Einführung in die Genetik Prof. Dr. Kay Schneitz (EBio Pflanzen) http://plantdev.bio.wzw.tum.de schneitz@wzw.tum.de Twitter: @PlantDevTUM, #genetiktum FB: Plant Development TUM Prof. Dr. Claus Schwechheimer (PlaSysBiol) http://wzw.tum.de/sysbiol claus.schwechheimer@wzw.tum.de
Einführung in die Genetik - Inhalte 1 Einführung 16. 10. 12 KS 2 Struktur von Genen und Chromosomen 23. 10. 12 KS 3 Genfunktion 30. 10. 12 KS 4 Transmission der DNA während der Zellteilung 06. 11. 12 KS 5 Vererbung von Einzelgenveränderungen 13. 11. 12 KS 6 Genetische Rekombination (Eukaryonten) 20. 11. 12 KS 7 Genetische Rekombination (Bakterien/Viren) 27. 11. 12 KS 8 Rekombinante DNA-Technologie 04. 12. 12 CS 9 Kartierung/Charakterisierung ganzer Genome 11. 12. 12 CS 10 Genmutationen: Ursache und Reparatur 18. 12. 12 CS 11 Veränderungen der Chromosomen 08. 01. 13 CS 12 Genetische Analyse biologischer Prozesse 15. 01. 13 CS 13 Transposons bei Eukaryonten 22. 01. 13 CS 14 Regulation der Genexpression 29. 01. 13 KS 15 Regulation der Zellzahl - Onkogene 05. 02. 13 CS
Transposons Genetics 13
Summary Discovery of transposons (Mais) Structure of transposons (simple vs. composite transposons) Replicative vs. conservative transposition Transposons and bacterial antibiotic resistance RNA transposons, retrotransposons (retrovirus, gag, pol, env) Experiment (retrotransposons, RNA intermediate) DNA transposons (P elements, Ac/Ds) Excision (short DNA repeat = footprint) Transposons and genome evolution Human genome (LINE and SINE RNA transposons; DNA transposons) Plant genomes (genome size and 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 I -, I s Y Y - P P - Z Z - O O c A A -
Generating partial diploids in E. coli
O c mutants: Synthesis of ß-galactosidase/permease
O c mutants: Synthesis of ß-galactosidase/permease
O c mutants: Synthesis of ß-galactosidase/permease
O c mutants: Synthesis of ß-galactosidase/permease
O c mutants: Synthesis of ß-galactosidase/permease
O c mutants: Synthesis of ß-galactosidase/permease O c is a constitutive mutation
O c mutants: Synthesis of ß-galactosidase/permease O c is a constitutive mutation Operator (O) is cis-acting
Interpretation
Interpretation Operators are cis-acting
I - mutants: Synthesis of ß- galactosidase/permease
I - mutants: Synthesis of ß- galactosidase/permease
I - mutants: Synthesis of ß- galactosidase/permease
I - mutants: Synthesis of ß- galactosidase/permease
I - mutants: Synthesis of ß- galactosidase/permease
I - mutants: Synthesis of ß- galactosidase/permease Repressor (I) is trans-acting
Interpretation
Interpretation Repressors are trans-acting
I s alleles: Synthesis of ß- galactosidase/permease
I s alleles: Synthesis of ß- galactosidase/permease
I s alleles: Synthesis of ß- galactosidase/permease
I s alleles: Synthesis of ß- galactosidase/permease
I s alleles: Synthesis of ß- galactosidase/permease I s : 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
Summary 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 gene are often clustered into operons on the chro and transcribed together into multigenic mrnas one cluster of regulatory sites per operon is sufficient to regulate expression of several genes Negative vs positive regulation 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 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
Summary Eukaryotic gene regulation resembles bacterial gene regulation 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 cis-regulatory elements, possibly located quite far away (>10-50kb) from promoter 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 eukaryotic genes are packed in chromatin activation/repression requires specific modifications to chromatin genes are mostly turned off and kept silent in part by nucleosomes and condensed chromatin histone code: pattern of posttranslational modifications of histone tails (acetylation, methylation, phosphorylation etc). histone code is an epigenetic mark involved in nucleosome positioning and chromatin condensation that can be altered by TFs Tfs recruit for example ATP-dependent chromatin remodelers (e.g., SWI-SNF)
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