Semi conservative DNA Replication

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