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Results for “structural genomics”

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PubMed1999

Genome structure and chromosomal mapping of the gene for Fe65L2 interacting with Alzheimer's beta-amyloid precursor protein.

Recently we cloned the cDNA encoding human Fe65L2, which interacts with Alzheimer's beta-amyloid precursor protein (APP). The protein has one WW domain and two PID elements (Neurosci. Lett. (1999) 261, 143-146.). Here, we report the entire genome structure and the chromosomal mapping of the Fe65L2 gene. The gene is composed of thirteen coding exons distributed over 6 kb and the genomic organization is similar to another Fe65 member, Fe65 gene. Two transcription start sites of the gene were found in a CpG island by primer extension analysis. Radiation hybrid mapping revealed that the Fe65L2 gene is on chromosome 5 between markers SHGC-9824 (D5S2374) and SHGC-8489 (D5S2569). Characterization of the Fe65L2 gene structure will be useful in the linkage analysis and search for mutations in the Fe65L2 gene in patients with Alzheimer's disease.

Alzheimer Disease↗

PubMed1997

Human XPMC2H: cDNA cloning, mapping to 9q34, genomic structure, and evaluation as TSC1.

XPMC2 is a Xenopus gene identified on the basis of its ability to correct a mitotic defect in fission yeast. Here we report the identification of cDNA clones for human XPMC2H, its mapping to the tuberous sclerosis gene TSC1 region on 9q34, determination of genomic structure, and identification of several coding region polymorphisms. The predicted protein has strong sequence similarity to the Xenopus gene. Through SSCP and heteroduplex analysis of genomic DNA, we found two intragenic polymorphisms but no evidence for significant mutations in patients with tuberous sclerosis in this gene.

Chromosome Mapping↗

PubMed2002

Molecular genetics and structural genomics of the human protein kinase C gene module.

BACKGROUND: Protein kinase C (PKC) has become a major focus among cell biologists interested in second-messenger signal transduction and much has been learned about differences in the cellular localization and function of its different isotypes. In this study we systematically address the genomic locations and gene structures of the human PKC gene module. RESULTS: We first carried out fine chromosomal mapping of all nine PKC genes by fluorescence in situ hybridization (FISH), using cosmid and BAC probes. The PKC genes are found to be dispersed throughout the genome, and in some positions distinct from those previously reported: PKCalpha is at 17q24, PKCbeta at 16p12, PKCgamma at 19q13.4, PKCdelta at 3p21.2, PKCepsilon at 2p21, PKCzeta at 1p36.3, PKCeta at 14q22-23, PKCtheta; at 10p15 and PKCiota at 3q26. For PKCiota, an additional FISH signal mapped on Xq21.3 revealed a pseudogene (derived by retrotransposition). PKCgamma, zeta, and theta; are found to map to the most distal positions on the chromosomes, potentially implicating telomere position effects in their expression. Using the complete human genome draft sequence and bioinformatics tools, we then carried out a systematic analysis of PKC gene structure, including determination of the occurrence of single-nucleotide polymorphisms corresponding to the PKC loci. CONCLUSION: This resource of genomic information now facilitates investigation of the PKC gene module in structural chromosomal abnormalities and human disease locus mapping studies.

Alternative Splicing↗

PubMed2006

The K+ channel gene, Kcnb1: genomic structure and characterization of its 5'-regulatory region as part of an overlapping gene group.

Kcnb1 expression is down-regulated in certain types of cardiomyopathy. As a first step towards understanding Kcnb1 regulation, we determined its genomic structure and characterized its 5'-regulatory region. Two species of Kcnb1 mRNA were found to arise from alternative usage of two highly GC-rich promoters (P1, P2). While transcripts arising from P1 were mainly detected in brain, P2 transcripts were highly expressed in heart and brain. Core regulatory regions were characterized for P1 and P2. The mutation of a potential Nur77/Nurr1/NOR-1 binding site, NBRE(Kcnb1), conserved in both human and mouse, resulted in a significant decrease in basal P2 promoter activity. Luciferase activities of the longest promoter-reporter construct reflected the level of endogenous Kcnb1 mRNA in myoblast, smooth muscle, and pituitary cell lines. Hyperosmolarity increased Kcnb1 mRNA concentration two-fold, mainly at the transcriptional level in clonal pituitary cells. These findings provide a basis for future studies of (post)transcriptional mechanism(s) down-regulating Kcnb1 expression in a variety of cardiomyopathies and point towards a possible involvement of Kcnb1 in pituitary cell excitability and secretory activity regulated by osmolarity.

Animals↗

PubMed2002

The use of bioinformatics to identify the genomic structure of the gene that encodes neutrophil antigen NB1, CD177.

The gene encoding neutrophil alloantigen NB1, CD177, is highly homologous to a gene overexpressed in polycythemia vera neutrophils, polycythemia rubra vera-1 (PRV-1). The cDNAs of both genes have been cloned, but their genomic structure is unknown. The purpose of this study was to determine the intron-exon organization of NB1 and PRV-1 and discern if they are separate members of a homologous gene family or alleles of the same gene. GenBank's human genome sequences were probed in silico with PRV-1's 1605-nucleotide coding sequence. Searches identified two adjacent bacterial artificial chromosomes (BACs) on chromosome 19q13.2: BC338531 and BC52850. BC338531 contained sequences 100% homologous to the first 654 nucleotides of PRV-1. Comparison of coding and genomic sequences allowed us to separate this region into exons 1 through 5, interrupted by five introns. BC52850 contained sequences 95% homologous to nucleotides 413 through 1605 of PRV-1, organized into exons 4 through 9. However, the orientation of PRV-1-homologous in the first BAC was plus-plus and of the second was plus-minus, indicating they could not be portions of the same gene. The GenBank sequence of BC338531 was incomplete, creating a sequence gap in chromosome 19q13.2. Evaluation of BC338531's unfinished sequences in Joint Genome Institute public databases allowed us to complete the gap and revealed that BC338531 contained sequences 98% homologous to all nine PRV-1 exons followed by a second gene consisting of exons 9 through 4. Most likely, NB1 and PRV-1 are alleles of the same gene, CD177, and the duplication of exons 4 through 9 is a pseudo gene.

Amino Acid Sequence↗

PubMed1993

Genomic structure of the locus encoding protein 4.1. Structural basis for complex combinational patterns of tissue-specific alternative RNA splicing.

Protein 4.1 (P4.1) is a multifunctional protein with heterogeneity in molecular weight, intracellular localization, tissue- and development-specific expression patterns. We have analyzed the genomic structure of the locus encoding mouse P4.1 and have systematically analyzed diverse P4.1 mRNA isoforms expressed in erythroid and nonerythroid tissues. Our results indicate that the mouse protein 4.1 gene, over 90 kilobases long, comprises at least 23 exons (13 constitutive exons, 10 alternative exons) interrupted by 22 introns. The donor and acceptor splice site sequences match the consensus sequences for the exon-intron boundaries of most eukaryotic genes. No significant sequence difference was observed between splice junctions of alternative and constitutive exons. Apparently, most alternative exon-encoded peptides are located within particular functional domains of the P4.1 protein: two peptides encoded by alternative exons 4 and 5 are located near or within the glycophorin/calmodulin binding domain, whereas three other alternative exon-encoded peptides (19-amino acid encoded by exon 14, 14-amino acid encoded by exon 15, and 21-amino acid encoded by exon 16) are located near or within the spectrin-actin binding domain. Selective use of exon 2', which carries an upstream translation initiation codon (AUG), may produce an elongated P4.1 isoform (135 kDa) that is predominantly expressed in nonerythroid tissues. Combinatorial splicing of these exons may generate different isoforms that exhibit complicated tissue-specific expression patterns.

3T3 Cells↗

PubMed1996

Genomic structure of the transforming growth factor beta type II receptor gene and its mutations in hereditary nonpolyposis colorectal cancers.

To characterize the tumorigenetic role of the transforming growth factor beta type II receptor (RII) gene, we defined its genomic structure, which consists of seven exons. The sequences of exon-intron junctions were determined to facilitate mutation analysis of each exon. Twenty-five carcinomas and five adenomas from hereditary nonpolyposis colorectal cancer patients were analyzed for mutations in the entire coding region. Four missense mutations (two in adenomas and two in carcinomas) were found in the 10 cases carrying the polyadenine deletions in one allele. These results indicate that RII shares the two-hit inactivation mechanism with tumor suppressor genes and that mutations of it may occur in the early stage of tumorigenesis.

Adult↗

PubMed1995

Characterization of the genomic structure and the promoter region of the human intestinal trefoil factor.

Trefoil proteins form a specific group of stable secretory polypeptides. They are expressed in a lot of human cancers and inflammatory processes of the gastrointestinal tract. Recently a new human trefoil protein, ITF/hP1.B was isolated. This gene is expressed mainly in globet cells of intestinum and colon. The genomic structure of the murine and rat homologues genes were described previously and a high nucleotide sequence conservation (78-95%) was found in the coding as well as the regulatory regions. We have isolated the human genomic ITF-gene region from a P1 library and a detailed analysis yielded in a similar gene structure with a three basepair displacement of the second exon intron boundary. The promoter region of the human gene interestingly is markedly different from rat and mouse.

Amino Acid Sequence↗

PubMed2004

Genome structure and transcriptional regulation of human coronavirus NL63.

BACKGROUND: Two human coronaviruses are known since the 1960s: HCoV-229E and HCoV-OC43. SARS-CoV was discovered in the early spring of 2003, followed by the identification of HCoV-NL63, the fourth member of the coronaviridae family that infects humans. In this study, we describe the genome structure and the transcription strategy of HCoV-NL63 by experimental analysis of the viral subgenomic mRNAs. RESULTS: The genome of HCoV-NL63 has the following gene order: 1a-1b-S-ORF3-E-M-N. The GC content of the HCoV-NL63 genome is extremely low (34%) compared to other coronaviruses, and we therefore performed additional analysis of the nucleotide composition. Overall, the RNA genome is very low in C and high in U, and this is also reflected in the codon usage. Inspection of the nucleotide composition along the genome indicates that the C-count increases significantly in the last one-third of the genome at the expense of U and G. We document the production of subgenomic (sg) mRNAs coding for the S, ORF3, E, M and N proteins. We did not detect any additional sg mRNA. Furthermore, we sequenced the 5' end of all sg mRNAs, confirming the presence of an identical leader sequence in each sg mRNA. Northern blot analysis indicated that the expression level among the sg mRNAs differs significantly, with the sg mRNA encoding nucleocapsid (N) being the most abundant. CONCLUSIONS: The presented data give insight into the viral evolution and mutational patterns in coronaviral genome. Furthermore our data show that HCoV-NL63 employs the discontinuous replication strategy with generation of subgenomic mRNAs during the (-) strand synthesis. Because HCoV-NL63 has a low pathogenicity and is able to grow easily in cell culture, this virus can be a powerful tool to study SARS coronavirus pathogenesis.

Amino Acid Sequence↗

PubMed2002

Human aquaporin adipose (AQPap) gene. Genomic structure, promoter analysis and functional mutation.

Aquaporin adipose (AQPap), which we identified from human adipose tissue, is a glycerol channel in adipocyte [Kishida et al. (2000) J. Biol. Chem. 275, 20896-20902]. In the current study, we determined the genomic structure of the human AQPap gene, and identified three AQPap-like genes that resembled (approximately 95%) AQPap, with little expression in human tissues. The AQPap promoter contained a putative peroxisome proliferator response element (PPRE) at -46 to -62, and a putative insulin response element (IRE) at -542/-536. Deletion of the PPRE abolished the pioglitazone-mediated induction of AQPap promoter activity in 3T3-L1 adipocytes. Deletion and single base pair substitution analysis of the IRE abolished the insulin-mediated suppression of the human AQPap gene. Analysis of AQPap sequence in human subjects revealed three missense mutations (R12C, V59L and G264V), and two silent mutations (A103A and G250G). The cRNA injection of the missense mutants into Xenopus oocytes revealed the absence of the activity to transport glycerol and water in the AQPap-G264V protein. In the subject homozygous for AQPap-G264V, exercise-induced increase in plasma glycerol was not observed in spite of the increased plasma noradrenaline. We suggest that AQPap is responsible for the increase of plasma glycerol during exercise in humans.

Aquaporins↗

PubMed2002

Establishment of the genomic structure and identification of thirteen single-nucleotide polymorphisms in the human RECK gene.

The human RECK gene, mapped at 9p13-->p12, is known as a tumor suppressor gene and as a key regulator of extracellular matrix integrity and angiogenesis. We have established the entire genomic structure of this gene, which spans more than 87 kb and consists of 21 exons and 20 introns, and identified thirteen single nucleotide polymorphisms (SNPs). Four SNPs were identified in the coding region of the gene (exons 1, 9, 13 and 15), and the remaining nine in introns 5, 8, 10, 12, 15 and 17. The availability of the genomic organization of the RECK gene and the identification of polymorphisms throughout its entire genome will facilitate the evaluation of its role in several disorders and also contribute to the assignment of genes to the several diseases mapped to this chromosomal region.

Base Sequence↗

PubMed1993

Human dystroglycan: skeletal muscle cDNA, genomic structure, origin of tissue specific isoforms and chromosomal localization.

Dystroglycan is a novel laminin binding component of the dystrophin-glycoprotein complex which provides a linkage between the subsarcolemmal cytoskeleton and the extracellular matrix. Here we report the cDNA and genomic structure of human dystroglycan. The human dystroglycan is encoded by a single gene (DAG1) mapped to chromosome 3 band p21. The coding sequence is organized into two exons, separated by a large intron. The predicted amino acid sequence of human and rabbit dystroglycan are 93% identical with predicted glycosylation sites being conserved. Human dystroglycan is expressed in a variety of fetal and adult tissues. Our data suggest that muscle and non-muscle isoforms of dystroglycan differ by carbohydrate moieties but not protein sequence. Therefore, we hypothesize that variable glycosylation of the conserved protein core might modulate laminin binding. The relationship of dystroglycan to human diseases is discussed.

Amino Acid Sequence↗

PubMed1996

Molecular cloning of human mitochondrial glycerophosphate dehydrogenase gene: genomic structure, chromosomal localization, and existence of a pseudogene.

cDNA of mitochondrial glycerophosphate dehydrogenase (mGPDH), a defect of which is a possible cause of non-insulin dependent diabetes mellitus, was cloned from a human insulinoma cDNA library. The deduced amino acid sequence showed 91% and 92% homology with those of rat and mouse mGPDH, respectively. The mGPDH gene was mapped to chromosome 2q23 by FISH analysis. Genomic clones for mGPDH were then isolated using mouse mGPDH cDNA and PCR products of human mGPDH cDNA as probes. Genomic structure was studied by sequencing the exon-intron boundaries and by PCR amplification of intronic regions using genomic clones as templates. The human mGPDH gene was shown to be composed of 15 coding exons, containing a (CA)n repeat region inside the gene, which was not polymorphic in the Japanese population. Genomic cloning also identified a pseudogene located on chromosome 19q13.4. These results provide information useful for analyzing the mGPDH gene in patients with non-insulin dependent diabetes mellitus.

Amino Acid Sequence↗

PubMed1997

Genome structure of cucumber leaf spot virus: sequence analysis suggests it belongs to a distinct species within the Tombusviridae.

The complete nucleotide sequence of cucumber leaf spot virus (CLSV) has been determined and the sizes and locations of predicted viral proteins deduced. The genome consists of 4432 nucleotides and contains five long ORFs. The 5' proximal ORF encodes a 25 kDa product that terminates in an amber codon which may be readthrough to produce an 84 kDa protein (ORF 2). ORF 3 codes for the 41 kDa coat protein (CP). ORFs 4 and 5 are completely overlapping at the 3' terminus and code for 27 and 17 kDa products, respectively. The CLSV genome structure is similar to that of tombusviruses and nearly identical to pothos latent virus (PoLV), a newly proposed, atypical, member of the Tombusviridae. It is proposed that CLSV and PoLV be considered strains of a new tombusvirus species. Amino acid sequence comparisons of the CLSV CP and the CPs of several small spherical plant viruses suggest that CLSV is most closely related to melon necrotic spot carmovirus (MNSV), red clover necrotic mosaic dianthovirus (RCNMV) and cucumber necrosis tombusvirus (CNV). These viruses, like CLSV, are transmitted by the soil inhabiting fungus, Olpidium bornovanus.

Amino Acid Sequence↗

PubMed2002

Genomic structure of the human UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase gene.

The UDP-GlcNAc:dolichol-P GlcNAc-1-P transferase catalyzes the first and committed step in the dolichol cycle, thus playing a fundamental role in the pathway for protein N-glycosylation. The structure of the GlcNAc-1-P transferase gene has been previously elucidated in mouse and hamster. Moreover, the human cDNA has been cloned. Using sequence database tools, we deduced the genomic structure of the human GlcNAc-1-P transferase gene, which was experimentally confirmed by sequence analysis. The gene is composed of 9 exons and spans 5.5 kb. All splice acceptor and donor sites conform to the canonical AG/GT rule. The 5'-end of the gene is different from previously reported, as, consequently, the N-terminal of the encoded protein, which is predicted to be 408 amino acids long. The transcription start site, determined by 5' RACE, occurs 180 nucleotides upstream of the translation initiation codon. Several potential transcription regulatory motifs, such as Sp-1, E4TF1 and ATF binding sites, were identified in the 5'-flanking region. A polyadenylation signal is located 466 bp downstream of the stop codon. The genomic organization of the gene is similar to that of the corresponding mouse and hamster genes, though extensive homology is restricted to the coding regions. Analysis of a panel of radiation hybrids led to the assignment of the GlcNAc-1-P transferase gene to chromosome 11, at 4.19 cR from NIB361, according to the location of the homologous sequences in the database at 11q23.

3' Untranslated Regions↗

PubMed1996

Genomic structure and organization of the human rBAT gene (SLC3A1).

Cystinuria is an autosomal recessive disorder of amino acid transport, manifesting as three phenotypes (I, II, and III). An amino acid transport gene, rBAT, is responsible for cystinuria. Mutation and linkage analyses have demonstrated the disease to be heterogeneous, with rBAT being the defective gene in type I cystinuria. The genomic structure of the human rBAT gene (HGMW-approved symbol SLC 3A1) has been established via two strategies: (i) construction of two different genomic libraries by subcloning the Mega-YAC921B6 (CEPH), containing rBAT, in Lambda ZAP and screening using rBAT cDNA and different PCR products; and (ii) generation and sequencing of genomic fragments by long PCR using rBAT cDNA-derived primers. The rBAT gene spans approximately 45 kb and consists of 10 exons. The introns range from 500 to 13,000 bp. All splice sites conform to the GT/AG rule. The promoter region has been further analyzed, and a predicted TATA box 98 bp upstream of the first coding ATG was identified. In addition an Alu repeat has been detected 72 bp upstream of the predicted TATA box.

Amino Acid Transport Systems, Basic↗

PubMed2005

Genomic structure of the proopiomelanocortin gene and expression during acute low-water stress in channel catfish.

Proopiomelanocortin (POMC) is an important gene involved in the stress response of the hypothalamic-pituitary-adrenal axis. It is a precursor of several peptide hormones including adrenocorticotropic hormone, melanocyte stimulating hormones, and beta-endorphin. Our study aims to determine genomic structure and expression of POMC gene during temporal stress in channel catfish (Ictalurus punctatus). The catfish POMC gene consisting of three exons and two introns has a similar structural organization to that of other species. The catfish and mammalian POMC promoters do not exhibit regions of conservation except that of one TATA box. Genomic Southern blot analysis indicated POMC is present as a single copy gene in the catfish genome. Real-time PCR allowed us to monitor temporal expression of the POMC mRNA in catfish pituitary during low-water stress. Plasma cortisol concentrations were also measured as an indicator of stress. Within 15 min after the onset of low-water stress, POMC mRNA expression was elevated 1.87-fold above the control value. The POMC mRNA level had declined after 30 min (1.29-fold) and 1h (1.1-fold) at which time stress was removed. After 1h recovery, a significant increase in the POMC mRNA expression was detected (2.44-fold, P<0.05) followed by a decline 2h later (1.52-fold) when the experiment was terminated. Plasma cortisol levels in stressed fish were significantly above the cortisol levels in control fish during stress application (t=15 min, t=30 min, and t=1h, P<0.05), which then returned to normal during recovery. We conclude that POMC and cortisol are both involved in the low-water stress response during which cortisol may serve as a negative regulator of POMC expression in catfish.

Adaptation, Physiological↗

PubMed1991

Genomic structure of the human prion protein gene.

Creutzfeld-Jacob disease and Gerstmann-Sträussler syndrome are rare degenerative disorders of the nervous system which have been genetically linked to the prion protein (PrP) gene. The PrP gene encodes a host glycoprotein of unknown function and is located on the short arm of chromosome 20, a region with few known genes or anonymous markers. The complete structure of the PrP gene in man has not been determined despite considerable interest in its relationship to these unusual disorders. We have determined that the human PrP gene has the same simple genomic structure seen in the hamster gene and consists of two exons and a single intron. In contrast to the hamster PrP gene the human gene appears to have a single major transcriptional start site. The region immediately 5' of the transcriptional start site of the human PrP gene demonstrates the GC-rich features commonly seen in housekeeping genes. Curiously, the genomic clone we have isolated contains a 24-bp deletion that removes one of five octameric peptide repeats predicted to form a B-pleated sheet in this region of the PrP. We have also identified 5' of the PrP gene an RFLP which has a high degree of heterozygosity and which should serve as a useful marker for the pter-12 region of human chromosome 20.

Amino Acid Sequence↗