Semiconservative Theory of DNA replication

Semiconservative Theory of DNA replication

Meselson and stahl Experiment

Watson and Crick discovered the DNA structure, but they had an important question, how can the preexisting DNA form the new DNA? Watson and Crick hypothesized the semi-conservation model of DNA replication. However, Semi conservative replication was not the only model of DNA replication proposed during the mid-1950s. Two other prominent hypotheses were also proposed, conservative replication and dispersive replication Different scientists proposed different models.

They are conservative model, dispersive model, semi-conservative model and.

• Conservative model: The complete DNA twisted around the histone proteins forms newly twisted DNA as it is.
• Dispersive model: The preexisting DNA is fragmented into many short nucleotide sequences and each synthesizes new short nucleotide sequences, finally forming the complete DNA.
• Semi-conservative: Preexisting DNA is divided into two strands, each strand acts as a template and synthesizes new DNA strands separately.
• According to the semi-conservative model, after one round of DNA replication, every new DNA double helix would be a hybrid that consists of one strand of old DNA bound to one strand of newly synthesized DNA. Then, during the second round of replication, the hybrids would separate, and each strand would pair with a newly synthesized strand. Afterwards, only half of the new DNA double helices would be hybrids; the other half would be completely new. Every subsequent round of replication therefore would result in fewer hybrids and more completely new double helices.

• According to the conservative model, after one round of DNA replication, half of the new DNA double helices would be composed of completely old, or the original DNA and the other half would be completely newly synthesized DNA double helices. Then, during the second round of replication, each double helix would be copied in its entire structure as it is. Afterwards, one-quarter, 1/4 (one of the four) of the double helices would be completely old, and three-quarters
• ¾ would be completely new (1:3). Thus, each subsequent round of replication would result in a greater proportion of completely new DNA double helices, while the number of completely original DNA double helices would remain constant.
• According to the dispersive model, every round of replication would result in hybrids or DNA double helices that are part of original DNA and part of new DNA. Each subsequent round of replication would then produce double helices with greater amounts of new DNA.

 

Many scientists confirmed that semi-conservative replication was the rule, not just in E. coli, but in every other species studied as well. To date, no one has found any evidence for either conservative or dispersive DNA replication.

 

Matthew Meselson and Franklin Stahl began their experiment with two isotopes of nitrogen, the common and lighter ¹⁴N, and the rare and heavier ¹⁵N (“heavy” nitrogen) with the caesium chloride (CsCl) equilibrium density gradient centrifugation technique, the sedimentation method. CsCl and DNA density would become equilibrium at one point. That point would be the aggregated band of all DNA molecules with equal density.

The scientists opted for nitrogen because it is an essential chemical component in DNA structure. Therefore, every the cell divides and its DNA replicates, it incorporates new N atoms into the DNA.

The principle of the experiment predicted by scientists at the beginning: “If several different density species of DNA would form, on culturing of different isotopes, each will form a band at the position where the density of the CsCl solution is equal to the buoyant density of that species. In this way, DNA labelled with heavy nitrogen (¹⁵N) may be resolved from unlabeled DNA”.

Culturing of Bacteria:

Step 1: Bacteria cultured in normal (without radio labelling) NH₄⁺ medium for many generations.

Step 2: Generation 0: Bacteria were cultured in ammonium containing the ¹⁵N radio-labelled NH₄⁺ medium for many generations.

Step 3: Generation1: Bacteria cultured in ¹⁵N labelled NH₄⁺ medium to ¹⁴N radio labelled NH₄⁺ medium for many generations.

Step 3: Generation 2: Bacterial culture continued in ¹⁴N labeled NH₄⁺ medium for many generations.

Collection of samples and Centrifuged with CsCl.

Samples from all the generations were collected and centrifuged in a “Density Gradient Centrifugal machine” with CsCl, at 30,000-50,000 rpm for 48-72 hours.

Observations:

1. Bacteria, cultured in Generation 0 obtained ¹⁵N DNA and settled the band at the bottom portion of the centrifugal tube.  DNA composed entirely of ¹⁵N-labeled DNA (i.e., DNA collected just before changing the culture from one containing only ¹⁵N to one containing only ¹⁴N) formed a single distinct band because both of its strands were made entirely in the “heavy” nitrogen medium.
2. Bacteria, cultured in Generation 1 contain ¹⁴N and ¹⁵N DNA molecules and formed the band above the ¹⁵N DNA band and below the band formed in Generation 2 (¹⁴N) in the centrifugal tube. This band is a “Hybrid band” and contains. The band is located halfway between “heavy” and “light”
3. Bacteria, cultured in Generation 2 contain equal proportions of ¹⁴N but low ¹⁵N as 50:50. This indicates the ¹⁵N DNA density is decreasing while the ¹⁴N is increasing. Hybrid band and other lighter bands above the hybrid band.
4. The next cultures continued in the same ¹⁴N labelled medium again increasing the ¹⁴N DNA and decreasing ¹⁵ N DNA densities by 25:75, and later in the same culture, it became 12: 88.

Conclusion:

• Twisted DNA molecule unwinds and separates during the replication, each strand acts as a template and synthesizes the new strand on it. It means every new DNA contains half of the old DNA. If DNA replicated conservatively, there should have been two distinct bands after a single round of replication; half of the new DNA would have migrated to the same position as it did before the culture was transferred to the ¹⁴N-containing medium (i.e., to the “heavy” position), and only the other half would have migrated to the new position (i.e., to the “light” position). According to the dispersive model, every round of replication would result in hybrids or DNA double helices that are part of the original DNA and part of the new DNA. Each subsequent round of replication would then produce double helices with greater amounts of new DNA.