Designing and reading the classic experiments

A question put to nature

An experiment is a question that is put to nature in a way that gives a clear answer. The first step is not to do the experiment. It is to write down what each possible answer would show. If two ideas give the same result, the experiment cannot separate them, and you must change it.

The experiments of Meselson and Stahl, of Taylor, of Avery and his team, and of Hershey and Chase are good examples. You know what they found. Now we look at how they were designed, and how to work out what they would show, with numbers.

Three models, with numbers

Start with heavy DNA (15N) and move the cells to light nitrogen (14N). We describe a molecule by its heavy share: $1$ for a molecule that is all heavy, $0$ for one that is all light. The place of a band in the tube depends on this share.

Read the table with care.

• Generation 1 separates the conservative model from the other two. Only the conservative model gives two bands.
• Generation 2 separates the semiconservative model from the dispersive model. The first gives two bands. The second gives one band that has moved up.

Experiments, like this one, are made to give a different picture for each hypothesis.

You should find that only model 3 never gives a second band. In model 2, the hybrid band stays at the same place, $0.5$, while its share falls.

Tests that add more evidence

Heat the DNA. Heating separates the strands. If replication is semiconservative, a hybrid molecule gives one heavy and one light strand. The tube then shows two bands of single strands. If replication were dispersive, each strand would be a mixture, and the tube would show one band between the two. This test does not need a second generation. The share of heavy strands after $g$ generations is $\dfrac{100}{2^g}$ per cent.

A control. Mix cells from a heavy culture and cells from a light culture and take out their DNA together. You get two bands, heavy and light, and no hybrid. So a hybrid band cannot come from the way that the DNA is taken out. It must be made by replication in a growing cell.

The other direction. Grow cells in 14N, and then move them to 15N. After one generation you see one hybrid band, as before. After two generations you see a hybrid band and a heavy band, in equal amounts. The experiment is the mirror of the first.

The chromatids of Taylor

In the experiment of Taylor, cells copied their chromosomes once in radioactive thymidine, and then grew in ordinary medium. Follow a labelled strand. It never disappears, but it is shared out at every division.

Purification, and an activity for each milligram

Avery and his team did not just add enzymes. They also purified the transforming principle. A good measure is the activity for each milligram, which means the units of transforming activity divided by the milligrams of material. It rises as the extract becomes purer.

• Purification is the activity for each milligram, divided by its value in the crude extract.
• Yield is the activity that is left, as a per cent of the first activity.

A good step raises the activity for each milligram a lot, and it keeps a high yield. You will work with such numbers in the problems. If the activity for each milligram stays the same after a step, the step has removed nothing that matters.

Try it in Python

The program follows every molecule in the three models, for the first three generations.

The next program heats the DNA of the semiconservative model, and counts the heavy strands.

Practise with new numbers

Common mistakes

• Saying that the first generation proves the semiconservative model. The dispersive model gives the same band. The second generation decides.
• Mixing up the share of molecules and the share of strands. After heating, the share of heavy strands is half the share of hybrid molecules.
• Saying that the label of Taylor is lost. It is shared out. The labelled strands are still there.
• Forgetting a control. Without a mixed culture, a hybrid band could come from the way the DNA is taken out.
• Reading the activity for each milligram as the yield. The first is the purity, and the second is how much of the activity is kept.

Remember these

• Write down the prediction of each model before you look at the result.
• Generation 1: only the conservative model gives two bands. Generation 2: semiconservative gives two bands, dispersive gives one that has moved.
• After heating, the heavy strands are $100 \div 2^g$ per cent of the strands.
• Labelled chromatids at metaphase $k$: $100 \div 2^{k-1}$ per cent.

Check yourself

Try each one on your own first. If you are stuck, open a hint. Then check with Show the answer.

1. Why can the first generation not tell the semiconservative model from the dispersive one?

2. What would be the result of mixing heavy and light cells before taking out the DNA, and why is it useful?

3. Cells that grew in 14N are moved to 15N. What does the tube show after two generations?

4. What is the heavy share of the strands after heating the DNA of generation 3, in the semiconservative model?

5. In a purification, the activity for each milligram rises from 20 to 600 units, and the total activity falls from 8000 to 7300 units. What are the purification and the yield?

References

• Meselson M. and Stahl F. W. (1958). The replication of DNA in Escherichia coli. Proceedings of the National Academy of Sciences, 44, 671 to 682.
• Taylor J. H., Woods P. S. and Hughes W. L. (1957). The organization and duplication of chromosomes as revealed by autoradiographic studies using tritium-labeled thymidine. Proceedings of the National Academy of Sciences, 43, 122 to 128.
• NCERT (2023). Biology, Textbook for Class XII, chapter 5, Molecular Basis of Inheritance.