Meselson and Stahl (1958) conducted experiments on E.coli and demonstrated that the semi conservative mode of replication is correct. In their experiments they used the following two basic principles.
(i) They cultured E.coli on a culture medium containing N15 (N15 is an isotope while normal nitrogen is N14). The bacteria replicated in this medium for a number of generations and as a result both the strands of the DNA molecule now contain N15.
In other words, the DNA of the bacterial population was now labeled with heavy nitrogen (N15). The labeled bacteria were then transferred to a culture medium containing N’4 and are allowed to replicate.
After the first generation of replication, the molecules of DNA each one possessed one strand with N’5 and the other strand with N14 (this should be the composition of the molecules if the mode of replication is semi conservative).
In these molecules the N15 strand represents a parental strand and the lighter N14 strand presents a newly synthesized strand thus each molecule can be regarded as a hybrid DNA (having both N15 andN14).
(ii) Meselson and Stahl used the principal of density gradient centrifugation and prepared gradients of Caesium chloride.
This density gradient is based on the fact that when heavy salt solutions are subjected to ultra centrifugation a continuous density gradient is set up (heavy would be at the bottom and lighter ones will be at the top with a range in between in the centrifuge tube).
When a substance having a density within the range of this gradient is dissolved in salt solution, the substance will find place at its own level of density. Thus with this technique even very slight differences in density can be detected.
Based on the principles mentioned above Messelson and Stahl subjected the bacterial cultures for density gradient centrifugation. At the first instance N15 labeled bacteria were allowed to replicate on a medium containing N14.
After the first division the centrifugation showed the hybrid DNA had an intermediate density and formed a band in-between N14 and N15 indicating clearly that each molecule contains one N14 and N15.
In the second generation two bands were found one having normal N14 and the other having a hybrid (N14 andN15)- This is expected because the labeled nitrogen – N15 is getting reduced as the medium has only N14 and the original labeled N15 gradually gets decreased in the molecules.
In the third generation 3/4ths of the DNA had N14 and l/4th of the DNA was hybrid. If the replication continues for some more generations N’5 would disappear.
In the above experiment if we assume that the replication is conservative there should be two bands in the centrifuge one with N15 and the other with N (newly formed molecule).
On the other hand if the mechanism is dispersive the molecule should spread all over the medium and should form one continuous band as both N14 and N15 in different combinations would be there in all the molecules.
If the mechanism is semi conservative the first generation would show only one layer having a mixture of N14 and N15 and in the second generation there would two layers one N14 and the other N15 and N14. Gradually N14 would show greater band width and N15 would decrease.
In the dispersive mode also only one band is formed in the first generation and it continues to be so for the second generation also where as in semi conservative mode two bands are formed in the second generation)
The above explanation clearly demonstrates that the semi conservative mode of replication proposed by Watson and Crick is the actual method of replication.
2. Cairn’s Auto Radiographic Experiment:
The semi conservative mode of DNA replication has been proved in the chromosomes of E.coli by auto radiographic experiments conducted by Cairns.
Autoradiography is based on the fact that decaying radioactive elements emit electrons which collide with the silver grains of a photographic plate and turn it back. Thus in an autoradiogram black dots represent positions of decaying radioactive elements.
DNA of an organism can be labeled selectively by growing cells in a medium containing radioactive thymidine. Radioactive thymidine is obtained by using H3– a heavy isotope of hydrogen known as tritium.
Cairns used tritiated thymidine as it selectively labels only DNA and not RNA, because thymidine base is found only in DNA and not in RNA.
By growing E.coli in the culture medium containing tritiated thymidine, radioactivity was incorporated in the daughter DNA molecules the material (DNA) was extracted and mounted on a photographic emulsion for autoradiography.
The labeled DNA exposes the film producing a diffused profile of DNA molecule. The duplicating molecule showed a replication fork.
After the replication the radioactivity was found incorporated in only one strands of DNA and in the second generation both the strands become labeled and this will be clearly seen on the photographic emulsion because of the presence of black dots.
This experiment also clearly shows the mode of replication is semi conservative because after the first replication, each molecule will have only one labeled and the other non tritiated strand.
In the second generation both the strands are tritiated in some molecules and in the other hybrid – one tritiated other non tritiated strands occur.