* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Level 3 Genes
RNA silencing wikipedia , lookup
Human genetic variation wikipedia , lookup
Epigenetics in learning and memory wikipedia , lookup
Primary transcript wikipedia , lookup
Transposable element wikipedia , lookup
X-inactivation wikipedia , lookup
Short interspersed nuclear elements (SINEs) wikipedia , lookup
Quantitative trait locus wikipedia , lookup
Gene nomenclature wikipedia , lookup
Gene therapy wikipedia , lookup
Gene desert wikipedia , lookup
Gene therapy of the human retina wikipedia , lookup
Minimal genome wikipedia , lookup
Epigenetics of neurodegenerative diseases wikipedia , lookup
Epigenetics of diabetes Type 2 wikipedia , lookup
Genomic imprinting wikipedia , lookup
Long non-coding RNA wikipedia , lookup
Biology and consumer behaviour wikipedia , lookup
Vectors in gene therapy wikipedia , lookup
Ridge (biology) wikipedia , lookup
Public health genomics wikipedia , lookup
Polycomb Group Proteins and Cancer wikipedia , lookup
Genome evolution wikipedia , lookup
Genetic engineering wikipedia , lookup
History of genetic engineering wikipedia , lookup
Mir-92 microRNA precursor family wikipedia , lookup
Site-specific recombinase technology wikipedia , lookup
Therapeutic gene modulation wikipedia , lookup
Nutriepigenomics wikipedia , lookup
Epigenetics of human development wikipedia , lookup
Gene expression programming wikipedia , lookup
Artificial gene synthesis wikipedia , lookup
Genome (book) wikipedia , lookup
Microevolution wikipedia , lookup
Information Storage and Processing in Biological Systems: A seminar course for the Natural Sciences Sept. 11 Biological Information, Sept 16 DNA, Gene regulation Sept 18 Translation and Proteins Sept 23 Enzymes and Signal Transduction Sept 25 Biochemical Networks Sept 30 Simple Genetic Networks (Dr. Jacob) Oct 2 Evolution, Evolvability and Robustness Oct 5 Adventures in multicellularity Operon-Operator Gene Regulation Model (Britten-Davidson) J. Holland: Adaptation in Natural and Artificial Systems 2 Genetic Networks (genetic regulatory networks) - a group of genes connected through transcription regulators encoded within the set of genes Promoter X gene X Promoter Y gene Y operator X 3 Genetic Networks (genetic regulatory networks) - a group of genes connected through transcription regulators encoded within the set of genes Promoter X gene X X Promoter Y gene Y operator X 4 Genetic Networks (genetic regulatory networks) - a group of genes connected through transcription regulators encoded within the set of genes Promoter X gene X X X gene Y operator X Y 5 Genetic Networks (genetic regulatory networks) By convention we simplify these diagrams as follows: Promoter X gene X X X X gene Y Y operator X Y 6 Genetic Networks (genetic regulatory networks) X Y Denotes positive regulation Y Denotes negative regulation Z 7 A photomicrograph of three cells showing the flagella filaments. Each filament forms an extend helix several cell lengths long. The filament is attached to the cell surface through a flexible ‘universal joint’ called the hook. Each filament is rotated by a reversible rotary motor, the direction of the motor is regulated in response to changing environmental conditions. The E. coli Flagellar Motor- a true rotary motor Rotationally averaged reconstruction of electron micrographs of purified hook-basal bodies. The rings seen in the image and labeled in the schematic diagram (right) are the L ring, P ring, MS ring, and C ring. (Digital print courtesy of David DeRosier, Brandeis University.) Regulation of flagella gene expression: A three tiered transcriptional hierarchy • 14 flagella operons • arranged in a regulatory cascade of three classes • Class 1 Operon / Gene: • encodes transcriptional activator of Class 2 operons • Class 2 Operons / Genes: • structural components of a rotary motor • transcriptional activator for Class 3 operons • Class 3 Operons / Genes: • flagellar filament structural genes • chemotaxis signal transduction system Checkpoint mechanism ensures that Class 3 genes are not transcribed before functional basal body-hook structures are completed. 10 Regulation of flagella gene expression: A three tiered transcriptional hierarchy Positive transcriptional regulators Alternative sigma factors Anti-sigma factors Temporal regulation 11 The “genetic network diagram” for the fla system A B C D E 12 The “genetic network diagram” for the fla system Level 1 flhCD fliL fliE fliF flgA flgB flhB n=6 flgM fliD flgK fliC meche mocha flgM fliA Level 2 n=6 Level 3 13 The Flagella Transcription Hierarchy 1. The Master Regulon CRP,H-NS,OmpR other? FlhCD 14 The Flagella Transcription Hierarchy 1. The Master Regulon 2. The FlhCD Regulon CRP,H-NS,OmpR other? FlhCD inside outside FlgM FliA Basal Body and Hook other? 15 The Flagella Transcription Hierarchy 1. The Master Regulon 2. The FlhCD Regulon CRP,H-NS,OmpR other? Chemotaxis proteins Motor proteins FlhCD inside outside FlgM FliA Basal Body and Hook other? 3. The FliA Regulon Filament 16 The flhDC promoter integrates inputs from multiple environmental signals flhDC ? CRP - catabolite repression, carbohydrate metabolism OmpR - osmolarity IHF - growth state of cell? HdfR - ? 17 FliA Regulation by FlgM FlhDC expression leads to activation of Level 2 genes including the alternative sigma factor FliA and an anti sigma factor FlgM FlgM accumulates in the cell and binds to FliA blocking its activity (i.e. interaction with RNA polymerase) preventing Level 3 gene expression. Level 3 Genes inside outside 18 FliA Regulation by FlgM Other level 2 genes required for Basal body and hook (BBH) assembly are made and begin to assemble in the membrane. Level 3 Genes inside outside Basal Body and Hook Assembly 19 FliA Regulation by FlgM The Basal body and hook assembly are completed. Level 3 Genes inside outside Completed Basal Body and Hook 20 FliA Regulation by FlgM The Basal body and hook assembly are completed. FlgM is exported through the Basal Body and Hook Assembly Level 3 Genes inside outside Completed Basal Body and Hook 21 FliA Regulation by FlgM Level 3 gene expression is initiated. FlgM is exported through the Basal Body and Hook Assembly. Level 3 Genes inside outside Completed Basal Body and Hook 22 FliA Regulation by FlgM Level 3 gene expression is initiated. FliA can interact with RNA polymerase and activate Level 3 gene expression. Level 3 Genes inside outside Completed Basal Body and Hook 23 FliA Regulation by FlgM Level 3 gene products are added to the motility machinery including the (1) flagella filament, (2) motor proteins and (3) chemotaxis signal transduction system. Chemotaxis proteins Motor proteins inside outside Filament 24 The “genetic network diagram” for the fla system A B C D E 25 The “genetic network diagram” for the fla system Class 1 Class 2 fliL fliE fliF flgA flgB flhB n=6 Level 1 flhCD flgM Class 3 fliD flgK fliC meche mocha flgM fliA Level 2 n=6 Level 3 26 How to Measure Gene Expression 1- Gene Expression Profiling With Real Promoters Modeling Genetic Networks - from small defined systems to genome wide Small Defined Networks High Throughput / High Quality Expression Profiling Modeling, Simulation 28 Using reporter genes to measure gene expression RNA polymerase Regulator Organization of operon on chromosome. flhD flhC flhDC promoter 29 Using reporter genes to measure gene expression RNA polymerase Regulator Organization of operon on chromosome. flhD flhC flhDC promoter Clone a copy of the promoter into a reporter plasmid. Reporter gene 30 Using reporter genes to measure gene expression RNA polymerase Regulator flhD flhC Both the flhDC genes and the reporter plasmid are regulated in the same way and thus the level of the reporter indicates the activity of the promoter. Reporter gene Note that the strain still has a normal copy of the genes. 31 Gene Expression in Populations Gene Expression in Single Cells Multi-well plate reader Video microscopy - sensitive, fast reading - high-throughput screening - liquid cultures - colonies - mixed cultures - “individuality” - cell cycle regulation - epigenetic phenomenon Automation: Both approaches are amenable to high throughput robotics 32 Gene Expression in Single Cells: Cell to Cell Variability Michael Elowitz, Rockefeller University 33 Fluorescence of flagella reporter strains as a function of time n ero Op s s Cla Fluorescence relative to max 0.6 0.1 0.01 0 Time [min] 600 34 The order of flagellar gene expression is the order of assembly Early Cluster 1 Class 1 flhDC Cluster 2 Late Cluster 3 Class 2 fliL Class 2 fliE Class 2 fliF Class 2 flgA Class 2 flgB Class 2 flhB Class 2 fliA Class 3 fliD Class 3 flgK Class 3 fliC Class 3 meche Class 3 mocha Class 3 flgM Master regulator Activator of class 3 35 Simple Mechanism for Temporal Expression Within a Regulon [protein] Induction of positive regulator Time Promoters with decreasing affinity for regulator 36 Simple Mechanism for Temporal Expression Within a Regulon [protein] 37 Using Expression Data to Define and Describe Regulatory Networks With the flagella regulon, current algorithms can distinguish Level 2 and Level 3 genes based on subtleties in expression patterns not readily distinguished by visual inspection. Using our methods for expression profiling (sensitive, good time resolution) we have been able to demonstrate more subtle regulation than previously described. Different mechanisms can give rise to different patterns- in this case temporal patterns arise by transcription hierarchies (I.e. Level 1 ‡ Level 2 ‡ Level 3) and by differences in binding site affinities within a level. “You can not infer mechanism from pattern.” 38 Methods such as the one described here or DNA microarrays can be used to measure expression of all the genes in a cell simultaneously. Reverse Engineering challenge – can we use expression data to infer genetic networks? C A B M D E F N X Y W V Z O U 39