Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.49 (reverse transcriptase)
 31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Avian myeloblastosis virus reverse transcriptase (AMV RT) is routinely used in the sequence analysis of RNA and DNA templates. We review the various methods for dealing with secondary structures that would otherwise result in premature termination or sequence compression. Based on our experience in sequencing the 11-kb single-stranded RNA genome of lymphocytic choriomeningitis virus, we have found that raising the reaction temperature above 47 degrees C is the simplest way to overcome template secondary structure, and the use of 98% formamide gels is the simplest way to overcome product secondary structure.
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PMID:Use of avian myeloblastosis virus reverse transcriptase at high temperature for sequence analysis of highly structured RNA. 247 18

Adherent cells display an important accessory role on normal T-cell colony formation. Since the in-vitro proliferation of T colony-forming cells (T-CFC) from AIDS patients is extremely impaired we studied the effect of patients' adherent cells on T-CFC growth. Patients' peripheral blood mononuclear cells (PBMC) were fractionated on the basis of rosette formation with sheep red blood cells and complement-mediated cytotoxicity with OKT3 monoclonal antibody (E-T3-). Both mature (E+) T cells and E-OKT3- cell fractions failed to generate T-cell colonies although colony growth could be obtained from unfractionated PBMC. In five out of 12 AIDS patients, adherent cell-depletion of PBMC enhanced the plating efficiency. Moreover, patients' but not normal adherent cells could inhibit normal T-cell colony growth in a dose-dependent manner. Media conditioned by patients' unstimulated adherent cells (LCM-A+p) also inhibit normal T-cell colony formation. In addition, LCM-A+p were capable of inhibiting interleukin 2-receptor (IL2-R) expression and interleukin 2 (IL2) production by normal mitogen-stimulated T cells. These LCM-A+p did not contain detectable reverse transcriptase activity nor could they infect the CEM T-cell line which is permissive to human immunodeficiency virus (HIV). Conversely, this adherent cell-derived inhibitory activity could be abrogated by heating or treatment with proteolytic enzymes. These findings indicate that the low T-cell colony formation in some AIDS patients could be due to adherent cell-derived inhibitory activity(ies).
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PMID:Peripheral blood adherent cells from AIDS patients inhibit normal T-colony growth through decreased expression of interleukin 2-receptors and production of interleukin 2. 311 67

Neurons have evolved strategies to evade immune surveillance that include an inability to synthesize the heavy chain of the class I major histocompatibility complex (MHC), proteins that are necessary for cytotoxic T lymphocyte (CTL) recognition of target cells. Multiple viruses have taken advantage of the lack of CTL-mediated recognition and killing of neurons by establishing persistent neuronal infections and thereby escaping attack by antiviral CTL. We have expressed a class I MHC molecule (Db) in neurons of transgenic mice using the neuron-specific enolase (NSE) promoter to determine the pathogenic consequences of CTL recognition of virally infected, MHC-expressing central nervous system (CNS) neurons. The NSE-Db transgene was expressed in H-2b founder mice, and transgene-derived messenger RNA was detected by reverse transcriptase-polymerase chain reaction in transgenic brains from several lines. Purified primary neurons from transgenic but not from nontransgenic mice adhered to coverslips coated with a conformation-dependent monoclonal antibody directed against the Dv molecule and presented viral peptide to CTL in an MHC-restricted manner, indicating that the Db molecule was expressed on transgenic neurons in a functional form. Transgenic mice infected with the neurotropic lymphocytic choriomeningitis virus (LCMV) and given anti-LCMV, MHC-restricted CTL displayed a high morbidity and mortality when compared with controls receiving MHC-mismatched CTL or expressing alternative transgenes. After CTL transfer, transgenic brains showed an increased number of CD8+ cells compared with nontransgenic controls as well as an increased rate of clearance of infectious virus from the CNS. Additionally, an increase in blood-brain barrier permeability was detected during viral clearance in NSE-Db transgenic mice and lasted several months after clearance of virus from neurons. In contrast, LCMV-infected, nontransgenic littermates and mice expressing other gene products from the NSE promoter showed no CNS disease, no increased intraparenchymal CTL, and no blood-brain barrier damage after the adoptive transfer of antiviral CTL. Our study indicates that viral infections and CTL-CNS interactions may induce blood-brain barrier disruptions and neurologic disease by a "hit-and-run" mechanism, triggering a cascade of pathogenic events that proceeds in the absence of continual viral stimulation.
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PMID:Consequences of cytotoxic T lymphocyte interaction with major histocompatibility complex class I-expressing neurons in vivo. 759 91

Infection of adult mice with lymphocytic choriomeningitis virus (LCMV), a non-cytopathic segmented RNA virus, leads initially to generalized infection, followed by clearance and subsequent life-long immunity. Indirect evidence has suggested that viral antigens may persist in lymphoid tissues during the phase of immunological memory, but viral genomic RNA has not been detected in previous studies. During a search for persistent virus in the spleen, we identified LCMV-specific sequences present as DNA by polymerase chain reaction (PCR) in mice over 200 days after infection. In vivo and in vitro studies revealed that reverse transcription of viral RNA into complementary DNA occurred after acute infection of cells of its natural hosts, mouse and hamster, but not of other species and could be inhibited in vitro by azidothymidine (AZT), indicating that this was mediated by endogenous reverse transcriptase activity. These findings reveal a surprising and new pathway of interaction between exogenous RNA viruses and endogenous retroviral, and perhaps other host components, that results in the persistence of virally determined DNA. We speculate that the latter may function in vivo as a form of DNA vaccine.
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PMID:A non-retroviral RNA virus persists in DNA form. 938 69

Lymphocytic choriomeningitis virus (LCMV) induces persistent infections in laboratory mice; is a known contaminant of biological materials, such as transplantable tumor cell lines; and is of great concern in animal facilities due to its zoonotic potential. Fluorogenic nuclease reverse transcriptase-polymerase chain reaction (fnRT-PCR) assays combine RT-PCR with an internal fluorogenic hybridization probe, thereby potentially enhancing specificity and eliminating post-PCR processing. An fnRT-PCR assay specific for LCMV was, therefore, developed by targeting primer and probe sequences to a unique region of the LCMV nucleocapsid (NP) gene. The LCMV fnRT-PCR assay detected only LCMV and did not detect other RNA viruses that naturally infect rodents. The fnRT-PCR assay detected as little as one picogram of LCMV RNA, but was 100-fold less sensitive when directly compared with the mouse antibody production test. The fnRT-PCR assay was also able to detect viral RNA in numerous tissues and in feces and cage swipe specimens collected from experimentally inoculated BALB/c mice, but did not detect any viral RNA in similar samples collected from age- and strain-matched mock-infected mice. In conclusion, the LCMV fnRT-PCR assay offers a potentially high-throughput diagnostic assay to detect LCMV in mice and contaminated biological materials.
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PMID:Detection of lymphocytic choriomeningitis virus by use of fluorogenic nuclease reverse transcriptase-polymerase chain reaction analysis. 1262 8

Cell-specific gene expression profiling from heterogeneous human tissues is confounded by cell purification limitations. Here, we describe a technique to generate gene expression profiles of pure populations of prostate cancer cells obtained from fresh-frozen prostatectomy specimens and small initial quantities of RNA by combining laser capture microdissection and microserial analysis of gene expression (LCM-microSAGE). Two microSAGE libraries were obtained from approximately 100,000 laser pulses, estimated to contain fewer than 3 x 10(5) cells and 20-30 ng mRNA. Two libraries were sequenced to a depth of 10,111 and 10,463 unique tags from normal and cancer cells, representing 6453 and 6923 genes, respectively. Most transcripts were expressed at similar levels, but cancer cells compared with normal cells had increased expression of 385 tags and decreased expression of 389 tags. A total of 20 genes were differentially expressed (P<0.05); five of these genes were upregulated and 15 were downregulated in cancer cells. Quantitative reverse transcriptase-polymerase chain reaction results from three selected genes corroborated the existence of cell-specific gene expression in LCM-microSAGE-derived libraries. In conclusion, the LCM-microSAGE approach demonstrates that large-scale expression profiles of known and unknown transcripts can be generated from pure populations of target cells obtained from human tissue samples comprised of heterogeneous mixtures of cell types.
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PMID:Combined laser capture microdissection and serial analysis of gene expression from human tissue samples. 1552 82