1. Coimmunoprecipitazione

2. Affinity co-purification
(pull down)

3. Sistema dei due ibridi

4. Sistema dei due ibridi

5. Sistema dei tre ibridi

6. Sistemi (sperimentali) di espressione
In vitro: - estratti cellulari
In vivo: - cellule procariotiche cellule eucariotiche in coltura
- animali transgenici

7. Gene reporter

9. Watson et al. , BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S. p
Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005

10. Watson et al. , BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S. p
Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005

11. Watson et al. , BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S. p
Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005

12. Trasfezione di cellule in coltura
transiente
stabile
vettori episomali
vettori virali

14. Watson et al. , BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S. p
Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005

16. RNA polymerases RNA polymerase I synthesizes rRNA in the nucleolus.
RNA polymerase II synthesizes mRNA in the nucleoplasm.
RNA polymerase III synthesizes small RNAs in the nucleoplasm.
All eukaryotic RNA polymerases have ~12 subunits and are aggregates of >500 kD.
Some subunits are common to all three RNA polymerases.
The largest subunit in RNA polymerase II has a CTD (carboxy- terminal domain) consisting of multiple repeats of an eptamer.

17. Promotori della pol IIIU6

18. Watson et al. , BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S. p
Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005

19. Watson et al. , BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S. p
Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005

21. I geni per gli rRNA sono ripetuti in tandem nei genomi eucariotici
ETS
18S
ITS
28S
NTS
RNA
RNA
DNA
DNA
gene
promoter
60/81 bp
repeats
spacer
other repetitive
elements
DNA

22. I precursori degli rRNA sono piu' lunghi della somma degli rRNA maturi

23. rRNA genes
Ribosomal RNA is coded by a large number of identical genes that are tandemly repeated to form a cluster(s).
Each rDNA cluster is organized so that transcription units giving a joint precursor to the major rRNAs alternate with nontranscribed spacers.

24. Watson et al. , BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S. p
Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005

25. Il complesso d'inizio della pol I
The structural organization of mammalian rDNA repeats and the basal factors required for transcription initiation. The diagram shows
the arrangement of tandemly repeated rRNA genes. The site of transcription initiation of 47S pre-rRNA (black arrow) and intergenic
transcripts from the spacer promoter (red arrow) are indicated. Terminator elements are located downstream of the transcription unit
(T1−10), downstream of the spacer promoter (TSP), and upstream of the gene promoter (To) (red boxes). Repetitive enhancer elements
(gray boxes) are located between the spacer promoter and major gene promoter. The ellipsoids show the factors that are associated with
the rDNA promoter and Pol I, respectively. TTF-I is associated with the upstream terminator To. Synergistic binding of UBF and
TIF-IB/SL1 to the rDNA promoter is required for the recruitment of RNA polymerase I (Pol I)—together with multiple Pol
I-associated factors—to the transcription start site to initiate pre-rRNA synthesis.

26. Il complesso d'inizio della pol I
UBF: inizio, allungamento
SL1: inizio
TIF-1A (RRN3): inizio
TTF-1: terminazione

27. Regolazione della sintesi dell'rRNA
Regulation of Pol I transcription in response to external signals. The bar diagrams show the relative levels of
pre-rRNA upon exposure of cells to amino acid starvation (left), exposure to oxidative stress (middle), and
growth factor stimulation (right).

28. Regolazione della sintesi dell'rRNA
Regulation of Pol I transcription during cell cycle progression. UBF is activated during interphase by
phosphorylation of serine 484 (S484) by Cdk4/cyclin D and phosphorylation of serine 388 (S388) by
Cdk2/cyclin E and A. At the entry into mitosis, phosphorylation of TAFI110 at threonine 852 (T852) by
Cdk1/cyclin B inactivates TIF-IB/SL1. At the exit from mitosis, Cdc14B dephosphorylates T852, leading to
recovery of TIF-IB/SL1 activity. Activating phosphorylations are marked in green, inhibiting ones in red.

29. Chemioterapici inibiscono la sintesi dell'rRNA

30. Gene reporter

31. Watson et al. , BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S. p
Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005

32. Watson et al. , BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S. p
Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005

33. Watson et al. , BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S. p
Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005

34. I promotori eucariotici sono costituiti
da una associazione variabile di “box” 1
-20
-40
-60
-80
-100 10
-120
-140
Ottamero
CAAT GC
SV40 (promotore precoce)
Timidina kinasi
Istone H2B

35. Attivatori costitutivi
Modulo
Attivatore
DNA occupato
Consenso
Distribuzione
CAAT box
CTF/NF1
22 bp
GGCCAATCT
ubiquitaria
GC box
SP1
20 bp
GGGCGG
Ottamero
Oct-1
ATTGCAT
Oct-2
23 bp
linfoidi kB
NFkB
10 bp
GGGACTTTCC
ATF
GTGACGT

36. Attivatori inducibili e elementi di risposta
Agente regolatore
Elemento di risposta
Consenso
Fattore
Dimensioni
(dalton)
Shock termico
HSE
CNNGCCNNTCCNNG
HSTF
93.000
Glucocorticoidi
GRE
TGGTACAAATGTTCT GR
94.000
Cadmio
MRE
CGNCCCGGNCNC
TPA
TRE
TGACTCA
AP1
39.000
Siero
SRE
CCATATTAGG
SRF
52.000

37. Esempio di promotore

38. Watson et al. , BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S. p
Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005

39. Watson et al. , BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S. p
Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005

40. Enhancers
Similar sequence elements are found in enhancers and promoters.
Enhancers form complexes of transcription factors that interact directly or indirectly with the promoter.

44. Watson et al. , BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S. p
Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005

45. Watson et al. , BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S. p
Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005

46. Watson et al. , BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S. p
Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005

47. Ruolo degli Isolatori

48. Watson et al. , BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S. p
Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005

49. Watson et al. , BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S. p
Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005

50. Watson et al. , BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S. p
Watson et al., BIOLOGIA MOLECOLARE DEL GENE, Zanichelli editore S.p.A. Copyright © 2005

52. LCR and insulators
An LCR is located at the 5 end of the domain and consists of several hypersensitive sites.
Insulators are specialized chromatin structures that have hypersensitive sites.
All known insulators are able to block passage of any activating or inactivating effects from enhancers, silencers, or LCRs.
In some cases, insulators have directionality, and may stop passage of effects in one direction but not the other.