Impact of five SNPs in dopamine-related genes on executive function.

OBJECTIVES: Dopamine neurotransmission is a critical factor for executive function, which is controlled by the prefrontal cortex in humans. Although the contribution of genetic factors to the regulation of brain dopaminergic activity is widely acknowledged, identification of a genotype-phenotype association has not yet been clearly established. In this study, we therefore evaluated the effects of five functional single-nucleotide polymorphisms (SNPs) in specific genes related to dopamine neurotransmission on executive function in a general population. MATERIALS AND METHODS: Participants of the health examination at the Shimane Institute of Health Science were recruited for this study (n = 964). To evaluate executive function, the Frontal Assessment Battery (FAB) was administered. SNPs were genotyped using the TaqMan method. RESULTS: A significant association was found between an SNP in the catechol-O-methyltransferase (COMT) gene (rs4680) encoding the low-activity Met allele and FAB score (P = 0.003). Of note, the flexibility subset of the FAB was associated with the SNP in COMT (P = 0.003) after adjustment for confounding factors. The generalized multifactor dimensionality reduction method identified that the combination of two SNPs in the COMT gene (rs4680) and the dopamine D4 receptor gene (rs1800955) had a significant effect on FAB score. CONCLUSIONS: Our study indicates a contribution of rs4680 in the COMT gene to the variability in executive function, as assessed by the FAB. In addition, we have indicated that a complex gene-gene interaction between SNPs in the genes related to dopamine neurotransmission may influence executive function in a general population.

Positional cloning of the gene for Nijmegen breakage syndrome.

Nijmegen breakage syndrome (NBS), also known as ataxia-telangiectasia (AT) variant, is an autosomal recessive disorder characterized by microcephaly, growth retardation, severe combined immunodeficiency and a high incidence of lymphoid cancers. Cells from NBS patients display chromosome instability, hypersensitivity to ionizing radiation and abnormal cell-cycle regulation after irradiation, all of which are characteristics shared with AT. Recently, the NBS locus was mapped at 8q21 by two independent approaches, complementation studies and linkage analysis. Here, we report the positional cloning of the NBS gene, NBS1, from an 800-kb candidate region. The gene comprises 50 kb and encodes a protein of 754 amino acids. The amino-terminal region of the protein shows weak homology to the yeast XRS2, MEK1, CDS1 and SPK1 proteins. The gene is expressed at high levels in the testes, suggesting that it might be involved in meiotic recombination. We detected the same 5-bp deletion in 13 individuals, and conclude that it is likely to be a founder mutation.

TSLC1 is a tumor-suppressor gene in human non-small-cell lung cancer.

The existence of tumor-suppressor genes was originally demonstrated by functional complementation through whole-cell and microcell fusion. Transfer of chromosome 11 into a human non-small-cell lung cancer (NSCLC) cell line, A549, suppresses tumorigenicity. Loss of heterozygosity (LOH) on the long arm of chromosome 11 has been reported in NSCLC and other cancers. Several independent studies indicate that multiple tumor-suppressor genes are found in this region, including the gene PPP2R1B at 11q23-24 (ref. 7). Linkage studies of NSCLC are precluded because no hereditary forms are known. We previously identified a region of 700 kb on 11q23.2 that completely suppresses tumorigenicity of A549 human NSCLC cells. Most of this tumor-suppressor activity localizes to a 100-kb segment by functional complementation. Here we report that this region contains a single confirmed gene, TSLC1, whose expression is reduced or absent in A549 and several other NSCLC, hepatocellular carcinoma (HCC) and pancreatic cancer (PaC) cell lines. TSLC1 expression or suppression is correlated with promoter methylation state in these cell lines. Restoration of TSLC1 expression to normal or higher levels suppresses tumor formation by A549 cells in nude mice. Only 2 inactivating mutations of TSLC1 were discovered in 161 tumors and tumor cell lines, both among the 20 primary tumors with LOH for 11q23.2. Promoter methylation was observed in 15 of the other 18 primary NSCLC, HCC and PaC tumors with LOH for 11q23.2. Thus, attenuation of TSLC1 expression occurred in 85% of primary tumors with LOH. Hypermethylation of the TSLC1 promoter would seem to represent the 'second hit' in NSCLC with LOH.

Detailed deletion mapping in squamous cell carcinomas of the esophagus narrows a region containing a putative tumor suppressor gene to about 200 kilobases on distal chromosome 9q.

We previously reported definition of a region containing a putative tumor suppressor gene for esophageal squamous cell carcinoma within an approximately 4-cM genomic segment at 9q31-q32. We have investigated this region further using six new microsatellite markers isolated from yeast artificial chromosome clones covering the deleted region and have narrowly defined the commonly deleted region to a segment between two loci, KM9.1 and D9S177. On the basis of the contig map of cosmid and yeast artificial chromosome clones, we estimate the physical size of the region of interest to be about 200 kb. Because the distal 9q region also has been implicated as the site of a tumor suppressor gene(s) related to squamous cell carcinomas of other tissues, our map provides useful information for attempts to identify a common gene for carcinomas of this cell type.

Regulation of binding of rhoB p20 to membranes by its specific regulatory protein, GDP dissociation inhibitor.

We have previously purified from bovine brain cytosol a novel regulatory protein for the GTP-binding proteins of the rho gene family. This regulatory protein, designated as rho GDP dissociation inhibitor (GDI), makes a complex stoichiometrically with the GDP-bound form of the rho gene products and thereby regulates the GDP/GTP exchange reaction by inhibiting the dissociation of GDP from and the subsequent binding of GTP to them. We show here that rho GDI also regulates the binding of rhoB p20, a member of the rho gene products, to various membranes including rat brain synaptic plasma membranes and human erythrocyte ghosts. Both the GDP- and GTP-bound forms of rhoB p20 bound to the membranes. This binding was not inhibited by prior treatment of the membranes with boiling or tryptic digestion, suggesting that a protein molecule of the membranes is not essential for the binding of rhoB p20 to the membranes. rho GDI inhibited the binding of the GDP-bound form of rhoB p20, but not that of the GTP-bound form, to the membranes. Moreover, rho GDI induced the dissociation of the GDP-bound form, but not that of the GTP-bound form, of rhoB p20 exogenously bound to the membranes from them. These results suggest that the rho gene products bind to the membranes, presumably in a reversible manner and that this binding is regulated by their specific GDI.

Post-translational modifications of the C-terminal region of the rho protein are important for its interaction with membranes and the stimulatory and inhibitory GDP/GTP exchange proteins.

We have recently found, by use of the rhoA p21 purified from bovine aortic smooth muscle, that it is similarly post-translationally processed as described for ras p21s: it is first geranylgeranylated at the cysteine residue in the C-terminal region followed by removal of the three C-terminal amino acids and the subsequent carboxyl methylation of the revealed C-terminal cysteine residue. In the present study, we investigated the function(s) of these post-translational modifications of the C-terminal region of rhoA p21 by use of the rhoA p21s purified from bovine aortic smooth muscle and rhoA p21-overexpressing Escherichia coli since the bacterial protein was not modified with a geranylgeranyl moiety. Bovine rhoA p21 bound to plasma membranes and phosphatidylserine-linked Affigel, but bacterial rhoA p21 did not bind to them. The inhibitory GDP/GTP exchange protein for rhoA p21, named GDP dissociation inhibitor (GDI), made a complex with the GDP-bound form of bovine rhoA p21 and thereby inhibited the dissociation of GDP from and the subsequent binding of GTP to it. However, rho GDI neither made a complex with the GDP-bound form of bacterial rhoA p21 nor affected these reactions of the bacterial protein. The stimulatory GDP/GTP exchange protein for rhoA p21, named GDP dissociation stimulator (GDS), stimulated the dissociation of GDP from bovine rhoA p21, but was inactive for the bacterial protein. In contrast, the GTPase activating protein for rhoA p21 is active not only for bovine rhoA p21 but also for the bacterial protein. These results suggest that the post-translational modifications of the C-terminal region of bovine rhoA p21, most presumably the geranylgeranylation, which are absent in bacterial rhoA p21, play important roles in its interaction with membranes and the stimulatory and inhibitory GDP/GTP exchange proteins but not with the GAP.

Sequence analysis of a total of three megabases of DNA in two regions of chromosome 8p.

Large-scale sequencing of genomic regions and in silico gene trapping together represent a highly efficient and powerful approach for identifying novel genes. We performed megabase-level sequence analyses of two genomic regions on human chromosome 8p (8p11.2 and 8p22-->p21.3), after covering those segments with sequence-ready contigs composed of 74 cosmids, 14 BACs, and three PAC clones. We determined continuous nucleotide sequences of 1,856,753 bases on 8p11.2 and 1,210,381 bases on 8p22-->p21.3 by combining the shotgun and primer-walking methods. In silico gene trapping identified four novel genes in the 8p11.2 region and, in the 8p22-->p21.3 region, six known genes (PRLTS, PCM1, MTAMR7, HCAT2, HFREP-1 and PHP) and three novel genes. The distribution of Alu and LINE1 repetitive elements and the densities of predicted exons were different in each region, and Alu-rich portions contained more exonic sequences than LINE1-rich areas.

Significant differences in the frequency of transcriptional units, types and numbers of repetitive elements, GC content, and the number of CpG islands between a 1010-kb G-band genomic segment on chromosome 9q31.3 and a 1200-kb R-band genomic segment on chromosome 3p21.3.

We determined the nucleotide sequence of the entire 1,010,525-bp insert contained in CEPH YAC clone 867e8. This human genomic segment was derived from chromosome 9q31.3 and corresponds to a G-band region. We compared this segment, in terms of structure, with a previously characterized 1,201,033-bp sequence in CEPH YAC936c1 that had come from a portion of human chromosome 3p21.3 corresponding to an R-band region. The two segments were significantly different with respect to the frequency of transcriptional units, the types and numbers of repetitive elements present, their GC content, and the number of CpG islands. Alu elements, GC content, and CpG islands all showed positive correlations with the abundance of exons, but the distribution of LINE1s did not. These observations might reflect an influence of the first three of these features on the functions or expression of genes in the respective regions. In addition to a novel gene (F36) lying at the centromeric end of the 9q segment, we found a cluster of placenta-specific genes within a small section (about 400 kb) on the telomeric side of YAC867e8. This cluster consisted of four apparently unrelated ESTs and two genes, pregnancy-associated plasma protein-A (PAPP-A) and a novel gene (tentatively named EST-YD1). Our characterization of the two chromosomal regions provided evidence that genes are not evenly distributed throughout the human genome, and that gene richness is correlated with the GC content and with the frequency of either Alu elements or CpG islands.

Characterization of a 1200-kb genomic segment of chromosome 3p22-p21.3.

We previously determined the nucleotide sequence and characterized the 685-kb proximal half of CEPH YAC936c1, which corresponds to a portion of human chromosome 3p21.3. In the study reported here, we characterized the remaining 515-kb of this YAC clone corresponding to the telomeric half of its human insert. The newly sequenced region contained a total of ten genes including six reported previously: phospholipase C delta 1 (PLCD1), human activin receptor type IIB (hActR-IIB), organic cation transporter-like 1 (OCTL1), organic cation transporter-like 2 (OCTL2), oxidative stress response 1 (OSR1), and human xylulokinase-like protein (XYLB). The remaining four genes present in the telomeric region included two known genes, MyD88 and ACAA, and two novel genes. One (designated ENGL) of the novel sequences was found to encode an amino-acid sequence homologous to the family of DNA/RNA endonucleases, especially endonuclease G. The other gene F56 revealed no significant homology to any known genes. These results disclosed complete physical and transcriptional maps of the 1200-kb region of 3p present in YAC 936c1.

Identification of tumor suppressor candidate genes by physical and sequence mapping of the TSLC1 region of human chromosome 11q23.

Loss of heterozygosity for a locus on human chromosome 11q22-23 is observed at high frequency in non-small cell lung carcinoma (NSCLC). Introduction of a 1.1 Mb fragmented yeast artificial chromosome (YAC) mapping to this region completely suppresses the tumorigenic properties of a human NSCLC cell line, A549. Smaller fragmented YACs give partial but not complete suppression. To further localize the gene(s) responsible for this partial suppression, a bacterial artificial chromosome (BAC) and P1-based artificial chromosome (PAC) contig was constructed, completely spanning the candidate region. End sequence generated in the construction of the BAC/PAC contig identified a previously unmapped EST and served to order genomic sequence contigs from the publicly available Celera Genomics (CG) and Human Genome Project (HGP) efforts. Comparison showed that CG provided larger contigs, while HGP provided more coverage. Neither CG nor HGP provided complete sequence coverage, alone or in combination. The sequence was used to map 110 ESTs and to predict new genes, including two GenScan gene predictions that overlapped ESTs and were shown to be differentially expressed in tumorigenic and suppressed A549 cell lines.

Development of a highly sensitive enzyme immunoassay for human calcitonin using solid phase coupled with multiple antibodies.

We have developed a highly sensitive chemiluminescent enzyme immunoassay for human calcitonin using three different mouse monoclonal antibodies that recognize the N-terminal, C-terminal and central portions of a human calcitonin molecule. The assay signal in a two-step sandwich enzyme immunoassay for human calcitonin using a solid phase coupled with a mixture of monoclonal antibodies. CT08 and OCT1, was 3.7-fold higher than when using either or both solid phases coupled with CT08 or OCT1, respectively. This enhancement is the result of improved avidity of immobilized antibodies and greater stability of the complex of immobilized antibodies and calcitonin in the first reaction, which resulted in greater reactivity of the immunocomplex with alkaline phosphatase-conjugate in the second reaction. The present assay showed a linear response up to 2.5 micrograms/L of human calcitonin and a high specificity for human calcitonin, but not for rat calcitonin, human calcitonin gene-related peptide and rat calcitonin gene-related peptide. The detection limit of human calcitonin was estimated to be 0.29 ng/L at zero (assay blank) + 3 SD. Interbatch coefficients of variation ranged from 2.2-26.7%.

Regioselective transglycosylation in the synthesis of oligosaccharides: comparison of beta-galactosidases and sialidases of various origins.

N-Acetyl-lactosamine(beta-D-Gal p-(1-->4)-D-Glc pNAc) was synthesized regioselectively with the aid of the transglycosylation activity of beta-galactosidase isolated from Diplococcus pneumoniae using p-nitrophenyl beta-D-galactopyranoside as the donor. Also, transglycosylation of the sialyl group in an alpha-(2-->8)-linked sialic acid dimer or p-nitrophenyl glycoside of sialic acid to N-acetyl-lactosamine was performed using sialidases of various origins. When sialidase from Clostridium perfringens, Arthrobacter ureafaciens, or Vibrio cholerae was used, alpha-(2-->6)-linked sialyl N-acetyl-lactosamine was obtained regioselectively. In contrast, when sialidase from newcastle disease virus was used, the alpha-(2-->3)-linked isomer was obtained regioselectively. The regioselectivity of the transglycosylation reaction using beta-galactosidase and sialidase was compared with hydrolysis specificity toward the same linkages.

Chemoenzymatic synthesis of the branched oligosaccharides which correspond to the core structures of N-linked sugar chains.

Synthetic routes are described to a partial structure common to all high mannose-type sugar chains and complex-type sugar chains based on a chemoenzymatic strategy which incorporates, (a) enzymatic synthesis of oligosaccharide blocks using glycosidases, and (b) chemical synthesis of the branching oligosaccharides via regioselective coupling. All reaction products correspond to key intermediates necessary for the construction of N-linked oligosaccharides and we have synthesized the branched tetra-manno-oligosaccharide high mannose-type sugar chain and the branched hexa-oligosaccharide complex-type sugar chain using this simple and direct method.

Enzymatic synthesis of mannobioses and mannotrioses by reverse hydrolysis using alpha-mannosidase from Aspergillus niger.

Various manno-oligosaccharides including alpha-D-man-(1 --> 2)-D man and alpha-D-man-(1 --> 2)-alpha-D-man-(1 --> 2)-D-man were formed when a highly concentrated mannose solution was incubated in the presence of alpha-mannosidase from Aspergillus niger. alpha-D-Man-(1 --> 2)-D-man and alpha-D- man-(1 --> 2)-alpha-D-man-(1 --> 2)-D-man were isolated by activated carbon chromatography followed by high performance liquid chromatography using an amino-silica column. In addition to the above oligosaccharides, alpha-D-man-(1 --> 3)-D-man, alpha-D-man-(1 --> 6)-D-man, and alpha-D-man-(1 --> 2)-alpha-D-man-(1 -->6)-D-man were also isolated.

[Enzyme immunoassay of CEA using monoclonal antibody].

Carcinoembryonic antigen (CEA) is important as one of the tumor markers, and enzyme-immunoassay using monoclonal anti-CEA, which is based on the Sandwich method and unnecessary for the treatment of CEA-extract from serum, was tried in this study. The standard curve obtained from this assay showed lineality on low CEA level. Sensitivity could allow to detect CEA below 5 ng/ml of CEA. Correlation between radioimmunoassay and this EIA was not found in especially high CEA-concentration. Reproducibility respect of Intra-assay and Inter-assay had good results. CEA value yielded by dilution of sample serum coincided with nearly expected value; this result indicated that dilution test was effective. CEA value determined by the assay to be necessary for treatment of extract was higher than that by the assay without treatment. Though the number of test samples used for assay of CEA in serum from various diseases were small, we considered it would be appropriate to set up a normal value range in 0.44-3.16 ng/ml (mean +/- 2 SD) and cut off value at 3.9 ng/ml (mean +/- 3 SD). We have been interested in cancer-specificity on monoclonal antibody, but we could not so for evaluate effects of the assay using monoclonal antibody.

Efficient guanine alkylation through cooperative heterodimeric formation of duocarmycin A and distamycin A.

Antitumor antibiotic Duocarmycin A alkylates adenines at the 3 end of sequences of three or more consecutive A or T base pairs through binding to the minor groove of DNA. In the presence of distamycin A, duocarmycin A was found to alkylate guanine residue in G-C rich sequences, which are not alkylated by duocarmycin A alone. Efficient guanine alkylation through cooperatively binding of a heterodimer in the minor groove of DNA will be discussed.

Allelic frequencies of twelve dinucleotide repeat marker loci on chromosome 13 in the normal Japanese population.

To establish a genotypic database for dinucleotide repeat marker loci in the Japanese population, we determined allelic frequencies of 12 such markers on chromosome 13 and compared them with data from Caucasians in the GDB archive. The average heterozygosity (79%) for the 12 loci was the same for the two populations. However, allelic distributions at two of the marker loci were quite different. These data will be useful for disease studies in the Japanese population that involve linkage or sibship-pair analyses, or association studies.

Molecular cloning and characterization of two novel genes on chromosome 8p21.3.

Through large-scale sequencing of genomic DNA from human chromosome 8p22-p21.3 we have isolated two novel genes, designated GK1 and G5. Their predicted products showed no significant similarity to any known proteins in public databases. A comparison of GK1 cDNA sequences, which encode a 1270-amino-acid protein, with corresponding genomic DNA sequences revealed that this gene consists of 15 exons and spans an approximately 113-kb genomic region. Northern blot analysis revealed ubiquitous expression of 7.0- and 4.4-kb transcripts; in addition, we detected a 5.0-kb skeletal muscle-specific transcript and a 4.0-kb transcript specifically expressed in heart and pancreas. Computer and immunocytochemical analyses of a GK1 Green fluolesent protein (GFP) fused construct indicated that the gene product, which contains putative leucine-zipper domains, was likely to be a mitochondrial protein. The other novel gene, G5, expressed four transcripts (4.2, 2.2, 1.7, and 1.0-kb) ubiquitously; the longer three transcripts, which differed only in the 3'-non coding region, encoded identical 397-amino-acid peptides. The G5 gene consists of 14 exons and spans approximately 52 kb of genomic DNA; its deduced 397-amino acid product appears to contain coiled-coil domains and a proline-rich region, and to be located in cytoplasm.