Difficulty: Introductory to Intermediate | Prerequisites: Basic cell biology, familiarity with DNA/protein structure from Chapter 1
This section traces how scientists figured out that DNA, not protein, carries genetic information. That conclusion was not obvious at the time. For decades, proteins were the leading candidate because they seemed more chemically complex. A series of elegant experiments between 1928 and 1953 gradually overturned that assumption and culminated in Watson and Crick's double helix model. If you are coming in cold, the key thing to know is that chromosomes contain both DNA and protein, and the central question here is: which one actually encodes heritable traits?
Researchers moved from complex organisms to simpler ones (bacteria, phage) to study genetics more efficiently. Through transformation experiments (Griffith, Avery), isotope labelling (Hershey-Chase), base-ratio analysis (Chargaff), and X-ray diffraction (Franklin), the field established that DNA is the genetic material. Watson and Crick then solved DNA's double-helix structure in 1953.
One gene – one enzyme hypothesis
The idea, proposed by Beadle and Tatum (1941), that each gene encodes a single enzyme. In simple terms, one stretch of DNA is responsible for producing one specific protein catalyst.
Transformation
The process by which non-virulent bacteria acquire virulence by taking up DNA from dead virulent bacteria. Think of it as one bacterium "picking up instructions" from another, even a dead one, and gaining a new trait.
Tetranucleotide hypothesis
Phoebus Levene's proposal (1910) that DNA was a simple, repeating four-nucleotide polymer with no informational capacity. This was wrong, but it dominated thinking for about 40 years and steered researchers towards proteins as the genetic material.
Chargaff's Rules
The observation that in any DNA sample, A = T and G = C (by percentage). In simple terms, the amount of adenine always equals thymine, and guanine always equals cytosine. This disproved the tetranucleotide hypothesis and hinted at base pairing.
Bacteriophage (phage)
A virus that infects bacteria. Phage T2, used in the Hershey-Chase experiment, has a DNA core surrounded by a protein coat (capsid).
Phosphodiester bond (3' to 5')
The covalent linkage that connects nucleotides in a DNA or RNA chain, joining the 3' carbon of one sugar to the 5' carbon of the next via a phosphate group.
Bacteria like E. coli are haploid, easy to grow, and divide roughly every 20 minutes.
From a single cell, 8 hours of growth yields about 16.8 million bacteria (N = 1 x 2^n), with several thousand mutants. This makes mutations far easier to find than in pea plants or fruit flies.
Mutation rate in bacteria is in the order of 10^-7 per base per generation (roughly 1 error in every 100 to 1,000 cells).
Studied the mould Neurospora crassa and irradiated spores with X-rays to create mutants.
Some mutant strains could not grow without the amino acid arginine being added to the medium.
They identified four arginine-dependent strains, each lacking a different enzyme in the arginine biosynthetic pathway.
The pathway is sequential: glutamate → ornithine → citrulline → argininosuccinate → arginine. A mutation in one enzyme blocks production downstream, but supplying the intermediate past the block rescues growth.
Nobel Prize in 1958. Established the one gene – one enzyme hypothesis, supporting Garrod's earlier work.
E. coli was adopted as a model organism because it is nonpathogenic, haploid, biochemically versatile, and fast-growing.
UV light induces mutations in biosynthetic pathways.
Replica plating is a technique to identify mutants: grow colonies on complete media, transfer to minimal media with velveteen, use penicillin to kill wild-type (rapidly growing) cells, then cycle back to complete media to enrich the mutant population.
In the 1800s, biochemists classified macromolecules. Lipids and carbohydrates are relatively simple (homopolymers or repetitive heteropolymers). Proteins, however, are non-repetitive heteropolymers built from 20 amino acids.
It seemed like only proteins had the complexity to carry information: 20 amino acids versus only 4 nucleotide bases.
Levene's tetranucleotide hypothesis reinforced this bias by treating DNA as a boring, repetitive molecule.
A key logical argument: building a 100-amino-acid protein would need 100 ordering enzymes, each of which would need its own enzymes, and so on. Enzymes alone cannot specify non-repetitive sequences. Therefore, information must come from genes.
Frederick Griffith worked on Streptococcus pneumoniae after the 1918 flu pandemic.
Two strains: Rough (R), non-pathogenic, lacking a capsule; Smooth (S), pathogenic, with a polysaccharide capsule protecting it from the immune system.
Heat-killed S bacteria mixed with live R bacteria transformed the R into virulent S in mice.
This was the first demonstration of transformation, showing that genetic traits are transferable and not permanently fixed.
After 10 years of work, they identified the transforming agent as DNA using cell-free extracts (no mice needed).
They treated S-strain extracts with enzymes that degrade lipids, sugars, proteins, and RNA, with no effect on transformation. Only deoxyribonuclease (DNase, from dog intestinal mucosa) destroyed the transforming ability.
Conclusion: DNA carries genetic specificity.
The experiment was largely ignored by the scientific community at the time; Erwin Chargaff and Hermann Muller were among the few who recognised its significance.
Used paper chromatography and UV spectrophotometry to measure base percentages.
Found A ≠ G and T ≠ C, but A ≈ T and G ≈ C across species. For example, in humans: A = 30.9%, T = 29.4%, G = 19.9%, C = 19.8%.
This disproved the tetranucleotide hypothesis and showed that base composition varies between species, consistent with DNA carrying genetic information.
Alfred Hershey and Martha Chase used bacteriophage T2, which has a DNA core and a protein capsid.
Labelled phage proteins with ^35S and DNA with ^32P.
Infected bacteria, then sheared off empty protein coats ("ghosts") by violent agitation.
Result: ^32P (DNA) was found inside bacteria and in progeny phage; very little ^35S (protein) entered the host.
Conclusion: DNA, not protein, is injected into the host and carries the genetic programme.
Note: this approach worked because phage separates its DNA from its coat upon injection. Animal viruses enter cells whole, so the same experiment would not have produced clear results.
Rosalind Franklin's Photo 51 confirmed DNA is helical and composed of two strands, and allowed key measurements of spacing.
Watson and Crick saw her data without her knowledge, providing a crucial piece of their structural model.
Alexander Todd showed that nucleotides in DNA are linked by 3' to 5' phosphodiester bonds.
Combined Chargaff's rules (A = T, G = C) and Franklin's diffraction data to build a structural model: two antiparallel, complementary helical strands.
The complementary structure immediately suggested a mechanism for replication: each strand could serve as a template for its partner.
Nobel Prize in 1962.
Bacterial growth: N_t = 1 x 2^n (where n = number of generations)
Mutation rate: ~10^-7 per base per generation
8 hours of growth from 1 cell ≈ 16,777,216 bacteria
Chargaff's Rule: %A ≈ %T; %G ≈ %C
Griffith's transformation is the conceptual ancestor of modern genetic engineering. When scientists today insert plasmid DNA into bacteria to produce insulin or other proteins, they are exploiting the same principle: cells can take up foreign DNA and express its genes. Chargaff's rules also underpin every DNA sequencing technology, since complementary base pairing is how sequencing reads are generated and aligned.
Students often assume Griffith knew DNA was the transforming substance. He did not. He showed transformation occurs but did not identify the molecule responsible.
The Hershey-Chase experiment is sometimes described as the definitive proof that DNA is the genetic material. It was strong evidence, but Avery's earlier work was the first direct chemical demonstration; Hershey-Chase confirmed and extended it.
Chargaff's rules (A = T, G = C) apply to double-stranded DNA, not to single-stranded DNA or RNA. Students sometimes apply them where they do not hold.
Watson and Crick did not discover DNA or perform experiments on it. They built a structural model by integrating other people's experimental data.
⚠️ Know the chronological order: Griffith (1928) → Avery (1944) → Chargaff (1949) → Hershey-Chase (1952) → Franklin (1952) → Watson and Crick (1953).
⚠️ Be able to explain what each experiment proved and what it did not prove.
⚠️ Understand why proteins were originally favoured (20 amino acids vs. 4 bases; tetranucleotide hypothesis).
⚠️ Replica plating and the logic of sequential biochemical pathways (blocking an enzyme, supplying intermediates) are commonly tested.
True or False: Griffith identified DNA as the transforming principle.
Fill in the blank: In Chargaff's analysis, the percentage of ______ always approximately equals the percentage of thymine.
True or False: In the Hershey-Chase experiment, ^35S labelled the DNA.
Fill in the blank: Beadle and Tatum's work led to the ______ hypothesis.
True or False: The tetranucleotide hypothesis correctly predicted that DNA has sufficient complexity to encode genetic information.
Answers: 1. False (he showed transformation but did not identify the molecule). 2. Adenine. 3. False (^35S labelled protein; ^32P labelled DNA). 4. One gene – one enzyme. 5. False (it incorrectly suggested DNA was too simple).
Q: Why did early geneticists favour proteins over DNA as the genetic material?
A: Proteins are non-repetitive heteropolymers composed of 20 different amino acids, which appeared to offer far more informational complexity than DNA's four nucleotide bases. Levene's tetranucleotide hypothesis further suggested DNA was a simple, repeating polymer with no capacity to encode information.
Q: In Avery's experiment, which enzyme treatment destroyed the transforming ability of S-strain extracts, and what did this demonstrate?
A: Deoxyribonuclease (DNase) destroyed the transforming ability. This demonstrated that DNA, not protein, lipid, or RNA, was the molecule responsible for transformation.
Q: In the Hershey-Chase experiment, why was bacteriophage T2 an ideal system for distinguishing between DNA and protein?
A: T2 phage injects its DNA into the host cell while leaving its protein coat outside. This physical separation allowed Hershey and Chase to label each component differently (^32P for DNA, ^35S for protein) and track which one entered the cell and appeared in progeny. Animal viruses would not have given such clean results because they enter cells whole.
Q: How does the sequential pathway logic in Beadle and Tatum's experiment work? If enzyme F is mutated, what happens?
A: The pathway runs glutamate → (E) → ornithine → (F) → citrulline → (G) → argininosuccinate → (H) → arginine. If enzyme F is mutated, ornithine accumulates and citrulline is not produced. Supplying citrulline (or anything downstream) rescues growth, but supplying ornithine does not.
This material connects directly to Chapter 1's discussion of how genetics moved from studying peas and fruit flies to microorganisms. It also sets the stage for Part 2 of these notes (DNA replication, Meselson-Stahl), since Watson and Crick's complementary structure immediately implied a replication mechanism. In a broader sense, the experiments here laid the groundwork for all of molecular biology, recombinant DNA technology, and modern genomics.
genetic material discovery, Griffith transformation experiment, Avery MacLeod McCarthy, Hershey-Chase experiment, Chargaff's rules, base pairing, tetranucleotide hypothesis, one gene one enzyme, Beadle and Tatum, Neurospora, bacteriophage T2, Rosalind Franklin Photo 51, Watson and Crick double helix, DNA structure history, replica plating, E. coli genetics, phosphodiester bonds, nucleic acids chapter 2