BIOLOGIA MOLECOLARE
2006-2007
10 crediti
Docenti:
Salvatore Oliviero
Esecitazioni:
Marina Rocchigiani
Monia bardelli
L’esame consiste in un compito scritto con
domande simili a quelle utilizzate nelle
esercitazioni del corso
Date delle prove d’esame:
• mercoledi’ 21 febbraio ore 9 aula 8
• giugno 4 settimana
• luglio 2-3 settimana
• settembre 3-4 settimana
Testi consigliati:
• file di ppt al sito http://www.unisi.it/oliviero_lab/
>teaching > Biologia Molecolare (Coordinatore Prof. Oliviero)
• Genes VIII - B. Lewin (ed. Oxford University press.)
• DNA ricombinante - Watson , Gilman, Witrowski , Zoller
( Ed. italiana Zanichelli) (introduzione alla BM)
•Dai Geni ai genomi - Dale, von Schantz (Ed.italiana EdiSES)
(metodi della Biologia Molecolare)
• Biologia Molecolare della cellula - H.Lodish e altri (ed. italiana Zanichelli)
• Il fago lamda - M.Ptashne (disponibile in biblioteca di biologia)
1953 (April) James Watson
and Francis Crick discover the double
helical structure of DNA (Nature).
Hypothetical model
Published in 1953
The Nobel Prize in Physiology or Medicine 1962
"for their discoveries concerning the molecular structure of nucleic acids
and its significance for information transfer in living material"
Francis Harry Compton Crick
James Dewey Watson
Maurice Hugh Frederick Wilkins
Institute of Molecular Biology
Cambridge, United Kingdom
b. 1916
Harvard University
Cambridge, MA, USA
b. 1928
University of London
London, United Kingdom
b. 1916
1/3 of the prize
United Kingdom
1/3 of the prize
USA
1/3 of the prize
United Kingdom
The difference between DNA and RNA is that DNA has a
deoxyribose sugar, with an H atom connected to
the 2’ position, whereas RNA has a ribose sugar with an -OH
group connected to the 2’ position.
DNA contains the four bases adenine, guanine, cytidine, and thymine;
RNA has uracil instead of thymine.
A nucleoside consists of a purine or
pyrimidine base linked to position 1
of a pentose sugar.
A nucleotide consists of a nucleoside
linked to a phosphate group
on either the 5’ or 3’ position
of the (deoxy)ribose.
Positions on the ribose ring
are described with a prime (’)
to distinguish them.
A nucleoside consists of a purine or
pyrimidine base linked to position 1
of a pentose sugar.
A nucleotide consists of a nucleoside
linked to a phosphate group
on either the 5’ or 3’ position
of the (deoxy)ribose.
Positions on the ribose ring
are described with a prime (’)
to distinguish them.
Successive (deoxy)ribose residues of a polynucleotide chain are joined
by a phosphate group between the 3’ position of one sugar and the
5 position of the next sugar.
DNA DOPPIA
ELICA ~10 bp
DNA-RNA
RNA-RNA
~11bp
ELICA
SINISTRORSA
Antiparallel strands of the double helix are organized in opposite
orientation, so that the 5’ end of one strand is aligned with the 3’ end
of the other strand.
The B-form of DNA is a double helix consisting of two polynucleotide
chains that run antiparallel.
The nitrogenous bases of each chain are flat purine or pyrimidine
rings that face inwards and pair with one another by hydrogen
bonding to form A-T or G-C pairs only.
The diameter of the double helix is 20 Å, and
there is a complete turn every 34 Å, with 10
base pairs per turn.
In B form each single stranded DNA is wound one around
The other at about ~10 bases per turn.
DNA with less than 10 bases per turn is supercoiled negatively
DNA with more than that 10 bases per turn is supercoiled positively
The linking number represents the degree of supercoiling
DNA molecules that differ only by the linking number are
topologcal isomeres
DL= DW + DT
L = linking number
W = writhing number (axis turn in space for a relaxed molecule
W = 0)
T = twisting number
total numbers of turn of one strand over the other
for circular flat DNA is total n.bases/n.bases per turn
Circular DNA or DNA fixed on the extremities the
linking number cannot change
Topoisomerase I introduces a transient break in one DNA strand
ATP independent
Topoisomerase II
double strand breack
In E. coli there are two type I
(topoisomerase I and III)
topoisomerases
And two type II topoisomerase
(girase and topoisomerase IV)
During trasnscription girase introduce negative supercoils
(or relieves the positive ahead of RNA polymerase) while
topoisomerase I and IV remove the negative supercoil
after the RNA polymerase
Denaturation of DNA or RNA describes its conversion from
the double-stranded to the single-stranded state; separation
of the strands is most often accomplished by heating.
Renaturation is the reassociation of denatured complementary
single strands of a DNA double helix.
Hybridization is the pairing of complementary DNA
•with DNA from other sources
•RNA and DNA strands to give an RNA-DNA hybrid.
Melting of DNA means its denaturation.
Melting temperature of DNA is the mid-point of the
transition when duplex DNA to denatured by heating to
separate into single strands.
Heating causes the two strands of a DNA
duplex to separate.
The Tm is the midpoint of the temperature
range for denaturation.
Complementary single strands can renature
when the temperature is reduced.
Denaturation and renaturation/hybridization
can occur with DNA-DNA, DNA-RNA, or
RNA-RNA
combinations, and can be intermolecular or
intramolecular.
The ability of two single-stranded nucleic
acid preparations to hybridize is a measure
of their complementarity.
In laboratorio si calcola in maniera emipirica la
temperatura di denaturazione
considerando che sia di
4 0C per ciascuna coppia G-C
e
2 0C per ciascuna coppia A-T
si applica solo a squenze brevi (10-20 nucleotidi)