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)

The synthesis of a protected form of the hypermodified nucleoside, N-[(9-beta-D-ribofuranosyl-2-methylthiopurin-6-yl)carbamoyl]threonine, (ms2t6A) is reported. The hypermodified nucleoside was subsequently elaborated to the protected nucleoside phosphophoramidite using a protecting group strategy compatible with standard RNA oligonucleotide chemistry. The phosphoramidite reagent was then used to synthesize the 17-nucleotide RNA hairpin having the sequence of the anticodon stem-loop (ASL) domain of human tRNA(Lys,3), the primer for HIV-1 reverse transcriptase. Introduction of the modification at position 37 of the tRNA ASL modestly decreases the thermodynamic stability of the RNA hairpin as has been seen previously for the prokaryotic t6A nucleoside lacking the 2-methylthio substituent. 2D NOESY NMR spectra of the ms2t6A containing tRNA ASL indicate that the threonyl side chain adopts a conformation similar to that seen in the solution structure of the analogous t6A containing E. coli tRNA(Lys), despite the presence of the bulky methylthio group. This synthetic approach allows for site-specific incorporation of the hypermodified nucleoside and should facilitate future studies directed at understanding the roles of nucleoside modification in modulating the stability and specificity of biologically important RNA-RNA interactions. Our synthesis of the ms2t6A containing RNAs demonstrates that this methodology is suitable for obtaining quantities of RNA required for structural studies of the HIV primer tRNA.
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PMID:Synthesis of the tRNA(Lys,3) anticodon stem-loop domain containing the hypermodified ms2t6A nucleoside. 1212 27

Cystathionine-beta-synthase (CBS) catalyzes the condensation of serine and homocysteine to form cystathionine, an intermediate step in the synthesis of cysteine. We previously described essential transactivating roles for specificity protein 1 (Sp1), Sp3, nuclear factor Y (NF-Y), and USF-1 in the regulation of the CBS-1b promoter. Differential binding of Sp1/Sp3 to the CBS-1b promoter due to differences in Sp1/Sp3 phosphorylation, and Sp1/Sp3 synergism with NF-Y might, in part, explain cell-specific patterns of CBS expression. In this report, the roles of various NF-YA isoforms in influencing cell-specific differences in CBS gene expression were determined in HT1080 and HepG2 cells. Seven unique NF-YA isoforms were detected in HT1080 by reverse transcriptase-PCR (RT-PCR) and DNA sequencing, characterized by deletions in the glutamine-rich and/or serine/threonine-rich domains. Only four of the seven NF-YA isoforms were found in HepG2 cells. The six alternatively spliced NF-YA isoforms all showed significantly less synergistic transactivation of the CBS-1b promoter with Sp1 than wild-type NF-YA, as determined by cotransfections in Drosophila SL2 cells with NF-YB and NF-YC. Further, all six alternatively spliced NF-YA isoforms inhibited the synergistic transactivation of the CBS-1b promoter by wild-type NF-Y and Sp1. Thus, the cellular distributions of these alternatively spliced NF-YA isoforms could impart an important cell-specific component to CBS transcriptional regulation, by virtue of their abilities to directly synergize with Sp1/Sp3 and interfere with transactivation of the CBS-1b promoter by wild-type NF-Y. Characterization of CBS promoter structure and function should clarify the molecular bases for variations in CBS gene expression in genetic diseases and the relationship between CBS and Down's syndrome (DS).
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PMID:Synergistic regulation of human cystathionine-beta-synthase-1b promoter by transcription factors NF-YA isoforms and Sp1. 1242 42

LeCTR1 was initially isolated by both differential display reverse transcriptase-polymerase chain reaction screening for tomato (Lycopersicon esculentum) fruit ethylene-inducible genes and through homology with the Arabidopsis CTR1 cDNA. LeCTR1 shares strong nucleotide sequence homology with Arabidopsis CTR1, a gene acting downstream of the ethylene receptor and showing similarity to the Raf family of serine/threonine protein kinases. The length of the LeCTR1 transcribed region from ATG to stop codon (12,000 bp) is more than twice that of Arabidopsis CTR1 (4,700 bp). Structural analysis reveals perfect conservation of both the number and position of introns and exons in LeCTR1 and Arabidopsis CTR1. The introns in LeCTR1 are much longer, however. To address whether this structural conservation is indicative of functional conservation of the corresponding proteins, we expressed LeCTR1 in the Arabidopsis ctr1-1 (constitutive triple response 1) mutant under the direction of the 35S promoter. Our data clearly show that ectopic expression of LeCTR1 in the Arabidopsis ctr1-1 mutant can restore normal ethylene signaling. The recovery of normal ethylene sensitivity upon heterologous expression of LeCTR1 was also confirmed by restored glucose sensitivity absent in the Arabidopsis ctr1-1 mutant. Expression studies confirm ethylene responsiveness of LeCTR1 in various tissues, including ripening fruit, and may suggest the evolution of alternate regulatory mechanisms in tomato versus Arabidopsis.
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PMID:LeCTR1, a tomato CTR1-like gene, demonstrates ethylene signaling ability in Arabidopsis and novel expression patterns in tomato. 1242 80

Comprehensive expression profiling of tumors using DNA microarrays has been used recently for molecular classification and biomarker discovery, as well as a tool to identify and investigate genes involved in tumorigenesis. Application of this approach to a cohort of benign and malignant adrenocortical tissues would be potentially informative in all of these aspects. In this study, we generated transcriptional profiles of 11 adrenocortical carcinomas (ACCs), 4 adrenocortical adenomas (ACAs), 3 normal adrenal cortices (NCs), and 1 macronodular hyperplasia (MNH) using Affymetrix HG_U95Av2 oligonucleotide arrays representing approximately 10,500 unique genes. The expression data set was used for unsupervised hierarchical cluster analysis as well as principal component analysis to visually represent the expression data. An analysis of variance on the three classes (NC, ACA plus MNH, and ACC) revealed 91 genes that displayed at least threefold differential expression between the ACC cohort and both the NC and ACA cohorts at a significance level of P < 0.01. Included in these 91 genes were those known to be up-regulated in adrenocortical tumors, such as insulin-like growth factor (IGF2), as well as novel differentially expressed genes such as osteopontin (SPP) and serine threonine kinase 15 (STK15). Increased expression of IGF2 was identified in 10 of 11 ACCs (90.9%) and was verified by quantitative reverse transcriptase-polymerase chain reaction. Select proliferation-related genes (TOP2A and Ki-67) were validated at the protein level using immunohistochemistry and adrenocortical tissue microarrays. Our results demonstrated significant and consistent gene expression changes in ACCs compared to benign adrenocortical lesions. Moreover, we identified several genes that represent potential diagnostic markers and may play a role in the pathogenesis of ACC.
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PMID:Distinct transcriptional profiles of adrenocortical tumors uncovered by DNA microarray analysis. 1254 10

Mixed lineage kinases (MLKs) belong to the family of mitogen activated protein kinase kinase kinase (MAPKKK) and cause neuronal cell death mediated through c-Jun, N-terminal kinase (JNK) pathway. Recently, genetic studies in Drosophila revealed the presence of an MLK termed slipper (slpr). However, its biochemical features like physiological substrate, role in different MAPK pathways and developmental and tissue-specific expression pattern were not reported. Here, we report cDNA cloning, expression analysis and biochemical characterization of a Drosophila mixed lineage kinase (dMLK) that is also known as slipper. The protein structure analysis of dMLK/slipper revealed, in addition to the conserved domains, a stretch of glutamine in the amino terminus and an asparagine-threonine stretch at the carboxy-terminus. In situ hybridization and reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed that dMLK is expressed in early embryonic stages, adult brain and thorax. Ectopic expression of dMLK either in Drosophila S2 or in mammalian HEK293 cells leads to activation of JNK, p38 and extracellular signal regulated kinase (ERK) pathways. Further, dMLK directly phosphorylates Hep, dMKK4 and also their mammalian counterparts, MKK7 and SEK1, in an in vitro kinase assay. Taken together, our results provide for the first time a comprehensive expression profile and new biochemical insight of dMLK/slipper.
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PMID:Drosophila mixed lineage kinase/slipper, a missing biochemical link in Drosophila JNK signaling. 1267 57

To better understand the emergence of HIV-1 variants in Barbados and the association with transmission modes, we analyzed phylogenetic relationships and genetic variability among HIV-1 strains collected in 1996 from 36 antiretroviral therapy-naive patients. Only subtype B variants were present in this sampling, based on analysis of HIV-1 envelope (env) C2V3, protease (PR), and reverse transcriptase (RT) sequences. The genetic diversity of env sequences was broad (13.9%; range, 5.9-24.9%), suggesting multiple introductions of distinct HIV-1 strains to the island. The frequency of subtype B HIV-1 variants with similar env V3 features, including the tetrameric tips, GPGR and GPGK, the threonine deletion at position 23, and the substitution of threonine to arginine at position 22, was comparable in heterosexual, bisexual, and homosexual patients. Analyses of amino acid variations in PR sequences revealed a lack of major drug resistance-conferring mutations and a high (90%) prevalence of secondary mutations at positions 36, 63, 71, and 77. While the occurrence of 361, 63P, and 71T mutations in Barbadian strains was similar to the global prevalence for subtype B variants, the frequency (64%) of the V77I mutation was more than three times that seen worldwide. Only two RT antiretroviral resistance mutations (M41L and T215Y) were observed, both from a single patient. This comprehensive genetic analysis documents a broad diversity within HIV-1 subtype B in Barbados and suggests a lack of association between particular subtype B variants and transmission modes.
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PMID:The molecular epidemiology and drug resistance determination of HIV type 1 subtype B infection in Barbados. 1281 80

Genome sequencing of C. trachomatis serovar D revealed the presence of three putative open reading frames (ORFs), CT145 (Pkn1), CT673 (Pkn5), and CT301 (PknD), encoding eukaryote-like serine/threonine kinases (Ser/Thr kinases). Two of these putative kinase genes, CT145 and CT301, were PCR amplified from serovar L2, cloned, and sequenced. Predicted translation products of the ORFs showed the presence of conserved kinase motifs at the N terminus of the proteins. CT145 and CT301 (encoding Pkn1 and PknD, respectively) were expressed in Escherichia coli as GST fusion proteins. In vitro kinase assays with Escherichia coli-derived glutathione S-transferase fusion proteins showed autophosphorylation of Pkn1 and PknD, indicating that they are functional kinases. Gene expression analysis of these kinase genes in Chlamydia by reverse transcriptase PCR indicated expression of these kinases at the early mid phase of the developmental cycle. Immunoprecipitated native chlamydial Pkn1 and PknD proteins also showed autophosphorylation in an in vitro kinase assay. Phosphoamino acid analysis by thin-layer chromatography confirmed that Pkn1 and PknD are phosphorylated on both serine and threonine residues. Interaction of Pkn1 and PknD with each other as well as interaction of Pkn1 with inclusion membrane protein G (IncG) was demonstrated by using a bacterial two-hybrid system. These interactions were further suggested by phosphorylation of the proteins in in vitro kinase assays. This report is the first description of the existence of functional Ser/Thr kinases in Chlamydia. The results of these findings should lead to a better understanding of how Chlamydia interact and interfere with host signaling pathways, since kinases represent potential mediators of the intimate host-pathogen interactions that are essential to the intracellular life cycle of Chlamydia.
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PMID:Identification of two eukaryote-like serine/threonine kinases encoded by Chlamydia trachomatis serovar L2 and characterization of interacting partners of Pkn1. 1450 Apr 99

Based on the recent studies of HBV strains with different replication efficiency, several new potential targets for anti-HBV replication have been presented. These include the viral and cellular regulatory factors associated with HBV replication and the process for encapsidation of viral genome and budding into endoplasmic reticulum (ER). A putative regulatory domain has been reported at the carboxyl-end of reverse transcriptase (RT) and when serine is substituted for proline at residue 652 of RT, replication efficiency of HBV is decreased. Substitution of proline for threonine at the 2798 nucleotide of the terminal protein (TP) gene, renders the mutant completely replication deficient. Expression of TP blocks the interferon (IFN) pathway and inhibits the responsive state of cells to interferons ( IFN) alpha and gamma. Interference of HBV capsid assembly drastically affects the encapsidation of viral genome, a crucial process for reverse transcription and viral DNA synthesis. Small molecules (bis-ANS) have been reported to act as a "wedge" to misdirect the polymerization of capsid, resulting in inhibition of virus replication. Another new group of compounds (HAP) has been shown to inhibit virus replication and also inhibit the assembly of viral capsid (core particle). Finally the capsids containing HBV genome are enveloped by budding into endoplasmic reticulum and release from virus infected cells, and this morphogenesis and secretion of HBV is dependent on glucosidases in the ER of host cells. Competitive inhibition of these glucosidases has been suggested as strategy against HBV replication.
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PMID:Exploiting new potential targets for anti-hepatitis B virus drugs. 1452 56

We have identified an alternatively spliced form of synaptotagmin I in Aplysia neurons. This isoform, synaptotagmin I C2B-beta, is generated by alternative exon usage in the C2B domain leading to nine amino acid changes in the C2B sequence from the previously characterized synaptotagmin I, now designated as synaptotagmin I C2B-alpha. Quantitative reverse transcriptase-polymerase chain reaction demonstrated that approximately 25% of mRNA encoding synaptotagmin I contained the C2B-beta exon in the nervous system. Synaptotagmin I C2B-beta showed greater resistance to digestion by chymotrypsin in the absence of calcium than did synaptotagmin I C2B-alpha, although both isoforms required the same amount of calcium to resist chymotrypsin digestion. The source of these changes in C2B properties was mapped to a single amino acid (threonine 358). We have also cloned SNAP 25 in Aplysia and show that it binds synaptotagmin I C2B-beta with a higher affinity than synaptotagmin I C2B-alpha. These results suggest that this splicing alters biochemical properties of the C2B domain, affecting a number of its important known interactions.
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PMID:Identification and characterization of a novel C2B splice variant of synaptotagmin I. 1505 79

Methionine at position 184 of human immunodeficiency virus type-1 (HIV-1) reverse transcriptase (RT) was changed to valine, isoleucine, threonine, or alanine in an HIV-1-based vector. The vectors were analyzed for replication capacity and for resistance to the nucleoside analog 2',3'-dideoxy-3'thiacytidine (3TC) using a single-cycle assay. Viruses containing the valine or isoleucine mutations were highly resistant to 3TC and replicated almost as well as the wild-type virus. The virus containing the threonine mutation was resistant to 3TC, but replicated about 30% as well as the wild-type. The alanine mutation conferred partial resistance to 3TC, but replicated poorly. The amounts of viral DNA synthesized decreased in 3TC-treated cells when the cells were infected with wild-type virus and the M184A mutant. The effect of these mutations on the generation of the ends of the linear viral DNA was determined using the sequence of the 2-LTR circle junctions. The M184T mutation increased the proportion of 2-LTR circle junctions containing a tRNA insertion, suggesting that the mutation affected the RNase H activity of RT.
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PMID:Mutations at position 184 of human immunodeficiency virus type-1 reverse transcriptase affect virus titer and viral DNA synthesis. 1506 12


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