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Einführung in die Genetik
Prof. Dr. Kay Schneitz (EBio Pflanzen)
http://plantdev.bio.wzw.tum.de
[email protected]
Twitter: @PlantDevTUM, #genetikTUM
FB: Plant Development TUM
Prof. Dr. Claus Schwechheimer (PlaSysBiol)
http://wzw.tum.de/sysbiol
[email protected]
Einführung in die Genetik - Inhalte
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Einführung
Struktur von Genen und Chromosomen
Genfunktion
Transmission der DNA während der Zellteilung
Vererbung von Einzelgenveränderungen
Genetische Rekombination (Eukaryonten)
Genetische Rekombination (Bakterien/Viren)
Rekombinante DNA-Technologie
Kartierung/Charakterisierung ganzer Genome
Genmutationen: Ursache und Reparatur
Veränderungen der Chromosomen
Genetische Analyse biologischer Prozesse
Transposons bei Eukaryonten
Regulation der Genexpression
Regulation der Zellzahl - Onkogene
16. 10. 12
23. 10. 12
30. 10. 12
06. 11. 12
13. 11. 12
20. 11. 12
27. 11. 12
04. 12. 12
11. 12. 12
18. 12. 12
08. 01. 13
15. 01. 13
22. 01. 13
29. 01. 13
05. 02. 13
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CS
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Transposons
Genetics 13
Summary
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Discovery of transposons (Mais)
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Replicative vs. conservative transposition
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Experiment (retrotransposons, RNA
intermediate)
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DNA transposons (P elements, Ac/Ds)
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Plant genomes (genome size and transposons)
Structure of transposons (simple vs.
composite transposons)
Transposons and bacterial antibiotic resistance
RNA transposons, retrotransposons
(retrovirus, gag, pol, env)
Excision (short DNA repeat = footprint)
Transposons and genome evolution
Human genome (LINE and SINE RNA
transposons; DNA transposons)
Safe havens (intergenic regions, introns, rRNA
genes, gene promoters)
Regulation of Gene
Expression
Genetics 14
Topics
Gene regulation in bacteria
Genetic analysis of gene induction
Basic logical and molecular principles of
genetic switches
Gene regulation in eukaryotes
Chromatin
Overview of transcription
E. coli: Initiation
E. coli: The promoter
Eukaryotes:
Initiation
Why?
Gene regulation in
bacteria
Genetic Switches
An/Ausschalter
Positive vs negative regulation
Modular functionality:
e.g., allosteric effectors
Model: the lac system of E. coli
Induction of ß-galactosidase
by the disaccharide lactose
Francois Jacob
André Lwoff
Jacques Monod
The inducer: lactose
Simplified lac operon model
No lactose present
Lactose present
Genetic dissection of
lac system
Genetic components
I
Y
P
Z
O
A
I-, Is
YPZOc
A-
Generating partial diploids
in E. coli
mutants: Synthesis
of ß-galactosidase/permease
c
O
mutants: Synthesis
of ß-galactosidase/permease
c
O
mutants: Synthesis
of ß-galactosidase/permease
c
O
mutants: Synthesis
of ß-galactosidase/permease
c
O
mutants: Synthesis
of ß-galactosidase/permease
c
O
mutants: Synthesis
of ß-galactosidase/permease
c
O
c
O
is a constitutive mutation
mutants: Synthesis
of ß-galactosidase/permease
c
O
c
O
is a constitutive mutation
Operator (O) is cis-acting
Interpretation
Interpretation
Operators are cis-acting
mutants: Synthesis of ßgalactosidase/permease
I
mutants: Synthesis of ßgalactosidase/permease
I
mutants: Synthesis of ßgalactosidase/permease
I
mutants: Synthesis of ßgalactosidase/permease
I
mutants: Synthesis of ßgalactosidase/permease
I
mutants: Synthesis of ßgalactosidase/permease
I
Repressor (I) is trans-acting
Interpretation
Interpretation
Repressors are trans-acting
alleles: Synthesis of ßgalactosidase/permease
s
I
alleles: Synthesis of ßgalactosidase/permease
s
I
alleles: Synthesis of ßgalactosidase/permease
s
I
alleles: Synthesis of ßgalactosidase/permease
s
I
alleles: Synthesis of ßgalactosidase/permease
s
I
s
I : repressor
is hyperactive
Interpretation
Interpretation
Repressor contains a lactose-binding site
Lactose present
Genetic analysis: I vs O
• both elements act in repressing lac operon
• they fundamentally differ in their mode of
action (cis vs trans mode)
• reveals important aspects of the molecular
mechanism of lac repression
Control of lac system:
Lactose vs glucose
Catabolite repression of
the lac operon
Catabolite repression of
the lac operon
Glucose is a
catabolite of
lactose
cAMP/CAP
complex is an
activator of
transcription
Control
of the lac
operon
Molecular anatomy of
the genetic switch
The operator is a
specific DNA sequence
The operator is a
specific DNA sequence
Very specific sequence! One base change
enough to eliminate O function
Many DNA binding
sites are symmetrical
Binding of CAP bends DNA
Binding of CAP bends DNA
Recognition of specific CAP-binding site
CAP/RNA pol: Binding sites
Helix-turn-helix is a
common DNA-binding motif
Helix-turn-helix is a
common DNA-binding motif
Specific contacts with bases in major groove
Helix-turn-helix is a
common DNA-binding motif
Dimers
Specific contacts with bases in major groove
AA side chains determine
specificity of DNA binding
Homeodomain
Repression
vs
activation
Genetic switches are often
part of a cascade mechanism
A
TF
B
C
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Summary •
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Cells respond to intrinsic and extrinsic signals by modulating
transcriptional control of certain genes
Gene activity is the result of the function of cis- and trans-acting
factors
Trans-acting proteins react to environmental signals by using built-in
sensors that continually monitor cellular conditions
Coordinated gene regulation in bacteria
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gene are often clustered into operons on the chro and
transcribed together into multigenic mRNAs
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one cluster of regulatory sites per operon is sufficient to regulate
expression of several genes
Negative vs positive regulation
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repressor proteins bind to DNA at operator site thereby
blocking transcription (e.g., lac operon)
•
activator proteins activate transcription by binding to DNA at the
promoter region (e.g., cAMP/CAP regulation of lac operon)
Molecular anatomy of genetic switch
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regulatory proteins have DNA-binding domains (e.g., HLH) and
protein-protein interaction domains (modular
•
specificity of gene regulation depends on specific protein-DNA
interactions mediated by the chemical interactions between aa
side chains and chemical groups of DNA bases
Gene regulation in
eukaryotes
Drosophila: MSL complex
and dosage compensation
Overview of transcriptional
regulation
Eukaryotic promoter
The yeast GAL system
The transcriptional
activator Gal4
The transcriptional
activator Gal4
TF: sequence-specific binding to regions
outside the promoters of target genes
TFs: Modular
Proteins
Transcriptional activators and
the transcription machinery
Enhancer action: Mechanism
Enhancer action: Mechanism
Cooperativity
Synergism
Disperse distribution of
enhancer elements
DPP of Drosophila
kb
Modular and
combinatorial control
eve
TATA
lacZ
Chromatin
Chromatin dynamics
Chromatin remodeling
e.g., by SWI-SNF complex
Histone modifications
and the histone code
Tup1, a histone deacetylase
from yeast, is a corepressor
Linking TFs and chromatin
dynamics
Enhanceosome
Enhanceosome
Cooperativity
Synergism
Enhanceosome
Cooperativity
Synergism
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Summary•
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Eukaryotic gene regulation resembles bacterial gene regulation
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trans-acting factors binding to cis-regulatory elements on the DNA
this regulatory factors determine the level of transcription by
regulating the binding of RNA pol II to the promoter of a gene
Enhancers/UAS
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cis-regulatory elements, possibly located quite far away (>10-50kb)
from promoter
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combinatorial interactions among different transcription factors
enhanceosome: complexes of regulatory proteins that interact in
cooperative and synergistic fashion --> high levels of transcription
through recruitment of RNA pol II
Gene regulation and chromatin
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eukaryotic genes are packed in chromatin
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histone code: pattern of posttranslational modifications of histone
tails (acetylation, methylation, phosphorylation etc).
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histone code is an epigenetic mark involved in nucleosome
positioning and chromatin condensation that can be altered by TFs
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Tfs recruit for example ATP-dependent chromatin remodelers
(e.g., SWI-SNF)
activation/repression requires specific modifications to chromatin
genes are mostly turned off and kept silent in part by nucleosomes
and condensed chromatin
THE END