- Matthew Meselson and Franklin Stahl performed an experiment in 1958 using isotopes of nitrogen (N15 and N14) to track DNA replication in E. coli bacteria. - They found that after one round of replication, the parental and newly synthesized DNA strands had an intermediate density, supporting Watson and Crick's semiconservative model where each new DNA molecule contains one original and one new strand. - Subsequent experiments by Meselson and Stahl and others confirmed that DNA replication is semiconservative, with the two strands of the double helix separating and each acting as a template for synthesis of a new complementary strand.

1. Semiconservative DNA replication
Matthew Meselson
and
Franklin Stahl
Experiment

2. DNA REPLICATION
• While proposing the double helical structure for DNA-----Watson
and Crick had immediately proposed a scheme for replication of
DNA..
• Semiconservative DNA replication model given by Watson and
Crick…
• But experimental proog given by Meselson and Stahl

3. Semiconservative DNA replication

4. Semiconservative DNA replication
• The scheme suggested that the two strands would separate and
act as a template for the synthesis of new complementary strands.
•

5. • After the completion of replication, each DNA molecule would
have one parental and one newly synthesised strand.
• This scheme was termed as semiconservative DNA replication..

6. The Experimental Proof of
semiconservative DNA replication
• It was shown first in Escherichia coli and
subsequently in higher organisms, such as plants
and human cells.
• Matthew Meselson and Franklin Stahl performed
the experiment in 1958,,

7. • They differentiated 15N & 14N DNA by centrifugation in a cesium
chloride (CsCl) density gradient.
• (Please note that 15N is not a radioactive isotope, and it
can be separated from 14N only based on densities).

8. • They grew E. coli in a medium containing 15NH4Cl (15N is the
heavy isotope of nitrogen) as the only nitrogen source for many
generations.

9. E. coli
N15
N14

10. • The result was that 15N was incorporated into newly synthesised
DNA (as well as other nitrogen containing compounds).

11. • Centrifugation is a technique used for the separation of
particles from a solution according to their size, shape,
density through spinning.

12. • Then they transferred the cells from N15 into a medium with
normal 14NH4Cl.
• They took DNA samples at various definite time intervals from
bacterial cells.

13. • The various DNA samples were separated
independently on CsCl gradients to measure the
densities of DNA..

16. After first generation ( 20min) in 14N medium
• The DNA that was extracted from the culture one
generation after the transfer from 15N to 14N medium
had a hybrid or intermediate density.

17. • DNA extracted from the culture after another
generation [that is after 40 minutes, II
generation] was composed of equal amounts of
this hybrid DNA and of ‘light’ DNA.

18. • In the 3rd generation (After 60min), 25% will be
hybrid and 75% will be light ..

19. • If E. coli was allowed to grow for 80 minutes then what
would be the proportions of light and hybrid densities
DNA molecule?
• In the fourth generation (After 80min) 12.5% will be
hybrid and 87.5% will be light strand.

20. • Taylor and colleagues performed similar experiments by
using radioactive thymidine on Vicia faba (faba beans) in
1958.
• The experiments proved that the DNA in chromosomes
also replicate semi conservatively.
•

21. The Machinery and the Enzymes
of DNA replication..

22. The Machinery and the Enzymes of DNA
replication..
• The main enzyme is referred to as DNA-dependent DNA
polymerase.
• DNA polymerase uses a DNA template to catalyse the
polymerisation of deoxynucleotides (dNTPs)..
• dATP, dGTP, dCTP, dTTP).

23. Deoxyribonucleoside triphosphates (dNTPs):
• dATP, dGTP, dCTP, dTTP.
• Deoxyribonucleoside triphosphates serve dual
purposes.
• In addition to acting as substrates, they provide energy
for polymerisation reaction.
• The two terminal phosphates in dNTPs are high-energy
phosphates.

25. DNA polymerase
• DNA polymerase enzymes are highly efficient, fast and
with high degree of accuracy.
• The DNA-dependent DNA polymerases catalyse
polymerisation only in one direction, that is 5’3‘
direction.

26. DNA polymerase
• It catalyse polymerisation of a large number of
nucleotides in a very short time.

27. • E. coli that has only 4.6 ×106 bp.
• The DNA polymerase completes the process of
replication within 38 minutes in E.coli.
• That means the average rate of polymerisation has to be
approximately 2000 bp per second.

28. • Any mistake during replication would result into
mutations..
• A mutation is a change in a DNA sequence.

29. origin of replication..
• There is a definite region in E. coli DNA where the
replication originates.
• Such regions are termed as origin of replication..

30. Replication fork.
• The two strands of DNA cannot be separated in its entire
length (due to very high energy requirement).
• The replication occur within a small opening of the DNA
helix, referred to as replication fork.

31. • The DNA-dependent DNA polymerases catalyse
polymerisation only in one direction, that is 53'.
• This creates some additional complications at the
replicating fork.
• The principle of complementarity governs the process of
Replication and Transcription.

33. • on one strand (the template with
polarity 35'), the replication is
continuous..
• while on the other (the template
with polarity 5’3'), it is
discontinuous.
• The discontinuously synthesised
fragments are later joined by the
enzyme DNA ligase..

34. • A piece of DNA propagated in to
vectors during recombinant DNA
procedure.
• The vectors provide the origin of
replication.

35. Polyploidy
• In eukaryotes, the replication of DNA takes place at S-phase of the
cell-cycle.
• A failure in cell division after DNA replication results into polyploidy
(a chromosomal anomaly).
• Anomaly = something different, abnormal

36. TRANSCRIPTION

37. Transcription
• The process of copying genetic information from one
strand of the DNA into RNA is termed as transcription.
DNA-dependent RNA polymerase

38. • The principle of complementarity governs the process of
transcription.
• (Remember complementarity does not mean identical)
• In transcription adenosine pair with uracil instead of
thymine.

39. • IN replication, total DNA of an organism gets duplicated,
• But in transcription only a segment of DNA and only one
of the strands is copied into RNA..

40. Both the strands are not copied during
transcription
• If both strands act as a template, they would code for
RNA molecule with different sequences..
• In turn, RNA molecules code for proteins, the sequence
of amino acids in the proteins would be different.

41. • Hence, one segment of the DNA would be coding for two
different proteins.
• This would complicate the genetic information transfer
machinery..

42. • Second, the two RNA molecules if produced
simultaneously would be complementary to each other.
• Hence would form a double stranded RNA.
• This would prevent RNA from being translated into
protein

43. Transcription Unit

44. Transcription unit
• A transcription unit in DNA is defined primarily by the
three regions in the DNA:
• (i) A Promoter
• (ii) The Structural gene
• (iii) A Terminator..

46. • DNA-dependent RNA polymerase also catalyse the
polymerisation in only one direction, that is, 5'→3‘.
• The strand that has the polarity 3'→5' acts as a template
strand.

47. • The other strand which has the polarity (5'→3') is
referred to as coding strand.
• Coding strand sequence same as RNA (except thymine at
the place of uracil)..
• Coding strand which does not code for anything..
Coding strand

48. 3' -ATGCATGCA-5' Template Strand
5' -TACGTACGT-3' Coding Strand

50. • The promoter and terminator flank the structural gene in
a transcription unit.
• The promoter is said to be located towards 5' -end
(upstream) of the structural gene (the reference is made
with respect to the polarity of coding strand).
• It is a DNA sequence that provides binding site for RNA
polymerase..

51. • The terminator is located towards 3' -end (downstream)
of the coding strand and it usually defines the end of the
process of transcription

52. Dr. HarinathaReddy Aswartha
Assistant professor
Department of Microbiology
ANDHRAPRADESH
INDIA