Data Analysis

Plot the activity data as a vertical stack of 24-h traces. This is called an actogram or actograph (see Fig. 1). Alternatevely, produce double- or triple-plotted actographs (Fig. 1) by stacking the data in overlapping 48- or 72-h traces. 2. Analyze the actogram by visual inspection in order to estimate the following parameters Amplitude The difference between peak and trough of activity. Circadian time (CT) Subjective time of the organism in which one circadian period is divided into 24 equal...

Kieron D Edwards and Andrew J Millar

Arabidopsis thaliana is the model organism for the study of the higher plant circadian clock. The physiological change in position of young leaves and cotyledons in Arabidopsis seedlings reveals an overt circadian rhythm. Measuring these leaf movements provides a simple and reliable assay of the plant circadian clock and, unlike systems based on the firefly luciferase reporter gene, requires no prior genetic manipulation of the plant. As such, leaf movement can be used to measure circadian...

Tominaga Yoshino Tomoko Ueyama and Hitoshi Okamura Summary

Brain slices prepared from early postnatal rodents can be maintained in culture from many weeks to months. In culture, brain slices retain their original characteristic cytoar-chitecture (organotypic) and continue to differentiate and mature in vitro resembling the characteristics of the original tissue in vivo. Therefore, this fascinating approach allows us to investigate fundamental issues of structure, function, and development of the central nervous system. This chapter introduces two...

Controls

Because of the possibility of nonspecific reactions caused by tissue antibody factors, several controls should be included in all ICC procedures in order to ensure the accuracy of the results. 3.5.1. Control for Nonspecific Staining by Primary Antibody When using a polyclonal antibody, an aliquot of serum can be absorbed with the antigen. Incubation with the preabsorbed serum, instead of the primary antibody, should reveal no staining. 3.5.2. Controls for Nonspecific Binding of Second and Third...

Introduction

In the mouse, the master core oscillator in the suprachiasmatic nucleus (SCN) is composed of interacting positive and negative transcription-translation feedback loops, which have a circadian (almost 24-h) rhythm and involve a set of clock genes (Clock, Bmal1, mCry1 2, mPer1 2 3, Rev-Erb a, CK e d) (1). A key step in this feedback loop is the shutdown of CLOCK BMAL1-driven transcription by mCRY proteins (2,3). Molecular oscillators also exist in peripheral tissues (liver, kidney, heart), where...

Northern Hybridization

Preparing Sense and Antisense frq Riboprobes and Cold Controls Here we describe the generation of RNA transcripts in vitro transcribed using a PCR amplification product as template (see also Note 11). The generation of the PCR template is described in Subheading 3.3.1.1. Riboprobes are produced when radioactively labeled nucleotides are incorporated during the transcription reaction (described in Subheading 3.3.1.2.). When no radioisotopes are present in the transcription reaction the...

Preparing Yeast Two Hybrid Data for Publication

As with all scientific experiments, the ultimate aim is to publish the data. Below we describe two of the most popular methods (streak and spot) used to present data obtained from yeast two-hybrid screens and interaction studies. The method chosen will depend on the amount of data to be presented and the way that most clearly demonstrates the protein-protein interaction differences detected. Additional methods include colony filter lift assay described in Subheading 3.2.4. and quantitative...

Kruno Sveric Moyra Mason Till Roenneberg and Martha Merrow Summary

As the molecular mechanism of circadian clocks has reached high complexity, the fungal model system, Neurospora crassa, is increasingly important for clock research. It offers the possibility of extensive biochemical experimentation and thorough description of circadian properties. Realization of the full potential is dependent on efficient, high-throughput methods. We have combined several protocols to develop abundant and inexpensive production of mutants, and subsequent identification of the...

References

O., and Botstein, D. (1999) Exploring the new world of the genome with DNA microarrays. Nat. Genet. 21, 33-37. 2. Hartwell, L. H., Hopfield, J. J., Leibler, S., and Murray, A. W. (1999) From molecular to modular cell biology. Nature 402, C47-C52. 3. Kitano, H. (2002) Systems biology a brief overview. Science 295, 1662-1664. 4. Lipshutz, R. J., Fodor, S. P., Gingeras, T. R., and Lockhart, D. J. (1999) High density synthetic oligonucleotide arrays. Nat. Genet. 21, 20-24. 5. Oltvai, Z....

Neurospora Growth and Induction of Kinase Expression

Inoculate 107 Neurospora conidia (originating from WT strain 74a transformed with pMYX2fK grown in solid flasks or from a frozen stock in water) in 200-mL flasks containing 100 mL of selective minimal media (see Notes 4 and 5). 2. Incubate the flasks at 28 C with constant shaking (150 rpm) for about 44 h. In these conditions Neurospora grows producing hyphae and at least 1.5 to 2 g of mycelia are usually obtained. 3. Filter the growing mycelia using a vacuum pump and filter paper and resuspend...

Light and Temperature Treatments and Fly Collection

The most commonly used conditions for synchronizing flies is a 12-h light 12-h dark regime at a constant temperature of 25 C. Two full light-dark cycles are sufficient to achieve synchronization. Flies can thus be collected on the third day, under the desired conditions (e.g., light-dark, constant darkness). Temperature can also be used as a synchronization cue, with, for example, 25 C for the day and 20 C for the night. At the time of collection, flies are transferred to 15-mL tubes chilled on...

Mutagenesis by Homologous Recombination

By this method, any gene on the chromosome of S. elongatus can be replaced by a modified homologous allele (Fig. 1A-D). When inactivating a cyanobacterial gene, the recombinant allele of interest is constructed in an E. coli vector (which will not replicate in the cyanobacterium) by inserting an antibiotic-resistance cassette or a transposon into the coding region of the gene, making sure that the insertion is flanked on each side by at least 300 base pairs of homologous genomic DNA ( 2,9 see...

Quantitative PCR

ABI PRISM 7900HT Sequence Detection System (Applied Biosystems). 2. Primer Express (Applied Biosystems). 3. Microsoft Excel (Microsoft). 4. Random primer (PROMEGA, cat. no. C1181) store at -20 C. 5. Super Script II RNaseH-Reverse Transcriptase 200 U L, 5X first-strand buffer, 0.1 M dithiothreitol (DTT Invitrogen, cat. no. 18064-014) store at -20 C. 6. 10 mM dNTP mix (Invitrogen, cat. no. 18427-013) store at -20 C. 7. 2X SYBR Green PCR Master Mix (Applied Biosystems, cat. no. 4309155) store at 4...

Acknowledgments

Mark Odell and Dr. Christian Heintzen for critical reading of the manuscript. This work was supported by the BBSRC and the Wellcome Trust. 1. Alwine, J. C., Kemp, D. J., and Stark, G. R. (1977) Method for detection of specific RNAs in agarose gels by transfer to diazobenzyloxymethyl-paper and hybridization with DNA probes. Proc. Natl. Acad. Sci. USA 74, 5350-5354. 2. McMaster, G. K., and Carmichael, G. G. (1977) Analysis of single- and double-stranded nucleic...

Estimating Periodicity and Strength of Rhythmicity

To estimate period, the concatenated data sets were analyzed using MESA and the results plotted using MATLAB software. Data were analyzed both with and without the use of a 4-h-cutoff low-pass two-pole Butterworth digital filter (see Note 10 and ref. 16). To report the best estimate, the three highest spectral peaks were read from the output files with MESPEAK. These results were then checked against the output plots and the autocorrelations to ensure that the peak best representing the...

Gene Chip

Affymetrix GeneChip technology equipment hybridization oven, operating software (GCOS), fluidics station 400, scanner 3000. 2.2.1. Preparation of Double-Stranded cDNA 1. T7-(dT)24 Primer (Amersham, cat. no. 72-0591-01) dissolve in water to 100 pmol L and store at -20 C. 2. Super ScriptII RNaseH-Reverse Transcriptase 200 U L, 5X first-strand buffer, 0.1 M DTT (Invitrogen, cat. no. 18064-014) store at -20 C. 3. 10 mM dNTP mix (Invitrogen, cat. no. 18427-013) store at -20 C . 4. 5X Second-strand...

Materials

Serum-containing medium (SCM) Schneider's cells medium, 10 fetal calf serum (FCS) heat-inactivated at 56 C for 30 min, 50 U penicillin G, 50 g mL streptomycin sulfate, filter-sterilized. Fig. 1. Original version of pAct5C. Additional description pactin, actin promoter region actin pA, poly adenilation signals from the actin locus. Ampicillin, gene conferring ampicillin resistance in Escherichia coli pBR322 ori, origin of replication in E. coli derived from plasmid pBR322. Arrows indicate the...

Establishing Primary Fibroblast Cell Line From Mouse Skin

As mentioned in Heading 1, the alterated period of the biological clock of a mCry knockout mouse can be visualized in vitro using cultured fibroblasts derived from those animals. It is therefore important to establish a simple protocol to obtain primary fibroblast cell lines from the skin of mice, particularly because the number of clock-gene knockout mice available is increasing very quickly. Moreover, a complex set of functional interactions (nuclear import, transcription inhibition,...

Irradiance or Radiance

Even with the definitions above, the appropriate application of irradiance or radiance measures is not always clear. The rule is that if the direction or position of the light source is important, then radiance measures are used. If the position of the light source is not important, then irradiance measures should be collected. A few examples below may help clarify this issue further. Measurement of electromagnetic energy within the optical spectrum, which includes ultraviolet radiation,...

Running the Experiment

Using the software provided by PerkinElmer, you must enter certain counting parameters before starting the experiment 1. The count delay (time each plate sits in the dark before being counted see Note 10). 2. Count time (time each well is counted see Note 10). 3. Specify which plates should be counted (i.e., the sample plates, and not the clear plates). 4. Specify which wells should be counted (see Note 1). 5. Start the assay. The duration of the experiment need not be entered at the beginning...

Preparing Glass Tubes for Locomotor Activity Monitor

In order to prepare the glass tubes to be placed in the locomotor activity monitor, it is necessary to provide each tube with a source of food and moisture, usually in the form of an agar-based Drosophila culture medium. 3.1.1. Preparing Drosophila Culture Medium Our laboratory currently employs the following formulation, but there are many variations on the theme, and each laboratory has its own favorite recipe (see, for example, refs. 10 and 11). 1. Weigh 44 g dried yeast, 44 g sugar, 12 g...

Description of LUC Vectors Used

Several luciferase reporter genes are currently available from Promega. We use the LUC+ gene that has been modified to improve its function as a genetic reporter. These modifications include the removal of the peroxisomal translocation sequence, resulting in the transport of luciferase to the cytoplasm and the removal of glycosylation sites. Together these changes produce a severalfold increase in the light signal. The promoter luciferase fusions were constructed in the plant binary vector...

Data Handling and Analysis

Upon completion of the analysis, the data (usually one data file per hour) are compressed and copied to a floppy disk. After extracting the data on a PC, it is loaded into I& A software (Microsoft Excel-based) developed in the laboratory of S. A. Kay (5). This software allows plotting the raw data of individual wells, generating group averages, and plotting data of several genotypes on a single graph (see Note 4). The software also allows exporting the data for quantitative analysis. For...

Wangjie Yu and Paul E Hardin

Circadian rhythms in metabolic, physiological, and behavioral processes are regulated by biological clocks. Many of these rhythmic processes can be measured over many days or weeks using automated recording devices, thus making it possible to precisely calculate period, phase, and amplitude values. With the advent of luciferase reporter genes and machines capable of quantifying luciferase-generated bioluminescence over long time frames, it is now possible to precisely monitor the rhythms in...

Notes

It is important not to leave the eggs for too long (longer than 5 min) in the working bleach solution because excess treatment can damage the embryos. 2. The time required for the pronase step to successfully dechorionate the embryos can vary from clutch to clutch of eggs. Ideally, watch the embryos under the microscope during this step and check when the chorion starts to become fragile. 3. Embryos usually separate from their chorion with gentle swirling during this rinsing step. 4. Cells from...

White Light or Monochromatic Light as Stimulus

It is difficult to make generalizations, but ideally, a standard monochromatic light stimulus, selected to coincide with the maximum (or near maximum) spectral response of the photosystem under examination, should be used for entrainment experiments. Monochromatic light has the advantage that it can be precisely defined and reproduced. Monochromatic light also avoids the complication of photoreversal, the biological phenomenon in which light at one wavelength drives a response in one direction,...

Amc1127

Bioluminescence from the dual-reporter stain, AMC1127, was measured using a cooled charge-coupled device camera. AMC1127 carries two promoter fusions in its chromosome PpsbAI luxAB in NS1 and PkaiBC luc in NS2. Cells were patched onto duplicate BG-11M plates. The upper plate was provided with gaseous -decanal to monitor transcription from the psbAI promoter, whereas the lower plate received 5 mM luciferin to examine the bioluminescence from the kaiBC promoter. Light produced from the...

Methods

A schematic overview of the RNA extraction method described in this chapter is presented in Fig. 1. The method is divided into three major phases. The first phase of the protocol describes the preparation of small, equal amounts of fresh Neurospora mycelium, so-called mycelial disks (see Subheading 3.1.). The second phase is the circadian experiment to be conducted (see Subheading 3.2.). The final phase in the protocol describes the actual extraction of total RNA from Neurospora mycelium, using...

Marta Muoz Llamosas Summary

Immunocytochemistry (ICC) is a sensitive and powerful method that is used to localize and identify cells containing a particular antigen. This chapter is dedicated to ICC of suprachiasmatic nucleus (SCN) slices. After a brief introduction to the technique, the materials and methods sections describe two different methods to obtain SCN slices the first one for fixed tissue, the second one for fresh frozen tissue followed by the description of two methods of antibody detection the indirect method...

EMS Mutagenesis of Arabidopsis Seeds

EMS is an excellent mutagen and consequently is highly carcinogenic your safety and that of others is therefore of utmost importance. EMS is volatile and all necessary safety precautions should be taken when conducting a mutagenesis experiment. Dress in full safety gear, including lab coat, appropriate double gloves, and safety glasses. The experiment should be carried out in a fully functional and inspected chemical fume hood. Inform all members of the laboratory about the experiment and post...

Fixation

Depending on the stability or accessibility of the antigen, various fixation protocols can be used. Methanol is an easy-to-use fixative, which is used mainly for cytoskeletal components, as it works really well for microtubules and can sometimes uncover epitopes left hidden by other methods. However, it frequently solubilizes and removes membrane-bound antigens, because it acts both as a fixative and as a permeabilizing agent, and provides only low structural preservation of samples. 1. Rinse...

Fourier Analysis

The previous section stated that the identification of rhythmically expressed genes is based on the maximum correlation coefficients between expression profiles and cosine waves. In this section, we describe an alternative approach by Fourier analysis. The Fourier transform decomposes an expression profile into a linear combination of sinusoids of different periods, and circadian rhyth-micity is measured by comparing the amplitude of the 24-h period sinusoid with that of other period sinusoids....

Separation of Proteins

Prior to the analysis of protein expression and abundance levels, proteins first have to be isolated into a purified state. Although there are a variety of chromatographic procedures for achieving this, two-dimensional (2D) gel elec-trophoretic separation has been the method of choice in the recent past. However, other new methodologies are now emerging, each methodology having complimentary strengths and weaknesses 1. Analysis of comparative expression once separated, it is then necessary to...

Case Study

One test of a mutagenesis protocol is the rediscovery of previously described mutant alleles. On screening the first 800 Basta-resistant strains, a mutant (KOMO 303) showed a phase of entrainment that was opposite that of wildtype in the 16-h temperature cycle protocol (22 to 27 C). In a light-dark cycle using 3 mol of photons m2 s, it completely failed to entrain, and in constant darkness, it was arrhythmic (see Fig. 5). These phenotypes resemble the null frequency mutant, frq9, and also the...

Analyzing Positive Interactions

False-positives are often generated in yeast two-hybrid experiments, and secondary criteria for distinguishing true positives should always be considered (see Note 18). Described below are protocols for (1) analysis of cDNA library inserts, (2) retransformation of the reporter yeast strain with an isolated cDNA library vector, and (3) DNA sequence analysis of unique clones. It may also be advantageous, although not strictly necessary, to place the bait protein in the prey vector and the prey...

Mauro A Zordan Clara Benna and Gabriella Mazzotta

In the 1970s, the intriguing discovery of autonomous circadian rhythmicity at the behavioral level in Drosophila set the starting point for one of the most remarkably rapid advancements in the understanding of the genetic and molecular bases of a complex behavioral trait. To this end, the design of appropriate electronic devices, apt to continuously monitor behavioral activity, has proven to be fundamental to such progress. In particular, most of the mutational screens performed to date in the...

Cas Kramer

In the filamentous fungus Neurospora crassa the production of asexual spores (conidia) is regulated by its circadian clock. When the fungus is grown on a thin layer of agar medium in long growth tubes (so-called race tubes), restricting its growth to one direction only, bright orange bands are clearly visible. This banding pattern persists with a periodicity of approx 24 h in the absence of any environmental stimuli. The bands are formed by alternating zones of nonsporulating mycelium and...

Monochromatic Light

There are five ways to produce narrow spectrum or monochromatic light. 1. Absorption filters are made of glass, gelatin, or liquids in which colored agents are dissolved or suspended. Gelatin filters are perhaps the most versatile type of filter and are made by mixing organic dyes in gelatin. They have the advantage of being relatively inexpensive and can be made very large, but are not stable over time (they can change their transmission spectrum with use and age) and tend to have fairly broad...

Reporters

The bacterial chloramphenicol acetyl transferase (CAT) enzyme transfers acetyl groups to chloramphenicol from acetyl coenzyme A. In a typical assay this reaction is monitored by 14C-labeled chloramphenicol, where acetylated and nonacetylated forms can be separated by thin-layer chromatography and quantitated in a scintillation counter. Because CAT is very stable (a couple of days in culture), it is not the reporter of choice when temporal changes in gene expression are studied. Bacterial...

Porous Membrane Culture

Porous membrane cultures are quite simple compared with those performed with the roller tube method. Slices are positioned on the membrane of well inserts of tissue-culture plates, and then culturing starts. 3.2.1. Preparation of Materials and Slices The preparation of materials and slices is generally the same as described for the roller tube cultures (see Subheadings 3.1.1.-3.1.4.). However, this procedure does not need cover slips, plasma, and thrombin solutions, but requires instead culture...

The Photon

It is critical to remember that photopigments absorb energy as photons, and hence act as photon counters. Thus all measures of light used in biological experiments should ideally be expressed as a photon flux. The energy of a photon is proportional to the reciprocal of its wavelength (1 X). This means that high-frequency short-wavelength light (blue light) has photons of higher energy than red light (see Note 1). Nonetheless, because photopigments act as photon counters, the biological...

Choogon Lee Summary

As with most other proteins, clock proteins physically interact with one another. Coimmunoprecipitation (colP) is the most straightforward technique to study proteinprotein interactions in vivo, if antibodies against the proteins of interest are available. To perform colP, first an antibody against a target protein is coupled to Sepharose beads through protein A or G, then the complexes containing the target protein are immunopre-cipitated with the antibody-coupled beads by centrifugation....

Scintillation Counter Measurement

A third method for detecting bioluminescence is the use of a liquid scintillation counter. This method is the most labor-intensive of all bioluminescence measurement techniques used in our laboratory, but allows monitoring of cir-cadian rhythmicity in growing liquid cultures of cyanobacteria that are being sampled for molecular rhythms in cyanobacterial extracts by RNA blots or immunoblot analysis, respectively (see Chapter 26). 1. Record the OD750 of a 3-mL liquid cell culture. 2. For luc...

Rhythmic Conidiation in Circadian Experiments

In Neurospora, the conidial banding pattern is strikingly visual and can be accurately assayed using race tubes, which makes race tubes such a versatile tool to study circadian biology. A wide variety of circadian experiments can be performed in race tubes, as circadian parameters for instance, the period and phase of the rhythm are easily examined. For each experiment, of course, the exact protocol will differ. However, some general conditions and considerations regarding the inoculation,...

Gianluca Tosini

Many of the behavioral parameters exhibited by an organism show daily fluctuations. These may persist under constant environmental conditions, demonstrating that they are governed by an endogenous (circadian) clock. The monitoring of locomotor activity in rodents is probably one of the most common methods to track this endogenous timing system. The analysis of locomotor activity rhythms can provide several parameters that may be used to describe the status of this endogenous clock. In the past...

Data Acquisition

Each camera should be focused directly onto the 12 seedlings, and the lens aperture should be adjusted to give the image with the highest contrast possible (see Notes 17 and 18). If phase information is required, data collection must be initiated immediately upon transfer of the seedlings to constant light. If only period information is required, the first 24 h of data are excluded from analysis to avoid any transient cycles following the LD entrainment data collection can then be initiated at...

Experimental Design

Because experimental design is an essential aspect of any empirical research, a brief discussion of assay design with respect to qPCR is provided here to address several areas of confusion. 1.2.1. Absolute and Relative Quantification First, is absolute or relative quantification required That is, are expression values to be presented as a value relative to an untreated control group (relative) or as a stand-alone number (absolute) For most biological research, relative quantification is the...

Biological Response

The biological response to light should be investigated using a series of monochromatic light stimuli, and for each wavelength, a range of irradiances should be used. These data are used to construct irradiance response curves (IRCs). The precise number of wavelengths, irradiance levels, and replicates per irradiance level will be a compromise based on such factors as the variability in response and the length of time it takes to undertake the experiments. The most important consideration,...

Activity Recording Setup

Soak specimen plate in 1 bleach for 1 h, then rinse repeatedly with hot tap water, then deionized water, then egg water. 2. In the afternoon or evening of the first recording day, transfer larvae to specimen plate wells. Use the baby tube to gently strain out larvae, then invert the tube and wash the larvae into clean egg water in a 100-mm Petri dish. 3. Fill the specimen plate wells with egg water. Use a large-bore pipet to transfer larvae to individual wells. Avoid transferring water with the...

Normalization

In spite of great care in keeping experimental conditions constant, random effects are unavoidable. In circadian research we usually use multiple chips (12 chips in our case) to measure temporal changes of mRNA expression. As stochastic variability is inevitable, proper mathematical procedures must be implemented to allow for cross-chip comparisons. Normalization is a term used to describe processes that reduce the impact of random effects on the data, with many methods having been proposed...

Applications

During the first 2 wk in culture the slices become thin and stable. Thus, slices must be maintained in vitro for at least 1 wk before starting experiments. We have successfully cultured slices of SCN that maintain rhythmic property in vitro with both the roller tube and the porous membrane methods. SCN slices cultured with the roller tube technique flatten to almost mono- or several layers and spread on cover slips but retain organotypic properties (ref. 3 Fig. 2). Using this approach, living...

How Many Chips

In circadian studies, animals or other organisms are first entrained to a 12 h 12 h light-dark (LD) cycle for days and then are released into free-running constant dark (DD) conditions. RNA is harvested during LD and or DD cycles, most commonly at 4-h intervals over 48 h (3-8). Twelve microarrays are therefore generally used for one experiment. Several studies, however, have suggested that it would be preferable to use more arrays over the course of an experiment. Panda et al.(6) used two...

Filippo Tamanini

The timing of both entry and permanence of core-clock proteins in the nucleus is critical to maintain the correct pace of the clock mechanism. Several such proteins, namely CRYPTOCHROMEs (CRY), PERIODs (PER), and BMAL1, were recently shown to contain nuclear transport signals that facilitate their shuttling between the nucleus and the cytoplasm. This type of dynamic intracellular movement not only regulates protein localization, but also often affects functions by determining interactive...

Preface

Rhythmicity is a pervasive feature of life. Most organisms, from bacteria to humans, have the ability to interpret and predict the daily cycles of our world, which indicates the presence of a timing device, a circadian (from the Latin circa diem, about a day) clock, able to synchronize the endogenous functions with the external environment. Furthermore, the ability to manipulate the temporal dimension offers ground to complexity, as the organisms have the opportunity to separate competing or...

Purification of a Soluble Protein of Interest

Two protein purification approaches have been used for studying circadian rhythms in cyanobacteria. The protein can be purified from cyanobacterial cells using an affinity purification tag (e.g., engineered 6-His) or immunoprecipita-tion (antibodies raised against the protein or epitope tag). In the case of epitope-tag-based purification, it is important to demonstrate genetically that the tag does not affect protein function. This is easily accomplished in Synechococcus elongatus by...

Natasha A Karp and Kathryn S Lilley Summary

Proteomics is the study of the complete set of proteins encoded by the genome. The study of the proteome involves the investigation of changes in protein abundance, localization, involvement in multiprotein complexes, and detection of different protein isoforms and posttranslational modifications under defined conditions, such as the circa-dian cycle. This type of approach complements comparative gene expression studies providing additional information with respect to posttranscriptional...

Loading Flies

Flies are aged 2 to 5 d and should be entrained to light-dark (LD) cycles. 2. Anesthetize the flies with CO2, pick the fly up by the wing with forceps, and place the fly onto the food in a well. 3. Carefully cover the fly with a dome made in Subheading 3.1. (see Note 3). 4. Load flies onto the plate in column order (e.g., A1, C1, E1, G1, then B2, D2, F2, H2, etc.). Record the order, group name, and date (see Note 5). 5. Place TopSeal-A film on top of the white Optiplate and poke a hole through...

Conclusions

The problem of providing an appropriate light stimulus in circadian experiments is greatly exaggerated because the photopigments that mediate photoentrainment have not been defined in most organisms. This issue is compounded by our poor understanding of the features of the light environment that provide the entrainment signal. In the absence of this information, circa-dian researchers should attempt to use light stimuli that can be accurately reproduced. For the reasons outlined above, light...

CoPurification of Interacting Proteins

These approaches are useful for identifying novel proteins that interact with a protein of interest, and for detecting time-specific interactions for proteins that have been shown by other methods to interact. Co-purification procedures are very similar to the purification protocols described in Subheading 3.3. However, co-purification should be performed using buffers with lower salt concentration than used in buffers in simple purification of the target protein. Relatively low salt...

Construction of Bait Plasmid and Yeast Transformation

The first step in a yeast two-hybrid screen is to construct a bait plasmid that expresses the protein of interest as a fusion to the DNA-BD. This plasmid is transformed into the appropriate yeast reporter strain and a series of control experiments are then performed to establish whether the bait is suitable for use in a library screen or interaction studies between two known proteins. The construction of a bait plasmid expressing the protein of interest is described in Subheadings 3.1.1-3.1.4....

Other Statistics

So far, we have demonstrated how to calculate the p value and fp value for the expression profile of each gene, which provides enough information to deter mine whether a gene exhibits rhythmic expression or not. This section describes how to calculate other useful statistics. The average expression of a gene over a time course provides useful information for evaluating the general level of expression in the samples. Generally, the expression profile of genes with a low average expression is...

Establishing Zebrafish Cell Cultures

The zebrafish is an excellent model for studying early vertebrate development because large numbers of embryos are immediately accessible and easily maintainable. Several protocols exist for establishing cell cultures from zebrafish embryos (8,9). We have routinely used the following protocol to establish cell cultures that can be efficiently transfected by electroporation. 1. Collect eggs 3 h after fertilization and wash three times with filtered E3 medium in a plastic tea strainer. 2....

Cas Kramer and Susan K Crosthwaite

In Northern analysis the presence of specific RNA transcripts is detected and their quantity can be estimated. RNA is separated using denaturing agarose gel electrophoresis and is subsequently transferred and fixed to a solid support, such as a nitrocellulose filter. When labeled probes are hybridized to these immobilized RNA molecules, their presence can be visualized by autoradiography. Here we describe Northern hybridization using radioactively labeled riboprobes to show circadian expression...

Note

Set up guillotine and surgery instruments for brain dissection. 2. Pour methyl butane into a stainless steel beaker and place in dry ice. Monitor temperature until it reaches -30 to -35 C. It is very important that temperature is kept within this range by placing the beaker back and forward in dry ice. Fig. 3. (opposite page) Detection of Perl mRNA by in situ hybridization in the rat suprachiasmatic nucleus. (A) Film autoradiography of a coronal hypothalamic section of a rat sacrificed during...

Library Screening

A variety of cDNA libraries are available commercially or directly from the laboratories where they have been constructed. Although constructing your own cDNA library does offers the opportunity to choose an mRNA source where your protein of interest may have a relevant biological role, cDNA library construction can be both time-consuming and costly. The authors used the MATCHMAKER library construction kit (Clontech) to generate a cDNA library from Arabidopsis thaliana seedlings. In this...

Charlotte Helfrich Forster Summary

This chapter describes immunohistochemistry on Drosophila heads (brains) with respect to antigens involved in the circadian system. Two different methods have been successfully performed in several labs immunolabeling on whole-mount brains and on cryostat sections of entire heads. Both methods are addressed here. The primary antisera can be detected by enzyme-labeled or fluorescence-labeled secondary antibodies. The advantages of the different methods are discussed. Key Words Antigen antiserum...

Generation of Mycelial Disks see also Fig steps

To obtain small pieces of vegetatively growing mycelium of equal size, a floating mat of mycelium (known as a hyphal mat or mycelial mat) is grown in standing liquid culture, from which small disks can be cut for experimental purposes (10,11). Two days prior to the intended start of the experiment 1. Make sure to have one or two fresh slants (3-10 d old) for each Neurospora strain to be used (see Note 4). 2. Add 30 mL of Vogel's minimal medium to a sterile Petri dish or cell culture dish (see...

Testing for Protein Interactions Between Two Known Proteins

The yeast two-hybrid system can also be used for the analysis of proteinprotein interactions between two known proteins or for determining what protein domains or amino acid residues mediate protein-protein interactions. To test for protein-protein interactions between two known proteins the MATCHMAKER pGADT7 AD vector (Fig. 2) is used in conjunction with the pGBKT7 DNA-BD vector. The pGADT7 harbors the T7 promoter, a hemagglutinin epitope tag, and an MCS and expresses proteins fused to the...

Immunostaining

The immunostaining procedure for whole mounts and cryosections is essentially the same, with the difference that the incubation times are reduced for cryosections. The protocol below refers to whole mounts. For cryosections, blocking solution and secondary antibodies should be applied for 2 h, whereas the primary antibody should be incubated overnight. Washes can be reduced to 5 min for each step. 3.3.1. Blocking and Incubation With Primary Antiserum 1. Block the specimens for at least 3 h in...

Methods 31 Race Tubes

What Is a Race Tube Purchasing Manufacturing Race tubes are long hollow glass tubes with a thin layer of agar medium, in which growth of Neurospora can be assayed accurately for several days (see Fig. 2). Race tubes are generally approx 400 mm in length, of which 45 to 50 mm on both ends are bent upward at an angle of 30 to 45 (see Note 5). Race tubes have an internal diameter of approx 14 mm and a glass thickness of about 1 mm. Uniformity of race tubes is an important parameter in...

Radiometry

Radiometry is the measurement of radiant energy in the form of electromagnetic radiation, which includes ultraviolet radiation, visible light, and infrared radiation. An ideal radiometric detector has a near flat spectral response (Fig. 1A), and the remaining irregularities may be compensated for by calibration. A range of radiometric quantities and units is used the two most commonly used in biology are irradiance and radiance (Fig. 2A,B). Irradiance is a radiometric measure of the amount of...

In Vitro Transcription

The detection of gene expression by in situ hybridization is carried out with RNA probes. Efficient RNA transcription requires the subcloning of the DNA fragment of interest into a vector. The multiple cloning site is flanked by phage-RNA-polymerase promoters for example, T3-, T7- or SP6-RNA polymerase, such as pBluescript II (Stratagene) or, for polymerase chain reaction products, pGEMTeasy, respectively (Promega Fig. 1A,B). The size of the cloned fragment should range between 0.5 and 3.0 kb....

Image Capture

Image acquisition can be achieved using a variety of scanners or digital imagers, most of which are based on photomultiplier tubes or charged-coupled devices. Several such systems are commercially available that are compatible with CyDye DIGE fluors, such as the ProXPRESS (PerkinElmer Life Sciences, Wellesley, MA), and Typhoon 9400 (GE Healthcare). Table 3 gives the excitation and emission parameters for all three fluors. When scanning DIGE gels, the following should be considered 1. When...

Reporter Assays M Fernanda Ceriani

Transcriptional feedback loops are at the core of the molecular clockworks. As single clock genes were cloned it was compelling to develop an assay that allowed simple and direct functional testing of putative activators or repressors of transcription. This chapter includes a general description and guidelines to carry out transcriptional assays in transiently transfected Schneider's cells. Key Words Transcriptional assays firefly luciferase Renilla luciferase S2 cells.

Paramecia Culture and Harvest see Note

Culture Paramecia in eight tubs in the fish colony room. Discard the oldest culture and start a new one every Monday, Wednesday, and Friday, or whenever a culture is used up. 2. Clean the culture tub with hot tap water, but no detergent. Wipe with 95 etha-nol and rinse with deionized water. 3. Fill the tub three-fourths full with egg water. Add 2 L of a culture that is growing at a high rate (7-10 d old, cloudy, yellow to pink, with many very active Paramecia). Fill the tub to within 1 cm of...

B

RNA extraction using a phenol-chloroform methodology is possible even with small tissue samples. (A) Coronal section showing staining of mPer2 by in situ hybridization, illustrating region of sampling and high mPer2 expression. (B) Quantitative real-time polymerase chain reaction data from six pooled suprachiasmatic nuclei punches, illustrating clear circadian rhythm in mPer2 expression. RNA was extracted in 0.5 mL monophasic lysis reagent. 3. Wear laboratory gloves at all times, and...

Homogenization

Break a big piece of frozen tissue into small pieces and transfer them into a 1.5-mL microcentrifuge tube (see Note 3). 2. Add 5 vol of EB to the tube. 3. Homogenize the tissue with 10 to 15 strokes (3-4 s stroke) using a mini-homog-enizer and plastic pestle on ice. 4. Spin at 12,000g for 15 min at 4 C. 5. Transfer the supernatant to a fresh tube. Try not to take any lipid from the surface layer or any precipitated particle from the bottom. They may interfere later with Western blotting and...

Preparation of Electrocompetent Cells

Grow the conidia on about 100 mL of solid minimal media after it has been autoclaved in a large (500-mL or 1-L) flask. Fig. 1. Transformation of Neurospora with a plasmid that yields Basta-resistant colonies. (A) pKSbar2 was used for insertional mutagenesis. It is the standard Bluescript vector pKS containing a Basta resistance cassette (see Note 5). (B) Two (or more, as shown here) days following transformation, resistant colonies are picked and transferred to slants. Fig. 1. Transformation of...

Transcriptional Assays

To analyze in depth the function of putative transcription factors, a reporter construct needs to be generated. Usually it takes the form of a full-length promoter or a portion therein where the major target sequences are included, driving the reporter gene selected (i.e., luciferase). This native regulatory region can also be replaced by a multimerized version of the core motifs (i.e., the E-box 12) flanked by the native sequences (from the per or tim promoters, ref. 1 see Note 2). These...

Homologous Recombination at Neutral Sites of S elongatus Chromosomes

S. elongatus possesses at least two sites on the chromosome, called neutral sites ( NS Neutral Site I and II see Note 11), where ectopic sequences can be inserted without generating any apparent phenotype. These loci have been developed as targeting sites for cloned genes. Any DNA of interest can be inserted into the S. elongatus NS sequences on a plasmid introduced into the cyanobacterium and, by homologous recombination, moved into the cyano-bacterial chromosome. The basic principles...

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 m

An agarose gel showing Hindlll restriction digest analysis of cDNA library plasmids recovered from 14 different positive clones. Based on their restriction pattern the clones were classified into different groups. Group A, clones 1, 3, 6 Group B, clones 2, 8 Group C, clones 4, 5 Group D, clones 7, 9 Group E, clones 10, 11 Group F, clones 12, 14 Group G, clone 13. 3.4.4. DNA Sequencing of cDNA Library Inserts Perform initial DNA sequencing of the inserts in the positive cDNA library...

Write Results to File

Entering analyzed data into a file that can be viewed and edited by spreadsheet software such as Microsoft Excel is useful for further analysis. In Mathematica, a tab-separated file in which each line consists of id list, average of expression, standard deviation of expression, peak time, max correlation, p value, and fp value can be written with the following code (see Note 8) tableForFile Table idList i , N avgs i , N sdvs i , N peakTimes i , maxCorrs i , N pValues i ,

Optimizing Transient Transfection of DNA Into Zebrafish Cells

Here, we describe a simple and reliable procedure for the introduction of DNA into zebrafish primary cultures by electroporation. Electroporation involves the exposure of cells to a pulsed electric field to create transient pores in the plasma membrane and thereby facilitate access of DNA (10,11). It is advisable to optimize the conditions for newly derived fish cell lines before embarking on stable transfections (see Notes 7 and 8). In order to assay electroporation efficiency we transiently...

Internal Controls

The matter of internal controls has been subject to intense debate within the scientific literature surrounding gene expression for a number of years. First, it should be stressed that there is no ideal internal control. All housekeeping genes used for this purpose possess merits and flaws. Although many people recommend the use of rRNAs (especially 18S rRNA), these are not ideal for a number of reasons, including the massive difference in expression between target Fig. 4. Melting curve (A) and...

Protein Extraction From Whole Heads

Samples must be kept as much as possible on dry ice until extraction actually begins. 2. Flies are decapitated by vigorously vortexing the 15-mL tubes twice for 15 s. 3. If the number of heads per time-point is small, heads are counted on a plastic mesh placed over dry ice and transferred into microcentrifuge tubes with a small brush. If the number of heads is large, brass sieves can be used to separate the heads from the bodies and other small body parts like legs and antennae. The top sieve...

Automated Imaging Using Cooled CCD Camera

For automated imaging we use an ORCA II camera (Hamamatsu) with a high-transmission lens (Xenon f 0.95, 25 mm). This camera is thermoelectrically cooled to -50 C and has low noise and a large dynamic range, making it ideal for luciferase imaging. Several other suitable cameras are available. The camera is set up as shown in Fig. 1. The camera is mounted on a dark box in a temperature-controlled room. The plants for imaging are placed approx 1 m away from the camera at this distance, four 12 x...

Calculate Relative Sensitivity

Once IRCs have been constructed and the EC50 calculated for each wavelength, the relative sensitivity may be calculated. As sensitivity is based on the photon flux required to elicit the same response, a low-photon flux is indicative of high sensitivity to this wavelength, and vice versa (i.e., there is a reciprocal relationship between photon flux required to elicit a 50 response and sensitivity). To calculate relative sensitivity, divide the lowest photon flux obtained by the photon flux...

Changing Emission Level Photon Flux of Light Source

There are two common methods for controlling the irradiance of a light source. The voltage applied to the light source can be varied and or the light source can be screened using neutral density filters. In almost all cases, changing the voltage applied to the light source will change its color temperature, and hence the spectral emission of the bulb (as described by black-body radiation see Fig. 3). In this case an associated change in spectral composition may invalidate comparisons at...

Protein Extraction Fractionation and Purification From Cyanobacteria

Golden Summary This chapter deals with methods of protein extraction from cyanobacterial cells based on work in the circadian model organism Synechococcus elongatus PCC 7942. Some of these techniques have already been used successfully for analysis of circadian rhythms in cyanobacteria, whereas others are heretofore unpublished, but may yield exciting results in the near future. Key Words cyanobacteria protein extraction protein purification protein interaction....

Choogon

Western blotting is one of the most commonly used biochemical techniques to detect a specific protein from a mixture of proteins such as tissue extracts. Antibodies to the specific antigen are used to detect the protein. The mixture of proteins is resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a membrane. A specific antigen immobilized on the membrane is detected and visualized by a primary antibody, a secondary antibody-peroxidase conjugate, and a...

Humana Press Totowa New Jersey

2007 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. Methods in Molecular Biology is a trademark of The Humana Press Inc. All papers, comments, opinions, conclusions, or recommendations are those of the author(s),...

Ambient Conditions

Experiments that aim at acquiring information regarding the patterns of cir-cadian activity shown by the organism of interest should be conducted under highly controlled environmental conditions. Such experiments should be conducted in strictly light-tight, possibly also soundproof, housings that should also allow the full control of temperature, humidity, and internal lighting conditions. Ideally, both temperature and light should be independently programmable. Better still would be to have...

Growth and Preparation of Plants

The stages below deal specifically with preparing A. thaliana seedlings for leaf movement. Other species of plant show rhythms in leaf movement, which can be assayed using the system outlined above, but these may require different preparation procedures and growth conditions (see Note 9). 1. Surface-sterilize Arabidopsis seed by soaking in 70 ethanol for 1 to 2 min, followed by soaking in 50 bleach for 8 min. 2. Remove the bleach and rinse two to three times in sterile distilled water, before...

Routine Passaging of Zebrafish Cell Cultures

Sterile, disposable tissue culture flasks and plates (no specialized coating), conical tubes, and pipets. 2. Laminar flow hood for sterile tissue culture work. 3. L15 complete culture medium L15 (Leibovitz) culture medium (Gibco), 15 fetal bovine serum (Biochrom KG), 100 U mL penicillin 100 mg mL streptomycin (Gibco), 50 mg mL gentamicin (Gibco). 4. 1X phosphate-buffered saline (PBS) without calcium and magnesium (Gibco). 5. 1X Trypsin-EDTA 10X stock (Gibco) diluted in 1X PBS.

General Protocols

It is a regular practice to counterstain tissue sections to reveal all the nuclei. When using a fluorescent label, counterstaining can be done with propidium iodide (red dye) or DAPI (blue dye), depending on the color of the fluoro-chrome used for immunostaining. If using an enzymatic label, the color of the chromogen willl determine which substance can be used to counterstain the sections. If the product of the immunoreaction is red, black, or brown, slides can be counterstained with...

Concepts

The amplification plot is the end result of real-time PCR no matter what platform or chemistry is used. The amplification plot is essentially a growth curve of fluorescence, which is proportional to the DNA concentration within Fig. 3. The end result of real-time polymerase chain reaction (PCR) is the amplification plot. The x-axis is the PCR cycle whereas the y-axis is the fluorescence that increases throughout the reaction. (A) Amplification plot on a linear scale, demonstrating appropriate...

Primer Testing

Details for primer design are largely dependent on the chemistry used. Probe-based systems such as TaqMan (Applied Biosystems) often have quite specific primer and probe requirements. For double-stranded DNA (dsDNA) binding dyes, the primers are the sole source of specificity, and as such it is not worth conducting a qPCR assay with suboptimal primers. Use of dsDNA binding dyes enables use of larger products, and this is also beneficial to allow the separation of primer-dimers and specific...

The Measurement of Light or Radiant Energy

Technically light and radiant energy are not the same. Radiant energy is considered to be all electromagnetic energy, whereas historically, light refers to the part of the electromagnetic spectrum that is visible to the human eye (between the wavelengths of 380 and 750 nm). Thus, the strict definition of light is based on human sensitivity to radiant energy. However, the term light is generally used to define radiant energy detected by physiological systems in humans and nonhumans, and the term...

Preparation of Extracts and Preclearing

Prepare the protein extract from the tissue of choice as described in Chapter 29. Usually 50 to 100 mg of tissue will yield enough samples to repeat the final Western blot three or four times. 2. Save 10 of the extract, mix with 1 vol of 2X SDS sample buffer, and boil at 95 C for 3 min. This will serve as the starting sample. 3. Prepare the beads. Take 20 L of beads per reaction (equivalent to 40 L of a 1 1 slurry the beads are normally sold as a 1 1 slurry in 20 EtOH) and equilibrate with 500...

Statistical Methods for Circadian Rhythms Rikuhiro Yamada and Hiroki R Ueda

Microarrays are promising tools that are increasingly being applied to the study of circadian rhythms. The large and complex datasets they generate, however, mean they require a new approach on how to design experiments, handle datasets, translate results, and derive conclusions. This technology also requires statistical methods for the correct interpretation of data generated by the microarrays. In this chapter, we provide an overview of analytical methods applied to microarray experiments for...