64 research outputs found
Characterisation of Transcriptional Regulation of the Human Telomerase RNA Gene
The human telomerase core enzyme consists of an essential structural RNA (hTERC) with a template domain for telomeric DNA synthesis and of a catalytic protein (hTERT) with reverse transcriptase activity. Expression of the hTERC and hTERT are essential for telomerase activity. Variation in telomerase activity is correlated with cellular senescence and tumour progression. Recent studies indicate that the regulation of telomerase activity is multifactorial in mammalian cells. The primary mode of control of hTERT appears to be transcriptional regulation but very little is known about the molecular mechanisms involved in the regulation of hTERC transcription. In this study, I have cloned and characterised the genomic sequences and promoter of the hTERC gene to provide evidence that transcriptional mechanisms are involved in hTERC gene regulation. Transient transfection with a series of 5'-deletion mutants demonstrated that between -5.0 kb and -51 by of the hTERC gene is responsible for high promoter activity, the minimal promoter region was defined as 176 by (-107 to +69 bp). With the aid of in vitro DNase I footprinting, electrophoretic mobility shift assays (EMSAs) and mutagenesis analysis, four Sp1 binding sites and one CCAAT-box bound by the transcription factor NF-Y were identified to be involved in regulation of hTERC transcription. Co-transfection experiments showed that Sp1 and the retinoblastoma protein (pRb) are activators of the hTERC promoter and Sp3 is a potent repressor. Mutation of the CCAAT-box or the coexpression of a dominant negative nuclear factor-Y (NF-Y) significantly attenuated the transactivation by pRb and Sp1, suggesting that NF-Y binding is a prerequisite for pRb and Sp1 to activate the hTERC promoter. The different transcriptional regulators appear to act in a species-specific manner. Whilst Sp1 and Sp3 act on the human, bovine and mouse TERC promoters, pRb activates only the human and bovine promoter and NF-Y is important for the human TERC gene. The hTERC gene is expressed during embryogenesis and then down-regulated during normal development but is re-expressed in tumour cells, the hTERC promoter activity was therefore further investigated and a higher promoter activity in immortal cells than in two mortal cell strains (WI38 and IMR90) was shown. In conclusion, hTERC promoter contains sequence elements that allow interactions with several different transcription factors. The interplay between NF-Y, pRb, Sp1 and Sp3 within the architecture of the hTERC promoter may combine to enable a wide variety of cell types from mortal to immortal to regulate hTERC expression through transcriptional control
MDM2 negatively regulates the human telomerase RNA gene promoter
BACKGROUND: We have previously demonstrated that NF-Y and Sp1 interact with the human telomerase RNA (hTR) promoter and play a central role in its regulation. We have also shown that pRB activates the hTR promoter, but the mechanism of pRb directed activation is unknown. It has recently been reported that pRB induces Sp1 activity by relieving inhibition mediated by mdm2. The aim was to investigate possible roles for mdm2 in hTR promoter regulation. METHODS: Chromatin immunoprecipitation was used to determine binding of mdm2 to the hTR promoter. Transfection and luciferase assays were used to investigate mdm2 repression of the promoter activity and interaction with known transcriptional modulators. RESULTS: Here we show using chromatin immunoprecipitation that mdm2 specifically binds the hTR promoter in vivo. Transient co-transfection experiments using an hTR promoter luciferase reporter construct show that hTR promoter activity is inhibited by over-expression of mdm2 in 5637 bladder carcinoma cells (p53 and pRB negative, low mdm2). Titration of mdm2 was able to antagonise activation of hTR promoter activity mediated by pRB or Sp1 over-expression, although in the presence of pRB, mdm2 could not repress promoter activity below basal levels. Using an Sp1 binding site mutation construct we showed that mdm2 repression did not absolutely require Sp1 binding sites in the hTR promoter, suggesting the possibility of pRB/Sp1 independent mechanisms of repression. Finally, we show that NF-Y mediated transactivation of the hTR promoter was also suppressed by mdm2 in a dose-dependent manner. CONCLUSIONS: These studies suggest that mdm2 may inhibit the hTR promoter by multiple mechanisms. Mdm2 may directly repress activation by both pRB and Sp1, or activation by NF-Y. Furthermore, the ability of mdm2 to interact and interfere with components of the general transcription machinery might partly explain the general repressive effect seen here. Elucidation of new regulators affecting hTR basal promoter activity in cancer cells provides a basis for future studies aimed at improving our understanding of the differential hTR expression between normal and cancer cells
Stability and infectivity of novel pandemic influenza A (H1N1) virus in blood-derived matrices under different storage conditions
Viremia Associated with Fatal Outcomes in Ferrets Infected with Avian H5N1 Influenza Virus
Avian H5N1 influenza viruses cause severe disease and high mortality in infected humans. However, tissue tropism and underlying pathogenesis of H5N1 virus infection in humans needs further investigation. The objective of this work was to study viremia, tissue tropism and disease pathogenesis of H5N1 virus infection in the susceptible ferret animal model. To evaluate the relationship of morbidity and mortality with virus loads, we performed studies in ferrets infected with the H5N1 strain A/VN/1203/04 to assess clinical signs after infection and virus load in lung, brain, ileum, nasal turbinate, nasal wash, and blood. We observed that H5N1 infection in ferrets is characterized by high virus load in the brain and and low levels in the ileum using real-time PCR. In addition, viral RNA was frequently detected in blood one or two days before death and associated with symptoms of diarrhea. Our observations further substantiate pathogenicity of H5N1 and further indicate that viremia may be a bio-marker for fatal outcomes in H5N1 infection
Identification of new, emerging HIV-1 unique recombinant forms and drug resistant viruses circulating in Cameroon
<p>Abstract</p> <p>Background</p> <p>The HIV epidemic in Cameroon is characterized by a high degree of viral genetic diversity with circulating recombinant forms (CRFs) being predominant. The goal of our study was to determine recent trends in virus evolution and emergence of drug resistance in blood donors and HIV positive patients.</p> <p>Methodology</p> <p>Blood specimens of 73 individuals were collected from three cities and a few villages in Cameroon and viruses were isolated by co-cultivation with PBMCs. Nested PCR was performed for gag p17 (670 bp) pol (840 bp) and Env gp41 (461 bp) genes. Sequences were phylogenetically analyzed using a reference set of sequences from the Los Alamos database.</p> <p>Results</p> <p>Phylogenetic analysis based on partial sequences revealed that 65% (n = 48) of strains were CRF02_AG, 4% (n = 3) subtype F2, 1% each belonged to CRF06 (n = 1), CRF11 (n = 1), subtype G (n = 1), subtype D (n = 1), CRF22_01A1 (n = 1), and 26% (n = 18) were Unique Recombinant Forms (URFs). Most URFs contained CRF02_AG in one or two HIV gene fragments analyzed. Furthermore, pol sequences of 61 viruses revealed drug resistance in 55.5% of patients on therapy and 44% of drug naïve individuals in the RT and protease regions. Overall URFs that had a primary HIV subtype designation in the pol region showed higher HIV-1 p24 levels than other recombinant forms in cell culture based replication kinetics studies.</p> <p>Conclusions</p> <p>Our results indicate that although CRF02_AG continues to be the predominant strain in Cameroon, phylogenetically the HIV epidemic is continuing to evolve as multiple recombinants of CRF02_AG and URFs were identified in the individuals studied. CRF02_AG recombinants that contained the pol region of a primary subtype showed higher replicative advantage than other variants. Identification of drug resistant strains in drug-naïve patients suggests that these viruses are being transmitted in the population studied. Our findings support the need for continued molecular surveillance in this region of West Central Africa and investigating impact of variants on diagnostics, viral load and drug resistance assays on an ongoing basis.</p
Comparative analysis of cell culture and prediction algorithms for phenotyping of genetically diverse HIV-1 strains from Cameroon
<p>Abstract</p> <p>Background</p> <p>With the advent of entry inhibitors, monitoring of viral tropism in the clinical setting is important. Conventional methods are cell-based and lengthy, therefore V3 sequence based prediction algorithms are becoming increasingly attractive as monitoring tools. Here we report a comparative analysis of viral tropism of strains circulating in Cameroon where diverse and emerging variant strains are prevalent.</p> <p>Methods</p> <p>Viruses were isolated from 17 HIV positive individuals from three cities in Cameroon. Ghost cell lines expressing either CCR5 or CXCR4 with CD4 or CD4 alone (NIH AIDS Reagent Program) were used to determine co-receptor usage. HIV replication was determined by measuring p24 antigen levels. Plasma viral load (VL) was determined using the Versant bDNA assay. Nucleotide sequencing was performed on the V3 region and sequences were edited, aligned and translated into amino acids as described in the algorithm. Bio-informatics tools based on the 11/25 and charge rule were used to predict co-receptor usage.</p> <p>Results</p> <p>The majority of patient isolates in our study were CRF02_AG or CRF02_AG containing recombinants. Tropism of these complex viruses based on the cell culture assay was determined to be R5 in 15/17 (88.2%) patients. However, two patient isolates were dual tropic R5X4 and had drug-specific mutations. Of these two patients, one was on antiretroviral treatment with a VL of 20,899 copies/ml and the other was drug-naïve with 141,198 copies/ml. Genotype based prediction was overall in good agreement with phenotype for R5 viruses, where 93% (14/15) of results were comparable, dual tropic viruses being reported as X4 viruses by prediction.</p> <p>Conclusion</p> <p>Our results indicate that most HIV strains in Cameroon were R5 tropic and some harbored drug-resistant mutations. V3 sequence based prediction compared well with cell based assays for R5 strains and may be useful even in settings where highly diverse strains are prevalent.</p
Multiplexed, rapid detection of H5N1 using a PCR-free nanoparticle-based genomic microarray assay
<p>Abstract</p> <p>Background</p> <p>For more than a decade there has been increasing interest in the use of nanotechnology and microarray platforms for diagnostic applications. In this report, we describe a rapid and simple gold nanoparticle (NP)-based genomic microarray assay for specific identification of avian influenza virus H5N1 and its discrimination from other major influenza A virus strains (H1N1, H3N2).</p> <p>Results</p> <p>Capture and intermediate oligonucleotides were designed based on the consensus sequences of the matrix (M) gene of H1N1, H3N2 and H5N1 viruses, and sequences specific for the hemaglutinin (HA) and neuraminidase (NA) genes of the H5N1 virus. Viral RNA was detected within 2.5 hours using capture-target-intermediate oligonucleotide hybridization and gold NP-mediated silver staining in the absence of RNA fragmentation, target amplification, and enzymatic reactions. The lower limit of detection (LOD) of the assay was less than 100 fM for purified PCR fragments and 10<sup>3 </sup>TCID<sub>50 </sub>units for H5N1 viral RNA.</p> <p>Conclusions</p> <p>The NP-based microarray assay was able to detect and distinguish H5N1 sequences from those of major influenza A viruses (H1N1, H3N2). The new method described here may be useful for simultaneous detection and subtyping of major influenza A viruses.</p
Absence of Detectable XMRV and Other MLV-Related Viruses in Healthy Blood Donors in the United States
BACKGROUND: Preliminary studies in chronic fatigue syndrome (CFS) patients and XMRV infected animals demonstrated plasma viremia and infection of blood cells with XMRV, indicating the potential risk for transfusion transmission. XMRV and MLV-related virus gene sequences have also been detected in 4-6% of healthy individuals including blood donors in the U.S. These results imply that millions of persons in the U.S. may be carrying the nucleic acid sequences of XMRV and/or MLV-related viruses, which is a serious public health and blood safety concern. METHODOLOGY/PRINCIPAL FINDINGS: To gain evidence of XMRV or MLV-related virus infection in the U.S. blood donors, 110 plasma samples and 71 PBMC samples from blood donors at the NIH blood bank were screened for XMRV and MLV-related virus infection. We employed highly sensitive assays, including nested PCR and real-time PCR, as well as co-culture of plasma with highly sensitive indicator DERSE cells. Using these assays, none of the samples were positive for XMRV or MLV-related virus. CONCLUSIONS/SIGNIFICANCE: Our results are consistent with those from several other studies, and demonstrate the absence of XMRV or MLV-related viruses in the U.S. blood donors that we studied
Sp1/3 and NF-1 mediate basal transcription of the human gene in megakaryoblastic MEG-01 cells-2
<p><b>Copyright information:</b></p><p>Taken from "Sp1/3 and NF-1 mediate basal transcription of the human gene in megakaryoblastic MEG-01 cells"</p><p>BMC Molecular Biology 2006;7():10-10.</p><p>Published online 10 Mar 2006</p><p>PMCID:PMC1464135.</p><p></p>nces used for primer extension analysis (Pet1-4) are indicated by horizontal arrows. End points of plasmid deletion constructs in this region are indicated by "p" and number above the sequence. Sequence numbering is relative to the transcription start site indicated as +1. The translation initiation ATG is boxed. The EMBL/Genbank accession number of this sequence is
- …