discovery of nucleic acids - unicamdocenti.unicam.it/tmp/218.pdfdiscovery of nucleic acids 1. 2. ......
TRANSCRIPT
DISCOVERY OFNUCLEIC ACIDS
1
2
Friedrich Miescher in 1869 isolated a ‘substance’ from salmon sperm and the pus of open wounds collected from bandages in a nearby clinic. Since it came from cell nuclei, Miescher named this new chemical, nuclein (actually, it was chromatin). He determined that nuclein was made up of hydrogen, oxygen, nitrogen and phosphorus and there was a unique ratio of nitrogen to phosphorus (N/P ≅ 1.67)
NOTE: He was the first to identify DNA as a distinct molecule.
F.M.I. in Basel
Felix Hoppe-Seyler laboratory at the Faculty of Natural Science in Tubingen
F. Miescher was given the task of researching the composition of white blood cells.
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. 3
Albrecht Kossel, Emeritus Professor of Physiology in the University of Heidelberg, identified DNA as a nucleic acid in 1881 and provided itspresent chemical name. Kossel's work would be rewarded in 1910 as the recipient of the first Nobel Prize (Physiology or Medicine) for research on Deoxyribonucleic Acid
In one of his first experiments he boiled nuclein in water to releasethe phosphorus, and then refined these experiments to show that
"among the soluble cleavage products of the nuclein whoseexamination has not yet been completed, one can demonstrate the
presence of hypoxanthine, which is not insignificant".
Kossel had one more contribution to make. In 1893 he was the first torecognize that nucleic acids also contained a carbohydrate.
He reported the presence of a reducing sugar in yeast nuclein which hedescribed as a pentose (i.e. with 5 carbon atoms in its structure).
He had found the first of the nitrogen-containing bases!
4
Rockfeller Institut (NY)
During the 1920s, biochemist Phoebus Aaron Levene analyzed the components of the DNA molecule.
He also figured out the sugar difference between DNA and RNA.
DNA uses deoxyribose sugar in its backbone; RNA uses ribose sugar.
He found it contained four nitrogenous bases: cytosine, thymine, adenine, and guanine; deoxyribose sugar; and a phosphate group. He concluded that the basic unit was composed of a base attachedto a sugar and that the phosphate also attached to the sugar (first definition of nucleotide: phosphate-sugar-base) .
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.
20 versus 4 building blocks HYPOTHESIS 5
123
9
67
4
8
10
13
5
1112
16
1415
17
1928
The genetic material can be transferredfrom one individualto another
6
Colonies of rough (R, the small colonies) and smooth(S, the large colonies) strains of Streptococcuspneumoniae.
S colonies are larger because of the gelatinous capsule on the S cells → they are VIRULENT !
7
R allele S allele
8
COMPETENCE OF BACTERIA - BACTERIA TRANSFORMATION
Preparation of antiserum raised against heat-killed bacteria, to be usedagainst pneumonia
9(1944)
10
Rockfeller Institut (NY)
11[ 4 building blocks are enough to ‘induce transformation ’ ]
The “Phage Club”: M. Delbruck, S. Luria, A. Hershey
T even series : T2, T4, T6
T odd series : T3, T5, T732P-labeling
35S-labeling
[ 4 building blocks are enough to ‘carry genetic information ’ ] 12
13
Viral DNA from T2, not the protein, is the genetic material
14
Tobacco Mosaic Virus: The Beginning of Plant Virology
This disease was observed in intensively cultivated tobacco fields in Colombia in the early 19th century.
The symptoms were described as a mixture of dark green and light green areas(A), reflecting typical symptoms of a Tobacco mosaic virus (TMV) infection on tobacco. This disease caused the plants to take on "a leaden-gray" color (B) and the tobacco "was extremely bitter to the taste"
15
RNA as a molecule carrying genetic informationGierer and Schramm (1956) They used phenol treated nucleic acids.
Upon injection of tobacco leaves with RNA, they produced viral infections
Fraenkel Conrat & Singer (1957) TMV studies
(Berkley)
123
9
67
4
8
10
13
5
1112
16
1415
17 16
17
All started with … X-Rays
Maltese cross
Erwin Chargaff (1951)
Analisys of DNA composition by paper chromatography
A/T ≅ 1 G/C ≅ 1
[Chargaff Rule]
Sourcemol % of bases Ratios
%GCA G C T A/T G/C
PhiX-174
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
Octopus 33.2 17.6 17.6 31.6 1.05 1.00 35.2
Chicken 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
Human 29.3 20.7 20.0 30.0 0.98 1.04 40.7
18
“ … a comparison of the molar proportions of the bases reveals certain striking regularities.”
DNA is not a simple repeating polymer !
P.A. Levene assumption was wrong
Diffraction pattern of B-DNA fibers from calf thymus(X-Ray data; 1952)
James Watson Linus PaulingFrancis Crick
Maurice Wilkins
Rosalind Franklin
King’s College (London)
Cavendish Laboratory (Cambridge)
19
(3-stranded helix – bases facing out)
SOLUBILITY ?
11
99
A
G
A T
G C
T
C
A = T
G C
Complementarity of NITROGEN BASES in DNA
20
“the twisted ladder"
21
22
Phosphate group (PO3) 5’ position
Hydroxyl group (- OH) 3’ positionH
(guanine)
23
24
hydrogen bonding
(2-3 Kcal/mol → this is about 1/2 of the energy of most hydrogen bonds, due to geometric constraints within the double helix)
base stacking interactions (hydrophobic interactions and Van der Waals forces between the planar rings of the aromatic bases) → ≅ 4 -15 Kcal/mol per dinucleotide
The DNA double helix is stabilized by:
25
A-DNA
Z-DNA
B-DNA
A-RNA
26
27
… “we wish to suggest a structure for the salt of deoxyribonucleic acid (DNA).
This structure has novel features which are of considerable biological interest.”
"It has not escaped our notice that the specificpairing we have postulated immediately suggestsa possible copying mechanism for the geneticmaterial." (J. Watson; F. Crick; Nature, 1953)
TESTABLE HYPOTHESIS
123
9
67
4
8
10
13
5
1112
16
1415
17 28
Suggestions from1953 article
Parental DNA double helix
DNA molecules after 1 replication cycle
semiconservative conservative dispersive
DNA REPLICATION
(6M)[500.000 X g 48 h]
1,65 g/cm3
1,90 g/cm3
29
3’
5’
5’
3’3’
Continuous synthesis
Discontinuous synthesis
…imagine DNA duplication as an unzipping process followed by the enzymatic synthesis of new DNA...
DNA polymerase
The replication fork is the region of DNA in which there isa transition from the unwound parental duplex to the newly replicated daughter duplexes
30
Experimental evidence of semiconservative DNA replication in E.coli
15N/15N DNA (heavy)
14N/14N DNA (light) 15N/14N DNA (hybrid) 31
32Cal-Tech
The sense strand (Crick strand) has the same nucleotide sequence of RNA in the 5’→ 3’ direction, substituting U for T in the RNA.
(Watson strand)
33
34
Exonucleases nibble from the ends
Endonucleases attack internal bonds
35
36
GENETIC SELECTION PROCEDURE
“ finding a needle in a haystack”
37Herpes Simplex Virus
38
123
9
67
4
8
10
13
5
1112
16
1415
17