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40 mld sekvencí
26.7.2004
Mus musculus
srovnávací biochemický a
fyziologický model s
lov kem
Drosophila melanogaster
Model pro studium exprese
jednotlivých gen
Husení ekk thall v (Arabidopsis
thaliana)
Základní rostlinný genetický
model
Zea mays
Zvláštní typ d di nosti
Escherichia coli
Kvasinka Saccharomyces
cerevisiae
hlístice Caenorhabditis
elegans
Nejlépe prostudovaný
mnohobun ný
organizmus (959 bun k)
HIV (Human
immunodeficiency virus)
virus replikující se p es RNA
SV40 (Simian virus 40)
virus zp sobující rakovinu
(onkovirus)
Bakteriofág ( )
virus infikující E. coli
Co je to gen?
Klasická, Mendelova definice: gen je jednotka
genetického materiálu, která má specifickou
funkci a která se p enáší z rodi na potomstvo.
Biochemická definice: gen je úsek DNA, který
kóduje jeden polypeptid
Molekulární definice: gen je sekvence DNA, která
je p episovaná jako jednotka a kóduje jeden nebo
skupinu polypeptid , gen je transkrip ní jednotka
Geny jsou jednotkami biologické informace
Kochovy postuláty
1884
R.Koch
1800
1850
1900
Kochovy postuláty
1. Patogen se nachází ve všech organizmech s danou
nemocí, ale nenachází se u zdravých organizm
2. Patogen by m l být izolován z nemocného organizmu a
m l by být namnožen v isté kultu e
3. Tento istý purifikát patogena by m l po inokulaci na
zdravý organizmus vyvolat stejné onemocn ní, jako
p vodní patogen
4. Patogen by m l být reizolován z experimentáln
inokulovaného organizmu
Geny jsou z DNA
Griffith, 1928
• Pneumococcus zp sobuje pneumonii u lov ka a myší
• Dv formy pneumokoka:
S forma – forma klinických izolát , tvo í na agaru
hladké kolonie, obalená polysacharidovým
obalem, patogenní
R forma – mutant bez obalu, tvo í drsné kolonie,
není patogenní
Petri
dish
agar
bacterial
colony
Griffith, 1928
This is the first
demonstration of
transformation,
the delivery of
functional DNA
to a new cell.
(Called
transfection in
mammalian systems)
Conclude:
Genetic material has passed
from dead S-type to live R-type.
But Griffith did
not know that he
was moving DNA
between cells.
objev d di nosti
1864
izolace DNA
1867
J.G.Mendel
1800
F.Miescher
1850
1900
Izolace DNA
1. Mechanické rozvoln ní bun k
2. Extrakce fenolem
3. Centrifugace a odebrání vodné fáze
4. Srážení ledovým etanolem
5. Centrifugace a vysušení sedimentu
sediment
Purifikace DNA - metody
1. CTAB
2. Wizard® Resin (nebo jiné absorp ní
metody)
3. Magnetic beads-based DNA purification
system
4. jiné…….
Ekvilibra ní hustotní centrifugace
p ed
centrifugací
DNA v
roztoku CsCl
po
centrifugaci
DNA
detekce pod
UV sv tlem
Centrifugace cca 24 hod. 500 000 x g
DNA migruje do pozice, kde má stejnou hustotu
jako gradient CsCl
Elektroforéza v agarózovém
gelu DNA z r zných vzork
GENES ARE MADE OF DNA
Avery et al., 1944
What was the active component of the heat-killed S?
Fractionated and purified the active component:
• Not affected by treatment with
trypsin (cleaves proteins)
chymotrypsin (cleaves proteins)
ribonuclease (cleaves RNA)
• Destroyed by treatment with deoxyribonuclease
• Elemental analysis was identical to that of DNA
Conclusion: DNA was the active component
GENES ARE MADE OF DNA
Hershey and Chase, 1952
Worked with a bacterial virus, T2
T2 Replication Cycle
Protocol:
1. Grow T2 in medium with
H332PO4 + H235SO4
(35S proteins and 32P DNA).
2. Infect bacterial with labeled T2.
3. After short period, shear off T2.
4. Pellet bacteria with T2 DNA (32P)
by centrifugation; T2 protein (35S)
stays in supernatant.
5. Resuspend bacteria in fresh
medium, allow T2 growth.
Results:
T2 produced without input protein.
2. Progeny phage have 32P but little 35S.
Conclusion:
DNA, not protein, carries
T2 info.
Molecular Structure of
DNA & Replication
GGG 291
Jessica Petersen
Outline
Watson & Crick
1953 – Discovery of Structure of DNA
Competition with Pauling
Contribution of Franklin
“Genetical Implications” of Structure
Meselson & Stahl
Semi-Conservative Replication
The Quest for DNA
What was known
DNA – Discovered in 1871 – Friedrich Miescher
DNA is unit of heredity – Avery, MacLoed,
McCarty (1944)
Chargaff’s Rule – 4 Nucleotides A:T, G:C
(1950)
1
Hershey & Chase – DNA is Genetic Info (1952)
Chargafovo pravidlo
Chargaff had found that the amounts of
adenine (A) and thymine (T) were
approximately equal and the amounts of
guanine (G) and cytosine (C ) were also
approximately equal.
This information gave Watson and Crick
the idea that the bases might be paired in
a specific way.
The Players
Linus Pauling
Caltech
Structural Chemist
Protein Structure
http://osulibrary.orst.edu/specialcollections/co
ll/pauling/dna/people/
Cavendish - 1951
Francis Crick
35 years old
Physicist
Grad Student Biophysics
Photos from: http://osulibrary.orst.edu/specialcollections/coll/pauling/dna/people/
James Watson
23 years old
Post Doc
http://www.virtuallaboratory.net/firstSe
ries/WhatisScience/section_11.html
King’s College
Rosalind Franklin
Maurice Wilkins
http://osulibrary.orst.edu/specialcollec
tions/coll/pauling/dna/people/
X-Ray Imaging of DNA
Raymond Gosling
http://www.kcl.ac.uk/depsta/iss/archives/dna/
Evidence for a Double Helix
Rosalind Franklin, working with Maurice
H.F. Wilkins, studied isolated fibers of
DNA by using the X-ray diffraction
technique, a procedure in which a beam of
parallel X rays is directed on a regular,
repeating array of atoms.
Watson saw pictures when Wilkins
showed them at a talk, without Franklin’s
knowledge.
Franklin’s Data
The diffraction patterns obtained by
directing X-rays along the length of drawnout fibers of DNA indicated that the
molecule is organized in a highly ordered,
helical structure.
The data showed DNA was a helical
structure which had two distinctive
regularities of 0.34 nm and 3.4 nm along
the axis of the molecule.
It looked like a double helix.
Early Models
Pauling and Corey
3 chains
Phosphates near axis
Bases outside
Fraser
3 strands
Helical structure
Bases Inside w/H-bonding
Watson & Crick Model
1st Try – 3 strands, Phosphates in core
Positive ions??
Invited Franklin to Discuss
Phosphates must be outside
Chargaff
Chargaff’s Rule
1:1 ratio of A:T, C:G
http://osulibrary.orst.edu/specialcollec
tions/coll/pauling/dna/people/
Photo 51
Wilkins Showed to
Watson – 1953
Max Perutz – shared
private report with
comments from Franklin
www.pbs.org/wgbh/ nova/photo51/pict-01.html
Corey saw photo – 1951
The first X-ray photograph of crystalline DNA in the A form.
Taken by Rosalind Franklin, 1952
Donohue
Not So Fast…
Jerry Donohue
Guanine/Thymine
Structures
Incorrect
Correct Structures
Allow H-Bonding
http://osulibrary.orst.edu/specialcollecti
ons/coll/pauling/dna/people/
Watson & Crick 1953
The Model?
"I have a very pretty
model, which is so pretty
that I am surprised that
no-one ever thought of it
before.“
- J. Watson
http://digitalcollections.library.oregonstate.edu/
THE Model
Double Helix
Position of
Phosphates and
Bases
A:T, C:G Bonding
Structure of DNA from Watson & Crick, 1953.
Success
Using the new forms, Watson discovered
that he could make two base pairs, one
consisting of adenine and thymine, and
the other consisting of guanine and
cytosine, that had exactly the same size.
They built the model and wrote the paper.
Watson and Crick discovered the structure
(or solved it) without direct
experimentation themselves.
They read, thought and talked their way to
a Nobel Prize.
Acknowledgements?
Contribution of Franklin
“We were not aware of the details of the
results presented there [in following
communications] when we devised our
structure…”
98 References in Nobel Lecture (1962) –
None are Franklin
“Genetical Implications”
Crick wanted to add implications to
initial paper
Purine-Pyrimidine pairing
Any sequence (A,T,C,G)
“. . . sequence of the bases is the
code which carries the genetical
information.”
“Genetical Implications”
One chain is complement of other
“. . . suggests how the deoxyribonucleic acid
molecule might duplicate itself.”
H-bonds broken
Unwind and separate
Pair of templates
Meselson & Stahl
Meselson at Caltech
PhD student, X-Ray Crystallography
Stahl to Caltech (1954)
Bacterial and Phage Genetics under Delbruck
Stahl declines position at Berkley –
Beautiful things ensue
Growth of E. coli
Grew on media with N15
Transferred to N14 media
Sampled and centrifuged at various
growth stages
No growth in 14N:
heavy/heavy
• 1 doubling in 14N:
heavy/light
• 2 doublings in 14N:
2 heavy/light & 2 light/light
• 4 doublings in 14N:
14 light/light & 2 heavy/light
Conclude:
DNA is replicated by a
semi-conservative mechanism
With increasing
numbers of doublings,
more and more
of the DNA bands
at the “light” position.
Results
Replication is Semi-Conservative!
Záv ry
1.
DNA je složena ze dvou
polynukleotidových et zc , které se
pravoto iv ovíjejí jeden kolem druhého
2 nm
Záv ry
3. Dva et zce jsou antiparalelní
(= mají opa nou polaritu)
et zce jsou orientovány opa ným
sm rem – jeden ve sm ru 5'- 3'
, druhý ve
sm ru 3'- 5'
.
DNA contains
two strands;
anti-parallel
orientation;
joined by
hydrogen
bonds and
other forces
Polymer:
polynucleotide chain
Repeating unit:
nucleoside
monophosphate;
also termed
nucleotide
Nucleotide =
base +
sugar +
phosphate group
Nucleotide = base + sugar + phosphate group
Note:
Sugar carbons
numbered 1’–5’
FOUR BASES
Adenine (A)
Guanine (G)
Thymine (T)
Cytosine (C)
O
3’ C – O – P – O – C 5’
OPhosphodiester Bond
Polynucleotide
Three components of each nucleotide
Four different nucleotides in DNA
The Solution (con’t)
4. Cukr-fosfátová kostra je vn šroubovice,
báze jsou orientovány sm rem dovnit
Báze jsou ploché molekuly uložené nad
sebou
DNA contains
two strands;
anti-parallel
orientation;
joined by
hydrogen
bonds and
other forces
The Solution (con’t)
5. Báze prolilehlých et zc jsou spolu vázány
relativn slabými vodíkovými vazbami
Specificky se párují A s T (2 vazbami) a G s C
(t emi vazbami)
A-T a G-C párování je jediné, které odpovídá
fyzikálním rozm r m modelu dvojšroubovice
Specifickému A-T a G-C párování se íká
komplementární párování, sekvence na jednom
et zci ur uje sou asn sekvenci na druhém
et zci
BASE PAIR RULES: strong hydrogen bonds form when
A pairs with T
G pairs with C
The Solution (con’t)
6.The base pairs are 0.34 nm apart in the
DNA helix.
A complete (360 degrees) turn of the helix
takes 3.4 nm; therefore, there are 10 base
pairs per turn.
Each base pair, then, is twisted 36
degrees clockwise with respect to the
previous pair.
The Solution
7. Because of the way the bases bond with
each other, the two sugar-phosphate
backbones of the double helix are not equally
spaced along the helical axis.
This results in grooves of unequal size
between the backbones called the major
groove (the wider groove of the two) and the
minor groove (the narrower groove of the
two).
Both of these grooves are large enough to
allow protein molecules to make contact with
the bases.
Right-handed helix
10 bp per turn
34 angstroms per turn
Minor
Groove
Major
Groove
20 angstroms diameter
1 angstrom = 10-10 meter
= 0.1 nm
RNA =
ribonucleic
acid
3’
2’
DNA:
• Storage
RNA:
• mRNA
• rRNA
• tRNA
• ribozymes
• storage
(viruses)
The doublestranded
structure of DNA
predicts how it can
be precisely
duplicated.
Each strand serves
as a template for its
new mate via basepairing rules.
Predicted by Watson and Crick (1953)
If thymine makes up 15
percent of the bases in a
certain DNA sample, what
percentage of bases must be
cytosine?
If thymine makes up 15
percent of the bases in a
certain DNA sample, what
percentage of bases must be
cytosine?
thymine = 15%, then adenine = 15%
A + T = 30%, then G + C = 70%
So, cytosine is 1/2 of 70% = 35%
A certain segment of DNA has
the following nucleotide
sequence in one strand:
5’ ATTGGCTCT 3’
What must be the sequence of
the other strand (label its 5’ and
3’ ends)?
A certain segment of DNA has the
following nucleotide sequence in one
strand:
5’ ATTGGCTCT 3’
What must be the sequence of the
other
strand (label its 5’ and 3’ ends)?
Writing in the same direction: 3’ TAACCGAGA
5’
Writing 5’ to 3’: 5’ AGAGCCAAT 3’
How many bases are there in 2
kb (2000 base pairs) of DNA?
How many bases are there in 2
kb (2000 base pairs) of DNA?
4000 bases in all.
!
A 3'
-TACGATCATAT-5'
B 3'
-ATGCTAGTATA-5'
C 3'
-AUGCUAGUAUA-5’
D 3'
-GCATATACGCG-5’
E 3'
-TATACTAGCAT-5'
"
B 3'
-ATGCTAGTATA-5'
5’-TACGATCATAT3’
3'
-ATGCTAGTATA5’
!
!
Meselson and Stahl (1958) proved that
DNA replicates by each strand serving as
template for the synthesis of a new strand.
+
They used Escherichia coli (E. coli)
as their experimental organism.
složení bakteriální bu ky
voda
70%
ionty
cukry
1%
3%
2%
tuky
proteiny
NK
15%
7%
složení bakteriální bu ky
voda
1
ionty
cukry
20
200
50
tuky
proteiny
NK
3000
1000
Jeden gen – jeden enzym
G.Beadle, E.Tatum – 1940
Pokusy s indukcí mutací rentgenovým
zá ením plísn Neurospora
Mutanty nerostou na minimální p d ,
pot ebují dodání aminokyselin nebo
mají poškozené ur ité enzymy
vitamín
jeden gen – jeden enzym
Geny jsou uspo ádány lineárn
Vzdálenost gen lze vypo ítat z frekvence
rekombinace
U bakterií lze ur it po adí gen transdukcí
fágem
Rekombinací fág bylo dokázáno, že
rekombina ní jednotkou je pár bází