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Q.Given below is a list of steps Meselson and Stahl carried out in their experiment to prove that DNA replication is semi-conservative. Select the option that gives the correct sequence of steps followed by them.

(i) Bacteria transferred to a N14 medium and sampled every 20 minutes.
(ii) All bacteria contain hybrid DNA (N14 DNA and N15 DNA).
(iii) Bacteria grown in N15 medium for many generations.
(iv) All bacteria contain N15 DNA.
(v) Bacteria contain either all N14 DNA or all hybrid DNA.
(a)
(ii) →
(iv) →
(iii) →
(i) →
(v)
(b)
(i) →
(ii) →
(v) →
(iv) →
(iii)
(c)
(iii) →
(iv) →
(i) →
(ii) →
(v)
(d)
(iv) →
(iii) →
(ii) →
(v) → (i)
CBSECBSE Class XII Board 2023MCQ· 1mImportance★★★★★
✓ Free question

Meselson and Stahl's experiment proved DNA replication is semi-conservative by tracking heavy nitrogen (15N) labeled DNA through successive generations in a light nitrogen (14N) medium.

The discovery of DNA's double helix structure by Watson and Crick in 1953 immediately raised a crucial question: how does this molecule replicate itself to pass genetic information accurately from one generation to the next? The structure itself, with its complementary base pairing (adenine always pairing with thymine, and guanine with cytosine), offered a strong hint. If the two strands of the double helix could separate, each strand could then serve as a template for the synthesis of a new complementary strand. This idea formed the basis of what is known as semi-conservative replication.

However, before Meselson and Stahl's groundbreaking work, there were three main hypotheses for how DNA might replicate:

  • Conservative Replication: In this model, the original double helix would remain intact after replication, and a completely new double helix would be synthesized from scratch. So, after one round, you'd have one old DNA molecule and one new DNA molecule.
  • Semi-conservative Replication: Here, each new DNA molecule would consist of one original (parental) strand and one newly synthesized strand. This is the model suggested by Watson and Crick.
  • Dispersive Replication: This model proposed that the parental DNA molecule would break into fragments, and new DNA would be synthesized in between these fragments. The resulting DNA molecules would be a mosaic of old and new DNA on both strands.

To definitively distinguish between these possibilities, Matthew Meselson and Franklin Stahl designed an elegant experiment in 1958 using Escherichia coli bacteria and isotopes of nitrogen. Their method relied on the fact that DNA contains nitrogen, and different isotopes of nitrogen have different atomic masses, which would affect the density of the DNA molecule.

Note

Isotopes are atoms of the same element that have different numbers of neutrons, and thus different atomic masses. 15N (heavy nitrogen) has one more neutron than 14N (light nitrogen), making DNA containing 15N denser than DNA containing 14N.

Here's how Meselson and Stahl carried out their experiment, following a precise sequence of steps:

  • Step 1: Labeling the parental DNA with heavy nitrogen.

    • (iii) Bacteria grown in N15 medium for many generations. They first grew E. coli in a culture medium where the only nitrogen source was 15NH4Cl (ammonium chloride containing heavy nitrogen). They allowed the bacteria to divide for many generations (multiple cell cycles).
    • (iv) All bacteria contain N15 DNA. After many generations in the 15N medium, virtually all the nitrogenous bases in the bacterial DNA incorporated 15N. This made the DNA molecules significantly denser than normal DNA. They confirmed this by centrifuging the DNA in a cesium chloride (CsCl) density gradient, where the heavy DNA settled at a lower position in the tube.
  • Step 2: Transferring to light nitrogen medium and observing the first generation.

    • (i) Bacteria transferred to a N14 medium and sampled every 20 minutes. They then transferred these 15N-labeled bacteria to a fresh culture medium containing only 14NH4Cl (light nitrogen). E. coli divides approximately every 20 minutes, so they took samples at this interval.
    • (ii) All bacteria contain hybrid DNA (N14 DNA and N15 DNA). After one generation (20 minutes), they extracted the DNA from the bacteria. When this DNA was centrifuged in a CsCl gradient, it formed a single band at an intermediate density, precisely between the positions of pure 15N DNA and pure 14N DNA. This "hybrid" band indicated that each DNA molecule contained both heavy (15N) and light (14N) nitrogen. This result immediately ruled out the conservative replication model, which would have predicted two distinct bands: one heavy and one light.
  • Step 3: Observing the second generation.

    • (v) Bacteria contain either all N14 DNA or all hybrid DNA. They allowed the bacteria to replicate for a second generation (another 20 minutes, totaling 40 minutes from the transfer to 14N medium). When DNA from this second generation was analyzed, it showed two distinct bands in the CsCl gradient: one band at the intermediate (hybrid) density and another band at the lighter (14N) density. The two bands were present in approximately equal proportions.
Important

The presence of two bands in the second generation (one hybrid and one light) was the definitive evidence. If replication were dispersive, all DNA molecules would still be a mosaic of 15N and 14N, resulting in a single band of intermediate density, but progressively lighter with each generation. The observation of distinct hybrid and light bands confirmed semi-conservative replication.

The results perfectly aligned with the semi-conservative model:

  • In the first generation, each original 15N strand served as a template for a new 14N strand, resulting in all DNA molecules being hybrids (15N-14N).
  • In the second generation, these hybrid molecules separated. The 15N strands again templated new 14N strands (forming more hybrids), while the 14N strands templated new 14N strands (forming entirely light DNA molecules). This led to 50% hybrid DNA and 50% light DNA.

Based on this detailed understanding, the correct sequence of steps followed by Meselson and Stahl is:

(iii) → (iv) → (i) → (ii) → (v)

This matches option (c).

✓Final answer

In short, Meselson and Stahl first labeled bacterial DNA with heavy nitrogen, then transferred it to a light nitrogen medium, observing the formation of hybrid DNA in the first generation and both hybrid and light DNA in the second, thereby proving semi-conservative replication. The correct sequence of steps is (c) (iii) → (iv) → (i) → (ii) → (v).

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