Protein synthesis simulation
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Process of evolution
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DNA Structure

3.4.1 Mechanism of DNA replication

[[image: width="446" height="418" align="left" caption="external image DNArep.gif"]]
1. The original double helix molecule.
2. Helicase enzyme breaks the hydrogen bonds between complementary base pairs. This unzips the double helix at a position called the replication fork.
3. There is an abundant supply of nucleotides in the nucleus for the formation of the new polynucleotides.
4. Nucleotides base pair to the bases in the original strands.
5. DNA polymerase joins together the nucleotides together with strong covalent phosphodiester bonds To form a new complementary polynucleotide strand.
6. The double strand reforms a double helix under the influence of an enzyme.
7 Two copies of the DNA molecule form behind the replication fork. These are the new daughter chromosomes.

Speed of replication:
  • DNA replication can take a few hours and this limits the speed of cell division.
  • Bacteria can replicate quickly because of the relatively small amount of DNA.
  • Eukaryotic organism's accelerate DNA replication by having thousands of replication forks along the length of the DNA molecule.
external image repfork.gif
external image repfork.gif


3.4.2 Conservation of base sequences in DNA

  • The significance of the mechanism outlined above is that the DNA molecule is copied precisely from one cell generation to the next.
  • In a unicellular organism this means that the total genome is successfully copied into each new generation.
  • In the multi-cellular organism all cells contain an exact copy of the total genome (even though not fully expressed).
  • Genes (base sequences) are faithfully passed from one generation to the next.
  • The genes (base sequences) which the reader possess have been passed from generation to generation until they arrived in you now. With minor and rare modification the base sequences copied by DNA replication and successfully passed on through sexual reproduction. Your base sequences have been copied for thousands of years.
  • A worked example is provided.
DNA Workshop

Meselson & Stahl Expt

3.4.3. Semi-conservative DNA replication

The mechanism of semi-conservative replication produces two descendent double helices that each contain one of the original polynucleotide chain. This need not have been the case as there are 3 possible mechanism for replication:
  • Conservative replication in which the original molecule is completely retained an a new molecule (two new polynucleotide chains are combined).
  • Semi-conservative replication in which each of the new double helices contains one of the original polynucleotide chains.
  • Dispersive replication in which fragment of the original double helix serve as templates for the new DNA . The fragments are randomly arranged.
In 1958 Matthew Meselson and Frank Stahl ( California Institute of technology) carried out the experiment which convinced everyone that the actual mechanism was semi-conservative as originally proposed by Watson & Crick.
Experiment : They used the bacterium E. coli together with the technique of density gradient centrifugation, which separates molecules on the basis of their density.
external image SEMICON.gif
external image SEMICON.gif

(1) This is a calibration step in which the bacterium is cultured in with Ammonium chloride (NH4Cl )
but the nitrogen was isotope N14. Therefore both polynucleotide chains contains nucleotides which contained N14. This produced in centrifugation the 'light',( N14N14 ) chains.

(2) This is another calibration step in which the bacterium is cultured in with Ammonium chloride (NH4Cl )
but the nitrogen was isotope N15. Therefore both polynucleotide chains contains nucleotides which contained N15. This produced in centrifugation the 'heavy',( N15N15)chains.
N15N15---> N15N14

(3) The 'heavy' ( N15N15) bacteria have been returned to a medium of ammonium chloride with N14. The DNA produced was neither 'heavy' or 'light' but exactly intermediate (N15N14). The reason is that one polynucleotide is composed of bases of N14 (new one) and the other N15 (old one). This step eliminates conservative replication as a mechanism.

N15N14 ---> N14N14, N15N14
(4) The hybrid bacterium N15N14is returned to the N14medium and after 40 minutes or two generations there are 'light' and 'intermediate' DNA. This step eliminates the dispersive replication hypothesis.

The conclusion of the experiment:
  • Step 3 eliminates the Conservative replication mechanism.
  • Step 4 eliminates the Dispersive replication mechanism.
  • In combination with step 3 with the formation of the intermediate DNA, and the elimination of conservative (step3) and dispersive (step4) replication the mechanism

Transcritio & translation

Animation on DNA Replication
Meselson & Stahl Expt
Replication Fork
Replication Dance

DNA Repair

Videoteaching onTranslation from Discovery
Vedios from Biointeractive

Gel Elecrophoresis
DNA Chip Technology