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PubMed2004

Identification and characterization of a nitrate transporter gene in Neurospora crassa.

The Neurospora crassa genome database was searched for sequence similarity to crnA, a nitrate transporter in Aspergillus nidulans. A 3.9-kb fragment (contig 3.416, subsequence 183190-187090) was cloned by PCR. The gene coding for this nitrate transporter was termed nit-10. The nit-10 gene specifies a predicted polypeptide containing 541 amino acids with a molecular mass of 57 kDa. In contrast to crnA, which is clustered together with niaD, encoding nitrate reductase, and niiA, encoding nitrite reductase, nit-10 is not linked to nit-3 (nitrate reductase), nit-6 (nitrite reductase), or to nit-2, nit-4 (both are positive regulators of nit-3), or nmr (negative regulator of nit-3) in Neurospora crassa. A nit-10 rip mutant failed to grow in the medium when nitrate (< 10 mM) was used as the sole nitrogen source, but grew similarly to wild type when nitrate concentration was 10 mM or higher. In addition, it showed strong sensitivity to cesium in the presence of nitrate and resistance to chlorate in the presence of alanine, proline, or hypoxanthine. The expression of nit-10 required nitrate induction and was subject to repression by nitrogen metabolites such as glutamine. Expression of nit-10 also required functional products of nit-2 and nit-4. The half-life of nit-10 mRNA was determined to be approximately 2.5 min.

Alanine↗

PubMed2004

Cloning and tissue distribution of the human B3GALT7 gene, a member of the beta1,3-Glycosyltransferase family.

We report here the cloning and tissue distribution of the human B3GALT7 gene, a member of the beta1,3-Glycosyltransferase family, structurally related to the beta1,3-Galactosyltransferase family and beta1,3- N -acetylglucosaminyltransferase family, isolated from a human lung cDNA library. B3GALT7 is mapped to chromosome 19q13.2 by browsing the UCSC genomic database. It contains an ORF with length of 1191bp, encoding a protein with a signal peptide sequence and galactosyl-T domain, and its molecular weight and isoelectric point is predicted to be 43.3 kDa and 8.67 respectively. The molecular weight of the protein when expressed in E. coli corresponded to that expected. Northern blotting showed that B3GALT7 was highly expressed in lung, throat and ileum, whereas the expression level was low in tongue, breast, uteri, testis. In addition, it was also demonstrated that B3GALT7 is differentially transcribed in human tumor cell lines.

Amino Acid Sequence↗

PubMed1997

Identification of genetic changes associated with drug resistance by reverse in situ hybridization.

The molecular cytogenetic techniques of comparative genomic hybridization (CGH) and reverse in situ hybridization (REVISH) allow the entire genomes of tumours to be screened for genetic changes without the requirement for specific probes or markers. In order to define the ability of REVISH to detect and map regions of amplification associated with drug resistance, we investigated a panel of cell lines selected for resistance to doxorubicin and intrinsic sensitivity to topoisomerase II-inhibitory drugs. We have defined a modified REVISH protocol, which involves double hybridizations with genomic DNA from the test cell lines and chromosome-specific whole chromosome paints to identify the chromosomes to which the amplicons localize. Sites of amplification are then mapped by fractional length measurements (Flpter), using published genome databases. Our findings show that amplification of the topoisomerase II alpha gene is readily detected and mapped, as is amplification of the MDR and MRP loci. Interestingly, REVISH detected a new amplicon in the doxorubicin-resistant lung cancer cell line, GLC4-ADR, which mapped to chromosome 1q. REVISH is therefore ideally suited to characterize genetic changes specific for drug resistance within a background of genetic anomalies associated with tumour progression.

Chromosome Mapping↗

PubMed2003

ATP-dependent reduction of cysteine-sulphinic acid by S. cerevisiae sulphiredoxin.

Proteins contain thiol-bearing cysteine residues that are sensitive to oxidation, and this may interfere with biological function either as 'damage' or in the context of oxidant-dependent signal transduction. Cysteine thiols oxidized to sulphenic acid are generally unstable, either forming a disulphide with a nearby thiol or being further oxidized to a stable sulphinic acid. Cysteine-sulphenic acids and disulphides are known to be reduced by glutathione or thioredoxin in biological systems, but cysteine-sulphinic acid derivatives have been viewed as irreversible protein modifications. Here we identify a yeast protein of relative molecular mass M(r) = 13,000, which we have named sulphiredoxin (identified by the US spelling 'sulfiredoxin', in the Saccharomyces Genome Database), that is conserved in higher eukaryotes and reduces cysteine-sulphinic acid in the yeast peroxiredoxin Tsa1. Peroxiredoxins are ubiquitous thiol-containing antioxidants that reduce hydroperoxides and control hydroperoxide-mediated signalling in mammals. The reduction reaction catalysed by sulphiredoxin requires ATP hydrolysis and magnesium, involving a conserved active-site cysteine residue which forms a transient disulphide linkage with Tsa1. We propose that reduction of cysteine-sulphinic acids by sulphiredoxin involves activation by phosphorylation followed by a thiol-mediated reduction step. Sulphiredoxin is important for the antioxidant function of peroxiredoxins, and is likely to be involved in the repair of proteins containing cysteine-sulphinic acid modifications, and in signalling pathways involving protein oxidation.

Adenosine Triphosphate↗

PubMed2002

Life and death in paradise.

Over 500 researchers participated in a recent American Association for Cancer Research special conference, entitled "Apoptosis and Cancer: Basic Mechanisms and Therapeutic Opportunities in the Post-Genomic Era" (February 13-17, 2002) in sunny Hawaii (Hilton Waikoloa village, Kona, Hawaii). The meeting participants presented the most recent findings on the mechanisms regulating cell death in cancer. In the past decade, apoptosis research has undergone a quantum leap, metamorphosing from a descriptive, phenomenological discipline into a molecularly defined, highly complex signalling field. This transformation was highlighted in the conference's opening talk by meeting co-organizer, John Reed (The Burnham Institute, La Jolla, CA). Reed and colleagues used published protein functional information and bio-informatic mining of the available human genome databases to tabulate the number of human proteins predicted to be involved in regulating apoptosis. The list includes 11 catalytically active caspases, 26 CARD (caspase associated recruitment domain)-, 32 DD (death domain)-, 12 DED (death effector domain)-, 8 BIR (baculovirus inhibitor of apoptosis protein region)-, 24 BH (Bcl-2 homology)-, and 34 PAAD/PYD (pyrin/PAAD)-containing sequences.

Animals↗

PubMed1996

CpG islands of chicken are concentrated on microchromosomes.

The chicken karyotype comprises 39 chromosome pairs of which at least 29 are 'microchromosomes'. Microchromosomes account for about 25% of the genomic DNA, but they are cytologically indistinguishable from one another (1). Due to technical limitations there is a strong bias of mapped genes within the chicken genome database ChickGBASE (2) towards macrochromosomes 1-6 and Z, with specific assignments to only one microchromosome (3,4). Several genes have, however, been assigned to the microchromosome group as a whole (3,5-9), demonstrating that these tiny chromosomes do not represent genetically inert DNA. To determine the overall chromosomal distribution of genes, as well as to provide a mapping resource, we prepared a CpG island library from chicken using differential binding to a methyl-CpG chicken using differential binding to a methyl-CpG binding column before and after de novo methylation (10). Surprisingly, we found that chicken CpG islands are highly concentrated on the microchromosomes, whereas macrochromosomes 1-6 are comparatively gene-poor by this assay. Our results raise the possibility that gene density on chicken microchromosomes approaches the maximum value known for vertebrates.

Animals↗

PubMed2002

High-throughput retroviral tagging to identify components of specific signaling pathways in cancer.

Genetic screens carried out in lower organisms such as yeast, Drosophila melanogaster and Caenorhabditis elegans have revealed many signaling pathways. For example, components of the RAS signaling cascade were identified using a mutant eye phenotype in D. melanogaster as a readout. Screening is usually based on enhancing or suppressing a phenotype by way of a known mutation in a particular signaling pathway. Such in vivo screens have been difficult to carry out in mammals, however, owing to their relatively long generation times and the limited number of animals that can be screened. Here we describe an in vivo mammalian genetic screen used to identify components of pathways contributing to oncogenic transformation. We applied retroviral insertional mutagenesis in Myc transgenic (E mu Myc) mice lacking expression of Pim1 and Pim2 to search for genes that can substitute for Pim1 and Pim2 in lymphomagenesis. We determined the chromosomal positions of 477 retroviral insertion sites (RISs) derived from 38 tumors from E mu Myc Pim1(-/-) Pim2(-/-) mice and 27 tumors from E mu Myc control mice using the Ensembl and Celera annotated mouse genome databases. There were 52 sites occupied by proviruses in more than one tumor. These common insertion sites (CISs) are likely to contain genes contributing to tumorigenesis. Comparison of the RISs in tumors of Pim-null mice with the RISs in tumors of E mu Myc control mice indicated that 10 of the 52 CISs belong to the Pim complementation group. In addition, we found that Pim3 is selectively activated in Pim-null tumor cells, which supports the validity of our approach.

Animals↗

PubMed2001

A Gr receptor is required for response to the sugar trehalose in taste neurons of Drosophila.

We recently identified from the Drosophila genome database a large family of G protein-coupled receptor genes, the Gr genes, and predicted that they encode taste receptors on the basis of their structure and specificity of expression. The expression of Gr genes in gustatory neurons has subsequently been confirmed and 56 family members have been reported. Here we provide functional evidence that one Gr gene, Gr5a, encodes a taste receptor required for response to the sugar trehalose. In two different mutants that carry deletions in Gr5a, electrophysiological and behavioral responses to trehalose were diminished but the response to sucrose was unaffected. Transgenic rescue experiments showed that Gr5a confers response to trehalose. The results correlate a particular taste ligand with a Gr receptor and indicate a role for G protein-mediated signaling in the transduction of sweet taste in Drosophila.

Action Potentials↗

PubMed2026

Association between smoking, tumor genetics, and outcomes in men with metastatic prostate cancer.

PURPOSE: Smoking has been associated with increased metastatic prostate cancer mortality, but the mechanisms behind this are largely unknown. We hypothesized that smoking increases the risk of genetic alterations associated with aggressive disease and/or the transformation to neuroendocrine prostate cancer (NEPC). PATIENTS AND METHODS: We utilized the Prostate Cancer Precision Medicine Multi-institutional Collaborative Effort (PROMISE) clinical genomic database for this retrospective analysis. We associated patient characteristics and tumor genetic data with smoking exposure at diagnosis (current, former, never and pack years) and with clinical outcomes, including overall survival (OS) from diagnosis or time to developing metastatic disease and NEPC status. RESULTS: We identified 2353 men with prostate cancer and next generation somatic tumor sequencing evaluable for analysis in PROMISE, including 8% current, 39% former, and 52% never smokers. Current smokers were more likely to be younger and to have metastatic (M1 or N1) disease at diagnosis, and less likely to have prior local therapy (all p < 0.001). Current smoking was associated with worse OS from diagnosis (99.9 mo vs 137.6 mo, HR 1.42, 95% CI 1.14-1.77), which remained significant after adjusting for disease characteristics. We found no difference in the percentage of NEPC at initial diagnosis or at any time between current, former, and never smokers (p = 0.8). We found positive associations between smoking status and genetic alterations in SPOP (current: 15%, former 6.7%, never 3.8%; p = 0.018), FGFR1 (current 10%, former 0.4%, never 1.1% p = 0.001), and ARID1A (current 5.1%, former 2.2%, never 0.4%; p = 0.035) in patients with metastatic androgen pathway modulator sensitive prostate cancer (APMS). CONCLUSION: Active smoking is associated with worse overall and prostate cancer specific survival as compared to never/former smoking and was associated with specific tumor genetic alterations but not small cell/NEPC transformation.

Journal Article↗

PubMed2003

Identification and characterisation of GPR100 as a novel human G-protein-coupled bradykinin receptor.

G-protein-coupled receptor 100 (GPR100) was discovered by searching the human genome database for novel G-protein-coupled peptide receptors. Full-length GPR100 was amplified from a cDNA library of the neuroendocrine cell line BON, which is derived from a human pancreas carcinoid. The open-reading frame, present on a single exon, coded for a protein of 374 amino acids with highest sequence identity (43%) to the human orphan somatostatin- and angiotensin-like peptide receptor. The analysis of chromosomal localisation mapped the GPR100 gene to chromosome 1q21.2-q21.3. The stable expression of GPR100 in Chinese hamster ovary cells together with aequorin as calcium sensor and the promiscuous G-protein subunit alpha16 as signal transducer revealed bradykinin and kallidin as effectors to elicit a calcium response. Dose-response curves yielded EC50 values for both ligands in the low nanomolar range, while the respective analogues without arginine at the carboxy-terminus were inactive. Calcium mobilisation was inhibited by the phospholipase C blocker U73122, but not by pertussis toxin, suggesting the involvement of the G-protein subunit alphaq and not alphai or alphao in signal transduction. In line with the main function of kinins as peripheral hormones, we found that GPR100 was expressed predominantly in tissues like pancreas, heart, skeletal muscle, salivary gland, bladder, kidney, liver, placenta, stomach, jejunum, thyroid gland, ovary, and bone marrow, but smaller amounts were also detected in the brain and in cell lines derived from tumours of various origins.

Amino Acid Sequence↗

PubMed2005

Intronic CA-repeat and CA-rich elements: a new class of regulators of mammalian alternative splicing.

We have recently identified an intronic polymorphic CA-repeat region in the human endothelial nitric oxide synthase (eNOS) gene as an important determinant of the splicing efficiency, requiring specific binding of hnRNP L. Here, we analyzed the position requirements of this CA-repeat element, which revealed its potential role in alternative splicing. In addition, we defined the RNA binding specificity of hnRNP L by SELEX: not only regular CA repeats are recognized with high affinity but also certain CA-rich clusters. Therefore, we have systematically searched the human genome databases for CA-repeat and CA-rich elements associated with alternative 5' splice sites (5'ss), followed by minigene transfection assays. Surprisingly, in several specific human genes that we tested, intronic CA RNA elements could function either as splicing enhancers or silencers, depending on their proximity to the alternative 5'ss. HnRNP L was detected specifically bound to these diverse CA elements. These data demonstrated that intronic CA sequences constitute novel and widespread regulatory elements of alternative splicing.

Alternative Splicing↗

PubMed1998

Isolation and characterization of a novel actin filament-binding protein from Saccharomyces cerevisiae.

We purified a novel actin filament (F-actin)-binding protein from the soluble fraction of Saccharomyces cerevisiae by successive column chromatographies by use of the 125I-labeled F-actin blot overlay method. The purified protein showed a minimum Mr of about 140 kDa on SDS-polyacrylamide gel electrophoresis and we named it ABP140. A search with the partial amino acid sequences of ABP140 against the Saccharomyces Genome Database revealed that the open reading frame of the ABP140 gene (ABP140) corresponded to YOR239W fused with YOR240W by the +1 translational frame shift. The encoded protein consisted of 628 amino acids with a calculated Mr of 71,484. The recombinant protein interacted with F-actin and showed the activity to crosslink F-actin into a bundle. Indirect immunofluorescence study demonstrated that ABP140 was colocalized with both cortical actin patches and cytoplasmic actin cables in intact cells. However, elimination of ABP140 by gene disruption did not show a deleterious effect on cell growth or affect the organization of F-actin. These results indicate that ABP140 is not required for cell growth but may be involved in the reorganization of F-actin in the budding yeast.

Actins↗

PubMed2004

Genomic annotation of the meningioma tumor suppressor locus on chromosome 1p34.

Meningioma is a frequently occurring tumor of the meninges surrounding the central nervous system. Loss of the short arm of chromosome 1 (1p) is the second most frequent chromosomal abnormality observed in these tumors. Previously, we identified a 3.7 megabase (Mb) region of consistent deletion on 1p33-p34 in a panel of 157 tumors. Loss of this region was associated with advanced disease and predictive for tumor relapse. In this report, a high-resolution integrated map of the region was constructed (CompView) to identify all markers in the smallest region of overlapping deletion (SRO). A regional somatic cell hybrid panel was used to more precisely localize those markers identified in CompView as within or overlapping the region. Additional deletion mapping using microsatellites localized to the region narrowed the SRO to approximately 2.8 Mb. The 88 markers remaining in the SRO were used to screen genomic databases to identify large-insert clones. Clones were assembled into a physical map of the region by PCR-based, sequence-tagged site (STS) content mapping. A sequence from clones was used to validate STS content by electronic PCR and to identify transcripts. A minimal tiling path of 43 clones was constructed across the SRO. Sequence data from the most current sequence assembly were used for further validation. A total of 59 genes were ordered within the SRO. In all, 17 of these were selected as likely candidates based on annotation using Gene Ontology Consortium terms, including the MUTYH, PRDX1, FOXD2, FOXE3, PTCH2, and RAD54L genes. This annotation of a putative tumor suppressor locus provides a resource for further analysis of meningioma candidate genes.

Chromosomes, Human, Pair 1↗

PubMed2000

Identification of the differential distribution patterns of mRNAs and consensus binding sequences for mouse DAF-16 homologues.

daf-16 is a forkhead-type transcription factor, functioning downstream of insulin-like signals, and is known to be critical to the regulation of life span in Caenorhabditis elegans. Mammalian DAF-16 homologues include AFX, FKHR and FKHRL1, which contain a conserved forkhead domain and three putative phosphorylation sites for the Ser/Thr kinase Akt/protein kinase B (PKB), as well as for DAF-16. To assess the function of the homologues, we examined tissue distribution patterns of mRNAs for DAF-16 homologues in mice. In the embryos, expressions of AFX, FKHR and FKHRL1 mRNAs were complementary to each other and were highest in muscle, adipose tissue and embryonic liver. The characteristic expression pattern remained in the adult, except that signals of FKHRL1 became evident in more tissues, including the brain. In order to clarify whether each DAF-16 homologue had different target genes, we determined the consensus sequences for the binding of DAF-16 and the mouse homologues. The binding sequences for all four proteins shared a core sequence, TTGTTTAC, daf-16 family protein-binding element (DBE) binding protein. However, electrophoretic mobility shift assay showed that the binding affinity of DAF-16 homologues to the core sequence was stronger than that to the insulin-responsive element in the insulin-like growth factor binding protein-1 promoter region, which has been identified as a binding sequence for them. We identified one copy of the DBE upstream of the first exon of sod-3 by searching the genomic database of C. elegans. Taken together, DAF-16 homologues can fundamentally regulate the common target genes in insulin-responsive tissues and the specificity to target genes of each protein is partially determined by the differences in their expression patterns.

Alternative Splicing↗

PubMed2007

Oligosaccharides, neoglycoproteins and humanized plastics: their biocatalytic synthesis and possible medical applications.

Glycobiology has become one of the fastest growing branches of the biological sciences. Glycomics, which is the study of an organism's entire array of oligosaccharides, is now emerging as the third informatics wave after genomics and proteomics. For example, it is possible to see this progress in the KEGG (Kyoto Encyclopedia of Genes and Genomes) database (http://www.genome.jp/kegg/pathway/map/map01110.html). The interest in this area stems from the realization that carbohydrates, especially oligosaccharides, and their interactions with proteins, play diverse informative roles in all organisms, and that more than half of all proteins are glycosylated. When the biological and pharmaceutical importance of glycoconjugates is considered, it is surprising how little glycobiotechnology has developed. This review reports the latest developments in the biocatalytic synthesis of oligosaccharides and glycoconjugates, with special attention paid to the glycosyltransferase approach. The second part of the review takes the 'conceptual approach' and covers possible medical applications of synthesized glycoconjugates. Various new examples of the conjugation of glyco-informative saccharide sequence to known pharmaceuticals or biomaterials are cited.

Catalysis↗

PubMed2004

Characterization of human brain nicotinamide 5'-mononucleotide adenylyltransferase-2 and expression in human pancreas.

NMNAT (nicotinamide 5'-mononucleotide adenylyltransferase; EC 2.7.7.1) catalyses the transfer of the adenylyl group from ATP to NMN to form NAD. We have cloned a novel human NMNAT cDNA, designated hNMNAT-2, from human brain. The cDNA contains a 924 bp open reading frame that encodes a 307 amino acid peptide that was expressed as a histidine-patch-containing thioredoxin fusion protein. Expressed hNMNAT-2 shared only 35% amino acid sequence homology with the human NMNAT enzyme (hNMNAT-1), but possessed enzymic activity comparable with hNMNAT-1. Using human genomic databases, hNMNAT-2 was localized to chromosome 1q25 within a 171 kb gene, whereas hNMNAT-1 is on chromosome 1p32-35. Northern blot analysis revealed highly restricted expression of hNMNAT-2 to brain, heart and muscle tissues, which contrasts with the wide tissue expression of hNMNAT-1; different regions of the brain exhibited differential expression of hNMNAT-2. Substitution mutations of either of two invariant residues, His-24 or Trp-92, abolished enzyme activity. Anti-peptide antibody to a unique epitope within hNMNAT-2 was produced, and immunohistochemical analysis of sections of normal adult human pancreas revealed that hNMNAT-2 protein was markedly expressed in the islets of Langerhans. However, the pancreatic exocrine cells exhibited weak expression of hNMNAT-2 protein. Sections of pancreas from insulinoma patients showed strong expression of hNMNAT-2 protein in the insulin-producing tumour cells, whereas acinar cells exhibited relatively low expression of hNMNAT-2 protein. These data suggest that the unique tissue-expression patterns of hNMNAT-2 reflect distinct functions for the isoforms in the regulation of NAD metabolism.

Amino Acid Sequence↗

PubMed2004

Putrescine biosynthesis in mammalian tissues.

L-ornithine decarboxylase provides de novo putrescine biosynthesis in mammals. Alternative pathways to generate putrescine that involve ADC (L-arginine decarboxylase) occur in non-mammalian organisms. It has been suggested that an ADC-mediated pathway may generate putrescine via agmatine in mammalian tissues. Published evidence for a mammalian ADC is based on (i) assays using mitochondrial extracts showing production of 14CO2 from [1-14C]arginine and (ii) cloned cDNA sequences that have been claimed to represent ADC. We have reinvestigated this evidence and were unable to find any evidence supporting a mammalian ADC. Mitochondrial extracts prepared from freshly isolated rodent liver and kidney using a metrizamide/Percoll density gradient were assayed for ADC activity using L-[U-14C]-arginine in the presence or absence of arginine metabolic pathway inhibitors. Although 14CO2 was produced in substantial amounts, no labelled agmatine or putrescine was detected. [14C]Agmatine added to liver extracts was not degraded significantly indicating that any agmatine derived from a putative ADC activity was not lost due to further metabolism. Extensive searches of current genome databases using non-mammalian ADC sequences did not identify a viable candidate ADC gene. One of the putative mammalian ADC sequences appears to be derived from bacteria and the other lacks several residues that are essential for decarboxylase activity. These results indicate that 14CO2 release from [1-14C]arginine is not adequate evidence for a mammalian ADC. Although agmatine is a known constituent of mammalian cells, it can be transported from the diet. Therefore L-ornithine decarboxylase remains the only established route for de novo putrescine biosynthesis in mammals.

Agmatine↗

PubMed2004

Glutamine availability up-regulates expression of the amino acid transporter protein ASCT2 in HepG2 cells and stimulates the ASCT2 promoter.

Glutamine transport into the human hepatoma cell line HepG2 is catalysed primarily by an ASCT2-type transporter identical in sequence with that cloned previously from JAR cells. An antibody raised against the C-terminus of the ASCT2 protein was shown to recognize ASCT2 on Western blots. Using this antibody, it was found that variation in cell growth rate did not affect ASCT2 expression, but both growth rate and ASCT2 expression were significantly reduced by glutamine deprivation. Expression of a number of other proteins was shown to be unaffected under these conditions. The sequence of the 5'-flanking region of the ASCT2 gene was derived from the human genome database. A 907 bp fragment of this sequence was directionally ligated into a luciferase reporter vector and was shown to exhibit promoter activity when transfected into HepG2 cells. Promoter activity was greatly reduced when transfection was performed in glutamine-free medium and was restored when glutamine was added post-transfection. The absence of other essential amino acids did not affect promoter activity, and glutamine deprivation did not affect the MCT1 (monocarboxylate transporter 1) promoter. These results indicate that both ASCT2 promoter activity and ASCT2 protein expression in these cells are dependent on glutamine availability.

Amino Acid Sequence↗