- Email: [email protected]
Molecular cloning of serotonin N-acetyltransferase gene from the mouse and its daily expression in the retina
Neuroscience Letters 250 (1998) 181–184
Molecular cloning of serotonin N-acetyltransferase gene from the mouse and its daily expression in the retina Katsuhiko Sakamoto, Norio Ishida* National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, MITI, Higashi 1-1, Tsukuba, Ibaraki 305-8566, Japan Received 25 May 1998; accepted 2 June 1998
Abstract The primary structure of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT: the rate-limiting enzyme in melatonin synthesis) in the mouse retina was deduced from the cDNA nucleotide sequence. The deduced protein consisted of 205 amino-acid residues with sequences highly conserved in AA-NATs of vertebrates, and was 96% identical to rat AA-NAT. Northern blot analysis of mouse retinal mRNA showed two obvious bands, of 1.5 kb and 4.5 kb in length. The levels of both transcripts were low at day and high at night, but the night-to-day ratios were ,2. These findings suggest that the expression mechanism of AA-NAT transcripts in the mouse retina may be different from those in other mammals, where a single transcript of AA-NAT is normally observed in Northern blots. 1998 Elsevier Science Ireland Ltd. All rights reserved
Keywords: Circadian rhythm; Arylalkylamine N-acetyltransferase; Melatonin; Retina; Mammals; Mouse
Circadian melatonin production in the retina, as in the pineal gland, has been observed in several vertebrates [5,18,19]. In mammals, pineal melatonin is considered to play an important role in circadian activity and seasonal changes in physiology [1,7], while melatonin produced in the retina seems to act as a local modulator in regulating various retinal functions [10,14], as in lower vertebrates [3,6,20]. Melatonin is made from serotonin, and rhythmic melatonin production is due primarily to changes in activity of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT: the rate-limiting enzyme in melatonin synthesis; [9]). AA-NAT cDNA has been isolated from several animals [4,8,13]. In the pineal gland of vertebrates, the relationships between the rhythm of the mRNA levels and the activity of AA-NAT were different from species to species [13]: for example, in the sheep, changes in the mRNA levels were much smaller than changes in AANAT activity [8], while in the rat, transcriptional events play an important role in regulating the AA-NAT activity * Corresponding author. Tel.: +81 298 546500; fax: +81 298 546095; e-mail: [email protected]
rhythm [15]. There are fewer studies on AA-NAT mRNA expression in the retina than in the pineal [2,15,16]. Yet, to our knowledge, the mouse AA-NAT cDNA sequence has not been published. The cloning of the mouse AA-NAT cDNA can be the first step to produce AA-NAT knockout mice. In this study, to investigate the molecular mechanism of melatonin production in the mammalian retina, the nucleotide sequence of AA-NAT cDNA from the mouse retina was determined to deduce the primary structure of the protein. We carried out polymerase chain reaction (PCR) with primers specific for rat AA-NAT cDNA using mouse retinal cDNA as a template, and obtained a mouse AA-NAT cDNA fragment. We also examined the expression of AA-NAT mRNA in the mouse retina under a daily light–dark cycle by Northern blot analysis. Adult male mice (BALB/cA Jcl; 10 weeks old; 25–30 g) were purchased from Clea Japan (Tokyo, Japan) and were housed in a 12 h light–12 h dark cycle (LD 12:12; lights on at zeitgeber time (ZT) 0) for at least 1 week before the experiment. A white fluorescent lamp was used as a source of light during the day (150–200 lux at the level of the cages).
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00462- 5
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K. Sakamoto, N. Ishida / Neuroscience Letters 250 (1998) 181–184
Twenty individuals were used at each time-point. Animals were decapitated, and retinas were dissected, quickfrozen and kept in liquid nitrogen until RNA extraction. In darkness, dissections were carried out under dim red light. Total RNA was isolated from the tissues by using ISOGEN (a guanidine HCl/phenol procedure; Nippon Gene, Tokyo, Japan). Messenger RNA was purified from the total RNA by using QuickPrep Micro mRNA Purification Kit (Pharmacia Biotech, USA). PCR was carried out to amplify a mouse AA-NAT cDNA fragment. We made oligonucleotide primers specific for rat AA-NAT cDNA (GenBank accession number U18913): RN-F (forward) 5′-GGGAGGGGTCAGTGGCCAGA-3′ and RN-R (reverse) 5′-CCATGGCCAAGTGCGCAGCTTG-3′; corresponding to nucleotides 178–97 and 823–44, respectively. These two primers enclosed the full length of the coding region of the rat AA-NAT cDNA. First strand cDNA was synthesized from total retinal RNA of mice sacrificed during the day, using the SuperScriptTM Preamplification System (GIBCO, Life Technologies, MD). In each PCR, a mixture containing 40 pM of each primer, 0.2 mM each of dNTP, 1 unit of Taq DNA polymerase (AmpliTaq DNA polymerase, Perkin Elmer, CT), 10× PCR buffer and water, was added to the template cDNA solution to give a final volume of 50 ml. For template DNA, we used one-twentieth of the cDNA which was made from 2 mg of the total RNA. To amplify DNA, we performed thermal cycling for 50 cycles of denaturation (94°C, 30 s), annealing (60°C, 30 s) and extension (72°C, 2 min). PCR products were cloned into a pCR2.1 vector using a TA Cloning Kit (Invitrogen, CA) for sequencing. For the amplified cDNA fragment, eight clones were isolated from a plural independent PCR reaction and were sequenced on the forward and reverse strands to confirm the nucleotide sequence of the cDNA. Messenger RNA was separated on a 1% agarose/0.7 M formaldehyde gel. Each lane contained 4 mg retinal mRNA from a pool of 40 retinas at each time-point. RNA was transferred to a nylon membrane (GeneScreen Plus, Du Pont, USA) by passive capillary transfer and probed with a 32P-labeled random primed probe. The cDNA probe was generated from the mouse AA-NAT cDNA fragment (667 bp) obtained in this study. Probes were hybridized to blots at 55°C, and the final wash was carried out at 55°C in 0.1× SSPE/1% SDS for 40 min (1× SSPE: 150 mM NaCl, 8.65 mM NaH2PO4, 1.25 mM EDTA, pH 7.4). Hybridized blots were imaged and analyzed by using BAS 2000 (Fuji Photo Film, Tokyo, Japan). Samples were normalized by determination of the amount of glyceraldehyde-3-phosphate dehydrogenase (rat GAPDH; GenBank accession number M17701) mRNA. After detection of AA-NAT mRNA, the blots were also probed with a 443-bp 32P-labeled probe generated from a rat GAPDH cDNA fragment (bases 133–575). To obtain a cDNA encoding AA-NAT of the mouse, we carried out PCR with primers specific for rat AA-NAT cDNA using mouse retinal cDNA as a template. The two
primers used enclosed the full length of the coding region of the rat AA-NAT cDNA. A 667-bp cDNA fragment was amplified. Fig. 1 shows the nucleotide and deduced amino-acid sequence. The cDNA sequence revealed an open reading frame of 618 bp starting from the first ATG and encoding 205 amino-acid residues with a calculated molecular mass of 23 kDa and an isoelectric point of 7.15. The deduced amino-acid sequence of the amplified cDNA was 96% identical to rat AA-NAT (GenBank accession number, U38306; Fig. 2), and shared 72–84% identity with avian and other mammalian AA-NATs (chick, ovine, bovine and human; GenBank accession numbers U46502, AD000742, U29663 and U40347, respectively). Like other AA-NATs, the protein had appropriate conserved aminoacid residues (Fig. 2, [13]). Putative motifs for acetyl coenzyme A binding (motifs A and B, [17]) were located between residues 119–138 and 163–175, respectively. Putative phosphorylation sites occurred in the protein: two sites for cyclic nucleotide-dependent protein kinase at Thr29 and Ser203, three sites for protein kinase C at Ser6, Thr127 and Ser192, and four sites for casein kinase II at Thr39, Ser45, Ser95 and Thr185. The five cysteine residues of which one or more have been thought to form structurally-important disulfide bonds in AA-NATs of vertebrates [13] had equivalents at positions 37, 61, 75, 158 and 177. On the basis of the similarities with AA-NATs of other animals, we concluded this protein is mouse AA-NAT. Northern blot analysis of mouse retinal mRNA showed two obvious bands, of 1.5 kb and 4.5 kb in length (Fig. 3). Normally a single transcript of AA-NAT is observed in Northern blots of mammals [13], although in the blots of night pineal mRNA of the rat there occurs an additional faint band of about 3 kb which might represent immature
Fig. 1. Nucleotide (upper) and deduced amino-acid (lower) sequence of mouse AA-NAT.
K. Sakamoto, N. Ishida / Neuroscience Letters 250 (1998) 181–184
183
Fig. 2. Comparison of the amino-acid sequences of AA-NAT form the mouse and the rat. Dots indicate sequence identity, and only where the two sequences differ is the amino acid indicated. The conserved potential motifs for acetyl coenzyme A binding (motifs A and B) are underlined. Putative phosphorylation sites in the mouse AA-NAT are shaded. pka, cyclic nucleotide-dependent protein kinase; pkc, protein kinase C; ck2, casein kinase II. Asterisks indicate cysteine residues of which one or more have been thought to form structurally-important disulfide bonds.
AA-NAT mRNA [15]. However, two transcripts of AANAT were clearly observed in the mouse retina both at day and at night. An additional faint band of 3 kb was also found. The ratios of the 4.5-kb band value to the 1.5kb band value were 0.8 at day and 1.0 at night. Multiple bands have been reported only in some fish [13]. The expression mechanism of AA-NAT mRNA in the mouse might differ from those in other mammals. In this study we could obtain only a single type of mouse AA-NAT cDNA by PCR. To characterize the mechanism of mouse AA-NAT expression, we require further experiments to isolate the two different transcripts of AA-NAT. In the mouse retina the levels of both transcripts of 1.5 kb and 4.5 kb were low at day (ZT 6) and high at night (ZT 18), but the night-to-day ratios were very low: 1.2 and 1.5, respectively (Fig. 3). Though we examined only at two points under a daily light–dark cycle and did not know in
detail the expression pattern of AA-NAT mRNA, the nightto-day ratios in the mouse retina seemed to be much lower than those observed in the rat retina: 10 in the Sprague– Dawley rat [15] and 3 in the Wistar rat [16]. The pineal glands of some strains of mice make melatonin, and show an overt melatonin-production rhythm, whereas other mice, including the BALB/c in this experiment, do not make pineal melatonin [12], probably because of a genetic defect in the activity of AA-NAT and/or hydroxyindole-O-methyltransferase [11]. Mice which show an overt rhythm in pineal melatonin may have a significant daily change in the retinal AA-NAT mRNA. The mouse is a good model in which to study the mechanism of AA-NAT mRNA expression in mammals. There is currently considerable interests in the question of whether the mammalian retina contains an endogenous clock that regulates rhythmic melatonin synthesis [15,16,19]. Comparisons of AA-NAT mRNA expression of several mouse strains may give us information about the molecular mechanism of circadian rhythm in melatonin production. We thank Dr. Philip Rodley for proofreading of the manuscript, and Mr. Kenta Sugimoto and Dr. Hitoshi Miyazaki for continuing support.
Fig. 3. Daily change of AA-NAT mRNA in retina. Mice were housed in LD 12:12 (lights on at ZT 0). Representative Northern blot analyses of AA-NAT and GAPDH mRNAs at day (ZT 6) and night (ZT 18) are shown here. (b) Quantification of AA-NAT mRNA levels from the Northern blots. The values shown in this figure are the averages of two values at each time-point from two independent experiments. The day value of the 1.5-kb band was expressed as 100%.
[1] Arendt, J., Melatonin and the Mammalian Pineal Gland, Chapman and Hall, London, 1995. [2] Bernard, M., Iuvone, P.M., Cassone, V.M., Roseboom, P.H., Coon, S.L. and Klein, D.C., Avian melatonin synthesis: photic and circadian regulation of serotonin N-acetyltransferase mRNA in the chicken pineal gland and retina, J. Neurochem., 68 (1997) 213–224. [3] Besharse, J.C. and Dunis, D.A., Methoxyindoles and photoreceptor metabolism: activation of rod shedding, Science, 219 (1983) 1341–1343. [4] Borjigin, J., Wang, M.M. and Snyder, S.H., Diurnal variation in mRNA encoding serotonin N-acetyltransferase in pineal gland, Nature, 378 (1995) 783–785. [5] Cahill, G.M. and Besharse, J.C., Circadian clock functions loca-
184
[6]
[7] [8]
[9]
[10] [11]
[12]
[13]
K. Sakamoto, N. Ishida / Neuroscience Letters 250 (1998) 181–184 lized in Xenopus retinal photoreceptors, Neuron, 10 (1993) 573–577. Cahill, G.M. and Besharse, J.C., Circadian rhythmicity in vertebrate retinas: regulation by a photoreceptor oscillator, Prog. Retin. Eye Res., 14 (1995) 267–291. Cassone, V.M., Effects of melatonin on vertebrate circadian systems, Trends Neurosci., 13 (1990) 457–464. Coon, S.L., Roseboom, P.H., Baler, R., Weller, J.L., Namboodiri, M.A.A., Koonin, E.V. and Klein, D.C., Pineal serotonin N-acetyltransferase: expression cloning and molecular analysis, Science, 270 (1995) 1681–1683. Deguchi, T., Circadian rhythms of indoleamines and serotonin N-acetyltransferase activity in the pineal gland, Mol. Cell. Biochem., 27 (1979) 57–66. Dubocovich, M.L., Melatonin is a potent modulator of dopamine release in the retina, Nature, 306 (1983) 782–784. Ebihara, S., Marks, T., Hudson, D.J. and Menaker, M., Genetic control of melatonin synthesis in the pineal gland of the mouse, Science, 231 (1986) 491–493. Goto, M., Oshima, I., Tomita, T. and Ebihara, S., Melatonin content of the pineal gland in different mouse strains, J. Pineal Res., 7 (1989) 195–204. Klein, D.C., Coon, S.L., Roseboom, P.H., Weller, J.L., Bernard, M., Gastel, J.A., Zatz, M., Iuvone, P.M., Rodriguez, I.R., Be´gay, V., Falc-n, J., Cahill, G.M., Cassone, V.M. and Baler, R., The melatonin rhythm-generating enzyme: molecular regulation of serotonin N-acetyltransferase in the pineal gland, Recent Prog. Horm. Res., 52 (1997) 307–358.
[14] Nowak, J.Z., Zurawska, E. and Zawilska, J., Melatonin and its generating system in vertebrate retina: circadian rhythm, effect of environmental lighting and interaction with dopamine, Neurochem. Int., 14 (1989) 397–406. [15] Roseboom, P.H., Coon, S.L., Baler, R., McCune, S.K., Weller, J.L. and Klein, D.C., Melatonin synthesis: analysis of the more than 150-fold nocturnal increase in serotonin N-acetyltransferase messenger ribonucleic acid in the rat pineal gland, Endocrinology, 137 (1996) 3033–3044. [16] Sakamoto, K. and Ishida, N., Circadian expression of serotonin N-acetyltransferase mRNA in the rat retina, Neurosci. Lett., 245 (1998) 113–116. [17] Tercero, J.C., Riles, L.E. and Wickner, R.B., Localized mutagenesis and evidence for post-transcriptional regulation of MAK3. A putative N-acetyltransferase required for doublestranded RNA virus propagation in Saccharomyces cerevisiae, J. Biol. Chem., 267 (1992) 20270–20276. [18] Thomas, K.B., Tigges, M. and Iuvone, P.M., Melatonin synthesis and circadian tryptophan hydroxylase activity in chicken retina following destruction of serotonin immunoreactive amacrine and bipolar cells by kainic acid, Brain Res., 601 (1993) 303–307. [19] Tosini, G. and Menaker, M., Circadian rhythms in cultured mammalian retina, Science, 272 (1996) 419–421. [20] Zawilska, J.B. and Nowak, J.Z., Regulatory mechanisms in melatonin biosynthesis in retina, Neurochem. Int., 20 (1992) 23–36.
Molecular cloning of serotonin N-acetyltransferase gene from the mouse and its daily expression in the retina Katsuhiko Sakamoto, Norio Ishida* National Institute of Bioscience and Human Technology, Agency of Industrial Science and Technology, MITI, Higashi 1-1, Tsukuba, Ibaraki 305-8566, Japan Received 25 May 1998; accepted 2 June 1998
Abstract The primary structure of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT: the rate-limiting enzyme in melatonin synthesis) in the mouse retina was deduced from the cDNA nucleotide sequence. The deduced protein consisted of 205 amino-acid residues with sequences highly conserved in AA-NATs of vertebrates, and was 96% identical to rat AA-NAT. Northern blot analysis of mouse retinal mRNA showed two obvious bands, of 1.5 kb and 4.5 kb in length. The levels of both transcripts were low at day and high at night, but the night-to-day ratios were ,2. These findings suggest that the expression mechanism of AA-NAT transcripts in the mouse retina may be different from those in other mammals, where a single transcript of AA-NAT is normally observed in Northern blots. 1998 Elsevier Science Ireland Ltd. All rights reserved
Keywords: Circadian rhythm; Arylalkylamine N-acetyltransferase; Melatonin; Retina; Mammals; Mouse
Circadian melatonin production in the retina, as in the pineal gland, has been observed in several vertebrates [5,18,19]. In mammals, pineal melatonin is considered to play an important role in circadian activity and seasonal changes in physiology [1,7], while melatonin produced in the retina seems to act as a local modulator in regulating various retinal functions [10,14], as in lower vertebrates [3,6,20]. Melatonin is made from serotonin, and rhythmic melatonin production is due primarily to changes in activity of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT: the rate-limiting enzyme in melatonin synthesis; [9]). AA-NAT cDNA has been isolated from several animals [4,8,13]. In the pineal gland of vertebrates, the relationships between the rhythm of the mRNA levels and the activity of AA-NAT were different from species to species [13]: for example, in the sheep, changes in the mRNA levels were much smaller than changes in AANAT activity [8], while in the rat, transcriptional events play an important role in regulating the AA-NAT activity * Corresponding author. Tel.: +81 298 546500; fax: +81 298 546095; e-mail: [email protected]
rhythm [15]. There are fewer studies on AA-NAT mRNA expression in the retina than in the pineal [2,15,16]. Yet, to our knowledge, the mouse AA-NAT cDNA sequence has not been published. The cloning of the mouse AA-NAT cDNA can be the first step to produce AA-NAT knockout mice. In this study, to investigate the molecular mechanism of melatonin production in the mammalian retina, the nucleotide sequence of AA-NAT cDNA from the mouse retina was determined to deduce the primary structure of the protein. We carried out polymerase chain reaction (PCR) with primers specific for rat AA-NAT cDNA using mouse retinal cDNA as a template, and obtained a mouse AA-NAT cDNA fragment. We also examined the expression of AA-NAT mRNA in the mouse retina under a daily light–dark cycle by Northern blot analysis. Adult male mice (BALB/cA Jcl; 10 weeks old; 25–30 g) were purchased from Clea Japan (Tokyo, Japan) and were housed in a 12 h light–12 h dark cycle (LD 12:12; lights on at zeitgeber time (ZT) 0) for at least 1 week before the experiment. A white fluorescent lamp was used as a source of light during the day (150–200 lux at the level of the cages).
0304-3940/98/$19.00 1998 Elsevier Science Ireland Ltd. All rights reserved PII S0304- 3940(98) 00462- 5
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K. Sakamoto, N. Ishida / Neuroscience Letters 250 (1998) 181–184
Twenty individuals were used at each time-point. Animals were decapitated, and retinas were dissected, quickfrozen and kept in liquid nitrogen until RNA extraction. In darkness, dissections were carried out under dim red light. Total RNA was isolated from the tissues by using ISOGEN (a guanidine HCl/phenol procedure; Nippon Gene, Tokyo, Japan). Messenger RNA was purified from the total RNA by using QuickPrep Micro mRNA Purification Kit (Pharmacia Biotech, USA). PCR was carried out to amplify a mouse AA-NAT cDNA fragment. We made oligonucleotide primers specific for rat AA-NAT cDNA (GenBank accession number U18913): RN-F (forward) 5′-GGGAGGGGTCAGTGGCCAGA-3′ and RN-R (reverse) 5′-CCATGGCCAAGTGCGCAGCTTG-3′; corresponding to nucleotides 178–97 and 823–44, respectively. These two primers enclosed the full length of the coding region of the rat AA-NAT cDNA. First strand cDNA was synthesized from total retinal RNA of mice sacrificed during the day, using the SuperScriptTM Preamplification System (GIBCO, Life Technologies, MD). In each PCR, a mixture containing 40 pM of each primer, 0.2 mM each of dNTP, 1 unit of Taq DNA polymerase (AmpliTaq DNA polymerase, Perkin Elmer, CT), 10× PCR buffer and water, was added to the template cDNA solution to give a final volume of 50 ml. For template DNA, we used one-twentieth of the cDNA which was made from 2 mg of the total RNA. To amplify DNA, we performed thermal cycling for 50 cycles of denaturation (94°C, 30 s), annealing (60°C, 30 s) and extension (72°C, 2 min). PCR products were cloned into a pCR2.1 vector using a TA Cloning Kit (Invitrogen, CA) for sequencing. For the amplified cDNA fragment, eight clones were isolated from a plural independent PCR reaction and were sequenced on the forward and reverse strands to confirm the nucleotide sequence of the cDNA. Messenger RNA was separated on a 1% agarose/0.7 M formaldehyde gel. Each lane contained 4 mg retinal mRNA from a pool of 40 retinas at each time-point. RNA was transferred to a nylon membrane (GeneScreen Plus, Du Pont, USA) by passive capillary transfer and probed with a 32P-labeled random primed probe. The cDNA probe was generated from the mouse AA-NAT cDNA fragment (667 bp) obtained in this study. Probes were hybridized to blots at 55°C, and the final wash was carried out at 55°C in 0.1× SSPE/1% SDS for 40 min (1× SSPE: 150 mM NaCl, 8.65 mM NaH2PO4, 1.25 mM EDTA, pH 7.4). Hybridized blots were imaged and analyzed by using BAS 2000 (Fuji Photo Film, Tokyo, Japan). Samples were normalized by determination of the amount of glyceraldehyde-3-phosphate dehydrogenase (rat GAPDH; GenBank accession number M17701) mRNA. After detection of AA-NAT mRNA, the blots were also probed with a 443-bp 32P-labeled probe generated from a rat GAPDH cDNA fragment (bases 133–575). To obtain a cDNA encoding AA-NAT of the mouse, we carried out PCR with primers specific for rat AA-NAT cDNA using mouse retinal cDNA as a template. The two
primers used enclosed the full length of the coding region of the rat AA-NAT cDNA. A 667-bp cDNA fragment was amplified. Fig. 1 shows the nucleotide and deduced amino-acid sequence. The cDNA sequence revealed an open reading frame of 618 bp starting from the first ATG and encoding 205 amino-acid residues with a calculated molecular mass of 23 kDa and an isoelectric point of 7.15. The deduced amino-acid sequence of the amplified cDNA was 96% identical to rat AA-NAT (GenBank accession number, U38306; Fig. 2), and shared 72–84% identity with avian and other mammalian AA-NATs (chick, ovine, bovine and human; GenBank accession numbers U46502, AD000742, U29663 and U40347, respectively). Like other AA-NATs, the protein had appropriate conserved aminoacid residues (Fig. 2, [13]). Putative motifs for acetyl coenzyme A binding (motifs A and B, [17]) were located between residues 119–138 and 163–175, respectively. Putative phosphorylation sites occurred in the protein: two sites for cyclic nucleotide-dependent protein kinase at Thr29 and Ser203, three sites for protein kinase C at Ser6, Thr127 and Ser192, and four sites for casein kinase II at Thr39, Ser45, Ser95 and Thr185. The five cysteine residues of which one or more have been thought to form structurally-important disulfide bonds in AA-NATs of vertebrates [13] had equivalents at positions 37, 61, 75, 158 and 177. On the basis of the similarities with AA-NATs of other animals, we concluded this protein is mouse AA-NAT. Northern blot analysis of mouse retinal mRNA showed two obvious bands, of 1.5 kb and 4.5 kb in length (Fig. 3). Normally a single transcript of AA-NAT is observed in Northern blots of mammals [13], although in the blots of night pineal mRNA of the rat there occurs an additional faint band of about 3 kb which might represent immature
Fig. 1. Nucleotide (upper) and deduced amino-acid (lower) sequence of mouse AA-NAT.
K. Sakamoto, N. Ishida / Neuroscience Letters 250 (1998) 181–184
183
Fig. 2. Comparison of the amino-acid sequences of AA-NAT form the mouse and the rat. Dots indicate sequence identity, and only where the two sequences differ is the amino acid indicated. The conserved potential motifs for acetyl coenzyme A binding (motifs A and B) are underlined. Putative phosphorylation sites in the mouse AA-NAT are shaded. pka, cyclic nucleotide-dependent protein kinase; pkc, protein kinase C; ck2, casein kinase II. Asterisks indicate cysteine residues of which one or more have been thought to form structurally-important disulfide bonds.
AA-NAT mRNA [15]. However, two transcripts of AANAT were clearly observed in the mouse retina both at day and at night. An additional faint band of 3 kb was also found. The ratios of the 4.5-kb band value to the 1.5kb band value were 0.8 at day and 1.0 at night. Multiple bands have been reported only in some fish [13]. The expression mechanism of AA-NAT mRNA in the mouse might differ from those in other mammals. In this study we could obtain only a single type of mouse AA-NAT cDNA by PCR. To characterize the mechanism of mouse AA-NAT expression, we require further experiments to isolate the two different transcripts of AA-NAT. In the mouse retina the levels of both transcripts of 1.5 kb and 4.5 kb were low at day (ZT 6) and high at night (ZT 18), but the night-to-day ratios were very low: 1.2 and 1.5, respectively (Fig. 3). Though we examined only at two points under a daily light–dark cycle and did not know in
detail the expression pattern of AA-NAT mRNA, the nightto-day ratios in the mouse retina seemed to be much lower than those observed in the rat retina: 10 in the Sprague– Dawley rat [15] and 3 in the Wistar rat [16]. The pineal glands of some strains of mice make melatonin, and show an overt melatonin-production rhythm, whereas other mice, including the BALB/c in this experiment, do not make pineal melatonin [12], probably because of a genetic defect in the activity of AA-NAT and/or hydroxyindole-O-methyltransferase [11]. Mice which show an overt rhythm in pineal melatonin may have a significant daily change in the retinal AA-NAT mRNA. The mouse is a good model in which to study the mechanism of AA-NAT mRNA expression in mammals. There is currently considerable interests in the question of whether the mammalian retina contains an endogenous clock that regulates rhythmic melatonin synthesis [15,16,19]. Comparisons of AA-NAT mRNA expression of several mouse strains may give us information about the molecular mechanism of circadian rhythm in melatonin production. We thank Dr. Philip Rodley for proofreading of the manuscript, and Mr. Kenta Sugimoto and Dr. Hitoshi Miyazaki for continuing support.
Fig. 3. Daily change of AA-NAT mRNA in retina. Mice were housed in LD 12:12 (lights on at ZT 0). Representative Northern blot analyses of AA-NAT and GAPDH mRNAs at day (ZT 6) and night (ZT 18) are shown here. (b) Quantification of AA-NAT mRNA levels from the Northern blots. The values shown in this figure are the averages of two values at each time-point from two independent experiments. The day value of the 1.5-kb band was expressed as 100%.
[1] Arendt, J., Melatonin and the Mammalian Pineal Gland, Chapman and Hall, London, 1995. [2] Bernard, M., Iuvone, P.M., Cassone, V.M., Roseboom, P.H., Coon, S.L. and Klein, D.C., Avian melatonin synthesis: photic and circadian regulation of serotonin N-acetyltransferase mRNA in the chicken pineal gland and retina, J. Neurochem., 68 (1997) 213–224. [3] Besharse, J.C. and Dunis, D.A., Methoxyindoles and photoreceptor metabolism: activation of rod shedding, Science, 219 (1983) 1341–1343. [4] Borjigin, J., Wang, M.M. and Snyder, S.H., Diurnal variation in mRNA encoding serotonin N-acetyltransferase in pineal gland, Nature, 378 (1995) 783–785. [5] Cahill, G.M. and Besharse, J.C., Circadian clock functions loca-
184
[6]
[7] [8]
[9]
[10] [11]
[12]
[13]
K. Sakamoto, N. Ishida / Neuroscience Letters 250 (1998) 181–184 lized in Xenopus retinal photoreceptors, Neuron, 10 (1993) 573–577. Cahill, G.M. and Besharse, J.C., Circadian rhythmicity in vertebrate retinas: regulation by a photoreceptor oscillator, Prog. Retin. Eye Res., 14 (1995) 267–291. Cassone, V.M., Effects of melatonin on vertebrate circadian systems, Trends Neurosci., 13 (1990) 457–464. Coon, S.L., Roseboom, P.H., Baler, R., Weller, J.L., Namboodiri, M.A.A., Koonin, E.V. and Klein, D.C., Pineal serotonin N-acetyltransferase: expression cloning and molecular analysis, Science, 270 (1995) 1681–1683. Deguchi, T., Circadian rhythms of indoleamines and serotonin N-acetyltransferase activity in the pineal gland, Mol. Cell. Biochem., 27 (1979) 57–66. Dubocovich, M.L., Melatonin is a potent modulator of dopamine release in the retina, Nature, 306 (1983) 782–784. Ebihara, S., Marks, T., Hudson, D.J. and Menaker, M., Genetic control of melatonin synthesis in the pineal gland of the mouse, Science, 231 (1986) 491–493. Goto, M., Oshima, I., Tomita, T. and Ebihara, S., Melatonin content of the pineal gland in different mouse strains, J. Pineal Res., 7 (1989) 195–204. Klein, D.C., Coon, S.L., Roseboom, P.H., Weller, J.L., Bernard, M., Gastel, J.A., Zatz, M., Iuvone, P.M., Rodriguez, I.R., Be´gay, V., Falc-n, J., Cahill, G.M., Cassone, V.M. and Baler, R., The melatonin rhythm-generating enzyme: molecular regulation of serotonin N-acetyltransferase in the pineal gland, Recent Prog. Horm. Res., 52 (1997) 307–358.
[14] Nowak, J.Z., Zurawska, E. and Zawilska, J., Melatonin and its generating system in vertebrate retina: circadian rhythm, effect of environmental lighting and interaction with dopamine, Neurochem. Int., 14 (1989) 397–406. [15] Roseboom, P.H., Coon, S.L., Baler, R., McCune, S.K., Weller, J.L. and Klein, D.C., Melatonin synthesis: analysis of the more than 150-fold nocturnal increase in serotonin N-acetyltransferase messenger ribonucleic acid in the rat pineal gland, Endocrinology, 137 (1996) 3033–3044. [16] Sakamoto, K. and Ishida, N., Circadian expression of serotonin N-acetyltransferase mRNA in the rat retina, Neurosci. Lett., 245 (1998) 113–116. [17] Tercero, J.C., Riles, L.E. and Wickner, R.B., Localized mutagenesis and evidence for post-transcriptional regulation of MAK3. A putative N-acetyltransferase required for doublestranded RNA virus propagation in Saccharomyces cerevisiae, J. Biol. Chem., 267 (1992) 20270–20276. [18] Thomas, K.B., Tigges, M. and Iuvone, P.M., Melatonin synthesis and circadian tryptophan hydroxylase activity in chicken retina following destruction of serotonin immunoreactive amacrine and bipolar cells by kainic acid, Brain Res., 601 (1993) 303–307. [19] Tosini, G. and Menaker, M., Circadian rhythms in cultured mammalian retina, Science, 272 (1996) 419–421. [20] Zawilska, J.B. and Nowak, J.Z., Regulatory mechanisms in melatonin biosynthesis in retina, Neurochem. Int., 20 (1992) 23–36.