Download Regulation of gene expression

Regulation of gene expression
Lecture plan
about 40 min to be given during digestion of plasmids
Concepts of DNA genes etc
Structure of a gene
Details of transcription
Transcription factors
Environment and transcription factors
Examples in the ear
Organ of Corti
Endolymph
Tectorial membrane
Inner hair cells
Stria
Vascularis
Outer hair cells
Spiral
Limbus
Supporting cells
Basilar membrane
Cell diversity
Different shapes
Different function
Different protein content
Inner hair cells
Outer hair cells
Supporting cells
Nuclei
DNA: same in all cells of an organism (with a few exceptions)
How can cells that have the same DNA express
different proteins?
Differences in protein content
• The human DNA consist of approximately 3x109
bases and contain about 100,000 genes.
• Any one cells may contain 5-10,000 different
proteins.
• Within a cell a protein may be present in about 10
copies while another protein may be present in
100,000 copies (even if there is only one DNA
sequence for each of these proteins).
• Within the same cell some proteins are present
throughout the life of the cell, while others are
present for only very limited periods.
• How can all this be possible?
Cooking a cell
Proteins
DNA
Proteins
Italian
Cuisine
Italian
Cuisine
Regulation of Gene Expression
• The cell can select:
– which genes to express
– when to express them
– how much to express them
Gene
Promoter
Exons
DNA
Introns
Transcription
gene
Exon 1
Intron 1 Exon 2
Intron 2
Exon 3
ACGTCTAGTACTGCATTAGCGATGCATACGATGCATGCAAAGGCATAC
TGCAGATCATGACGTAATCGCTACGTATGCTACGTACGTTTCCGTATG
DNA
ACGTCTAGTACTGCATTAGCGATGCATACGATGCATGCAAAGGCATAC
RNA polymerase
RNA
GUAC
TGCAGATCATGACGTAATCGCTACGTATGCTACGTACGTTTCCGTATG
nuclear factors
hRNA
GUACUGCAUUAGCGAUGCAUACGAUGCAUGCAAAGGCAUAC
(heteronuclear)
splicing
mRNA
(messenger)
GUACUGCAUUCAUACGGGCAUACAAAAAAAAAAAA
polyA tail
Alternative splicing
hRNA
GUAGAUGAUUAGCGAUGCAUACGAUGCAUGCAAAGGCUAAC
GUAGAUGAUUCAUACGGGCUAAC
Met Ile His Thr Gly Stop
GUAGAUGAUUGGCUAAC
Met Ile Gly Stop
The Promoter
nuclear factors
elongation factors
complex
RNA
pol.
exon1
TAAATA
Responsive elements
RNA polymerase
binding site
DNA
The Promoter
nuclear factors
RNA
pol.
TAAATA
DNA
RNA
The Promoter
RNA
DNA
Nuclear Factors
• proteins that bind to specific DNA sequences and
that influence gene expression
• also known as: transcription factors
regulatory factors
• the DNA sequences that they bind to are known
as:
transcription element
regulatory elements
regulatory consensus sequences
• the regulation of expression and activity of
nuclear factors determines which proteins will be
expressed in the cell
selective gene expression
GeneA
GeneA
Exon1
Exon1
GeneB
Exon1
Exon1
GeneB
selective gene expression
RNA
Pol.
GeneA
Exon1
Exon1
GeneB
RNA
Pol.
GeneA
Exon1
GeneB
Exon1
Signal transduction to the nucleus
G
adenylate cyclase
cAMP
nucleus
PKA
CREB
P
P
CREB
CREB
CREB = cAMP responsive element
binding protein
RNA
Gene expression
Signals can induce
gene expression in
specific cells
P
CREB
GeneA
GeneA
RNA
Pol.
Exon1
Exon1
GeneB
Exon1
Exon1
GeneB
Time scale of gene expression
Gene C
Gene B
Gene A
Time
Birth
Death
Gene gene interaction
Precursor
Cell type A
Differentiated
Cell type A
Precursor
Cell Type B
Differentiated
Cell type B
Time
Gene expression assays
Western Blot
Protein levels
Immunocytochemstry
ACGTA
Northern Blots
RNA levels
UGCAU
PCR
InSitu Hybridization
ACGTA
UGCAU
Probe hybridisation
A-T C-G
G
A
T
C
G
Probe
C
T
T
A
T
C
Probe hybridisation
A
T
C
A
G
G
A
C
C
C
T
T
G
T
T
A
C
T
G
Probe hybridisation
T
C
G
T
G
G
T
T
C
T
G
C
T
G
T
A
C
G
C
T
A
T
C
C
C
T
A
T
C
T
G
T
T
A
C
T
G
T
A
C
A
C
T
A
hybridisations
C
G
A
T
G
C
G
T
C
T
A
T
A
G
C
In situ hybridisation
• fix tissue
T
• Cut thin sections (10-50 um)
• hybridise with probe
• wash unbound probe
• visualize probe
C
T
T
T
A
Northern Blot
• RNA extraction
• gel electrophoresis
• blot to nitrocellulose
• hybridise with probe
• wash unbound probe
• visualize probe
C
RT-PCR
reverse transcriptase-polymerase chain reaction
UGGGUACAAUGGGUACAAUGGGUAC
Reverse trancriptase
TGGGTACAATGGGTACAATGGGTAC
ACCCATGTTACCCATGTTACCCATG
RT-PCR
Denaturation
94°C
1007550250-25-
TGGGTACAATGGGTACAATGGGTAC
ACCCATGTTACCCATGTTACCCATG
RT-PCR
Annealing 55-60°C
1007550250-25-
TGGGTACAATGGGTACAATGGGTAC
TACCC
TACAA
ACCCATGTTACCCATGTTACCCATG
RT-PCR
Elongation
( Taq DNA polymerase)
72°C
1007550250-25-
TGGGTACAATGGGTACAATGGGTAC
ACCCATGTTACCCATGTTACCC
TACAATGGGTACAATGGGTAC
ACCCATGTTACCCATGTTACCCATG
RT-PCR-2nd cycle
94°C
1007550250-25-
TGGGTACAATGGGTACAATGGGTAC
ACCCATGTTACCCATGTTACCC
TACAATGGGTACAATGGGTAC
ACCCATGTTACCCATGTTACCCATG
RT-PCR (2nd cycle)
55-60°C
1007550250-25-
TGGGTACAATGGGTACAATGGGTAC
TACCC
TACAA
ACCCATGTTACCCATGTTACCC
TACAATGGGTACAATGGGTAC
TACCC
TACAA
ACCCATGTTACCCATGTTACCCATG
RT-PCR (2nd cycle)
72°C
1007550250-25-
TGGGTACAATGGGTACAATGGGTAC
ACCCATGTTACCCATGTTACCC
TACAATGGGTACAATGGG
ACCCATGTTACCCATGTTACCC
TACAATGGGTACAATGGGTAC
ATGTTACCCATGTTACCC
TACAATGGGTACAATGGGTAC
ACCCATGTTACCCATGTTACCCATG
RT-PCR 3rd cycle
TGGGTACAATGGGTACAATGGGTAC
94°C
ACCCATGTTACCCATGTTACCC
TACAATGGGTACAATGGG
ACCCATGTTACCCATGTTACCC
TACAATGGGTACAATGGGTAC
ATGTTACCCATGTTACCC
TACAATGGGTACAATGGGTAC
ACCCATGTTACCCATGTTACCCATG
1007550250-25-
RT-PCR 3rd cycle
TGGGTACAATGGGTACAATGGGTAC
TACCC
TACAA
ACCCATGTTACCCATGTTACCC
TACAATGGGTACAATGGG
TACCC
TACAA
ACCCATGTTACCCATGTTACCC
TACAATGGGTACAATGGGTAC
TACCC
TACAA
ATGTTACCCATGTTACCC
TACAATGGGTACAATGGGTAC
TACCC
TACAA
ACCCATGTTACCCATGTTACCCATG
55-60°C
1007550250-25-
4th cycle
Summary of gene expression detection techniques
Technique
Sensitivity
Specificity
Anatomical
information
Northern Blot
Medium
High
Adjustable
Limited
RT-PCR
Very High
Very High
Limited
InSitu Hybridisation
High
High
High
Study of gene expression
• the regulation of expression and activity of
nuclear factors determines which proteins will be
expressed in the cell
• nuclear factors can be modulated by external
and/or internal signals
• the expression of one gene often affects the
expression of other genes, both in the same cell
and in other cells.
• the characterisation of the ”signal to gene” and
“gene to gene talk” is one of the key focus of
molecular biology.