Discovery and History
After the brilliant work of describing the DNA structure, Watson and Crick also proposed a hypothesis that the DNA replication process is semi-conservative. This hypothesis was strengthened by the experiment of Meselson and Stahl in which they elucidated the nature of replication of DNA.
The cell was first discovered to be dividing by Hugo Von Mohl in 1835. Later, cell division was filmed and captured by Kurt Michel in 1943, who was renowned for micro-cinematography. It became evident that when the cell divides the nucleus also divides, and whatever material is present in the nucleus also undergoes division. So, the DNA present in the nucleus must undergo division. This division of DNA was named DNA replication. There were a lot of other hypotheses explaining the nature of DNA replication, but the Meselson-Stahl Experiment stood out the most as it supported the already existing hypothesis of Watson and Crick.
- Discovery and History
- The Background of Replication of DNA
- Semi-conservative Replication
- Hypotheses Regarding DNA replication
- Experimental Process and Protocol
- Importance and Application of Meselson and Stahl Experiment
The Background of Replication of DNA
DNA is the basic code of life. When the cell divides it also divides and is transferred to the daughter cells. Just like the production of daughter cells, there are production daughter DNA strands. This production is simply the copying of the hereditary material and is called DNA replication. The process begins at specific sites where there is a characteristic nucleotide sequence.
Several enzymes have been found to assist in this process most important of which is DNA polymerase. This enzyme is of a single type in the case of eukaryotes while there are three types (DNA polymerase I, II, and III) in the case of prokaryotes.
The DNA polymerase I is relatively a short sized enzyme that plays only a supportive role, but DNA polymerase III is the main enzyme that causes replication in E. coli. DNA polymerase III has a large size, almost 10 times larger than DNA polymerase I. It moves along the DNA strand, adding nucleotides at a very quick rate of 1000 per second. However, DNA polymerase cannot itself initiate DNA replication. It requires the enzyme primase to create RNA primer, a complementary sequence of 10 nucleotides. DNA polymerase III identifies this and starts further adding nucleotides to complete the new daughter strand.
In this replication, it is cleared that when the DNA helix replicates it unwinds itself and turns out to create two strands. This unwinding is done by Helicase enzyme. One of the parent strands act as a template strand and on this, the complementary base pair attach to form a new stand. After the formation of this new strand or daughter strand, they wind together to form a supercoiled DNA double Helix with the help of topoisomerase enzymes. The same process happens with the other parent strand of DNA.
Thus, it was concluded that the new copies or replicas of the parent DNA helix consist of half (one of the strands) of the parent DNA. That is why it is called semi-conservative as it contains fifty percent of older or parent DNA.
Hypotheses Regarding DNA replication
Three hypotheses were tried to elucidate the DNA replication process up to their own extent.
- The Dispersive hypothesis
- The Conservative Hypothesis
- The Semi-conservative Hypothesis
The Dispersive Hypothesis
This hypothesis was based on the model, which was put forward by Max Delbruck, a German – American biophysicist. He introduced the concepts of physics at the molecular level in biology. According to the dispersive hypothesis, the parent DNA is digested into around ten or more segments. These segments are used as templates to make new segments. The old and new segments then mix and coil to form the daughter DNA strands.
Simply, this model suggested that the parent DNA strand would disintegrate, and the new strands formed will be the blend of the newly formed nucleotides (made by polymerase enzymes) and older nucleotides (from the parent DNA).
The Conservative Hypothesis
This hypothesis considers the DNA a template as a whole. The histone proteins bind to the DNA and revolve around it, producing a new DNA copy. In simple terms, the DNA duplex will remain the same. It will not be unwound and will create completely new copies.
The Semi Conservative
As described earlier, it supports the hypothesis of Watson and Crick. Each of the parent DNA strands acts as a template and the new bases or nucleotides will form. This means it will be half new and half old i.e. semi-conservative replication.
In all of the above hypotheses, there is a prognosis about how the parent DNA would distribute itself. In essence, the conservative hypothesis tells us that the parent DNA will not mix up with the daughter DNA, but it will retain its identity. However, the daughter DNA might be new, but it will have the same sequence as the parent DNA.
The semi conservative hypothesis tells us that after the replication process the new DNA duplex formed will have fifty percent of the parent material and fifty percent new formed or daughter material (nucleotides), as complete single strands.
Whereas, the dispersive theory explains that the whole DNA duplex will divide itself in about 10 nucleotides in size and the old fragments will join randomly with the newly formed fragment or more precisely the nucleotides.
Experimental Process and Protocol
All three hypotheses were assessed by Mathew Meselson and Franklin Stahl. They performed experiments in California. As their experiment was highly based on the studies and hypotheses of Watson and Crick. They realized that DNA is made up of nucleotides. These nucleotides are further comprised of a phosphate group, deoxyribose sugar and most importantly nitrogenous base.
The nitrogenous bases are present in each nucleotide of DNA. All the bases present in those nucleotides have nitrogen. They realized it would be greatly helpful to tag the parent DNA.
The best way to tag it was to change the nitrogen atoms present in the parent DNA. They used a nitrogen isotope to make a difference between the daughter and parent strand. Due to this remarkable use of isotopes and biophysics, this experiment is regarded as the most beautiful experiment.
Procedure and Protocol followed by Meselson and Stahl
The experiment began with the preparation of a culture media for the bacteria. The microorganism chosen was E. coli. The culture media consisted of NH4CL and 15N isotope of nitrogen. This isotope of nitrogen has a higher molecular weight than the normal existing nitrogen isotope 14N.
When the E. coli was allowed to grow in the culture containing high molecular weight isotope of nitrogen, the bacteria started to incorporate 15N atoms of nitrogen into its DNA. So, after several generations, the DNA of the bacteria grown in 15N culture becomes denser than the normal bacteria grown in normal culture.
The other E. coli ware grown in a normal culture media containing the common isotope of nitrogen, the 14N nitrogen. Thus, the DNA of this bacterial culture was less dense than 15N culture media.
After completing enough generations, they transferred the bacteria from the 15N media to 14N media. They allowed the bacteria containing 15N in their DNA to grow in a culture containing 14N nitrogen.
Results and Observation
They obtained DNA from the mixture and dissolved it in solution containing cesium chloride. Later, they centrifuged this mixture at high speed i.e. using the ultracentrifugation technique. The high centrifugal forces cause the ions of CsCl to produce a density gradient. The gradient is produced as the ions migrate to the bottom of the tube due to ultra-centrifugation. A constant density is established throughout the solution. The DNA samples start to float in the solution. After keeping the tube at rest for a while, the different DNAs of different densities maintain a specific position according to their densities.
As the DNA with 15N isotope is heavier, it finds its place at the bottom of the tube. On the other hand, the DNA with 14N isotopes of nitrogen finds its place on an elevated level in the tube. However, a type of DNA is also found in between the elevated level and bottom level.
Ruling out Conservative Hypothesis
Both the scientists concluded that if conservative hypothesis were to be true, then there would be DNAs of only two densities. As the parent DNA would have maintained its integrity and gave birth to a new daughter strand. However, they found three types of densities regarding DNA in their experiment.
Ruling out Dispersive Hypothesis
According to the semi-conservative replication hypothesis, there would be a hybrid density DNA. As one strand will be of 14N and one strand will be of 15N isotope. However, concerning dispersive hypothesis the new DNA formed will be of intermediate density just like the semi-conservative. But, each of the strands from the double helix will not solely contain either 14N or 15N nitrogen, it will be a mixture of both isotopes.
To rectify these confusions, Meselson and Stahl sampled the second replication of 15N bacteria in the 14N medium. They followed the same procedure and protocol and put the DNA in the test tube with Cesium Chloride and established a density gradient.
It was found that there were DNAs of two different densities, unlike the first time when there were three. One of the two had intermediate density just like in the previous experiment and the second one had the density of pure 14N isotope.
This result did not support the dispersive hypothesis of replication as it would have given one DNA having a density lower than the intermediate density found in the experiment.
Importance and Application of Meselson and Stahl Experiment
This experiment provided modern biology with the knowledge of DNA replication. This knowledge granted the vision to peek into hereditary diseases and disorders. It is a simple yet provoking experiment that has been accepted by a lot of scientists.
Despite the affirmative result provided by the experiment of the Meselson-Stahl, it took a few years for acceptance by the scientific community. The Meselson and Stahl experiment did not only provide evidence for the semi-conservative theory which was put forward by Watson and Crick, this experiment also confirmed the Watson and Crick model of DNA structure. Thus, it strengthened the standpoint of Watson and Crick which was taking years to get accepted.
In 1952, a scientist studied the experiment by implementing it on the cancer researches. He did not negate the semi-conservative replication hypothesis. This further supported the experiment.
However, the only thing that fell short in this experiment was the proper explanation about the DNA subunits. This discovery of Meselson and Stahl allowed the scientists to discover the mode of transmission of DNA and follow up on the genetic disorders.
This experiment is simply based on tagging the DNA and separating them based on their densities relative to the solution created by using Cesium Chloride.
It was a known fact by then that DNA is made up of nucleotides and these nucleotides contained nitrogen.
Meselson and Stahl utilized the common nitrogen by replacing it with a heavier isotope so that they can identify the parent and daughter DNA in solution by mixing the DNAs of different densities.
They experimented using simple techniques as ultra-centrifugation and density grading.
This experiment is a great milestone in modern biology as those were early times when atomic physics was applied in biological studies. Meselson and Stahl made three major outcomes.
- DNA is made up of two strands, which was based on the Watson and Crick Model
- If the parent DNA has two strands, then each of the strand acts as a template and retains its integrity and forms a daughter strand. Thus, the semi-conservation is evident
- Each of the parent DNA strand is shared by two daughter DNA.
In any case, the investigation helped researchers to clarify legacy by indicating how DNA saves hereditary data through all the progressive DNA replication cycles as a cell grows, divides and repeats the cycle.
- John Cairns to Horace F Judson, in The Eighth Day of Creation: Makers of the Revolution in Biology (1979). Touchstone Books, ISBN 0-671-22540-5. 2nd edition: Cold Spring Harbor Laboratory Press, 1996 paperback: ISBN 0-87969-478-5.
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