Search results for "RNA-Directed DNA Polymerase"
showing 10 items of 13 documents
Manganese Ions Individually Alter the Reverse Transcription Signature of Modified Ribonucleosides
2020
Reverse transcription of RNA templates containing modified ribonucleosides transfers modification-related information as misincorporations, arrest or nucleotide skipping events to the newly synthesized cDNA strand. The frequency and proportion of these events, merged from all sequenced cDNAs, yield a so-called RT signature, characteristic for the respective RNA modification and reverse transcriptase (RT). While known for DNA polymerases in so-called error-prone PCR, testing of four different RTs by replacing Mg2+ with Mn2+ in reaction buffer revealed the immense influence of manganese chloride on derived RT signatures, with arrest rates on m1A positions dropping from 82% down to 24%. Additi…
Machine learning of reverse transcription signatures of variegated polymerases allows mapping and discrimination of methylated purines in limited tra…
2020
AbstractReverse transcription (RT) of RNA templates containing RNA modifications leads to synthesis of cDNA containing information on the modification in the form of misincorporation, arrest, or nucleotide skipping events. A compilation of such events from multiple cDNAs represents an RT-signature that is typical for a given modification, but, as we show here, depends also on the reverse transcriptase enzyme. A comparison of 13 different enzymes revealed a range of RT-signatures, with individual enzymes exhibiting average arrest rates between 20 and 75%, as well as average misincorporation rates between 30 and 75% in the read-through cDNA. Using RT-signatures from individual enzymes to trai…
Variability of reverse transcriptase and overlapping S gene in hepatitis B virus isolates from untreated and lamivudine-resistant chronic hepatitis B…
2009
Background The high degree of diversity of the hepatitis B virus (HBV) quasispecies in chronically infected individuals raises the possibility that HBV genetic variants favouring resistance to nucleoside/nucleotide analogues (NAs) might pre-exist to treatment. The aim of this study was to investigate the genetic variability of the entire HBV reverse transcriptase (RT) domain and of the overlapping S gene in a large series of untreated hepatitis B surface antigen carriers and in lamivudine (3TC)-resistant patients. Methods Sequencing analysis of the entire HBV RT domain of isolates from 100 untreated (treatment- naive group) and 59 3TC-resistant (3TC-resistant group) consecutive patients wit…
Inducible nitric oxide synthase in skeletal muscle of patients with chronic heart failure
1998
Abstract Objectives. The expression and localization of inducible nitric oxide (NO) synthase (NOS II) was evaluated as a source of NO which has been shown to affect muscle contraction. Background. Advanced stages of chronic heart failure are associated with systemic activation of cytokines which have been shown to stimulate the expression of NOS II in various cell types, including myocytes. We hypothesized that systemic cytokine activation could lead to expression of NOS II in skeletal muscle of patients with chronic heart failure. Methods. Skeletal muscle specimens were obtained by percutaneous needle biopsy in six normal volunteers and eight patients with heart failure (New York Heart Ass…
Atherogenic properties of enzymatically degraded LDL: selective induction of MCP-1 and cytotoxic effects on human macrophages.
1998
Abstract —The mechanisms underlying the selective accumulation of macrophages in early atherosclerotic lesions are poorly understood but are likely to be related to specific properties of altered low density lipoprotein (LDL) deposited in the subendothelium. Enzymatic, nonoxidative degradation of LDL converts the lipoprotein to a potentially atherogenic moiety, enzymatically altered LDL (E-LDL), which activates complement and is rapidly taken up by human macrophages via a scavenger receptor–dependent pathway. Immunohistological evidence indicates that E-LDL is present in an extracellular location in the early lesion. We report that E-LDL causes massive release of monocyte chemotactic prote…
EWS/FLI-1 rearrangement in small round cell sarcomas of bone and soft tissue detected by reverse transcriptase polymerase chain reaction amplificatio…
1994
Recent cloning of the t(11;22) region has led to the detection of a number of sequences involved in the breakpoints by substituting a sequence which encodes a putative RNA binding domain for that of the DNA binding domain of the human homologue of murine FLI-1. Several tumours display consistent translocation at t(11;22) (q24;q12), a finding that suggests these fusion transcripts could be expressed and detected by reverse transcriptase polymerase chain reaction amplification. To date, only a small number of Ewing's sarcomas (Es) and peripheral neuroectodermal tumours (pPNET) of bone have been tested with this novel molecular biology approach. In this study, we confirmed the presence of the …
Termination of transcription in an ‘in vitro’ system is dependent on a polyadenylation sequence
1991
Using HeLa cell nuclear extract as a source of the different transcription and polyadenylation factors and reverse transcription to analyze the levels of RNA 5' and 3' to the cleavage-polyadenylation site, an in vitro assay has been established to study polyadenylation coupled to transcription directed by different adenovirus promoters. The levels of transcription 5' and 3' to the cleavage site in the L3 polyadenylation region are practically the same as described previously, however, the level of transcription 3' to the cleavage site in the SV40 early polyadenylation region decreases immediately after the cleavage site indicating a termination of the transcription.
A method for rapid generation of competitive standard molecules for RT-PCR avoiding the problem of competitor/probe cross-reactions.
1995
The analysis of gene expression is a widespread issue in a growing number of fields such as molecular genetics, immunology, and medical diagnostics. The ideal method for mRNA detection should be fast, inexpensive, sensitive, and reliable. Well-elaborated standard methods such as Northern hybridization, Sl-mapping, and RNAse protection are useful and recommended, but only reverse transcription PCR (RT-PCR) gives the highest possible sensitivity required. For many issues it is necessary not only to detect a distinct mRNA but to compare changes in mRNA levels. The use of RT-PCR for such semiquantitative and quantitative approaches resolves problems attributable to the intrinsic property of PCR…
Reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) as a user-friendly system to detect SARS-CoV-2 infection: a multicentric study
2021
Although reverse transcriptase quantitative PCR remains the gold standard to perform viral detection, reverse transcriptase loop-mediated isothermal amplification (RT-LAMP) is already used to perform diagnosis of various infections. This work reports the results of a multicentric study performed in Sicily to evaluate the diagnostic power of an RT-LAMP kit for the diagnosis of SARS-CoV-2 infection on a total of 551 samples collected in January and February 2021, revealing sensitivity, specificity, accuracy, positive and negative predictive values ≥95%. Our results suggest the potential employment of this kit as a screening test to be used where fast and reliable results are demanded without …
Expression of C1q, a subcomponent of the rat complement system, is dramatically enhanced in brains of rats with either Borna disease or experimental …
1995
In situ hybridization, RT-PCR and Northern blot analysis as well immunohistochemistry were used to examine the expression of C1q, a subcomponent of the rat complement system, in brains of rats infected with Borna disease virus (BDV) and rats afflicted with experimental allergic encephalomyelitis (EAE) induced by the adoptive transfer of myelin basic protein specific T cells. C1q mRNA, which was not detected in normal brain, became clearly detectable using RT-PCR analysis by d14 post infection (p.i.) with BDV. Maximal levels of C1q mRNA were reached 21 days p.i. when inflammatory reactions in the brain were also at a peak. Similarly, C1q mRNA was elevated when the clinical symptoms of EAE be…