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.
Front Cover 1
Circadian Rhythms and Biological Clocks, A 4
Copyright 5
Contents 6
Contributors 12
Preface 18
Part I: Organismal Rhythms as Read-Outs of Clock Function 20
Chapter 1: Studying Circadian Rhythm and Sleep Using Genetic Screens in Drosophila 22
1. Introduction: Studying Circadian Behavior in the Fruit Fly, Drosophila melanogaster 23
2. Screening for Circadian Rhythm and Sleep Mutants 24
2.1. History of circadian rhythm screens 24
2.2. History of sleep screens 26
3. Screening Techniques 29
3.1. EMS mutagenesis 29
3.1.1. X-linked EMS screen for sleep mutants 29
3.2. Transposon mutagenesis 31
3.3. Tools for conditional transgene expression 32
3.4. Drosophila RNAi libraries and screens 33
3.4.1. RNAi screen for suppressors and enhancers of shaggy 34
3.4.2. Neuronal RNAi screen for sleep mutants 37
3.5. Advantages and drawbacks of screening with RNAi in comparison to chemical and transposon mutagenesis 38
Acknowledgments 40
References 41
Chapter 2: Dissecting the Mechanisms of the Clock in Neurospora 48
1. Introduction 48
1.1. Methods of analysis of circadian rhythms in Neurospora crassa 49
1.2. Circadian rhythms in other fungi 52
2. Molecular Mechanism of the Neurospora Circadian Oscillator 53
3. Core Clock Components 56
3.1. The FRQ/FRH complex 56
3.2. The White Collar Complex 62
3.3. The input and output of the clock 63
4. Conclusion 65
References 66
Chapter 3: High-Throughput and Quantitative Approaches for Measuring Circadian Rhythms in Cyanobacteria Using Bioluminescence 72
1. Theory 73
2. Build a Computer-Controlled Turntable 74
2.1. Materials 75
2.2. Programs 77
2.3. Protocol 77
3. Use a Computer-Controlled Turntable 79
3.1. Programs 79
3.2. Protocol 80
4. Analyzing Data from Turntable 82
4.1. Programs 82
4.2. Protocol 82
5. Steps to Extract Reliable Quantitative Information from Bioluminescence Levels 83
5.1. Equipment 84
5.2. Programs 85
5.3. Protocol 85
Acknowledgments 90
References 90
Chapter 4: Using Circadian Entrainment to Find Cryptic Clocks 92
1. Introduction 93
1.1. Entrainment protocols 94
1.1.1. Photoperiod-Longer or shorter days, shorter or longer nights 94
1.1.2. Zeitgeber strength 95
1.1.3. Dawn and dusk transitions 96
1.1.4. T cycles and phase angles 96
2. Methods 97
2.1. Saccharomyces cerevisiae 97
2.1.1. Identification of the optimal dilution rate 97
2.1.2. T cycles 100
2.1.3. Zeitgeber strength and entrainment of yeast 102
2.1.4. Constant conditions: Free-running rhythm? 102
2.2. Caenorhabditis elegans 103
3. Discussion 106
Acknowledgments 109
References 109
Chapter 5: Wavelet-Based Analysis of Circadian Behavioral Rhythms 114
1. Introduction 115
2. Fourier and Wavelet Methods for Time Series Analysis 117
2.1. Discrete Fourier transform 117
2.1.1. Background and theory 117
2.1.2. Applications to chronobiology 119
2.2. Short-time Fourier transform 119
2.2.1. Background and theory 119
2.2.2. Applications to chronobiology 120
2.3. Analytic wavelet transform 121
2.3.1. Background and theory 121
2.3.2. Applications to chronobiology 125
2.4. Discrete wavelet transform 126
2.4.1. Background and theory 126
2.4.2. Applications to chronobiology 131
2.5. Example with wavelet analysis of a behavioral record 131
2.6. Implications of the uncertainty principle for time-frequency analysis 132
3. Computations 135
4. Concluding Remarks 135
References 136
Chapter 6: Genetic Analysis of Drosophila Circadian Behavior in Seminatural Conditions 140
1. Introduction 141
2. Considerations for Studies Outside 145
3. Simulating Natural Conditions in the Laboratory 147
Acknowledgments 151
References 151
Part II: Characterization of Molecular Clock Components 154
Chapter 7: Methods to Study Molecular Mechanisms of the Neurospora Circadian Clock 156
1. Introduction 156
2. Description of Methods 159
2.1. Purification of epitope-tagged proteins and interacting partners from Neurospora extracts 159
2.2. Identification of phosphorylated residues of clock proteins 161
2.2.1. Mapping in vitro phosphorylation sites 161
2.2.2. Mapping in vivo phosphorylation sites 161
2.2.3. MS analyses 161
2.2.4. Quantitative MS 162
2.3. Isolation of Neurospora nuclei to analyze localization of clock proteins 162
2.4. Chromatin immunoprecipitation 163
2.5. Monitoring bioluminescence reporter expression during the circadian cycle 164
2.6. Analysis of protein conformation changes by limited digestion and freeze-thaw cycles 166
2.6.1. Limited protease digestion 166
2.6.2. Freeze-thaw assay 167
3. Concluding Remarks 167
Acknowledgment 167
References 167
Chapter 8: Detecting KaiC Phosphorylation Rhythms of the Cyanobacterial Circadian Oscillator In Vitro and In Vivo 172
1. Theory 174
2. Equipment 174
3. Materials 176
3.1. Solutions and buffers 176
4. Protocol 180
4.1. Duration 180
4.2. Preparation 180
5. Step 1: Expression of KaiA or KaiB in E. coli 180
5.1. Overview 180
5.2. Duration 180
5.3. Tip 181
6. Step 2: Expression of KaiC in E. coli 181
6.1. Overview 181
6.2. Duration 181
6.3. Tip 181
7. Step 3: Purification of KaiA or KaiB 181
7.1. Overview 181
7.2. Duration 181
7.3. Tip 183
7.4. Tip 183
7.5. Tip 183
8. Step 4: Purification of KaiC 183
8.1. Overview 183
8.2. Duration 183
8.3. Tip 184
8.4. Tip 184
9. Step 5: In vitro oscillation reaction 184
9.1. Overview 184
9.2. Duration 184
9.3. Tip 185
10. Step 6: SDS-PAGE 185
10.1. Overview 185
10.2. Duration 185
10.3. Tip 185
10.4. Tip 186
11. Step 7: Densitometry 186
11.1. Overview 186
11.2. Duration 186
11.3. Tip 186
11.4. Tip 186
12. Detection of protein phosphorylation forms from in vivo cell extracts 187
13. Equipment 187
14. Materials 188
14.1. Solutions and buffers 189
15. Protocol 189
15.1. Duration 189
16. Step 1: Preparation 189
16.1. Overview 189
16.2. Tip 190
16.3. Tip 190
17. Step 2: Electrophoresis and Blotting 191
17.1. Overview 191
17.2. Tip 191
Acknowledgments 191
References 192
Chapter 9: The Role of Casein Kinase I in the Drosophila Circadian Clock 194
1. Introduction 195
2. Expression of Mutant Forms of DBT with the GAL4/UAS Binary Expression Method 197
3. Expression of DBT in Drosophila S2 Cells for Analysis of DBT Kinase Activity 199
4. Proteomic Approaches 200
4.1. Isolation of DBT-containing complexes 200
4.1.1. Expression and purification of DBT-MYC from S2 cells by immunoprecipitation 204
4.1.2. Expression and purification of DBT-MYC from fly heads 206
4.2. Isolation of DBT for analysis of its autophosphosphorylation 207
4.2.1. Tandem affinity expression and purification of DBT-MYC-HIS from Drosophila S2 cells for analysis of its phosphoryl... 208
4.3. Highly sensitive methods for LC-tandem-MS 209
References 211
Chapter 10: Purification and Analysis of PERIOD Protein Complexes of the Mammalian Circadian Clock 216
1. General Strategy 217
2. Extraction and Characterization of PER Complexes from Mouse Tissues 220
2.1. Extraction methods 220
2.1.1. Materials and methods for isolation of cell nuclei from mammalian tissues 220
2.1.2. Materials and methods for extraction of nuclear PER complexes 221
2.2. Characterizing the size distribution of nuclear PER complexes 222
2.2.1. Materials and methods for gel filtration chromatography analysis of native PER complexes in nuclear extracts 222
2.2.2. Materials and methods for BN-PAGE analysis of PER complexes 223
2.3. Preparative purification of PER complexes from mouse tissues 224
2.3.1. Materials and methods for preparative purification of PER complexes from tissues of FH-Per1 or Per2-FH mouse lines 225
2.4. Chromatin immunoprecipitation analysis of the recruitment of PER complex proteins to circadian target genes 226
2.4.1. Materials and methods for ChIP of PER complex proteins 227
References 229
Chapter 11: Best Practices for Fluorescence Microscopy of the Cyanobacterial Circadian Clock 230
1. Introduction 230
2. Materials 231
3. Methods 232
3.1. Generating fusions to fluorescent proteins 232
3.2. Validating fusions 233
3.3. Imaging fluorescent fusion proteins 235
3.3.1. Image cells over a circadian time course via time-point sampling 236
3.3.2. Time-lapse imaging of cells 236
3.3.3. Investigation of a fluorescent fusion to FtsZ 237
Acknowledgments 239
References 239
Chapter 12: Structural and Biophysical Methods to Analyze Clock Function and Mechanism 242
1. Introduction 243
2. Kai Protein Overexpression, Purification, Complex Formation, and Analysis by Denatured and Native Polyacrylamide Gel E... 247
2.1. Protein expression and purification 247
2.2. Denatured and native polyacrylamide gel electrophoresis 248
3. Analytical Ultracentrifugation 250
4. Dynamic Light Scattering 251
5. Thin Layer Chromatography 252
6. Mass Spectrometry 253
7. Site-Directed Mutagenesis 254
8. Fluorescence Techniques (Labeled Proteins, Anisotropy, and Fluorescence Resonance Energy Transfer) 255
9. Electron Microscopy 257
9.1. Negative stain EM 257
9.2. Cryo EM 259
10. X-ray Crystallography 260
11. Small-Angle X-ray and Neutron Scattering 264
12. Nuclear Magnetic Resonance 268
13. Hydrogen-Deuterium Exchange 270
14. MD Simulations 272
15. Modeling the In Vitro Oscillator 274
16. Summary and Outlook 275
Acknowledgments 278
References 278
Chapter 13: Identification of Small-Molecule Modulators of the Circadian Clock 286
1. Introduction 287
2. Cell-Based Circadian Assay 287
2.1. Luciferase reporter genes 287
2.2. Reporter cells 289
3. High-Throughput Screening System 289
3.1. Liquid handling apparatus 289
3.2. Plate readers 292
3.3. Data analysis software 293
4. Circadian Screening 293
4.1. Assay optimization and validation 293
4.2. High-throughput chemical screening 295
5. Conclusion 297
References 299
Part III: Circadian Regulation of Gene and Protein Expression 302
Chapter 14: ChIP-seq and RNA-seq Methods to Study Circadian Control of Transcription in Mammals 304
1. Critical Factors 307
1.1. Antibody 307
1.2. Cross-linking/fixation 308
1.3. Sonication 309
1.4. Detergents 309
1.5. Bioinformatics 309
2. ChIP-seq Method for Mouse Liver 309
2.1. Tissue sampling 309
2.2. ChIP-seq 310
2.3. Library preparation for ChIP-seq 313
2.4. Equipment and reagents needed 313
2.5. Buffers and enzyme mixes recipes 313
2.6. Adapters and primers 315
2.7. Detailed protocol 315
2.7.1. End repair 315
2.7.2. Bead based size selection 316
2.7.3. A-tailing 317
2.7.4. Y-shaped adapter ligation 317
2.7.5. Double-bead cleanup 317
2.7.6. PCR amplification 319
2.7.7. Double-bead cleanup 319
2.8. Quality control 320
2.9. Quantification of libraries 321
2.10. Normalizing and pooling libraries for sequencing 321
2.11. Data analysis for ChIP-seq 321
3. RNA-Seq Method for Mouse Liver 322
3.1. Overview of RNA-seq strategy 322
3.2. Library preparation for RNA-Seq 324
3.3. Equipment and reagents needed 324
3.4. Buffers and enzyme mixes recipes 324
3.5. Adapters and primers 327
3.6. Detailed protocol 328
3.6.1. mRNA isolation from total RNA 328
References 338
Chapter 15: ChIPping Away at the Drosophila Clock 342
1. Introduction 343
2. Equipment 345
3. Solutions 347
4. Protocol 352
4.1. Step 1. Isolating fly heads 352
4.2. Step 2. X-Nuclei preparation 354
4.3. Step 3. Sonication 355
4.4. Step 4. IP and washes 357
4.5. Step 5. Elution and DNA extraction 359
4.6. Step 6. qPCR analysis 360
5. Discussion 363
References 364
Chapter 16: Considerations for RNA-seq Analysis of Circadian Rhythms 368
1. Introduction 369
2. Results 372
2.1. Overview 372
2.2. Sample density 373
2.3. Alignment algorithm and splice form detection 375
2.4. Read-depth normalization 375
2.5. Read depth 376
2.6. Cycling detection algorithms 379
2.7. False discovery correction 380
2.8. Validation and follow-up 380
3. Conclusions 380
4. Methods 381
Acknowledgments 382
References 382
Chapter 17: RNA-seq Profiling of Small Numbers of Drosophila Neurons 388
1. Introduction 389
2. Results/Methods 390
2.1. Isolating neurons of interest 390
2.2. Amplification of mRNA 392
2.3. Amplification of miRNA 397
3. Discussion 401
References 404
Chapter 18: Analysis of Circadian Regulation of Poly(A)-Tail Length 406
1. Introduction 407
2. Measurement of Poly(A)-Tail Length at a Genomewide Level 408
2.1. Poly(A)-tail size RNA fractionation 409
2.2. 3/-End labeling assay 411
2.3. Microarray analysis 413
3. Measurement of Poly(A)-Tail Length at a Single-Gene Level 414
3.1. Poly(A) tail (PAT) assay 414
3.2. Potential issues with PAT assays 417
3.3. LM-PAT assay 418
4. Materials 420
4.1. Poly(A)-tail size RNA fractionation 420
4.2. 3/-End labeling assay 420
5. Concluding Remarks 420
Acknowledgments 421
References 421
Chapter 19: Sample Preparation for Phosphoproteomic Analysis of Circadian Time Series in Arabidopsis thaliana 424
1. Introduction 425
2. Materials and Methods 427
2.1. Plant material 427
2.2. Protein extraction for buffer optimization and the RapiGest SF experiment 428
2.3. Fractionation with polyethylene glycol 428
2.4. Protein precipitation by TCA/acetone 429
2.5. Tryptic digest 429
2.6. Detergent removal by ethyl acetate for sample OG ethyl acetate and SDS ethyl acetate 430
2.7. Removal of RapiGest SF by acidification 430
2.8. Cleanup of digests 430
2.9. Phosphopeptide enrichment 430
2.10. Mass spectrometry 431
2.11. Data analysis 432
2.12. Gene ontology enrichment analysis 432
3. Results 434
3.1. Choice of extraction buffer and detergent removal method affects number of detected proteins and phosphopeptides 434
3.2. Fractionation with PEG does not increase numbers of identified peptides 440
3.3. The acid-labile detergent RapiGest does not increase the number of detected phosphopeptides 441
4. Discussion 442
4.1. Extraction with a nonionic detergent and precipitation with TCA/acetone outperforms other strategies 443
4.2. The nonionic detergent IGEPAL extracts more membrane- and chloroplast-related proteins 445
4.3. Fractionation by density using PEG is not superior to increasing replicate number 445
4.4. Alternative strategies 447
5. Conclusions 447
Acknowledgments 448
References 448
Author Index 452
Subject Index 476
Color Plate 490
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.
| Erscheint lt. Verlag | 30.1.2015 |
|---|---|
| Sprache | englisch |
| Themenwelt | Studium ► 1. Studienabschnitt (Vorklinik) ► Physiologie |
| Naturwissenschaften ► Biologie ► Biochemie | |
| Naturwissenschaften ► Biologie ► Humanbiologie | |
| Naturwissenschaften ► Biologie ► Zoologie | |
| Naturwissenschaften ► Physik / Astronomie ► Angewandte Physik | |
| ISBN-10 | 0-12-801341-9 / 0128013419 |
| ISBN-13 | 978-0-12-801341-0 / 9780128013410 |
| Haben Sie eine Frage zum Produkt? |
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