Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Glycolipid transfer protein (GLTP) is a small (23-24 kDa), basic protein (pI congruent with 9.0) that accelerates the intermembrane transfer of various glycolipids. Here, we report the first cloning of cDNAs that encode the bovine and porcine GLTPs. The cDNA open reading frame for bovine GLTP was constructed by bridge-overlapping extension polymerase chain reaction (PCR) after obtaining partial coding cDNA clones by hot start, seminested, and rapid amplification of cDNA ends-PCR. The cDNA open reading frame for porcine GLTP was constructed by reverse transcriptase-PCR. The encoded amino acid sequences in the full-length bovine and porcine cDNAs were identical, consisting of 209 amino acid residues, and were nearly the same as the published sequence determined by Edman degradation. The cDNA encoded one additional amino acid at the N terminus (methionine), arginine at positions 10 and 200 instead of lysine, and threonine at position 65 instead of alanine. Expression of GLTP-cDNA in Escherichia coli using pGEX-6P-1 vector resulted in glutathione S-transferase (GST)-GLTP fusion protein. Regulation of growth and induction conditions led to approximately 50% of expressed fusion protein being soluble and active. Proteolytic cleavage of GST-GLTP fusion protein (bound to GST-Sepharose) and affinity purification resulted in fully active GLTP. Northern blot analyses of bovine tissues showed a single transcript of approximately 2.2 kilobases and the following hierarchy of mRNA levels: cerebrum > kidney > spleen congruent with lung congruent with cerebellum > liver > heart muscle. Reverse transcriptase-PCR analyses of mRNA levels supported the Northern blot results.
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PMID:Cloning and expression of glycolipid transfer protein from bovine and porcine brain. 1067 54

Plant phosphoenolpyruvate carboxylase (PEPc) activity and allosteric properties are regulated by PEPc kinase (PPcK) through reversible phosphorylation of a specific serine (Ser) residue near the N terminus. We report the molecular cloning of PPcK from the facultative Crassulacean acid metabolism (CAM) common ice plant (Mesembryanthemum crystallinum), using a protein-kinase-targeted differential display reverse transcriptase-polymerase chain reaction approach. M. crystallinum PPcK encodes a minimal, Ca(2+)-independent Ser/threonine protein kinase that is most closely related to calcium-dependent protein kinases, yet lacks both the calmodulin-like and auto-inhibitory domains typical of plant calcium-dependent protein kinase. In the common ice plant PPcK belongs to a small gene family containing two members. McPPcK transcript accumulation is controlled by a circadian oscillator in a light-dependent manner. McPPcK encodes a 31.8-kD polypeptide (279 amino acids), making it among the smallest protein kinases characterized to date. Initial biochemical analysis of the purified, recombinant McPPcK gene product documented that this protein kinase specifically phosphorylates PEPc from CAM and C(4) species at a single, N-terminal Ser (threonine) residue but fails to phosphorylate mutated forms of C(4) PEPc in which this specific site has been changed to tyrosine or aspartate. McPPcK activity was specific for PEPc, Ca(2+)-insensitive, and displayed an alkaline pH optimum. Furthermore, recombinant McPPcK was shown to reverse the sensitivity of PEPc activity to L-malate inhibition in CAM-leaf extracts prepared during the day, but not at night, documenting that PPcK contributes to the circadian regulation of photosynthetic carbon flux in CAM plants.
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PMID:A minimal serine/threonine protein kinase circadianly regulates phosphoenolpyruvate carboxylase activity in crassulacean acid metabolism-induced leaves of the common ice plant. 1093 63

Sequence analysis of human immunodeficiency virus type 1 (HIV-1) from 74 persons with acute infections identified eight strains with mutations in the reverse transcriptase (RT) gene at positions 41, 67, 68, 70, 215, and 219 associated with resistance to the nucleoside analogue zidovudine (AZT). Follow-up of the fate of these resistant HIV-1 strains in four newly infected individuals revealed that they were readily replaced by sensitive strains. The RT of the resistant viruses changed at amino acid 215 from tyrosine (Y) to aspartic acid (D) or serine (S), with asparagine (N) as a transient intermediate, indicating the establishment of new wild types. When we introduced these mutations and the original threonine (T)-containing wild type into infectious molecular clones and assessed their competitive advantage in vitro, the order of fitness was in accord with the in vivo observations: 215Y < 215D = 215S = 215T. As detected by real-time nucleic acid sequence-based amplification with two molecular beacons, the addition of AZT or stavudine (d4T) to the viral cultures favored the 215Y mutant in a dose-dependent manner. Our results illustrate that infection with nucleoside analogue-resistant HIV leads in newly infected individuals to mutants that are sensitive to nucleoside analogues, but only a single mutation removed from drug-resistant HIV. Such mutants were shown to be transmissible, stable, and prone to rapid selection for resistance to AZT or d4T as soon as antiretroviral therapy was administered. Monitoring of patients for the presence of new HIV-1 wild types with D, S, or N residues at position 215 may be warranted in order to estimate the threat to long-term efficacy of regimens including nucleoside analogues.
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PMID:Establishment of new transmissible and drug-sensitive human immunodeficiency virus type 1 wild types due to transmission of nucleoside analogue-resistant virus. 1113 72

The mutant catalase purified previously from acatalasemic dog liver was heat-labile but possessed normal activity, suggesting a mutation within the coding region distal from the catalytic site. The nucleotide and deduced amino acid sequences of acatalasemic beagle dog catalase were determined by analysis of cDNA obtained by 5'- and 3'-RACE and reverse transcriptase-polymerase chain reaction (RT-PCR) methods. Comparative analysis of cDNA sequences of normal and acatalasemic dog catalases indicated a single nucleotide difference where alanine(327) (G macro CT) was substituted with threonine (ACT). The mutant catalase, which was overexpressed in COS-1 cells, was heat-labile as previously observed with the purified enzyme from acatalasemic dog liver, indicating that this amino acid substitution can lead to structural instability. No catalase protein and activity were detected by immunoblotting and spectrophotomeric assay in acatalasemic dog reticulocytes although almost the same level of mRNA expression as that in the normal reticulocytes was observed. Pulse-labeling and immunoprecipitation examination indicated that the level of catalase synthesis in the acatalasemic dog reticulocytes was almost the same (approximately 80%) as that in the normal reticulocytes. On the other hand, the synthesized mutant catalase in reticulocytes was rapidly degraded (t(1/2): 1.8 h) compared with the normal catalase (t(1/2): 14.0 h) and this degradation was almost completely inhibited by lactacystin (LC). These results suggested that the proteolytic degradation mediated most likely by proteasome might be involved in disposing of the mutant catalase in acatalasemic erythroid cells.
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PMID:cDNA cloning of mutant catalase in acatalasemic beagle dog: single nucleotide substitution leading to thermal-instability and enhanced proteolysis of mutant enzyme. 1113 58

Smad proteins are essential intracellular signal transducers of the transforming growth factor-beta (TGF-beta) superfamily. The TGF-beta superfamily signals through phosphorylation and activation of R-Smad proteins, receptor-regulated Smads, by heteromeric complexes of ligand-specific type I and type II serine/threonine kinase receptors. R-Smads receive a signal from the activated receptor complex and transmit it to the nucleus. A cDNA was isolated that encodes a 649-amino acid protein found to be homologous to members of R-Smad subfamily with highest homology scored to clawed African frog and human Smad2. The Schistosoma mansoni homologue (SmSmad2) was overexpressed in bacteria as a Sj26-GST fusion protein and used to raise specific antibodies. The IgG fraction of the immunized rabbit serum identified 70- and 72-kDa protein bands in Western analysis of schistosome extracts. Treatment with alkaline phosphatase removed the 72-kDa band, which indicates that this band represents the phosphorylated form of schistosome Smad2. SmSmad2 was localized in the subtegument, parenchymal cells, and sex organs in both male and female worm cryosections. Similar results were also obtained from the analysis of the Smad2 mRNA distribution pattern revealed by in situ hybridization of adult worm pair paraffin sections. SmSmad2 mRNA levels were determined by reverse transcriptase-polymerase chain reaction in different mammalian host developmental stages and found to be constitutively expressed. SmSmad2 was also found to interact with a previously identified SmTbetaR-I, a serine/threonine type I kinase receptor. Furthermore, SmSmad2 was shown to undergo phosphorylation by constitutively active forms of SmTbetaR-I in vitro. In addition, SmSmad2 localized in the nuclei of mink lung epithelial cells upon treatment with TGF-beta(1). These data indicate that the SmSmad2 responds to the TGF-beta signals by interaction with receptor I, which phosphorylates it, whereupon it translocates into the nucleus presumably to regulate target gene transcription and consequently elicit a specific TGF-beta effect.
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PMID:Identification and characterization of a Smad2 homologue from Schistosoma mansoni, a transforming growth factor-beta signal transducer. 1115 51

Applying 10 pmol of okadaic acid (OA), a specific inhibitor of type 1 or type 2A serine/threonine protein phosphatases, to the orchid (Phalaenopsis species) stigma induced a dramatic increase in ethylene production and an accelerated senescence of the whole flower. Aminoethoxyvinylglycine or silver thiosulfate, inhibitors of ethylene biosynthesis or action, respectively, effectively inhibited the OA-induced ethylene production and retarded flower senescence, suggesting that the protein phosphatase inhibitor induced orchid flower senescence through an ethylene-mediated signaling pathway. OA treatment induced a differential expression pattern for the 1-aminocyclopropane-1-carboxylic acid synthase multigene family. Accumulation of Phal-ACS1 transcript in the stigma, labelum, and ovary induced by OA were higher than those induced by pollination as determined by "semiquantitative" reverse transcriptase-polymerase chain reaction. In contrast, the transcript levels of Phal-ACS2 and Phal-ACS3 induced by OA were much lower than those induced by pollination. Staurosporine, a protein kinase inhibitor, on the other hand, inhibited the OA-induced Phal-ACS1 expression in the stigma and delayed flower senescence. Our results suggest that a hyper-phosphorylation status of an unidentified protein(s) is involved in up-regulating the expression of Phal-ACS1 gene resulting in increased ethylene production and accelerated the senescence process of orchid flower.
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PMID:Differential expression of 1-aminocyclopropane-1-carboxylate synthase genes during orchid flower senescence induced by the protein phosphatase inhibitor okadaic acid. 1135 Oct 88

An amino acid mutation at residue 284 (Ala to Thr) in the VP2 protein of infectious bursal disease viruses (IBDVs) has been correlated with the ability to replicate in cell culture. In this study, we designed a molecular test for this mutation. The reverse transcriptase/polymerase chain reaction (RT/PCR) was used to amplify a 743-bp region of the VP2 gene that contained the codon for amino acid 284. The restriction endonuclease NgoMIV was selected for this study because the first three nucleotides of its six-base recognition sequence are the codon responsible for the amino acid alanine at residue 284. The RT/PCR products from 10 known pathogenic and 16 vaccine strains of IBDV were examined for the presence or absence of the NgoMIV site. We also examined 189 field strains of IBDV for the NgoMIV site. All 10 known pathogenic IBDV strains contained the NgoMIV site, indicating they contained alanine at residue 284. None of the vaccine strains had the NgoMIV site, suggesting they had threonine or another amino acid at residue 284. The results suggest that the presence of this NgoMIV site can be used as a marker for the identification of wild-type (nonvaccine) IBDV strains. The RT/PCR products from 152 (80.4%) of the field strains had the NgoMIV site and thus have the potential to be wild-type pathogenic viruses. The RT/PCR products from 37 (19.6%) of the field strains were not cleaved by NgoMIV and thus are potentially attenuated vaccine strains. Molecular diagnostic assays have been used to place IBDV strains into genetically related groups. The identification of this genetic marker now makes it possible to identify viruses that are wild-type strains that have the potential to be pathogenic viruses.
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PMID:Use of a genetic marker for wild-type potentially pathogenic infectious bursal disease viruses. 1156 47

Human endogenous retroviruses (HERVs) have been implicated as causative agents in diseases characterized by inflammation and macrophage activation, such as multiple sclerosis. Because monocyte activation and differentiation influence retroviral transcription and replication, we investigated the contribution of these processes to the expression of four HERV families (HERV-W, HERV-K, HERV-E, and HERV-H) in human monocytes, and autopsied brain tissue from patients with brain diseases associated with increased macrophage activity. Reverse transcriptase-polymerase chain reaction analysis of primary macrophages and U937 monocytoid cells stimulated with phorbol-12-myristate-13-acetate or lipopolysaccharide revealed three- to ninefold increases in HERV-W, HERV-K, and HERV-H RNA levels. In addition, elevated reverse transcriptase activity and HERV RNA were detectable in supernatants from PMA-stimulated U937 cultures, properties that could be attenuated with the inhibitor of monocyte differentiation threonine-lysine-proline. In contrast, stimulation of monocytes decreased or had no effect on HERV-E expression. Compared with controls, HERV-W and HERV-K expression was increased in brain tissue from patients with multiple sclerosis or human immunodeficiency virus infection or AIDS, with concomitant elevated tumor necrosis factor-alpha levels. Similarly, elevated HERV-W levels were detected in patients with Alzheimer's dementia only when tumor necrosis factor-alpha expression was also evident (2 of 6 cases). The detection of several HERVs in inflammatory brain diseases and the capacity to augment HERV expression in monocytes with compounds that influence cellular activity suggest that increased expression of these viruses is a consequence of increased immune activity rather than causative of distinct diseases.
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PMID:Monocyte activation and differentiation augment human endogenous retrovirus expression: implications for inflammatory brain diseases. 1160 92

Phosphorylation/dephosphorylation of proteins is a general mechanism of hormonal signal transduction, including ABA, and serine/threonine protein phosphatases 2C (PP2C, EC 3.1.3.16) have been suggested to play an important role in this process. By means of differential reverse transcriptase-polymerase chain reaction (RT-PCR) and further screening of a cDNA library made from mRNA of ABA-treated Fagus sylvatica L. seeds, a full-length cDNA clone (FsPP2C2) encoding a putative PP2C was obtained. Comparison to the databases revealed high homology to plant PP2C and most features of these enzymes, but unusual characteristics were found within the catalytic domain and the N-terminal region of the amino acid sequence. The coding region of FsPP2C2 was expressed in Escherichia coli as histidine tag fusion protein and shows Mg2+-dependent in vitro phosphatase activity. Transcription of the FsPP2C2 gene is low during seeds stratification at 4 degrees C or under gibberellic acid (GA3) treatment and clearly increases when seeds are treated with ABA and calcium (Ca2+) together, while the addition of calcium chelators (EGTA or TMB-8) decreases its expression. Furthermore, FsPP2C2 is only expressed in ABA-treated tissues, preferentially in seeds, which suggests that this PP2C is specifically induced by ABA in dormant seeds, in a Ca2+-dependent manner, and also in other ABA-treated tissues.
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PMID:Molecular cloning of a functional protein phosphatase 2C (FsPP2C2) with unusual features and synergistically up-regulated by ABA and calcium in dormant seeds of Fagus sylvatica. 1206 Feb 71

Alterations in gene expression represent key events in carcinogenesis. We have studied HPV-induced cervical carcinogenesis, using an HPV-33-positive cell line (UT-DEC-1) established from a low-grade vaginal dysplasia (VAIN-I). Early-passage cells contained HPV-33 in episomal form, but these were superseded at later passages by cells carrying only integrated virus. To gain insight into the biologic significance of HPV integration, we compared the level of gene expression in normal vaginal keratinocytes, early-passage and late-passage UT-DEC-1 cells, using cDNA microarrays. Total RNA was isolated from cells by CsCl-gradient centrifugation, reverse-transcribed with MMLV reverse transcriptase and labeled with alpha-(32)P ATP. A cDNA microarray expression profile analysis was performed with Clontech's Human Cancer 1.2 cDNA expression array kit. The 16 upregulated genes (cut-off 2-fold), identified by comparing both cell types to control keratinocytes, appeared to support cell-cycle progression or to be functional in mitosis. These included, e.g., MCM4 DNA replication licensing factor, cdc2p34 and chromatin assembly factor 1 p48 subunit. Downregulated genes (44 altogether) interfered with apoptosis and cell adhesion, including the apoptosis-inducing genes FRAP, Bik and caspase-9 precursor. The most significant differences between the late and early passages (29 and 46 constantly up- and downregulated genes without any fluctuation) were overexpression of the transcription factors E2F5 with its dimerization partner DP1, NF-kappa B and serine/threonine kinases and underexpression of enzymes of the MAPK pathway. Acquisition of a selective growth advantage after viral integration might be explained by a major shift from a MAPK pathway to cell-cycle dysregulation (G(2)/M).
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PMID:Transcriptional profiling of a human papillomavirus 33-positive squamous epithelial cell line which acquired a selective growth advantage after viral integration. 1211 47


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