Home About us Contact
|
Home About us Contact | ||
|
|
||
Coding Sequence (coding + sequence)
Kinds of Coding Sequence Selected AbstractsRobust support for tardigrade clades and their ages from three protein-coding nuclear genesINVERTEBRATE BIOLOGY, Issue 2 2004Jerome C. Regier Abstract. Coding sequences (5,334 nt total) from elongation factor-1,, elongation factor-2, and the largest subunit of RNA polymerase II were determined for 6 species of Tardigrada, 2 of Arthropoda, and 2 of Onychophora. Parsimony and likelihood analyses of nucleotides and amino acids yielded strong support for Tardigrada and all internal nodes (i.e., 100% bootstrap support for Tardigrada, Eutardigrada, Parachela, Hypsibiidae, and Macrobiotidae). Results are in agreement with morphology and an earlier molecular study based on analysis of 18S ribosomal sequences. Divergence times have been estimated from amino acid sequence data using an empirical Bayesian statistical approach, which does not assume a strict molecular clock. Divergence time estimates are pre-Vendian for Tardigrada/Arthropoda, Vendian or earlier for Eutardigrada/Heterotardigrada, Silurian to Ordovician for Parachela/Apochela, Permian to Carboniferous for Hypsibiidae and Macrobiotidae, and Mesozoic for Isohypsibius/Thulinia (both within Hypsibiidae) and Macrobiotus/Richtersius (both within Macrobiotidae). [source] Ectopic activation of the transcription promoter for the testis-specific mouse Pgk-2 gene on elimination of a cis -acting upstream DNA regionDEVELOPMENT GROWTH & DIFFERENTIATION, Issue 4 2000Hiroshi Ando Transgenic mice carrying the coding sequence of ,-galactosidase, for which expression was driven by various upstream regions including the transcription promoter of the testis-specific mouse Pgk-2 gene, were generated. Expression of ,-galactosidase mRNA driven by the region between nucleotide positions , 1404 and + 61, with respect to the transcription initiation site numbered + 1, was examined by reverse transcription-mediated polymerase chain reaction, blot hybridization and in situ hybridization, and compared with that of endogenous Pgk-2 mRNA. The results revealed that the 1.4 kb DNA region is sufficient for determining the organ-specific, developmental stage-specific and spermatogenic stage-specific transcription of the mouse Pgk-2 gene. When the region between , 684 and + 61 was used to generate transgenic mice, ,-galactosidase mRNA was detectable not only in the testis, but also in other organs such as brain and lung. However, the timing and cell-type specificity of testicular expression of ,-galactosidase mRNA were retained in these mice. Because the region between , 1404 and , 685 repressed the Pgk-2 promoter in somatic cell-derived cell lines, it is suggested that the organ specificity of Pgk-2 transcription is achieved at least partly by negative regulation. [source] The Tol2kit: A multisite gateway-based construction kit for Tol2 transposon transgenesis constructsDEVELOPMENTAL DYNAMICS, Issue 11 2007Kristen M. Kwan Abstract Transgenesis is an important tool for assessing gene function. In zebrafish, transgenesis has suffered from three problems: the labor of building complex expression constructs using conventional subcloning; low transgenesis efficiency, leading to mosaicism in transient transgenics and infrequent germline incorporation; and difficulty in identifying germline integrations unless using a fluorescent marker transgene. The Tol2kit system uses site-specific recombination-based cloning (multisite Gateway technology) to allow quick, modular assembly of [promoter],[coding sequence],[3, tag] constructs in a Tol2 transposon backbone. It includes a destination vector with a cmlc2:EGFP (enhanced green fluorescent protein) transgenesis marker and a variety of widely useful entry clones, including hsp70 and beta-actin promoters; cytoplasmic, nuclear, and membrane-localized fluorescent proteins; and internal ribosome entry sequence,driven EGFP cassettes for bicistronic expression. The Tol2kit greatly facilitates zebrafish transgenesis, simplifies the sharing of clones, and enables large-scale projects testing the functions of libraries of regulatory or coding sequences. Developmental Dynamics 236:3088,3099, 2007. © 2007 Wiley-Liss, Inc. [source] A common gene exclusion mechanism used by two chemosensory systemsEUROPEAN JOURNAL OF NEUROSCIENCE, Issue 4 2009Luca Capello Abstract Sensory coding strategies within vertebrates involve the expression of a limited number of receptor types per sensory cell. In mice, each vomeronasal sensory neuron transcribes monoallelically a single V1R pheromone receptor gene, chosen from a large V1R repertoire. The nature of the signals leading to this strict receptor expression is unknown, but is apparently based on a negative feedback mechanism initiated by the transcription of the first randomly chosen functional V1R gene. We show, in vivo, that the genetic replacement of the V1rb2 pheromone receptor coding sequence by an unrelated one from the odorant receptor gene M71 maintains gene exclusion. The expression of this exogenous odorant receptor in vomeronasal neurons does not trigger the transcription of odorant receptor-associated signalling molecules. These results strongly suggest that despite the different odorant and vomeronasal receptor expression sites, function and transduction cascades, a common mechanism is used by these chemoreceptors to regulate their transcription. [source] Episodic ataxia type 1 mutations in the KCNA1 gene impair the fast inactivation properties of the human potassium channels Kv1.4-1.1/Kv,1.1 and Kv1.4-1.1/Kv,1.2EUROPEAN JOURNAL OF NEUROSCIENCE, Issue 11 2006Paola Imbrici Abstract Episodic ataxia type 1 (EA1) is an autosomal dominant neurological disorder characterized by constant muscle rippling movements (myokymia) and episodic attacks of ataxia. Several heterozygous point mutations have been found in the coding sequence of the voltage-gated potassium channel gene KCNA1 (hKv1.1), which alter the delayed-rectifier function of the channel. Shaker -like channels of different cell types may be formed by unique hetero-oligomeric complexes comprising Kv1.1, Kv1.4 and Kv,1.x subunits. Here we show that the human Kv,1.1 and Kv,1.2 subunits modulated the functional properties of tandemly linked Kv1.4-1.1 wild-type channels expressed in Xenopus laevis oocytes by (i) increasing the rate and amount of N-type inactivation, (ii) slowing the recovery rate from inactivation, (iii) accelerating the cumulative inactivation of the channel and (iv) negatively shifting the voltage dependence of inactivation. To date, the role of the human Kv1.4-1.1, Kv1.4-1.1/Kv,1.1 and Kv1.4-1.1/Kv,1.2 channels in the aetiopathogenesis of EA1 has not been investigated. Here we also show that the EA1 mutations E325D, V404I and V408A, which line the ion-conducting pore, and I177N, which resides within the S1 segment, alter the fast inactivation and repriming properties of the channels by decreasing both the rate and degree of N-type inactivation and by accelerating the recovery from fast inactivation. Furthermore, the E325D, V404I and I177N mutations shifted the voltage dependence of the steady-state inactivation to more positive potentials. The results demonstrate that the human Kv,1.1 and Kv,1.2 subunits regulate the proportion of wild-type Kv1.4-1.1 channels that are available to open. Furthermore, EA1 mutations alter heteromeric channel availability which probably modifies the integration properties and firing patterns of neurones controlling cognitive processes and body movements. [source] Expression of trunk Hox genes in the centipede Strigamia maritima: sense and anti-sense transcriptsEVOLUTION AND DEVELOPMENT, Issue 3 2006Carlo Brena SUMMARY We report the coding sequence and embryonic expression of the four trunk Hox genes Antennapedia (Antp), Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B) in the geophilomorph centipede Strigamia maritima. In geophilomorph centipedes, all leg-bearing segments (LBS) are generated during embryogenesis, allowing us to define expression in relation to the full extent of the forming trunk. Persistent Antp expression characterizes the maxillipedal (poison claw) segment, whereas all LBS express the three Hox genes Antp, Ubx, and abd-A. Abd-B is never detectably expressed in segmented tissue, but is restricted to a zone around the proctodaeum that contributes to the hindgut. Expression of all these Hox genes initiates in the unsegmented tissue of the blastodisc, with expression of Antp respecting a sharply defined anterior border before the appearance of morphological segmentation in the trunk. The accumulation of Hox gene transcripts is strongly modulated by the maturing segment pattern, suggesting regulatory interactions with multiple levels of the segment patterning machinery. For one of these genes, Ubx, we detect both sense and anti-sense transcripts. The anti-sense transcripts originate 3, to the Ubx coding sequence and overlap the homeobox exon; they are expressed earlier than the Ubx coding transcripts and persistently, in an axially restricted pattern comparable to but distinct from those of the Hox coding transcripts. The pattern of accumulation of Ubx sense and anti-sense transcripts is strikingly complementary, suggesting the possibility of anti-sense regulation of Ubx expression. [source] MBP-1 is efficiently encoded by an alternative transcript of the ENO1 gene but post-translationally regulated by proteasome-dependent protein turnoverFEBS JOURNAL, Issue 20 2010Jrhau Lung The c-myc promoter-binding protein-1 (MBP-1) is a transcriptional suppressor of tumorigenesis and thought to be the product of alternative translation initiation of the ,-enolase (ENO1) transcript. In the present study, we cloned a 2552-bp novel cDNA with a putative coding sequence of MBP-1 and functionally examined its ability to encode the MBP-1 protein. Similarly to ENO1, the obtained MBP-1 was widely and differentially expressed in a variety of normal tissues and cancer cells. Experiments using MBP-1 promoter-driven luciferase reporter assays, biochemical cell fractionation followed by RT-PCR detection of the cytoplasmic mRNA, and transcription/translation-coupled reactions, consistently demonstrated that this novel transcript was alternatively transcribed from intron III of the ENO1 gene and was feasible for MBP-1 production. Hypoxia treatments significantly increased the transcriptional activation of the MBP-1 gene. Blocking the proteasomal degradation by MG132 stabilized the MBP-1 protein in cells. Compared with the translation efficiency for production of the MBP-1 protein, the MBP-1 transcript was 17.8 times more efficient than the ENO1 transcript. Thus, we suggest that this newly discovered transcript is a genuine template for the protein synthesis of MBP-1 in cells, and optimal expression of this gene in tumors may lead to effective clinical therapies for cancers. [source] Reduced FAS transcription in clones of U937 cells that have acquired resistance to Fas-induced apoptosisFEBS JOURNAL, Issue 2 2009Jeanette Blomberg Susceptibility to cell death is a prerequisite for the elimination of tumour cells by cytotoxic immune cells, chemotherapy or irradiation. Activation of the death receptor Fas is critical for the regulation of immune cell homeostasis and efficient killing of tumour cells by apoptosis. To define the molecular changes that occur during selection for insensitivity to Fas-induced apoptosis, a resistant variant of the U937 cell line was established. Individual resistant clones were isolated and characterized. The most frequently observed defect in the resistant cells was reduced Fas expression, which correlated with decreased FAS transcription. Clones with such reduced Fas expression also displayed partial cross-resistance to tumour necrosis factor-, stimulation, but the mRNA expression of tumour necrosis factor receptors was not decreased. Reintroduction of Fas conferred susceptibility to Fas but not to tumour necrosis factor-, stimulation, suggesting that several alterations could be present in the clones. The reduced Fas expression could not be explained by mutations in the FAS coding sequence or promoter region, or by silencing through methylations. Protein kinase B and extracellular signal-regulated kinase, components of signalling pathways downstream of Ras, were shown to be activated in some of the resistant clones, but none of the three RAS genes was mutated, and experiments using chemical inhibitors could not establish that the activation of these proteins was the cause of Fas resistance as described in other systems. Taken together, the data illustrate that Fas resistance can be caused by reduced Fas expression, which is a result of an unidentified mode of regulation. [source] Cloning, chromosomal localization and characterization of the murine mucin gene orthologous to human MUC4FEBS JOURNAL, Issue 13 2002Jean-Luc Desseyn We report here the full coding sequence of a novel mouse putative membrane-associated mucin containing three extracellular EGF-like motifs and a mucin-like domain consisting of at least 20 tandem repeats of 124,126 amino acids. Screening a cosmid and a BAC libraries allowed to isolate several genomic clones. Genomic and cDNA sequence comparisons showed that the gene consists of 25 exons and 24 introns covering a genomic region of ,,52 kb. The first intron is ,,16 kb in length and is followed by an unusually large exon (, 9.5 kb) encoding Ser/Thr-rich tandemly repeated sequences. Radiation hybrid mapping localized this new gene to a mouse region of chromosome 16, which is the orthologous region of human chromosome 3q29 encompassing the large membrane-anchored mucin MUC4. Contigs analysis of the Human Genome Project did not reveal any other mucin on chromosome 3q29 and, interestingly, our analysis allowed the determination of the genomic organization of the human MUC4 and showed that its exon/intron structure is identical to that of the mouse gene we cloned. Furthermore, the human MUC4 shares considerable homologies with the mouse gene. Based on these data, we concluded that we isolated the mouse ortholog of MUC4 we propose as Muc4. Expression studies showed that Muc4 is ubiquitous like SMC and MUC4, with highest levels of expression in trachea and intestinal tract. [source] BJ46a, a snake venom metalloproteinase inhibitorFEBS JOURNAL, Issue 10 2001Isolation, characterization, cloning, insights into its mechanism of action Fractionation of the serum of the venomous snake Bothrops jararaca with (NH4)2SO4, followed by phenyl-Sepharose and C4 -reversed phase chromatographies, resulted in the isolation of the anti-hemorrhagic factor BJ46a. BJ46a is a potent inhibitor of the SVMPs atrolysin C (class P-I) and jararhagin (P-III) proteolytic activities and B. jararaca venom hemorrhagic activity. The single-chain, acidic (pI 4.55) glycoprotein has a molecular mass of 46 101 atomic mass units determined by MALDI-TOF MS and 79 kDa by gel filtration and dynamic laser light scattering, suggesting a homodimeric structure. mRNA was isolated from the liver of one specimen and transcribed into cDNA. The cDNA pool was amplified by PCR, cloned into a specific vector and used to transform competent cells. Clones containing the complete coding sequence for BJ46a were isolated. The deduced protein sequence was in complete agreement with peptide sequences obtained by Edman degradation. BJ46a is a 322-amino-acid protein containing four putative N-glycosylation sites. It is homologous to the proteinase inhibitor HSF (member of the fetuin family, cystatin superfamily) isolated from the serum of the snake Trimeresurus flavoviridis, having 85% sequence identity. This is the first report of a complete cDNA sequence for an endogenous inhibitor of snake venom metalloproteinases (SVMPs). The sequence reveals that the only proteolytic processing required to obtain the mature protein is the cleavage of the signal peptide. Gel filtration analyses of the inhibitory complexes indicate that inhibition occurs by formation of a noncovalent complex between BJ46a and the proteinases at their metalloproteinase domains. Furthermore, the data shows that the stoichiometry involved in this interaction is of one inhibitor monomer to two enzyme molecules, suggesting an interesting mechanism of metalloproteinase inhibition. [source] Promoters of crystal protein genes do not control crystal formation inside exosporium of Bacillus thuringiensis ssp. finitimus strain YBT-020FEMS MICROBIOLOGY LETTERS, Issue 1 2009Fang Ji Abstract Most Bacillus thuringiensis parasporal crystals separate from spores after sporulation. A special phenomenon called spore-crystal association (SCA) occurs in a few subspecies (e.g. ssp. finitimus) where enclosed crystals are associated with spores. In this study, the involvement of crystal protein gene promoters in SCA was investigated. Two crystal protein genes, cry26Aa and cry28Aa, were isolated from subspecies finitimus strain YBT-020, and each or both were then transferred to acrystalliferous B. thuringiensis strain BMB171 and the plasmid-cured derivative of strain YBT-020. SCA was not observed with any recombinant strain, implying that the crystal protein genes are not sufficient to cause SCA. When the typical crystal protein gene cry1Ca was introduced into strain YBT-020, free bipyramidal crystals formed in addition to SCA. Recombinant genes containing the promoter of cry26Aa or cry28Aa fused with the coding sequence (CDS) of cry1Ca were introduced into strain YBT-020, and the typical cry1Ca phenotype was observed. Another two fusion genes consisting of the promoter of cry1Ca and the CDS of cry26Aa or cry28Aa were also transferred to strain YBT-020. Only enclosed crystals formed. These results indicate that the promoters of the crystal protein genes are not the key factor determining the crystal location in strain YBT-020. [source] Localization of gene products using a chromosomally tagged GFP-fusion library in the fission yeast Schizosaccharomyces pombeGENES TO CELLS, Issue 2 2009Aki Hayashi We constructed a library of chromosomally-tagged green fluorescent protein (GFP) fusions in the fission yeast Schizosaccharomyces pombe. This library contains 1058 strains. In each strain, the coding sequence of GFP is integrated at the 3,-end of a particular chromosomal ORF such that the full-length GFP fusion construct is expressed under the control of the original promoter. Integration of the GFP coding sequence at the authentic chromosomal location of each gene was confirmed by PCR. Microscopic screening of these strains detected sufficient levels of GFP signal in 710 strains and allowed assignment of these GFP-fusion gene products with their intracellular localization: 374 proteins were localized in the nucleus, 65 proteins in the nucleolus, 34 proteins at the nuclear periphery, 27 proteins at the plasma membrane and cytoplasmic membranous structures, 24 proteins at the spindle pole body and microtubules, 92 proteins at cytoplasmic structures, and 94 proteins were uniformly distributed throughout the cytoplasm. [source] Deletions of SCN1A 5, genomic region with promoter activity in Dravet syndrome,HUMAN MUTATION, Issue 7 2010Tojo Nakayama Abstract Mutations involving the voltage-gated sodium channel ,I gene SCN1A are major genetic causes of childhood epileptic disorders, as typified by Dravet syndrome. Here we investigated the upstream regions of the SCN1A 5, noncoding exons and found two major regions with promoter activity. These two major promoters were simultaneously active in various brain regions and in most neurons. Using multiplex ligation-dependent probe amplification (MLPA) assays with probes for the 5, noncoding exons, their upstream regions, and all coding exons of SCN1A, we investigated 130 epileptic patients who did not show any SCN1A mutations by sequence analysis of all coding exons and exon,intron boundaries. Among 71 Dravet syndrome patients, we found two patients with heterozygous microdeletions removing the 5, noncoding exons and regions with promoter activity but not affecting the coding exons. We also identified four patients with deletions/duplication in the coding region. One patient with symptomatic focal epilepsy also showed a deletion in the coding region. This study provides the first case of microdeletion limited to the SCN1A 5, promoter region with the coding sequence preserved, and indicates the critical involvement of this upstream region in the molecular pathology of Dravet syndrome. Hum Mutat 31:,11, 2010. © 2010 Wiley-Liss, Inc. [source] Molecular characterization of novel progranulin (GRN) mutations in frontotemporal dementia,HUMAN MUTATION, Issue 4 2008Odity Mukherjee Abstract Frontotemporal dementia (FTD) is a clinical term encompassing dementia characterized by the presence of two major phenotypes: 1) behavioral and personality disorder, and 2) language disorder, which includes primary progressive aphasia and semantic dementia. Recently, the gene for familial frontotemporal lobar degeneration (FTLD) with ubiquitin-positive, tau-negative inclusions (FTLD-U) linked to chromosome 17 was cloned. In the present study, 62 unrelated patients from the Washington University Alzheimer's Disease Research Center and the Midwest Consortium for FTD with clinically diagnosed FTD and/or neuropathologically characterized cases of FTLD-U with or without motor neuron disease (MND) were screened for mutations in the progranulin gene (GRN; also PGRN). We discovered two pathogenic mutations in four families: 1) a single-base substitution within the 3, splice acceptor site of intron 6/exon 7 (g.5913A>G [IVS6,2A>G]) causing skipping of exon 7 and premature termination of the coding sequence (PTC); and 2) a missense mutation in exon 1 (g.4068C>A) introducing a charged amino acid in the hydrophobic core of the signal peptide at residue 9 (p.A9D). Functional analysis in mutation carriers for the splice acceptor site mutation revealed a 50% decrease in GRN mRNA and protein levels, supporting haploinsufficiency. In contrast, there was no significant difference in the total GRN mRNA between cases and controls carrying the p.A9D mutation. Further, subcellular fractionation and confocal microscopy indicate that although the mutant protein is expressed, it is not secreted, and appears to be trapped within an intracellular compartment, possibly resulting in a functional haploinsufficiency. Hum Mutat 29(4), 512,521, 2008. © 2008 Wiley-Liss, Inc. [source] Impact of mutant p53 functional properties on TP53 mutation patterns and tumor phenotype: lessons from recent developments in the IARC TP53 database,,HUMAN MUTATION, Issue 6 2007Audrey Petitjean Abstract The tumor suppressor gene TP53 is frequently mutated in human cancers. More than 75% of all mutations are missense substitutions that have been extensively analyzed in various yeast and human cell assays. The International Agency for Research on Cancer (IARC) TP53 database (www-p53.iarc.fr) compiles all genetic variations that have been reported in TP53. Here, we present recent database developments that include new annotations on the functional properties of mutant proteins, and we perform a systematic analysis of the database to determine the functional properties that contribute to the occurrence of mutational "hotspots" in different cancer types and to the phenotype of tumors. This analysis showed that loss of transactivation capacity is a key factor for the selection of missense mutations, and that difference in mutation frequencies is closely related to nucleotide substitution rates along TP53 coding sequence. An interesting new finding is that in patients with an inherited missense mutation, the age at onset of tumors was related to the functional severity of the mutation, mutations with total loss of transactivation activity being associated with earlier cancer onset compared to mutations that retain partial transactivation capacity. Furthermore, 80% of the most common mutants show a capacity to exert dominant-negative effect (DNE) over wild-type p53, compared to only 45% of the less frequent mutants studied, suggesting that DNE may play a role in shaping mutation patterns. These results provide new insights into the factors that shape mutation patterns and influence mutation phenotype, which may have clinical interest. Hum Mutat 28(6), 622,629, 2007. Published 2007 Wiley-Liss, Inc. [source] Mutation frequencies of X-linked mental retardation genes in families from the EuroMRX consortium,,HUMAN MUTATION, Issue 2 2007Arjan P.M. de Brouwer Abstract The EuroMRX family cohort consists of about 400 families with non-syndromic and 200 families with syndromic X-linked mental retardation (XLMR). After exclusion of Fragile X (Fra X) syndrome, probands from these families were tested for mutations in the coding sequence of 90 known and candidate XLMR genes. In total, 73 causative mutations were identified in 21 genes. For 42% of the families with obligate female carriers, the mental retardation phenotype could be explained by a mutation. There was no difference between families with (lod score >2) or without (lod score <2) significant linkage to the X chromosome. For families with two to five affected brothers (brother pair=BP families) only 17% of the MR could be explained. This is significantly lower (P=0.0067) than in families with obligate carrier females and indicates that the MR in about 40% (17/42) of the BP families is due to a single genetic defect on the X chromosome. The mutation frequency of XLMR genes in BP families is lower than can be expected on basis of the male to female ratio of patients with MR or observed recurrence risks. This might be explained by genetic risk factors on the X chromosome, resulting in a more complex etiology in a substantial portion of XLMR patients. The EuroMRX effort is the first attempt to unravel the molecular basis of cognitive dysfunction by large-scale approaches in a large patient cohort. Our results show that it is now possible to identify 42% of the genetic defects in non-syndromic and syndromic XLMR families with obligate female carriers. © 2007 Wiley-Liss, Inc. [source] Genetic variability, haplotypes, and htSNPs for exons 1 at the human UGT1A locus,,HUMAN MUTATION, Issue 7 2006Sushma S. Thomas Abstract UDP-Glucuronosyltransferases (UGTs) are a superfamily of enzymes responsible for glucuronidation of xenobiotics and endobiotics. Genetic polymorphisms have been identified in the promoter and exonic regions of several UGT genes. The UGT1As on chromosome 2q37 have unique exons 1 but share the remainder of their coding sequence. We screened exon 1 of each of the nine functional UGT1As in Asians (n=46) and Caucasians (n=92) with the aim of determining linkage disequilibrium (LD) and haplotypes across the entire locus in both populations. For polymorphisms in UGT 1A3, 1A4, 1A5, 1A7, and 1A8, we observed significant differences in the allele frequency between the two populations. The haplotype block structure across the UGT1A locus was constructed using all 83 polymorphisms and showed four and five haplotype blocks in Caucasians and Asians, respectively. There was long-distance LD between UGT pairs: 1A8 and 1A10; 1A1 and 1A3; 1A1 and 1A6; 1A6 and 1A7; and 1A7 and 1A9. Whereas both ethnic groups shared some haplotype-tagging SNPs (htSNPs), Caucasians and Asians also had unique htSNPs. This was partly due to the fact that rare variants (<5% allele frequency) were included in our analyses. Haplotypes with frequencies >5% represented only 60% of Caucasian and 65% of Asian UGT1A haplotypes. Differences in haplotype distribution patterns suggest individual and ethnic differences in glucuronidation capacity. Published 2006 Wiley-Liss, Inc. [source] Megalencephalic leukoencephalopathy with subcortical cysts: an update and extended mutation analysis of MLC1,HUMAN MUTATION, Issue 6 2006P. K. Ilja Boor Abstract Megalencephalic leukoencephalopathy with subcortical cysts (MLC) is an autosomal recessive cerebral white matter disorder in children. This disease is histopathologically characterized by myelin splitting and intramyelinic vacuole formation. MLC is caused by mutations in the gene MLC1, which encodes a novel protein, MLC1. Since the first report, 50 mutations in this gene have been found. Mutations occur throughout the entire coding region and include all different types: 11 splice-site mutations; one nonsense mutation; 24 missense mutations; and 14 deletions and insertions. Until now, six polymorphisms within the coding sequence of MLC1 had been reported. In about 20% of the patients with a typical clinical and MRI picture, no mutations in the MLC1 gene are found. Several of the families, in which no mutations are found, also do not show linkage with the MLC1 locus, which suggests a second gene involved in MLC. The absence of mutations may also be the consequence of performing standard mutation analysis that can miss heterozygous deletions, mutations in the promoter, 3, and 5, untranslated regions (UTRs), and intron mutations, which may influence the amino acid composition of the end product. In this work we describe 13 novel mutations, including those found with extended mutation analysis on MLC patients. This study shows that extended mutation analysis is a valuable tool to identify at least some of the missing mutations. Therefore, we suggest extended mutation analysis for the MLC1 gene, if no mutations are found during standard analysis. Hum Mutat 27(6), 505,512, 2006. © 2006 Wiley-Liss, Inc. [source] Prioritizing regions of candidate genes for efficient mutation screening,HUMAN MUTATION, Issue 2 2006Terry A. Braun Abstract The availability of the complete sequence of the human genome has dramatically facilitated the search for disease-causing sequence variations. In fact, the rate-limiting step has shifted from the discovery and characterization of candidate genes to the actual screening of human populations and the subsequent interpretation of observed variations. In this study we tested the hypothesis that some segments of candidate genes are more likely than others to contain disease-causing variations and that these segments can be predicted bioinformatically. A bioinformatic technique, prioritization of annotated regions (PAR), was developed to predict the likelihood that a specific coding region of a gene will harbor a disease-causing mutation based on conserved protein functional domains and protein secondary structures. This method was evaluated by using it to analyze 710 genes that collectively harbor 4,498 previously identified mutations. Nearly 50% of the genes were recognized as disease-associated after screening only 9% of the complete coding sequence. The PAR technique identified 90% of the genes as containing at least one mutation, with less than 40% of the screening resources that traditional approaches would require. These results suggest that prioritization strategies such as PAR can accelerate disease-gene identification through more efficient use of screening resources. Hum Mutat 27(2), 195,200, 2006. © 2006 Wiley-Liss, Inc. [source] A genetic polymorphism in the coding region of the gastric intrinsic factor gene (GIF) is associated with congenital intrinsic factor deficiency,HUMAN MUTATION, Issue 1 2004Marilyn M. Gordon Abstract Congenital intrinsic factor (IF) deficiency is a disorder characterized by megaloblastic anemia due to the absence of gastric IF (GIF, GenBank NM_005142) and GIF antibodies, with probable autosomal recessive inheritance. Most of the reported patients are isolated cases without genetic studies of the parents or siblings. Complete exonic sequences were determined from the PCR products generated from genomic DNA of five affected individuals. All probands had the identical variant (g.68A>G) in the second position of the fifth codon in the coding sequence of the gene that introduces a restriction enzyme site for Msp I and predicts a change in the mature protein from glutamine5 (CAG) to arginine5 (CGG). Three subjects were homozygous for this base exchange and two subjects were heterozygous, one of which was apparently a compound heterozygote at positions 1 and 2 of the fifth codon ([g.67C>G] + [g.68A>G]). The other patient, heterozygous for position 2, had one heterozygous unaffected parent. Most parents were heterozygous for this base exchange, confirming the pattern of autosomal recessive inheritance for congenital IF deficiency. cDNA encoding GIF was mutated at base pair g.68 (A>G) and expressed in COS-7 cells. The apparent size, secretion rate, and sensitivity to pepsin hydrolysis of the expressed IF were similar to native IF. The allelic frequency of g.68A>G was 0.067 and 0.038 in two control populations. This sequence aberration is not the cause of the phenotype, but is associated with the genotype of congenital IF deficiency and could serve as a marker for inheritance of this disorder. Hum Mutat 23:85,91, 2004. © 2003 Wiley-Liss, Inc. [source] The major antennal chemosensory protein of red imported fire ant workersINSECT MOLECULAR BIOLOGY, Issue 3 2009D. González Abstract Some chemosensory proteins (CSPs) are expressed in insect sensory appendages and are thought to be involved in chemical signalling by ants. We identified 14 unique CSP sequences in expressed sequence tag (EST) libraries of the red imported fire ant, Solenopsis invicta. One member of this group (Si-CSP1) is highly expressed in worker antennae, suggesting an olfactory function. A shotgun proteomic analysis of antennal proteins confirmed the high level of Si-CSP1 expression, and also showed expression of another CSP and two odorant-binding proteins (OBPs). We cloned and expressed the coding sequence for Si-CSP1. We used cyclodextrins as solubilizers to investigate ligand binding. Fire ant cuticular lipids strongly inhibited Si-CSP1 binding to the fluorescent dye N-phenyl-naphthylamine, suggesting cuticular substances are ligands for Si-CSP1. Analysis of the cuticular lipids showed that the endogenous ligands of Si-CSP1 are not cuticular hydrocarbons. [source] Identification and function of Abdominal-A in the silkworm, Bombyx moriINSECT MOLECULAR BIOLOGY, Issue 2 2009M-H. Pan Abstract Abdominal-A (adb-A) is a key gene in the development of insects. To understand its function in the silkworm, we cloned 1193 bp of the abd-A gene of Bombyx mori (Bmabd-A), including the complete coding sequence and part of the 3, untranslated region sequence. Bmabd-A has at least three mRNA splice variants with coding sequences of lengths 1032, 1044 and 1059 bp, encoding 343, 347 and 352 amino acids, respectively. Each splice variant of Bmabd-A has three exons and differs only in second exon size. Bmabd-A was expressed at low levels in unfertilized eggs, but increased gradually in fertilized eggs after laying 22 h. Bmabd-A expression decreased in ant silkworms (newly hatched silkworms). After RNA interference for Bmabd-A, the embryos had two mutant phenotypes, either completely or partially absent abdominal feet from the third to sixth abdominal segments, suggesting that Bmabd-A is responsible for normal development of the third to sixth abdominal segments during embryonic development. [source] Development of an orally infectious Sindbis virus transducing system that efficiently disseminates and expresses green fluorescent protein in Aedes aegyptiINSECT MOLECULAR BIOLOGY, Issue 2 2003D. J. Pierro Abstract We have constructed an orally infectious Sindbis virus, ME2/5,2J/GFP, that expresses green fluorescent protein (GFP) in the midgut of Aedes aegypti and in other tissues as the virus disseminates. This virus has two unique features that are improvements over the SIN-based expression systems currently used in mosquitoes. First, a subgenomic RNA promoter and GFP coding sequence is located 5,- to the second subgenomic promoter and structural genes of the virus. Second, the E2 glycoprotein gene of TE/5,2J/GFP is replaced with the E2 gene of MRE16 SIN virus. The first feature enhances virus genome stability during virus dissemination from the midgut to other tissues and the second allows efficient virus entry into the midgut epithelial cells and then spread of the virus throughout the mosquito. [source] CLONING AND EXPRESSION OF SPODOPTERA LITURA UBIQUITIN GENEINSECT SCIENCE, Issue 1 2003LI Zhao-fei Abstract Ubiquitin (UBI) plays a very important role in regulated non-lysosomal ATP dependent protein degradation. In the present work, the coding sequence of Spodoptera litura UBI gene was isolated (GenBank Accession No. AF436066). The length of this ORF is 228bp, encoding a protein with Mr of 8.56 kD and isoelectric point of 6.56. Multiple sequence alignment indicated that S. litura UBI is very similar to the homologous proteins of other eukaryotic species and it has 84% identity with S. litura nucleopolyhedrovirus (SpltMNPV) UBI at amino acid level. RT-PCR analysis showed that S. litura UBI gene is ubiquitously expressed in larva tissues which are susceptible to SpltMNPV infection. By constructing E. coli expression vector, S. litura UBI was highly expressed and the recombinant protein was purified using Ni-NTA resin column, and currently further study on the function of S. litura UBI in SpltMNPV infection is underway. [source] Analysis of the human APC mutation spectrum in a saccharomyces cerevisiae strain with a mismatch repair defectINTERNATIONAL JOURNAL OF CANCER, Issue 5 2003Kazunori Otsuka Abstract Somatic APC mutations in colorectal tumors with an RER phenotype reflect excessive frameshift mutations, especially in simple repetition tracts within the coding sequence. Because this type of mutation is characteristic of cells with a deficient DNA MMR system, the APC mutation signature of RER tumors may be attributable to a defect in the MMR system. However, there is little experimental evidence to prove that the spectrum of mutations and the APC gene distribution are directly influenced by MMR system defects. We therefore examined the mutation spectrum of the MCR of the APC gene after transfection into both MMR-proficient and MMR-deficient yeast strains and compared it with a previously reported human APC mutation database. Small insertions or deletions in mono- or dinucleotide repeats were more common in the MMR-deficient than in the MMR-proficient strain (91.2% vs. 38.1%, Fisher's exact test p < 0.0001). Furthermore, the 2 mutation hot spots, 4385,4394(AG)5 and 4661,4666(A)6, found in the yeast system corresponded with those in human tumors. Combining our data with those from human tumors, there appears to be hypermutable mutations in specific simple repetitive sequences within the MCR, which are more prevalent in MMR-deficient cells and RER tumors than in MMR-proficient cells and non-RER tumors. We therefore consider that the differences in the spectra of RER and non-RER tumors are attributable at least in part to the MMR system of the host cells. © 2002 Wiley-Liss, Inc. [source] Novel variants within the coding regions of the Slc11A1 gene identified in Bos taurus and Bos indicus breedsJOURNAL OF ANIMAL BREEDING AND GENETICS, Issue 1 2008R. Martínez Summary Although in the cow the genetic resistance to brucellosis has been previously attributed to the Slc11A1 gene encoding Nramp1 protein, none of the mutations described to date seems to be the cause. To be able to associate another polymorphism of the gene to brucellosis resistance, we characterized the gene and identified in different breeds of Bos taurus and Bos indicus, six new variants among a total of 11 single nucleotide mutations, of which five occurred in the coding sequence (three are missense mutations), one in the promoter region and five in introns. The allelic and genotypic frequencies calculated revealed differences (p < 0.05) among the breeds studied. [source] Simultaneous genotyping to detect myostatin gene polymorphism in beef cattle breedsJOURNAL OF ANIMAL BREEDING AND GENETICS, Issue 6 2002M. E. Miranda Summary The myostatin gene codes for a growth factor involved in muscle development, and polymorphism in this gene can have important economic consequences. Nine mutations affecting the amino-acidic sequence have already been described, six of which are disruptive, inactivating the protein and causing bovine muscular hypertrophy. As the number of known mutations grows, it is necessary to develop a simple, routinely usable technique able to screen individuals in all populations. The oligonucleotide ligation assay (OLA) is proposed here for the rapid genotyping of the nine mutations known affecting the coding sequence in the main breeds of beef cattle. This technique showed its ability to reveal the genotype of individuals being a good tool to determine the frequency of each mutation in a population. The procedure is very flexible as new mutations can be added and removed at any time. Depending on the genotype of each individual, the technique allows breeders to make quick decisions on matings and general selection tendencies. Zusammenfassung Simultane Genotypisierung von Polymorphismen im Myostatin-Gen in Fleischrinderrassen Das Myostatin-Gen kodiert für einen Wachstumsfaktor, der in die Muskelentwicklung eingebunden ist und Polymorphismen in diesem Gen können daher wichtige ökonomische Konsequenzen haben. Bisher wurden neun Mutationen, die Auswirkungen auf die Aminosäuresequenz haben, beschrieben. Sechs davon inaktivieren das Protein und verursachen bovine muskuläre Hypertrophie. Da die Anzahl der bekannten Mutationen in diesem Gen steigt, ist es notwendig, eine einfache, in der Routine einsetzbare Methode zu entwickeln, um Individuen in allen Populationen untersuchen zu können. Zur schnellen Genotypisierung der neun bekannten Mutationen, welche die kodierende Sequenz in den Hauptfleischrinderrassen betreffen, wird hier der Oligo-Ligationsassay (OLA) vorgeschlagen. Durch diese Technik ist es möglich, den Genotyp jedes Individuums und die Frequenz jeder einzelnen Mutation in der Population festzustellen. Die Prozedur ist sehr flexibel, da zu jedem Zeitpunkt neue Mutationen hinzugefügt bzw. weggelassen werden können. Diese Methode erlaubt dem Züchter, in Abhängigkeit vom Genotyp jedes Individiums schnelle Entscheidungen über die Anpaarung und die allgemeine Selektionsrichtung zu treffen. [source] Preonset Studies of Spondyloepiphyseal Dysplasia Tarda Caused by a Novel 2-Base Pair Deletion in SEDL Encoding Sedlin,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 12 2001Steven Mumm Abstract Spondyloepiphyseal dysplasia tarda (SEDT), an X-linked recessive skeletal disorder, presents with disproportionate short stature and "barrel-chest" deformity in affected (hemizygous) adolescent boys. In four reported families to date, mutations in a gene designated SEDL (spondyloepiphyseal dysplasia late) cosegregate with SEDT. We diagnosed SEDT in a short-stature, kyphotic 15-year-old boy because of his characteristic vertebral malformations. Clinical manifestations of SEDT were evident in at least four previous generations. A novel 2-base pair (bp) deletion in exon 5 of SEDL was found in the propositus by polymerase chain reaction (PCR) amplification and sequencing of all four coding exons. The mutation ATdel241-242 cosegregated with the kindred's skeletal disease. The deletion is adjacent to a noncanonical splice site for exon 5 but does not alter splicing. Instead, it deletes 2 bp from the coding sequence, causing a frameshift. A maternal aunt and her three young sons were investigated subsequently. Radiographs showed subtle shaping abnormalities of her pelvis and knees, suggesting heterozygosity. X-rays of the spine and pelvis of her 8-year-old son revealed characteristic changes of SEDT, but her younger sons (aged 6 years and 3 years) showed no abnormalities. SEDL analysis confirmed that she and only her eldest boy had the 2-bp deletion. Molecular testing of SEDL enables carrier detection and definitive diagnosis before clinical or radiographic expression of SEDT. Although there is no specific treatment for SEDT, preexpression molecular testing of SEDL could be helpful if avoiding physical activities potentially injurious to the spine and the joints proves beneficial. [source] Differential Expression Patterns of Runx2 Isoforms in Cranial Suture MorphogenesisJOURNAL OF BONE AND MINERAL RESEARCH, Issue 5 2001Mi-Hyun Park Abstract Runx2 (previously known as Cbfa1/Pebp2,A/AML3), a key transcription factor in osteoblast differentiation, has at least two different isoforms using alternative promoters, which suggests that the isoforms might be expressed differentially. Haploinsufficiency of the Runx2 gene is associated with cleidocranial dysplasia (CCD), the main phenotype of which is inadequate development of calvaria. In spite of the biological relevance, Runx2 gene expression patterns in developing calvaria has not been explored previously, and toward this aim we developed three probes: pRunx2, which comprises the common coding sequence of Runx2 and hybridizes with all isoforms; pPebp2,A, which specifically hybridizes with the isoform transcribed with the proximal promoter; and pOsf2, which hybridizes with the isoform transcribed with the distal promoter. These probes were hybridized with tissue sections of mouse calvaria taken at various time points in development. Runx2 expression was localized to the critical area of cranial suture closure, being found in parietal bones, osteogenic fronts, and sutural mesenchyme. Pebp2,A and Osf2 showed tissue-specific expression patterns. The sites of Pebp2,A expression were almost identical to that of pRunx2 hybridization but expression was most intense in the sutural mesenchyme, where undifferentiated mesenchymal cells reside. The Osf2 isoform was strongly expressed in the osteogenic fronts, as well as in developing parietal bones, where osteopontin (OP) and osteocalcin (OC) also were expressed. However, in contrast to Pebp2,A, Osf2 expression did not occur in sutural mesenchyme. Pebp2,A also was expressed prominently in primordial cartilage that is found under the sutural mesenchyme and is not destined to be mineralized. Thus, Osf2 isoforms contribute to events later in osteoblast differentiation whereas the Pebp2,A isoform participates in a wide variety of cellular activities ranging from early stages of osteoblast differentiation to the final differentiation of osteoblasts. [source] Evidence for a Single Nucleotide Polymorphism in the KCNQ1 Potassium Channel that Underlies Susceptibility to Life-Threatening ArrhythmiasJOURNAL OF CARDIOVASCULAR ELECTROPHYSIOLOGY, Issue 11 2001TOMOYUKI KUBOTA M.D. Ion Channel Polymorphism and Cardiac Arrhythmia. Introduction: Congenital long QT syndrome (LQTS) is a genetically heterogeneous arrhythmogenic disorder caused by mutations in at least five different genes encoding cardiac ion channels. It was suggested recently that common polymorphisms of LQTS-associated genes might modify arrhythmia susceptibility in potential gene carriers. Methods and Results: We examined the known LQTS genes in 95 patients with definitive or suspected LQTS. Exon-specific polymerase chain reaction single-strand conformation polymorphism and direct sequence analyses identified six patients who carried only a single nucleotide polymorphism in KCNQ1 that is found in , 11% of the Japanese population. This 1727G> A substitution that changes the sense of its coding sequence from glycine to serine at position 643 (G643S) was mostly associated with a milder phenotype, often precipitated by hypokalemia and bradyarrhythmias. When heterologously examined by voltage-clamp experiments, the in vitro cellular phenotype caused by the single nucleotide polymorphism revealed that G643S- KCNQ1 forms functional homomultimeric channels, producing a significantly smaller current than that of the wild-type (WT) channels. Coexpression of WT- KCNQ1 and G643S- KCNQ1 with KCNE1 resulted in , 30% reduction in the slow delayed rectifier K+ current IKs without much alteration in the kinetic properties except its deactivation process, suggesting that the G643S substitution had a weaker dominant-negative effect on the heteromultimeric channel complexes. Conclusion: We demonstrate that a common polymorphism in the KCNQ1 potassium channel could be a molecular basis for mild IKs dysfunction that, in the presence of appropriate precipitating factors, might predispose potential gene carriers to life-threatening arrhythmias in a specific population. [source] | |||||||||||||||||||||||||||||||||||||||||||