This section has been subdivided into two parts. The first part deals with the isolation and characterisation o f 5’-upstream region of the human DRD3 and the second part deals with the screening the 5’-upstream region for polymorphisms in association w ith schizophrenia.
3.3.1 ISOLATION AND CHARACTERISATION OF THE 5 ’ UPSTREAM
REGION OF THE HUMAN DRD3
3.3.1.1 INTRODUCTION
The organisation o f the coding region o f the five human dopamine receptor genes is well understood, but little is known about the molecular events that regulate the expression o f these genes (D RD l, DRD2, DRD3, DRD4 and DRD5, see section 1.9.3 and table 1.2). Characterisation o f the promoters o f the human D R D l (Minowa et a l, 1992), rat D R D l (Zhou et a l, 1992), rat DRD2 (Minowa et a l, 1992), 600bp o f the 5 ’-untranslated region o f the human DÆD2 (Gandelman et a l, 1991) and human DRD5 (Beischlag et a l, 1995) genes have shed some light on the nature o f the promoters that regulate the tissue specific expression o f these genes, although the exact mechanism is not yet clearly understood (see section 1.10). The tissue specific expression o f these receptors, as discussed in section 1.9.3.6 (see table 1.2), suggests that there is a complex and specific mechanism governing transcription o f these genes. The promoter region o f the human D2-like dopamine receptor genes remains to be characterised in greater depth. The DRD3 promoter in particular has not been characterised to date and investigated for variants in association with schizophrenia. The DRD3 is an important candidate gene for the disease since it combines the dopamine receptor hypothesis and the limbic system hypothesis o f schizophrenia (see section 1.8.1 and 1.8.2). Therefore elucidation o f factors involved in the transcriptional regulation o f the DRD3 will assist our understanding o f how alterations in the expression or regulation o f expression o f this receptor could cause various pathologic states. This section will deal with the isolation and characterisation o f the human DRD3. The mutation analysis o f the 5 ’-upstream region is discussed in section 3.3.2.
The D2-like dopamine receptors
3.3.1.2 METHODS
3.3.1.2.1 The strategy followed to isolate and characterise the 5^-upstream region
of the human DRD3
As discussed in section 2.12, several different walking PCR techniques have been reported that are based on similar functional principles. These have been employed b y various researchers to isolate uncharacterised regions starting from a site o f known sequence with increasingly greater degrees o f success (refer to the references cited in section 2.12). Therefore, this approach was chosen to isolate the 5 ’-upstream region o f the DRD3 from human and from pig, chicken and mouse. Attempts were made to isolate the 5 ’-upstream region o f the DRD3 from the pig, chicken and mouse genomes in order to perform cross species comparison o f the promoter region. Cross species comparison may reveal conserved sequences o f transcription control elements that are involved in regulating the expression o f the respective dopamine D3 receptor genes, thus facilitating the characterisation o f the human promoter region.
The initial aim was to isolate a clone containing the DRD3 and apply an appropriate walking PCR technique to isolate the 5’-upstream region. However due to the number o f failures encountered in either isolating a clone o f interest or the application o f walking PCR techniques, it became necessary to adopt a number o f different approaches, involving a number o f genomic templates and walking PCR techniques. This study was initiated with screening the human ICI Y AC pool for a clone containing the DRD3. Subsequently, the following libraries were screened to isolate a clone: human genomic cosmid library, human genomic PAC library, mouse genomic phage library, mouse genomic PI library, pig and chicken genomic cosmid libraries (see section 2.11.1).
3.3.1.2.2 Genomic templates
Human genomic DNA and mouse genomic DNA served as templates to PCR amplify parts o f the DRD3, DNA fragments which were then used as probes to screen the human genomic PAC, human genomic cosmid, mouse genomic P I, mouse genomic phage, pig genomic cosmid and chicken genomic cosmid libraries, respectively. The libraries were screened to isolate clones containing the corresponding DRD3. The human ICI Y AC library was screened directly using the recombinant Y AC clones as the template for PCR amplification. For details o f the various libraries screened refer to section 2.11.1. Human
The D2-like dopamine receptors genomic DNA was extracted from lOmls of whole blood drawn from individuals working in the lab using the Nucleon II™ DNA extraction kit (see section 2.3.1). Mouse genomic DNA was extracted from a BALB/c strain. The genomic DNA was extracted from liver tissue using the protocol outlined in section 2.3.2.
3.3.1.2.3 PCR amplification of the exons of the human and mouse DRD3
The standard PCR amplification protocol using NH4 reaction buffer and BioTaq DN A polymerase was used to amplify regions o f the human DRD3 and the mouse orthologue (see section 2.4.2). The primers for the human and mouse DRD3 exons were designed based on the published cDNA and genomic sequence using the guide line outlined in section 2.4.1 (human DRD3 cDNA sequence- GenBank accession number- U32499 and U25441 and mouse DRD3 cDNA sequence- GenBank accession number- X67274). The exon/intron boundaries o f the human gene have not been characterised, but the organisation o f the rat and mouse genes are known. Primers were designed complementary to exonl o f both genes and also to PCR amplify the seventh transmembrane domain (TMD) o f the human gene. A list o f the primers designed, annealing temperatures used for each primer pair and the size o f the expected amplified DNA fragment are summarised in table 3.10. Refer to figure 3.6 for the nucleotide sequence o f exonl and the seventh TMD o f the human DRD3, exonl o f the mouse orthologue and for the location o f the primers designed.
Table 3.10: List of primers designed and used to PCR amplify coding regions of the human and mouse DRD3. P r im e r s e q u e n c e (S' - 3 ’) a n d T m (°C ) Prim er Pair Ta ( ° Q Size o f product (bp) Human
D3ExlA+; GTC TCC TCA CAG GAA GCC GCT TG (80) D 3 E x lA + /D 3 E x lA - 65 s 1.9Kb
D 3ExlB-; CCG CTC CTT CAG CAC AGC CAT G (84) D 3 E x lA + /D 3 E x lB - 70 2 1 9 b p
D3EX1C-: GAG GGC ATA GTA GGC ATG TGG G (68) D 3 E x lA + /D 3 E x lC - 65 1 48bp
D3EX1A-; CTG GCT GTA CAC ATC ATG ACA TC (72)