Learning Unit 3

MOLECULAR STRUCTURE AND REPLICATION OF THE GENETIC MATERIAL

Chap9 page 221

MRS AE KANEME
 Introduction
• Molecular genetcc – the study of DNA structure and
functon at the molecular level.
• Goal – use the knowledge of DNA structure to understand
how DNA functons at the molecular level.
• Molecular techniques – examples DNA extracton, PCR etc
• Value of molecular techniques – determined the
organizaton of genes.
• This helped with understanding the expression of genes
that govern the outcome of an individuals inherited traits.
Identifcation of DNA as the Genetic
Material
• It is known that genetc material makes us alive. It governs an individual’s traits and is transferable during the
process of reproducton.
• But to qualify anything as genetc material it must meet all the criteria listed below;
• 1. Informaton: Genetc material should contain the informaton needed to construct an entre organism.
• 2. Trancmiccion: During reproducton, the genetc material must be passed from parent to ofspring.
• 3. Replicaton: Genetc material should be copied. This is the only way it can be transmited from parent to
ofspring, mother cell to daughter cell.
• 4. Variaton: There must be some variability within any species. Genetc material must have variaton that can
account for the known phenotypic diferences within each species.
• These 4 criteria was sufcient to characterize the nature of the genetc material. However, almost nothing was
known about the chemical structure of genetc material.
• In the 1880s August Weismann and Carl Nageli pioneered the idea that the nature of genetc material was a
chemical.
• It was then that the theory of the chromosome was born. The experiment showed that the chromosomes
were the carriers of the genetc material. The informaton was not complete, and more work had to be done.
• However, this was not enough because the chromosome as we know it contained both proteins and DNA and
RNA is also found within the cell.
Experimental researches to
Identify genetic materials.
• Further research was needed to precisely identfy the genetc
materials.
• Understanding how DNA was discovered to be the foundaton of
genetc material requires one to understand a series of experiments
carried out by Grifths, Avery, MacLeod and McCarty.
 Griffiths Experiments tith
Pneumococcus
• Grifths studied the bacterium pneumococci.
• Certain strains secrete a polysaccharide capsule whereas some do not.
• When grown on a petri dish on solid medium, the capsule-secretng strains
have a smooth colony morphology and those that can't, have a rough
appearance.
• This also afects their virulence (ability of a microorganism to cause disease or
damage to the host cells).
• Smooth strain infects mouse – capsule allows the bacteria to escape atack by
the mouse’s immune system. As a results, the bacteria can grow and
eventually kills the mouse.
• Non-capsulated strain infects mouse – they are destroyed by the animal's
immune system and the mouse survives.
 Unexpected result
• Live R (Non-virulent) bacteria was mixed with heat killed type S
(virulent)bacteria.
• Mouse died and furthermore, extracts from the tssues of the dead mouse
were found to contain living type S bacteria!!!!
• Why?
• It means that something from the dead type S bacteria was transforming
the type R bacteria in type S.
• The process was called transformaton.
• The unidentied substances causing this to occur was termed the
transformaton principle.
• Diccovery of trancformaton
•  Grifths experiment (1928) showed that something from the virulent S strain of Streptococcus
pneumoniae ‘transformed’ the non-virulent R strain.
•  The transformed bacteria acquired the informaton to make a smooth capsule.
•  The genetc material for the smooth capsule trait must have been replicated so that it could be
transmited from mother to daughter cells during cell division
•  Grifth’s experiment showed that some genetc material from the dead bacteria had been transferred
to the living bacteria and provided them with a new trait (namely a type smooth capsule).

• What ic the trancforming material?

• At this point, Grifths experiment showed that genetc material from the dead smooth capsule (virulent)
strain of S. pneumonia had been transferred into the rough capsule (non-virulent) strain of S.
pneumonia. It was stll not clear what chemical was responsible for the transformaton.
Transforming principle- a substance that could be transferred from non-living cells to living cells, causing the living cell to
show characteristics of the non-living cell.
 Summary of experiments
• The aim of this experiment was to determine what is the genetc material.
• Twelve years afer Grifths experiments, 3 scientsts gathering proof to show that DNA was the
foundaton of genetc material used Grifths observatons as a strategy to identfy genetc material.
• At this point in tme DNA, RNA, proteins and carbohydrates were known to be major consttuents of
a cell.
• However, Avery, MacLeod and McCarty being biochemists they knew how to prove their point.
• They did this with enzymes that selectvely hydrolysed (hydro=water, lysis=separaton) DNA, RNA or
protein in puriied cell extracts of virulent S. pneumoniae.
• By doing this they could prove that only hydrolysis of DNA inhibited transformaton – therefore
DNA must have been responsible for the transformaton process.
• DNA is the genetc code – it carries the data/informaton that determines what a living organism is
and how it behaves
Avery, MacLeod and McCarty
Experiments
 Experiment by Hershey and
Chase
• Evidence that DNA is the genetc material of T2 phage.
• Studied the virus known as T2.
• Virus that infects E.coli (bacteria) cells and therefore known as a
bacteriophage.
 Summary of experimental
fndings
• DNA is found inside the head of T2 capsid.
• Simple virus composed of two types of macromolecules i.e DNA and proteins.
• Viruses cannot synthesis new viruses instead it introduces its genetc material
into the cytoplasm of a living cell.
• This involves atachment of the tail ibers to the bacterial cell wall and the
injecton of the genetc material into the cytoplasm of the host cell.
• The bacterial cytoplasm provides all the machinery to make viral proteins and
DNA. These assemble and make new viruses.
• The viral DNA and protein assembles to make new viruses that are released
from the cell by lycic –( cell breakage).
 An account of the Hershey and Chase experiment using radioactive medium

 Hershey and Chase conducted experiments on bacteriophage to prove that DNA is the genetc material.

 Bacteriophage viruses were grown on a medium that contained radioactve phosphorus P32 and some in another medium with
radioactve sulfur S35.

 Viruses that grow in radioactve phosphorus contain radioactve DNA.

 Similarly, viruses grown in the presence of radioactve sulfur contained radioactve protein.

 Both the radioactve virus types were allowed to infect E. coil separately.

 The infected bacterial cells were agitated in a blender to remove viral coats from the bacteria.

 It was observed that only radioactve phosphorous was found to be within the bacterial cell. In contrast, radioactve sulfur was
only found in the surrounding medium, not the bacterial cell.

 This proves that only DNA and not protein coat entered the bacterial cell.

 Thus, DNA is the genetc material.

Evidence that DNA is the genetic
material in phage
 RNA functions as the genetic
material in some viruses
• All organism have genetc material.
• So do viruses – Hershey and Chase concluded from their experiments that this
genetc material is DNA.
• This is correct for the T2 phage.
• However, many viruses use RNA rather than DNA as the genetc material.
• RNA has been isolated from tobacco mosaic virus which infects plants.
• When applied to plant tssue, the plants developed the same types of lesions
that occurred when there were exposed to intact TMV.
• Conclusion – viral genome of TMV (tobacco mosaic virus) is composed of RNA
rather than DNA.
 Nucleic acid structure
pg228
• Nucleic acids – DNA and RNA
• Four levels of complexity in
structure:
• Nucleotdes
• Strands
• Double helix
• 3D structure of DNA from folding.
 Nucleotides are the building
blocks of Nucleic acids
• Deoxyribose (DNA)
and ribose (RNA).
• Purines and
Pyrimidines
• A, T, C , G
• U in RNA instead of T.
• Nucleoside – base +
sugar
 Nucleotides are linked together
to form a strand
• Phosphodiester linkages
– links nucleotdes in the
RNA and DNA strand.
• Directonality – base on
the orientaton of the
sugar molecules within
that strand.
• Directon is either 5’ – 3’
or 3’ -5’
• 5’ – TACG- 3’
 Discovery of the Double Helix
structure
• James Watson and Francis Crick
in 1953.
• They set out to determine the
structure of DNA because they
felt it would be important in
understanding the functoning
of genes.
• Other researchers contributed
as well, Rosalind Franklin and
Linus Pauling
 Chargaffs Rule that DNA is
symmetrical
• Biochemical compositon of
DNA
• The amounts of A = T and C =
G
• For every adenine there is a
thymine (complementary
bases).
• For every cytosine there is a
guanine (complementary
bases).
Chargaffc Experiment

• 1. He extracted the chromosomes from various cells and then treated them with protease to separate the
DNA from the chromosomal proteins.
• 2. He then treated the DNA with a strong acid that cleaved the bond between the deoxyribose sugar and the
bases thus releasing the individual bases.
• 3. This mixture of bases was then subjected to paper chromatography to separate the four types
• 4. The quantty of each base was then determined spectroscopically

• His discovery was very important because it proved that A always binds to T and G always binds to C which
was crucial in understanding the nature of DNA
 Watson and Crick deduce the
double helical structure
Key featurec of DNA includec;
• Helical structure
• Two strands
• 10 bases per turn
• Chargaf’s rule
• Linked in a linear fashion
 The molecular structure of the
DNA double helix
• Key features:
• Two strands form a right handed double helix
• Bases hydrogen bond A-T and C-G
• Two strands are antparallel in directon with regards
to their 5’ – 3’ or 3’ -5’ directonality .
• 10.0 nucleotdes in each strand per complete 360⁰
turn of the helix.
• Complementary
• Antparallel- Refers to opposite orientaton of
two DNA molecules.
• Grooves (major and minor)
 DNA can form alternative types
of double helices
• A DNA – right handed helices
• B DNA – right handed helices
• Z DNA – lef handed conformaton
 DNA can form Triplex DNA
• Formed in vitro using pieces of DNA that
were made synthetcally.
• Potental tool as inhibit certain genes.
• Synthetc DNA binds to a gene and inhibits
transcripton.
• Hence, gene silencing.
• More research is required.
 RNA Molecules
• Can form diference helical structures due to their complementary
regions.
• This base pairing can form double stranded regions.
• These structural paterns include bulge loops, internal loops,
multbranched junctons and stem loops ( hairpin loops).
 Tutorial questions
• What was the purpose of Griffith’s experiments 1 and 2 respectively:

• Experiment 1, in which he injected a mouse with live R cells?

 To illustrate the effect of R cells on mice-R cells are non-virulent and did not kill the mice when injected.

 experiment 2, in which he injected a mouse with live S cells?

 To illustrate the effect of S cells in mice-S cells are virulent and killed the mice.

What happened in experiment 3, in which he injected a mouse with heat-killed S cells?

 The effect of heat-killed S cells on mice-Heat-killed S cells do not affect the mice.

• What happened in experiment 4, in which he injected a mouse with a mixture of heat-killed S cells and live R cells?

 The effect of a mixture of heat-killed S cells and live R cells- The mixture killed the mice. Some factor or biomolecule from the heat-killed S bacteria had entered the living
R bacteria, as a result, this factor "transformed" the R bacteria into S bacteria.

• Why can an S strain of bacteria cause disease, but an R strain cannot?

 capsule allows the bacteria to escape attack by the mouse’s immune system. As a result, the bacteria can grow and eventually kills the mouse.

 Non-capsulated strain infects mice – the animal’s immune system destroys them.

 OR

The s- strain is virulent while the R strain is non-virulent.