Download Slide 1 Molecules of Genetic Inheritance

Shikha Yashveer1, Jayanti Tokas2, Shalini Jain3 and Hariom Yadav4
1Department
of Molecular Biology and Biotechnology, 2Department of Biochemistry
CCS HAU, Hisar, Haryana, India
3Postgraduate Institute of Medical Education and Research, Chandigarh, India
4National Agri-Food Biotechnology Institute, Mohali, Punjab, India
Email: [email protected]
The period from the early 1900s to World
War II has been considered as the "golden
age" of genetics
 Scientists still had not determined that
DNA or the protein, was the hereditary
material.
Friedrich Meischer in 1869 isolated DNA
from fish sperm and the pus of open wounds
Since it came from nuclei, Meischer named
this new chemical, nuclein.
Subsequently the name was changed
to nucleic acid and lastly to deoxyribonucleic
acid (DNA).
Robert Feulgen, in 1914, discovered that
fuchsin dye stained DNA
 DNA was then found in the nucleus of
all eukaryotic cells.
During the 1920s, biochemist P.A. Levene
analyzed the components of the DNA molecule.
He found it contained four nitrogenous bases:
cytosine, thymine, adenine, and guanine;
deoxyribose sugar; and a phosphate group
He concluded that the basic unit (nucleotide)
was composed of a base attached to a sugar and
that the phosphate also attached to the sugar
He (unfortunately) also erroneously concluded
that the proportions of bases were equal and that
there was a tetra nucleotide that was the
repeating structure of the molecule
 The nucleotide, however, remains as the
fundamental unit (monomer) of the nucleic acid
polymer
There are four nucleotides: those with cytosine
(C), those with guanine (G), those with adenine
(A), and those with thymine (T).
During the early 1900s, the link between
Mendel's work and that of cell biologists resulted
in the chromosomal theory of inheritance
Garrod proposed the link between genes and
"inborn errors of metabolism"
Now the question was formed: what is a gene?
The answer came from the study of a deadly
infectious disease: pneumonia.
Frederick Griffith in 1928
Experimental proof that DNA is the genetic material
Worked with 2 strains of the bacterium
Streptococcus pneumoniae ----- R & S
Gelatinous capsule
Capsular
polysaccharide
absent
Small
colonies
Rough
surface
Avirulent
Makes colony large
Smooth appearance
Protects the bacterium
from defense mechanism of
infected animal
Virulent
Causes pneumonia
R
S
Died
Mouse remains healthy
Mouse remains healthy
Mice died of pneumonia
Transforming principle
Chemical nature not known
DNA
TRANSFORMATION
R colonies & few S
colonies
Culture of R cells
S cells extract
Culture of R cells
R colonies only
Transforming activity most likely
DNA
Their evidence was strong but not totally
conclusive
 The then-current favorite for the
hereditary material was protein; DNA was
not considered by many scientists to be a
strong candidate.
The breakthrough in the quest to determine the hereditary
material came from the work of Max Delbruck and Salvador
Luria with Bacteriophages in the 1940s
Bacteriophages are a type of virus that attacks bacteria
 The viruses that Delbruck and Luria worked with were
those attacking Escherichia coli
Bacteriophages consist of protein coats covering DNA
making them ideal to resolve the nature of the hereditary
material.
•E.Coli cells grown in ³²P
– containing medium
(DNA
becomes
radioactive)
•Infection with nonradioactive T₂ phage
•DNA labeled progeny
phages
•E.Coli cells grown in ³S•containing medium
•Infection with nonradioactive T₂ phage
•Protein labeled progeny
phages
DNA had been proven as the genetic material by the
Hershey-Chase experiments, but how DNA served as genes
was not yet certain
TO SERVE AS GENE:
 DNA must carry information from parent cell to daughter
cell
 It must contain information for replicating itself
It must be chemically stable, relatively unchanging
However, it must be capable of mutational change Without
mutations there would be no process of evolution.
Many scientists were interested in deciphering
the structure of DNA
 Among them were Francis Crick, James
Watson, Rosalind Franklin, and Maurice
Wilkens
 Franklin took X-ray diffraction photomicrographs
of crystalline DNA extract
These studies of Rosalind Franklin & Maurice
Wilkens show very clear picture of X-ray diffraction of
DNA
These lines show
the helical nature
of DNA molecule
Dark structures
show regularities
or periodicity in
helical structure
( Recurring bases)
Base equivalence rule given by Chargaff
He analyzed the base sequence (molar proportions
of bases) of different cells, tissues and also from
different species
Source of DNA
A
Bovine thymus 28.2
Bovine spleen
27.9
Bovine sperm
28.7
Rat bonemarrow 28.6
Herring testes
27.9
Wheat germ
27.3
Yeast
31.3
E.Coli
26.0
X174
24.3
G
21.5
22.7
22.2
21.4
19.5
22.7
18.7
24.9
24.5
C
T
5meC
21.2
20.8
20.7
20.4
21.5
16.8
17.1
25.2
18.2
27.8
27.3
27.2
28.4
28.2
27.1
32.9
23.9
32.3
1.3
1.3
1. 3
1.1
2.8
6.0
-
A & T are present in equimolar amounts
G & C are also present in equimolar amounts
Thus, Chargaff made this observation that
G=C
A=T &
But this quantity varies from species to species
This equivalence is of utmost importance in relation to
the formation of DNA helix and may be referred to as
Chargaff’s rule
Also, Sum of Purines = Sum of Pyrimidines
Sum of amino base = Sum of keto (oxo) bases
(A + C)
(G +T)
Major deviations from the rule:
1. In wheatgerm DNA G = C but if 5-methyl
cytosine is added to it, the scarcity of cytosine is
compensated and so it could be explained
2. In ϴX174 DNA, A = T & G = C this is because
ϴX174 DNA is single stranded
These rules can only be applied to double
stranded DNA and not to RNA & single stranded DNA
Watson and Crick gathered all available data in an attempt to develop a model of
DNA structure
Main
of their
are:& Crick proposed a secondary structure of DNA
From Features
the available
data,model
Watson
Other
pairings
of
bases
don't
fit
the
double
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has
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groovesmajor
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&
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DNAhelix
Each
The
Three
two
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DNA
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is
acan
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is
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&
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are
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deoxyribose
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the
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isthe
atheof
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right
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The
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various
surrounding
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the
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attached
isaxis
3.4
toand
nm
theheld
sugartogether by Hbonds between the bases
P
P
S
A
T
S
P
P
S
G
C
S
P
P
S
P
P
C
G
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P
S
T
A
S
P
The diameter of DNA helix is 20 A° & in this
diameter a base pair made up of one purine and
one pyrimidine can fit easily
A base pair made up of two purines would be too
large to fit inside the helix and a base pair made up
of two pyrimidines would be too far apart to form
stable hydrogen bond
So to maintain regularity in structure only one
purine can bind to one pyrimidine
DNA was proven as the hereditary material and
Watson et al. had deciphered its structure.
Now the question was how DNA copies its
information and how that was expressed in the
phenotype as cells multiply and give rise to new
cells, the genome must be accurately duplicated so
that information is passed to each new generation
with minimal error.
Watson and Crick while describing the discovery
of a model structure for B-DNA gave a famous
statement:
“It has not escaped our notice that the specific
pairing we have postulated immediately suggests a
possible copying mechanism for the genetic
material.”
Three possible modes of replication could be
hypothesized based on Watson and Crick’s model
for the structure of the DNA double helix:
Semiconservative,
Conservative
Dispersive.
SEMI-CONSERVATIVE REPLICATION
CONSERVATIVE REPLICATION
DISPERSIVE REPLICATION
Watson and Crick proposed the hypothesis of
semiconservative replication
 This hypothesis was proved by experiments
carried out by Matthew Meselson and Franklin
Stahl in 1957.
Meselson and Stahl grew E. coli cells for many
generations in a medium in which the sole
nitrogen source (NH₄Cl) contained ¹⁵N, the
“heavy” isotope of nitrogen, instead of the
normal, more abundant “light” isotope,¹⁴N
The DNA isolated from these cells had a
density about 1% greater than that of normal
[¹⁴N]DNA and a mixture of heavy [¹⁵N]DNA and
light [¹⁴N]DNA can be separated by
centrifugation to equilibrium in a cesium chloride
density gradient
The E. coli cells grown in the ¹⁵N medium were
transferred to a fresh medium containing only the
¹⁴N isotope, where they were allowed to grow until
the cell population had just doubled
The DNA isolated from these first-generation
cells formed a single band in the CsCl gradient at a
position indicating that the double helical DNA
molecules of the daughter cells were hybrids
containing one new ¹⁴N strand and one parent ¹⁵N
strand
The semi conservative replication hypothesis
was further supported in the next step of the
experiment
Cells were again allowed to double in number in
the ¹⁴N medium
The isolated DNA product of this second cycle
of replication exhibited two bands in the density
gradient, one with a density equal to that of light
DNA and the other with the density of the hybrid
DNA observed after the first cell doubling