Associations have been reported of the seven-repeat (7R) allele of the human dopamine receptor D4 (DRD4) gene with both attentiondeficit͞hyperactivity disorder and the personality trait of novelty seeking. This polymorphism occurs in a 48-bp tandem repeat in the coding region of DRD4, with the most common allele containing four repeats (4R) and rarer variants containing 2-11. Here we show by DNA resequencing͞haplotyping of 600 DRD4 alleles, representing a worldwide population sample, that the origin of 2R-6R alleles can be explained by simple one-step recombination͞mutation events. In contrast, the 7R allele is not simply related to the other common alleles, differing by greater than six recombinations͞mutations. Strong linkage disequilibrium was found between the 7R allele and surrounding DRD4 polymorphisms, suggesting that this allele is at least 5-10-fold ''younger'' than the common 4R allele. Based on an observed bias toward nonsynonymous amino acid changes, the unusual DNA sequence organization, and the strong linkage disequilibrium surrounding the DRD4 7R allele, we propose that this allele originated as a rare mutational event that nevertheless increased to high frequency in human populations by positive selection.

There is an increased prevalence of the 7-repeat (7R) allele of the dopamine receptor D4 (DRD4) gene in attention-deficit/hyperactivity disorder (ADHD). However, the population prevalence of the 7R allele varies considerably across ethnicity and is very low in Asians. To test whether this 7R allele/ADHD association still held in a Chinese clinical sample, 32 Han Chinese children with a confirmed ADHD diagnosis and normal IQ who were methylphenidate-responders were genotyped. None of them had a DRD4 7R allele. Instead, we observed a significantly increased prevalence of the 2-repeat (2R) allele in this clinical sample (33%) compared to ethnically-matched controls (20%) (chi(2)(1d.f.) = 5.90, P = 0.015). This approximately 1.65-fold increase of the 2R allele in our probands is close to the observed increase of the 7R allele in European-ancestry ADHD children. Recent genetic studies have indicated that the 2R allele in Asians is likely derived from the 7R allele. Further, available biochemical data indicate that both the 2R and 7R protein have blunted responses to dopamine compared to the 4R protein. Based on these results, we propose that the observed increased prevalence of the 2R allele in our Han Chinese ADHD probands is still consistent with the 7R allele hypothesis of ADHD in European-ancestry children. Recent studies have suggested that any variant from the conserved ancestral 4R allele might potentially alter biochemistry/phenotype. We hypothesize that an increased frequency of any non-4R allele may define the association of the DRD4 gene with ADHD that holds across ethnicity. The present findings, however, obtained with a small ADHD sample size, should be replicated.

Accession no. X07806 From a Xqtri cDNA library derived from Drosophila embryo, a 4,500 bp cDNA clone was isolated and sequenced by the dideoxy rnethod (1). DNA sequence analysis revealed an open 2 AAGTGGAAGAGATCC

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We isolated and sequenced a Drosophila genomic DNA sequence that encodes the entire coding region of the laminin B2 chain. The 11,464-bp genomic sequence contains a 2.1-kb of 5'-flanking DNA, ten exons, nine introns, and the 3'-flanking region. The first exon encodes a 5' untranslated region; the ATG translational start codon is in exon 2. The entire translated region is within a 8.3-kb Eco RI fragment. The Drosophila laminin B2 gene differs substantially in size and exon pattern from those of the human B1 and B2 genes. However, as in the case of the human B1 gene, the overall exon pattern of the Drosophila B2 gene does not correlate well with the highly conserved structural domains and internal repeats of the B2 polypeptide chain. Unlike the human and mouse B1 and B2 genes, the 2.1-kb 5'-flanking region of the Drosophila B2 gene contains a TATA box and two CAAT boxes. Other potential transcriptional regulatory sequences include two reverse complementary cAMP response element sequences; two sequences that are homologous to the retinoic acid response element motifs of the mouse B1 gene; and sequences homologous to the binding sites for transcription factors dFRA and dJRA, zeste, and possibly GAGA. When transfected into Drosophila SL-2 cells, pCAT plasmid containing 2,090 bp of 5'-flanking region shows a 3.0- to 3.5-fold increase in chloramphenicol acetyltransferase activity after induction with retinoic acid and/or 8-bromo-cAMP. These results suggest that this 5'-flanking promoter region may contain DNA sequences that can regulate the expression of the laminin B2 gene.

We report here a modified protocol for double stranded DNA sequencing (1) and a simple method for synthetic oligonucleotides purification. Primers were synthesized with a Pharmacia Gene Assembler using cyanoethyl phosphoramidites and modified T7 DNA polymerase (SequenaseTM) was used in sequencing reaction mixtures. Sequencing results as good as single stranded DNA sequencing can be routinely produced ( fig.1). .A Primer preparation: a After synthesis, incubate detritylated primers from the DNA synthesizer with NH40H at 55°C for 16 hours. b Deprotected primer in NH40H is loaded directly onto a Pharmacia Mono Q 5/5 column equilibrated with 10 mM CAPS (3-[Cyclohexylaminoj-1l-propane-OA " -wsulfonic acid) , pH 11, and elute with a NaCI gradient. Set the flow rate and gradient at 0.5 -as ml/min and 10 mM NaCI/min respectively. This al2 gradient can give single base separation for most primers shorter than 30 bases. A chromatogram 2 for the separation of a 18 bases long primer is presented in figure 2. Fig. 2 c. Primer from the column is diluted to desired concentration with water and use directly in annealing step. B Annealing step: a To 2 tg of CsCI-EtBr purified supercoiled DNA, add NaOH to 0.2 M (total volume 10p1), incubate at room temperature for 5 min. b. Add 10 pmoles of primer (7 RI), mix by vortexing. c Neutralize by adding 3 IL of 3 M sodium acetate (pH 4.5), then precipitate the template/primer with 75 g1 of 100% ethanol at -700C for 10 minutes. d Pellet the template/primer by centrifugation, then wash the pellet with 75% ethanol. Recover the pellet by centrifugation at room temperature for 10 min. e Draw away the ethanol, and dry the template/primer in a Sped Vac concentrator. t Resuspend the template/primer in 8 IL H20. -C Sequencing reaction: See SequenaseTM Manual (2) for details and buffer composition. a The reaction mixture consists of: 8 ;1I tamplate/primer in H20, 2 pLI Sequencing buffer, 1 1l 0.1 M DTT, 2 p1 5 fold diluted Labeling Mix, 1 p1 [y-35SJ dATP (20-M, 10 1LCi/p1), and 2 p1t 8 fold diluted SequenaseT. Incubate at room temperature for 5 min after mixing. _= b. Carry out the termination reaction as in (2) at 370C for 5 min.

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