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Results for “structural genomics”

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PubMed1999

Genomic structure of rat 3alpha-hydroxysteroid/dihydrodiol dehydrogenase (3alpha-HSD/DD, AKR1C9).

Rat liver 3alpha-hydroxysteroid/dihydrodiol dehydrogenase (3alpha-HSD/DD) is a member of the aldo-keto reductase (AKR) superfamily. It is involved in the inactivation of steroid hormones and the metabolic activation of polycyclic aromatic hydrocarbons (PAH) by converting trans-dihydrodiols into reactive and redox-active o-quinones. The structure of the 5'-flanking region of the gene and factors involved in the constitutive and regulated expression of this gene have been reported [H.-K. Lin, T.M. Penning, Cloning, sequencing, and functional analysis of the 5'-flanking region of the rat 3alpha-hydroxysteroid/dihydrodiol dehydrogenase gene, Cancer Res. 55 (1995) 4105-4113]. We now describe the complete genomic structure of the rat type 1 3alpha-HSD/DD gene. Charon 4A and P1 genomic clones contained at least three rat genes (type 1, type 2 and type 3 3alpha-HSD/DD) each of which encoded for the same open reading frame (ORF) but differed in their exon-intron organization. 5'-RACE confirmed that the type 1 3alpha-HSD/DD gene encodes for the dominant transcript in rat liver and it was the regulation of this gene that was previously studied. The rat type 1 3alpha-HSD/DD gene is 30 kb in length and consists of nine exons and eight introns. Exon 9 encodes +931 to 966 bp of the ORF and the 1292 bp 3'-UTR implicated in mRNA stability. This genomic structure is nearly identical to the homologous human genes, type 1 3alpha-HSD (chlordecone reductase/DD4, AKR1C4), type 2 3alpha-HSD (AKR1C3) and type 3 3alpha-HSD (bile-acid binding protein, AKR1C2) genes. Three different cDNA's containing identical ORFs for 3alpha-HSD have been reported suggesting that all three genes may be expressed in rat liver. Using 5' primers corresponding to the 5'-UTR's of the three different cDNA's only one PCR fragment was obtained and corresponded to the type 1 3alpha-HSD/DD gene. These data suggested that the type 2 and type 3 3alpha-HSD/DD genes are not abundantly expressed in rat liver. It is unknown whether the type 2 and type 3 3alpha-HSD/DD genes represent pseudo-genes or whether they represent genes that are differentially expressed in other rat tissues.

3' Untranslated Regions↗

PubMedDate not supplied

Genomic structure and expression analysis of the spastic paraplegia gene, SPG7.

SPG7 is a newly identified gene involved in an autosomal recessive form of hereditary spastic paraplegia (HSP), a genetically heterogeneous group of neurodegenerative disorders. This gene encodes a protein characterized as a nuclear-encoded mitochondrial metalloprotease. The present report describes the genomic structure of the SPG7 gene. It is organized into 17 exons ranging from 78 to 242 bp and spans approximately 52 kb within three overlapping cosmids. The exon/intron boundaries and all splice junctions are consistent with the published consensus sequences for donor and acceptor sites. The provided genomic structure of SPG7 should facilitate the screening for mutations in this gene in patients with HSP and other related mitochondrial disease syndromes. SPG7 has been mapped to chromosome 16q24.3, a region of frequent loss of heterozygosity (LOH) seen in sporadic breast and prostate cancer. We have performed single-strand conformation polymorphism analysis of ten exons of this gene in a number of sporadic breast cancer samples showing LOH at 16q24.3. No mutations were detected; only single nucleotide polymorphisms were observed in exon 11, intron 7, intron 10 and intron 12. An expression analysis study has revealed the differential expression of SPG7 mRNA in various tissues and at different developmental stages.

ATPases Associated with Diverse Cellular Activitie↗

PubMed2004

DNA microarrays: from structural genomics to functional genomics. The applications of gene chips in dermatology and dermatopathology.

The human genome project was successful in sequencing the entire human genome and ended earlier than expected. The vast genetic information now available will have far-reaching consequences for medicine in the twenty-first century. The knowledge gained from the mapping and sequencing of human genes on a genome-wide scale--commonly referred to as structural genomics--is prerequisite for studies that focus on the functional aspects of genes. A recently invented technique, known as gene chip, or DNA microarray, technology, allows the study of the function of thousands of genes at once, thereby opening the door to the new field of functional genomics. At its core, the DNA microarray utilizes a unique feature of DNA known as complementary hybridization. As such, it is not different from Southern (DNA) blot or northern (RNA) blot hybridizations, or the polymerase chain reaction, with the exception that it allows expression profiling of the entire human genome in a single hybridization experiment. The article highlights the principles, technology, and applications of DNA microarrays as they pertain to the field of dermatology and dermatopathology. The most important applications are the gene expression profiling of skin cancer, especially of melanoma. Other potential applications include gene expression profiling of inflammatory skin diseases, the mutational analysis of genodermatoses, and polymorphism screening, as well as drug development and chemosensitivity prediction. cDNA microarrays will shape the diagnostic approach of the dermatology and the dermatopathology of the future and may lead to new therapeutic options.

Dermatology↗

PubMed2000

Genomic structure and chromosomal localization of the human hepatocyte growth factor activator inhibitor type 1 and 2 genes.

Hepatocyte growth factor activator inhibitor type 1 (HAI-1) and type 2 (HAI-2) are recently discovered Kunitz-type serine protease inhibitors which can be purified and cloned from human stomach cancer cell line MKN45 as specific inhibitors against hepatocyte growth factor activator (HGFA). HAI-2 was identical with the protein originally reported as placental bikunin. Both proteins contain two Kunitz inhibitor domains (KDs), of which the first domain (KD1) is mainly responsible for the inhibitory activity against HGFA, and are expressed ubiquitously in various tissues. In this study, we cloned the genes coding for these two structurally similar proteins by screening of human genomic bacterial artificial chromosome (BAC) library and their genomic structures were compared. HAI-1 and -2 genes consist of 11 and 8 exons spanning 12 kbp and 12.5 kbp, respectively. Three exons were inserted between KD1 and KD2 of each gene, of which the middle one was the low-density lipoprotein (LDL) receptor-like domain (HAI-1) and the testis specific exon (HAI-2). Apparently homologous regions between HAI-1 and -2 were not found in 5'-flanking region and neither TATA nor CAAT box was present. The genes were mapped to chromosome 15q15 (HAI-1) and 19q13.11 (HAI-2). These results suggested that although HAI-1 and -2 genes might be derived from same ancestor gene, they acquired distinctive in vivo roles during their evolution.

Amino Acid Sequence↗

PubMed2001

Genomic structure of the human MAD2 gene and mutation analysis in human lung and breast cancers.

Some of the many human cancers that exhibit chromosomal instability also carry mutations in mitotic checkpoint genes and/or reveal reduced expression of some of those genes, such as hMAD2. To facilitate investigation of alterations of hMAD2, we determined its genomic structure and intronic primers designed to amplify the entire coding region. Since general impairment of the mitotic checkpoint is frequently reported in lung cancers, and reduced expression of hMAD2 has been reported in breast cancers as well, we searched for mutations throughout the coding sequence of this gene in the genomic DNA of 30 primary lung tumors, 30 lung-cancer cell lines and 48 primary breast cancers. Our approach, which involved polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis and direct sequencing, revealed nucleotide variants in only two of the 108 specimens. One was a cytosine-to-adenine substitution 3 bp upstream of exon 4 that occurred in one lung cancer cell line and one primary breast tumor, a change that did not alter transcriptional sequence. The other was an adenine-to-guanine substitution within exon 4, of the same lung cell line; this change already had been reported as a polymorphism. The results suggested that the hMAD2 gene is not commonly mutated in either lung nor breast cancers. Further studies should focus on other mechanisms that might account for reduced expression of the hMAD2 gene, and/or pursue analyses of other mitotic checkpoint genes for mutations in human cancer. Nevertheless, the genomic structure, the intronic primer sequences, and polymorphisms of the hMAD2 gene presented here will facilitate future studies to determine the full spectrum and frequency of the genetic events that can affect expression of the hMAD2 gene in human tumors.

Breast Neoplasms↗

PubMed2001

Genomic structure of three CLN3-like genes in Caenorhabditis elegans.

The genome of Caenorhabditis elegans is predicted to carry three genes similar to CLN3, the gene underlying juvenile neuronal ceroid lipofuscinosis. All three genes are transcribed and the genomic structure has been determined. The number and position of exons for two of the genes differ from that predicted from the genomic sequence, but no discrepancies with the genomic nucleotide sequence were found. Gene F07B10.1 (cln-3.1) is predicted to have 7 exons and to encode a protein of 424 amino acids. Gene C01G8.2 (cln-3.2) has 9 exons and encodes a protein of 435 amino acids. Gene ZC190.1 (cln-3.3) is predicted to have 9 exons and to encode a protein of 416 amino acids.

Animals↗

PubMed2003

[Study on the genome structure of human telomeric repeat binding factor 1 and its pseudogenes].

OBJECTIVE: To determine the genome structure of human telomeric repeat binding factor 1 (TERF1) and its pseudogenes. METHODS: Sequences were obtained from GenBank and analyzed using the BLAST program and other relevant biology program (Sequencher, DNA Strider and Autoassembler, etc) to determine the genome and pseudogenome structure of TERF1. PCR and sequencing were performed to verify the results. RESULT: TERF1 gene which mapped to 8q13 was divided into 10 exons. It had four processed pseudogenes located on chromosome 13, 18, 21 and X respectively (Psi TERF1-13 Psi TERF1-18 Psi TERF1-21 and Psi TERF1-X ). They were entire intronless TERF1 genes which lacked some exons. Three homologous fragments of at least 60 kb on the flanking region of Psi TERF1-13, Psi TERF1-18 and Psi TERF1-21, respectively were noted. CONCLUSION: TERF1 gene has 10 exons. It has four processed pseudogenes which are located on chromosome 13, 18, 21, and X, respectively. Large homologous fragments that belong to the recently duplicated segments are transchromosomal duplications.

Chromosome Mapping↗

PubMed2005

Genomic structure and transcriptional studies on the mouse ribosomal protein S3 gene: expression of U15 small nucleolar RNA.

Ribosomal protein S3 (rpS3) is a multifunctional ribosomal protein (RP) which is known to function as a DNA repair endonuclease as well as an RP. Recently, it was reported that rpS3 is involved in apoptosis. We identified the complete 4760 base pair genomic structure of the mouse rpS3 gene, which is composed of 7 exons and 6 introns. Promoter study revealed that transcription of the mouse rpS3 gene started at two C residues embedded in the 5'-terminal oligopyrimidine tract (5'-TOP); this was then compared with the human counterpart. Functional U15 small nucleolar RNAs (snoRNAs) were expressed from the first and the fifth introns. About 300 base pairs (bps) upstream of the 5'-untranslated region (5'-UTR) of the mouse rpS3 gene was sufficient to show maximum transcription activity. This report shows the conservation of the genomic structure of the rpS3 gene in vertebrates and characteristics of its promoter similar to those of promoters of other mammalian RPs.

5' Untranslated Regions↗

PubMed2003

Genomic structure and promoter analysis of PKC-delta.

Protein kinase C-delta (PKC-delta) is a ubiquitously expressed kinase involved in a variety of cellular signaling pathways including cell growth, differentiation, apoptosis, tumor promotion, and carcinogenesis. While signaling pathways downstream of PKC-delta are well studied, the regulation of the gene has not been extensively analyzed. A mouse genomic DNA fragment containing the PKC-delta gene was sequenced by the primer-walking method, and the subsequent DNA sequence data were used as a query to clone Caenorhabditis elegans and human genomic homologs from the publicly available genomic databases. The genomic structures of C. elegans, mouse, rat, and human PKC-delta were analyzed, and the result revealed that PKC-delta genes comprise 12, 18, 19, and 18 exons for C. elegans, mouse, rat, and human, respectively. The translation start methionine resides in the second exon in mouse and human and in the third exon in rat. The first intron between the first exon and the exon with the translation start methionine in mammalian genes represents a very large gap, as long as 17 kb in human, indicating a complexity involved in gene splicing. Overall exon-intron genomic structure is highly conserved among mammals, while significantly diverged in C. elegans. Putative transcription factor binding sites on the 1.7-kb promoter region of the mouse gene suggest that PKC-delta might be involved in spermatogenesis, embryogenesis, development, brain generation, immune response, oxidative environment, and oncogenesis. Studies on the promoter and subsequent biological testing on mouse keratinocytes indicate that tumor necrosis factor (TNF)-alpha increases the expression of PKC-delta, and this correlates with the time of NFkappaB nuclear translocation and activation. This TNF-alpha-mediated upregulation of PKC-delta is repressed in keratinocytes that are preinfected with IkappaB superrepressor adenovirus, suggesting that NFkappaB is involved directly in PKC-delta expression.

Animals↗

PubMed2000

SLC7A7 genomic structure and novel variants in three Japanese lysinuric protein intolerance families.

Lysinuric protein intolerance (LPI) is a rare inherited disease caused by defective transport of the dibasic amino acids at the basolateral membranes of epithelial cells in the renal tubules and small intestine. The metabolic defect leads to brain dysfunction caused by hyperammonemia with a functional impairment of the urea cycle. Recently, mutations in the human SLC7A7 cDNA coding for y(+)LAT-1, which express dibasic amino acid transport activity, were reported to be responsible for LPI. In the present study, we examined the genomic structure of SLC7A7 by DNA sequencing of PCR products, and determined that the gene had 11 exons and 10 introns spanning about 18 kb of genomic DNA. We also identified an alternative RNA splicing at the 5' untranslated region of the SLC7A7 mRNA in human peripheral blood leukocytes, cultured lymphoblasts, and fibroblasts. As a result of mutational analysis of SLC7A7 in three Japanese LPI families, we found a nonsense mutation (R410X), a splicing mutation(911+1G>A) in intron 4, and four silent polymorphisms (201C/T, 445A/G, 784C/T, 946T/C). Identification of the genomic structure of SLC7A7 may provide a molecular basis for a genetic survey for LPI.

5' Untranslated Regions↗

PubMed2006

Genomic structure and expression of uncoupling protein 2 genes in rainbow trout (Oncorhynchus mykiss).

BACKGROUND: Uncoupling protein 2 (UCP2) belongs to the superfamily of mitochondrial anion carriers that dissociate the respiratory chain from ATP synthesis. It has been determined that UCP2 plays a role in several physiological processes such as energy expenditure, body weight control and fatty acid metabolism in several vertebrate species. We report the first characterization of UCP2s in rainbow trout (Oncorhynchus mykiss). RESULTS: Two UCP2 genes were identified in the rainbow trout genome, UCP2A and UCP2B. These genes are 93% similar in their predicted amino acid sequences and display the same genomic structure as other vertebrates (8 exons and 7 introns) spanning 4.2 kb and 3.2 kb, respectively. UCP2A and UCP2B were widely expressed in all tissues of the study with a predominant level in macrophage-rich tissues and reproductive organs. In fry muscle we observed an increase in UCP2B expression in response to fasting and a decrease after refeeding in agreement with previous studies in human, mouse, rat, and marsupials. The converse expression pattern was observed for UCP2A mRNA which decreased during fasting, suggesting different metabolic roles for UCP2A and UCP2B in rainbow trout muscle. Phylogenetic analysis including other genes from the UCP core family located rainbow trout UCP2A and UCP2B with their orthologs and suggested an early divergence of vertebrate UCPs from a common ancestor gene. CONCLUSION: We characterized two UCP2 genes in rainbow trout with similar genomic structures, amino acid sequences and distribution profiles. These genes appeared to be differentially regulated in response to fasting and refeeding in fry muscle. The genomic organization and phylogeny analysis support the hypothesis of a common ancestry between the vertebrate UCPs.

Amino Acid Sequence↗

PubMed1996

Genomic structure of a Y-specific ribonucleic acid binding motif-containing gene: a putative candidate for a subset of male infertility.

The genetic basis of infertility remains unclear in a majority of infertile men. Deletion mapping studies suggest that genes on the long arm of the Y-chromosome (Yq) may be important in the spermatogenic process and may play a pathogenetic role in a subset of infertile men. Complementary DNA sequences of two Y-specific genes that contain ribonucleic acid binding motifs and, therefore, referred to as RBM genes (previously named YRRM) were published recently. To develop a PCR-single strand conformation polymorphism strategy for detection of point mutations in the RBM gene(s) in infertile men, we determined the genomic structure and flanking sequences at the intron-exon junctions. Two separate strategies were used in parallel to isolate the genomic fragment bearing the RBM gene. The first strategy employed screening of a P1 genomic library using PCR primers corresponding to the sequences in the 5'- and 3'-ends of the published RBM-1 complementary DNA sequence. The second strategy used subcloning of the YAC clone 925D10 (that contained the RBM gene described here) into cosmids. The P1 and cosmid clones were further restriction mapped and subcloned for DNA sequencing. Because the sequences contained in the P1 and cosmid clones were identical, the sequence information was pooled. A 15-kilobase genomic segment includes the entire RBM gene. The genomic structure of this RBM gene is characterized by 12 exons and 11 introns. There is considerable homology among exons VII, VIII, IX, and X; each encodes one of the SRGY boxes. Several introns also have a high degree of homology among them (introns VI, VII, VIII, and IX). Eleven of the 12 exons have complete sequence homology with the RBM-1 sequence. There is 1 base difference in exon IV at position 495 (a T in the previously published DNA sequence vs. an A in the sequence reported here). The exonic sequences of this gene are distinct from that of the RBM-2 gene. The flanking sequences at the exon-intron junctions were also determined and are reported. Reverse transcription-PCR analysis, using human testis ribonucleic acid suggests that this gene is either not expressed in the testis or, more likely, the single base difference from RBM1 represents a polymorphism in the YAC clone. A high degree of homology between intronic and exonic sequences within the same gene and between different members of the RBM gene family (data not reported in this paper) suggests origin from common ancestral sequences; this also indicates that development of a single strand conformation polymorphism approach for detection of point mutations is likely to prove difficult for some of the exons of this gene.

Base Sequence↗

PubMed1998

Genomic structure and expression of murine poly(A) binding protein II gene.

The genomic structure and expression of the murine poly(A) binding protein II (mPABII) gene were analyzed by using genomic DNA and cDNA clones. The expression level of the mPABII gene varied among tissues. Besides two transcripts detected in all tissues, an additional transcript was detected in testis. The mPAB gene has pseudogenes or related genes in its genome.

Amino Acid Sequence↗

PubMed1999

Genomic structure and chromosomal mapping of the murine and human Mbd1, Mbd2, Mbd3, and Mbd4 genes.

DNA methylation is essential for murine development and is implicated in the control of gene expression. MeCP2, MBD1, MBD2, MBD3, and MBD4 comprise a family of mammalian, nuclear proteins related by the presence in each of an amino acid motif called the methyl-CpG binding domain (MBD). Each of these proteins, with the exception of MBD3, is capable of binding specifically to methylated DNA. MeCP2, MBD1 and MBD2 can also repress transcription. We describe the genomic structure and chromosomal localization of the human and murine Mbd1, Mbd2, Mbd3, and Mbd4 genes. We find that the highly similar MBD2 and MBD3 proteins are encoded by genes that map to different chromosomes in humans and mice but show a similar genomic structure. The Mbd1 and Mbd2 genes, in contrast, map together to murine and human Chromosomes (Chrs)18. The Mbd3 and Mbd4 genes map to murine Chrs 10 and 6, respectively, while the human MBD3 and MBD4 genes map to Chrs 19 and 3, respectively.

Animals↗

PubMed2000

Protein NMR spectroscopy in structural genomics.

Protein NMR spectroscopy provides an important complement to X-ray crystallography for structural genomics, both for determining three-dimensional protein structures and in characterizing their biochemical and biophysical functions.

Animals↗

PubMed2000

Human protein tyrosine phosphatase-like gene: expression profile, genomic structure, and mutation analysis in families with ARVD.

The mouse protein tyrosine phosphatase-like gene (Ptpla) was recently cloned and data suggested that it plays a role in myogenesis and cardiogenesis. The human homologue (PTPLA) was mapped to chromosome 10p13-14, a region where we have mapped a locus responsible for arrhythmogenic right ventricular dysplasia (ARVD). As a positional candidate gene, we characterized PTPLA by determining its tissue expression, its genomic structure, and we also screened for mutations in the ARVD patients. Northern analysis demonstrated PTPLA is preferentially expressed in both adult and fetal heart. A much lower expression was detected in skeletal and smooth muscle tissues. Virtually no expression was observed in other tissues. The protein-encoding sequences of PTPLA consist of seven exons. A sequence variation (Lys64Gln) was found in all the affecteds in a large ARVD family. However, the same variant was also detected in normal control subjects (three alleles/100 chromosomes). Thus, the variant (Lys64Gln) is not responsible for ARVD in our family and is a benign polymorphism. Nevertheless, its tissue-specific expression in the developing and adult heart suggest PTPLA has a role in regulating cardiac development, differentiation, or other cellular events. The genomic structure and intragenic polymorphism of PTPLA should be useful for further clinical and genetic studies such as gene targeting of PTPLA.

Arrhythmogenic Right Ventricular Dysplasia↗

PubMed1997

cDNA cloning, genomic structure, and chromosome mapping of the human epithelial membrane protein CL-20 gene (EMP1), a member of the PMP22 family.

CL-20 is a novel gene encoding a protein that is structurally related to but distinct from the peripheral myelin protein PMP22. Like PMP22, CL-20 is likely to play important roles in the regulation of cell proliferation, differentiation, and cell death. In this study, we describe the cloning and sequencing of a cDNA encoding the human homologue of CL-20 and characterize the genomic structure of this gene. The hCL-20 gene (HGMW-approved symbol EMP1) encodes a protein of 157 amino acids that exhibits 76% identity to the rabbit CL-20 and to the rat EMP-1, which have been described recently, and 39% identity to human PMP22. CL-20 contains four hydrophobic domains, suggesting that it is an integral membrane protein. In particular the second hydrophobic domain encoded within the fourth exon is highly conserved among CL-20, EMP-1, and PMP22, suggesting a functional role for this region. CL-20 mRNA is abundant in squamous-differentiated bronchial epithelial cells; however, low levels of CL-20 mRNA can be detected in several human tissues by Northern analysis. Retinoic acid, which inhibits squamous differentiation, represses CL-20 expression in normal human bronchial epithelial cells. The genomic structure of the hCL-20 gene was analyzed using a P1 vector containing this gene. The hCL-20 gene contains five exons about 0.2, 0.12, 0.1, 0.14, and 2.2 kb and four introns about 15, 1.9, 0.1, and 0.7 kb. We have mapped the hCL-20 gene to chromosome 12p12 by fluorescence in situ hybridization.

Amino Acid Sequence↗

PubMed2003

Directed evolution approach to a structural genomics project: Rv2002 from Mycobacterium tuberculosis.

One of the serious bottlenecks in structural genomics projects is overexpression of the target proteins in soluble form. We have applied the directed evolution technique and prepared soluble mutants of the Mycobacterium tuberculosis Rv2002 gene product, the wild type of which had been expressed as inclusion bodies in Escherichia coli. A triple mutant I6TV47MT69K (Rv2002-M3) was chosen for structural and functional characterizations. Enzymatic assays indicate that the Rv2002-M3 protein has a high catalytic activity as a NADH-dependent 3alpha, 20beta-hydroxysteroid dehydrogenase. We have determined the crystal structures of a binary complex with NAD(+) and a ternary complex with androsterone and NADH. The structure reveals that Asp-38 determines the cofactor specificity. The catalytic site includes the triad Ser-140Tyr-153Lys-157. Additionally, it has an unusual feature, Glu-142. Enzymatic assays of the E142A mutant of Rv2002-M3 indicate that Glu-142 reverses the effect of Lys-157 in influencing the pKa of Tyr-153. This study suggests that the Rv2002 gene product is a unique member of the SDR family and is likely to be involved in steroid metabolism in M. tuberculosis. Our work demonstrates the power of the directed evolution technique as a general way of overcoming the difficulties in overexpressing the target proteins in soluble form.

Alcohol Oxidoreductases↗