—ORIGINAL—

Determination and Variability of Nucleotide Sequences for D4Dopamine Receptor Genes (DRD4) in Genus Equus

Telhisa HASEGAWA*, Fumio SATO and Nobushige ISHIDAEquine Research Institute, Japan Racing Association, 321–4 Tokami-Cho, Utsunomiya 320-0856, Japan

J. Equine Sci.Vol. 13, No. 2pp. 57–62, 2002

The D4 dopamine receptor gene (DRD4) polymorphic region, which is possibly related to thepersonality trait known as Novelty Seeking in humans, was examined in equids. Partialnucleotide sequences in exon 3 of DRD4 gene were compared among several species in genusEquus. Inter- and intra-species differences were observed, as well as between two domestichorse breeds, Thoroughbred and Kiso pony (Japanese native pony). In equids, thesevariations were consisted of as many as 9 fold repeats of 18 bp variable number of tandemrepeat (VNTR) type polymorphisms and single nucleotide polymorphisms (SNPs).Key words: D4 dopamine receptor, genus Equus, polymorphisms

The fast development of the human race was significance to study the genetic background of the

facilitated by the domestication of useful livestock. Inthe course of domestication, the animals chosen for usewere those that had behavioural nature intrinsicallyadapted to such environmental control, and those thathad required physical properties. Behaviouralproperties was undoubtedly resolved the selection andretention of horses. It is possible that the behaviouraltraits necessary for a domestic animal, such asobedience, have been further developed during thethousands years of selection when morphologicalcharacteristics were also improved [15].

Horses had played a direct role in accelerating socialprocesses and political developments. Horses has beenused so widely as a means of transport in war and peace,for communications and agricultural progress, as wellas the dogs that has been used widely in sport andrecreation. The highly developed ability of the wildhorse to run fast and for a long distance was possiblythe major attraction for domestication. Although thereexist seven member species in the genus Equus, onlytwo species, horses (Equus caballus) and donkeys (Equusasinus), had been subjected for domestication. Theother species include wild asses and zebras have beenthought not to be suitable for domestication because oftheir untamable nature. Thus, there is considerable

This article was accepted June 3, 2002.*Corresponding author. e-mail: telhisah@center.equinst.go.jp

behavioural traits of equids.The dopamine D4 receptor is a member of five

receptors known to function in mammaliandopaminergic pathways. The DNA sequence of thehuman dopamine D4 receptor gene (DRD4: OMIM126452) has previously been investigated and found tobe highly polymorphic at both the DNA and amino acidsequences, exhibiting at least 25 alleles [13]. Thisvariation results from a variable number of tandemrepeat (VNTR) type polymorphisms, which differs inthe number and DNA sequence of a 48-bp (16-aminoacid) repeat unit in the coding region of DRD4 [9, 14].These genetic polymorphisms are proposed to beassociated with human personality traits [1, 3]. Niimi etal. reported the polymorphisms of the DRD4 exon 3among dogs (Canis familialis) [8]. In the present study,the partial nucleotide sequence of the equine DRD4exon 3 was determined in order to compare the speciesdifferences among Equidae, and to survey its potency foran index of the breed and individual characteristics ofthe domestic horses.

Materials and Methods

The peripheral blood samples of Thoroughbredhorse (Equus caballus) were drawn from experimentalhorse raised in the Equine Research Institute, the Japan

T. HASEGAWA, F. SATO, N. ISHIDA58

Racing Association, Utsunomiya. The blood samples ofMountain zebra (Equus zebra) and Donkey (E. asinus)were obtained through the courtesy of YumemigasakiZoological Park, Kawasaki. The blood samples ofGrevy’s zebra (E. grevyi), and Plains zebra (E. burchelli)were kindly gift from Tama Zoological Park, Hino,Japan, Toyohashi Zoo and Botanical Park, Toyohashi,respectively. The blood samples of Mongolian wildhorse (E. przewalskii; International Studbook Number880) and a Kiso pony, Japanese native horse breed,were from the Equestrian Park of the Japan RacingAssociation, Tokyo. Genomic DNA was isolated fromeach peripheral blood sample by using the standardtechniques [10]. DNA samples for Asiatic wild ass (E.hemionus onager) were kindly provided from Dr. G. F.Stranzinger, Swiss Federal Institute of Technology,Zurich.

A pair of PCR primers D4F (5’ -TTCTTCCTACCCTGCCCGCTCATG-3’) and D4R (5’-GACCACCACCGGCAGGACCCTCAT-3’), originallydesigned by Niimi et al. for canine study [8] were usedto amplify the polymorphic region of the exon 3 in theDRD4 gene, in this study. 50 ng of template genomicDNA, 30 pmoles of each primer, 25 µl of 2x GC bufferII, 8 µl of dNTP mix (2.5 mM each) and 2.5 units of LA-Taq polymerase (Takara Shuzo, Kyoto) were mixed in50 µl of reaction mixture. After initial incubation at94°C for 5 min, PCR amplification was performed for35 cycles composed of denaturation at 94°C for 60 sec,annealing at 70°C for 60 sec, and extension at 72°C for60 sec. This was followed by a final extension at 72°Cfor 7 min. The PCR products were analyzed byelectrophoresis in 2% agarose gel. PCR products werecloned into TA cloning vector pCR2.1 (Invitrogen, SanDiego) and determined their nucleotide sequence by acombination of Big-dye terminater FS cycle sequencingkit and ABI PRISM 310 Genetic Analyzer (AppliedBiosystems, Urayasu). A pair of primers D4F-2 (5’-CCGCTCATGCTGCTGCTCTACTGG-3’) and D4R-2(5’-TGCGCTCCCGGCCGGTGATCTT-3’) weredesigned on the basis of common region of equineDRD4 sequence, and these primers were used for thesubsequent PCR. All the determined sequence data ofequine DRD4 PCR products were compared betweenindividuals, breeds and species by personal computerbased sequence analysis software package, DNASIS-Macver. 3.6 (Hitachi Software Engineering, Yokohama).

Results and Discussion

The DRD4 had been cloned as the fourth member ofhuman dopamine receptor [13], as well as thedopamine D5 receptor gene (DRD5) as the fifth [12].The DRD4 is also known as the first reportedcatecholamine receptor gene that displays polymorphicvariation in the human population [14]. Therefore,the surveys were conducted with this gene sequencevariations in human [5] or primates [6]. In human, ithas come from the interests in the association betweenthis gene and schizophrenia, because of its high affinitywith antipsycotic clozapin [14] and its increase in theschizophrenia [11]. On the other hand, these geneticpolymorphisms are proposed to be associated withhuman personality. The association between humanpersonality trait of Novelty Seeking and the sequencerepeat length polymorphisms in the third exon ofDRD4 had been first but simultaneously reported in1996 from 2 distinct research groups [1, 3]. Even afterthese publications, there are still many investigationson the DRD4 polymorphisms in a field of psychiatrics[4]. In dogs, Niimi et al. tried to determine thenucleotide sequence of canine DRD4 [8], and toelucidate the relat ionship between caninecharacter ist ics trai ts of each breed and thepolymorphisms in DRD4 [7]. It is interesting toinvestigate inter- and intra-species differences of thisgene in domestic animals, both in terms of evolutionand breeding strategy of its times gone by.

In the present study, the partial nucleotide sequenceof the equine DRD4 was determined in order to surveyits potency for an index of the individual or breedcharacteristics of horses. Partial nucleotide sequenceof the equine DRD4 exon 3 was determined by cloningof PCR products amplified with a pair of primersderived from human sequence. Determinednucleotide sequence of PCR product from equine (E.caballus) genomic DNA was 598 base pairs in lengthincluding a pair of primer sequences, D4F and D4R,and it showed high similarity with those of human andcanine counterparts especially nearby primers, but itwas inconsistent in the polymorphic repeat region atthe center of the PCR product (Fig. 1). However, thereseemed low similarity in the middle piece of the PCRproduct, where the VNTR-type polymorphisms hadbeen reported in humans [2], non-human primates [5,6] and dogs [8]. These lower similarities in the midpieces are also found between human and caninesequences [8], thus it might come from inter-species

59EQUINE D4 DOPAMINE RECEPTOR GENE POLYMORPHISMS

differences in the region of VNTR-type polymorphisms.In dogs, the VNTR-type polymorphic region could bedivided into 12 parts, that consisted of 7 times repeatsof 39 bp unit, 4 times repeats of 12 bp unit and a 27 bpunit [8], where the variation between 2 to 11 timesrepeats of 48 bp unit exists in humans [2].

A new pair of primers, D4F-2 and D4R-2, weredesigned from the nucleotide sequences just inside ofthe primers as equine specific amplification to comparethese parts among equid species. Total 10 nucleotidesequences were determined in 7 species and aligned inFig. 2, as well as their deduced amino acid sequences.Most of the sequences, both in nucleotide and aminoacid, were similar except several single nucleotidepolymorphisms (SNPs) and the amplified fragmentlength polymorphisms (AFLPs). The lengths ofamplified fragments were varied from the shortest 442bp (E. asinus) to the longest 550 bp al leles(Thoroughbred, E. przewalskii, E. grevyi, and E. zebra).These AFLPs were caused by polymorphisms of repeat

Fig. 1. Nucleotide sequence for equine DRD4 exon 3 inucleotide sequence for equine DRD4 was determprimers, D4F and D4R. The nucleotides matched wprimers, D4F-2 and D4R-2, were designed from thiand species.

numbers of 18 bases repeated structures, and thelongest alleles contained 9 repeat units whilst theshortest allele contained 3. These repeat units were notcompletely identical with each other, and were smallerthan that of human (48 bp) or canine (39 bp) ones. Noframe shifts or stop codons were detected among thesespecies, and deduced amino acid sequences were alsopolymorphic in the repeat numbers of 6 amino acidsunits. In these, two breeds of domestic horse (E.caballus) were tested. The Thoroughbred horseshowed the longest 9 folds repeat pattern, while theKiso pony showed 18 bp shorter, 8 folds repeat. So, it issuggested that there are intra-specific variations in theVNTR-type polymorphisms of the equine DRD4. Inaddition to this, Kiso pony contained 4 SNPs withThoroughbred. Further investigations are necessary toelucidate whether these polymorphisms have comefrom inter-breed differences or individual variations.The Mongolian wild horse (E. przewalskii) showed quitehigh similarity and same length as in Thoroughbred,

n comparison with human and canine sequences. Theined by cloning after PCR amplification using a pair of

ith equine sequence were indicated with shadows. A pair ofs data, and used for subsequent comparison among breeds

T. HASEGAWA, F. SATO, N. ISHIDA60

Fig. 2. Alignments of the nucleotide (A) and amino acid (B) sequences for 2 equine breeds and 6 equid species. Unmatched basesor amino acid residues were highlighted with dark characters. The lacks of bases or amino acid residues were also indicatedwith dash (–). The repeat units were consisted with 18 nucleotides and 6 amino acids.

61EQUINE D4 DOPAMINE RECEPTOR GENE POLYMORPHISMS

whereas only an exception of SNP (G316A) has beenfound. Two thymidine residues (295T and 398T) wereobserved only, but in both domestic and wild horses.One of the donkeys (E. asinus) showed the shortestform of 3 folds repeat, which is 6 folds less than thelongest form, but the another individual had the 8 foldsrepeat, which was only 1 fold less than the longest onesof horses. An SNP (A525C) was found in both twoindividuals of the domestic donkey, and the lack of theseventh unit of the VNTR was also observed in commonamong the individuals of domestic donkey and wild assspecies. One of the two individuals of Asiatic wild ass(onager: E. hemionus onager) lacked the first two repeatunits in addition to the seventh repeat. Two thymidineresidues (64T and 249T) were observed in commonbetween two individuals of this species. Three zebraspecies (E. burchelli, E. grevyi and E. zebra) showedsmaller differences with horses than donkeys, whileonly the plains zebra (E. burchelli) lacked the first 5repeat units and it was the second shortest among thespecies tested. The variations of DRD4 exon 3 in theequid species and breeds were not completelyelucidated in this study, because the number of thesamples was quite limited. There may exist morevariations among equid species and breeds.

This i s the f irs t report to invest igate thepolymorphisms in DRD4 of Perissodactyla. In thispreliminary study, we demonstrated DRD4 showsVNTR-type polymorphisms. The size and sequence ofthe repeat unit was different from those of the human[5] or canine [8] ones. The results suggested that thereshould exist a different evolutional pathway in DRD4 ofPerissodactyla in comparison with those in bothPrimates and Carnivora. Thus, the analysis ofpolymorphisms in DRD4 exon 3 should be a usefulmeans for the studies of evolution and behavioursciences in an aspect of species (breed) or individualdifferences. For this purpose, it is necessary toaccumulate more data of sequence variations byanalyzing increased number of individuals in all thespecies or breeds for analysis.

Acknowledgments

The authors would like to thank Dr. M. Inoue-Murayama, Faculty of Agriculture, Gifu University, forcanine DRD4 information and useful suggestions. Thenucleotide sequence data determined in this study willappear in the DDBJ/EMBL/GenBank nucleotide

databases with the accession numbers AB080626(Thoroughbred), AB080627 (Kiso pony), AB080628 (E.przewalskii), AB080629 and AB080630 (E. asinus),AB080631 and AB080632 (E. hemionus), AB080633 (E.burchelli), AB080634 (E. grevyi), and AB080635 (E. zebra).

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