regulation of gene expression. lecture plan about 40 min to be given during digestion of plasmids...
TRANSCRIPT
Regulation of gene expression
Lecture planabout 40 min to be given during digestion of plasmids
Concepts of DNA genes etcStructure of a geneDetails of transcriptionTranscription factorsEnvironment and transcription factorsExamples in the ear
Organ of Corti
Outer hair cellsInner hair cells
Tectorial membrane
Basilar membrane
Supporting cellsSpiralLimbus
Stria Vascularis
Endolymph
Cell diversity
Outer hair cellsInner hair cells
Supporting cells
Different shapesDifferent functionDifferent protein content
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
Italian Cuisine
Italian Cuisine
DNA
Proteins
Proteins
Regulation of Gene Expression
• The cell can select:– which genes to express– when to express them– how much to express them
DNA
Promoter
Exons Introns
Gene
Transcription
hRNA(heteronuclear)
GUACUGCAUUAGCGAUGCAUACGAUGCAUGCAAAGGCAUAC
RNA polymeraseACGTCTAGTACTGCATTAGCGATGCATACGATGCATGCAAAGGCATAC
TGCAGATCATGACGTAATCGCTACGTATGCTACGTACGTTTCCGTATGGUAC
nuclear factors
RNA
Exon 1 Intron 1
DNA
gene
ACGTCTAGTACTGCATTAGCGATGCATACGATGCATGCAAAGGCATACTGCAGATCATGACGTAATCGCTACGTATGCTACGTACGTTTCCGTATG
Exon 2 Exon 3Intron 2
mRNA(messenger)
splicing
GUACUGCAUUCAUACGGGCAUACAAAAAAAAAAAApolyA tail
Alternative splicinghRNA
GUAGAUGAUUAGCGAUGCAUACGAUGCAUGCAAAGGCUAAC
GUAGAUGAUUCAUACGGGCUAAC GUAGAUGAUUGGCUAAC
Met Ile His Thr Gly Stop Met Ile Gly Stop
The Promoter
RNApol.
nuclear factors elongation factorscomplex
RNA polymerasebinding site
DNA
exon1
TAAATA
Responsive elements
The Promoter
TAAATA
nuclear factors
RNApol.
DNARNA
The Promoter
RNADNA
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 GeneBExon1 Exon1
GeneA GeneBExon1 Exon1
selective gene expression
GeneA GeneBExon1 Exon1
GeneA GeneBExon1 Exon1
RNA Pol.
RNA Pol.
Signal transduction to the nucleus
adenylate cyclase
cAMP
PKA
CREB CREB
P PCREB
CREB = cAMP responsive element binding protein
nucleus
G
RNA
GeneA GeneBExon1 Exon1
GeneA GeneBExon1 Exon1
Gene expression
RNA Pol.
P
CREB
Signals can induce gene expression in specific cells
Time scale of gene expression
Birth Death
Time
Gene A
Gene B
Gene C
Gene gene interaction
Precursor Cell type A
Precursor Cell Type B
Time
Differentiated Cell type A
Differentiated Cell type B
Gene expression assays
Protein levelsWestern Blot
Immunocytochemstry
Northern Blots
PCR
InSitu Hybridization
RNA levels
ACGTA
UGCAU
UGCAU
ACGTA
Probe hybridisation
AT CT TC G
ATCG
Probe
A-T C-G
Probe hybridisation
AT CT TC G
ATCGATCGATCG
Probe hybridisation
AT CT TC G
ATC
T TC G
AT CT T
C G
T
CG
AC
T TC GT
A
A
T TC CGTG
ATCG
hybridisations
ATCG ATC
T TC
G
ATCG ATC
T TC
G
Northern Blot• RNA extraction• gel electrophoresis• blot to nitrocellulose• hybridise with probe• wash unbound probe• visualize probe
In situ hybridisation• fix tissue• Cut thin sections (10-50 um)• hybridise with probe• wash unbound probe• visualize probe
RT-PCRreverse transcriptase-polymerase chain reaction
TGGGTACAATGGGTACAATGGGTAC
UGGGUACAAUGGGUACAAUGGGUAC
ACCCATGTTACCCATGTTACCCATG
Reverse trancriptase
RT-PCR
TGGGTACAATGGGTACAATGGGTAC
ACCCATGTTACCCATGTTACCCATG
94°CDenaturation
100-75-50-25-
0--25-
RT-PCR
TGGGTACAATGGGTACAATGGGTAC
ACCCATGTTACCCATGTTACCCATG
55-60°C
TACCCTACAA
Annealing
100-75-50-25-
0--25-
RT-PCR
TGGGTACAATGGGTACAATGGGTAC
ACCCATGTTACCCATGTTACCCATG
72°C
TACAATGGGTACAATGGGTAC
Elongation( Taq DNA polymerase)
ACCCATGTTACCCATGTTACCC
100-75-50-25-
0--25-
100-75-50-25-
0--25-
RT-PCR-2nd cycle
TGGGTACAATGGGTACAATGGGTAC
ACCCATGTTACCCATGTTACCCATG
94°C
TACAATGGGTACAATGGGTAC
ACCCATGTTACCCATGTTACCC
RT-PCR (2nd cycle)
TGGGTACAATGGGTACAATGGGTAC
ACCCATGTTACCCATGTTACCCATG
55-60°C
TACAATGGGTACAATGGGTAC
ACCCATGTTACCCATGTTACCC
TACCCTACAA
TACCCTACAA
100-75-50-25-
0--25-
RT-PCR (2nd cycle)
TGGGTACAATGGGTACAATGGGTAC
ACCCATGTTACCCATGTTACCCATG
72°C
TACAATGGGTACAATGGGTAC
ACCCATGTTACCCATGTTACCC
ATGTTACCCATGTTACCCTACAATGGGTACAATGGGTAC
ACCCATGTTACCCATGTTACCCTACAATGGGTACAATGGG
100-75-50-25-
0--25-
RT-PCR 3rd cycleTGGGTACAATGGGTACAATGGGTAC
ACCCATGTTACCCATGTTACCCATG
94°C
TACAATGGGTACAATGGGTAC
ACCCATGTTACCCATGTTACCC
ATGTTACCCATGTTACCC
ACCCATGTTACCCATGTTACCC
TACAATGGGTACAATGGG
TACAATGGGTACAATGGGTAC
100-75-50-25-
0--25-
RT-PCR 3rd cycleTGGGTACAATGGGTACAATGGGTAC
ACCCATGTTACCCATGTTACCCATG
TACAATGGGTACAATGGGTAC
ACCCATGTTACCCATGTTACCC
ATGTTACCCATGTTACCC
ACCCATGTTACCCATGTTACCC
TACAATGGGTACAATGGG
TACCCTACAA
TACCC
TACCC
TACAA
TACAA
TACAA
TACAATGGGTACAATGGGTACTACCC
55-60°C
100-75-50-25-
0--25-
4th cycle
Summary of gene expression detection techniques
Technique Sensitivity Specificity Anatomicalinformation
Northern Blot Medium High LimitedAdjustable
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.