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DNA Copies (dna + copy)
Terms modified by DNA Copies Selected AbstractsCharacterization of genomic DNA encoding cecropins from an Aedes albopictus mosquito cell lineINSECT MOLECULAR BIOLOGY, Issue 1 2002D. Sun Abstract We used cDNA probes from Aedes albopictus mosquito cecropins AalCecA, B, and C to obtain genomic DNA copies and flanking DNA. Two gene copies (AalCecA1 and A2, AalCecB1 and B2, AalCecC1 and C2) encoding each of the three mature cecropin peptides were recovered. All these genes had a similar organization, into two exons interrupted by a single short intron. AalCecA1 and AalCecA2 encode mature protein products that differ by one amino acid residue, while AalCecB1 and AalCecB2, AalCecC1 and AalCecC2 encode identical mature cecropin peptides, respectively. The AalCecB and C gene pairs each share a common intergenic region of approximately 1 kb, with the two coding regions transcribed in opposite directions. With the exception of small insertions/deletions, the intergenic spacer region was highly conserved between the B1/C1 and B2/C2 clones. In transfected cells, 0.8 kb of upstream sequence was sufficient for inducible expression of AalCecA1. Within this region, a 28 bp sequence at positions ,192 to ,165 upstream of the transcription initiation site was found to contain a potential regulatory element. In electrophoretic mobility shift assays, synthetic double-stranded DNA containing this 28 bp sequence retarded protein in cytoplasmic and nuclear extracts from C7-10 cells. [source] A real-time PCR assay for detection and quantification of Mycoplasma agalactiae DNAJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2007A. Lorusso Abstract Aims:, The aim of this study was to develop a rapid, sensitive, specific tool for detection and quantification of Mycoplasma agalactiae DNA in sheep milk samples. Methods and Results:, A real-time polymerase chain reaction (PCR) assay targeting the membrane-protein 81 gene of M. agalactiae was developed. The assay specifically detected M. agalactiae DNA without cross-amplification of other mycoplasmas and common pathogens of small ruminants. The method was reproducible and highly sensitive, providing precise quantification of M. agalactiae DNA over a range of nine orders of magnitude. Compared with an established PCR assay, the real-time PCR was one-log more sensitive, detecting as few as 101 DNA copies per 10 ,l of plasmid template and 6ˇ5 × 100 colour changing units of reference strain Ba/2. Conclusions:, The real-time PCR assay is a reliable method for the detection and quantification of M. agalactiae DNA in sheep milk samples. The assay is more sensitive than gel-based PCR protocols and provides quantification of the M. agalactiae DNA contained in milk samples. The assay is also quicker than traditional culture methods (2,3 h compared with at least 1 week). Significance and Impact of the Study:, The established real-time PCR assay will help study the patterns of shedding of M. agalactiae in milk, aiding pathogenesis and vaccine efficacy studies. [source] Quantitation of cytomegalovirus (CMV) DNA by real-time PCR for occurrence of CMV disease in HIV-infected patients receiving highly active antiretroviral therapyJOURNAL OF MEDICAL VIROLOGY, Issue 3 2003Karine Gourlain Abstract In HIV-infected patients treated with highly active antiretroviral therapy (HAART) included in the Predivir cohort, we have evaluated the usefulness of CMV DNA quantitation by a TaqManŽ PCR assay from peripheral blood leukocytes (PBLs) to predict CMV disease occurrence. In parallel with the immune restoration after treatment by HAART, the percentage of positive samples decreased progressively from 7.3% at Day 0 to 3.5% at Month 12. Among the CMV markers, the smallest concordance with PBL CMV TaqManŽ PCR, as evaluated by kappa, was observed with pp65 antigenemia, whereas concordance with all other CMV markers was high. Among the 16 patients with CMV DNA copies at least once >100/150,000 cells, CMV disease occurred in six during follow-up, whereas among the 159 patients with CMV DNA copies always <10/150,000 cells, CMV disease occurred in three and among the seven patients with CMV DNA copies >10 and <100 occurred in only one. In univariate Cox models, all the CMV markers including PBL CMV TaqManŽ PCR >10/150,000 cells (RR: 27.6, IC95: 7.1,107.2), the CD4 cell count <75 cells/mm3 and the HIV viral load >100,000 copies/ml were predictive for CMV disease. In a stepwise multivariate analysis, which should be interpreted with caution due to the small number of events (n = 10), three covariates were associated independently with CMV disease: pp65 antigenemia >100 nuclei/200,000, PBL CMV TaqManŽ PCR >10 copies/150,000 cells and HIV viral load remaining or increasing >100,000 copies/ml. J. Med. Virol. 69:401,407, 2003. Š 2003 Wiley-Liss, Inc. [source] Self-association of an amphipathic helix peptide inhibitor of HIV-1 integrase assessed by electro spray ionization mass spectrometry in trifluoroethanol/water mixturesRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 5 2001S. Fermandjian Establishing the auto-associative properties of a molecule in solution can be important for determination of its structure and function. EAA26 (VESMNEELKKIIAQVRAQAEHLKTAY) has been designed to inhibit HIV-1 integrase via formation of a stable coiled-coil structure with a nearly homologous segment in the enzyme. The latter catalyzes the permanent incorporation of a DNA copy of the retrovirus genome into host cell DNA, and is thus essential to the life of the retrovirus. This makes integrase an obvious drug target in the therapy of AIDS. The present work has demonstrated, using electrospray ionization mass spectrometry (ESI-MS), that EAA26 is monomeric in pure water, and tetrameric and dimeric at respectively low and medium concentrations of 2,2,2-trifluoroethanol (TFE), and again monomeric at higher TFE concentrations. Thus, the apolar solvent TFE may contribute to either stabilization or disruption of the intermolecular hydrophobic contacts depending on its concentration in aqueous solution. Previous NMR and ultracentifugation results are thus confirmed, indicating the reliability of ESI-MS for defining the self-association state of biologically relevant peptides in both water and organic-water solutions. Copyright Š 2001 John Wiley & Sons, Ltd. [source] Enhancer-promoter Activity of Human Papillomavirus Type 16 Long Control Regions Isolated from Cell Lines SiHa and CaSki and Cervical Cancer BiopsiesCANCER SCIENCE, Issue 3 2000Takuyo Kozuka Expression of human papillomavirus 16 (HPV-16) oncogenes is markedly higher in cervical cancer cells than in precancerous cells, and the elevated expression is believed to be required for the malignant phenotypes. We compared cancer cell lines CaSki (with 200 to 400 copies of HPV-16 DNA per cell) and SiHa (with one to two copies of HPV-16 DNA per cell) for the E7 expression in cells and the enhancer-promoter activity of the isolated viral long control region (LCR). Although these parameters per cell were 10-fold higher in CaSki than in SiHa, the levels of the E7 mRNA and protein per HPV DNA copy were 10- to 20-fold higher in SiHa than in CaSki. Characterization of the isolated LCRs showed that, whereas the LCR from CaSki resembled the prototype in structure and activity, the LCR from SiHa, with a deletion of 38 base pairs, enhanced transcription from P97 as assayed by using a plasmid capable of expressing luciferase. The upregulation appeared to be due to removal of one of the silencer YY1-binding sites. Furthermore, we isolated and characterized LCRs from 51 cervical cancer patients' biopsies. Among them, one with a deletion including YY1-binding sites and the other with a substitution in a YY1-motif were found to enhance the transcription. These findings suggest that mutation affecting YY1-motifs in the LCR is one of the mechanisms enhancing the viral oncogene expression in the course of progression of cancer cells. [source] | ||||||||||