Sehgal | Circadian Rhythms and Biological Clocks Part A | E-Book | sack.de
E-Book

E-Book, Englisch, Band Volume 551, 488 Seiten

Reihe: Methods in Enzymology

Sehgal Circadian Rhythms and Biological Clocks Part A


1. Auflage 2015
ISBN: 978-0-12-801341-0
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark

E-Book, Englisch, Band Volume 551, 488 Seiten

Reihe: Methods in Enzymology

ISBN: 978-0-12-801341-0
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark



Two new volumes of Methods in Enzymology continue the legacy of this premier serial with quality chapters authored by leaders in the field. Circadian Rhythms and Biological Clocks Part A and Part B is an exceptional resource for anybody interested in the general area of circadian rhythms. As key elements of timekeeping are conserved in organisms across the phylogenetic tree, and our understanding of circadian biology has benefited tremendously from work done in many species, the volume provides a wide range of assays for different biological systems.  Protocols are provided to assess clock function, entrainment of the clock to stimuli such as light and food, and output rhythms of behavior and physiology.  This volume also delves into the impact of circadian disruption on human health.  Contributions are from leaders in the field who have made major discoveries using the methods  presented here. - Continues the legacy of this premier serial with quality chapters authored by leaders in the field - Covers research methods in biomineralization science - Keeping with the interdisciplinary nature of the circadian rhythm field, the volume includes diverse approaches towards the study of rhythms, from assays of biochemical reactions in unicellular organisms to monitoring of behavior in humans.

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1;Front Cover;1
2;Circadian Rhythms and Biological Clocks, A;4
3;Copyright;5
4;Contents;6
5;Contributors;12
6;Preface;18
7;Part I: Organismal Rhythms as Read-Outs of Clock Function;20
7.1;Chapter 1: Studying Circadian Rhythm and Sleep Using Genetic Screens in Drosophila;22
7.1.1;1. Introduction: Studying Circadian Behavior in the Fruit Fly, Drosophila melanogaster;23
7.1.2;2. Screening for Circadian Rhythm and Sleep Mutants;24
7.1.2.1;2.1. History of circadian rhythm screens;24
7.1.2.2;2.2. History of sleep screens;26
7.1.3;3. Screening Techniques;29
7.1.3.1;3.1. EMS mutagenesis;29
7.1.3.1.1;3.1.1. X-linked EMS screen for sleep mutants;29
7.1.3.2;3.2. Transposon mutagenesis;31
7.1.3.3;3.3. Tools for conditional transgene expression;32
7.1.3.4;3.4. Drosophila RNAi libraries and screens;33
7.1.3.4.1;3.4.1. RNAi screen for suppressors and enhancers of shaggy;34
7.1.3.4.2;3.4.2. Neuronal RNAi screen for sleep mutants;37
7.1.3.5;3.5. Advantages and drawbacks of screening with RNAi in comparison to chemical and transposon mutagenesis;38
7.1.4;Acknowledgments;40
7.1.5;References;41
7.2;Chapter 2: Dissecting the Mechanisms of the Clock in Neurospora;48
7.2.1;1. Introduction;48
7.2.1.1;1.1. Methods of analysis of circadian rhythms in Neurospora crassa;49
7.2.1.2;1.2. Circadian rhythms in other fungi;52
7.2.2;2. Molecular Mechanism of the Neurospora Circadian Oscillator;53
7.2.3;3. Core Clock Components;56
7.2.3.1;3.1. The FRQ/FRH complex;56
7.2.3.2;3.2. The White Collar Complex;62
7.2.3.3;3.3. The input and output of the clock;63
7.2.4;4. Conclusion;65
7.2.5;References;66
7.3;Chapter 3: High-Throughput and Quantitative Approaches for Measuring Circadian Rhythms in Cyanobacteria Using Bioluminescence;72
7.3.1;1. Theory;73
7.3.2;2. Build a Computer-Controlled Turntable;74
7.3.2.1;2.1. Materials;75
7.3.2.2;2.2. Programs;77
7.3.2.3;2.3. Protocol;77
7.3.3;3. Use a Computer-Controlled Turntable;79
7.3.3.1;3.1. Programs;79
7.3.3.2;3.2. Protocol;80
7.3.4;4. Analyzing Data from Turntable;82
7.3.4.1;4.1. Programs;82
7.3.4.2;4.2. Protocol;82
7.3.5;5. Steps to Extract Reliable Quantitative Information from Bioluminescence Levels;83
7.3.5.1;5.1. Equipment;84
7.3.5.2;5.2. Programs;85
7.3.5.3;5.3. Protocol;85
7.3.6;Acknowledgments;90
7.3.7;References;90
7.4;Chapter 4: Using Circadian Entrainment to Find Cryptic Clocks;92
7.4.1;1. Introduction;93
7.4.1.1;1.1. Entrainment protocols;94
7.4.1.1.1;1.1.1. Photoperiod-Longer or shorter days, shorter or longer nights;94
7.4.1.1.2;1.1.2. Zeitgeber strength;95
7.4.1.1.3;1.1.3. Dawn and dusk transitions;96
7.4.1.1.4;1.1.4. T cycles and phase angles;96
7.4.2;2. Methods;97
7.4.2.1;2.1. Saccharomyces cerevisiae;97
7.4.2.1.1;2.1.1. Identification of the optimal dilution rate;97
7.4.2.1.2;2.1.2. T cycles;100
7.4.2.1.3;2.1.3. Zeitgeber strength and entrainment of yeast;102
7.4.2.1.4;2.1.4. Constant conditions: Free-running rhythm?;102
7.4.2.2;2.2. Caenorhabditis elegans;103
7.4.3;3. Discussion;106
7.4.4;Acknowledgments;109
7.4.5;References;109
7.5;Chapter 5: Wavelet-Based Analysis of Circadian Behavioral Rhythms;114
7.5.1;1. Introduction;115
7.5.2;2. Fourier and Wavelet Methods for Time Series Analysis;117
7.5.2.1;2.1. Discrete Fourier transform;117
7.5.2.1.1;2.1.1. Background and theory;117
7.5.2.1.2;2.1.2. Applications to chronobiology;119
7.5.2.2;2.2. Short-time Fourier transform;119
7.5.2.2.1;2.2.1. Background and theory;119
7.5.2.2.2;2.2.2. Applications to chronobiology;120
7.5.2.3;2.3. Analytic wavelet transform;121
7.5.2.3.1;2.3.1. Background and theory;121
7.5.2.3.2;2.3.2. Applications to chronobiology;125
7.5.2.4;2.4. Discrete wavelet transform;126
7.5.2.4.1;2.4.1. Background and theory;126
7.5.2.4.2;2.4.2. Applications to chronobiology;131
7.5.2.5;2.5. Example with wavelet analysis of a behavioral record;131
7.5.2.6;2.6. Implications of the uncertainty principle for time-frequency analysis;132
7.5.3;3. Computations;135
7.5.4;4. Concluding Remarks;135
7.5.5;References;136
7.6;Chapter 6: Genetic Analysis of Drosophila Circadian Behavior in Seminatural Conditions;140
7.6.1;1. Introduction;141
7.6.2;2. Considerations for Studies Outside;145
7.6.3;3. Simulating Natural Conditions in the Laboratory;147
7.6.4;Acknowledgments;151
7.6.5;References;151
8;Part II: Characterization of Molecular Clock Components;154
8.1;Chapter 7: Methods to Study Molecular Mechanisms of the Neurospora Circadian Clock;156
8.1.1;1. Introduction;156
8.1.2;2. Description of Methods;159
8.1.2.1;2.1. Purification of epitope-tagged proteins and interacting partners from Neurospora extracts;159
8.1.2.2;2.2. Identification of phosphorylated residues of clock proteins;161
8.1.2.2.1;2.2.1. Mapping in vitro phosphorylation sites;161
8.1.2.2.2;2.2.2. Mapping in vivo phosphorylation sites;161
8.1.2.2.3;2.2.3. MS analyses;161
8.1.2.2.4;2.2.4. Quantitative MS;162
8.1.2.3;2.3. Isolation of Neurospora nuclei to analyze localization of clock proteins;162
8.1.2.4;2.4. Chromatin immunoprecipitation;163
8.1.2.5;2.5. Monitoring bioluminescence reporter expression during the circadian cycle;164
8.1.2.6;2.6. Analysis of protein conformation changes by limited digestion and freeze-thaw cycles;166
8.1.2.6.1;2.6.1. Limited protease digestion;166
8.1.2.6.2;2.6.2. Freeze-thaw assay;167
8.1.3;3. Concluding Remarks;167
8.1.4;Acknowledgment;167
8.1.5;References;167
8.2;Chapter 8: Detecting KaiC Phosphorylation Rhythms of the Cyanobacterial Circadian Oscillator In Vitro and In Vivo;172
8.2.1;1. Theory;174
8.2.2;2. Equipment;174
8.2.3;3. Materials;176
8.2.3.1;3.1. Solutions and buffers;176
8.2.4;4. Protocol;180
8.2.4.1;4.1. Duration;180
8.2.4.2;4.2. Preparation;180
8.2.5;5. Step 1: Expression of KaiA or KaiB in E. coli;180
8.2.5.1;5.1. Overview;180
8.2.5.2;5.2. Duration;180
8.2.5.3;5.3. Tip;181
8.2.6;6. Step 2: Expression of KaiC in E. coli;181
8.2.6.1;6.1. Overview;181
8.2.6.2;6.2. Duration;181
8.2.6.3;6.3. Tip;181
8.2.7;7. Step 3: Purification of KaiA or KaiB;181
8.2.7.1;7.1. Overview;181
8.2.7.2;7.2. Duration;181
8.2.7.3;7.3. Tip;183
8.2.7.4;7.4. Tip;183
8.2.7.5;7.5. Tip;183
8.2.8;8. Step 4: Purification of KaiC;183
8.2.8.1;8.1. Overview;183
8.2.8.2;8.2. Duration;183
8.2.8.3;8.3. Tip;184
8.2.8.4;8.4. Tip;184
8.2.9;9. Step 5: In vitro oscillation reaction;184
8.2.9.1;9.1. Overview;184
8.2.9.2;9.2. Duration;184
8.2.9.3;9.3. Tip;185
8.2.10;10. Step 6: SDS-PAGE;185
8.2.10.1;10.1. Overview;185
8.2.10.2;10.2. Duration;185
8.2.10.3;10.3. Tip;185
8.2.10.4;10.4. Tip;186
8.2.11;11. Step 7: Densitometry;186
8.2.11.1;11.1. Overview;186
8.2.11.2;11.2. Duration;186
8.2.11.3;11.3. Tip;186
8.2.11.4;11.4. Tip;186
8.2.12;12. Detection of protein phosphorylation forms from in vivo cell extracts;187
8.2.13;13. Equipment;187
8.2.14;14. Materials;188
8.2.14.1;14.1. Solutions and buffers;189
8.2.15;15. Protocol;189
8.2.15.1;15.1. Duration;189
8.2.16;16. Step 1: Preparation;189
8.2.16.1;16.1. Overview;189
8.2.16.2;16.2. Tip;190
8.2.16.3;16.3. Tip;190
8.2.17;17. Step 2: Electrophoresis and Blotting;191
8.2.17.1;17.1. Overview;191
8.2.17.2;17.2. Tip;191
8.2.18;Acknowledgments;191
8.2.19;References;192
8.3;Chapter 9: The Role of Casein Kinase I in the Drosophila Circadian Clock;194
8.3.1;1. Introduction;195
8.3.2;2. Expression of Mutant Forms of DBT with the GAL4/UAS Binary Expression Method;197
8.3.3;3. Expression of DBT in Drosophila S2 Cells for Analysis of DBT Kinase Activity;199
8.3.4;4. Proteomic Approaches;200
8.3.4.1;4.1. Isolation of DBT-containing complexes;200
8.3.4.1.1;4.1.1. Expression and purification of DBT-MYC from S2 cells by immunoprecipitation;204
8.3.4.1.2;4.1.2. Expression and purification of DBT-MYC from fly heads;206
8.3.4.2;4.2. Isolation of DBT for analysis of its autophosphosphorylation;207
8.3.4.2.1;4.2.1. Tandem affinity expression and purification of DBT-MYC-HIS from Drosophila S2 cells for analysis of its phosphoryl...;208
8.3.4.3;4.3. Highly sensitive methods for LC-tandem-MS;209
8.3.5;References;211
8.4;Chapter 10: Purification and Analysis of PERIOD Protein Complexes of the Mammalian Circadian Clock;216
8.4.1;1. General Strategy;217
8.4.2;2. Extraction and Characterization of PER Complexes from Mouse Tissues;220
8.4.2.1;2.1. Extraction methods;220
8.4.2.1.1;2.1.1. Materials and methods for isolation of cell nuclei from mammalian tissues;220
8.4.2.1.2;2.1.2. Materials and methods for extraction of nuclear PER complexes;221
8.4.2.2;2.2. Characterizing the size distribution of nuclear PER complexes;222
8.4.2.2.1;2.2.1. Materials and methods for gel filtration chromatography analysis of native PER complexes in nuclear extracts;222
8.4.2.2.2;2.2.2. Materials and methods for BN-PAGE analysis of PER complexes;223
8.4.2.3;2.3. Preparative purification of PER complexes from mouse tissues;224
8.4.2.3.1;2.3.1. Materials and methods for preparative purification of PER complexes from tissues of FH-Per1 or Per2-FH mouse lines;225
8.4.2.4;2.4. Chromatin immunoprecipitation analysis of the recruitment of PER complex proteins to circadian target genes;226
8.4.2.4.1;2.4.1. Materials and methods for ChIP of PER complex proteins;227
8.4.3;References;229
8.5;Chapter 11: Best Practices for Fluorescence Microscopy of the Cyanobacterial Circadian Clock;230
8.5.1;1. Introduction;230
8.5.2;2. Materials;231
8.5.3;3. Methods;232
8.5.3.1;3.1. Generating fusions to fluorescent proteins;232
8.5.3.2;3.2. Validating fusions;233
8.5.3.3;3.3. Imaging fluorescent fusion proteins;235
8.5.3.3.1;3.3.1. Image cells over a circadian time course via time-point sampling;236
8.5.3.3.2;3.3.2. Time-lapse imaging of cells;236
8.5.3.3.3;3.3.3. Investigation of a fluorescent fusion to FtsZ;237
8.5.4;Acknowledgments;239
8.5.5;References;239
8.6;Chapter 12: Structural and Biophysical Methods to Analyze Clock Function and Mechanism;242
8.6.1;1. Introduction;243
8.6.2;2. Kai Protein Overexpression, Purification, Complex Formation, and Analysis by Denatured and Native Polyacrylamide Gel E...;247
8.6.2.1;2.1. Protein expression and purification;247
8.6.2.2;2.2. Denatured and native polyacrylamide gel electrophoresis;248
8.6.3;3. Analytical Ultracentrifugation;250
8.6.4;4. Dynamic Light Scattering;251
8.6.5;5. Thin Layer Chromatography;252
8.6.6;6. Mass Spectrometry;253
8.6.7;7. Site-Directed Mutagenesis;254
8.6.8;8. Fluorescence Techniques (Labeled Proteins, Anisotropy, and Fluorescence Resonance Energy Transfer);255
8.6.9;9. Electron Microscopy;257
8.6.9.1;9.1. Negative stain EM;257
8.6.9.2;9.2. Cryo EM;259
8.6.10;10. X-ray Crystallography;260
8.6.11;11. Small-Angle X-ray and Neutron Scattering;264
8.6.12;12. Nuclear Magnetic Resonance;268
8.6.13;13. Hydrogen-Deuterium Exchange;270
8.6.14;14. MD Simulations;272
8.6.15;15. Modeling the In Vitro Oscillator;274
8.6.16;16. Summary and Outlook;275
8.6.17;Acknowledgments;278
8.6.18;References;278
8.7;Chapter 13: Identification of Small-Molecule Modulators of the Circadian Clock;286
8.7.1;1. Introduction;287
8.7.2;2. Cell-Based Circadian Assay;287
8.7.2.1;2.1. Luciferase reporter genes;287
8.7.2.2;2.2. Reporter cells;289
8.7.3;3. High-Throughput Screening System;289
8.7.3.1;3.1. Liquid handling apparatus;289
8.7.3.2;3.2. Plate readers;292
8.7.3.3;3.3. Data analysis software;293
8.7.4;4. Circadian Screening;293
8.7.4.1;4.1. Assay optimization and validation;293
8.7.4.2;4.2. High-throughput chemical screening;295
8.7.5;5. Conclusion;297
8.7.6;References;299
9;Part III: Circadian Regulation of Gene and Protein Expression;302
9.1;Chapter 14: ChIP-seq and RNA-seq Methods to Study Circadian Control of Transcription in Mammals;304
9.1.1;1. Critical Factors;307
9.1.1.1;1.1. Antibody;307
9.1.1.2;1.2. Cross-linking/fixation;308
9.1.1.3;1.3. Sonication;309
9.1.1.4;1.4. Detergents;309
9.1.1.5;1.5. Bioinformatics;309
9.1.2;2. ChIP-seq Method for Mouse Liver;309
9.1.2.1;2.1. Tissue sampling;309
9.1.2.2;2.2. ChIP-seq;310
9.1.2.3;2.3. Library preparation for ChIP-seq;313
9.1.2.4;2.4. Equipment and reagents needed;313
9.1.2.5;2.5. Buffers and enzyme mixes recipes;313
9.1.2.6;2.6. Adapters and primers;315
9.1.2.7;2.7. Detailed protocol;315
9.1.2.7.1;2.7.1. End repair;315
9.1.2.7.2;2.7.2. Bead based size selection;316
9.1.2.7.3;2.7.3. A-tailing;317
9.1.2.7.4;2.7.4. Y-shaped adapter ligation;317
9.1.2.7.5;2.7.5. Double-bead cleanup;317
9.1.2.7.6;2.7.6. PCR amplification;319
9.1.2.7.7;2.7.7. Double-bead cleanup;319
9.1.2.8;2.8. Quality control;320
9.1.2.9;2.9. Quantification of libraries;321
9.1.2.10;2.10. Normalizing and pooling libraries for sequencing;321
9.1.2.11;2.11. Data analysis for ChIP-seq;321
9.1.3;3. RNA-Seq Method for Mouse Liver;322
9.1.3.1;3.1. Overview of RNA-seq strategy;322
9.1.3.2;3.2. Library preparation for RNA-Seq;324
9.1.3.3;3.3. Equipment and reagents needed;324
9.1.3.4;3.4. Buffers and enzyme mixes recipes;324
9.1.3.5;3.5. Adapters and primers;327
9.1.3.6;3.6. Detailed protocol;328
9.1.3.6.1;3.6.1. mRNA isolation from total RNA;328
9.1.4;References;338
9.2;Chapter 15: ChIPping Away at the Drosophila Clock;342
9.2.1;1. Introduction;343
9.2.2;2. Equipment;345
9.2.3;3. Solutions;347
9.2.4;4. Protocol;352
9.2.4.1;4.1. Step 1. Isolating fly heads;352
9.2.4.2;4.2. Step 2. X-Nuclei preparation;354
9.2.4.3;4.3. Step 3. Sonication;355
9.2.4.4;4.4. Step 4. IP and washes;357
9.2.4.5;4.5. Step 5. Elution and DNA extraction;359
9.2.4.6;4.6. Step 6. qPCR analysis;360
9.2.5;5. Discussion;363
9.2.6;References;364
9.3;Chapter 16: Considerations for RNA-seq Analysis of Circadian Rhythms;368
9.3.1;1. Introduction;369
9.3.2;2. Results;372
9.3.2.1;2.1. Overview;372
9.3.2.2;2.2. Sample density;373
9.3.2.3;2.3. Alignment algorithm and splice form detection;375
9.3.2.4;2.4. Read-depth normalization;375
9.3.2.5;2.5. Read depth;376
9.3.2.6;2.6. Cycling detection algorithms;379
9.3.2.7;2.7. False discovery correction;380
9.3.2.8;2.8. Validation and follow-up;380
9.3.3;3. Conclusions;380
9.3.4;4. Methods;381
9.3.5;Acknowledgments;382
9.3.6;References;382
9.4;Chapter 17: RNA-seq Profiling of Small Numbers of Drosophila Neurons;388
9.4.1;1. Introduction;389
9.4.2;2. Results/Methods;390
9.4.2.1;2.1. Isolating neurons of interest;390
9.4.2.2;2.2. Amplification of mRNA;392
9.4.2.3;2.3. Amplification of miRNA;397
9.4.3;3. Discussion;401
9.4.4;References;404
9.5;Chapter 18: Analysis of Circadian Regulation of Poly(A)-Tail Length;406
9.5.1;1. Introduction;407
9.5.2;2. Measurement of Poly(A)-Tail Length at a Genomewide Level;408
9.5.2.1;2.1. Poly(A)-tail size RNA fractionation;409
9.5.2.2;2.2. 3/-End labeling assay;411
9.5.2.3;2.3. Microarray analysis;413
9.5.3;3. Measurement of Poly(A)-Tail Length at a Single-Gene Level;414
9.5.3.1;3.1. Poly(A) tail (PAT) assay;414
9.5.3.2;3.2. Potential issues with PAT assays;417
9.5.3.3;3.3. LM-PAT assay;418
9.5.4;4. Materials;420
9.5.4.1;4.1. Poly(A)-tail size RNA fractionation;420
9.5.4.2;4.2. 3/-End labeling assay;420
9.5.5;5. Concluding Remarks;420
9.5.6;Acknowledgments;421
9.5.7;References;421
9.6;Chapter 19: Sample Preparation for Phosphoproteomic Analysis of Circadian Time Series in Arabidopsis thaliana;424
9.6.1;1. Introduction;425
9.6.2;2. Materials and Methods;427
9.6.2.1;2.1. Plant material;427
9.6.2.2;2.2. Protein extraction for buffer optimization and the RapiGest SF experiment;428
9.6.2.3;2.3. Fractionation with polyethylene glycol;428
9.6.2.4;2.4. Protein precipitation by TCA/acetone;429
9.6.2.5;2.5. Tryptic digest;429
9.6.2.6;2.6. Detergent removal by ethyl acetate for sample OG ethyl acetate and SDS ethyl acetate;430
9.6.2.7;2.7. Removal of RapiGest SF by acidification;430
9.6.2.8;2.8. Cleanup of digests;430
9.6.2.9;2.9. Phosphopeptide enrichment;430
9.6.2.10;2.10. Mass spectrometry;431
9.6.2.11;2.11. Data analysis;432
9.6.2.12;2.12. Gene ontology enrichment analysis;432
9.6.3;3. Results;434
9.6.3.1;3.1. Choice of extraction buffer and detergent removal method affects number of detected proteins and phosphopeptides;434
9.6.3.2;3.2. Fractionation with PEG does not increase numbers of identified peptides;440
9.6.3.3;3.3. The acid-labile detergent RapiGest does not increase the number of detected phosphopeptides;441
9.6.4;4. Discussion;442
9.6.4.1;4.1. Extraction with a nonionic detergent and precipitation with TCA/acetone outperforms other strategies;443
9.6.4.2;4.2. The nonionic detergent IGEPAL extracts more membrane- and chloroplast-related proteins;445
9.6.4.3;4.3. Fractionation by density using PEG is not superior to increasing replicate number;445
9.6.4.4;4.4. Alternative strategies;447
9.6.5;5. Conclusions;447
9.6.6;Acknowledgments;448
9.6.7;References;448
9.7;Author Index;452
9.8;Subject Index;476
9.9;Color Plate;490


Chapter One Studying Circadian Rhythm and Sleep Using Genetic Screens in Drosophila
Sofia Axelrod; Lino Saez; Michael W. Young1    Laboratory of Genetics, The Rockefeller University, New York, USA
1 Corresponding author: email address: michael.young@rockefeller.edu Abstract
The power of Drosophila melanogaster as a model organism lies in its ability to be used for large-scale genetic screens with the capacity to uncover the genetic basis of biological processes. In particular, genetic screens for circadian behavior, which have been performed since 1971, allowed researchers to make groundbreaking discoveries on multiple levels: they discovered that there is a genetic basis for circadian behavior, they identified the so-called core clock genes that govern this process, and they started to paint a detailed picture of the molecular functions of these clock genes and their encoded proteins. Since the discovery that fruit flies sleep in 2000, researchers have successfully been using genetic screening to elucidate the many questions surrounding this basic animal behavior. In this chapter, we briefly recall the history of circadian rhythm and sleep screens and then move on to describe techniques currently employed for mutagenesis and genetic screening in the field. The emphasis lies on comparing the newer approaches of transgenic RNA interference (RNAi) to classical forms of mutagenesis, in particular in their application to circadian behavior and sleep. We discuss the different screening approaches in light of the literature and published and unpublished sleep and rhythm screens utilizing ethyl methanesulfonate mutagenesis and transgenic RNAi from our lab. Keywords Behavior Drosophila Sleep Circadian rhythm Biological clock Genetic screen RNAi Mutagenesis Neuroscience Review 1 Introduction: Studying Circadian Behavior in the Fruit Fly, Drosophila melanogaster
Drosophila exhibits a multitude of innate and adaptive behaviors that allow researchers to study complex behaviors in a genetically tractable organism. Fruit flies, like all animals, need to correctly interpret and respond to their environment. All life on earth is subject to the changes in light and temperature due to the earth's rotation. Many animals and plants exhibit diurnal or nocturnal behavior depending on their habitat and lifestyle. French scientist Jean-Jaques d’Ortous de Mairan discovered in 1729 that the daily opening and closing of plant leaves persisted in a dark room, indicating that this circadian behavior was not merely a reaction to light, but was effected by internal processes (de Mairan, 1729). It was not until over 200 years later that Konopka and Benzer analyzed the role of endogenous forces—genes—on the daily eclosion rhythm of the fruit fly Drosophila melanogaster (Konopka & Benzer, 1971). Since then, studies in Drosophila have played a prominent role in elucidating the genes and molecular mechanisms driving circadian behavior (Blau et al., 2007; Stanewsky, 2003). Analogous studies in mammals have revealed that these genes and mechanisms are largely conserved through evolution, indicating that these mechanisms are fundamental and underlie the conservation of animal behavior across evolution (Wager-Smith & Kay, 2000). Insights from Drosophila continue to have a broad impact on our understanding of circadian biology in vertebrates, including mechanisms of human circadian dysfunction that alter core clock components homologous to those characterized in Drosophila (Toh, Jones, He, Eide, & Hinz, 2001; Xu, Padiath, Shapiro, Jones, & Wu, 2005). More recently, Drosophila has been used to study sleep, a behavior that is functionally linked to the circadian clock. Like other invertebrates that have been carefully examined (Campbell & Tobler, 1984), Drosophila displays the key behavioral attributes of sleep (Hendricks, Finn, Panckeri, & Chavkin, 2000; Shaw, Cirelli, Greenspan, & Tononi, 2000). These attributes include postural changes specific to sleep, immobility correlated with an increased arousal threshold, a homeostatic rebound in sleep duration and intensity after sleep deprivation, changes in brain electrical activity during sleep (Nitz, van Swinderen, Tononi, & Greenspan, 2002), and alterations in sleep by stimulants and hypnotics that parallel their effects in mammals (Hendricks et al., 2000; Shaw et al., 2000). Recently, it has been suggested that sleep in fruit flies, like that of humans, has different stages of depth during the sleep cycle (van Alphen, Yap, Kirszenblat, Kottler, & van Swinderen, 2013). Although the adoption of Drosophila as a model organism to study sleep is relatively recent, considerable enthusiasm exists for its potential impact on our understanding of the molecular underpinnings of sleep regulation and function. Despite intensive studies over the past several decades, many aspects of sleep have remained elusive. How sleep is regulated by circadian inputs and in a homeostatic manner (Borbély, 1982) is one focus of investigation. A second focus concerns the essential functions of sleep, as well as how sleep or lack thereof affects other physiological and behavioral processes. Theories for the functions of sleep invoke memory consolidation, synaptic downscaling, cell repair, metabolic and immune augmentation, and removal of toxins from the brain (Crocker & Sehgal, 2010; Xie et al., 2013). How sleep might function within the brain and somatic tissues to achieve these functions is still unclear, particularly at a molecular and cellular level, and these questions are the subject of several studies in Drosophila. The impact of Drosophila in studies of circadian rhythms and sleep, as in other areas of biology, stems from the ability to perform large-scale and unbiased forward genetic screens and from powerful genetic tools that enable the fruits of these screens to be exploited (St Johnston, 2002). This chapter reviews recent genetic screens to gain further insight into the molecular basis circadian rhythm and sleep. We touch briefly on prior screens for rhythm and sleep mutants and proceed to the genetic screens for circadian rhythm and sleep that have been performed in recent years with an emphasis on transgenic mutagenesis in comparison with classical methods of genomic mutagenesis. 2 Screening for Circadian Rhythm and Sleep Mutants
2.1 History of circadian rhythm screens
In their landmark 1971 study, Konopka and Benzer isolated the first mutants altering the rhythmicity of Drosophila circadian behavior (Konopka & Benzer, 1971). They conducted a screen with the goal of identifying genes for so-called free-running behavior in constant darkness (dark:dark, DD) and described mutants of a locus they named period (per), which shortened, lengthened, or abolished the rhythmicity of eclosion and locomotor activity in constant darkness. The cloning of the per gene in 1984 (Bargiello, Jackson, & Young, 1984; Zehring et al., 1984) marked the onset of a “clockwork explosion” in genetic screens identifying the genetic basis and molecular characteristics of the circadian clock. It has been over 15 years since most of these screens were completed and uncovered the majority of the circadian components. Extensive review of these earlier screens is not the subject of this review and can be found elsewhere (Blau et al., 2007, Price, 2005, Stanewsky, 2003). While the first rhythm screens utilized measurement of eclosion behavior to identify mutants, later higher throughput screens monitored the rhythmicity of locomotor behavior in individual animals and its persistence in free-running conditions (Stanewsky, 2003). Drosophila means “dew-loving,” and when put in a 12 h light–12 h dark cycle (12:12 LD), flies are indeed most active during dawn and dusk, and sleep most of the day and night (Fig. 1). In free-running conditions without any light or temperature cues, flies continue to wake at the beginning of the subjective day and sleep during the subjective night. Mutants deficient in clock components cannot maintain wild-type (~ 24 h) rhythmicity in DD and, depending on the type of mutation, display shortened or lengthened rhythms, or become completely arrhythmic. Figure 1 Workflow of three recent screens for circadian behavior. The workflow depicts differences and similarities in the screening process. We employed different strategies to obtain genetic nulls and hypomorphs, either in the whole fly or in specific cell types. Depending on the goal of the screen, behavioral assays were focused either on recording sleep, which is carried out in LD, or rhythmic behavior, which is conducted in DD. While circadian rhythms can be detected with data collections every 5 min or even every 30 min, measuring sleep requires a higher data resolution of at least 1 min bins. In all three screens, data were acquired in 1 min bins. Candidate genes were subjected...



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