Introduction to Molecular

Genetics
Akn Pala OM
http://akin.houseofpala.com
akinpala@gmail.com
Twitter: @akinpala
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 Molecular Genetics
How things were invented?
Important to understand the mechanisms
Underlying theory should be understood to
reach correct conclusions.
By learning how mechanisms were figured
out by other scientists in the past, one can
learn how to figure out new things.
As opposed to the imitation game
The view: it will be invented abroad is bad

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 Molecular Genetics
Although genes were known to exist on
chromosomes, chromosomes are composed
of both protein and DNA, and we did not
know which of these is responsible for
inheritance.
In 1928, Frederick Griffith discovered the
phenomenon of transformation. Griffith's
experiment showed that dead bacteria could
transfer genetic material to "transform" other
still-living bacteria.

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 Griffith's experiment
This was an experiment done in 1928 by
Frederick Griffith.
It was one of the first experiments
showing that bacteria can get DNA
through a process called transformation.
Griffith used two strains of Pneumococcus
(Streptococcus pneumoniae).
These bacteria infect mice.

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 Griffith's experiment
Griffith used a type III-S (smooth) and type
II-R (rough) strain.
The III-S strain covers itself with a
polysaccharide capsule
The capsule protects it from the host's immune
system.
The host dies.
The mutant II-R strain does not have that
protective shield around it
The rough strain is killed by the host's immune
system.

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 Griffith's experiment
In this experiment, bacteria from the III-S
strain were killed by heat
He boiled them
The boiled bacteria, as expected, did not kill
the mice.

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Griffith's experiment

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 Griffith's experiment
Then, just to be thorough, Griffith added
boiled bacteria remains to live mutant II-R
strain bacteria.
Neither heat killed-smooth nor rough
strain killed mice on their own, each was
harmless,
But the blend of the two killed the mice
And live wild type Pneumoniae were found in
the mice.

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 Griffith's experiment
Griffith was also able to get both live II-R
and live III-S strains of pneumoniae from
the blood of these dead mice.
He concluded that the type II-R had been
"transformed" into the lethal III-S strain
by a "transforming principle" that was
somehow part of the dead III-S strain
bacteria.

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 Griffith's experiment
Today, we know that the "transforming
principle" Griffith saw was the DNA of the
III-S strain bacteria.
The genes of the wild type survived the
boiling and infiltrated the live mutants.
Transforming the harmless bacteria into
the killer wild type.

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 Griffith's experiment
While the bacteria had been killed, the DNA
had survived the heating process and was
taken up by the II-R strain bacteria.
The III-S strain DNA contains the genes that
form the shielding polysaccharide part from
attack.
Armed with this gene, the former II-R strain
bacteria were now protected from the host's
immune system and could kill the host.

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Griffith's experiment discovering the "transforming principle" in
pneumoniae bacteria.

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 Oswald Averys Investigation
16 years later, in 1944, the Avery
MacLeodMcCarty experiment identified
DNA as the molecule responsible for
transformation.
Avery and his colleagues showed that DNA
was the key component of Griffith's
experiment
mice are injected with dead bacteria of one
strain and live bacteria of another, and
develop an infection of the dead strain's type
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 AveryMacLeodMcCarty
experiment
In 1944 it was reported that the DNA is
the substance that causes bacterial
transformation.
Back then it had been widely believed that it
was proteins that served the function of
carrying genetic information.
The word protein itself was coined to indicate
a belief that its function was primary (from
greek adjective for first rank)
And the Earth was flat

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 AveryMacLeodMcCarty
experiment
It was the culmination of research in the 1930s
and early 1940s at the Rockefeller Institute for
Medical Research to purify and characterize the
"transforming principle" responsible for the
transformation phenomenon first described in
Griffith's experiment of 1928.
Avery and his colleagues suggested that DNA,
rather than protein as widely believed at the time,
may be the hereditary material of bacteria.
Studies on the Chemical Nature of the Substance Inducing
Transformation of Pneumococcal Types: Induction of Transformation by
a Deoxyribonucleic Acid Fraction Isolated from Pneumococcus Type III.
1944, Journal of Experimental Medicine.

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 AveryMacLeodMcCarty
experiment
Avery boiled plenty of bacteria,
precipitated, extracted, centrifuged,
analyzed, over and over, until he had pure
genetic material.
He removed various organic compounds
from bacteria,
if the remaining organic compounds were still
able to cause R strain bacteria to transform,
then the substances removed could not be
the carrier of genes.

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 AveryMacLeodMcCarty
experiment
S-strain bacteria first had the large cellular
structures removed.
They were treated with protease enzymes,
which removed the proteins from the cells
the remainder was placed with R strain
bacteria.
The R strain bacteria transformed,
meaning that proteins did not carry the
genes causing the disease.

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 AveryMacLeodMcCarty
experiment
The remnants of the R strain bacteria
were treated with a deoxyribonuclease
enzyme which removed the DNA.
The R strain bacteria no longer
transformed after the enzyme treatment.
This indicated that DNA was the carrier of
genes in cells.

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When he announced his results in 1940, few
scientists believed him.

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 The HersheyChase
experiments
The HersheyChase experiment in 1952
confirmed that DNA (rather than protein)
is the genetic material of the viruses that
infect bacteria
This provided further evidence that DNA is the
molecule responsible for inheritance.

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 HersheyChase experiments
Alfred Hershey and Martha Chase.
Not the makers of Hershey chocolate
The experiments were to make sure that
DNA was the genetic material which had
been discovered by the Swiss physician
Friedrich Miescher in his experiments on
white blood cells/leukocytes in 1869.
Hershey shared the 1969 Nobel Prize in
Physiology or Medicine for his discoveries
concerning the genetic structure of viruses.

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 HersheyChase Experiments
Methodology
Hershey and Chase used T2 phage, a
bacteriophage, for their experiments.
The phage infects a bacterium by attaching to it
and injecting its genetic material into it.
They put labels on phage DNA with
radioactive Phosphorus-32.
They then followed the phages while they
infected E. coli. They found that the
radioactive element was only in the bacteria,
and not in the phage.

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 HersheyChase Experiments
Methodology
In a second experiment, they put labels on
the phage protein with radioactive Sulfur-35.
After the phage was attached to the
bacterium, the radioactive element was
found in the phage, but not in the bacteria.
This showed them that genetic material
which infects the bacteria is DNA.
Hershey A.D. and Chase M. (1952) Independent functions of viral protein and
nucleic acid in growth of bacteriophage. J Gen Physiol. 36:39-56.

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HersheyChase experiments

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 Hmmerlings algae
Dr. Joachim August Wilhelm Hmmerling:
Danish-German biologist funded by Nazi
Germany.
The role of the nucleus as the repository of
genetic information in eukaryotes had been
established by Hmmerling in 1943 in his
work on the single celled algae Acetabularia.
In 1930's J. Hmmerling was wondering
where the genetic traits were stored inside
the eukaryotic cell.

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 Hmmerlings algae
Acetabularia
In 1944, Avery et al. showed that the traits
were stored in a chemical called DNA - but
that was in prokaryotic cells.
1953, Hmmerling, J. : Nucleo-cytoplasmic
relationships in the development of
Acetabularia. J. Intern. Rev. Cytol. 2: 475-
498: reported his work done in the 1930s.
The same great year that Watson and Crick
published their double helix paper.
This was one of the most elegant and simple
experiments.

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 Hmmerlings algae
Acetabularia has three basic parts
Rhizoid, a short set of root-like appendages. The
"foot" or base which contains the nucleus,
Median stalk, which accounts for most of its
length
Apex, where the cap forms.
Acetabularia are among the largest single-
celled organisms, having also a remarkably
large nucleus.
Great model organism to study, just like C.
Elegans

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 Model Organism
A model organism is a non-human species
that is extensively studied to understand
particular biological phenomena
The expectation is that discoveries made
in the organism model will provide insight
into the workings of other organisms

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 Model Organism

Escherichia coli is a
prokaryotic model Drosophila melanogaster
organism
Saccharomyces
cerevisiae (bakers
yeast), one of the
most intensively
studied eukaryotic
model organisms
in molecular and
cell biology
Genetically selected (diabetic mice)

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 Hmmerling algae
During sexual reproduction, the
nucleus undergoes multiple
rounds of mitosis, forming many
daughter nuclei all within one
nuclear membrane.
These nuclei undergo meiosis
and are transported to the tips of
the branches, the sporangia,
where they are released as
gametes.

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 Hmmerlings Experiment
Each Acetabularia cell is composed of
three segments: the root (nucleus), the
stalk, and the cap.
If the "traits" were bundled up in some
manner and not distributed all throughout
the cell,
just cutting the cells in half and seeing which part
could regenerate would be a pretty good hint as to
where the "traits" resided in the cell.

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 Hmmerlings Experiment
The heads of the algae withered when
detached, and only the feet regenerated.
Hence, he was pretty sure that the "traits"
were somewhere stored in the feet of
these algae.

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 Hmmerlings Experiment
Hmmerling exchanged caps between
individuals from two species, A.
mediterranea and A. crenulata.
A. mediterranea has a smooth, disc
shaped cap
A. crenulata has a branched, flower-like
cap.

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 Hmmerlings Experiment
After switching, no parts of the
morphological hybrids withered, but over
time the top halves slowly changed or
"morphed" into the form that was dictated
by the "traits'" instructions in the
respective bases.
This showed that the nucleus controlled
the form of the cap.

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 Hmmerlings Experiment
He used his microscope to
make sure that the feet
contained that thing called a
nucleus.
Hammerling concluded that
the genes or traits resided in
the nucleus - all of the cell's
traits, and not just some of
them.
Why all? Because the cells
could do all the steps of their
life-cycles.
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 Hmmerlings Experiment
In another experiment, Hmmerling
inserted a nucleus from one species of
Acetabularia into an intact Acetabularia of
a different species.
The Acetabularia then produced a hybrid
cap with characteristics of both species.

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 Hmmerlings Experiment
This showed that both nuclei influenced
the form of the cap.
Hammerling's results showed that the
nucleus of a cell contains the genetic
information that directs cellular
development.

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 Watson & Crick
James Watson and Francis
Crick determined the structure of DNA in
1953, using the X-ray
crystallography work of Rosalind
Franklin and Maurice Wilkins that indicated
DNA had a helical structure (shaped like a
corkscrew).
Nobel Prize in 1962 "for their discoveries concerning
the molecular structure of nucleic acids and its
significance for information transfer in living material"

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 Watson & Crick
Their double-helix model had two strands
of DNA with the nucleotides pointing
inward, each matching a complementary
nucleotide on the other strand to form
what looks like rungs on a twisted ladder.
This structure showed that genetic
information exists in the sequence of
nucleotides on each strand of DNA.

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 Watson & Crick
The structure also suggested a simple
method for replication:
if the strands are separated, new partner
strands can be reconstructed for each based
on the sequence of the old strand.
This property is what gives DNA its semi-
conservative nature where one strand of
new DNA is from an original parent
strand.
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DNA model built
by Crick and
Watson in 1953,
on display in the
Science
Museum,
London.

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 James Watson
Watson has repeatedly supported genetic
screening and genetic engineering in public
lectures and interviews, arguing that stupidity
is a disease and the "really stupid" bottom
10% of people should be cured.
He also suggested that beauty could be
genetically engineered, saying in 2003,
"People say it would be terrible if we made
all girls pretty. I think it would be great.

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 Molecular Genetics
Although the structure of DNA showed
how inheritance works, it was still not
known how DNA influences the behavior
of cells.
In the following years, scientists tried to
understand how DNA controls the process
of protein production.

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 DNA-RNA
It was discovered that the cell uses DNA as a
template to create matching messenger RNA
(mRNA).
Transcription is the first step of gene expression,
in which a particular segment of DNA is copied
into RNA by the enzyme RNA polymerase
The nucleotide sequence of a mRNA is used
to create an amino acid sequence in protein;
this translation between nucleotide
sequences and amino acid sequences is
known as the genetic code.

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 Sanger Sequencing
With the newfound molecular
understanding of inheritance came a surge
of research.
Now interest is measured using # of papers
published on a subject.
One important development was chain-
termination DNA sequencing in 1977
by Frederick Sanger.

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 Sanger
Frederick Sanger, (1918 2013) was a
British biochemist who won the Nobel
Prize for Chemistry twice (1958 & 1980),
one of only two people to have done so in
the same category.
1958 for his work on the structure of proteins,
especially that of insulin.
1980 the determination of base sequences in
nucleic acids.

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 Sanger Sequencing
This technology allows us to read the
nucleotide sequence.
It was the most widely used sequencing
method for approximately 25 years (up to
2002).
In 2004, companies such as 454 (now
Roche) started replacing Sanger sequencing
with the "Next-Gen" sequencing methods.
Now Illumina (Miseq, Hiseq, Hiseq 10) and
Life technologies (Ion torrent) desktop
sequencers etc.

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 More advanced technologies
but smaller companies
Quantum biosystems
Each chip has a nanogap through which only
a single strand of DNA or RNA passes, and
the device uses nanoelectrodes
There is one molecule-wide gap between
electrodes
It measures tunnel current, and looks at the
current differences.

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 Quantum Biosystems
Complete sequencing system on a chip:
The device incorporates on-chip sample
preparation to denature and linearize the
DNA.
The molecule is first denatured by
microheaters and then driven through an
array of nanopillars to ensure linearization
before it reaches the sensor.

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Quantum Biosystems

It is not a desktop machine.

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 Quantum biosystems
Bias free (PCR free)
The polymerase chain reaction (PCR)
amplifies one or several copies of a piece
of DNA by several orders of magnitude, to
generate up to millions of copies of that
DNA sequence.
PCR can cause mutations or primer dimers
as the enzymes copy sequences.

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 Quantum biosystems
Current DNA sequencing technology depends
on PCR, resulting in inherent accuracy issues
due to PCR generated errors.
Current technologies use pre-treatments such
as labeling, PCR, or detection with
fluorescent reagents, which are widely used
in current DNA sequencing.
For Illumina, it takes at least 2 hours of lab work
before the sample is put to the sequencing
machine.
You dont have to do PCR before hand, but
Illumina machine runs sort of a PCR itself.

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 More advanced technologies
but smaller companies
MinION USB size sequencing.
Nanopores perform a complete single-
molecule sensing experiment.
Plugging directly into a laptop or desktop
computer through a USB port, it is a self-
contained device to deliver real-time
experimental data.
Accuracy is about 80 %

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MinION, USB size sequencing

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 Sequencing
These have been used especially for large-
scale, automated genome analyses.
However, the Sanger method remains in
wide use, for smaller-scale projects,
validation of Next-Gen results and for
obtaining especially long contiguous DNA
sequence reads (>500 nucleotides).

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 Sanger Sequencing
If 2-3 samples out of 100 samples put in an
Illumina miseq machine does not produce
good results (many missing/unknown),
because their PCR did not work, or for some
other reason, the small sample size can be
run on a Sanger machine (cheaper)
For higher sample sizes or # of genes, next
generation sequencing methods are cheaper
per nucleotide.

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 Molecular Genetics
In 1983, Kary Banks Mullis developed
the polymerase chain reaction, providing a
quick way to isolate and amplify a specific
section of DNA from a mixture.
The efforts of the Human Genome Project,
Department of Energy, NIH, and parallel
private effort by Celera Genomics led to
the sequencing of the human genome in
2003.

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 Molecular Genetics
Epigenetics has been the popular study,
the cutting edge science.
In 2013, an MIT graduate student used
CRISPR transfection technique to make
transgenic mice more precisely, and in a
shorter time without backwards
crossbreeding for homozygous animals.
Transformation in prokaryotes, transfection in
mammalian cells.

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 Homework 4, paper
Using your own words, write about history
of Genetics.
You are free to start at any point of
history.
Your paper should use at least 200 words.
You can use your word processor to count,
but should be about half an A4 page.

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