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Identification of putative rat ribonuclease III by differential display: a novel rat mRNA expressed in a circadian manner in the rat suprachiasmatic nucleus
Molecular Brain Research 131 (2004) 51 – 57 www.elsevier.com/locate/molbrainres
Research report
Identification of putative rat ribonuclease III by differential display: a novel rat mRNA expressed in a circadian manner in the rat suprachiasmatic nucleus Ranjit K. Bhogala,1, Alexander L. Mitchellb,2, Clive W. Coena,* b
a School of Biomedical Sciences, King’s College London, London SE1 1UL, UK Department of Neuroscience, Institute of Psychiatry, King’s College London, London SE5 8AF, UK
Accepted 27 July 2004
Abstract The suprachiasmatic nucleus (SCN) of the hypothalamus constitutes the principal site responsible for the generation and entrainment of circadian rhythms in mammals. The mechanisms of the circadian clock involve periodic gene expression. Here we report the use of differential display reverse transcriptase polymerase chain reaction to identify a novel rat mRNA sequence which is highly homologous to human ribonuclease III. Analysis of its expression in the rat brain by in situ hybridization histochemistry showed this transcript to be expressed at differing intensities at various sites. Temporal variation in expression was observed in the SCN, with a peak at circadian time (CT) 2 and a nadir at CT14. No significant changes in its expression were detected across the cycle within the supraoptic nucleus, cingulate cortex or caudate putamen. D 2004 Elsevier B.V. All rights reserved. Theme: Neural basis of behaviour Topic: Biological rhythms and sleep Keywords: Circadian rhythms; Suprachiasmatic nucleus; Ribonuclease III; Differential display
1. Introduction Daily rhythms of biological activity in almost every living organism are driven by self-sustaining, endogenous systems called circadian clocks. The suprachiasmatic nucleus (SCN) of the hypothalamus contains the principal endogenous oscillator in mammals and consists of paired nuclei lying adjacent to the third cerebral ventricle in the anterior hypothalamus, immediately dorsal to the optic
* Corresponding author. Tel.: +44 20 7848 6205; fax: +44 20 7848 6220. E-mail address: [email protected] (C.W. Coen). 1 Present address: Unilever Research and Development, Colworth, Sharnbrook, Bedford MK44 1LQ, UK. 2 Present address: European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK. 0169-328X/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molbrainres.2004.07.023
chiasm [20]. Various genes involved in the mammalian circadian clock have been identified; these include Clock [14], Bmal1 [11], three Period isoforms mPer1, mPer2 and mPer3 [3,26,29,31–33,37] and two Cryptochrome isoforms mCry1 and mCry2 [16,19,34]. The products of these genes have been assembled into a model of a molecular oscillator based on interlocking feedback loops [2,13,25]. Identification of these clock genes does not preclude the existence of additional clock-related genes. Various approaches have been employed to identify such genes, including mutant screening [24], microarrays [1,23] and differential display [8,12,27]. Differential display is a sensitive mRNA screening technique that enables comparison of reverse transcribed and arbitrarily amplified cDNAs from two or more cell or tissue types. The isolated cDNAs can be identified by sequencing and interrogation of databases; they can subsequently be cloned and used as probes
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Serial coronal brain sections (300 Am) containing the SCN were cut and mounted onto RNase-free glass slides. The slides were placed on a microdissecting stage attached to a freezing device [9] which allows the sections to remain frozen during dissection. Using a microdissecting needle (internal diameter 400 Am; Fine Science Tools, Vancouver, Canada), the bilateral SCN from two serial coronal sections was punched out. The dissected SCN from individual animals were immediately stored at 70 8C for subsequent RNA extraction. Total RNA was isolated from SCN tissue using the Total RNA Extraction System (Promega, Southampton, UK) according to the manufacturer’s instructions.
points: CT0, CT2, CT6, CT10, CT12, CT14, CT18 and CT22. The differential display protocol employed here was an adaptation [4] of the original methodology [17] using the Hieroglyph mRNA Profile Kit (Beckman Instruments Fullerton, USA). The procedure involved reverse transcription of total RNA using anchored primers designed to select subpopulations of mRNA based on the poly(A)+ tail and the two bases upstream of the tail. The resulting singlestranded subpopulations of cDNAs were then amplified by PCR using the same anchored primer together with an arbitrary primer in the presence of a radiolabelled deoxynucleotide. Briefly, total RNA (200 ng) was denatured at 65 8C for 10 min with the anchored primer (2 AM) to a final volume of 8 Al. The denatured total RNA was reverse transcribed in the presence of the following at final concentrations: 1 SuperScript II RT buffer (0.05 mM Tris–HCl, 0.075 M KCl, 3 mM MgCl2), 10 mM DTT, 25 AM dNTP mixture (dGTP, dATP, dTTP and dCTP each at 25 AM), 1 unit RNasin (Promega), 2 units SuperScript II RT enzyme (Gibco Life Technologies, Paisley, UK) and DEPCtreated H2O to a final volume of 20 Al at 42 8C for 60 min; the reaction was terminated by heating to 95 8C for 5 min. cDNA templates (2 Al) were amplified by PCR in duplicate in the presence of the following at final concentrations: 1 PCR buffer (10 mM Tris–HCl, 0.05 M KCl, 1.5 mM MgCl2), 20 AM dNTP mixture (dGTP, dATP, dTTP and dCTP each at 20 AM), 0.2 AM anchored primer (as used in the RT reaction), 0.2 AM arbitrary primer, 0.05 units Amplitaq DNA polymerase (Perkin Elmer, Cambridge, UK), 1 ACi [a-33P]dATP (NEN, Hounslow, UK) and DEPC (Sigma, Dorset, UK) treated H2O to final volume of 20 Al. The reactions were placed in a pre-heated (95 8C) thermal cycler and incubated as follows: 95 8C for 2 min; 4 cycles of 92 8C for 15 s, 46 8C for 30 s, 72 8C for 2 min; 30 cycles of 92 8C for 15 s, 60 8C for 30 s, 72 8C for 2 min; 72 8C for 7 min. Aliquots of each PCR reaction were separated on a 4.5% denaturing polyacrylamide gel by electrophoresis at 800 V, 100 W and 40 C for 16 h. Dried gels were apposed to X-ray film. Bands representing mRNA transcripts were assessed by eye and those displaying differences in intensity between the eight time points across the 24-h cycle were isolated from the gel and reamplified by PCR for sub-cloning. This paper presents data concerning one such transcript, amplified using anchored primer 5V-TTTTTTTTTTTTGA-3V and arbitrary primer 5V-CGACTCCAAG-3V. The reamplified product was sub-cloned into the pGEM-T Easy Vector (Gibco Life Technologies) and sequenced using primers located outside the multiple cloning sites (Lac reverse primer 5V-GGAAACAGCTATGACCATG-3V and Lac 40 primer 5VGTTTTCCCAGTCACGACG-3V).
2.3. Differential display
2.4. In situ hybridization histochemistry (ISHH)
Differential display reverse transcriptase polymerase chain reaction (RT PCR) was carried out on total RNA from the bilateral SCN obtained from individual rats at eight time
Coronal sections (15 Am), containing the mid-rostrocaudal SCN, were obtained with a cryostat from 5 rats at each time point. They were thaw-mounted onto glass slides
for downstream analysis with quantitative in situ hybridization histochemistry. Here we report the use of differential display in the identification of a novel rat mRNA sequence that is highly homologous to human ribonuclease III; this transcript cycles in a circadian manner in the rat SCN as confirmed using in situ hybridization histochemistry.
2. Materials and methods 2.1. Animals Male Lister Hooded rats (~250 g; Harlan Sera Laboratory, Loughborough, UK) were maintained in a 12:12-h light/dark (LD) schedule at 22F2C for a minimum of four weeks. Darkness (DD conditions) consisted of constant dim red light providing b2 lx at cage level. The emission spectrum of this light source was between 600 and 700 nM, a range within the red part of the spectrum outside the sensitivity curve for a circadian phase shifting response as determined in golden hamsters [30]. Food and water were available ad libitum. In order to determine up- and down-regulated mRNA transcripts in the rat SCN over a 24-h cycle in the absence of Zeitgeber cues, lights were not switched on at the usual time of transition from dark to light; this time point was designated as circadian time (CT) 0. The animals were then maintained under dark:dark (DD) conditions for 48 h (two cycles) and sacrificed by decapitation under constant dim red light at eight time points (CT 0, 2, 6, 10, 12, 14, 18 and 22) during the third cycle in DD. To reduce variation in sampling time, no more than three animals were sacrificed on a single occasion. The optic nerves were severed and the brain was immediately removed, rapidly frozen in powdered dry ice and stored at 70 8C. 2.2. SCN Microdissection and RNA extraction
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and air dried for ~30 min. Sections were then pretreated (4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) for 15 min; 0.1 M PBS rinse; 0.1 M triethanolamide (TEA) for 1 min; 0.1 M TEA, 0.25 mM acetic anhydride for 10 min; 70% EtOH, 80% EtOH, 95% EtOH, 100% absolute alcohol for 5 min each; chloroform for 10 min; absolute alcohol and 95% EtOH for 5 min each) prior to hybridization. Antisense riboprobes were produced by linearisation of the vector using NcoI; control sense probes were produced using SpeI. Riboprobes were synthesised by run-off transcription using ~1 Ag linearised plasmid template in the presence of the following at final concentrations: 1 transcription buffer (40 mM Tris pH 7.9, 6 mM MgCl2, 2 mM spermidine, and 10 mM NaCl), 5 mM rNTP mixture (rATP, rGTP and rCTP each at 5 mM), 25 mM DTT, 2 Units RNasin, 5 ACi [a-35S] UTP (NEN), 1 unit RNA polymerase (SP6 for antisense probe and T7 for sense probe; Promega) and DEPC-treated H2O to a final volume of 20.0 Al. The reaction was carried out at 37 8C for 30 min after which a second unit of RNA polymerase was added and the reaction was incubated for a further 45 min. DNase I (0.25 units) was then added and the reaction continued at 37 8C for 15 min. The radioactively labelled riboprobe was purified from unlabelled riboprobe using sephadex G50 RNA columns (Boehringer Mannheim, East Sussex, UK) and DTT added to the labelled probe to a final concentration of 20 mM. Riboprobes were diluted to 1105, 5105, 1106 and 5106 cpm in hybridization buffer at the following final concentrations: 50% deionised formamide, 1 Denhart’s solution [0.02% bovine serum albumin, 0.2% polyvinylpyrrolidone and 0.2% Ficoll], 8% dextran sulphate, 4 SSC, 120 Ag/Al tRNA, 120 Ag/Al herring sperm DNA, 0.05 M DTT and 0.03% SDS). Sections were hybridised with labelled probe for 18 h in a humidifed chamber (2 SSC, 50% formamide). Following hybridization sections were washed in SSC at increasing stringency, dried and apposed to Kodak BioMax MR X-ray film at 4 8C. The autoradiograph film was developed under safelight conditions for 3 min in Kodak GBX developer, rinsed in ddH2O, fixed for 5 min in Kodak GBX fixer, washed in running water for at least 15 min and then left to dry prior to image analysis as described below.
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Under safelight conditions, the slides were coated in K-5 nuclear emulsion diluted 1:1 with ddH2O and stored in light-tight slide boxes at 4 8C for four times longer than the time period over which they were apposed to X-ray film. The sections were then developed under safelight conditions using Kodak D19 developer, fixed with Kodak RA300 fixer and counter-stained with toluidine blue. 2.5. Image analysis Quantification of signals in film autoradiographs was carried out using Scion Image (version 4.02; Scion, Frederick, MD, USA). The relative optical density (ROD) value was obtained bilaterally at the mid-rostro-caudal level of the SCN for the SCN, supraoptic nucleus (SON), cingulate cortex and caudate putamen of each animal (2 sections/animal); for each section, this value was corrected for non-specific signal by subtraction of the ROD derived from an area of tissue displaying the lowest signal level, namely the corpus callosum. The corrected values were converted into mean nCi/g of tissue equivalent using a standard curve created from the 14C microscale (Amersham, Buckinghamshire, UK). All values obtained fell within the linear range of the calibration curve. The values for each animal (n=5 per time point) were used to calculate the group mean and standard error of mean (S.E.M.) assessed by analysis of variance (ANOVA); post hoc comparisons were performed using the Tukey Kramer multiple comparison test. Statistical significance was defined as pb0.05.
3. Results 3.1. Identification of a novel rat mRNA sequence using differential display Various transcripts were identified as differentially expressed in the SCN over a 24-h period under DD conditions. Only one of the isolated transcripts, initially designated SCN A (GenBank accession number AY373464), is discussed here. Differential display indicated differential expression of a band (designated SCN A) at approximately 1 kb (Fig. 1); a signal was detected only at CT2. SCN A was found to be
Fig. 1. Differential display images showing (A) the differentially expressed transcript SCN A and (B) a non-differentially expressed transcript; they were amplified from bilateral suprachiasmatic nuclei micropunched from individual rats killed at circadian time (CT) 0 to 22 during the third circadian cycle under constant dim red light. Each sample was run in duplicate.
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[35]. The particularly high homology (87%) between SCN A and human ribonuclease III lies over a stretch of 864 bp at the 3V-end coding region of human ribonuclease III mRNA. The full-length mRNA sequence of human ribonuclease III is 4763 nucleotides in length; this suggests that SCN A is a partial sequence for the full-length rat ribonuclease III mRNA sequence. Furthermore, the putative amino acid sequence for this rat transcript is highly homologous (98%) to the amino acid sequence for human ribonuclease III. 3.2. mRNA expression of a novel mRNA in the rat brain Fig. 2. Film autoradiograph generated from a coronal section of a rat brain collected at circadian time (CT) 2 and hybridized to the antisense riboprobe for rat ribonuclease III (SCN A). SCN=suprachiasmatic nucleus; SON= supraoptic nucleus; Cg=cingulate cortex; CPu=caudate putamen.
995 bp in length. Sequence comparison using various databases revealed SCN A to be highly similar to a sequence annotated as a human ribonuclease III mRNA, AF189011
Coronal rat brain sections (at the level of the mid-rostrocaudal SCN) hybridized with an antisense riboprobe for rat ribonuclease III showed expression in various regions including the SCN (Fig. 2). The most intense signals were in the piriform cortex; signals were also present in the rest of the cerebral cortex and in the SON. Levels in the caudate putamen and globus pallidus were, respectively, low and negligible. Control procedures involving RNase-treated
Fig. 3. Expression of rat ribonuclease III mRNA in the bilateral (A) suprachiasmatic nucleus (SCN), (B) supraoptic nucleus (SON), (C) cingulate cortex and (D) caudate putamen of male rats killed around the circadian cycle following a minimum of 48 h in constant dim red light. Hybridization signals from film autoradiograms at the mid-rostro-caudal level of the SCN were analyzed and expressed as mean (FS.E.M.) nCi/g tissue (n=5 animals per time point with two sections analyzed from each animal). The data at CT0 have been replotted at CT24. Asterisks indicate levels that are significantly higher than the level indicated by the linked plus sign. *pb0.05, **pb0.01, ***pb0.001 (ANOVA followed by Tukey multiple comparison test).
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sections hybridized with an antisense riboprobe or a sense riboprobe hybridized to normal brain sections resulted in a total absence of signal. 3.3. Circadian mRNA expression of a novel mRNA in the rat SCN Quantification of the in situ hybridization signal for rat ribonuclease III mRNA over eight time points across a circadian cycle in the SCN, SON, cingulate cortex and caudate putamen showed this transcript to be expressed with a rhythmic pattern in the SCN (Fig. 3A). One-way ANOVA indicated a highly significant variation at this site ( F 7,32=7.0, pb0.001); the post hoc multiple comparison test demonstrated that rat ribonuclease III mRNA was significantly lower at CT14 compared with CT2, CT6, CT18 and CT22, and at CT12 compared with CT2 and CT6. The maximum SCN A mRNA level at CT2 was more than twofold higher than the minimum level at CT14. Although a similar temporal pattern of mRNA expression levels was apparent in the SON over the cycle, with lowest levels at CT14 and highest levels at CT2 and CT22 (Fig. 3B), there were no significant differences between any of the levels measured at this site. Within the cingulate cortex and caudate putamen there was also no significant variation (Fig. 3C,D). Rat ribonuclease III mRNA signals within the SCN in emulsioncoated sections showed relatively high levels of silver grains at CT2 as compared with CT14 (Fig. 4).
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4. Discussion Differential display is a useful method for identifying genes the expression of which may be up- or downregulated in one sample relative to another. Following its introduction [17], the methodology has been widely used and improved [4,5,10,15,28]. One of the products identified by our differential display analysis of the rat SCN across a circadian cycle was found to be elevated at CT2 but below detectibility at other time points. On the basis of this finding, the transcript, initially designated SCN A, was selected for further neuroanatomical and quantitative investigation. This rat transcript showed 87% homology with the mRNA for human ribonuclease III over a stretch of 864 bp at the 3V-end coding region. Since the putative amino acid sequence for this rat transcript is 98% homologous to the amino acid sequence for human ribonuclease III, it seems probable that SCN A is a partial sequence for the full-length rat ribonuclease III mRNA sequence. Analysis of rat ribonuclease III mRNA expression by ISHH at the coronal level of the mid-rostro-caudal SCN showed this transcript to be expressed at differing intensities in various regions of the rat brain. Circadian variation in expression was observed in the SCN; the peak level at CT 2 was more than twofold higher than the nadir level at CT14. No significant changes in expression were detected across the cycle within the SON, cingulate cortex or caudate putamen.
Fig. 4. Representative photomicrographs of signals for rat ribonuclease III mRNA in the suprachiasmatic nucleus (SCN) of emulsion-coated hybridized sections. Darkfield images of the SCN at CT2 (A) or CT14 (B). Higher power brightfield images of the silver grains detected at CT2 (AV) or CT14 (BV) in the areas within the SCN indicated by the boxes in the respective darkfield images (A and B). Scale bars indicate 200 Am in A and B and 20 Am in AV and BV.
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These observations indicate the utility of the differential display screening procedure in identifying a differentially expressed transcript in the SCN. They also illustrate the imprecision that can confound this approach. The time at which a signal had been detected by the differential display, CT2, corresponded to the time at which the highest ISHH signal was subsequently observed; nevertheless, the elevated levels detected in the SCN by ISHH both before and after CT2 had not been detected during the differential display stage. This emphasises the provisional nature of the differential screening procedure and the importance of testing its findings by the more precise technique of quantitative ISHH. A cautionary approach is especially important when the initial screening is applied to an extremely small site for which even the most careful microdissection techniques cannot guarantee complete reproducibility. When RIGUI (mPer1) was identified, it was proposed [29] that putative mammalian circadian regulators should have a number of characteristics; they should be expressed in the SCN, their mRNA expression should oscillate with a 24-h rhythm, their circadian expression should persist under constant conditions and their rhythm of expression should be reset by changes in the phase of environmental cues. Thus, the circadian rhythm of rat ribonuclease III mRNA expression within the SCN fulfils some of the putative criteria for genes involved in circadian functions. The present findings do not indicate regionalised changes in ribonuclease III expression within the SCN across the cycle. In this respect, its expression at this site appears to be similar to that of Bmal1 [22], mCry1 [16], dbp [18] and pk2 [7]. In contrast, circadian changes in Per1 and Per2 expression have been shown to vary within the subdivisions of the SCN [36]. Bacterial ribonuclease III has been shown to participate in various RNA maturation and decay pathways [21]. In E. coli, it has been found to regulate its own message by cleaving a region in the stem of a hairpin loop in its mRNA [6]. The role of eukaryotic ribonuclease III is less well studied, but inhibition of human ribonuclease III causes cell death, suggesting an essential role [35]. Furthermore, human ribonuclease III has been shown to be involved in preribosomal RNA processing, similar to its actions in bacteria [35]. Panda et al. [23] reported various oscillating transcripts in the SCN that are involved in protein synthesis. It remains to be determined whether rat ribonuclease III participates in processes underlying the synthesis of proteins involved in the mammalian clock mechanism. The present evidence for a rat ribonuclease III transcript which is differentially expressed across the circadian cycle in the rat SCN provides a novel perspective on this molecule.
Acknowledgements This study was supported by a studentship from the BBSRC and by additional funding from the BBSRC and the
Wellcome Trust. The authors wish to thank Dr. F.R.A. Cagampang for his assistance in the initial stages of the research and also Dr. P. Marsh for technical expertise.
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Research report
Identification of putative rat ribonuclease III by differential display: a novel rat mRNA expressed in a circadian manner in the rat suprachiasmatic nucleus Ranjit K. Bhogala,1, Alexander L. Mitchellb,2, Clive W. Coena,* b
a School of Biomedical Sciences, King’s College London, London SE1 1UL, UK Department of Neuroscience, Institute of Psychiatry, King’s College London, London SE5 8AF, UK
Accepted 27 July 2004
Abstract The suprachiasmatic nucleus (SCN) of the hypothalamus constitutes the principal site responsible for the generation and entrainment of circadian rhythms in mammals. The mechanisms of the circadian clock involve periodic gene expression. Here we report the use of differential display reverse transcriptase polymerase chain reaction to identify a novel rat mRNA sequence which is highly homologous to human ribonuclease III. Analysis of its expression in the rat brain by in situ hybridization histochemistry showed this transcript to be expressed at differing intensities at various sites. Temporal variation in expression was observed in the SCN, with a peak at circadian time (CT) 2 and a nadir at CT14. No significant changes in its expression were detected across the cycle within the supraoptic nucleus, cingulate cortex or caudate putamen. D 2004 Elsevier B.V. All rights reserved. Theme: Neural basis of behaviour Topic: Biological rhythms and sleep Keywords: Circadian rhythms; Suprachiasmatic nucleus; Ribonuclease III; Differential display
1. Introduction Daily rhythms of biological activity in almost every living organism are driven by self-sustaining, endogenous systems called circadian clocks. The suprachiasmatic nucleus (SCN) of the hypothalamus contains the principal endogenous oscillator in mammals and consists of paired nuclei lying adjacent to the third cerebral ventricle in the anterior hypothalamus, immediately dorsal to the optic
* Corresponding author. Tel.: +44 20 7848 6205; fax: +44 20 7848 6220. E-mail address: [email protected] (C.W. Coen). 1 Present address: Unilever Research and Development, Colworth, Sharnbrook, Bedford MK44 1LQ, UK. 2 Present address: European Bioinformatics Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SD, UK. 0169-328X/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molbrainres.2004.07.023
chiasm [20]. Various genes involved in the mammalian circadian clock have been identified; these include Clock [14], Bmal1 [11], three Period isoforms mPer1, mPer2 and mPer3 [3,26,29,31–33,37] and two Cryptochrome isoforms mCry1 and mCry2 [16,19,34]. The products of these genes have been assembled into a model of a molecular oscillator based on interlocking feedback loops [2,13,25]. Identification of these clock genes does not preclude the existence of additional clock-related genes. Various approaches have been employed to identify such genes, including mutant screening [24], microarrays [1,23] and differential display [8,12,27]. Differential display is a sensitive mRNA screening technique that enables comparison of reverse transcribed and arbitrarily amplified cDNAs from two or more cell or tissue types. The isolated cDNAs can be identified by sequencing and interrogation of databases; they can subsequently be cloned and used as probes
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Serial coronal brain sections (300 Am) containing the SCN were cut and mounted onto RNase-free glass slides. The slides were placed on a microdissecting stage attached to a freezing device [9] which allows the sections to remain frozen during dissection. Using a microdissecting needle (internal diameter 400 Am; Fine Science Tools, Vancouver, Canada), the bilateral SCN from two serial coronal sections was punched out. The dissected SCN from individual animals were immediately stored at 70 8C for subsequent RNA extraction. Total RNA was isolated from SCN tissue using the Total RNA Extraction System (Promega, Southampton, UK) according to the manufacturer’s instructions.
points: CT0, CT2, CT6, CT10, CT12, CT14, CT18 and CT22. The differential display protocol employed here was an adaptation [4] of the original methodology [17] using the Hieroglyph mRNA Profile Kit (Beckman Instruments Fullerton, USA). The procedure involved reverse transcription of total RNA using anchored primers designed to select subpopulations of mRNA based on the poly(A)+ tail and the two bases upstream of the tail. The resulting singlestranded subpopulations of cDNAs were then amplified by PCR using the same anchored primer together with an arbitrary primer in the presence of a radiolabelled deoxynucleotide. Briefly, total RNA (200 ng) was denatured at 65 8C for 10 min with the anchored primer (2 AM) to a final volume of 8 Al. The denatured total RNA was reverse transcribed in the presence of the following at final concentrations: 1 SuperScript II RT buffer (0.05 mM Tris–HCl, 0.075 M KCl, 3 mM MgCl2), 10 mM DTT, 25 AM dNTP mixture (dGTP, dATP, dTTP and dCTP each at 25 AM), 1 unit RNasin (Promega), 2 units SuperScript II RT enzyme (Gibco Life Technologies, Paisley, UK) and DEPCtreated H2O to a final volume of 20 Al at 42 8C for 60 min; the reaction was terminated by heating to 95 8C for 5 min. cDNA templates (2 Al) were amplified by PCR in duplicate in the presence of the following at final concentrations: 1 PCR buffer (10 mM Tris–HCl, 0.05 M KCl, 1.5 mM MgCl2), 20 AM dNTP mixture (dGTP, dATP, dTTP and dCTP each at 20 AM), 0.2 AM anchored primer (as used in the RT reaction), 0.2 AM arbitrary primer, 0.05 units Amplitaq DNA polymerase (Perkin Elmer, Cambridge, UK), 1 ACi [a-33P]dATP (NEN, Hounslow, UK) and DEPC (Sigma, Dorset, UK) treated H2O to final volume of 20 Al. The reactions were placed in a pre-heated (95 8C) thermal cycler and incubated as follows: 95 8C for 2 min; 4 cycles of 92 8C for 15 s, 46 8C for 30 s, 72 8C for 2 min; 30 cycles of 92 8C for 15 s, 60 8C for 30 s, 72 8C for 2 min; 72 8C for 7 min. Aliquots of each PCR reaction were separated on a 4.5% denaturing polyacrylamide gel by electrophoresis at 800 V, 100 W and 40 C for 16 h. Dried gels were apposed to X-ray film. Bands representing mRNA transcripts were assessed by eye and those displaying differences in intensity between the eight time points across the 24-h cycle were isolated from the gel and reamplified by PCR for sub-cloning. This paper presents data concerning one such transcript, amplified using anchored primer 5V-TTTTTTTTTTTTGA-3V and arbitrary primer 5V-CGACTCCAAG-3V. The reamplified product was sub-cloned into the pGEM-T Easy Vector (Gibco Life Technologies) and sequenced using primers located outside the multiple cloning sites (Lac reverse primer 5V-GGAAACAGCTATGACCATG-3V and Lac 40 primer 5VGTTTTCCCAGTCACGACG-3V).
2.3. Differential display
2.4. In situ hybridization histochemistry (ISHH)
Differential display reverse transcriptase polymerase chain reaction (RT PCR) was carried out on total RNA from the bilateral SCN obtained from individual rats at eight time
Coronal sections (15 Am), containing the mid-rostrocaudal SCN, were obtained with a cryostat from 5 rats at each time point. They were thaw-mounted onto glass slides
for downstream analysis with quantitative in situ hybridization histochemistry. Here we report the use of differential display in the identification of a novel rat mRNA sequence that is highly homologous to human ribonuclease III; this transcript cycles in a circadian manner in the rat SCN as confirmed using in situ hybridization histochemistry.
2. Materials and methods 2.1. Animals Male Lister Hooded rats (~250 g; Harlan Sera Laboratory, Loughborough, UK) were maintained in a 12:12-h light/dark (LD) schedule at 22F2C for a minimum of four weeks. Darkness (DD conditions) consisted of constant dim red light providing b2 lx at cage level. The emission spectrum of this light source was between 600 and 700 nM, a range within the red part of the spectrum outside the sensitivity curve for a circadian phase shifting response as determined in golden hamsters [30]. Food and water were available ad libitum. In order to determine up- and down-regulated mRNA transcripts in the rat SCN over a 24-h cycle in the absence of Zeitgeber cues, lights were not switched on at the usual time of transition from dark to light; this time point was designated as circadian time (CT) 0. The animals were then maintained under dark:dark (DD) conditions for 48 h (two cycles) and sacrificed by decapitation under constant dim red light at eight time points (CT 0, 2, 6, 10, 12, 14, 18 and 22) during the third cycle in DD. To reduce variation in sampling time, no more than three animals were sacrificed on a single occasion. The optic nerves were severed and the brain was immediately removed, rapidly frozen in powdered dry ice and stored at 70 8C. 2.2. SCN Microdissection and RNA extraction
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and air dried for ~30 min. Sections were then pretreated (4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS) for 15 min; 0.1 M PBS rinse; 0.1 M triethanolamide (TEA) for 1 min; 0.1 M TEA, 0.25 mM acetic anhydride for 10 min; 70% EtOH, 80% EtOH, 95% EtOH, 100% absolute alcohol for 5 min each; chloroform for 10 min; absolute alcohol and 95% EtOH for 5 min each) prior to hybridization. Antisense riboprobes were produced by linearisation of the vector using NcoI; control sense probes were produced using SpeI. Riboprobes were synthesised by run-off transcription using ~1 Ag linearised plasmid template in the presence of the following at final concentrations: 1 transcription buffer (40 mM Tris pH 7.9, 6 mM MgCl2, 2 mM spermidine, and 10 mM NaCl), 5 mM rNTP mixture (rATP, rGTP and rCTP each at 5 mM), 25 mM DTT, 2 Units RNasin, 5 ACi [a-35S] UTP (NEN), 1 unit RNA polymerase (SP6 for antisense probe and T7 for sense probe; Promega) and DEPC-treated H2O to a final volume of 20.0 Al. The reaction was carried out at 37 8C for 30 min after which a second unit of RNA polymerase was added and the reaction was incubated for a further 45 min. DNase I (0.25 units) was then added and the reaction continued at 37 8C for 15 min. The radioactively labelled riboprobe was purified from unlabelled riboprobe using sephadex G50 RNA columns (Boehringer Mannheim, East Sussex, UK) and DTT added to the labelled probe to a final concentration of 20 mM. Riboprobes were diluted to 1105, 5105, 1106 and 5106 cpm in hybridization buffer at the following final concentrations: 50% deionised formamide, 1 Denhart’s solution [0.02% bovine serum albumin, 0.2% polyvinylpyrrolidone and 0.2% Ficoll], 8% dextran sulphate, 4 SSC, 120 Ag/Al tRNA, 120 Ag/Al herring sperm DNA, 0.05 M DTT and 0.03% SDS). Sections were hybridised with labelled probe for 18 h in a humidifed chamber (2 SSC, 50% formamide). Following hybridization sections were washed in SSC at increasing stringency, dried and apposed to Kodak BioMax MR X-ray film at 4 8C. The autoradiograph film was developed under safelight conditions for 3 min in Kodak GBX developer, rinsed in ddH2O, fixed for 5 min in Kodak GBX fixer, washed in running water for at least 15 min and then left to dry prior to image analysis as described below.
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Under safelight conditions, the slides were coated in K-5 nuclear emulsion diluted 1:1 with ddH2O and stored in light-tight slide boxes at 4 8C for four times longer than the time period over which they were apposed to X-ray film. The sections were then developed under safelight conditions using Kodak D19 developer, fixed with Kodak RA300 fixer and counter-stained with toluidine blue. 2.5. Image analysis Quantification of signals in film autoradiographs was carried out using Scion Image (version 4.02; Scion, Frederick, MD, USA). The relative optical density (ROD) value was obtained bilaterally at the mid-rostro-caudal level of the SCN for the SCN, supraoptic nucleus (SON), cingulate cortex and caudate putamen of each animal (2 sections/animal); for each section, this value was corrected for non-specific signal by subtraction of the ROD derived from an area of tissue displaying the lowest signal level, namely the corpus callosum. The corrected values were converted into mean nCi/g of tissue equivalent using a standard curve created from the 14C microscale (Amersham, Buckinghamshire, UK). All values obtained fell within the linear range of the calibration curve. The values for each animal (n=5 per time point) were used to calculate the group mean and standard error of mean (S.E.M.) assessed by analysis of variance (ANOVA); post hoc comparisons were performed using the Tukey Kramer multiple comparison test. Statistical significance was defined as pb0.05.
3. Results 3.1. Identification of a novel rat mRNA sequence using differential display Various transcripts were identified as differentially expressed in the SCN over a 24-h period under DD conditions. Only one of the isolated transcripts, initially designated SCN A (GenBank accession number AY373464), is discussed here. Differential display indicated differential expression of a band (designated SCN A) at approximately 1 kb (Fig. 1); a signal was detected only at CT2. SCN A was found to be
Fig. 1. Differential display images showing (A) the differentially expressed transcript SCN A and (B) a non-differentially expressed transcript; they were amplified from bilateral suprachiasmatic nuclei micropunched from individual rats killed at circadian time (CT) 0 to 22 during the third circadian cycle under constant dim red light. Each sample was run in duplicate.
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[35]. The particularly high homology (87%) between SCN A and human ribonuclease III lies over a stretch of 864 bp at the 3V-end coding region of human ribonuclease III mRNA. The full-length mRNA sequence of human ribonuclease III is 4763 nucleotides in length; this suggests that SCN A is a partial sequence for the full-length rat ribonuclease III mRNA sequence. Furthermore, the putative amino acid sequence for this rat transcript is highly homologous (98%) to the amino acid sequence for human ribonuclease III. 3.2. mRNA expression of a novel mRNA in the rat brain Fig. 2. Film autoradiograph generated from a coronal section of a rat brain collected at circadian time (CT) 2 and hybridized to the antisense riboprobe for rat ribonuclease III (SCN A). SCN=suprachiasmatic nucleus; SON= supraoptic nucleus; Cg=cingulate cortex; CPu=caudate putamen.
995 bp in length. Sequence comparison using various databases revealed SCN A to be highly similar to a sequence annotated as a human ribonuclease III mRNA, AF189011
Coronal rat brain sections (at the level of the mid-rostrocaudal SCN) hybridized with an antisense riboprobe for rat ribonuclease III showed expression in various regions including the SCN (Fig. 2). The most intense signals were in the piriform cortex; signals were also present in the rest of the cerebral cortex and in the SON. Levels in the caudate putamen and globus pallidus were, respectively, low and negligible. Control procedures involving RNase-treated
Fig. 3. Expression of rat ribonuclease III mRNA in the bilateral (A) suprachiasmatic nucleus (SCN), (B) supraoptic nucleus (SON), (C) cingulate cortex and (D) caudate putamen of male rats killed around the circadian cycle following a minimum of 48 h in constant dim red light. Hybridization signals from film autoradiograms at the mid-rostro-caudal level of the SCN were analyzed and expressed as mean (FS.E.M.) nCi/g tissue (n=5 animals per time point with two sections analyzed from each animal). The data at CT0 have been replotted at CT24. Asterisks indicate levels that are significantly higher than the level indicated by the linked plus sign. *pb0.05, **pb0.01, ***pb0.001 (ANOVA followed by Tukey multiple comparison test).
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sections hybridized with an antisense riboprobe or a sense riboprobe hybridized to normal brain sections resulted in a total absence of signal. 3.3. Circadian mRNA expression of a novel mRNA in the rat SCN Quantification of the in situ hybridization signal for rat ribonuclease III mRNA over eight time points across a circadian cycle in the SCN, SON, cingulate cortex and caudate putamen showed this transcript to be expressed with a rhythmic pattern in the SCN (Fig. 3A). One-way ANOVA indicated a highly significant variation at this site ( F 7,32=7.0, pb0.001); the post hoc multiple comparison test demonstrated that rat ribonuclease III mRNA was significantly lower at CT14 compared with CT2, CT6, CT18 and CT22, and at CT12 compared with CT2 and CT6. The maximum SCN A mRNA level at CT2 was more than twofold higher than the minimum level at CT14. Although a similar temporal pattern of mRNA expression levels was apparent in the SON over the cycle, with lowest levels at CT14 and highest levels at CT2 and CT22 (Fig. 3B), there were no significant differences between any of the levels measured at this site. Within the cingulate cortex and caudate putamen there was also no significant variation (Fig. 3C,D). Rat ribonuclease III mRNA signals within the SCN in emulsioncoated sections showed relatively high levels of silver grains at CT2 as compared with CT14 (Fig. 4).
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4. Discussion Differential display is a useful method for identifying genes the expression of which may be up- or downregulated in one sample relative to another. Following its introduction [17], the methodology has been widely used and improved [4,5,10,15,28]. One of the products identified by our differential display analysis of the rat SCN across a circadian cycle was found to be elevated at CT2 but below detectibility at other time points. On the basis of this finding, the transcript, initially designated SCN A, was selected for further neuroanatomical and quantitative investigation. This rat transcript showed 87% homology with the mRNA for human ribonuclease III over a stretch of 864 bp at the 3V-end coding region. Since the putative amino acid sequence for this rat transcript is 98% homologous to the amino acid sequence for human ribonuclease III, it seems probable that SCN A is a partial sequence for the full-length rat ribonuclease III mRNA sequence. Analysis of rat ribonuclease III mRNA expression by ISHH at the coronal level of the mid-rostro-caudal SCN showed this transcript to be expressed at differing intensities in various regions of the rat brain. Circadian variation in expression was observed in the SCN; the peak level at CT 2 was more than twofold higher than the nadir level at CT14. No significant changes in expression were detected across the cycle within the SON, cingulate cortex or caudate putamen.
Fig. 4. Representative photomicrographs of signals for rat ribonuclease III mRNA in the suprachiasmatic nucleus (SCN) of emulsion-coated hybridized sections. Darkfield images of the SCN at CT2 (A) or CT14 (B). Higher power brightfield images of the silver grains detected at CT2 (AV) or CT14 (BV) in the areas within the SCN indicated by the boxes in the respective darkfield images (A and B). Scale bars indicate 200 Am in A and B and 20 Am in AV and BV.
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These observations indicate the utility of the differential display screening procedure in identifying a differentially expressed transcript in the SCN. They also illustrate the imprecision that can confound this approach. The time at which a signal had been detected by the differential display, CT2, corresponded to the time at which the highest ISHH signal was subsequently observed; nevertheless, the elevated levels detected in the SCN by ISHH both before and after CT2 had not been detected during the differential display stage. This emphasises the provisional nature of the differential screening procedure and the importance of testing its findings by the more precise technique of quantitative ISHH. A cautionary approach is especially important when the initial screening is applied to an extremely small site for which even the most careful microdissection techniques cannot guarantee complete reproducibility. When RIGUI (mPer1) was identified, it was proposed [29] that putative mammalian circadian regulators should have a number of characteristics; they should be expressed in the SCN, their mRNA expression should oscillate with a 24-h rhythm, their circadian expression should persist under constant conditions and their rhythm of expression should be reset by changes in the phase of environmental cues. Thus, the circadian rhythm of rat ribonuclease III mRNA expression within the SCN fulfils some of the putative criteria for genes involved in circadian functions. The present findings do not indicate regionalised changes in ribonuclease III expression within the SCN across the cycle. In this respect, its expression at this site appears to be similar to that of Bmal1 [22], mCry1 [16], dbp [18] and pk2 [7]. In contrast, circadian changes in Per1 and Per2 expression have been shown to vary within the subdivisions of the SCN [36]. Bacterial ribonuclease III has been shown to participate in various RNA maturation and decay pathways [21]. In E. coli, it has been found to regulate its own message by cleaving a region in the stem of a hairpin loop in its mRNA [6]. The role of eukaryotic ribonuclease III is less well studied, but inhibition of human ribonuclease III causes cell death, suggesting an essential role [35]. Furthermore, human ribonuclease III has been shown to be involved in preribosomal RNA processing, similar to its actions in bacteria [35]. Panda et al. [23] reported various oscillating transcripts in the SCN that are involved in protein synthesis. It remains to be determined whether rat ribonuclease III participates in processes underlying the synthesis of proteins involved in the mammalian clock mechanism. The present evidence for a rat ribonuclease III transcript which is differentially expressed across the circadian cycle in the rat SCN provides a novel perspective on this molecule.
Acknowledgements This study was supported by a studentship from the BBSRC and by additional funding from the BBSRC and the
Wellcome Trust. The authors wish to thank Dr. F.R.A. Cagampang for his assistance in the initial stages of the research and also Dr. P. Marsh for technical expertise.
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