Dead S strain proteins → mice live

No transformation

  • Live R strain + Dead S strain RNA → mice live

No transformation

  • Live R strain + Dead S strain DNA → mice die

R bacteria transformed into S bacteria

Then, they did a final test to confirm it was DNA:

  • They killed the DNA with an enzyme DNase

  • With the DNA destroyed, no transformation occured when dead S Strain injected

  • DNA is the transforming principle

Therefore,

  • DNA is the molecule of heredity

Hershey and Chase (1952): Conclusive Proof

  • Used bacteriophages (viruses that infect bacteria).

  • Viruses contain only protein and DNA.

  • Gave each radioactive atoms to track them

    • Protein with radioactive sulfur (³⁵S)

    • DNA with radioactive phosphorus (³²P)

What happened:

  • Phosphorus (DNA) entered bacterial cells.

  • Sulfur (protein) did not.

Meaning:

  • DNA enters the cell and directs viral replication.

  • DNA is definitively the hereditary molecule.

Chargaff (1949): Chemical Patterns in DNA

  • analyzed chemical composition of DNA

  • DNA is made of four nucleotides:

    • Adenine (A)

    • Thymine (T)

    • Cytosine (C)

    • Guanine (G)

  • Found consistent ratios:

    • %A = %T

    • %C = %G

  • Purines vs. Pyrimidines

    • Purines: A, G (two rings)

    • Pyrimidines: C, T (one ring)

  • Suggested base pairing, which was essential for discovering DNA’s structure.

Franklin and Wilkins: X-ray Crystallography

Franklin:

  • Used X-ray crystallography to photograph DNA.

  • Turned DNA into crystal then put through x-ray

  • Image showed:

    • DNA is helical

    • Uniform diameter

    • Repeating structure

Wilkins:

  • Shared Franklin’s data with Watson and Crick

Watson and Crick (1953): Double Helix Model

  • DNA is a double helix

  • Sugar-phosphate backbone on the outside

  • Nitrogen bases on the inside

  • Complementary base pairing:

    • A–T

    • C–G

    • This allows each DNA strand to serve as a template during replication, ensuring accurate copying of genetic information

  • Explained:

    • How DNA stores information

    • How DNA replicates

    • How mutations can occur

Structure of DNA

  • A nucleotide makes up DNA

  • Each nucleotide has 3 parts:

    1. Phosphate group (P)

      • Makes up the backbone of DNA

      • Connects to the sugar of the next nucleotide

    2. Sugar (S)

      • DNA = deoxyribose (missing an oxygen on carbon 2)

    3. Nitrogenous base (A, T, G, C in DNA)

      • Carries the genetic information

      • Forms connection of the DNA ladder by pairing with a complementary base

  • Double stranded

    • Two strands run antiparallel:

      • One strand: 5’ → 3’

      • Other strand: 3’ → 5’

    • Strands are connected by hydrogen-bonded base pairs

    • 3’ end → sugar with free OH on carbon 3

    • 5’ end → phosphate on carbon 5

    • DNA replication and enzymes work in this 5’ → 3’ direction

DNA Replication

  • Replication is semiconservative: each new DNA double helix has one parent strand and one new strand.

  • Each DNA molecule has two antiparallel strands: one 3′ → 5′, other 5′ → 3′.

  • Helicase unzips DNA

  • Replication bubble forms

  • Replication forks form at both ends

  • Topoisomerases (DNA gyrase) relieves supercoiling

  • SSBs (Single-strand binding proteins) prevent strands from rejoining

  • DNA polymerase adds nucleotides only to the 3′ end

  • RNA primase creates RNA primers

  • Primers provide a starting point for DNA synthesis

  • DNA polymerase III builds new DNA

  • Leading strand

    • Continuous synthesis

    • One primer

  • Lagging strand

    • Discontinuous synthesis

    • Multiple primers

    • Forms Okazaki fragments

  • DNA polymerase I

    • Removes RNA primers

    • Replaces them with DNA

  • DNA ligase

    • Joins Okazaki fragments

  • DNA polymerase III proofreads

  • DNA polymerase I and II repair errors

  • On the lagging strand, the final Okazaki fragment is copied

  • When the RNA primer is removed by DNA polymerase I, it is not replaced with DNA

  • Result: small sections of DNA are lost with each replication

  • Replication completes

  • Two identical DNA molecules formed

Eukaryotic cells and DNA Replication

  • Telomeres are repeating, non-coding DNA sequences at the ends of eukaryotic chromosomes

  • Telomeres protect important coding regions from being lost

  • Each time DNA is copied, telomeres become shorter

  • When telomeres become too short, the cell can no longer divide, hits hayflick limit (the maximum number of times a cell can divide)

  • The cell enters senescence

  • Senescence is a state where a cell is alive but permanently stops dividing

Aging

  • Aging occurs as more cells enter senescence

  • Aging results in reduced ability to repair tissues

  • Telomerase is an enzyme that can restore telomere length

  • Higher telomerase levels are found in populations that tend to live longer

Cancer Cells and Telomerase

  • Cancer cells divide uncontrollably

  • Normal cells enter senescence when telomeres become too short

  • Cancer cells continue dividing despite shortened telomeres

  • Cancer cells produce high levels of telomerase

  • Telomerase prevents telomere shortening

  • Cancer cells do not enter senescence

  • Cancer cells are considered “immortal”

Eukaryotic and Prokaryotic

  • Prokaryotic chromosomes consist of a single circular DNA molecule (Plasmids) and a single replication origin and bubble

  • Eukaryotic chromosomes are linear and appear X-shaped during cell division and and thousands of replication origins and replication bubbles

How DNA are chromosomes

  • Each chromosome consists of two chromatids, one inherited from each parent

  • Each chromatid is a long strand of chromatin made of DNA tightly coiled and wrapped

  • Chromatin is DNA wrapped around positively charged histone proteins, forming nucleosomes

  • Six nucleosomes coil together to form a solenoid