Blast analysis of public databases reveals a large number of coding sequences with significant similarity to Xenopus nocturnin (xNoc). As we originally reported [36], XNOC protein is similar to a large, C-terminal domain of the transcriptional regulator, CCR4, but appears to be lacking in several regulatory domains critical to CCR4 function. Recently, however, mouse and human cDNAs encoding homologues (Fig. 1A) of the same domain of CCR4, but comparable in size to XNOC [38,39] have been reported (Accession number # AAD56547 and AAD56548). Furthermore, availability of the complete genome ofDrosophila melanogaster [40] has revealed a coding sequence (AAF54601.1) with significant similarity to XNOC. In addition to these, we have recently added a complete coding sequence of mouse nocturnin cDNA from retina (mNoc) along with partial coding sequences for human (hNoc), bovine (bNoc), rat (rNoc) and chicken (cNoc).

Among the sequences illustrated in Figure 1, NOCTURNIN shows a high level of conservation throughout its coding sequence. As aligned, xNoc is 66% and 65% identical to HNOC and MNOC respectively. The aa identity drops to 36% when compared to DNOC. Among the vertebrate species this conservation is particularly striking beginning at aa 67. This methionine (not aligned in the Drosophila sequence) corresponds to the fourth codon of Xenopus [36] and mouse exon II [39]; the ATG at this site meets the Kozak [41] consensus for translation initiation in all three species and represents a possible alternative site of translation initiation. In the region beginning at the start of exon II XNOC is 78% identical to HNOC. The short coding sequence of XNOC exon I (22 aa) aligns poorly with a longer amino terminal region in the other species. In addition to the high sequence similarity, Xenopus [36] and mouse [39] genes have a simple 3 exon structure with very similar boundaries of the second and third exons.

Previously, an unusual leucine zipper-like domain was identified in Xenopus nocturnin [36]. The third leucine in the Xenopus sequence is either a tyrosine (mouse and rat) or a phenylalanine (human, cow and chicken) in other vertebrates (see bar in Fig. 1). Furthermore, the alanine in position 4 of the second heptad is replaced by a proline in all five species. The latter proline is adjacent to a conserved proline identified earlier in XNOC (Fig. 1). These proline residues are not compatible with the coiled-coil structure charateristic of leucine zipper motifs [42]. However, conservation of this region of the protein suggests that it is functionally important, perhaps serving as a protein interaction domain as is the case for a leucine rich-region in CCR4 [32,37,43]

The principal goal of this study was to analyze mNoc mRNA expression. We used C3H/He mice because they are useful for studying clock activity based on rhythmic release of melatonin; C3H/He is one of the few mouse strains that synthesizes melatonin rhythmically [29,44]. In Northern analysis using single stranded probes generated from the mNoc 3' UTR or from exon II, we found that in contrast to our prior work in Xenopus, mNoc is expressed as a single mRNA of about 3 kb (Fig. 2). The only variations from this pattern was diffuse hybridization of the probe above the 3 kb position when gels contained higher levels of mNoc mRNA (Fig. 2, liver and kidney; Fig. 3, 4 and 5 at ZT 12) and diffuse hybridization below the 3 kb band specifically in spleen (Figs. 2, 5C); the latter may reflect RNA degradation.

mNoc is expressed (Fig. 2) in retina, brain, heart, liver, lung, kidney, ovary, skeletal muscle (data not shown), pineal gland (data not shown), testis and thymus. It appears to be expressed at the highest levels in liver, then testis, kidney and retina. Lung has the lowest expression level of those tested. In addition, mNoc mRNA is expressed at early embryonic stages (Fig. 2).

Northern analysis of retinal RNA shows that, as was the case in Xenopus, mNoc exhibits a rhythm of mRNA abundance. Peak expression occurs at the time of light offset (Fig. 3A). However, the amplitude of the rhythm is approximately 2 fold compared to the greater than 10 fold amplitude seen in Xenopus retina. Rhythmicity with a similar peak at ZT12 is also seen in heart (Fig. 3B), spleen (Fig. 3C), kidney (Fig. 3D), and liver (Fig. 4). The amplitudes of the rhythms in heart, spleen and kidney, as determined by phosphor imaging, reflect 2 to 5 fold changes between minimum and maximum. In contrast, the magnitude of the day-night difference in liver represents a nearly 30 fold change (see Fig. 4). Although the overall pattern of rhythmicity is similar in these tissues, baseline expression during the day is evident in retina, heart, and spleen and in part accounts for the lower amplitude in these tissues.

In order to investigate the endogenous rhythmicity of mNoc expression, C3H/He mice were maintained in constant darkness for 36 hours before sampling for rhythmic changes in DD. Samples were then taken in darkness at 6 time points referenced to the normal LD cycle in which they had been maintained (referred to as Zeitgeber Time). mNoc from all five tissues shows rhythmic changes in mRNA abundance (Figs. 4 and 5). Furthermore, liver tissue exhibits a high amplitude rhythm with virtually no mRNA detectable in the day-time as was the case in LD (Fig. 4B). The other four tissues all exhibit a higher level of daytime expression than in LD (Fig. 5). Unlike other tissues, spleen RNA exhibits a diffuse zone of hybridization centered at 1.0-1.2 kb, which may reflect RNA degradation.

mNoc mRNA appeared to reach higher levels at ZT12 in DD than LD in all tissues except retina (compare Figs 3,4,5), suggesting that light may suppress mNoc mRNA level. This was particularly striking in liver where the ratio of mNoc/β-actin as determined by phosphorimaging was greater at ZT12 in DD than in LD (see Fig. 4D). This difference appears to be significant in that it was reproducible in an independent replication of the experiment in which LD and DD samples were analyzed on the same blot.

Laboratory strains of mice are heterogeneous in the presence of a transposable intracisternal A-particle (IAP) element in the nocturnin/CCR4 gene. During the course of our work on the mNoc cDNA it was reported [38,39] that a transposable IAP of viral origin, present in about 1000 copies throughout the mouse genome, is found in the first intron of the mNoc/CCR4 gene. In DBA/2, BALB/c, C57Bl/6 and C57Bl/10 mice, transcriptional read through from the IAP insert to the mNoc/CCR4 open reading frame resulted in hybrid transcripts (3, 6 and 10 kb) whose abundance increased in aging mice. Apparently, insertion of the IAP element in the first intron occurred relatively recently because the insert was found to be lacking in some strains of mice [39]. We confirmed the lack of an IAP element in 129/SV and C3H/He (the strain used in the rhythmic analysis above) mice through a combination of genomic PCR and partial sequencing of a genomic clone. As shown in Figure 6, genomic PCR using primers from sites in the intron adjacent to the IAP position revealed only intronic sequence in C3H/He and 129/SV mice. This was further confirmed for 129/SV mice by the lack of an IAP element in genomic sequence from this region as well (data not shown). Although the IAP element is absent in these two strains of mice, we confirmed the presence of the IAP sequence by PCR in BALB/c (Fig. 6B) and C57/Bl6 (data not shown) mice as reported [39].

We tested the hypothesis that the IAP element in Intron I of BALB/c mice ([14,36] see Fig. 6) would disrupt rhythmic expression of mNoc. Six week old BALB/c mice were kept in our animal facilities for two weeks and liver and kidney tissues were obtained for Northern analysis at 6 hour intervals through a light-dark cycle. As shown in Figure 7, mNoc in BALB/c mouse liver and kidney clearly exhibits rhythmicity similar to that seen in C3H/He mice with a prominent mRNA band at approximately 3 kb. Less abundant larger bands are also seen above 4 kb (liver and kidney) and 8 kb (liver only). Although larger mRNA bands have been reported to reflect hybrid transcripts including components of the IAP element in aging BALB/c mice, it is unclear whether this would explain the larger bands in Figure 7. The bands in Figure 7 are smaller than the 6 and 10 kb bands reported in old mice [38,39] and follow a rhythmic pattern similar to that of the 3 kb band. The larger bands could reflect splicing intermediates that are seen only during the period of maximal transcription of mNoc. Although it is possible that altered transcription of mNoc as a consequence of an interaction between aging and the IAP insert may alter the rhythmic pattern of expression [39], our data clearly indicate that the mere presence of the IAP element in mice 8 weeks of age has little or no impact on rhythmic expression. Comparable results have been obtained using C57/Bl6 and CBA/J mice (data not shown).

As shown in Figure 2, mNoc mRNA is expressed in tissue from the mid-brain, which contains the hypothalamus including the suprachiasmatic nucleus. In several attempts at temporal Northern analysis in LD using samples excised from the midbrain of C3H/He and C57/Bl6 mice, we saw hints of low amplitude rhythmicity, but the results were variable and could have resulted from sampling error (data not shown). We, therefore, examined brain expression of mNoc mRNA further using in situ hybridization of tissue from C57/Bl6 mice fixed at ZT 4, ZT 12, and ZT 20. mNoc transcripts were detected (Fig. 8) in the suprachiasmatic nucleus (SCN), the ventral hypothalamic nucleus, arcuate nucleus (Arc), the piriform cortex (Pir), the hippocampus (Hip), the cerebellum, the subiculum, the internal granule layer of the olfactory bulbs and the pineal gland. Although we saw variability at different sample times (Table 1) in the intensity of the hybridization in several brain regions, the observations were of a qualitative nature and the magnitude of the changes was not great. Although the in situ hybridization data suggest low amplitude rhythmicity in these brain regions (including the SCN), we have not detected rhythmic expression in the brain comparable to the high amplitude rhythmicity detected in peripheral tissues.

C3H/He mice, wild type (+/+) at the rd locus, were originally obtained from Dr. Michael Menaker at University of Virginia and then maintained as a breeding colony in ventilated environmental compartments within a temperature-controlled animal facility (24 -25°C) on a 12 hour light:12 hour dark cycle (LD), except as noted. BALB/c, CBA/J and C57/Bl6 mice were purchased from Charles River Laboratories (Wilmington, MA) or Jackson Laboratories and maintained under similar conditions. Experimental protocols were approved by the Institutional Animal Care and Use Committee and follow all federal guidelines. Mice were sacrificed by cervical dislocation following exposure to carbon dioxide or an overdose halothane anesthesia. Tissues for RNA extraction in LD were collected at ZT0, ZT6, ZT12 and ZT18 in light (standard room fluorescent light) or dim red light (Kodak Wratten #2, filters). Those for constant dark (DD) experiments were collected in dim red light at ZT0, ZT4, ZT8, ZT12, ZT16 and ZT20 referenced to the LD cycle immediately before DD treatment. Bovine, chicken, rat (Sprague Dawley obtained from the ARC) and human retinal tissue (obtained from the Eye Research Institute at Medical College of Wisconsin) were immediately frozen on dry ice after dissection and stored at -80°C.

Total RNA was extracted by the TRIZOL reagent protocol (GIBCO/BRL, Rockville, MD), and then dissolved in DEPC-treated water before storage at -80°C. QIAamp Tissue Kits (Qiagen Inc., Santa Claita, CA,) were used for genomic DNA extraction from liver tissues according to the kit protocol.

Mouse nocturnin cDNA (mNoc) clones were obtained by screening a mouse BALB/c retinal cDNA library from ATCC (ATCC# 77448, Rockville, MD; reference [51]) using the standard protocol published in Sambrook, et al, 1989 [52] with probes from a human EST (T87026) purchased from Research Genetics, Incorporated (Huntsville, AL). mNoc clones were custom sequenced by Sequetech Corporation (Mountain View, CA). The human EST (T87026) and additional human clones obtained by PCR were sequenced manually using T7 Sequenase 2.0 DNA sequencing kit (Amersham, Piscataway, NJ) or using a BigDye terminator cycle sequencing kit on an ABI Prism 310 capillary sequencer (PE Applied Biosystems, Foster City, CA). MacVector software was used for sequence analysis in this study.

mNoc bacterial artifical chromosome (BAC) clones were obtained from Research Genetics, Incorporated (Huntsville, AL) by custom screening of a 129/SV BAC library with the "whole cell PCR" protocol. BAC DNA was isolated by a Qiagen Plasmid Maxi Kit and sequenced using the ABI Prism 310. For the genomic PCR in Figure 6 the forward primer F1 (5'-AGTGACTGTCCTTCCTCTGT-3) is located upstream (5') and the reverse primer, R1 (5'-AACACAGTGAGACGCTGTCT-3') is located downstream (3') of an intracisternal A-particle (IAP) element (reference [39]) identified in the nocturnin/CCR4 gene. The forward primer F2 (5'-TGATGTCCAGGGCGTCAATA-3') is located in the IAP element itself [39]. The sequences for F1 and R1 are based on sequences from the BAC clone characterized in Figure 6A while the sequence of F2 is from the IAP element (reference [39]). These three primers were used for genomic PCR with Taq DNA polymerase (Promega). All of the resulting PCR products were cloned into pCRII-TOPO (Invitrogen, Carlsbad, CA) and sequenced as above.

Single strand PCR probes for Northern hybridization representing 553 bp of 3' UTR or Exon II of mNoc were generated using a modification of the single-strand DNA protocol of Bednarczuk, et al. [53], including ³²P-dCTP (40 mCi/ml; NEN Life Science Products, Boston, MA) in the reaction mixture. The primers for the 3'UTR probe were 5'-AACCATGCAGGTACAGTC-3' (bp 1557-1575 of the mNoc cDNA, forward) and 5'-GTTTGGAAGAGGCTTCAAC-3' (bp 2128-2147, reverse); for the Exon II probe they were 5'-ACCAGTCGACTCTACAGTGC-3' (bp 355-374, forward) and 5'-GGCTGGAAGGTGTCAAAG-3' (bp741-759, reverse). Random primed probes were prepared using the Random Primers DNA Labeling Kit (GIBCO/BRL, Rockville, MD). Radioactive probes were purified through NucTrap gel filtration columns (Stratagene, La Jolla, CA).

Ten μg (or less as specified) of each RNA sample was separated on 1.0% formaldehyde-agarose gels using standard procedures [35]. Northern blot analysis was carried out according to QuikHyb hybridization solution protocol (Stratagene, La Jolla, CA). Nylon membranes were stripped by washing twice for 10 min in boiling 0.01X SSPE (0.18 M NaCl/10 mM phosphate, pH 7.4/1 mM EDTA, 0.5% SDS) and rehybridized with probes made from mouse β-actin cDNA [54]. Hybridization signals were quantitated using a Storm PhosporImager and ImageQuant software (Molecular Dynamics) using a previously described method [36].

Total RNA used as a template in 5'-RACE and RT-PCR was treated with RNase-free DNase I (Promega, Madison, WI) and subsequently phenol-chloroform extracted. RNasin Ribonuclease Inhibitor (Promega) was used in both 5'-RACE and RT-PCR reactions. 5'-RACE was performed according to kit protocol (GIBCO/BRL, Rockville, MD). The reverse Transcription System (Promega) coupled with Taq DNA Polymerase (Promega) was used for RT-PCR.

Degenerative PCR was carried out using Taq DNA Polymerase (Promega) with 5'-GATGGGAAAC(A/G)GCACCAG(C/T)(A/C)GAC-3' and 5'-GC(G/C)AG(A/G)ATGTTCCACTGCAT(G/C)AC-3' as forward and reverse primers respectively. The resulting PCR products were cloned into pCRII-TOPO and sequenced with an ABI Prism 310 sequencer.

C57/Bl6 mice were decapitated following an overdose of halothane anesthesia. The brain was removed, frozen on dry ice and stored at -80° until sectioning. In situ hybridization followed the protocol of Fukuhara, et al. [55]. {α-³⁵S} UTP (1250 Ci/mmol; NEN Life Science Products, Boston, MA) labeled probes were obtained by in vitro transcription. A mouse nocturnin cDNA fragment (450 bp) cloned into the pBluescript KS (+) vector (Stratagene) was linearized with XhoI or XbaI for antisense or sense probes, and radiolabeled using T7 or T3 RNA polymerase respectively. Serial coronal cryostat sections (20 μm thick) were hybridized overnight at 55°C and washed at 57°C. Slides were exposed to Kodak Biomax film for 6 days at room temperature.

Sequences completed for this work have been placed in GenBank. Newly assigned GenBank Accession numbers for these sequences are AF199491 (mNoc), AF199492 (hNoc, genomic fragment), AF199493 (hNoc, RT-PCR product), AF199494 (hNoc, EST T87026), AF199495 (rNoc, 5' RACE product), AF199496 (rNoc, RT-PCR product), AF199497 (bNoc) and AF199498 (cNoc).