DNA Semiconservative

BIO1400/BIO1412
 Objective
 1. Explain the basic principles of molecular biology and
genetics.
 2. Describe the role of gene regulation in cellular function.
 3. Explain the chromosomal theory of inheritance.
 4. Discuss classical Mendelian and post-Mendelian genetics
 Molecular Biology
 DNA replication in prokaryotes: Semi-conservative replication,
 Transcription: mRNA, tRNA and rRNA transcription in prokaryotes
(Initiation, elongation and termination)
 Translation: Protein synthesis; properties of the genetic code;
 Regulation of gene expression: Significance; the Lac operon
 Mutagens
 GENETICS:
 Chromosomal theory of inheritance
 Cell division
 Introduction to genetics
 Mendelian Genetics
 Gene interactions
 Sex determination and sex-linkage
 Gene Mutations
Objectives
• You should be able to:
• 1) define DNA replication.
• 2) Describe the mechanisms proposed to explain DNA
replication.
• 3) Describe the mechanisms
• 4) Explain the roles of various enzymes
Replication
• The process involves the making of new DNA strands of DNA
using old DNA.
• The new strands are complementary in sequence to the
original strand.
• The process of making new strands is catalyzed by DNA
polymerases.
• These add nucleotides to each nascent DNA strand.
• Polymerases use bases on the template strand as guides to
form the Watson and Crick pairs where A and T form double
bonds and C and G form triple bonds.
• DNA polymerases catalyzed reactions are slow.
• Add more or less than 100 nucleotides per second to a growing
strand.
• Imagine a chromosome containing 100 million bp.
• It would take 1 million seconds to get from one end to the
other.
• This is roughly 11 days!
• The replication process in eukaryotes take about 6-8 hours and
occurs in the S phase of the cell cycle.
• This is made possible by using thousand of origins of
replication, ori.
• The ori is the site where DNA synthesis can begin.
• Thousands of ori sites are activated simultaneously.
 Three models were originally proposed.
 These were conservative, dispersive and semi-conservative replication.
History
• Semi-conservative model, the two parental strands separate
and each makes a copy of itself.
• The two daughter molecules each comprises one old and one
new strand after one replication cycle.
• After two cycles, two of the DNA molecules consist only of new
material, while the other two contain one old and one new
strand.
History
• In the conservative model, the parental molecule directs
synthesis of an entirely new double-stranded molecule.
• After one round of replication, one molecule is conserved as
two old strands.
• This is repeated in the second round.
History
• In the dispersive model, material in the two parental strands is
distributed more or less randomly between two daughter
molecules.
• In the model old material is distributed symmetrically between
the two daughters molecules.
• Other distributions are possible.
“The Most Beautiful Experiment in
Biology”
Meselson & Stahl:
Semiconservative Replication of DNA
Matthew Meselson
(Molecular Biologist & Geneticist)
• Born May 24th, 1930 in Denver,

Colorado
• Labs in garage & basement
• Graduated high school 2 years early
• University of Chicago (chemistry),
California Institute of Technology (Ph.D.
under Linus Pauling x-ray
crystallography, density centrifugation)
• Since 1960-Professor at Harvard
Franklin Stahl
(Molecular Biologist & Geneticist)
• Born October 8th, 1929 in Boston,

Massachusetts
• Studied at Harvard (A.B.) &
University of Rochester (Ph.D.)
• Researched at California Institute
Technology & University of
Missouri
• Currently professor at University
of Oregon
Woods Hole
• 1954: Meselon went to Woods Hole to be a teaching assistant

• Stahl was a post-doctoral fellow at Woods Hole, where he was
studying molecular biology techniques
• Throughout the summer, Meselson and Stahl had discussions on
theory and experiments
– Shared similar ideas and were keen to work together
– Stahl received a postdoctoral position in Caltech
– Particularly interested in building off of Watson and Crick’s DNA
model
The DNA Model
• Watson and Crick’s DNA Model (1953):

– Double-stranded helix
– Right-handed
– Nucleotides bases form interior; sugar phosphate backbone
• Aligning with Chargaff’s rule each purine paired with a pyrimidine (A-
T, G-C sequences)
*Watson and Crick speculated that the specific base pairings were indicative of a precise mode of DNA replication
Meselson and Stahl sought to prove Watson and Crick’s hypothesis.

3 Major Hypotheses for
DNA Replication
Semiconservative: each strand serves as

template for replication
Conservative: intact double helix is copied in

its entirety
Dispersive: replication results in hybrids

consisting of old and new DNA
Meselson and Stahl’s Experiment
(1958)
Overview
• Utilization of isotope labels to differentiate

between parent and daughter DNA
– Chose 14N (common, lighter) and 15N (rare,
heavier)key element of DNA
• Technique of cesium chloride equilibrium density gradient centrifugation
– Separation of molecules based on density
• Goal: detect whether new nitrogen atoms appear on 1 or both daughter strands
Methodology
• Grew 14 generations E. coli in 15N (dense, would be at bottom of

centrifuge)
• Changed medium to 14Nfrom this point forward all DNA replicated
in this medium
• Periodically sampled the DNA grown in the 14N medium by means of
equilibrium density gradient centrifugation to compare the
percentage of 15N to 14N
– Samples mixed with cesium chloride, centrifuged and allowed to settle
– http://www.phschool.com/science/biology_place/biocoach/dnarep/cscl
.html
Results
1) DNA made up of only 15N formed a single band

2) First replication band located at midpoint between 15N and 14N
3) Second generation had two bands (1 at midpoint, 1 at 14N); same
two bands seen subsequently
Semiconservative Replication
Meselson and Stahl elucidated the core mechanism of DNA replication

Contributions to Biotechnology
• “Most Beautiful Experiment” forming predictions based on
various models and pinpointing which matches experimental
data
– Addressed all models simultaneously
• Demonstrated effective, unique mode of isotope visualization
– Labeling not based on radioactivity but rather density (gradient
analyzed by means of centrifugation)
– Heavy isotopes are now widely used to label and analyze
molecules
Advancing the Field of Molecular Biology
• Semiconservative DNA replication has been confirmed in

every other studied species
• Understanding semiconservative DNA replication is crucial
to elucidating functionality of cells and organisms
– Genetic material must replicate completely and accurately
during divisions
• Furthered knowledge on mechanisms of DNA and its
interactions with enzymes (i.e. helicase)
• Having a grasp on proper DNA replication allows for
identification of abnormalities underlying defects
• Insights into heredity.

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BIO1400/BIO1412

• Born May 24th, 1930 in Denver,

• Born October 8th, 1929 in Boston,

• 1954: Meselon went to Woods Hole to be a teaching assistant

• Watson and Crick’s DNA Model (1953):

Meselson and Stahl sought to prove Watson and Crick’s hypothesis.

Semiconservative: each strand serves as

Conservative: intact double helix is copied in

Dispersive: replication results in hybrids

• Utilization of isotope labels to differentiate

– Separation of molecules based on density

• Grew 14 generations E. coli in 15N (dense, would be at bottom of

1) DNA made up of only 15N formed a single band

Meselson and Stahl elucidated the core mechanism of DNA replication

• Semiconservative DNA replication has been confirmed in

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