Identification of the Genetic material

Objectives of the lesson:

At the end of this lesson students will be
able to:
Identify the genetic material
List evidences that sounds DNA as a
genetic material
Provide justifications why proteins are no
more considered as a genetic material
Identify components of the chromosomes

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 Identification of the genetic material

Chromosomes were the organs of

heredity, because
1. They formed the only link between two
generations
2. They occurred in every organism in
specific number and had specific
morphology for particular species
3. They carried linearly arranged genes

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 Identification…

4. Any variation in their number or

morphology affected the phenotype of
the species
 varies attempts were made by early
molecular geneticists to identify the
physical and chemical nature of a
gene.

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 Identification….

But the gene were found so minute

structures that their physical identity
remained almost impossible.
However, the extensive chemical
analysis of chromosomes of different
organisms revealed that chromosomes
contain protein and nucleic acids.

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 Identification….

It was thought that genes might have

either proteins or nucleic acids as
their component molecules
Early molecular geneticists have
assigned the informational role of
genes to the chromosomal protein
because they found nucleic acids too
simple to carry genetic information
(tetra nucleotide hypothesis)

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 Identification..

The controversy about assignment of

genetic role either to chromosomal
protein or chromosomal DNA existed
up to 1949
 A.Mirsky and H.Ris had found that
all cells of an organism appeared to
contain the same amount of DNA
Different cell types contained quite
different amount and kind of protein

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 Identification...

Its constancy favored DNA as a genetic

material.
Around 1953, it was universally accepted
that DNA is the genetic substance
Later on RNA was found to be the genetic
material of some viruses
The concept that DNA or RNA is the genetic
material has been developed and supported
by the following evidence

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 Direct evidence

The most conclusive evidence in support

of DNA as a genetic material came from
the following three approach on
microorganisms:
1. Transformation of Bacteria
2. Mode of infection of bacteriophage
3. Conjugation of bacteria

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 Direct….

 In 1928, Frederick Grift encountered a

phenomenon now called genetic
transformation
 Colonies of virulent strains of pneumonia,
Diplococcus pneumonia that grow on
nutrient agar have a smooth (S) glistering
appearance due to the presence of a type
specific polysaccharide
The avirulent lack this capsule and they
produce dull, rough(R) colonies

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 Direct….

Smooth(S) and Rough (R) characteristics are

directly related to presence or absence of a
capsule and this character is to be
determined genetically
 Smooth forms sometimes mutate to rough
forms, but this change has not been found
reversible

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 Direct….

Griffith injected laboratory mice with live R-II

Pneumoococci; the mice suffered no illness
 when the mice were injected with S-III
Pneumoococci the mice suffered from
pneumonia and died.
 He injected a mixture of living avirulent R-II
and heat killed S-III virulent to a mice and
unexpected symptoms of pneumonia
appeared and high mortality resulted.

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 Direct….

By postmorteming the dead mouce, it

was found that their heart blood has
both R-II and S-III pneumonococci

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 Direct….
 From this result Griffith concluded
that the presence of the heat killed S-
III strain must have caused a
transformation of the living R-II so
as to restore to them the capacity of
capsule formation they had earlier
lost by gene mutation , this was
called Griffith effect or more popularly
bacterial transformation

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 Direct….

Live S-III injected to live mouse Dead mouse

Dead S-III injected to live mouse Live mouse

Live R-II injected to Live mouse Live mouse

Dead S-III+ live R-II injected to Dead Mouse

live mouse

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 Direct….

Griffith could not understand the cause of

bacterial transformation
That is identified by Oswald Avery, Colin
MacLeod and MaclynMcCarty (1944)
They identified the transforming principle
from the cell extract(I.e. cell free extractor S-
III bacteria) and demonstrated that it was
DNA
They isolated DNA from S-III and added this
DNA to a live R-II bacteria culture
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 Direct....

 After a period of time they placed a sample of S-III containing

R- II bacteria culture on agar surface and allowed it to form
colonies
 Some of the colonies (1 in 10, 000) that grew were SIII type
 To show that this was a permanent genetic change, they
disperse the newly formed S-III colonies on a second agar
surface.
 The resulting colonies were again S-III type

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 Direct…
purified polysaccharide coat around each S-
III bacterium to transform was tested but
no transformation was observed
 Evidence that extracted transforming
principle contains DNA was provided By:-
1. Chemical analysis showed that the major
component was dioxyribose containing
nucleic acids
2. Physical measurements showed that the
sample contains highly viscous substance

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 Direct…
3. Transforming activity is not lost by reaction
with either a) purified proteolytic enzyme,
b/ ribonuclease
4. DNAase (DNA-depolymerizing) inactivated
the teransforming principle.

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 The Blender Experiment

was performed by Alfered Henshey and

Martha Chase in 1952
is an experiment with E. coli phage T2.
 a kitchen waring blender was used as a
major piece of apparatus
 they demonstrated as the DNA injected by a
phage particle contains all the information
required to synthesize progeny phage
particle

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 The Blender…

The phage DNA contains

phosphorus, the protein coat
contains sulfur
 Phage DNA was made radioactive
by growing infected bacteria on a
medium containing radioactive phosphorus
(32Po4) and radioactive 35SO4

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 The Blender…

such differential labeling will help in

identifying DNA and protein
with out performing any chemical test
 They allowed both kinds of labeled phage
particles to infect E.coli
 the bacteria were immediately agitated in a
waring blender

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 The Blender...
After shaking, only radioactive 32P was found
associated with bacterial cell and 35S was
found only in surrounding medium and
not in bacterial cell
 when phage progeny was studied for
radioactivity it was found that the phage
progeny carried labeled only with 32P
 The progeny was not labeled with 35S
Thus DNA entering the host cell carries all
information for synthesis of new phage
particles

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 Bacterial conjugation
Laderberg and Tatum 1964 found that
when F+ strain of E.coli conjugates with F-
cell the latter will convert in to F+ or male
strain
The F factor was found to be a fragment of
DNA molecule which occurred in
cytoplasm of bacterial cell.

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 Indirect evidence

came from higher organisms which are not

easy to manipulate
 the fact that DNA is a genetic material in
higher organisms can be supported by:-
1. The Feulgen techniques has shown that
DNA entirely remains restricted to the
chromosomes and it forms one of the
major components of chromosomes

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 Indirect evidence

2. Various quantitative measurements of

amounts of DNA in different cells have
shown that there is a correlation between
the amount of DNA and the number of
chromosomes sets (ploidy)
3. Diploid cells contain twice as much DNA as
do hypoid cells of the same species.

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 Evidence for RNA

RNA is a genetic material for some RNA

viruses .
The evidence or this is:-
 A.Gierer and G. Schramm (1956) showed
that tobacco plants could be inoculated
with purified RNA from TMV
TMV like lesions could latter be identified
on the tobacco leaves

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 Evidence for RNA...

A different approach was taken by H.

Fraaenkel Conrat and B. Singer in 1957
 they separated the TMV in to protein and
RNA
 They tested RNA and protein separately for
infecting tobacco
 RNA alone was able to cause infection
 They formed reconstituted viruses
(chimeras) protein from one mutant strain of
TMV Virus and RNA from another or vice
versal
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 Evidence for RNA

 the chimeras were allowed to infect tobacco plant

they examine the progeny of the chimeras
 in all cases the progeny were the parental RNA type
and not the parental protein type
Type A RNA Type B

←protein→

Progeny was
Progeny was type B
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Biolt 611 N. Berhane
 Chemical nature of Genetic material

DNA extracted from a wide Varity of

organisms is complex-macro molecular or
polymeric chemical compound that
contains four kinds of smaller building
blocks (monomers) called
deoxyribonucleotides

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 Chemical nature of Genetic material

The monomer units of DNA are nucleotides

 Each nucleotide consists of a 5-carbon
sugar (2 deoxyribose),
 a phosphate group.
 a pyramidine or purine nitrogenous bases
 There are four different types of nitrogenous
bases

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 Chemical nature
 Two are heterocyclic two ringed purins, Adenine (A)
andGuanine (G)
 two are one ringed pyramidines cytosine (C) and
Thiamine (T)
The four deoxyribosenucliotides also occur in nucleoplasm
and cytoplasm, but in their triphosphate forms
 dATP
dGTP
dCTP
TTP
 the significance of this occurrence in triphosphate forms
is during DNA replication , the DNA polymerase enzyme can
act only on triphosphate of deoxyribonucleotides

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 General structure of a nucleotide

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 Nitrogenous bases

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 Deoxyribose Sugar

The deoxyribose sugar of the DNA backbone has

5 carbons and 3 oxygens.
The carbon atoms are numbered 1', 2', 3', 4', and
5' to distinguish from the numbering of the
atoms of the purine and pyrmidine rings.
The hydroxyl groups on the 5'- and 3'- carbons
link to the phosphate groups to form the DNA
backbone.
Deoxyribose lacks an hydroxyl group at the 2'-
position when compared to ribose, the sugar
component of RNA.
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 Nucleosides

A nucleoside is one of the four DNA bases covalently

attached to the C1' position of a sugar.
The sugar in deoxynucleosides is 2'-deoxyribose.
The sugar in ribonucleosides is ribose.
Nucleosides differ from nucleotides in that they lack
phosphate groups.
The four different nucleosides of DNA are
deoxyadenosine (dA)
deoxyguanosine (dG),
deoxycytosine (dC), and
(deoxy)thymidine (dT, or T).

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 Nucleotides

A nucleotide is a nucleoside with one or more

phosphate groups covalently attached to the 3'- and/or
5'-hydroxyl group(s).

DNA Backbone
The DNA backbone is a polymer with an alternating
sugar-phosphate sequence.
The deoxyribose sugars are joined at both the 3'-
hydroxyl and 5'-hydroxyl groups to phosphate groups
in ester links, also known as phosphodiester bonds.

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 Features of DNA back bone

 Alternating backbone of deoxyribose and

phosphodiester groups
 Chain has a direction (known as polarity), 5'- to
3'- from top to bottom
 Oxygens with in phosphates are polar and
negatively charged A, G, C, and T bases
can extend away from chain, and stack
atop each other
 Bases are hydrophobic

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 DNA Double Helix

DNA is normally double stranded

macromolecule.
Two polynucleotide chains, held together by
weak thermodynamic forces, form a DNA
molecule.
Features of the DNA Double Helix
Two DNA strands form a helical spiral, winding
around a helix axis in a right-handed spiral
 The two polynucleotide chains run in
opposite directions
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 DNA Double Helix

The sugar-phosphate backbones of the two

DNA strands wind around the helix axis like
the railing of a sprial staircase
 The bases of the individual nucleotides are on
the inside of the helix, stacked on top of each
other like the steps of a spiral staircase.

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 Structure of DNA

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 Base Pairs

Within the DNA double helix:

 A forms 2 hydrogen bonds with T on the
opposite strand,
 G forms 3 hyrdorgen bonds with C on the
opposite strand.
 dA-dT and dG-dC base pairs are the same
length, and occupy the same space within a
DNA double helix. dA-dT and dG-dC base
pairs can occur in any order within DNA
molecules

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 DNA Helix Axis

The helix axis is most apparent from a view

directly down the axis.
The sugar-phosphate backbone is on the
outside of the helix where the polar
phosphate groups can interact with the polar
environment.
The nitrogen containing bases are inside,
stacking perpendicular to the helix axis.

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 The equivalent rule

Chargaff (1950) discovered the equivalent rule which suggests

that despite wide compositional variations exhibited by
different types of DNA the total amount of purins equals the
total amount of pyramidines (A+G=T+C)

The amount of Adenine equals the amount of thiamine (A=T)

 The amount of guanine equals the amount of cytosine (G=C)
 Applied almost in all organisms
 However DNA isolated from plant and animal is rich in
adenine and thymine and relatively poor in guanine and
cytosine

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 The equivalent...

DNA isolated from microorganisms is rich in

guanine and cytosine and relatively poor in
adenine and thiamine
 This difference reflects the difference in
genetic information carried by this
hereditary molecule
 Has phylogenetic , evolutionary and
taxonomical importance

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 Next topic will be on Mechanism of DNA
replication

Assignment :
 differentiate between transformation
and transduction
 What is alternate form of DNA double
helix

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