School of Biosciences and Veterinary Medicine
University of Camerino
MOLECULAR BIOLOGY, GENERAL
GENETICS
Molecular Biology Course
Attilio Fabbretti,
Fabbretti, PhD
What is molecular biology?
SCIENTIFIC METHOD
OBSERVATIONAL BIOLOGY
MOLECULAR BIOLOGY
PREDICTIVE/EXPERIMENTAL
BIOLOGY
Timeline
The history of the discovery of
DNA
C. Darwin
1859
G. Mendel
1866
F. Miescher
1869
A. Kossel
1881
W. Sutton
1902
P.A. Levene
1920
F. Griffith
1928
O.T. Avery
1944
E. Chargaff
1951
A.D. Harsey
1952
R. Franklin
1952
J. Watson
1953
The history of the discovery of
DNA
Charles Darwin
Alfred Wallace: patterns in the
geographical distribution of living and
fossil species could be explained if
every new species always came into
existence near an already existing,
closely related species
On the Origin of Species, published on November 1859
Evolutionary theory:
theory:
all species of organisms arise and develop through the
natural selection of small, inherited variations that
increase the individual's ability to compete, survive, and
reproduce.
The history of the discovery of
DNA
Gregor Mendel 1866
Fundamental laws of inheritance. He deduced that
genes come in pairs and are inherited as distinct
units, one from each parent
The history of the discovery of
DNA
Felix HoppeHoppe-Seyler laboratory at
the Faculty of Natural Science in
Tubingen
Friedrich Miescher was given the
task of researching the composition
of white blood cells.
The history of the discovery of
DNA
Friedrich Miescher in 1869 isolated a ‘substance’ from the pus of open
wounds collected from bandages in a nearby clinic.
The history of the discovery of
DNA
Miescher protocol:
Collect used bandages from clinic daily
Separate cells via soaking in (9:1 water : sodium
sulfate) solution
Found 5 proteinprotein-like entities (via solubility properties).
Also found other substance, unlike any known protein
Tried to stain new nucleus material with iodine, which
causes proteins to turn yellow
The history of the discovery of
DNA
Conclusion:
Mysterious substance is not protein
New material from nuclei had unexpected properties:
precipitated by acidifying solution and rere-dissolved by
making solution more alkaline ( basic )
The history of the discovery of
DNA
Further study implied
need for increased
purity of mysterious
substance
Developed complicated
protocols involving,
e.g., warm alcohol;
acid in pig’
pig’s stomach,
which contained
enzyme pepsin;
ether……
The history of the discovery of
DNA
He determined that nuclein was made up of carbon,
hydrogen, oxygen, nitrogen and an unexpected element:
phosphorus..
phosphorus
He was the first to identify DNA as a distinct molecule.
The history of the discovery of
DNA
Albrecht Kossel,
Kossel, Emeritus
Professor of Physiology in the
University of Heidelberg
Heidelberg,,
identified DNA as a nucleic acid in
1881 and provided its present
chemical name
name..
The history of the discovery of
DNA
In one of his first experiments he boiled nuclein in
water to release the phosphorus,
phosphorus, and then refined
these experiments to show that "among the soluble
cleavage products of the nuclein whose examination
has not yet been completed
completed,, one can demonstrate
the presence of hypoxanthine
hypoxanthine,, which is not
insignificant".
insignificant
".
The history of the discovery of
DNA
In 1893 he was the first to recognize that nucleic
acids also contained a carbohydrate
carbohydrate..
He reported the presence of a reducing sugar in
yeast nuclein which he described as a pentose (i.e.
with 5 carbon atoms in its structure).
structure).
In 1910 ……
The first Nobel Prize (Physiology or Medicine) for
research on Deoxyribonucleic Acid
The history of the discovery of
DNA
Phoebus Levene
chemist
Levene made important contributions to understanding
nuclein,, now universally called
Miescher’’s nuclein
Miescher
deoxyribonucleic acid (DNA)
Miescher determined relative amounts of five elements in
DNA, but not chemical structure.
The history of the discovery of
DNA
…….with more modern chemistry techniques
Levene characterized chemical structure of DNA;
contained four bases: adenine, guanine, thymine,
cytosine, as well as deoxyribose
deoxyribose,, and phosphate
group
The history of the discovery of
DNA
In addition to determining chemical constituents of DNA,
Levene also concluded that the basic unit was composed of
a base attached to a sugar and that the phosphate also
attached to the sugar (first definition of nucleotide
nucleotide::
phosphatephosphate
-sugar
sugar-base) .
He also figured out the sugar difference between DNA and
RNA.
DNA uses deoxyribose sugar in its backbone; RNA uses
ribose sugar.
The history of the discovery of
DNA
He (unfortunately) also erroneously concluded that the
proportions of bases were equal, and that there was a
tetranucleotide that was the repeating structure of
the molecule.
Tetranucleotide
Led to conclusion: DNA could not carry genetic information. Thus, biologists
thought protein was basis of heredity
The history of the discovery of
DNA
Frederick Griffith 1928
New progress started from unlikely source:
Medical officer of British Ministry of Health,
Frederick Griffith
His specialty: Pneumonia
His concern: Significance in spread of disease of
different pneumococcal types
The history of the discovery of
DNA
Griffith studied varieties of different types
pneumonia;; examined for his 1928 report on 278
pneumonia
cases.
Also conducted large number of experiments on mice
of various strains of pneumococcal types under wide
variety of conditions and prepre-treatments of various
strains of pneumococci
The history of the discovery of
DNA
Some colonies of pneumococci had rough surfaces ( R form,
small); these pneumococci were generally not virulent
Some colonies had smooth surfaces ( S form, large) and
were virulent.
S colonies are larger because of the gelatinous capsule on
the S cells → they are VIRULENT !
The history of the discovery of
DNA
Big surprise: R forms convertible to S forms when mixed
with killed (via heating) S forms.
What was “Transforming Principle”
Principle”?
The history of the discovery of
DNA
Griffith’s results were subject of comment and
Griffith’
inference, but little or no new experimentation, mostly
near repetition……….
Oswald Theodore Avery, MacLeod, and McCarty in
1943….
They decided to seek and to study chemical nature
of substance(s) inducing transformation
(=conversion) of pneumococcal types
The history of the discovery of
DNA
How did Avery et al. proceed?
Approach:
Isolate, purify, and test chemical agent
responsible for transformation in vitro, to
better control
Procedure: S form of bacteria treated
essentially following Miescher protocol.
The history of the discovery of
DNA
The history of the discovery of
DNA
The history of the discovery of
DNA
Detailed chemical basis for actions of DNA in
this study were wholly unknown. But this
1944 paper through its careful experimental
basis and detailed discussion considered
landmark in biology: it seemed to establish
with little doubt relevance of DNA, as
opposed to proteins, to secret of heredity.
The history of the discovery of
DNA
Erwin Chargaff in 1951……..
Analisys of DNA composition
by paper chromatography
The history of the discovery of
DNA
Sourc
e
mol % of bases
Ratios
%GC
A
G
C
T
A/T
G/C
PhiX174
24.0
23.3
21.5
31.2
0.77 ¦
1.08
44.8
Maize
26.8
22.8
17.0 *
27.2
0.99
0.98
46.1
Octopu
s
33.2
17.6
17.6
31.6
1.05
1.00
35.2
Chicke
n
28.0
22.0
21.6
28.4
0.99
1.02
43.7
Rat
28.6
21.4
20.5
28.4
1.01
1.00
42.9
Huma
n
29.3
20.7
20.0
30.0
0.98
1.04
40.7
DNA is not a simple repeating polymer !
P.A. Levene assumption was wrong
A/T ≅ 1
G/C ≅ 1
[Chargaff Rule
Rule]]
The history of the discovery of
DNA
Alfred Hershey and Martha Chase
in 1952.......
What is responsible for heredity – protein or DNA?
Use radioactive tracers, 32P (DNA) and 35S (protein), in
virus (= DNA wrapped in protein) that infects bacteria – no
phosphorus in protein; no sulfur in DNA. Therefore, means
to distinguish two possibilities
The history of the discovery of
DNA
The “Phage Club”: M. Delbruck, S.
Luria, A. Hershey
The phage group started around 1940 and was an informal network of biologists centered
on Max Delbrück that contributed heavily to bacterial genetics and the origins of
molecular biology in the mid-20th century. The phage group takes its name
from bacteriophages, the bacteria-infecting viruses that the group used as
experimental model organisms.
The history of the discovery of
DNA
DNA from T2, not the protein
protein,, is the genetic material!
The history of the discovery of
DNA
Free download
The history of the discovery of
DNA
All started with … X-Rays
Maltese cross
The history of the discovery of
DNA
1953 Maurice Wilkins and Rosalind
Franklin (both radiologists)
X-ray data that
showed the repeating
units of DNA
The history of the discovery of
DNA
You already know
the end of this
story…..
1953- Watson and Crick determined
1953the double helix arrangement of
DNA (Cambridge, England)
England)
The DNA double helix model was
intorduced to the world in the
magazine Nature in 1953.
The history of the discovery of
DNA
Microbiology and
Cell biology
Timeline
Welcome into
MOLECULAR BIOLOGY!!!