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Convenient synthesis of oligodeoxyribonucleotides bearing arabinofuranosyl pyrimidine derivatives and its duplex formation with complementary DNA.

The oligodeoxyribonucleotides bearing 2,2'-anhydro-beta-D-arabinofuranosyluracil derivatives were synthesized and the modified residue was converted to beta-D-arabinofuranosyluracil derivatives or beta-D-arabinofuranosylisocytosine derivatives by post-synthetic modification method. The melting profiles of their ODNs with complementary DNA were studied.

Arabinose↗

The p53 gene in breast cancer: prognostic value of complementary DNA sequencing versus immunohistochemistry.

BACKGROUND: Mutations in the p53 tumor suppressor gene (also known as TP53) have been detected in a wide variety of human cancers. In breast cancer, the presence of p53 gene alterations has been associated with worse prognosis. PURPOSE: We compared a complementary DNA (cDNA)-based sequencing method and an immunohistochemical (IHC) method for their abilities to detect p53 mutations in breast cancer specimens. In addition, we determined the prognostic value of information obtained when these two methods were used. METHODS: Specimens from 316 primary breast tumors were evaluated for the presence of mutant p53 protein by use of the mouse monoclonal antibody Pab 1801 (that recognizes both wild-type and mutant forms of p53) and standard IHC methods. In addition, the entire coding region of p53 genes expressed in these tumors was screened for mutations by combining reverse transcription, the polymerase chain reaction, and DNA sequencing. Probabilities for overall survival (OS), breast cancer-corrected survival (BCCS; death from breast cancer is the considered event), and relapse-free survival (RFS) were estimated by use of the Kaplan-Meier method, and survival curves for different patient subgroups were compared by use of the logrank method. All reported P values are from two-sided tests. RESULTS: Sixty-nine (22%) of 316 tumors had p53 gene mutations detected by the cDNA-based sequencing method; only 31 (45%) of these mutations were located in evolutionarily conserved portions of the p53 coding region. Sixty-four tumors (20% of the total) had elevated levels of p53 protein as detected by IHC, suggesting the presence of mutations. Of the sequencing-positive tumors (i.e., p53 mutant), 23 exhibited negative IHC reactions, indicating that IHC failed to detect 33% of the mutations. Furthermore, 19 of the IHC-positive tumors were sequencing negative (i.e., p53 wild-type), suggesting a 30% false-positive frequency with IHC. Four tumors (1.3% of the total) could not be analyzed by the cDNA-based sequencing method, and three tumors (1% of the total) could not be analyzed by IHC. The 5-year estimates for RFS, BCCS, and OS were significantly shorter for patients with p53 sequencing-positive tumors than for patients with sequencing-negative tumors (P = .001, P = .01, and P = .0003, respectively). Patients with IHC-positive tumors showed reduced survival in all three categories when compared with those with IHC-negative tumors, but the differences were not statistically significant. CONCLUSIONS: Use of a cDNA-based sequencing method to determine the status of the p53 gene in primary breast cancers yielded better prognostic information than IHC performed with the Pab 1801 monoclonal antibody.

Adult↗

Complementary DNA cloning and immunologic characterization of a new Penicillium citrinum allergen (Pen c 3).

BACKGROUND: Penicillium citrinum has been identified as the most prevalent airborne Penicillium species in the Taipei area. It is important to understand the allergenic composition of this ubiquitous fungal species. OBJECTIVE: The complementary DNA (cDNA) clone of an allergen from P citrinum was isolated and expressed in Escherichia coli as a fusion protein. mAbs were prepared with the recombinant protein as antigen. The corresponding natural allergen in the fungal extracts was identified with the mAbs. METHODS: A Uni-Zap XR P citrinum cDNA library was screened with sera from asthmatic patients. An IgE-binding cDNA clone was isolated and expressed as a glutathione-S-transferase fusion protein. The frequency of IgE binding to the expressed protein was analyzed by immunoblotting. Spleen cells from BALB/c mice immunized with the recombinant protein were fused with NS-1 cells for mAb generation. RESULTS: A P citrinum cDNA library was screened with a mixture of serum samples from 4 asthmatic patients. An IgE-binding cDNA clone was obtained and designated as PCE2. PCE2 has a 694-bp insert that contains a 167 amino acids open reading frame. The deduced amino acid sequence of the encoded protein has 82.6% (138 amino acids) identity with an Aspergillus fumigatus peroxisomal membrane protein allergen (Asp f 3). PCE2 was expressed in E coli as a fusion protein and designated as Pen c 3. Sera from 13 (46%) of the 28 Penicillium-sensitized asthmatic patients demonstrated IgE binding to Pen c 3. In addition, 11 of the 13 Pen c 3-positive serum samples have IgE immunoblot reactivity to recombinant Asp f 3. The presence of IgE cross-reactivity between Pen c 3 and Asp f 3 was also detected by immunoblot inhibition. Four of the 6 mAbs generated against Pen c 3 cross-react with Asp f 3. The presence of the corresponding 18-k natural allergens in the crude extracts of P citrinum and A fumigatus were detected by immunoblot with use of the mAbs and sera from asthmatic patients. CONCLUSION: Results obtained suggest that the peroxisomal membrane protein (Pen c 3) is an important allergen of P citrinum. PCE2 is a full-length cDNA clone encoding this allergen. In addition, the mAbs generated may be useful in standardizing the diagnostic allergenic extracts.

Adult↗

Widespread presence, in chickens, of DNA complementary to the RNA genome of avian leukosis viruses.

DNA-RNA hybridization experiments have demonstrated the widespread presence in chickens of DNA complementary to the RNA of avian myeloblastosis virus. All apparently normal chicken embryos, or adult chickens that were tested, contained viral DNA in amounts ranging from 1.7 to 4.6 viral genome equivalents per cell. Embryos that were negative or positive for the group-specific antigen of avian leukosis viruses contained the same amount of viral DNA. Embryos from a strain of chickens free of leukosis viruses of groups A and B that develop 40% fewer spontaneous leukotic tumors than the original strain contained an average of 2.1 viral DNA equivalents per cell, whereas the original strain contained 3.2 viral DNA equivalents. By comparison, leukemic cells and cells infected with avian myeloblastosis virus or Rous sarcoma virus contained between 4 and 13 viral DNA equivalents. This study provides direct biochemical evidence for the presence in normal chicken cells of avian leukosis virus DNA that is inheritable and that represents either complete or incomplete viral genomes.

Animals↗

Isolation, molecular cloning and in vitro expression of rhesus monkey (Macaca mulatta) prominin-1.s1 complementary DNA encoding a potential hematopoietic stem cell antigen.

Human prominin-1 (CD133 or AC133) is an important cell surface marker used to isolate primitive hematopoietic stem cells. The commercially available antibody to human prominin-1 does not recognize rhesus prominin-1. Therefore, we isolated, cloned and characterized the complementary DNA (cDNA) of rhesus prominin-1 gene and determined its coding potential. Following the nomenclature of prominin family of genes, we named this cDNA as rhesus prominin-1.s1. The amino acid sequence data of the putative rhesus prominin-1.s1 could be used in designing antigenic peptides to raise antibodies for use in isolation of pure populations of rhesus prominin-1(+) hematopoietic cells. To the best of our knowledge, there has been no previously published report about the isolation of a prominin-1 cDNA from rhesus monkey (Macaca mulatta).

AC133 Antigen↗

Introduction of human erythropoietin receptor complementary DNA by retrovirus-mediated gene transfer into murine embryonic stem cells enhances erythropoiesis in developing embryoid bodies.

To evaluate the role of the erythropoietin (Epo) receptor (R) in erythropoiesis in more primitive stem cells, we assessed the influence of retrovirus-mediated gene transfer of human (h) EpoR complementary DNA (cDNA) into murine embryonic stem (ES) cells on erythroid differentiation of these cells. The hEpoR cDNA was efficiently transduced into ES cells, forming hEpoR that stably expressed ES (ES-hEpoR) cells. Expression of hEpoR cDNA was confirmed in ES-hEpoR cells by reverse transcriptase-polymerase chain reaction and Northern blot analysis. Colony assays demonstrated that definitive erythroid and primitive erythroid colonies were significantly increased from ES-hEpoR cells, when compared with mock virus-transduced ES (ES-Neo) cells, during the time course of differentiation induced by withdrawal of leukemia inhibitory factor, in either the presence or the absence of Epo. Multipotential colony-forming units (CFU-Mix) were also increased in ES-hEpoR cells at different stages of differentiation, but no changes were detected for CFU-granulocyte-macrophage colonies (CFU-GM). Time course studies by Northern blot analysis demonstrated elevated levels of expression of beta-H1 and beta-Major globin genes in embryoid bodies derived from ES-hEpoR cells stimulated with Epo, when compared with similar expression from ES-Neo cells. Expression of the GATA-1 gene was enhanced in ES-hEpoR cells, when compared with ES-Neo cells, beginning immediately after initiation of the cultures until 8 days of differentiation. These data indicate that primitive and definitive erythropoiesis in differentiating embryoid bodies can be enhanced by retrovirus-mediated gene transfer of an hEpoR gene.

Animals↗

Purification of DNA complementary to the env gene of avian sarcoma virus and analysis of relationships among the env genes of avian leukosis-sarcoma viruses.

The env gene of avian leukosis-sarcoma viruses encodes a glycoprotein that determines the host range and surface antigenicitiy of virions. We have purified radioactive DNA (cDNAgp) complementary to at least a portion of the env gene for viral subgroups A and C; complementary DNA was synthesized with purified virions of wild-type avian sarcoma virus, and RNA from a mutant with a deletion in env was used to select DNA specific to env by molecular hybridization. The genetic complexity of cDNAgp for subgroup A (ca. 2,000 nucleotides) was sufficient to represent the entire deletion and most or all of the env cistron. The deletions in env in two independently isolated strains of virus (Bryan and rdNY8SR) overlap, and cDNAgp represents nucleotide sequences common to both deletions. By contrast, we could detect no overlap between deletions in env and deletions in the adjacent viral gene src. Laboratory stocks of viral subgroups A, B, C, D and E do not contain detectable amounts of env deletions when tested by molecular hybridization; hence, segregation of deletions in env is a less frequent event that the segregation of deletions in the viral transforming gene src (Vogt, 1971). We found extensive homology among the nucleotide sequences encoding the env genes of virus strains indigenous to chickens (subgroups A, B, C, D, and E) although subgorups B, D and E appear to differ slightly from subgroups A and C at the env locus. By contrast, viruses obtained from pheasant cells (subgroups F and G) have env genes with little or no relationship to env genes of chikcen viruses. According to available data, viruses of subgroup F arose by recombination between an avarian sarcoma virus and viral genes in the genome of ring-necked pheasants, whereas subgroup G viruses may be entirely endogenous to golden pheasants.

Alpharetrovirus↗

Expression of murine dihydrofolate reductase complementary DNA in oocytes of Xenopus laevis.

1. Murine dihydrofolate reductase was produced efficiently in a Xenopus laevis oocyte that received microinjection of the dihydrofolate reductase mRNA. 2. Murine dihydrofolate reductase was produced under the direction of the plasmid pAdD26SVpA, which contains adenovirus major late promoter and the simian virus 40 T antigen gene for transcription initiation and murine dehydrofolate reductase complementary DNA fragment. 3. Injection of the plasmid pDHFR26, which contains murine dihydrofolate reductase complementary DNA and normal transcription initiation site segment in the 5' untranslational region, failed to produce proper translational products due to improper transcription.

Animals↗

Structural properties of hybrid triplex of polycation deoxyribonucleic S-methylthiourea (DNmt) strands with a complementary DNA strand, probed by nanosecond molecular dynamics.

The replacement of phosphodiester linkages of the polyanion DNA with S-methylthiourea linkers provides the polycation deoxyribonucleic S-methylthiourea (DNmt). Molecular dynamics studies to 1,220 ps of the hybrid triplex formed from octameric DNmt strands d(Tmt)8 with a complementary DNA oligomer strand d(Ap)8 have been carried out with explicit water solvent and Na+Cl- counterions under periodic boundary conditions using the CHARMM force field and the Ewald summation method. The Watson-Crick and Hoogsteen hydrogen-bonding patterns of the A/T tracts remained intact without any structural restraints for triplex structures throughout the simulation. The duplex portion of the triplex structure equilibrated at a B-DNA conformation in terms of the helical rise and other helical parameters. The dynamic structures of the DNmt x DNA x DNmt triplex were determined by examining histograms from the last 800 ps of the dynamics run. These included the hydrogen-bonding pattern (sequence recognition), three-centered bifurcating occurrences, minor groove width variations, and bending of tracts for the hybrid triplex structures. Together with the Watson-Crick hydrogen-bondings, the strong Hoogsteen hydrogen-bondings, the partially maintained three-centered bifurcatings in the Watson-Crick pair, and the medium-strength three-centered bifurcatings in the Hoogsteen pair suggest that the hybrid triplex is energetically favorable as compared to a duplex with similar base stacking, van der Waals interactions, and helical parameters. This is in agreement with our previously reported thermodynamic study, in which only triplex structures were observed in solution. The bending angle measured between the local axis vectors of the first and last helical axis segments is about 20 degrees for the Watson-Crick portion of the averaged structure. Propeller twist (associated with three-centered hydrogen-bonding) up to -30 degrees, native to DNA AT base pairing, was also observed for the triplex structure. The sugar pseudorotation phase angles and the ring rotation angles for the DNA strand are within the C3'-endo domain and C2'-endo domain for the DNmt strand. Water spines are observed in both minor and major grooves throughout the dynamics run. The molecular dynamics simulations of the structural properties of DNmt x DNA x DNmt hybrid triplex is compared to the DNG x DNA x DNG hybrid triplex (In DNG the -O-(PO2-)-O- linkers in DNA is replaced by -NH-C(=N+H2)-NH-).

Base Pairing↗

Molecular cloning of complementary DNA encoding maize nitrite reductase: molecular analysis and nitrate induction.

Complementary DNA has been isolated that codes for maize nitrite reductase (NiR) by using the corresponding spinach gene (E Back et al. 1988 Mol Gen Genet 212:20-26) as a heterologous probe. The sequences of the complementary DNAs from the two species are 66% homologous while the deduced amino acid sequences are 86% similar when analogous amino acids are included. A high percentage of the differences in the DNA sequences is due to the extremely strong bias in the corn gene to have a G/C base in the third codon position with 559/569 codons ending in a G or C. Using a hydroponic system, maize seedlings grown in the absence of an exogenous nitrogen source were induced with nitrate or nitrite. Nitrate stimulated a rapid induction of the NiR mRNA in both roots and leaves. There is also a considerable induction of this gene in roots upon the addition of nitrite, although under the conditions used the final mRNA level was not as high as when nitrate was the inducer. There is a small but detectable level of NiR mRNA in leaves prior to induction, but no constitutive NiR mRNA can be seen in the roots. Analysis of genomic DNA supports the notion that there are at least two NiR genes in maize.

Journal Article↗

Complementary DNA sequences encoding the rat MHC class II RT1-Bu and RT1-Du alpha and beta chains.

The rat major histocompatibility complex loci RT1-B and RT1-D are equivalent to the human leucocyte antigens HLA-DQ and HLA-DR respectively. Here we describe the complementary DNA (cDNA) sequence encoding the alpha and beta chains of both the RT1-B and RT1-D locus genes of the rat RT1u haplotype. We have found entire sequence identity between five different inbred rat strains of the RT1u haplotype, which differs from previously published, incomplete sequences. This information is of considerable value for experimental studies of transplantation immunity and autoimmune disease.

Animals↗

Oligonucleotide-poly-L-ornithine conjugates: binding to complementary DNA and RNA.

On the basis of the reported enhanced antisense activity of polylysine-oligonucleotide conjugates, a synthetic 12-mer oligodeoxyribonucleotide has been coupled at its 5' terminus to a series of positively charged (delta-ornithine)n cysteine peptides. Binding between the nucleic acid-peptide conjugate and its complementary DNA target sequence was detected by the impact of complexation on the melting temperature (Tm). It was found that the Tm for the nucleic acid-peptide gradually increased with increasing net charge on the conjugated peptide. Site-directed cleavage with RNase H demonstrates that the peptide-modified oligomer also hybridizes with its RNA target sequence. Increased affinity for target mRNA with net charge was shown by a cell-free translation arrest assay.

Base Sequence↗

Cloning and expression of complementary DNA coding for an allergen with common antibody-binding specificities with three allergens of the house dust mite Blomia tropicalis.

The mite Blomia tropicalis is a potent source of allergens in tropical and subtropical regions. So far, most of these allergens have only been studied by immunoblotting. To characterize them at the molecular level, a lambda gt11 complementary DNA library was constructed from messenger RNA isolated from whole B. tropicalis mites. This library was screened by using pooled sera from patients allergic to B. tropicalis in a plaque IgE radioimmunoassay. A B. tropicalis IgE-positive clone (Bt-M) was selected for immunologic studies. After subcloning into pBluescript (Stratagene, La Jolla, Calif.), it produced a sequence of 310 bp, with a probable amino acid sequence of 72 residues for the expressed peptide. The recombinant protein was transferred to nitrocellulose filters and probed with 100 sera from patients allergic to B. tropicalis. Forty-seven percent of sera reacted with the recombinant allergen. Immunoblottings performed with allergic serum and B. tropicalis-affinity-purified IgE demonstrated that the recombinant protein shares allergenic epitopes with the 11-13, 14, and 16 kd native allergens of B. tropicalis, which are known to be important allergens of this mite.

Adolescent↗

Complementary DNA cloning and constitutive expression of cytochrome P450 1C1 in the gills of carp (Cyprinus carpio).

Cytochrome P450 (CYP) enzymes constitute a multigene family of many endogenous and xenobiotic substances. The CYP1 family is of particular interest in environmental toxicology because its members are dominant in the metabolism of polycyclic aromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and aryl amines. A new complementary DNA of the CYP1C subfamily encoding CYP1C1 was isolated from carp liver after intraperitoneal injection of beta-napthoflavone (BNF). The full-length cDNA obtained contained a 5' noncoding region of 244 bp, an open reading frame of 1572 bp coding for 524 amino acids, a stop codon, and a 3' noncoding region of 965 bp. The predicted molecular weight of the protein was approximately 59.3 kDa. The deduced amino acid sequence of this cDNA was 82.1% and 80.2% similar to Japanese eel and scup CYP1C1 sequences, respectively, while it exhibited a similarity of 74.9% with the scup CYP1C2 sequence. The deduced amino acid sequence of carp CYP1C1 showed similarities with those of the reported CYP1B1s of teleosts and mammals of 48.4, 48.8, 48.2, 48.6, 45.3, and 45.5% for carp CYP1B1, carp CYP1B2, plaice CYP1B1, and human, rat, and mouse CYP1B1, respectively. The phylogenetic tree constructed using fish and mammalian CYP1 sequences suggested a closer relationship of the CYP1C subfamily to CYP1B than to CYP1A. The tree showed the possibility of the existence of CYP1C subfamily genes in mammalian species. Northern blot analysis for the liver, intestine, gills, and kidney showed no detectable induced expression but constitutive expression in the gill organs.

Amino Acid Sequence↗

Structural investigations of the major allergen Phl p I on the complementary DNA and protein level.

Until now investigations of group I grass allergens have mainly been performed on ryegrass allergen (Lol p I). We studied this major allergen grass group with timothy grass pollen (Phl p I), a very common and important cause of type I allergy, to determine intraspecific and interspecific variations among different grass species. By immunoscreening a timothy grass pollen complementary DNA library we obtained three full-length clones. They revealed identical nucleotide sequences in the coding regions consisting of 262 amino acids, including a leader sequence of 23 amino acid residues. The comparison of our data with the amino acid sequences deduced from Lol p I and Hol 1 I clones showed sequence identities of greater than 85% and homologies of greater than 90%, indicating a high degree of sequence conservation. Despite the high degree of homology, amino acid differences were in immunodominant positions, which may be responsible for the differing immune response to group I allergens of different grass species.

Allergens↗

Molecular cloning of complementary DNA for a novel human hepatoma-derived growth factor. Its homology with high mobility group-1 protein.

A novel hepatoma-derived growth factor was purified from the conditioned medium of human hepatoma-derived cell line, HuH-7, by the assay of [3H]thymidine incorporation into Swiss 3T3 cells. Molecular cloning of a complementary DNA from the cDNA library of HuH-7 cells was done on the basis of the N-terminal amino acid sequence. This protein was acid- and heat-labile heparin-binding protein and inactivated by reducing condition. The cDNA is 2.4 kilobase pairs long, and the deduced amino acid sequence from cDNA contained 240 amino acids without a signal peptide-like N-terminal hydrophobic sequence. Heparin column-eluted fraction of the conditioned medium of Cos-7 cells transfected by the cDNA stimulated the DNA synthesis. Northern blot analysis revealed its ubiquitous expression in several tumor-derived cell lines, as well as in normal tissues. The primary sequence shares homology with the high mobility group (HMG)-1 protein (23.4% amino acid identity and 35.6% similarity). However, its hydrophobic profile was distinct from that of HMG-1 except for the C-terminal acidic region, and it contained no apparent HMG box motif. Immunofluorescence study showed that it is localized in the cytoplasma of hepatoma cells. These findings suggest that this factor is a novel heparin-binding protein, with mitogenic activity for fibroblasts.

Amino Acid Sequence↗

Increased expression of protein kinase Calpha, interleukin-1alpha, and RhoA guanosine 5'-triphosphatase in osteoblasts expressing the Ser252Trp fibroblast growth factor 2 receptor Apert mutation: identification by analysis of complementary DNA microarray.

Apert (Ap) syndrome is a craniofacial malformation characterized by premature fusion of cranial sutures (craniosynostosis). We previously showed that the Ser252Trp fibroblast growth factor receptor 2 (FGFR-2) mutation in Ap syndrome increases osteoblast differentiation and subperiosteal bone matrix formation, leading to premature calvaria ossification. In this study, we used the emerging technology of complementary DNA (cDNA) microarray to identify genes that are involved in osteoblast abnormalities induced by the Ser252Trp FGFR-2 mutation. To identify the signaling pathways involved in this syndrome, we used radioactively labeled cDNAs derived from two sources of cellular messenger RNAs (mRNAs) for hybridization: control (Co) and mutant Ap immortalized osteoblastic cells. Among genes that were differentially expressed, protein kinase Ca (PKC-alpha), interleukin-1alpha (IL-1alpha), and the small guanosine-5'-triphosphatase (GTPase) RhoA were increased in FGFR-2 mutant Ap cells compared with Co cells. The validity of the hybridization array was confirmed by Northern blot analysis using mRNAs derived from different cultures. Furthermore, immunochemical and Western blot analyses showed that mutant Ap cells displayed increased PKC-alpha, IL-1alpha, and RhoA protein levels compared with Co cells. Treatment of Co and Ap cells with the PKC inhibitor calphostin C decreased IL-1alpha and RhoA mRNA and protein levels in Ap cells, indicating that PKC is upstream of IL-1alpha and RhoA. Moreover, SB203580, a specific inhibitor of p38 mitogen-activated protein kinase (MAPK), and PD-98059, a specific inhibitor of MAPK kinase (MEKK), also reduced IL-1alpha and RhoA expression in Ap cells. These data show that the Ser252Trp FGFR-2 mutation in Ap syndrome induces constitutive overexpression of PKC-alpha, IL-1alpha, and small GTPase RhoA, suggesting a role for these effectors in osteoblast alterations induced by the mutation. The cDNA microarray technology appears to be a useful tool to gain information on abnormal gene expression and molecular pathways induced by genetic mutations in bone cells.

Acrocephalosyndactylia↗

Human aminopeptidase B (rnpep) on chromosome 1q32.2: complementary DNA, genomic structure and expression.

Aminopeptidase B (APB) is a Zn(2+)-metalloexopeptidase, which selectively removes Arg and/or Lys residues from the N-terminus of several peptide substrates. Several data strongly support the hypothesis that this enzyme could participate in the final stages of precursor processing mechanisms and/or in particular inflammatory processes and tumor developments. Therefore, we have cloned the complementary DNA encoding the human APB, a 658-residues protein, containing the canonical "HEXXH(X(18))E", a signature allowing its classification in the M1 family of metallopeptidases. The genomic structure of the human APB gene (rnpep; 1q32.1-q32.2) was also determined. rnpep is bracketed by pre-protein translocase of the inner mitochondrial membrane gene and ETS family transcription factor ELF3 gene. It spans more than 24 kbp and contains 11 exons ranging from 109 to 574 bp. Finally, expression of the human APB messenger RNA (mRNA) was investigated using a pre-made dot-blot. This mRNA seems to be ubiquitous although its expression level varies depending of the cells or tissues considered.

Amino Acid Sequence↗