D2Like Dopamine Receptor Genes 221 D2 Dopamine Receptor Genes 2211 Gene Structure and Organization

The cloning of the rat D2 dopamine receptor was a major breakthrough in the neuroscience field [22]. The cloning strategy that was employed utilized the coding region of the hamster ^-adrenergic receptor [23]. This genomic DNA sequence was used to probe a rat genomic library for the presence of homologous fragments in Southern blot analysis. Under low-stringency hybridization conditions several clones were found, and one clone (clone RGB-2) was further characterized. This clone consisted of a 0.8 kb EcoRI-PstI fragment that revealed a high degree of similarity to the nucleotide sequence of the putative transmembrane domain of the hamster ^-adrenergic receptor. The 0.8 kb EcoRI-PstI fragment of RGB-2 was then used to probe a rat brain cDNA library. A full-length cDNA of 2,455 bases was isolated that encoded a protein of 415 amino acids. A hydrophobicity plot of this amino acid sequence indicated that it belonged to the family of G protein-coupled receptors, as it consisted of seven putative transmembrane domains [24]. Subsequently, the human pituitary cDNA (hPITD2) was cloned using rat brain D2 dopamine receptor cDNA as a hybridization probe [25]. The human and rat nucleotide sequences were found to be 90% identical and they indicated 96% homology at the amino acid level. When hPITD2 cDNA was expressed in mouse Ltk- cells, the protein showed a pharmacological profile which was essentially identical to that obtained with the cloned rat D2 dopamine receptor [22]. However, the human pituitary D2 dopamine receptor encoded a protein of 444 amino acids, 29 amino acids longer than the rat D2 dopamine receptor. DNA sequence analysis showed that the coding sequence had seven exons interrupted by six introns and that the additional amino acid sequence was encoded by a single exon (exon 5) of 87 base pairs, which was present in the putative third cytoplasmic loop of the receptor. These D2 dopamine receptors of different sizes from the two species were referred to as the D2L (long) and D2S (short) forms and it was postulated that they were produced by alternative splicing of mRNA [25]. The human D2 dopamine receptor gene was found to localize to chromosome 11q23-24 [26].

The structure and organization of the rat D2 dopamine receptor gene was subsequently further delineated when demonstrated that the gene contains eight exons and spans at least 50 kb [27]. This research group identified seven coding exons (numbered as exons 2-8), including the alternatively expressed exon (exon 6), clustered in approximately 13 kb of the genome which revealed a similar structure to the human D2 dopamine receptor gene. Furthermore, they also identified a non-coding exon termed as exon 1, thus generating a different exon numbering system to that previously used for the human D2 dopamine receptor gene [25]. Additionally, the same research group consequently analysed the structure of the human D2 dopamine receptor [28]. Like the rat D2 dopamine receptor gene, the human D2 dopamine receptor gene was found to contain at least eight exons and spans at least 52 kb. The coding exons 2-8 are clustered within 14 kb and the non-coding exon 1 is separated from exon 2 by at least 38 kb. Similarly, the mouse D2 dopamine receptor gene was found to span at least 30 kb with the coding exons 2-8 clustered in ~ 11 kb and the non-expressed exon 1 located at least 18 kb away from exon 2 revealing an analogous organization to the rat and human D2 dopamine receptor genes [29]. Each intron/exon boundary was also sequenced in the mouse D2 dopamine receptor gene and compared with the rat and human species [29]. The position of all boundaries was conserved in all three species except for intron 4 which contains a variant donor splice site (a GC dinucleotide instead of the canonical GT) in the mouse and rat but not in the human D2 dopamine receptor gene [27].

Other studies also demonstrated that alternative splicing produces the expression of two rat D2 dopamine receptor isoforms [27, 30-32]. Furthermore, similar investigations were performed on both the rat and human D2 dopamine receptor isoforms [31], on the rat and bovine D2 dopamine receptor isoforms [33-36] and for the mouse D2 dopamine receptor isoforms [29]. In the literature the long form of the D2 dopamine receptor was referred to as the D2L, D2(long), D2A, D2(444) or D2-in, whereas the short form was termed D2S, D2(short), D2B, D2(4i5) or D2-o.

2.2.1.2 Promoter Structure and Transcriptional Regulation

A short fragment of 500 bp from the translational start site of the rat D2 dopamine receptor gene was initially sequenced [27]. No transcriptional elements such as CCAAT or TATA boxes were found but the region was 78% GC rich and consisted of several Sp1-like binding sites. Subsequently, the analysis of the promoter region of the rat D2 dopamine receptor gene was comprehensively determined [37]. This analysis included cloning of exon 1, identification of its 5'-end, determination of the transcription start sites and the ability of D2 promoter deletion mutants to transcribe the reporter gene chloramphenicol acetyltransferase (CAT) in various cell lines. The rat D2 dopamine receptor gene spans at least 50 kb with coding exons 2-7 clustered in approximately 13 kb of genome, revealing that intron 1 is very long and over 20 kb [27]. A 21-mer oligonucleotide probe consisting of exon 1 sequences [27] was used to screen a rat genomic library [37]. A 1.3 kb region including all of exon 1, its 5'-flanking region and part of intron 1 was sequenced. S1 nuclease analysis indicated three consecutive nucleotides as the main transcription start sites and several weaker sites also noted upstream from the 3'-end of exon 1. The results also reveal no exon further upstream to the non-coding exon 1 in the D2 dopamine receptor gene. The +1 was designated as the adenine that corresponds to one of the strong S1 signals and to one of the 5'-cDNA ends generated by RACE (rapid amplification of cDNA ends). The promoter region of the D2 dopamine receptor gene was found to lack TATA and CCAAT boxes and is rich in GC content (reaching 80% in some portions) with several putative binding sites for the transcription factor Sp1. An initiator-like sequence was sited between nucleotides -6 and +11, suggesting transcription initiation from this position. Transient expression assays using 5'-deletion mutant constructs controlling transcription of the CAT gene were determined in murine neuroblastoma cells (NB41A3) that endogenously express the

D2 dopamine receptor gene. Strongest transcriptional activity was found between nucleotides -75 and -30 and silencing activity was present between nucleotides -217 and -76. DNase I footprinting studies using nuclear extract from NB41A3 cells suggested Sp1 binding to its consensus sequence at nucleotide -48 but inhibition of Sp1 binding at nucleotide -86 by the extract. The D2 promoter showed no transcription activity of the heterologous CAT gene in rat glioma C6, mouse embryonal NIH 3T3 and human hepatoblastoma Hep G2 cells, indicating that it is regulated in a tissue-specific manner.

Subsequently, another study demonstrated the transcription of the rat D2 dopamine receptor gene from two promoter regions [38]. Using single-stranded ligation to single-stranded cDNA (SLIC), the gene was found to contain two transcription start sites: the major one located about 320 bp upstream from the 3'-end of the first exon and a minor site 70 bp further upstream. Transient expression assays with fusion constructs consisting of fragments of the rat D2 promoter region and the luciferase reporter gene confirmed the presence of two independent TATA-lacking promoter regions. Both promoters independently induced transcription of the luciferase gene in C6 glioma cells, fibroblasts and GH3 and MMQ rat pituitary cell lines, although only the MMQ cells express the D2 dopamine receptor. The transcriptional activity was enhanced in the presence of both promoters and modified by the upstream sequences. These data differ from that derived by [37] and were suggested to be due to the use of different reporter gene assays with varying sensitivities and/or the utilization of different cell lines [38].

The negative modulator of the rat D2 dopamine receptor gene was further analysed [39]. In this study, a small deletion series within the negative modulator fused with the CAT reporter gene was used to transfect the D2-expressing cells, NB41A3. The results identified two c/s-acting functional DNA sequences. The first is a 41 bp segment between nucleotides -116 and -76 (D2Neg-B) and the second is a 26 bp segment between nucleotides -160 and -135 (D2Neg-A). D2Neg-B decreased transcription from the D2 promoter by 45%, whereas D2Neg-A in the presence of the downstream negative modulator reduced transcription down to the level of a promoterless vector. Furthermore, DNase I footprinting, gel mobility shift and competitive cotransfection experiments suggested that D2Neg-A functions without trans-acting factors, while D2Neg-B interacts with nuclear factors at its Sp1 binding sequences. Gel supershift assays with anti-Sp1 antibody and UV cross-linking experiments revealed that a novel 130 kDa factor as well as Sp1 interacts with D2Neg-B in NB41A3 cells. The novel protein that recognizes Sp1 binding sequences in the D2 gene negative modulator was also found to be present in rat striatum nuclear extract.

In the case of the human D2 dopamine receptor gene only a small fragment of the 5'-flanking region was isolated and sequenced which enabled screening of genetic variants [40]. A significant polymorphism in the D2 promoter is the -141C Ins/Del (insertion/deletion), where one or two cytosines are found as part of a putative binding site for the transcription factor Sp1. Constructs consisting of the -141C Del allele cloned into a plasmid with the luciferase reporter gene demonstrated lower transcriptional activity in human retinoblastoma Y-79 cells (that express D2) and human kidney 293 cells (D2 non-expressing) compared to the -141C Ins allele [40]. Interestingly, it was additionally demonstrated in case-control studies that the -141C Del allele was significantly lower in schizophrenic patients than in control subjects in Japanese and Swedish populations [40,41].

The promoter region of the D2 dopamine receptor has been found to be regulated by other transcription factors including retinoids [42, 43], AP1 [44], Sp1/Sp3 [45] and Zif268 in the rat [46], by nuclear factor-KB in human [47] and by dopamine receptor regulating factor (DRRF) in mouse [48]. The latter report showed that DRRF is a zinc finger transcription factor that binds to GC and GT boxes in the D2 dopamine receptor promoters and effectively displaces Sp1 and Sp3 from these sequences. Highest levels of DRRF mRNA were found in mouse brain in areas including olfactory bulb and tubercle, nucleus accumbens, striatum, hippocampus, amygdala and frontal cortex. Interestingly, these brain regions also express abundant levels of dopamine receptors, indicating the importance of DRRF in regulating dopaminergic neurotransmission. In the D2-expressing NB41A3 cells, DRRF potently inhibited transcription from the D2 promoter, whereas it was found to activate the D2 promoter in NS20Y and TE671 cells. In vivo experiments show that DRRF mRNA is significantly altered in striatum and nucleus accumbens brain regions in mice treated with acute and chronic doses of cocaine and haloperidol. Furthermore, in situ hybridization studies in mice have shown that DRRF mRNA is expressed uniquely during development with high levels observed at E12, E14 and E16 in various tissues [49]. DRRF expression during development is also found in particular brain regions such as the neopallial cortex, olfactory lobe and corpus striatum. This pattern of DRRF distribution during embryogenesis overlaps with that found in the adult brain and with the expression profile of dopamine receptors both in adult and during development. Additionally, the promoter region of murine DRRF was characterized, revealing tissue-specific activity, suggesting that it shares structural and functional similarities with the dopamine receptor genes that it regulates [50]. More recently, it has been found that DRRF auto-regulates its own promoter by competing with Sp1 and that both AP1 and AP2 modulate its expression [51]. Additionally a small segment of the rat D2 promoter has been found to be regulated by corticosterone and oestrogen in NB41A3 cells in vitro [52, 53].

Moreover, DNA methylation has been demonstrated within the promoter region of the human D2 dopamine receptor gene, suggesting that this transcription regulatory mechanism plays a role in controlling human D2 dopamine receptor gene expression [54]. Lately, the 5'-regulatory region of the human D2 dopamine receptor gene has been found to have methylated cytosines mainly in three clusters [55].

2.2.2 D3 Dopamine Receptor Genes 2.2.2.1 Gene Structure and Organization

The molecular cloning of the rat D3 dopamine receptor gene was performed using reverse transcription polymerase chain reaction (RT-PCR) [56]. Genomic and cDNA

libraries were screened using a probe derived from the D2 dopamine receptor sequence published earlier [22]. The positive clone that was obtained coded for a protein of 446 amino acid residues, and hydrophobicity analysis of the clone indicated seven putative transmembrane regions characteristic of G protein-coupled receptors. The D3 dopamine receptor gene contained introns similar to the D2 dopamine receptor gene, and 75% homology existed between the rat D2S and D3 dopamine receptor genes within the transmembrane regions. Consistent with this high sequence homology, the pharmacological properties of the D3 receptor were similar to but distinct from those of the D2 dopamine receptor. The rat D3 dopamine receptor gene contains six coding exons separated by five introns. In humans, this gene is located on chromosome 3, band 3q13.3 [57], with a coding region consisting of six exons over 53 kb and an open reading frame of only 400 amino acids. This difference of 46 amino acid residues between the rat and human D3 dopamine receptors is located within the third cytoplasmic loop of the protein [58].

The polymerase chain reaction amplification of mRNA from rat brain revealed the existence of two shorter isoforms of the D3 dopamine receptor in addition to the D3 dopamine receptor itself [59]. The isoforms were suggested to result from different processes of alternative splicing. One form was produced by splicing of an exon whose absence deletes the third transmembrane domain, resulting in a protein (termed D3(TM3-del)) having no dopaminergic ligand binding activity. The second isoform resulted from splicing at a receptor site that coded for half the second extracellular loop and part of the fifth transmembrane domain (referred to as D3(O2-del)). Other research groups have also demonstrated splice variants of the D3 dopamine receptor in the rat and human brain [60-62], but none of the truncated proteins encoded by these variants has dopamine receptor activity. However, the alternatively spliced short isoform of the mouse D3 dopamine receptor that lacks 63 nucleotides in the third cytoplasmic loop was found to bind dopaminergic lig-ands [63]. This alternative splicing reflects the presence of a sixth intron found in the mouse D3 receptor gene [64, 65]. The functional and physiological role of the truncated forms of the D3 dopamine receptors is not known, but it has been suggested that they could be formed for controlling the amount of active D3 dopamine receptors [59]. Defects in the regulation of alternative splicing of the receptor could result in formation of inactive D3 dopamine receptors and may be associated with psychiatric disorders.

2.2.2.2 Promoter Structure and Transcriptional Regulation

The D3 dopamine receptor gene has been implicated in neuropsychiatric disorders and found to be regulated following antipsychotic drug treatment [66, 67]. To begin with only a short segment of the 5'-untranslated region of the D3 dopamine receptor gene was described in the mouse [65] and human [62]. However, a comprehensive investigation of the gene's transcriptional control was elucidated when the 5'-flanking region was characterized by isolating the 5'-end of its cDNA as well as 4.6 kb of genomic sequence [68]. Analysis of this region revealed the presence of two new (untranslated) exons of 196 bp and 120 bp, designated exon 1 and exon

2, that are separated by an 855-bp intron located several kilobases (at least 4 kb) upstream of the previously published coding exons. This evidence shows that the rat D3 dopamine receptor gene is organized into eight exons and is comparable to the structure of the rat D2 dopamine receptor gene. The rat D2 dopamine receptor gene has a single non-coding exon located at least 35 kb upstream from its first of seven coding exons [27, 37, 38]. However, sequence comparison between the 5'-UTR and 5'-flanking regions of the rat D2 and D3 dopamine receptor genes shows substantial homology. On the other hand, the DiA dopamine receptor gene has been found to have a different organization with a non-coding exon separated from a single coding exon by a small intron [69, 70], which is 116 bp in humans (see Section 2.3). There is no sequence homology between the 5'-flanking region of the D1A and the rat D3 dopamine receptor genes.

The transcription initiation site of the rat D3 dopamine receptor gene determined by primer extension analysis and repeated rounds of 5'-RACE (rapid amplification of cDNA ends) was found to consist of a pyrimidine-rich consensus "initiator" sequence, similar to the rat D2 dopamine receptor gene [37, 68]. The promoter region of the rat D3 dopamine receptor gene did not reveal any TATA and CCAAT boxes but unlike that of D1 and D2 dopamine receptor genes has only 52% GC content. These results demonstrate that the rat D3 dopamine gene has similarities with the rat D2 dopamine receptor gene as both are transcribed from a TATA-less promoter that has an initiator element. Functional studies of rat D3 promoter deletion mutants fused to the CAT reporter gene were carried out in a human medul-loblastoma cell line (TE671 cells) [68]. These cells endogenously express the D3 dopamine receptor [71]. Strongest transcriptional activity was determined within 36 nucleotides upstream of the transcriptional start site and a potent silencer identified between bases -37 and -86 which extends to -537 as transcriptional activity is noticeably and gradually reduced with the addition of sequences between -36 and -537 until complete inhibition. There was a small recovery of reporter gene activity with the addition of nucleotides -538 to -782, suggesting the presence of a potential activator. Additionally, another weaker silencer is located between nucleotides -783 and-1,046. These data suggest that the rat D3 dopamine receptor gene is under intense negative regulation and similar to that observed with the rat D2 dopamine receptor gene. Interestingly, none of the D3 deletion mutant constructs showed any transcriptional activity in COS-7 (African green monkey kidney) or C6 rat glioma cells, which are not known to express the D3 dopamine receptor gene endogenously. However, the shortest construct having 36 nucleotides from the transcriptional start site also showed significant transcriptional activity in OK (opossum kidney) and HepG2 (human hepatoblastoma) cells even though the evidence suggests that these cells do not express the D3 dopamine receptor mRNA. However, unlike the potent silencing effect of the longer upstream regulatory regions of the D3 gene in TE671 cells, these fragments showed only weak inhibition in OK cells or strong activation in HepG2 cells. Thus although the core promoter of the rat D3 dopamine receptor gene is active in these three different cell types, its regulation by the upstream elements varies in D3- and non-D3-expressing cells. The presence of specific transcription factors in the different cell lines could help explain the complex differential regulation of the rat D3 dopamine receptor gene. Interestingly, the transcription factor dopamine receptor regulating factor (DRRF) was found to activate the regulatory region of the rat D3 dopamine receptor gene in TE671 cells [48].

Subsequently, a 9 kb genomic fragment of the human D3 dopamine receptor gene was isolated upstream from the translational start site [72]. Studies using 5'-RACE identified three additional exons, and transcriptional activity was found in two putative 500 bp 5'-regions derived from brain tissue and lymphoblast cells following transfection in human cell lines. However, interpretations of these findings were ambiguous as the transcriptional start site was not accurately determined and no series of deletion constructs tested for transcriptional activity. The main focus of this report appeared to be identification of single nucleotide polymorphisms in the 5'-region of the gene, but no association was found with schizophrenia.

2.2.3 D4 Dopamine Receptor Genes 2.2.3.1 Gene Structure and Organization

The human D4 dopamine receptor was cloned and characterized after screening various cell lines for other D2-like dopamine receptors [73]. The cloning strategy employed in the discovery of this receptor utilized the D2 dopamine receptor sequence that encoded for the sixth and seventh putative transmembrane regions [22]. This fragment of the rat D2 dopamine receptor served as a probe for screening genomic and cDNA libraries under low- and high-stringency conditions. The genomic intron-exon organization of the human D4 dopamine receptor gene indicated the presence of five coding exons for a protein of 387 amino acids. Hydrophobicity analysis of the protein sequence indicated seven putative transmembrane regions, which suggested that it belonged to the family of G protein-coupled receptors. The pharmacological characteristics of the D4 dopamine receptor resembled that of the D2 and D3 dopamine receptors. However, the "atypical neuroleptic" drug clozapine had higher affinity for D4 than for D2 and D3 dopamine receptors.

The rat analogue of the human D4 dopamine receptor gene was cloned, also revealing high affinity for clozapine [74]. This rat gene was found to have only four coding exons which encoded a protein of 368 amino acids, suggesting an additional splice site in the human D4 dopamine receptor gene. However, despite the differences in gene structure the rat gene shares a high homology of 73% and 77% with the human D4 gene at the amino acid and nucleic acid level, respectively. Interestingly, the amino acid homology of the gene between the two species increased to 89-96% when only the transmembrane regions were considered. The majority of the differences observed amongst the rat and human D4 dopamine receptor genes occurred within the third cytoplasmic loop, which have only 50% amino acid identity. In the human gene, this region contains an unusual splice site within intron 3 (a donor/acceptor site of TC/CT is present instead of the GT/AG). Additionally, the rat D4 dopamine receptor mRNA was detected in the cardiovascular system in addition to the brain, suggesting that this receptor is an important dopamine receptor in the peripheral nervous system [74]. Similar to the rat D4 gene, the murine D4 dopamine receptor gene was found to have four coding exons that span over 30 kb [75]. The gene encodes a 387 amino acid protein displaying 80% and 95% homology with the human and rat D4 dopamine receptors, respectively, at the amino acid level. Likewise, at the nucleotide level the mouse D4 dopamine receptor gene revealed 79% and 93% homology with the human and rat D4 dopamine receptor genes, respectively. The most conserved regions were seen within the transmembrane domains that are thought to form the ligand binding site.

Three polymorphic forms of the D4 dopamine receptor in humans were further discovered [76]. These were the three most common variants, having 2-, 4-or 7-fold imperfect repeats of a 48 bp sequence in the putative third cytoplasmic loop of the receptor, located in exon 3 of the gene. This was the first example of polymorphic variation observed in catecholamine receptors. Although the different forms of the receptor showed slightly different pharmacological profiles with drugs spiperone and clozapine, they all coupled to G proteins [76]. Similarly, the same group later found that the polymorphic repeat sequences conferred only small differences in pharmacological binding properties [77, 78] and also in functional properties to inhibit cyclic adenosine monophosphate [79]. Therefore, it was concluded from the evidence that there was no direct relationship between length of the polymorphism and changes in these activities. The D4 dopamine receptor variants were also capable of coupling to several G protein (Gi a) subtypes, but no evidence of any quantitative difference in G protein coupling related to repeat length was observed [80]. However, transcriptional differences were observed when the repeat variants were cloned downstream from the luciferase gene in expression vectors and tested in a somatomammotrophic (GH4C1) cell line [81]. Constructs having 7 repeat sequences significantly suppressed expression of the reporter gene compared to those consisting of the 2 and 4 repeats, which was suggested to be via mechanisms involving RNA stability or translational efficiency. More recently, dopamine was found to be more potent at D4 receptors having 2 and 7 repeats than those with 4 repeats, suggesting that the actions of dopamine and therapeutic drugs on D4 dopamine receptors may vary amongst individuals depending on the variants they have [82].

At least 19 different repeat unit sequences, used in 25 different haplotypes that code for 18 different unique receptor variants, were identified in the human D4 dopamine receptor gene [83]. More recently, an extra 35 different alleles have been detected in the population having single nucleotide polymorphisms [84]. The 7-repeat allele was initially reported to be associated with the personality of novelty seeking [85, 86] and then more lately with attention deficit hyperactivity disorder (ADHD) [87-90].

2.2.3.2 Promoter Structure and Transcriptional Regulation

The 5'-flanking region of the human D4 dopamine receptor gene was isolated and sequenced, revealing a transcription initiation region located between -501 and -436 bp relative to the first nucleotide of the translational codon [91]. A CpG island spanned the region from -900 to +500 bp (which is over 50% GC rich) but no TATA or CCAAT boxes were present in the 5'-flanking region. However, the region was found to have consensus binding sites for transcription factors including Sp1. These properties are similar in the 5'-regulatory sequences of the Di, D2, D3 and D5 dopamine receptor genes. Functional analysis of deletion constructs fused to the CAT reporter gene and transiently transfected into IMR32 (neuroblastoma) and Y-79 (retinoblastoma) cells demonstrated promoter activity in the region around -591 and -123 bp and the presence of a negative modulator between -770 and -679 bp. However, no transcriptional activity was observed in human epithelial (HeLa) cells, suggesting cell-specific regulation of the human D4 dopamine receptor gene.

The 5'-flanking region of the human D4 dopamine receptor contains several polymorphisms [92]. One of these is a -521 C/T polymorphism that showed weak association to schizophrenia. Functional studies demonstrated that this promoter polymorphism had reduced transcriptional activity compared to the C allele in human neuroblastoma cells (Y-79) [93]. However, more lately the -521 C/T polymorphism showed no significant differences in transcriptional activity in three human neuroblastoma (SK-N-F1, IMR32 and Y-79) cell lines [94]. The discrepancy in results between the two studies was suggested to be due to the use of different reporter vectors and variation in the length of the cloned fragment that harboured the polymorphism. Interestingly, the transcriptional regulation of the human D4 dopamine receptor gene was also further analysed in SK-N-F1, Y-79, IMR32 and HeLa cells [94]. The highest transcriptional activity was observed between nucleotides -668 and -389 from the translational start site and a putative silencer region was located from -1,571 to -800 bp. These results together with the previous findings on the regulatory region of the D4 dopamine receptor gene [91] suggest that the gene may possess two negative modulators, one of which was not functional in the cell systems utilized in the study by [94].

A tandem duplication of 120 bp located 1.2 kb upstream from the initiation codon and approximately 850 bp upstream from the transcription start site was identified in the human D4 dopamine receptor gene [95]. This polymorphism has been found to be associated with ADHD [96-98]. Transient transfection in human neuroblas-toma cells and other human cell lines with coupled luciferase reporter gene assays demonstrated that the duplication had lower transcriptional activity in human neuroblastoma cells (SK-N-MC, SH-SY5Y), human embryonic kidney cells (HEK293) and HeLa cells compared to the non-duplicated form [99]. These data also suggested that the D4 dopamine receptor gene may have an alternative promoter in an intron region, as the tandem duplication revealed promoter activity. This is yet to be confirmed, but this speculation is in agreement with a previous report that suggested that the promoter region characterized by [91] could be in an intron region due to the discrepancy in size of mRNA from previous studies [100]. They also suggested that a large intron could separate the coding exons of the D4 dopamine receptors from potential untranslated exons similar to that observed for the other D2-like receptors [100], and the interpretation of findings from the functional studies of the tandem duplication were discussed in light of these considerations [99].

A recent study has verified these functional data, also showing that the duplicated allele but this case in constructs comprising the major part of the 5'-regulatory region showed lower transcriptional activity in human cell lines Y-79, SK-N-F1 and HeLa compared to the non-duplicated form [101]. They also identified a 4-repeat allele of this polymorphism which showed dose-dependent functional effects with the lowest transcriptional activity in the cell lines tested.

Capillary electrophoretic mobility shift assays were used to compare the binding capacity of the transcription factor Sp1 to the polymorphic 120 bp sequence in the human D4 dopamine receptor gene [102]. The data suggest enhanced binding capacity of the transcription factor to the duplicated form using HeLa nuclear extracts. However, these data have not been verified independently using other cell culture systems and/or other techniques such as standard electrophoretic mobility shift assays (EMSA).

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Understanding And Treating ADHD

Understanding And Treating ADHD

Attention Deficit Disorder or ADD is a very complicated, and time and again misinterpreted, disorder. Its beginning is physiological, but it can have a multitude of consequences that come alongside with it. That apart, what is the differentiation between ADHD and ADD ADHD is the abbreviated form of Attention Deficit Hyperactive Disorder, its major indications being noticeable hyperactivity and impulsivity.

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