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Complementary DNA (complementary + dna)
Terms modified by Complementary DNA Selected AbstractsCloning of PRL and VIP cDNAs of the Java sparrow (Padda oryzivora)ANIMAL SCIENCE JOURNAL, Issue 2 2009Gen HIYAMA ABSTRACT Complementary DNA (cDNA) of prolactin (PRL) and vasoactive intestinal polypeptide (VIP) of the Java sparrow were cloned and sequenced. The proximal region of the PRL promoter was also identified. Java sparrow PRL was found to have 88.3, 88.3, and 89.1% sequence identity at the cDNA level to PRL of chicken, turkey, and duck, respectively. The predicted amino acid sequence had an overall similarity with a comparable region of chicken (91.4%), turkey (88.9%) and duck (92.0%) PRL. Based on the cDNA sequence and genomic structure of the chicken PRL gene, the proximal promoter was characterized. Sequence analysis of the proximal region of Java sparrow PRL promoter revealed a high degree of similarity to that of chicken, turkey and duck PRL promoters. Moreover, cDNA of prepro-VIP was also cloned and sequenced. Java sparrow prepro-VIP shows high similarity to chicken and turkey prepro-VIP. However, the region upstream of the 5, untranslated region of Java sparrow prepro-VIP did not show similarity to that of chicken. These results suggest that the mechanisms, which regulate expression of the VIP gene, may be different between precocial and altricial birds, but expression of the PRL gene may be widely conserved in avian species. [source] Expression of Ht2 -related genes in response to the HT-Toxin of Exserohilum turcicum in MaizeANNALS OF APPLIED BIOLOGY, Issue 1 2010H. Wang Complementary DNA amplified fragment length polymorphism (cDNA-AFLP) analysis was conducted to analyze differential expression of Ht2 -related genes between maize (Zea mays) near-isogenic lines (NILs), Huangzaosi (HZS) and HuangzaosiHt2 (HZSHt2), following treatment with a crude extract of the HT-toxin. Twenty-one transcript-derived fragments (TDFs), designated H1 to H21, were specifically expressed or upregulated in HZSHt2 following exposure to the HT-toxin. Among them, 4, 7, 4, 2, 2 and 2 TDFs were detected at 3, 6, 12, 24, 48 and 72 h after treatment, respectively. BLAST analysis showed that H1, H11, H13 and H15 are related to regulation of the defence response to environmental stresses. H3, H6 and H10 are associated with energy metabolism. H5, H17 and H18 are involved in photosynthesis. H9 is similar to ubiquitin-like domain containing CTD phosphatase. H8, H9, H16 and H20 are probably transcription factors. The genes associated with basal energy metabolism and signal of stress tolerance were mainly expressed at 3 h after treatment. Transcription factor and most genes for stress tolerance were expressed at 6 h after treatment. RT-PCR analysis demonstrated that H8 was upregulated in HZSHt2 only at 6 h after exposure to the HT-toxin and H13 was upregulated at 6 and 12 h. The full length cDNAs of H8 (GenBank accession number FJ600319) and H13 (FJ600320) were cloned. The deduced protein encoded by H8 cDNA showed 77% homology to the Plus-3 domain containing protein, which is found in yeast gene Rtf1. H13 cDNA encodes a QM-like protein, which is an important protein in plant tolerance to environmental stress. The mechanism regulating the resistance of Ht2 to the HT-toxin might involve a translation elongation factor or an upregulated QM-like protein. [source] HCV-RNA In Sural Nerve From Hcv Infected Patients With Peripheral NeuropathyJOURNAL OF THE PERIPHERAL NERVOUS SYSTEM, Issue 1 2001L De Martino Objective: Evaluation of hepatitis C virus (HCV) by reverse transcription-polymerase chain reaction (RT-PCR) in peripheral nerve tissues from HCV infected patients with peripheral neuropathy. METHODS: RT-PCR was performed on homogenates of nerve biopsies from 17 consecutive HCV-positive patients with peripheral neuropathy, with or without mixed cryoglobulinemia, hospitalised from 1996 to 2000. Sural nerve specimens were frozen in iso-pentane pre-cooled in liquid nitrogen and stored at ,80°C until use. RNA was extracted from ten 7-,m thick cryostatic sections or from a nerve trunk specimen of about 3 mm length, collected from each biopsy. Three different protocols of RNA extraction were tested (1,3). Complementary DNAs (cDNAs) were obtained without or with RNasin (Promega, Madison, WI) addition in the reaction mixture to inhibit residual RNase activity. Two sets of commercially available PCR primers for the outer and the nested reaction were used. PCR products were analysed by agarose gel electrophoresis and ethidium bromide staining. Serum samples and liver specimens from proven HCV positive patients served as positive controls, whereas sera from healthy subjects were negative controls. RESULTS: Sufficient amount of RNA could be obtained either by cryostatic sections or by in toto nerve specimens. Extraction by Trizol (Gibco-BRL) allowed the best concentration and purity of RNA as assessed by biophotometry. The presence of RNasin didn't improve the cDNA synthesis. The resulting amplification product of the nested PCR was 187 bp long. We have always observed this product in our positive controls and never in the negative. Six samples from patients either with or without cryoglobulinemia resulted positive; 7 were negative. Four samples gave variable results. CONCLUSIONS: While 40% of the nerves in our series were undoubtedly HCV positive, the cause(s) of negative and variable results in the remaining samples is likely more complex than variations in the detection protocols and deserve further investigations. REFERENCES: 1) Chomczynski P, Sacchi N (1987). Anal Biochem 162:156. 2) Marquardt O et al. (1996). Med Microbiol Lett 5:55. 3) Chomczynski P (1993). Bio/Techniques 15:532. [source] Global gene expression analysis of the shoot apical meristem of maize (Zea mays L.)THE PLANT JOURNAL, Issue 3 2007Kazuhiro Ohtsu Summary All above-ground plant organs are derived from shoot apical meristems (SAMs). Global analyses of gene expression were conducted on maize (Zea mays L.) SAMs to identify genes preferentially expressed in the SAM. The SAMs were collected from 14-day-old B73 seedlings via laser capture microdissection (LCM). The RNA samples extracted from LCM-collected SAMs and from seedlings were hybridized to microarrays spotted with 37 660 maize cDNAs. Approximately 30% (10 816) of these cDNAs were prepared as part of this study from manually dissected B73 maize apices. Over 5000 expressed sequence tags (ESTs) (about 13% of the total) were differentially expressed (P < 0.0001) between SAMs and seedlings. Of these, 2783 and 2248 ESTs were up- and down-regulated in the SAM, respectively. The expression in the SAM of several of the differentially expressed ESTs was validated via quantitative RT-PCR and/or in situ hybridization. The up-regulated ESTs included many regulatory genes including transcription factors, chromatin remodeling factors and components of the gene-silencing machinery, as well as about 900 genes with unknown functions. Surprisingly, transcripts that hybridized to 62 retrotransposon-related cDNAs were also substantially up-regulated in the SAM. Complementary DNAs derived from the LCM-collected SAMs were sequenced to identify additional genes that are expressed in the SAM. This generated around 550 000 ESTs (454-SAM ESTs) from two genotypes. Consistent with the microarray results, approximately 14% of the 454-SAM ESTs from B73 were retrotransposon-related. Possible roles of genes that are preferentially expressed in the SAM are discussed. [source] Abnormal myelin formation in rhizomelic chondrodysplasia punctata type 2 (DHAPAT-deficiency)DEVELOPMENTAL MEDICINE & CHILD NEUROLOGY, Issue 7 2000László Sztriha MD PhD The case of a Yemeni girl with isolated peroxisomal acyl-CoA:dihydroxyacetonephosphateacyltransferase (DHAPAT) deficiency is reported. She had rhizomelic chondrodysplasia punctata, microcephaly, failure to thrive, delayed motor and mental development, and spastic quadriplegia. Deficient de novo plasmalogen synthesis in her fibroblasts as a result of low DHAPAT activity was found, while her very-long-chain fatty acid profile, phytanic acid concentration, alkyl-dihydroxyacetonephosphate synthase (alkyl-DHAP synthase) activity, and peroxisomal 3-ketoacyl-CoA thiolase protein were normal. A mutation in her DHAPAT complementary DNA resulted in the substitution of an arginine residue in the protein at position 211 by a histidine (R211H). Magnetic resonance imaging showed abnormal white matter signal in the centrum semiovale involving the arcuate fibers, while the corpus callosum was normal. DHAPAT and alkyl-DHAP synthase initiate the synthesis of plasmalogens, which are major constituents of myelin phospholipids. The reported girl's abnormal formation of myelin is probably related to the inadequacy of plasmalogen biosynthesis, which is likely to be due to deficient DHAPAT activity. [source] Microarrays: The Technology, Analysis and ApplicationENGINEERING IN LIFE SCIENCES (ELECTRONIC), Issue 3 2005A. Kumar Abstract DNA microarray analysis represents one of the major advances leading to the development of functional genomics and proteomics. It involves the fabrication of DNA either by in situ or on-chip photolithographic synthesis or by inkjet or microjet deposition, as microspots immobilized on the surface of miniaturized substrates like glass or membranes. The immobilized DNA molecules are then allowed to hybridize with labeled complementary DNA. The hybrid DNA so formed is read through scanning devices, such as fluorescence and radioactivity. Further, computational approaches, for example, normalization and clustering allow thousands of genetic parameters in a single experiment to be simultaneously analyzed. This review discusses the fundamental principles and data analysis of the microarray technology, while focusing on its application in gene expression analysis, genotyping for point mutation and diseases diagnostics. [source] Development of a fish reporter gene system for the assessment of estrogenic compounds and sewage treatment plant effluentsENVIRONMENTAL TOXICOLOGY & CHEMISTRY, Issue 9 2002Gabriele E. Ackermann Abstract This study reports on the development and application of a fish-specific estrogen-responsive reporter gene assay. The assay is based on the rainbow trout (Oncorhynchus mykiss) gonad cell line RTG-2 in which an acute estrogenic response is created by cotransfecting cultures with an expression vector containing rainbow trout estrogen receptor a complementary DNA (rtER, cDNA) in the presence of an estrogen-dependent reporter plasmid and an estrogen receptor (ER) agonist. In a further approach, RTG-2 cells were stably transfected with the rtER, cDNA expression vector, and clones responsive to 17,-estradiol (E2) were selected. The estrogenic activity of E2, 17,-ethinylestradiol, 4-nonylphenol, nonylphenoxy acetic acid, 4- tert -octylphenol, bisphenol A, o,p,-DDT, p,p,-DDT, o,p,-2,2-bis(chlorophenyl)-1,1-dichloroethylene (o,p,-DDE), p,p,-DDE, o,p,-2,2-bis(chlorophenyl)-1,1-di-chloroethane (o,p,-DDD), p,p,-DDD, and p,p,-2,2-bis(chlorophenyl)acetic acid (p,p,-DDA) was assessed at increasing concentrations. All compounds except o,p,-DDT, p,p,-DDE, and p,p,-DDA showed logistic dose-response curves, which allowed the calculation of lowest-observed-effect concentrations and the concentrations at which half-maximal reporter gene activities were reached. To check whether estrogen-responsive RTG-2 cells may be used to detect the estrogenic activity of environmental samples, an extract from a sewage treatment plant (STP) effluent was assessed and found to have estrogenic activity corresponding to the transcriptional activity elicited by 0.05 nM of E2. Dose-response curves of nonylphenol, octylphenol, bisphenol A, and o,p,-DDD revealed that the RTG-2 reporter gene assay is more sensitive for these compounds when compared to transfection systems recombinant for mammalian ERs. These differences may have an effect on the calculation of E2 equivalents when estrogenic mixtures of known constitution, or environmental samples, such as STP effluents, are assessed. [source] Screening the Structural Space of Bicyclo-DNA: Synthesis and Thermal Melting Properties of bc4,3 -DNAEUROPEAN JOURNAL OF ORGANIC CHEMISTRY, Issue 8 2009Andrea Stauffiger Abstract In attempts to screen the structural and functional properties of bicyclo-DNA, in which the ribose C(3,) and C(5,) centers are integrated into an additional five-membered carbocyclic ring ([3.3.0]-series) we have now synthesized and investigated a ring enlarged analogue in which C(5,) and C(3,) are spanned by a six-membered carbocyclic ring ([4.3.0]-series). The synthesis of the corresponding bc4,3 -T nucleoside 13 was performed in 12 steps by starting from known allyl furanose 1. X-ray analysis of its benzyl protected precursor 12 showed the cyclohexane ring to adopt a chair conformation with the O(5,) substituent in an axial position. The furanose part shows clearly S-type sugar pucker. This nucleoside was converted into the corresponding phosphoramidite building block 15 and incorporated into oligodeoxynucleotides by standard phosphoramidite chemistry. The thermal stabilities of oligonucleotides with single or double incorporations of bc4,3 -T residues, paired to complementary DNA or RNA, were found to be similar to those of unmodified oligonucleotides (,2.3 to +0.7 °C per modification) and to those with the known bc-T modifications. We also found that mismatch discrimination in the bc4,3 -T series was similar to that of the natural series but less discriminative in comparison to the known bc-T series.(© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009) [source] Expression of one sponge Iroquois homeobox gene in primmorphs from Suberites domuncula during canal formationEVOLUTION AND DEVELOPMENT, Issue 3 2003Sanja Perovi SUMMARY Sponges (Porifera) represent the evolutionary oldest multicellular animals. They are provided with the basic molecules involved in cell,cell and cell,matrix interactions. We report here the isolation and characterization of a complementary DNA from the sponge Suberites domuncula coding for the sponge homeobox gene, SUBDOIRX-a. The deduced polypeptide with a predicted Mr of 44,375 possesses the highly conserved Iroquois-homeodomain. We applied in situ hybridization to localize Iroquois in the sponge. The expression of this gene is highest in cells adjacent to the canals of the sponge in the medulla region. To study the expression of Iroquois during development, the in vitro primmorph system from S. domuncula was used. During the formation of these three-dimensional aggregates composed of proliferating cells, the expression of Iroquois depends on ferric iron and water current. An increased expression in response to water current is paralleled with the formation of canal-like pores in the primmorphs. It is suggested that Iroquois expression is involved in the formation of the aquiferous system, the canals in sponges and the canal-like structures in primmorphs. [source] Hev b 9, an enolase and a new cross-reactive allergen from Hevea latex and moldsFEBS JOURNAL, Issue 24 2000Purification, characterization, cloning, expression Natural rubber latex allergy is an IgE-mediated disease that is caused by proteins that elute from commercial latex products. A complementary DNA (cDNA) coding for Hev b 9, an enolase (2-phospho- d -glycerate hydrolyase) and allergen from latex of the rubber tree Hevea brasiliensis, was amplified by PCR. The PCR primers were designed according to conserved regions of enolases from plants. The obtained cDNA amplification product consisted of 1651 bp and encoded a protein of 445 amino-acid residues with a calculated molecular mass of 47.6 kDa. Sequence comparisons revealed high similarities of the Hevea latex enolase to mold enolases that have been identified as important allergens. In addition, the crucial amino-acid residues that participate in the formation of the catalytic site and the Mg2+ binding site of enolases were also conserved. Hevea latex enolase was produced as a recombinant protein in Escherichia coli with an N-terminal hexahistidyl tag, and purified by affinity chromatography. The yield amounted to 110 mg of purified Hev b 9 per litre of bacterial culture. The recombinant allergen bound IgE from latex, as well as mold-allergic patients, in immunoblot and ELISA experiments. The natural enolase was isolated from Hevea latex by (NH4)2SO4 precipitation and ion exchange chromatography. The natural and the recombinant (r)Hev b 9 showed equivalent enzymatic activity. Patients' IgE-antibodies preincubated with rHev b 9 lost their ability to bind to natural (n) Hev b 9, indicating the identity of the B-cell epitopes on both molecules. Cross-reactivity with two enolases from Cladosporium herbarum and Alternaria alternata was determined by inhibition of IgE-binding to these enolases by rHev b 9. Therefore, enolases may represent another class of highly conserved enzymes with allergenic potentials. [source] Altered gene expression in acute systemic inflammation detected by complete coverage of the human liver transcriptomeHEPATOLOGY, Issue 2 2004Cédric Coulouarn The goal of the current study was to provide complete coverage of the liver transcriptome with human probes corresponding to every gene expressed in embryonic, adult, and/or cancerous liver. We developed dedicated tools, namely, the Liverpool nylon array of complementary DNA (cDNA) probes for approximately 10,000 nonredundant genes and the LiverTools database. Inflammation-induced transcriptome changes were studied in liver tissue samples from patients with an acute systemic inflammation and from control subjects. One hundred and fifty-four messenger RNAs (mRNA) correlated statistically with the extent of inflammation. Of these, 134 mRNA samples were not associated previously with an acute-phase (AP) response. The hepatocyte origin and proinflammatory cytokine responsiveness of these mRNAs were confirmed by quantitative reverse-transcription polymerase chain reaction (Q-RT-PCR) in cytokine-challenged hepatoma cells. The corresponding gene promoters were enriched in potential binding sites for inflammation-driven transcription factors in the liver. Some of the corresponding proteins may provide novel blood markers of clinical relevance. The mRNAs whose level is most correlated with the AP extent (P < .05) were enriched in intracellular signaling molecules, transcription factors, glycosylation enzymes, and up-regulated plasma proteins. In conclusion, the hepatocyte responded to the AP extent by fine tuning some mRNA levels, controlling most, if not all, intracellular events from early signaling to the final secretion of proteins involved in innate immunity. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html). (HEPATOLOGY 2004;39:353,364.) [source] Cancer-associated molecular signature in the tissue samples of patients with cirrhosis,HEPATOLOGY, Issue 2 2004Jin Woo Kim Several types of aggressive cancers, including hepatocellular carcinoma (HCC), often arise as a multifocal primary tumor. This suggests a high rate of premalignant changes in noncancerous tissue before the formation of a solitary tumor. Examination of the messenger RNA expression profiles of tissue samples derived from patients with cirrhosis of various etiologies by complementary DNA (cDNA) microarray indicated that they can be grossly separated into two main groups. One group included hepatitis B and C virus infections, hemochromatosis, and Wilson's disease. The other group contained mainly alcoholic liver disease, autoimmune hepatitis, and primary biliary cirrhosis. Analysis of these two groups by the cross-validated leave-one-out machine-learning algorithms revealed a molecular signature containing 556 discriminative genes (P < .001). It is noteworthy that 273 genes in this signature (49%) were also significantly altered in HCC (P < .001). Many genes were previously known to be related to HCC. The 273-gene signature was validated as cancer-associated genes by matching this set to additional independent tumor tissue samples from 163 patients with HCC, 56 patients with lung carcinoma, and 38 patients with breast carcinoma. From this signature, 30 genes were altered most significantly in tissue samples from high-risk individuals with cirrhosis and from patients with HCC. Among them, 12 genes encoded secretory proteins found in sera. In conclusion, we identified a unique gene signature in the tissue samples of patients with cirrhosis, which may be used as candidate markers for diagnosing the early onset of HCC in high-risk populations and may guide new strategies for chemoprevention. Supplementary material for this article can be found on the HEPATOLOGY website (http://interscience.wiley.com/jpages/0270-9139/suppmat/index.html). (HEPATOLOGY 2004;39:518,527.) [source] Ribosome Display and Dip-Pen Nanolithography for the Fabrication of Protein Nanoarrays,ADVANCED MATERIALS, Issue 17 2008Jung Dong Kim Protein nanochip fabrication is performed via immobilizing proteins by dip-pen nanolithography (DPN) and the ribosome display technique. Protein,ribosome,mRNA fusion molecules permit the simultaneous immobilization of functional proteins without purification through hybridization to complementary DNA that has been immobilized on a nanometer scale on the surface via DPN. [source] DFT study of the electronic properties of DNA,DNA and PNA,DNA double strandsINTERNATIONAL JOURNAL OF QUANTUM CHEMISTRY, Issue 15 2006Takayuki Natsume Abstract The electronic properties of DNA,DNA and PNA,DNA double strands having 3,6 base pairs (bp) were investigated by density functional theory (DFT) calculations. The binding energies and the highest occupied molecular orbital,lowest unoccupied molecular orbital (HOMO,LUMO) energy gaps for the PNA,DNA hybrids in the vapor phase are found to be greater than those for the DNA,DNA hybrids, regardless of the number of base pairs involved. The study supports the experimental finding that PNA displays high affinity toward a complementary DNA and that PNA,DNA strands are much more thermodynamically stable than their DNA,DNA counterparts. The results suggest much higher sensitivity in DNA sequencing with the arrays of PNA than with those of DNA. © 2006 Wiley Periodicals, Inc. Int J Quantum Chem, 2006 [source] The long and winding road to RNA editing in plant mitochondria: The Tübingen-Berlin chapter,IUBMB LIFE, Issue 12 2009Axel Brennicke Abstract It took several independent observations of C-to-T differences between genomic mtDNA sequences and corresponding complementary DNA (cDNA) sequences before RNA editing in plant mitochondria was accepted as a fact by the group at Tübingen and later Berlin (Hiesel et al., Science246 (1989) 1632,1634). The first such deviating sequence runs were critically viewed in the lab as being errors of some kind, most likely cloning artifacts, which occur only too frequently. Several such cDNA-mtDNA differences identified in independent cDNA clones in different libraries and finally CGG to TGG codon changes dispelled the skeptical view, and this phenomenon was finally recognized as plant mitochondrial RNA editing of a type similar to the apolipoprotein B RNA editing in mammals. © 2009 IUBMB IUBMB Life, 61: 1105,1109, 2009. [source] Purification of Matrix Gla Protein From a Marine Teleost Fish, Argyrosomus regius: Calcified Cartilage and Not Bone as the Primary Site of MGP Accumulation in Fish,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 2 2003DC Simes Abstract Matrix Gla protein (MGP) belongs to the family of vitamin K-dependent, Gla-containing proteins, and in mammals, birds, and Xenopus, its mRNA was previously detected in extracts of bone, cartilage, and soft tissues (mainly heart and kidney), whereas the protein was found to accumulate mainly in bone. However, at that time, it was not evaluated if this accumulation originated from protein synthesized in cartilage or in bone cells because both coexist in skeletal structures of higher vertebrates and Xenopus. Later reports showed that MGP also accumulated in costal calcified cartilage as well as at sites of heart valves and arterial calcification. Interestingly, MGP was also found to accumulate in vertebra of shark, a cartilaginous fish. However, to date, no information is available on sites of MGP expression or accumulation in teleost fishes, the ancestors of terrestrial vertebrates, who have in their skeleton mineralized structures with both bone and calcified cartilage. To analyze MGP structure and function in bony fish, MGP was acid-extracted from the mineralized matrix of either bone tissue (vertebra) or calcified cartilage (branchial arches) from the bony fish, Argyrosomus regius,, separated from the mineral phase by dialysis, and purified by Sephacryl S-100 chromatography. No MGP was recovered from bone tissue, whereas a protein peak corresponding to the MGP position in this type of gel filtration was obtained from an extract of branchial arches, rich in calcified cartilage. MGP was identified by N-terminal amino acid sequence analysis, and the resulting protein sequence was used to design specific oligonucleotides suitable to amplify the corresponding DNA by a mixture of reverse transcription-polymerase chain reaction (RT-PCR) and 5,rapid amplification of cDNA (RACE)-PCR. In parallel, ArBGP (bone Gla protein, osteocalcin) was also identified in the same fish, and its complementary DNA cloned by an identical procedure. Tissue distribution/accumulation was analyzed by Northern blot, in situ hybridization, and immunohistochemistry. In mineralized tissues, the MGP gene was predominantly expressed in cartilage from branchial arches, with no expression detected in the different types of bone analyzed, whereas BGP mRNA was located in bone tissue as expected. Accordingly, the MGP protein was found to accumulate, by immunohistochemical analysis, mainly in the extracellular matrix of calcified cartilage. In soft tissues, MGP mRNA was mainly expressed in heart but in situ hybridization, indicated that cells expressing the MGP gene were located in the bulbus arteriosus and aortic wall, rich in smooth muscle and endothelial cells, whereas no expression was detected in the striated muscle myocardial fibers of the ventricle. These results show that in marine teleost fish, as in mammals, the MGP gene is expressed in cartilage, heart, and kidney tissues, but in contrast with results obtained in Xenopus and higher vertebrates, the protein does not accumulate in vertebra of non-osteocytic teleost fish, but only in calcified cartilage. In addition, our results also indicate that the presence of MGP mRNA in heart tissue is due, at least in fish, to the expression of the MGP gene in only two specific cell types, smooth muscle and endothelial cells, whereas no expression was found in the striated muscle fibers of the ventricle. In light of these results and recent information on expression of MGP gene in these same cell types in mammalian aorta, it is likely that the levels of MGP mRNA previously detected in Xenopus, birds, and mammalian heart tissue may be restricted toregions rich in smooth muscle and endothelial cells. Our results also emphasize the need to re-evaluate which cell types are involved in MGP gene expression in other soft tissues and bring further evidence that fish are a valuable model system to study MGP gene expression and regulation. [source] Osteoclast Inhibitory Peptide 2 Inhibits Osteoclast Formation via Its C-Terminal FragmentJOURNAL OF BONE AND MINERAL RESEARCH, Issue 10 2001Sun Jin Choi Abstract Osteoclast inhibitory peptide 2 (OIP-2) is a novel autocrine/paracrine factor produced by osteoclasts (OCLs) that inhibits bone resorption and OCL formation in vitro and in vivo. It is identical to the asparaginyl endopeptidase legumain. During maturation of OIP-2, a signal peptide and a 17-kDa C-terminal fragment (CTF) are cleaved to produce the mature enzyme. To determine if enzyme activity is required for inhibition of OCL formation or if only the CTF is responsible for these effects, we synthesized His-tagged complementary DNA (cDNA) constructs for the CTF of OIP-2, the proform of OIP-2, and the "mature enzyme" form of OIP-2. The proform or the CTF portion of OIP-2 inhibited OCL formation in a dose-dependent manner in murine bone marrow cultures stimulated with 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. The mature form of OIP-2, which was enzymatically active, did not inhibit OCL formation. In addition, OIP-2 inhibited OCL formation in cultures of highly purified human OCL precursor cells or RAW264.7 cells stimulated with 10 ng/ml of receptor activator of NF-,B (RANK) ligand. Binding studies with His-tagged OIP-2 showed expression of a putative OIP-2 receptor on RAW264.7 cells treated with RANK ligand for 4 days and human marrow cultures treated with 1,25(OH)2D3 for 3 weeks. These data show that the CTF of OIP-2, rather than the mature enzyme, mediates the inhibitory effects of OIP-2 through a putative receptor on OCL precursors. [source] Canine COL1A2 Mutation Resulting in C-Terminal Truncation of Pro-,2(I) and Severe Osteogenesis ImperfectaJOURNAL OF BONE AND MINERAL RESEARCH, Issue 6 2001Bonnie G. Campbell Abstract RNA and type I collagen were analyzed from cultured skin fibroblasts of a Beagle puppy with fractures consistent with type III osteogenesis imperfecta (OI). In a nonisotopic RNAse cleavage assay (NIRCA), the proband's RNA had a unique cleavage pattern in the region of COL1A2 encoding the C-propeptide. DNA sequence analyses identified a mutation in which nucleotides 3991-3994 ("CTAG") were replaced with "TGTCATTGG." The first seven bases of the inserted sequence were identical to nucleotides 4002-4008 of the normal canine COL1A2 sequence. The resulting frameshift changed 30 amino acids and introduced a premature stop codon. Reverse-transcription polymerase chain reaction (RT-PCR) with primers flanking the mutation site amplified two complementary DNA (cDNA) fragments for the proband and a single product for the control. Restriction enzyme digestions also were consistent with a heterozygous mutation in the proband. Type I procollagen labeled with [3H]proline was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Increased density of pC-,2(I) suggested comigration with the similarly sized pro-,2(I) derived from the mutant allele. Furthermore, ,-chains were overhydroxylated and the ratio of ,1(I):,2(I) was 3.2:1, consistent with the presence of ,1(I) homotrimers. Analyses of COL1A2 and type I collagen were both consistent with the described heterozygous mutation affecting the pro-,2(I) C-propeptide and confirmed a diagnosis of OI. [source] Cloning, Sequencing, and Functional Characterization of the Rat Homologue of Receptor Activator of NF-,B Ligand,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 11 2000Jiake Xu Abstract A complementary DNA (cDNA) encoding the rat homologue of receptor activator of NF-,B ligand/osteoprotegerin ligand/osteoclast differentiation factor/tumor necrosis factor (TNF)-related activation-induced cytokine (RANKL/OPGL/ODF/TRANCE) was cloned and sequenced from tibias of ovariectomized (OVX) rats. The predicted amino acid sequence of rat RANKL (rRANKL) has 84% and 96% identity to that of human and mouse RANKL, respectively, and 35% and 37% similarity to that of human and mouse TNF-related apoptosis-inducing ligand (TRAIL), respectively. RANKL transcripts were expressed abundantly in the thymus and bone tissues of OVX rats. rRANKL has a single hydrophobic region between residues 53 and 69, which is most likely to serve as a transmembrane domain. The long C-terminal region containing ,-sheet-forming sequences of the TNF-like core is considered the extracellular region. Three truncated domains within the TNF-like core region were expressed as glutathione S-transferase (GST) fusion proteins and investigated for their ability to induce osteoclastogenesis. The results showed that GST-rRANKL (aa160-318) containing the full TNF-like core region had the highest capability to induce the formation of osteoclast-like cells from RAW264.7 cells. GST-rRANKL (aa239-318 and aa160-268) had lesser degrees of osteoclast inductivity. Furthermore, the GST-rRANKL (aa160-318) is capable of (1) inducing osteoclast formation from rat spleen cells in the presence of macrophage colony-stimulating factor (M-CSF), (2) stimulating mature rat osteoclast polarization and bone resorption ex vivo, and (3) inducing systemic hypercalcemia in vivo; thus the full TNF-like core region of rRANKL is an important regulator of calcium homeostasis and osteoclastic function. [source] Synthesis, characterization and hybridization studies of new nucleo-,-peptides based on diaminobutyric acidJOURNAL OF PEPTIDE SCIENCE, Issue 12 2006G. N. Roviello Abstract In the present work, we report the synthesis and the characterization of a new chiral nucleoaminoacid, in which a diaminobutyric moiety is connected to the DNA nucleobase by an amidic bond, and its oligomerization to give the corresponding nucleo-,-peptide. The ability of this synthetic polymer to bind complementary DNA was studied in order to explore its possible use in antigene/antisense or diagnostic applications. Our interest in the presented DNA analogue was also supported by the importance of ,-aminoacid-containing compounds in natural products of biological activity and by the known stability of ,-peptides to enzymatic degradation. Furthermore, our work could contribute to the study of the role of nucleopeptides as prebiotic material in a PNA world that could successively lead to the actual DNA/RNA/protein world, as recently assumed. Copyright © 2006 European Peptide Society and John Wiley & Sons, Ltd. [source] Fine Mapping of Polymorphic Alcohol-Related Quantitative Trait Loci Candidate Genes Using Interval-Specific Congenic Recombinant MiceALCOHOLISM, Issue 11 2002Marissa A. Ehringer Background The inbred long-sleep (ILS) and inbred short-sleep (ISS) strains of mice are widely studied as a model of initial sensitivity to alcohol. Recently, a large comparative DNA sequencing study of candidate genes located within the four Lore quantitative trait loci (QTLs) associated with the ethanol-induced loss of righting reflex in ILS and ISS mice has identified eight genes that contain coding region differences corresponding to amino acid changes. Here, recently developed interval-specific congenic recombinant mice (ISCRs) have been used to map these genes in relationship to newly narrowed QTL regions. Methods Regions of candidate genes containing DNA differences corresponding to previously identified amino acid changes between ISS and ILS mice were amplified from either genomic DNA or complementary DNA from ISCR mice using polymerase chain reaction. The products were purified and directly sequenced to determine the genotypes for each polymorphism. On the basis of these genotypic data, each candidate gene was determined to be located either within or outside of recently narrowed Lore QTL intervals. Results Of these eight candidates with protein-coding differences, five are now excluded from their respective Lore intervals. The other three (Znf142, Ptprn, and Znf133) have been localized to the narrowed QTL intervals. Conclusions These three central nervous system genes (Znf142, Ptprn, and Znf133) represent promising candidates for involvement in the differential sensitivity to alcohol exhibited between ILS and ISS mice. This study also demonstrates how the combination of high-throughput comparative gene sequencing and concomitant genetic fine mapping of QTL regions with ISCRs can be an effective tool for accelerating the process of moving from QTL to gene. [source] Mutation Causing von Willebrand's Disease in Scottish TerriersJOURNAL OF VETERINARY INTERNAL MEDICINE, Issue 1 2000Patrick J. Venta Von Willebrand's Disease (vWD) in the Scottish Terrier breed is a serious, often fatal, hereditary bleeding disorder. Elimination of the mutated gene by selective breeding is an important goal for the health of this breed. Although the standard protein-based tests are accurate for identification of affected Scottish Terriers, they are not reliable for the identification of carriers of the mutant gene unless multiple replicate assays are performed. A simple, highly accurate test for carriers of the disease is needed so that veterinarians can counsel clients on which animals to use in their breeding programs. The complete coding region of von Willebrand factor (vWF) complementary DNA (cDNA) was sequenced from an affected animal, and a single base deletion in the codon for amino acid 85 of the prepro-vWF cDNA that leads to Scottish Terrier vWD was identified. A highly accurate polymerase chain reaction assay was developed that can distinguish homozygous normal animals from those that are homozygous affected or heterozygous. In a voluntary survey of 87 animals provided by Scottish Terrier owners, 15 were carriers and 4 were affected with vWD, 2 of which had previously been shown to have undetectable vWF. The determination of the complete canine vWF cDNA sequence should facilitate the identification of additional vWD alleles in other breeds and other species. [source] Complementary DNA cloning of rat spetex-1, a spermatid-expressing gene-1, encoding a 63 kDa cytoplasmic protein of elongate spermatidsMOLECULAR REPRODUCTION & DEVELOPMENT, Issue 4 2004Hiroshi Iida Abstract We used differential display in combination with complementary DNA (cDNA) cloning approach to isolate a novel rat gene designated as spetex-1, which had an open reading frame of 1,668-length nucleotides encoding a protein of 556 amino acids. Spetex-1 mRNA was highly expressed in testis, and weekly expressed in lung, intestine, and spleen. Spetex-1 expression in the rat testes was detected first at 3 weeks in postnatal development and continued to be detected up to adulthood. A search in the databases showed that the amino acid sequence of spetex-1 was 82% identical to that of its mouse homologue found in the databases. Both rat spetex-1 and the mouse homologue contained Ser-X (X,=,His, Arg, or Asn) repeats in the middle portion of the proteins. In situ hybridization revealed that spetex-1 mRNA was expressed in haploid spermatids of step 7,18 within the seminiferous epithelium. Immunohistochemical analysis with confocal laser-scanning microscopy demonstrated that spetex-1 protein was not expressed in spermatogonia, spermatocytes, and round spermatids in adult rat testis, but was specifically detected in the residual cytoplasm of elongate spermatids of step 15,18 as well as in residual bodies engulfed by Sertoli cells. We interpreted these data as a potential role of spetex-1 in spermatogenesis, especially in cell differentiation from late elongate spermatids to mature spermatozoa. Mol. Reprod. Dev. 68: 385,393, 2004. © 2004 Wiley-Liss, Inc. [source] CDNA microarray analysis of gene expression in fibroblasts of patients with x-linked Emery,Dreifuss muscular dystrophyMUSCLE AND NERVE, Issue 6 2002Toshifumi Tsukahara PhD Abstract To clarify the molecular nature of the pathogenesis in X-linked Emery,Dreifuss muscular dystrophy (EDMD), we monitored the expression of 2400 genes in control and EDMD fibroblasts by using complementary DNA (cDNA) microarray techniques. A total of 60 genes whose expression was altered in EDMD fibroblasts when compared with control fibroblasts were identified. Twenty-eight genes whose expression was altered with the emerin deficiency were rescued by infection with a recombinant adenovirus expressing emerin. The altered expression in five genes, including the lamin A/C gene, was confirmed by reverse transcription,polymerase chain reaction. Our preliminary results suggest a correlation between disease similarity and gene expression. We conclude that the cDNA microarray is a very efficient tool to clarify genetic and pathological features of diseases. © 2002 Wiley Periodicals, Inc. Muscle Nerve 25: 898,901, 2002 [source] Molecular fingerprinting of TGFß-treated embryonic maxillary mesenchymal cellsORTHODONTICS & CRANIOFACIAL RESEARCH, Issue 4 2003M.M. Pisano Abstract The transforming growth factor-ß (TGFß) family represents a class of signaling molecules that plays a central role in normal embryonic development, specifically in development of the craniofacial region. Members of this family are vital to development of the secondary palate where they regulate maxillary and palate mesenchymal cell proliferation and extracellular matrix synthesis. The function of this growth factor family is particularly critical in that perturbation of either process results in a cleft of the palate. While the cellular and phenotypic effects of TGFß on embryonic craniofacial tissue have been extensively cataloged, the specific genes that function as downstream mediators of TGFß in maxillary/palatal development are poorly defined. Gene expression arrays offer the ability to conduct a rapid, simultaneous assessment of hundreds to thousands of differentially expressed genes in a single study. Inasmuch as the downstream sequelae of TGFß action are only partially defined, a complementary DNA (cDNA) expression array technology (Clontech's AtlasTM Mouse cDNA Expression Arrays), was utilized to delineate a profile of differentially expressed genes from TGFß-treated primary cultures of murine embryonic maxillary mesenchymal cells. Hybridization of a membrane-based cDNA array (1178 genes) was performed with 32P-labeled cDNA probes synthesized from RNA isolated from either TGFß-treated or vehicle-treated embryonic maxillary mesenchymal cells. Resultant phosphorimages were subject to AtlasImageTM analysis in order to determine differences in gene expression between control and TGFß-treated maxillary mesenchymal cells. Of the 1178 arrayed genes, 552 (47%) demonstrated detectable levels of expression. Steady state levels of 22 genes were up-regulated, while those of 8 other genes were down-regulated, by a factor of twofold or greater in response to TGFß. Affected genes could be grouped into three general functional categories: transcription factors and general DNA-binding proteins; growth factors/signaling molecules; and extracellular matrix and related proteins. The extent of hybridization of each gene was evaluated by comparison with the abundant, constitutively expressed mRNAs: ubiquitin, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ornithine decarboxylase (ODC), cytoplasmic beta-actin and 40S ribosomal protein. No detectable changes were observed in the expression levels of these genes in response to TGFß treatment. Gene expression profiling results were verified by Real-Time quantitative polymerase chain reaction. Utilization of cDNA microarray technology has enabled us to delineate a preliminary transcriptional map of TGFß responsiveness in embryonic maxillary mesenchymal cells. The profile of differentially expressed genes offers revealing insights into potential molecular regulatory mechanisms employed by TGFß in orchestrating craniofacial ontogeny. [source] Alterations in Mitochondrial and Apoptosis-regulating Gene Expression in Photodynamic Therapy-resistant Variants of HT29 Colon Carcinoma Cells,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 2 2005Xiao Yun Shen ABSTRACT Photodynamic therapy (PDT) is a novel cancer therapy inducing irreversible photodamage to tumor tissue via photosensitizer-mediated oxidative cytotoxicity. The cellular and molecular responses associated with PDT are only partially understood. We have reported previously the generation of several photosensitizer-specific PDT-resistant cell variants of HT29 human colon adenocarcinoma cells by selecting cells from sequential PDT treatment using different photosensitizers. In this report, we describe the use of messenger RNA (mRNA) differential display to identify genes that were differentially expressed in the parental HT29 cells compared with their resistant variants. In comparison with parental HT29 cells, mRNA expression was increased in the PDT-resistant cell variants for BNIP3, estrogen receptor-binding fragmentassociated gene 9, Myh-1c, cytoplasmic dynein light chain 1, small membrane protein I and differential dependent protein. In contrast, expression in the PDT-resistant variants was downregulated for NNX3, human HepG2 3,region Mbol complementary DNA, glutamate dehydrogenase, hepatomaderived growth factor and the mitochondrial genes coding for 16S ribosomal RNA (rRNA) and nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 4. The reduction for mitochondrial 16S rRNA in the PDT-resistant variants was confirmed by Northern blotting, and the elevated expression of the proapoptotic BNIP3 in the PDT-resistant variants was confirmed by Northern and Western blotting analysis. We also examined the expression of some additional apoptosis-regulating genes using Western blotting. We show an increased expression of Bcl-2 and heat shock protein 27 and a downregulation of Bax in the PDT-resistant variants. In addition, the mutant p53 levels in the parental HT29 cells were reduced substantially in the PDT-resistant variants. We suggest that the altered expression in several mitochondria1 and apoptosisregulating genes contributes to PDT resistance. [source] Expressed Sequence Tag Analysis of the Dinoflagellate Lingulodinium polyedrum During Dark Phase,PHOTOCHEMISTRY & PHOTOBIOLOGY, Issue 1 2004Naomi Tanikawa ABSTRACT To collect information on gene expression during the dark period in the luminous dinoflagellate Lingulodinium polyedrum, normalized complementary DNA (cDNA) libraries were constructed from cells collected during the first hour of night phase in a 12:12 h light-dark cycle. A total of 4324 5,-end sequence tags were isolated. The sequences were grouped into 2111 independent expressed sequence tags (EST) from which 433 groups were established by similarity searches of the public nonredundant protein database. Homology analysis of the total sequences indicated that the luminous dinoflagellate is more similar to land plants and animals (vertebrates and invertebrates) than to prokaryotes or algae. We also isolated three bioluminescence-related (luciferase and two luciferinbinding proteins [LBP]) and 37 photosynthesis-related genes. Interestingly, two kinds of LBP genes occur in multiple copies in the genome, in contrast to the single luciferase gene. These cDNA clones and EST sequence data should provide a powerful resource for future genome-wide functional analyses for uncharacterized genes. [source] Enhanced expression of genes for ACC synthase, ACC oxidase, and NAC protein during high-temperature-induced necrosis of young inflorescences of CymbidiumPHYSIOLOGIA PLANTARUM, Issue 3 2006Satoru Mita Growing Cymbidium under high-temperature conditions (25,30°C) results in the necrosis of young inflorescences. An increase in the evolution of ethylene was correlated with the necrosis. To study the molecular aspects of high-temperature-induced necrosis of Cymbidium floral buds, we isolated complementary DNA (cDNA) clones for proteins that are likely to be involved in the biosynthesis of ethylene during high-temperature-induced necrosis of young inflorescences, namely, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (CyACS1) and ACC oxidase (CyACO1). In addition, a cDNA (CyNAC1) encoding an NAC protein whose expression is modulated during high-temperature treatment was isolated by differential display. High levels of expression of CyACS1, CyACO1 and CyNAC1 were observed in the necrotic inflorescences of wild-type Cymbidium at high temperatures. Bud necrosis was not observed in the mericlone mutant (nhn, non,high-temperature-induced necrosis) of Cymbidium. Ethylene evolution was lower in nhn than in wild-type, but application of exogenous ACC or ethephon to the young inflorescences of nhn restored the high-temperature necrosis response. Expression of CyACS1, CyACO1 and CyNAC1 did not increase with high-temperature treatment in the nhn mutant. Expression levels of CyACS1, CyACO1 and CyNAC1 in necrotic inflorescences of nhn treated with 5.0 mM ACC were much lower than in necrotic inflorescences of wild-type at high temperatures, but CyACS1 and CyNAC1 were stimulated by ACC treatment. These results suggest that ethylene is involved in high-temperature necrosis of young inflorescences of Cymbidium and that an NAC protein may be involved in the regulatory mechanisms of genes that are regulated during necrosis. [source] Sequence polymorphisms in porcine homologs of murine coat colour-related genesANIMAL GENETICS, Issue 2 2010N. Okumura Summary Herein, we report the variability among 57 porcine homologs of murine coat colour-related genes. We identified single nucleotide polymorphisms (SNPs) and insertions/deletions (InDels) within 44 expressed gene sequences by aligning eight pig complementary DNA (cDNA) samples. The sequence alignment revealed a total of 485 SNPs and 15 InDels. The polymorphisms were then validated by performing matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with reference DNA samples obtained from 384 porcine individuals. Of the 384 individuals, three parents of the experimental F2 family were included to detect polymorphisms between them for linkage mapping. We also genotyped previously reported polymorphisms of 12 genes, and one SNP each in three genes that were detected by performing a BLAST search of the Trace database. A total of 211 SNPs and three InDels were successfully genotyped from our porcine DNA panel. We detected SNPs in 33 of the 44 genes among the parents of an experimental F2 family and then constructed a linkage map of the 33 genes for this family. The linkage assignment of each gene to the porcine chromosomes was consistent with the location of the BAC clone in the porcine genome and the corresponding gene sequence. We confirmed complete substitutions of EDNRB and MLPH in the Jinhua and Clawn miniature breeds, respectively. Furthermore, we identified polymorphic alleles exclusive to each pig group: 13 for Jinhua, two for Duroc, three for Meishan, four for the Japanese wild boar, one for the Clawn miniature pig and four for the Potbelly pig. [source] A novel Nav1.7 mutation producing carbamazepine-responsive erythromelalgia,ANNALS OF NEUROLOGY, Issue 6 2009Tanya Z. Fischer MD Objective Human and animal studies have shown that Nav1.7 sodium channels, which are preferentially expressed within nociceptors and sympathetic neurons, play a major role in inflammatory and neuropathic pain. Inherited erythromelalgia (IEM) has been linked to gain-of-function mutations of Nav1.7. We now report a novel mutation (V400M) in a three-generation Canadian family in which pain is relieved by carbamazepine (CBZ). Methods We extracted genomic DNA from blood samples of eight members of the family, and the sequence of SCN9A coding exons was compared with the reference Nav1.7 complementary DNA. Wild-type Nav1.7 and V400M cell lines were then analyzed using whole-cell patch-clamp recording for changes in activation, deactivation, steady-state inactivation, and ramp currents. Results Whole-cell patch-clamp studies of V400M demonstrate changes in activation, deactivation, steady-state inactivation, and ramp currents that can produce dorsal root ganglia neuron hyperexcitability that underlies pain in these patients. We show that CBZ, at concentrations in the human therapeutic range, normalizes the voltage dependence of activation and inactivation of this inherited erythromelalgia mutation in Nav1.7 but does not affect these parameters in wild-type Nav1.7. Interpretation Our results demonstrate a normalizing effect of CBZ on mutant Nav1.7 channels in this kindred with CBZ-responsive inherited erythromelalgia. The selective effect of CBZ on the mutant Nav1.7 channel appears to explain the ameliorative response to treatment in this kindred. Our results suggest that functional expression and pharmacological studies may provide mechanistic insights into hereditary painful disorders. Ann Neurol 2009;65:733,741 [source] | ||||||||||||||||