Semiconservative replication and the Meselson-Stahl… | StemPy Lab
Course 23.2·Lesson 1 of 3
Semiconservative replication and the Meselson-Stahl experiment
Before a cell divides, all of its DNA is copied. Watson and Crick suggested that each strand is a template for a new one, and Meselson and Stahl proved it with heavy nitrogen and a density gradient. After one generation the DNA is hybrid, and after two generations half is hybrid and half is light. So replication is semiconservative.
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Copying the plan
Before a cell divides, it makes a second copy of all its DNA, so that each daughter cell gets a full set. How can a molecule copy itself?
Watson and Crick had an answer in 1953. The two strands are held together by base pairing, and each base has only one partner. So if the strands come apart, each one can serve as a template, a pattern, for a new strand. The new strand is made by pairing each base of the template with its partner.
After the copying, each double helix has one old strand and one new strand. This scheme is called semiconservative replication. It is half conserved, because half of each new molecule is old.
Three ways in which DNA could be copied
This part is not in the NEET syllabus, but A-Level and IB ask it. We need it to see why the experiment was done. There are three possible ways to copy.
A grid of three rows and three columns that shows the three models of replication, with DNA molecules drawn as pairs of vertical bars, dark blue for an old 15N strand and pale blue for a new 14N strand. The columns are the parent, generation 1 and generation 2. Conservative row: the parent is two dark strands. In generation 1 there is one all-dark molecule and one all-pale molecule. In generation 2 there is one all-dark and three all-pale molecules. Semiconservative row: generation 1 has two molecules, each with one dark strand and one pale strand. Generation 2 has two such hybrid molecules and two all-pale molecules. Dispersive row: in generation 1 both molecules have strands that are half dark and half pale, and in generation 2 all four molecules have strands that are one quarter dark and three quarters pale. A caption under each row says what the tube would show in generation 1.
Model
What the daughter molecules hold
Conservative
The old molecule stays whole, and a new molecule is made entirely of new strands
Semiconservative
Each daughter has one old strand and one new strand
Dispersive
Old and new pieces are mixed along the strands of both daughters
How could anyone tell them apart? The old and new DNA had to be told apart in a tube. In 1958, two scientists found a way.
The experiment of Meselson and Stahl (1958)
Matthew Meselson and Franklin Stahl used E. coli, a bacterium that divides about every 20 minutes.
They grew the bacteria for many generations in a medium with 15NH4Cl. The only nitrogen that the cells could use was 15N, a heavy isotope of nitrogen. It is not radioactive. All the DNA became heavy.
They moved the cells to a medium with ordinary nitrogen, 14NH4Cl. New DNA was now made from the light nitrogen.
They took samples of the cells at fixed times, and took out the DNA.
They spun the DNA in a solution of caesium chloride (CsCl). In a long spin, the salt forms a density gradient, so that the solution is denser at the bottom of the tube. Each DNA settles at the place where its density equals that of the solution. Heavy DNA lies lower, and light DNA lies higher.
Five centrifuge tubes in a row, drawn as tall rounded tubes, that show where the DNA lies in a gradient of caesium chloride after spinning. Heavy DNA lies low in the tube, light DNA high, and hybrid DNA in the middle. Tube 1, the start in the 15N medium: one dark band low in the tube, 100 per cent. Tube 2, one generation in 14N after 20 minutes: one band in the middle, 100 per cent hybrid. Tube 3, two generations after 40 minutes: a hybrid band in the middle and a light band above it, 50 per cent each. Tube 4, three generations: hybrid 25 per cent and light 75 per cent. Tube 5, four generations: hybrid 12.5 per cent and light 87.5 per cent.
Time in the 14N medium
The tube shows
Start
One heavy band
After 20 min (1 generation)
One band in the middle, a hybrid of 15N and 14N
After 40 min (2 generations)
Two bands, hybrid and light, in equal amounts
Later
The light band gets stronger, and the hybrid band weakens
Reading the result
After one generation there is only one band, and it lies between heavy and light. So every molecule has one heavy and one light strand. The conservative model would give two bands, a heavy one and a light one, so it is wrong.
After two generations there are two bands. A dispersive model would still give a single band, a little lighter than before, so it is wrong too.
Only the semiconservative model fits both results.
Why exactly two hybrid molecules?
Start with one heavy molecule. It has two old strands. In every generation, each strand is copied, but the old strands never disappear. They stay, one in each of two molecules. So there are always 2 hybrid molecules, and all the others are light.
After n generations there are 2n molecules. The share of the hybrid is
hybrid=2n2
You should find that the hybrid share halves in each generation, and that the light DNA grows to nearly all of the DNA, but the two hybrid molecules never disappear.
Chromosomes copy in the same way
In 1957, J. Herbert Taylor and his colleagues used root tips of the broad bean, Vicia faba. NCERT gives the year as 1958. They gave the cells radioactive thymidine, a building block that only DNA uses. The labelled chromosomes showed that the DNA of chromosomes is also copied semiconservatively. After the first division both chromatids of a chromosome were labelled. After the second division, in ordinary medium, only one of the two chromatids was labelled.
When does the copying take place?
The DNA is copied in the S phase of the cell cycle. The copying and the division of the cell must be well timed. If the DNA is copied but the cell does not divide, the cell gets extra sets of chromosomes. Such a cell is polyploid.
Try it in Python
The program lists the heavy, hybrid and light DNA for the first generations.
Practise with new numbers
Common mistakes
Saying that the 15N is radioactive. It is a heavy isotope, and it is not radioactive.
Saying that the hybrid DNA disappears. The two hybrid molecules stay for ever, but their share becomes small.
Saying that one generation gives two bands. It gives one band, in the middle. Two bands are the sign of the conservative model.
Forgetting the time. One generation of E. coli takes about 20 minutes, so two generations take 40 minutes.
Mixing up the heavy and the light. The heavy DNA lies lower in the tube.
Remember these
Semiconservative replication: every new double helix has one old and one new strand.
Meselson and Stahl (1958): 15N, then 14N, caesium chloride gradient. After 20 min one hybrid band. After 40 min hybrid and light in equal amounts.
After n generations, the hybrid share is 2÷2n.
Taylor used Vicia faba and radioactive thymidine to show the same in chromosomes.
Check yourself
Try each one on your own first. If you are stuck, open a hint. Then check with Show the answer.
Why did Meselson and Stahl use caesium chloride in the centrifuge?
What did the tube show after one generation in the 14N medium, and what does this rule out?
After two generations, what did the tube show?
What share of the DNA is hybrid after three generations in the 14N medium?
What did the experiment of Taylor show?
References
NCERT (2023). Biology, Textbook for Class XII, chapter 5, Molecular Basis of Inheritance.
Clark M. A. et al. (2018). Biology 2e, OpenStax, chapter 14, section 14.3, Basics of DNA Replication.
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.
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