BIO 112

MOLECULAR BIOLOGY AND

GENETICS
 METHOD OF LEARNING

Lectures Four hours per week

Tutorials One hour per week
Laboratory sessions Three hours per week
 ASSESSMENT
Continous Assessment 40%
2 Tests 20%
Practicals 15%
Theory Quiz 5%
Final Examination 60%

Prescribed reading
• Elliot, W. H. and Elliot, D. C. (2004). Biochemistry and
Molecular Biology. 3rd Edition. Oxford.
• Taylor, D. J. et al. (1997). Biological Science. London:
Cambridge University Press.
 LECTURE 1

DNA REPLICATION IN
PROKARYOTES/EURKAYOTES
 Historical perspective
• 1953, James Watson and Francis Crick
published a two-page paper in the journal
Nature entitled “Molecular Structure of
Nucleic Acids: A Structure for Deoxyribose
Nucleic Acid.”
• Once the structure of the genetic material
had been determined, an understanding of its
method of replication and its functioning
quickly followed.
 DNA Replication

• Occurs during interphase of cell cycle

• When a eukaryotic cell divides, the process is
called mitosis (cell splits) into two identical
cells)
- the DNA must be replicated so that each
daughter cell has a copy.
• DNA replication is a means to produce new
DNA molecules that have the same sequence.
• DNA replication involves several processes:
- first, the DNA must be unwound, separating
the two strands
- the single strands then act as templates for
synthesis of the new strands, which are
complimentary in sequence
- bases are added one at a time until two new
DNA strands that exactly duplicate the original
DNA are produced.
 Semi-conservative replication
• DNA replication is semi-conservatively.
• The parent DNA strand separates into two
• Each strand serves as a template for formation
of a new complementary strands
• The new double helix is half original because
one strand of each daughter DNA comes from
the parent DNA and one strand is new
• Semi-conservative was the accepted model
after Meselson and Stahl did experiments
(Meselson & Stahl 1958)
 Other Replication Models

• Conservative model, the parental

molecule directs synthesis of an entirely
new double-stranded molecule, such that
after one round of replication, one
molecule is conserved as two old strands.
• This is repeated in the second round.
• Dispersive model, material in the two parental
strands is distributed more or less randomly
between two daughter molecules.
• In this model, old material is distributed
symmetrically between the two daughters
molecules.
• Two templates, each of which carries all the
information of the original molecule.
 The Meselson - Stahl Experiment

• Proof of the replication model of DNA as we

know it today came from the experiments of
Meselson and Stahl.
• They grew E. coli is a medium using ammonium
ions (NH4+) as the source of nitrogen for DNA
synthesis.
• 14N is the common isotope of nitrogen, but they
could also use ammonium ions that were
enriched for a rare heavy isotope of nitrogen,
15N.
• After growing E. coli for several generations in
a medium containing 15NH4+, they found that
the DNA of the cells was heavier than normal
(because of the 15N atoms in it).
• The difference could be detected by extracting
DNA from the E. coli cells and spinning it in an
ultracentrifuge.
• The density of the DNA determines where it
accumulates in the tube.
• Then they transferred more living cells that
had been growing in 15NH4+ to a medium
containing ordinary ammonium ions (14NH4+)
and allowed them to grow for four
generations.
• At each generation (which takes about 20
minutes to grow), a DNA sample is taken and
analyzed by centrifugation in a CsCl gradient
• The DNA in this new generation of cells was
exactly intermediate in density between that
of the previous generation and the normal
• Generation 0; 100% of DNA in nitrogen-15
band.
• Generation 1; 100% of DNA in a band
intermediate in position between nitrogen-14
and nitrogen-15 bands.
• Generation 2; 50% of DNA in a band
intermediate in position between nitrogen-14
and nitrogen-15 bands. 50% of DNA in nitrogen-
14 band.
• Generation 3; 25% of DNA in a band
intermediate in position between nitrogen-14
and nitrogen-15 bands. 75% of DNA in
nitrogen-14 band.
• Generation 4; 12% of DNA in a band
intermediate in position between nitrogen-14
and nitrogen-15 bands. 88% of DNA in
nitrogen-14 band.
Generation 0
• DNA isolated from cells at the start of the
experiment “generation 0,” just before the
switching to the light normal ammonium ions
(14NH4+) produced a single band after
centrifugation.
• This result made sense because the DNA
should have contained only heavy 15N
isotope.
Generation 1
• DNA isolated after one generation (one round
of DNA replication) also produced a single
band when centrifuged.
• However, this band was higher, intermediate
in density between the heavy 15N DNA and
light 14N DNA.
• The intermediate band told Meselson and Stahl
that the DNA molecules made in the first round of
replication was a hybrid of light and heavy DNA.
• This result fit with the dispersive and semi-
conservative models, but not with the
conservative model.
• The conservative model would have predicted
two distinct bands in this generation (a band for
the heavy original molecule and a band for the
light, newly made molecule).
• Generation 2
• Information from the second generation let
Meselson and Stahl determine which of the
remaining models (semi-conservative or
dispersive) was actually correct.
• When second-generation DNA was centrifuged, it
produced two bands.
• One was in the same position as the
intermediate band from the first generation,
while the second was higher (appeared to be
labeled only with 14N .
• This result told Meselson and Stahl that the
DNA was being replicated semi-conservatively.
• The pattern of two distinct bands—one at the
position of a hybrid molecule and one at the
position of a light molecule—is just what we'd
expect for semi-conservative replication (as
illustrated in the diagram below).
• In contrast, in dispersive replication, all the
molecules should have bits of old and new
DNA, making it impossible to get a "purely
light" molecule.
• Generations 3 and 4
• In the semi-conservative model, each hybrid
DNA molecule from the second generation
would be expected to give rise to a hybrid
molecule and a light molecule in the third
generation, while each light DNA molecule
would only yield more light molecules.
• Thus, over the third and fourth generations,
we'd expect the hybrid band to become
progressively fainter (because it would
represent a smaller fraction of the total DNA)
and the light band to become progressively
stronger (because it would represent a larger
fraction).
• As we can see in the figure, Meselson and
Stahl saw just this pattern in their results,
confirming a semi-conservative replication
model.
• As the figure indicates, each original strand
remains intact as it builds a complementary
strand from the nucleotides available to it.
• This is called semi-conservative replication
because each daughter DNA molecule is half
"old" and half "new".
END OF LECTURE!
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