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Genomic Sequencing (genomic + sequencing)
Selected AbstractsDNA methylation of Sleeping Beauty with transposition into the mouse genomeGENES TO CELLS, Issue 8 2005Chang Won Park The Sleeping Beauty transposon is a recently developed non-viral vector that can mediate insertion of transgenes into the mammalian genome. Foreign DNA elements that are introduced tend to invoke a host-defense mechanism resulting in epigenetic changes, such as DNA methylation, which may induce transcriptional inactivation of mammalian genes. To assess potential epigenetic modifications associated with Sleeping Beauty transposition, we investigated the DNA methylation pattern of transgenes inserted into the mouse genome as well as genomic regions flanking the insertion sites with bisulfite-mediated genomic sequencing. Transgenic mouse lines were created with two different Sleeping Beauty transposons carrying either the Agouti or eGFP transgene. Our results showed that DNA methylation in the keratin-14 promoter and Agouti transgene were negligible. In addition, two different genomic loci flanking the Agouti insertion site exhibited patterns of DNA methylation similar to wild-type mice. In contrast, high levels of DNA methylation were observed in the eGFP transgene and its ROSA26 promoter. These results indicate that transposition via Sleeping Beauty into the mouse genome may result in a significant level of de novo DNA methylation. This may depend on a number of different factors including the cargo DNA sequence, chromosomal context of the insertion site, and/or host genetic background. [source] Myoclonus-dystonia: significance of large SGCE deletions,,HUMAN MUTATION, Issue 2 2008A. Grünewald Abstract Myoclonus-dystonia (M-D) is an autosomal-dominant movement disorder caused by mutations in SGCE. We investigated the frequency and type of SGCE mutations with emphasis on gene dosage alterations and explored the associated phenotypes. We tested 35 M-D index patients by multiplex ligation-dependent probe amplification (MLPA) and genomic sequencing. Mutations were found in 26% (9/35) of the cases, all but three with definite M-D. Two heterozygous deletions of the entire SGCE gene and flanking DNA and a heterozygous deletion of exon 2 only were detected, accounting for 33% (3/9) of the mutations found. Both large deletions contained COL1A2 and were additionally associated with joint problems. Further, we discovered one novel small deletion (c.771_772delAT, p.C258X) and four recurrent point mutations (c.289C>T, p.R97X; c.304C>T, p.R102X; c.709C>T, p.R237X; c.1114C>T, p.R372X). A Medline search identified 22 articles on SGCE mutational screening. Sixty-four unrelated M-D patients were described with 41 different mutations. No genotype,phenotype association was found, except in patients with deletions encompassing additional genes. In conclusion, a rigorous clinical preselection of patients and careful accounting for non-motor signs should precede mutational tests. Gene dosage studies should be included in routine SGCE genetic testing. © 2007 Wiley-Liss, Inc. [source] Splice mutations in the p53 gene: case report and review of the literature,,HUMAN MUTATION, Issue 1 2003R. Holmila Abstract Splice mutations in the p53 gene (TP53) are described as rare events that occur at a frequency of less than 1%. Using a functional assay based on the transcriptional activity of p53 and using RNA as starting material, we describe here a p53 splice mutation that could not be detected by genomic sequencing. This lack of detection is due to a deletion of the region complementary to primers commonly used for amplification. Reviewing the literature, we show that p53 splice mutations have been certainly underestimated and that careful strategy should be used for a complete mutational analysis of the p53 gene. Furthermore, some p53 gene mutations described as "neutral" due to the absence of any amino-acid change are truly deleterious, as they affect gene splicing. © 2002 Wiley-Liss, Inc. [source] N-MYC Downstream-Regulated Gene 1 Is Mutated In Hereditary Motor And Sensory Neuropathy-LOMJOURNAL OF THE PERIPHERAL NERVOUS SYSTEM, Issue 1 2001L Kalaydjieva Hereditary motor and sensory neuropathies, to which Charcot-Marie-Tooth (CMT) disease belongs, are a common cause of disability in adulthood. Growing awareness that axonal loss, rather than demyelination per se, is responsible for the neurological deficit in demyelinating CMT disease has focused research on the mechanisms of early development, cell differentiation, and cell-cell interactions in the peripheral nervous system. Autosomal recessive peripheral neuropathies are relatively rare but are clinically more severe than autosomal dominant forms of CMT, and understanding their molecular basis may provide a new perspective on these mechanisms. Here we report the identification of the gene responsible for hereditary motor and sensory neuropathy-Lom (HMSNL). HMSNL shows features of Schwann-cell dysfunction and a concomitant early axonal involvement, suggesting that impaired axon-glia interactions play a major role in its pathogenesis. The gene was previously mapped to 8q24.3, where conserved disease haplotypes suggested genetic homogeneity and a single founder mutation. We have reduced the HMSNL interval to 200 kb and have characterized it by means of large-scale genomic sequencing. Sequence analysis of two genes located in the critical region identified the founder HMSNL mutation: a premature-termination codon at position 148 of the N-myc downstream-regulated gene 1 (NDRG1). NDRG1 is ubiquitously expressed and has been proposed to play a role in growth arrest and cell differentiation, possibly as a signaling protein shuttling between the cytoplasm and the nucleus. We have studied expression in peripheral nerve and have detected particularly high levels in the Schwann cell. Taken together, these findings point to NDRG1 having a role in the peripheral nervous system, possibly in the Schwann-cell signaling necessary for axonal survival. [source] Fluorescence in situ hybridization analysis with a tissue microarray: ,FISH and chips' analysis of pathology archivesPATHOLOGY INTERNATIONAL, Issue 8 2010Haruhiko Sugimura Practicing pathologists expect major somatic genetic changes in cancers, because the morphological deviations in the cancers they diagnose are so great that the somatic genetic changes to direct these phenotypes of tumors are supposed to be correspondingly tremendous. Several lines of evidence, especially lines generated by high-throughput genomic sequencing and genome-wide analyses of cancer DNAs are verifying their preoccupations. This article reviews a comprehensive morphological approach to pathology archives that consists of fluorescence in situ hybridization with bacterial artificial chromosome (BAC) probes and screening with tissue microarrays to detect structural changes in chromosomes (copy number alterations and rearrangements) in specimens of human solid tumors. The potential of this approach in the attempt to provide individually tailored medical practice, especially in terms of cancer therapy, is discussed. [source] Molecular characterization of sickle cell anemia in the Northern Brazilian state of ParáAMERICAN JOURNAL OF HUMAN BIOLOGY, Issue 5 2010Greice De Lemos Cardoso To assess ,+-thalassemia deletion alleles, ,-thalassemia mutations and haplotypes linked to the HBB*S cluster in a sample of 130 unrelated sickle cell anemia (SCA) patients (55% female) from Belém, Pará State, for their possible effects on the patients' survival. -,3.7, -,42, -,20.5, and ,MED ,+-thalassemia deletion alleles were investigated using multiplex gap-PCR method. Characterization of ,-thalassemia mutations was made by direct genomic sequencing of the ,-globin gene amplified through polymerase chain reaction (PCR). Haplotypes were determined by analysis of six polymorphic restriction sites [(1) XmnI-5,,G, (2) HindIII-,G, (3) HindIII-,A, (4) HincII-,,, (5) HincII-3,,,, and (6) HinfI-5,,] followed by restriction digestion and agarose gel electrophoresis. Twenty-one patients (16%) presented -,3.7 thalassemia. Sixteen of those (76%) were heterozygous (-,3.7/,,) and 5 (24%) were homozygous (-,3.7/-,3.7). -,4.2, -,20.5 and ,MED deletions were not found. Nine cases of sickle cell-, thalassemia were found and four different ,-thal mutations were identified: ,+ ,88 (C>T), 3.8%; ,+ codon 24 (T > A), 1.5%; ,+ IVSI-110 (G > A), 0.7% and , (IVSI-1 (G > A), 0.7%. No differences according to age were observed in -,3.7 deletion, ,-thalassemia and HHB*S haplotypes distribution. Our results suggest that although ,- and ,-thalassemia and ,S haplotypes may have modulating effect on clinical expression and hematological parameters of SCA, these genetic variables probably have little influence on the subjects' survival. Am. J. Hum. Biol. 22:573,577, 2010. © 2010 Wiley-Liss, Inc. [source] Consistent transcriptional silencing of 35S-driven transgenes in gentianTHE PLANT JOURNAL, Issue 4 2005Kei-ichiro Mishiba Summary In this study, no transgenic gentian (Gentiana triflora × Gentiana scabra) plants produced via Agrobacterium -mediated transformation exhibited transgene (GtMADS, gentian-derived MADS-box genes or sGFP, green fluorescent protein) expression in their leaf tissues, despite the use of constitutive Cauliflower mosaic virus (CaMV) 35S promoter. Strikingly, no expression of the selectable marker gene (bar) used for bialaphos selection was observed. To investigate the possible cause of this drastic transgene silencing, methylation-specific sequences were analysed by bisulfite genomic sequencing using tobacco transformants as a control. Highly methylated cytosine residues of CpG and CpWpG (W contains A or T) sites were distinctively detected in the promoter and 5, coding regions of the transgenes 35S- bar and 35S- GtMADS in all gentian lines analysed. These lines also exhibited various degrees of cytosine methylation in asymmetrical sequences. The methylation frequencies in the other transgene, nopaline synthase (NOS) promoter-driven nptII, and the endogenous GtMADS gene coding region, were much lower and were variable compared with those in the 35S promoter regions. Transgene methylation was observed in the bialaphos-selected transgenic calluses expressing the transgenes, and methylation sequences were distributed preferentially around the as-1 element in the 35S promoter. Calluses derived from leaf tissues of silenced transgenic gentian also exhibited transgene suppression, but expression was recovered by treatment with the methylation inhibitor 5-aza-2,-deoxycytidine (aza-dC). These results indicated that cytosine methylation occurs exclusively in the 35S promoter regions of the expressed transgenes during selection of gentian transformants, causing transcriptional gene silencing. [source] Persea americana (avocado): bringing ancient flowers to fruit in the genomics eraBIOESSAYS, Issue 4 2008André S. Chanderbali The avocado (Persea americana) is a major crop commodity worldwide. Moreover, avocado, a paleopolyploid, is an evolutionary "outpost" among flowering plants, representing a basal lineage (the magnoliid clade) near the origin of the flowering plants themselves. Following centuries of selective breeding, avocado germplasm has been characterized at the level of microsatellite and RFLP markers. Nonetheless, little is known beyond these general diversity estimates, and much work remains to be done to develop avocado as a major subtropical-zone crop. Among the goals of avocado improvement are to develop varieties with fruit that will "store" better on the tree, show uniform ripening and have better post-harvest storage. Avocado transcriptome sequencing, genome mapping and partial genomic sequencing will represent a major step toward the goal of sequencing the entire avocado genome, which is expected to aid in improving avocado varieties and production, as well as understanding the evolution of flowers from non-flowering seed plants (gymnosperms). Additionally, continued evolutionary and other comparative studies of flower and fruit development in different avocado strains can be accomplished at the gene expression level, including in comparison with avocado relatives, and these should provide important insights into the genetic regulation of fruit development in basal angiosperms. BioEssays 30:386,396, 2008. © 2008 Wiley Periodicals, Inc. [source] | |||||||||||||