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cDNA Sequencing (cdna + sequencing)
Selected AbstractsIdentification of some human genes oppositely regulated during esophageal squamous cell carcinoma formation and human embryonic esophagus developmentDISEASES OF THE ESOPHAGUS, Issue 3 2010M. V. Zinovyeva SUMMARY Here we directly compared gene expression profiles in human esophageal squamous cell carcinomas and in human fetal esophagus development. We used the suppression subtractive hybridization technique to subtract cDNAs prepared from tumor and normal human esophageal samples. cDNA sequencing and reverse transcription polymerase chain reaction (RT-PCR) analysis of RNAs from human tumor and the normal esophagus revealed 10 differentially transcribed genes: CSTA, CRNN, CEACAM1, MAL, EMP1, ECRG2, and SPRR downregulated, and PLAUR, SFRP4, and secreted protein that is acidic and rich in cysteine upregulated in tumor tissue as compared with surrounding normal tissue. In turn, genes up- and downregulated in tumor tissue were down- and upregulated, respectively, during development from the fetal to adult esophagus. Thus, we demonstrated that, as reported for other tumors, gene transcriptional activation and/or suppression events in esophageal tumor progression were opposite to those observed during development from the fetal to adult esophagus. This tumor ,embryonization' supports the idea that stem or progenitor cells are implicated in esophageal cancer emergence. [source] Molecular analysis of mutations at the HPRT and TK loci of human lymphoblastoid cells after combined treatments with 3,-azido-3,-deoxythymidine and 2,,3,-dideoxyinosine,ENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 4 2002Quanxin Meng Abstract Combinations of antiretroviral drugs that include nucleoside reverse transcriptase inhibitors (NRTIs) are superior to single-agent regimens in treating or preventing HIV infection, but the potential long-term health hazards of these treatments in humans are uncertain. In earlier studies, our group found that coexposure of TK6 human lymphoblastoid cells to 3,-azido-2,,3,-dideoxythymidine (AZT) and 2,,3,-dideoxyinosine (ddI), the first two NRTIs approved by the FDA as antiretroviral drugs, produced multiplicative synergistic enhancement of DNA incorporation of AZT and mutagenic responses in both the HPRT and TK reporter genes, as compared with single-drug exposures (Meng Q et al. [2000a]: Proc Natl Acad Sci USA 97:12667,12671). The purpose of the current study was to characterize the mutational specificity of equimolar mixtures of 100 ,M or 300 ,M AZT + ddI at the HPRT and TK loci of exposed cells vs. unexposed control cells, and to compare the resulting mutational spectra data to those previously found in cells exposed to AZT alone (Sussman H et al. [1999]: Mutat Res 429:249,259; Meng Q et al. [2000b]: Toxicol Sci 54:322,329). Molecular analyses of HPRT mutant clones were performed by reverse transcription,mediated production of cDNA, PCR amplification, and cDNA sequencing to define small DNA alterations, followed by multiplex PCR amplification of genomic DNA to define the fractions of deletion events. TK mutants with complete gene deletions were distinguished by Southern blot analysis. The observed HPRT mutational categories included point mutations, microinsertions/microdeletions, splicing-error mutations, and macrodeletions including partial and complete gene deletions. The only significant difference or shift in the mutational spectra for NRTI-treated cells vs. control cells was the increase in the frequency of complete TK gene deletions following exposures (for 3 days) to 300 ,M AZT,ddI (P = 0.034, chi-square test of homogeneity); however, statistical analyses comparing the observed mutant fraction values (measured mutant frequency × percent of a class of mutation) between control and NRTI-treated cells for each class of mutation showed that the occurrences of complete gene deletions of both HPRT and TK were significantly elevated over background values (0.34 × 10,6 in HPRT and 6.0 × 10,6 in TK) at exposure levels of 100 ,M AZT,ddI (i.e., 1.94 × 10,6 in HPRT and 18.6 × 10,6 in TK) and 300 ,M AZT,ddI (i.e., 5.6 × 10,6 in HPRT and 34.6 × 10,6 in TK) (P < 0.05, Mann,Whitney U -statistic). These treatment-related increases in complete gene deletions were consistent with the spectra data for AZT alone (ibid.) and with the known mode of action of AZT and ddI as DNA chain terminators. In addition, cotreatments of ddI with AZT led to substantial absolute increases in the mutant fraction of other classes of mutations, unlike cells exposed solely to AZT [e.g., the frequency of point mutations among HPRT mutants was significantly increased by 130 and 323% over the background value (4.25 × 10,6) in cells exposed to 100 and 300 ,M AZT,ddI, respectively]. These results indicate that, at the same time that AZT,ddI potentiates therapeutic or prophylactic efficacy, the use of a second NRTI with AZT may confer a greater cancer risk, characterized by a spectrum of mutations that deviates from that produced solely by AZT. Environ. Mol. Mutagen. 39:282,295, 2002. Published 2002 Wiley-Liss, Inc. [source] Complete subunit sequences, structure and evolution of the 6 × 6-mer hemocyanin from the common house centipede, Scutigera coleoptrataFEBS JOURNAL, Issue 13 2003Kristina Kusche Hemocyanins are large oligomeric copper-containing proteins that serve for the transport of oxygen in many arthropod species. While studied in detail in the Chelicerata and Crustacea, hemocyanins had long been considered unnecessary in the Myriapoda. Here we report the complete molecular structure of the hemocyanin from the common house centipede Scutigera coleoptrata (Myriapoda: Chilopoda), as deduced from 2D-gel electrophoresis, MALDI-TOF mass spectrometry, protein and cDNA sequencing, and homology modeling. This is the first myriapod hemocyanin to be fully sequenced, and allows the investigation of hemocyanin structure,function relationship and evolution. S. coleoptrata hemocyanin is a 6 × 6-mer composed of four distinct subunit types that occur in an approximate 2 : 2 : 1 : 1 ratio and are 49.5,55.5% identical. The cDNA of a fifth, highly diverged, putative hemocyanin was identified that is not included in the native 6 × 6-mer hemocyanin. Phylogenetic analyses show that myriapod hemocyanins are monophyletic, but at least three distinct subunit types evolved before the separation of the Chilopoda and Diplopoda more than 420 million years ago. In contrast to the situation in the Crustacea and Chelicerata, the substitution rates among the myriapod hemocyanin subunits are highly variable. Phylogenetic analyses do not support a common clade of Myriapoda and Hexapoda, whereas there is evidence in favor of monophyletic Mandibulata. [source] Characterization of a human alternatively spliced truncated reduced folate carrier increasing folate accumulation in parental leukemia cellsFEBS JOURNAL, Issue 3 2000Stavit Drori Human CEM-7A cells established by gradual deprivation of leucovorin from the growth medium, display 100-fold overexpression of methotrexate transport activity. We found that this was associated with 10-fold reduced folate carrier gene amplification and 50-fold overexpression of both the principal 3 kb reduced folate carrier transcript and, surprisingly, a novel truncated 2 kb reduced folate carrier mRNA poorly expressed in parental CEM cells. The molecular basis for the generation of this truncated reduced folate carrier transcript and its potential functional role in folate accumulation were studied. Reduced folate carrier genomic and cDNA sequencing revealed that the truncated transcript had an internal deletion of 987 nucleotides which was a result of an alternative splicing utilizing a cryptic acceptor splice site within exon 6. This deletion consisted of the 3,-most 480 nucleotides of the reduced folate carrier ORF and the following 507 nucleotides of the 3,-UTR. These resulted in a truncated reduced folate carrier protein, which lacks the C-terminal 160 amino acids, but instead contains 58 new C-terminal amino acids obtained from reading through the 3,-UTR. Consequently, a truncated reduced folate carrier protein is generated that lacks the 12th transmembrane domain and contains a new and much shorter C-terminus predicted to reside at the extracellular face. Western analysis with plasma-membrane fraction from CEM-7A cells revealed marked overexpression of both a broadly migrating , 65,90 kDa native reduced folate carrier and a , 40,45 kDa truncated reduced folate carrier, the core molecular masses of which were confirmed by in vitro translation. However, unlike the native reduced folate carrier, the truncated reduced folate carrier protein failed to bind the affinity labels NHS-[3H]MTX and NHS-[3H]folic acid. Stable transfection of the truncated reduced folate carrier cDNA into mouse L1210 leukemia cells: increased folate accumulation, decreased their leucovorin and folic acid growth requirements, and increased their sensitivity to methotrexate. This constitutes the first documentation of an expressed alternatively spliced truncated reduced folate carrier that, when coexpressed along with the native carrier, augments folate accumulation and consequently decreases the cellular folate growth requirement. The possible mechanisms by which the truncated reduced folate carrier may increase folate accumulation and/or metabolism in cells coexpressing the truncated and native reduced folate carrier are discussed. [source] Immune-privileged embryonic Swiss mouse STO and STO cell-derived progenitor cells: major histocompatibility complex and cell differentiation antigen expression patterns resemble those of human embryonic stem cell linesIMMUNOLOGY, Issue 1 2006Katherine S. Koch Summary Embryonic mouse STO (S, SIM; T, 6-thioguanine resistant; O, ouabain resistant) and 3(8)21-enhanced green fluorescent protein (EGFP) cell lines exhibit long-term survival and hepatic progenitor cell behaviour after xenogeneic engraftment in non-immunosuppressed inbred rats, and were previously designated major histocompatibility complex (MHC) class I- and class II-negative lines. To determine the molecular basis for undetectable MHC determinants, the expression and haplotype of H-2K, H-2D, H-2L and I-A proteins were reassessed by reverse transcriptase,polymerase chain reaction (RT-PCR), cDNA sequencing, RNA hybridization, immunoblotting, quantitative RT-PCR (QPCR), immunocytochemistry and flow cytometry. To detect cell differentiation (CD) surface antigens characteristic of stem cells, apoptotic regulation or adaptive immunity that might facilitate progenitor cell status or immune privilege, flow cytometry was also used to screen untreated and cytokine [interferon (IFN)-,]-treated cultures. Despite prior PCR genotyping analyses suggestive of H-2q haplotypes in STO, 3(8)21-EGFP and parental 3(8)21 cells, all three lines expressed H-2K cDNA sequences identical to those of d-haplotype BALB/c mice, as well as constitutive and cytokine-inducible H-2Kd determinants. In contrast, apart from H-2Ld[LOW] display in 3(8)21 cells, H-2Dd, H-2Ld and I-Ad determinants were undetectable. All three lines expressed constitutive and cytokine-inducible CD34; however, except for inducible CD117[LOW] expression in 3(8)21 cells, no expression of CD45, CD117, CD62L, CD80, CD86, CD90·1 or CD95L/CD178 was observed. Constitutive and cytokine-inducible CD95[LOW] expression was detected in STO and 3(8)21 cells, but not in 3(8)21-EGFP cells. MHC (class I+[LOW]/class II,) and CD (CD34+/CD80,/CD86,/CD95L,) expression patterns in STO and STO cell-derived progenitor cells resemble patterns reported for human embryonic stem cell lines. Whether these patterns reflect associations with mechanisms that are regulatory of immune privilege or functional tissue-specific plasticity is unknown. [source] Unexpected complexity of the budding yeast transcriptomeIUBMB LIFE, Issue 12 2008Takashi Ito Abstract The genome of the budding yeast Saccharomyces cerevisiae was sequenced over a decade ago and has been annotated to encode ,6,000 genes. However, recent high throughput studies using tiling array hybridization and cDNA sequencing have revealed an unexpectedly large number of previously undescribed transcripts. They largely lack protein-coding capacity and are transcribed from both strands of intragenic and intergenic regions in the genome. Accordingly, pervasive transcription leading to a plethora of noncoding RNAs, which was first revealed for mammalian genomes to attract intense attentions, is likely an intrinsic feature of eukaryotic genomes. Although it is not clear what fraction of these transcription events are functional, some were shown to induce transcriptional interference or histone modifications to regulate gene expression. The budding yeast may serve as an excellent model to study pervasive transcription and noncoding RNAs. © 2008 IUBMB IUBMB Life, 60(12): 775,781, 2008 [source] Characterization of the 3p12.3-pcen region associated with tumor suppression in a novel ovarian cancer cell line model genetically modified by chromosome 3 fragment transferMOLECULAR CARCINOGENESIS, Issue 12 2009Neal A.L. Cody Abstract The genetic analysis of nontumorigenic radiation hybrids generated by transfer of chromosome 3 fragments into the tumorigenic OV-90 ovarian cancer cell line identified the 3p12.3-pcen region as a candidate tumor suppressor gene (TSG) locus. In the present study, polymorphic microsatellite repeat analysis of the hybrids further defined the 3p12.3-pcen interval to a 16.1 Mb common region containing 12 known or hypothetical genes: 3ptel - ROBO2-ROBO1-GBE1-CADM2-VGLL3-CHMP2B-POU1F1-HTR1F-CGGBP1-ZNF654-C3orf38-EPHA3 -3pcen. Seven of these genes, ROBO1, GBE1, VGLL3, CHMP2B, CGGBP1, ZNF654, and C3orf38, exhibited gene expression in the hybrids, placing them as top TSG candidates for further analysis. The expression of all but one (VGLL3) of these genes was also detected in the parental OV-90 cell line. Mutations were not identified in a comparative sequence analysis of the predicted protein coding regions of these candidates in OV-90 and donor normal chromosome 3 contig. However, the nondeleterious sequence variants identified in the transcribed regions distinguished parent of origin alleles for ROBO1, VGLL3, CHMP2B, and CGGBP1 and cDNA sequencing of the hybrids revealed biallelic expression of these genes. Interestingly, underexpression of VGLL3 and ZNF654 were observed in malignant ovarian tumor samples as compared with primary cultures of normal ovarian surface epithelial cells or benign ovarian tumors, and this occurred regardless of allelic content of 3p12.3-pcen. The results taken together suggest that dysregulation of VGLL3 and/or ZNF654 expression may have affected pathways important in ovarian tumorigenesis which was offset by the transfer of chromosome 3 fragments in OV-90, a cell line hemizygous for 3p. Mol. Carcinog. © 2009 Wiley-Liss, Inc. [source] The proteome survey of an electricity-generating organ (Torpedo californica electric organ)PROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 4 2007Javad Nazarian Abstract Torpedo californica is a species in class Chondrichthyes. Electric rays have evolved the electric organ, which is similar to the mammalian neuromuscular junction (NMJ). Here, we took a combined cDNA sequencing and proteomic approach to define the molecular constituents of the T. californica electric organ. For soluble proteins, 2-DE was used and 224 protein spots were mapped. Plasma membrane fractions were analyzed using the shotgun approach (LC-MS/MS). A Torpedo cDNA library was constructed and 607 cDNA clones were sequenced. Identification of electric organ proteins was done using cross-species comparisons, and a custom database was constructed from cDNA translations. We unambiguously identified 121 proteins and transcripts, 103 of which were novel additions to the existing databases of Torpedo fish. Fifteen proteins of known function, but not previously associated with either the electroplaque or NMJ, were present at high abundance. These included the heat shock and oxidative stress proteins, annexin V (calelectrin), and plectin 1. Most interesting were the unambiguous matches to 11 human ORFs of unknown function, including four potential RNA splicing proteins, a vacuolar sorting protein, and a tetraspanin containing protein. This analysis identified proteins that may play a role in the higher vertebrate neuromuscular junction or other electrical synapses. [source] Transcriptionally active transposable elements in recent hybrid sugarcaneTHE PLANT JOURNAL, Issue 5 2005Paula G. de Araujo Summary Transposable elements (TEs) are considered to be important components of the maintenance and diversification of genomes. The recent increase in genome sequence data has created an opportunity to evaluate the impact of these active mobile elements on the evolution of plant genomes. Analysis of the sugarcane transcriptome identified 267 clones with significant similarity to previously described plant TEs. After full cDNA sequencing, 68 sugarcane TE clones were assigned to 11 families according to their best sequence alignment against a fully characterized element. Expression was further investigated through a combined study utilizing electronic Northerns, macroarray, transient and stable sugarcane transformation. Newly synthesized cDNA probes from flower, leaf roll, apical meristem and callus tissues confirm previous results. Callus was identified as the tissue with the highest number of TEs being expressed, revealing that tissue culture drastically induced the expression of different elements. No tissue-specific family was identified. Different representatives within a TE family displayed differential expression patterns, showing that each family presented expression in almost every tissue. Transformation experiments demonstrated that most Hopscotch clone-derived U3 regions are, indeed, active promoters, although under a strong transcriptional regulation. This is a large-scale study about the expression pattern of TEs and indicates that mobile genetic elements are transcriptionally active in the highly polyploid and complex sugarcane genome. [source] A Mutation that Creates a Pseudoexon in SOD1 Causes Familial ALSANNALS OF HUMAN GENETICS, Issue 6 2009Paul N. Valdmanis Summary Amyotrophic lateral sclerosis (ALS) is an adult onset neurodegenerative disease which targets motor neurons of the cortex, brainstem and spinal cord. About 5,10% of all amyotrophic lateral sclerosis cases are familial (FALS), and 15,20% of FALS cases are caused by mutations in the zinc-copper superoxide dismutase gene (SOD1). We identified a large family from France with ten members affected with ALS. Linkage was established to the SOD1 locus on chromosome 21 and genomic and cDNA sequencing was performed for the SOD1 gene. This revealed an activated pseudoexon between exons 4 and 5 that was present in two tested members of the family. Translation of this 43 base pair exon results in the introduction of seven amino acids before a stop codon is present, leading to a prematurely truncated SOD1 protein product of 125 amino acids. Sequencing intron 4 in a patient revealed a heterozygous change 304 bp before exon 5 (c.358 , 304C > G), but only 5 bp after the cryptic exon, thus causing this alternative splice product. This mutation segregated in all affected individuals of the family. This adds an additional genetic mechanism for developing SOD1 -linked ALS and is one which can be more readily targeted by gene therapy. [source] | ||||||||||||||||