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cDNA Analysis (cdna + analysis)
Selected AbstractsLeukocyte cDNA Analysis of NSD1 Derived from Confirmed Sotos Syndrome PatientsANNALS OF HUMAN GENETICS, Issue 6 2007M. Duno Summary Background: Haploinsufficiency of the NSD1 gene leads to Sotos syndrome (Sos), which is characterised by excessive growth, especially during childhood, distinct craniofacial features and variable degree of mental impairment. A wide spectrum of NSD1 mutations have been described in Sos patients, ranging from more than 100 different single nucleotide changes, to partial gene deletions, and to microdeletions of various sizes comprising the entire NSD1 locus. Objective: To investigate the NSD1 cDNA sequence in genetically confirmed Sos patients harbouring truncating and missense mutations. Method: Total RNA was isolated from a 250 ,l standard EDTA blood sample from nine genetically verified Sos patients, and subsequent reverse-transcribed into cDNA followed by PCR and direct sequencing of specific NSD1 cDNA sequences. Results: All nine mutations, including missense, nonsense and whole exon deletions, previously identified in genomic DNA, could confidently be detected in cDNA. Several NSD1 transcript splice variants were detected. Conclusion: Despite the fact that Sos is caused by haploinsufficiency, NSD1 transcripts containing nonsense and frame shift mutations can be detected in leukocyte-derived cDNA. The possibility therefore exists that certain NSD1 mutations are expressed and contribute to the phenotypic variability of Sos. NSD1 cDNA analysis is likely to enhance mutation detection in Sos patients. [source] Neuropeptide and neurohormone precursors in the pea aphid, Acyrthosiphon pisumINSECT MOLECULAR BIOLOGY, Issue 2010J. Huybrechts Abstract Aphids respond to environmental changes by developing alternative phenotypes with differing reproductive modes. Parthenogenetic reproduction occurs in spring and summer, whereas decreasing day lengths in autumn provoke the production of sexual forms. Changing environmental signals are relayed by brain neuroendocrine signals to the ovarioles. We combined bioinformatic analyses with brain peptidomics and cDNA analyses to establish a catalogue of pea aphid neuropeptides and neurohormones. 42 genes encoding neuropeptides and neurohormones were identified, of which several were supported by expressed sequence tags and/or peptide mass analyses. Interesting features of the pea aphid peptidome are the absence of genes coding for corazonin, vasopressin and sulfakinin and the presence of 10 different genes coding insulin related peptides, one of which appears to be very abundantly expressed. [source] Comprehensive survey of carapacial ridge-specific genes in turtle implies co-option of some regulatory genes in carapace evolutionEVOLUTION AND DEVELOPMENT, Issue 1 2005Shigehiro Kuraku Summary The turtle shell is an evolutionary novelty in which the developmental pattern of the ribs is radically modified. In contrast to those of other amniotes, turtle ribs grow laterally into the dorsal dermis to form a carapace. The lateral margin of carapacial primordium is called the carapacial ridge (CR), and is thought to play an essential role in carapace patterning. To reveal the developmental mechanisms underlying this structure, we systematically screened for genes expressed specifically in the CR of the Chinese soft-shelled turtle, Pelodiscus sinensis, using microbead-based differential cDNA analysis and real-time reverse transcription-polymerase chain reaction. We identified orthologs of Sp5, cellular retinoic acid-binding protein-I (CRABP-I), adenomatous polyposis coli down-regulated 1 (APCDD1), and lymphoid enhancer-binding factor-1 (LEF-1). Although these genes are conserved throughout the major vertebrate lineages, comparison of their expression patterns with those in chicken and mouse indicated that these genes have acquired de novo expression in the CR in the turtle lineage. In association with the expression of LEF-1, the nuclear localization of ,-catenin protein was detected in the CR ectoderm, suggesting that the canonical Wnt signaling triggers carapace development. These findings indicate that the acquisition of the turtle shell did not involve the creation of novel genes, but was based on the co-option of pre-existing genes. [source] Evaluation of somatic alterations of Pcdh-, transcripts in the brain by cDNA analysis without PCRGENES TO CELLS, Issue 1 2006Teruyoshi Hirayama No abstract is available for this article. [source] Similar gene structure of two Sox9a genes and their expression patterns during gonadal differentiation in a teleost fish, rice field eel (Monopterus albus)MOLECULAR REPRODUCTION & DEVELOPMENT, Issue 3 2003Rongjia Zhou Abstract The Sox9 gene encodes a transcription factor that is critical for testis determination and chondrogenesis in vertebrates. Mutations in human SOX9 cause campomelic dysplasia, a dominant skeletal dysmorphology syndrome often associated with male to female sex reversal. Here we show that the Sox9a gene was duplicated during evolution of the rice field eel, Monopterus albus, a freshwater fish which undergoes natural sex reversal from female to male during its life, and has a haploid genome size (0.6,0.8 pg) that is among the smallest of the vertebrates. The duplicated copies of the gene (named Sox9al and Sox9a2) fit within the Sox9 clade of vertebrates, especially in the Sox9a subfamily, not in the Sox9b subfamily. They have similar structures as revealed by both genomic and cDNA analysis. Furthermore, both Sox9al and Sox9a2 are expressed in testis, ovary, and ovotestis; and specifically in the outer layer (mainly gonocytes) of gonadal epithelium with bipotential capacity to form testis or ovary, suggesting that they have similar roles in gonadal differentiation during sex reversal in this species. The closely related gene structure and expression patterns of the two sox9a genes in the rice field eel also suggest that they arose in recent gene duplication events during evolution of this fish lineage. Mol. Reprod. Dev. 66: 211,217, 2003. © 2003 Wiley-Liss, Inc. [source] High-resolution Melting Facilitates Mutation Screening of PYGM in Patients with McArdle DiseaseANNALS OF HUMAN GENETICS, Issue 3 2009Morten Duno Summary Mutations in PYGM, encoding the muscle-specific glycogen phosphorylase (myophosphorylase), are responsible for McArdle disease. Among Caucasians, a large proportion of patients are homozygous for the R50X mutation, but other mutations can affect all the 20 exons of PYGM, making mutation detection laborious. We have developed a high-resolution melting (HRM) assay for mutation detection in PYGM. Twelve McArdle patients were investigated, in whom pre-screening had ruled out homozygosity or compound heterozygosity for the two common G205S and R50X mutations. In total, we identified 16 different variations. Thirteen of these are pathogenic, and three were classified as polymorphisms. Nine variations had not previously been described. One of the novel mutations, c.2430C > T, was initially predicted to result in a silent G810G change, but cDNA analysis demonstrated that the mutation led to abnormal mRNA processing. The HRM protocol reduced the need for direct sequencing by approximately 85%, and is a good approach to search for new mutations in PYGM. [source] Leukocyte cDNA Analysis of NSD1 Derived from Confirmed Sotos Syndrome PatientsANNALS OF HUMAN GENETICS, Issue 6 2007M. Duno Summary Background: Haploinsufficiency of the NSD1 gene leads to Sotos syndrome (Sos), which is characterised by excessive growth, especially during childhood, distinct craniofacial features and variable degree of mental impairment. A wide spectrum of NSD1 mutations have been described in Sos patients, ranging from more than 100 different single nucleotide changes, to partial gene deletions, and to microdeletions of various sizes comprising the entire NSD1 locus. Objective: To investigate the NSD1 cDNA sequence in genetically confirmed Sos patients harbouring truncating and missense mutations. Method: Total RNA was isolated from a 250 ,l standard EDTA blood sample from nine genetically verified Sos patients, and subsequent reverse-transcribed into cDNA followed by PCR and direct sequencing of specific NSD1 cDNA sequences. Results: All nine mutations, including missense, nonsense and whole exon deletions, previously identified in genomic DNA, could confidently be detected in cDNA. Several NSD1 transcript splice variants were detected. Conclusion: Despite the fact that Sos is caused by haploinsufficiency, NSD1 transcripts containing nonsense and frame shift mutations can be detected in leukocyte-derived cDNA. The possibility therefore exists that certain NSD1 mutations are expressed and contribute to the phenotypic variability of Sos. NSD1 cDNA analysis is likely to enhance mutation detection in Sos patients. [source] | ||||||||||