Journal of Heredity 2003:94(1)
© 2003 The American Genetic Association 94:49-56
Isolation and Characterization of the Canine Serotonin Receptor 1A Gene (htr1A)
From the Department of Clinical Sciences of Companion Animals, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 8, 3584 CM Utrecht, the Netherlands.
Address correspondence to L. van den Berg or B. van Oost at their respective addresses above, or e-mail: L.vandenBerg{at}vet.uu.nl or b.vanOost{at}vet.uu.nl.
 |
Abstract |
|---|
 Top Abstract Materials and MethodsResultsDiscussionReferences |
|---|
Although the serotonergic system and htr1A have been studied extensively, little is known about the canine serotonin receptor 1A. We are interested in this receptor in the dog because it is likely to be involved in behavioral disorders such as anxiety. Therefore, we isolated a canine bacterial artificial chromosome (BAC) clone containing htr1A, and, with the help of this clone, the complete canine coding sequence of this gene was determined. Radiation hybrid (RH) mapping showed that htr1A is part of a conserved linkage group also including the survival of motor neuron 1 (smn1) gene. Htr1A is estimated to be located about 7.3 Mb from smn1 on cfa02. In addition, we report a possible breed-specific variant of the gene in four golden retrievers.
Having survived more than 750 million years of evolution, serotonin (5-hydroxytryptamine, 5-HT) is expected to be of great importance in many living organisms (Peroutka 1995). 5-HT functions as a hormone, a mitogen, and a neurotransmitter. Its significance is not only evident from its evolutionary age but also demonstrated by the fact that it is used by one of the most extensive signaling systems found in the brain: from the raphe nuclei of the brain stem, serotonergic neurons project widely throughout the central nervous system, and 14 receptors have already been identified that mediate its biological effects (Hoyer et al. 1994). There has always been much interest in the serotonergic system because it plays a role in central nervous system processes regulating fear, anxiety, aggression, control of sleep, and modulation of feeding behaviors (Gingrich and Hen 2001).
One of the 14 serotonin receptors known to date is serotonin receptor 1A. Its activity is mediated by G-proteins that inhibit adenylate cyclase activity, and it can be found as an autoreceptor on serotonergic cell bodies and dendrites in the raphe nuclei, as well as on postsynaptic targets of serotonin release in a number of limbic structures (see Barnes and Sharp 1999 for a review). Stimulation of 5-HT1A autoreceptors inhibits cell firing and serotonin release, which enables the receptor to modulate the activity of the serotonergic system. (Corley et al. 1992; Jolas et al. 1995; Sprouse and Aghajanian 1987). Although results are conflicting (Olivier et al. 2001; Pattij 2002), the 5-HT1A receptor has often been associated with anxiety, depression, aggression, and stress response. In humans as well as animals, (partial) 5-HT1A receptor agonists have been reported to have anxiolytic effects (Cervo et al. 2000; DeVry 1995; File et al. 1996; Oshima 2001), and several strains of htr1A knockout mice show heightened anxiety, stress response, and an antidepressant-like phenotype. (Heisler et al. 1998; Parks et al. 1998; Ramboz et al. 1998).
Kobilka et al. (1987) first cloned and mapped the human gene encoding serotonin receptor 1A (htr1A). Currently, the complete coding sequence of htr1A is known for several organisms, including human (Homo sapiens), mouse (Mus musculus), rat (Rattus norvegicus), Caenorhabditis elegans, and Drosophila melanogaster. The htr1A gene is intronless, has seven predicted transmembrane-spanning domains, and contains sites for glycosylation and phosphorylation. It is localized on the human chromosome 5q11.2-q13.
This study on canine htr1A was performed in the context of a research project involving canine fear and aggression. Extreme levels of fear and aggression in dogs can result in biting incidents, with serious implications for the victim (obviously) and for the dog, because some owners consider euthanasia for their pets in such cases (Galac and Knol 1997). In order to reveal the etiology of these behavioral problems, we are studying parts of the canine serotonergic system. Although an enormous number of studies have been performed on the serotonergic system in general and the 5-HT1A receptor in particular, little is known about the canine serotonin receptor 1A. In this article we describe the isolation of a canine bacterial artificial chromosome (BAC) clone containing htr1A, the complete coding sequence of this gene, and analysis of its position on the physical map of the dog. In addition, we report a possible breed-specific variant of the gene in four golden retrievers.
|
Materials and Methods |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
DNA Isolation and Dogs
Human and golden retriever genomic DNA was isolated from whole blood lymphocytes by the salt extraction method of Miller et al. (1988). Great Dane genomic DNA was extracted from spleen.
The four golden retrievers used in this study were privately owned, unrelated dogs. The retrievers visited our clinic in the context of a research project involving canine fear and aggression, but their owners did not consider the dogs to be either aggressive or anxious.
BAC Library Screening
The human coding sequence of htr1A (GenBank accession no. X57829) was used to design primers 1 and 2 (Table 1). These primers amplified a 635 bp fragment in a PCR using 200 ng human genomic DNA, 0.5 µM primers, 0.2 mM dNTPs, 1.5 mM MgCl2, and 2.5 units Taq DNA polymerase in 1x Gibco-BRL buffer in a 100 µl reaction volume. This 635 bp probe (htr1A-635) was labeled with [
-32P] dATP with a megaprime DNA labeling kit (Amersham, Piscataway, NJ).
|
The canine genomic BAC library RPCI-81, derived from a Doberman pinscher (Li et al. 1999), was screened with probe htr1A-635 as previously described (van de Sluis et al. 1999). This resulted in only one positive colony: BAC 160O12. For further applications, the alkaline lysis method as described on the BacPac Web site (http://www.chori.org/bacpac/) was used for BAC DNA isolation.
Southern Blot
To confirm the identity of the BAC clone, genomic golden retriever DNA, genomic Great Dane DNA and BAC clone 160O12 DNA were digested with EcoRI. The resulting fragments were separated on a 0.7% agarose gel, transferred to Hydrobond N+ filter (Amersham) and hybridized at 65°C with the htr1A-635 probe.
Sequence Analysis of the Canine htr1A
The major part of the canine htr1A sequence was determined by sequencing subcloned BAC 160O12 fragments. BAC 160O12 DNA was digested with Sau3AI; the resulting fragments were ligated into Bam HI digested pZeROtm–1 vector with T4 DNA ligase, transformed into TOP10F' bacterial cells, and plated on low-salt LB plates with 50 µg/ml zeocin. Zeocin-resistent colonies were transferred to Hydrobond N+ filters that were hybridized with the htr1A-635 probe. Positive colonies were picked and grown overnight. DNA was isolated from these cultures by miniprepping with a Promega DNA purification system. The subcloned DNA was sequenced with T7 (5'-TAA TAC GAC TCA CTA TAG GG-3') and SP6 (5'-ATT TAG GTG ACA CTA TAG-3') primers using an ABI 310 Genetic Analyzer (Applied Biosystems, Foster City, CA) with BigDye Termination Mix.
The first 50 bp of the gene were sequenced by means of direct BAC DNA sequencing. Twenty pmol of a reverse primer (5'-GAG GTG ATC ACT TGG TAG CTG-3') was used in a 30 µl tercycle reaction with 4 mM MgCl2, 12 µl Big Dye Terminator Ready Reaction Mix, and 750 ng BAC DNA. The tercycle consisted of 5 min at 95°C, followed by 34 cycles of 30 s at 95°C, 10 s at 56°C, and 4 min at 60°C. Tercycle products were purified with multiscreen 96-well filtration plates (Millipore) and sequenced in 10 µl distilled water with an ABI 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA)
Three primer pairs (3 and 4, 5 and 6, 7 and 8; see Table 1) were designed based on the preliminary canine coding sequence. These primers amplified adjacent fragments in PCRs, using BAC clone 160O12 DNA as a template, and the PCR products were sequenced several times. The first and last 50 bp of the exon were checked by direct BAC DNA sequencing. In this way, every base in the preliminary sequence was checked at least once.
Homologies with human and murine sequences were calculated with SEQMAN and MEGALIGN (DNA Star Software). Sequences retrieved have the following GenBank accession numbers: X57829, AB041403, XM_003692, AF498978 (human), U39391, S67168, XM_122644, and NM_008308 (mouse). Predicted positions of the transmembrane regions in the protein were derived from the SWISSPROT Web site (http://srs6.ebi.ac.uk/srs6bin/cgi-bin/wgetz?-newId+-e+[libs%3d{SWALL_SP_REMTREMBL}-acc:P08908]).
Radiation Hybrid Mapping
The canine chromosome segments corresponding to HSA05 were studied in order to select two genes flanking htr1A in the human genome: cartilage linking protein 1 (crtl1) and survival of motor neuron 1, telomeric (smn1) (Figure 1). The commercially available canine whole genome radiation hybrid (RH) panel T72 (3000 RAD, Research Genetics, http://www.resgen.com/products/CRH.php3) was used to determine linkage between htr1A and both of these genes. Primers 9 and 4 were used to establish retention of htr1A in the hybrids, and primers 10 and 11 and 12 and 13 were used for crtl1 and smn1, respectively (Table 1).
|
PCR reactions contained 25 ng DNA, 0.53 µM primers, 0.2 mM dNTPs, 1.5 mM MgCl2, 0.6 units Platinum Taq Polymerase (Invitrogen), and 1x Gibco-BRL buffer in a total reaction volume of 15 µl. We used the following PCR program: 3 min 94°C initial denaturation, followed by 35 cycles of 30 s at 94°C, 30 s at TA, and 30 s at 72°C. The final step was 4 min at 72°C. The primers were first tested on genomic canine, hamster, and 2:1 hamster/canine DNA in order to confirm their specificity for dog DNA.
A two-point LOD score was computed for both htr1A/smn1 and htr1A/crtl1 with RH2PT software (RHMAP, version 3.0; Boehnke et al. 1996).
Partial Sequence Analysis of htr1A in Four Golden Retrievers
Analysis of htr1A sequences in genomic DNA of four golden retrievers was performed by means of PCR product sequencing, using primers 14 and 15 and 16 and 17. Reliable sequencing results were obtained for base pairs 115–1250 in each of these dogs. Note that there is a mismatch in primer 14: the eighth base is a T, where the corresponding position in the exon shows a G, but this apparently did not affect the specificity of the primer.
|
Results |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
BAC Library Screening and Southern Blot
BAC library screening resulted in one positive BAC clone: 160O12. Both this clone and genomic dog DNA showed a single 9.4 kb band in a Southern blot analysis using a 635 bp human probe (Figure 2). Because the htr1A exon contains no EcoRI sites, it was not considered necessary to repeat the Southern blotting procedure with a full-length probe. These results imply that we have isolated a genomic clone of canine htr1A and that the htr1A gene is single copy in the canine genome.
|
Sequence Analysis
With the help of the 160O12 BAC clone, we have managed to reveal the entire coding sequence of htr1A in the domestic dog, including 22 bp at the 5'-flanking side and 45 bp at the 3'-flanking side. The gene consists of only one exon, which is 1272 bp long (Figure 3). In dogs, the gene is longer than in humans (1269 bp) and mice (1266 bp). Homology of the exonic nucleotide sequence with human and murine sequences is high (89% and 85% sequence identity, respectively). The corresponding protein consists of 423 amino acids. Amino acid homology reached 92% with human and 85% with mouse.
|
Several regions in the protein were even more homologous between the species (Figure 4). The seven hydrophobic (transmembrane) regions consist of amino acid residues 37–62, 74–98, 110–132, 153–178, 192–217, 346–367 and 379–403 in humans, mice, and dogs (where the initiator ATG codon is designated number 1). Amino acid composition of these seven regions is 100% identical between dog and human. The murine amino acid composition is different at two positions (residues 177 and 379). At amino acid positions 147–151 and 227–232 two sites for phosphorylation by protein kinase C can be found in the human, canine, and murine protein. These regions are 100% conserved. Furthermore, three asparagine residues (10, 11, and 24) that were indicated as potential sites for glycosylation by Kobilka et al. (1987) can be found at identical positions in human, mouse, and dog.
|
Radiation Hybrid Mapping
htr1A was retained in 26 hybrids (28.5%). The calculated distance between htr1A and smn1 was 34.8 cR3000 (LOD score = 9.7). Because 1 cR is estimated to be 210 kb for this RH panel (van de Sluis et al. 2000) and because smn1 has been mapped to cfa02, htr1A is expected to be localized about 7.3 Mb from smn1 on canine chromosome 2. These results imply that htr1A will be positioned either between microsatellite markers REN154C04 and REN115P05, or between microsatellite marker C02.466 and gene-based marker FGF1 on the 2001 integrated dog map, assuming that 1 cR5000 corresponds to 100 kb (Breen et al. 2001; see Figure 5). Not surprisingly then, no linkage (LOD score = 0) was found between htr1A and crtl1, which has been mapped to canine chromosome 3.
|
Partial Sequence Analysis of htr1A in Four Golden Retrievers
Sequence analysis of base pairs 115–1250 in four golden retrievers revealed that all of these dogs had cytosine instead of adenine at position 808 in their htr1A sequence. Amino acid 270 will therefore be glutamine instead of lysine in these dogs (Figures 3 and 4). This finding might point to breed-specific differences in the structure of serotonin receptor 1A. No other differences were found between the BAC 160O12 sequence and the genomic golden retriever sequence.
|
Discussion |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
We have isolated a canine BAC clone containing the gene encoding serotonin receptor 1A, and we have determined the entire coding sequence of this gene. The 1272 bp intronless gene is very similar to human and murine htr1A, especially in transmembrane regions and at the sites for phosphorylation and glycosylation. Canine htr1A is more homologous to human htr1A than to murine htr1A; 89% of the canine nucleotides are identical to the human sequence, whereas human and mouse share only 85% of their nucleotides. At the amino acid level, dog and human are 92% identical, whereas mouse and human are 88% the same.
In the short stretch of 5'-flanking region that we have sequenced, several promoter elements can be recognized. Both human and mouse 5'-flanking regions of htr1A have been shown to contain complex TATA-less promoters (Parks and Shenk 1996). It will be very interesting to find out more about the htr1A promoter region in the dog by sequencing farther in the 5' direction.
Radiation hybrid mapping showed that htr1A resides on cfa02, at a distance of 34.8 cR3000 from smn1. These results are in accordance with the human situation, where htr1A and smn1 are separated by about 6 Mb on hsa05 (Human Map Viewer, http://www.ncbi.nlm.nih.gov/cgi-bin/Entrez/maps.cgi?org=hum&chr=5). Additional detailed mapping will reveal whether htr1A is positioned proximally or distally from smn1.
An A
C substitution was found in four golden retrievers, resulting in a different amino acid at position 270. As can be seen in Figure 4, this amino acid is not situated in a very conserved region of the protein. The region forms one of the extracellular loops of the receptor, and, because the ligand binding site of the 5-HT1A receptor is localized in the membrane domains (Shih et al. 1991), it is not expected that this substitution of residue 270 results in altered ligand-binding properties of the receptor. It will be very interesting to determine the rest of the htr1A sequence in these golden retrievers and to compare these sequences with those in other breeds. Moreover, a comparison between anxious or aggressive members of a certain dog breed and "normal" dogs of the same breed will be useful.
In conclusion, we have cloned, characterized, and mapped one of the genes of the serotonergic system in the dog. These data are valuable for candidate gene studies of behavioral disorders in dogs, and they provide a new Type I marker on the canine genome map. Future studies will be directed at determination of the htr1A sequence and its promoter region sequence in several dog breeds and in dogs with behavioral disorders.
|
Acknowledgments |
|---|
The Jubileumfonds Hoogleraren Diergeneeskunde and ID-Lelystad supported this work. We would like to thank Camiel van Lenteren, Jesse Willemse, Marijke Kwant, and Monique van Wolferen for technical assistance; Bart van de Sluis, Sandra Imholz, and Polona Stabej for their useful comments; Peter Leegwater, Matthijs Schilder, and Bart Knol for carefully reading through the manuscript; Harry van Engelen for his help with the collection of blood samples; and the golden retriever owners for their willingness to cooperate with our project. This paper was delivered at the Advances in Canine and Feline Genomics symposium, St. Louis, MO, May 16–19, 2002.
|
Footnotes |
|---|
Corresponding Editor: Urs Giger
Received August 15, 2002
Accepted October 19, 2002
|
References |
|---|
|
TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
-
Barnes NM, Sharp T, 1999. A review of central 5-HT receptors and their function. Neuropharmacology. 38:1083-1152.[CrossRef][ISI][Medline]
Boehnke M, Lange K, Cox DR, 1991. Statistical methods for multipoint radiation hybrid mapping. Am J Hum Genet. 49:1174-1188.[ISI][Medline]
Breen M, Jouquand S, Renier C, Mellersh CS, Hitte C, Holmes NG, Cheron A, Suter N, Vignaux F, Bristow AE, Priat C, McCann E, Andre C, Boundy S, Gitsham P, Thomas R, Bridge WL, Spriggs HF, Ryder EJ, Curson A, Sampson J, Ostrander EA, Binns MM, Galibert F, 2001. Chromosome-specific single-locus FISH probes allow anchorage of an 1800-marker integrated radiation-hybrid/linkage map of the domestic dog genome to all chromosomes. Genome Res. 11:1784-1795.
Cervo L, Mocaer E, Bertaglia A, Samanin R, 2000. Roles of 5-HT(1A) receptors in the dorsal raphe and dorsal hippocampus in anxiety assessed by the behavioral effects of 8-OH-DPAT and S 15535 in a modified Geller-Seifter conflict model. Neuropharmacology. 39:1037-1043.[CrossRef][ISI][Medline]
Corley KC, Singh VB, Phan TH, Boadle-Biber MC, 1992. Effect of gepirone on increases in tryptophan hydroxylase in response to sound stress. Eur J Pharmacol. 213:417-425.[CrossRef][ISI][Medline]
De Vry J, 1995. 5-HT1A receptor agonists: recent developments and controversial issues. Psychopharmacol Ser (Berl). 121:1-26.
File SE, Gonzalez LE, Andrews N, 1996. Comparative study of pre- and postsynaptic 5-HT1A receptor modulation of anxiety in two ethological animal tests. J Neurosci. 16:4810-4815.
Galac S, Knol BW, 1997. Fear-motivated aggression in dogs: patient characteristics, diagnosis and therapy. Anim Welf. 6:9-15.
Gingrich JA, Hen R, 2001. Dissecting the role of the serotonin system in neuropsychiatric disorders using knockout mice. Psychopharmacol Ser (Berl). 155:1-10.
Heisler LK, Chu HM, Brennan TJ, Danao JA, Bajwa P, Parsons LH, Tecott LH, 1998. Elevated anxiety and antidepressant-like responses in serotonin 5-HT1A receptor mutant mice. Proc Natl Acad Sci USA. 95:15049-15054.
Hoyer D, Clarke DE, Fozard JR, Hartig PR, Martin GR, Mylecharane EJ, Saxena PR, Humphrey PP, 1994. International Union of Pharmacology classification of receptors for 5-hydroxytryptamine (serotonin). Pharmacol Rev. 46:157-203.[Abstract]
Jolas T, Schreiber R, Laporte AM, Chastanet M, De Vry J, Glaser T, Adrien J, Hamon M, 1995. Are postsynaptic 5-HT1A receptors involved in the anxiolytic effects of 5-HT1A receptor agonists and in their inhibitory effects on the firing of serotonergic neurons in the rat? J Pharmacol Exp Ther. 272:(2): 920-929.
Kobilka BK, Frielle T, Collins S, Yang-Feng T, Kobilka TS, Francke U, Lefkowitz RJ, Caron MG, 1987. An intronless gene encoding a potential member of the family of receptors coupled to guanine nucleotide regulatory proteins. Nature. 329:75-79.[CrossRef][Medline]
Li R, Mignot E, Faraco J, Kadotani H, Cantanese J, Zhao B, Lin X, Hinton L, Ostrander EA, Patterson DF, de Jong PJ, 1999. Construction and characterization of an eightfold redundant dog genomic bacterial artificial chromosome library. Genomics. 58:9-17.[CrossRef][ISI][Medline]
Miller SA, Dykes DD, Polesky HF, 1988. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 16:1215.
Olivier B, Pattij T, Wood SJ, Oosting R, Sarnyai Z, Toth M, 2001. The 5-HT1A receptor knockout mouse and anxiety. Behav Pharmacol. 12:439-450.[ISI][Medline]
Oshima T, Kasuya Y, Terazawa E, Nagase K, Saitoh Y, Dohi S, 2001. The anxiolytic effects of the 5-hydroxytryptamine-1A agonist tandospirone before otolaryngologic surgery. Anesth Analg. 93:1214-1216.
Parks CL, Robinson PS, Sibille E, Shenk T, Toth M, 1998. Increased anxiety of mice lacking the serotonin 1A receptor. Proc Natl Acad Sci USA. 95:10734-10739.
Parks CL, Shenk T, 1996. The serotonin 1A receptor gene contains a TATA-less promoter that responds to MAZ and Sp1. J Biol Chem. 271:4417-4430.
Pattij T, 2002. 5-HT1A receptor knockout mice and anxiety: behavioral and physiological studies (PhD dissertation). Utrecht, the Netherlands: Utrecht University. 151 pp.
Peroutka SJ, 1995. 5-HT receptors: past, present and future. Trends Neurosci. 18:68-69.[CrossRef][ISI][Medline]
Ramboz S, Oosting R, Amara DA, Kung HF, Blier P, Mendelsohn M, Mann JJ, Brunner D, Hen R, 1998. Serotonin receptor 1A knockout: an animal model of anxiety-related disorder. Proc Natl Acad Sci USA. 95:14476-14481.
Shih JC, Yang W, Chen K, Gallaher T, 1991. Molecular biology of serotonin (5-HT) receptors. Pharmacol Biochem Behav. 40:1053-1058.[CrossRef][ISI][Medline]
Sprouse JS, Aghajanian GK, 1987. Electrophysiological responses of serotonergic dorsal raphe neurons to 5-HT1A and 5-HT1B agonists. Synapse. 1:3-9.[CrossRef][ISI][Medline]
Van de Sluis BJ, Breen M, Nanji M, van Wolferen M, de Jong P, Binns MM, Pearson PL, Kuipers J, Rothuizen J, Cox DW, Wijmenga C, van Oost BA, 1999. Genetic mapping of the copper toxicosis locus in Bedlington terriers to dog chromosome 10, in a region syntenic to human chromosome region 2p13-p16. Hum Mol Genet. 8:501-507.
Van de Sluis BJ, Kole S, van Wolferen M, Holmes NG, Pearson PL, Rothuizen J, van Oost BA, Wijmenga C, 2000. Refined genetic and comparative physical mapping of the canine copper toxicosis locus. Mamm Genome. 11:455-460.[CrossRef][ISI][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  L. van den Berg, L. Kwant, M. S. Hestand, B. A. van Oost, and P. A. J. Leegwater Structure and Variation of Three Canine Genes Involved in Serotonin Binding and Transport: The Serotonin Receptor 1A Gene (htr1A), Serotonin Receptor 2A Gene (htr2A), and Serotonin Transporter Gene (slc6A4) J. Hered., November 1, 2005; 96(7): 786 - 796. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
A. V. Kukekova, L. N. Trut, I. N. Oskina, A. V. Kharlamova, S. G. Shikhevich, E. F. Kirkness, G. D. Aguirre, and G. M. Acland A Marker Set for Construction of a Genetic Map of the Silver Fox (Vulpes vulpes) J. Hered., May 1, 2004; 95(3): 185 - 194. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||