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:2.7.7.48 (transcriptase)
9,479 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. Messenger activity of phage f2 RNA modified with methoxyamine under non-denaturing conditions was studied in E. coli-free system. The incorporation of amino acids into phage polypeptides was decreased, and the synthesis of phage-specific proteins was diminished. The RNA replicase synthesis was more affected than synthesis of coat protein. The impaired messenger activity of the methoxyamine-modified f2 RNA was due to the blocking of elongation process by modified cytosines present in RNA chain. 2. Specificity of f2 RNA to stimulate ribosomal binding predominantly at the coat protein initiation site was not affected by methoxyamine-treatment, as demonstrated by unchanged binding of f[3H]Met-tRNA and [14C]alanyl-tRNA to ribosomes. 3. Unfolding of f2 RNA molecule on treatment with methoxyamine in the presence of guanidine-HCl resulted in a significant increase of RNA capacity to direct fMet-tRNA binding to ribosomes. Sucrose-density gradient profiles revealed the formation of polysome-like initiation complexes indicating that ribosomes were able to bind at many hitherto inaccessible initiation codons in RNA molecules. fMet-tRNA bound to ribosomes in the presence of unfolded RNA was found to be fully reactive with puromycin.
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PMID:Activity of methoxyamine-modified f2 RNA in initiation and elongation steps of protein synthesis. 78 15

Cross-linking of ribonucleoside triphosphates (NTPs) to specific binding sites on the poliovirus RNA-dependent RNA polymerase has been performed by ultraviolet irradiation and by reduction of oxidized nucleotide-protein complexes. The latter method approached a cross-linking efficiency of 1 NTP/molecule of enzyme. Nucleotide competition experiments suggested that the same binding site is occupied by all NTPs. Analysis of peptides produced by proteinase Glu-C and trypsin digestion and labeled with [32P]GTP indicated that a lysine residue between Met-189 and Lys-228 in the polymerase was cross-linked to NTP. Nucleotide binding was exploited for rapid purification of the enzyme by GTP-agarose affinity chromatography. In addition, a set of cloned, modified polymerase molecules with reduced or absent polymerization activity was analyzed for binding efficiency to a GTP-agarose column. Some mutations eliminated GTP binding, whereas others generated proteins with varying affinities for GTP. Incubation of the poliovirus polymerase with high concentrations of NTP, particularly GTP, resulted in a dramatic protection against heat denaturation and activity loss. These data suggest that nucleotide binding results in an alteration of the enzyme conformation or the stabilization of an ordered conformation.
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PMID:Nucleotide binding by the poliovirus RNA polymerase. 132 24

The two variants of influenza A/Victoria/35/72 (H3N2) virus resistant simultaneously to remantadine, deitiforin, adapromine and amantadine were obtained while passaging the virus in presence of remantadine or deitiforin. Both variants differed from the parental strain in optimal pH for hemolysis, transcriptase activity and in amino acid sequence of M2 protein. Maximal hemolytic activity of the parental strain is registered at pH 5.2, for the variants cultured in the presence of remantadine or deitiforin at pH 5.5 and 5.8, respectively. In contrast to NH4OH, remantadine and deitiforin do not exert inhibition of virus-induced hemolysis. Transcriptase activity of resistant variants is about 50% higher as compared with parental strain (enzyme source--whole virus particles or RNP). The M2 protein of the remantadine variant has 2 amino acid substitutions: 31 (Ser----Asn) and 59 (Met----Leu); the deitiforin variant has 3 substitutions: 14 (Met----Leu), 30 (Ala----Val) and 59 (Met----Leu). The phenotypic resistance of the virus seems to be determined by the mutations in the hydrophobic protein region (30,31); the other substitutions (14,59) may modify conformational structure and functional activity of the viral proteins.
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PMID:[The change in functional activity and primary structure of the M2 protein in variants of the influenza virus resistant to remantadine and deitiforin: common and individual differences from the original strain]. 281

Previous data demonstrated that reovirus mRNA, synthesized in vitro with the particulate RNA transcriptase of reovirus cores, efficiently directs the synthesis of polypeptides in vitro. The present studies indicate that all of the three size classes of reovirus mRNA produced in vitro can form protein initiation complexes with rat liver [(36)S]Met-tRNA(F) and incubated 40S and 60S ribosomal subunits, which had been washed in 0.5 M KCl of mouse fibroblast L-929 cells. Mild prior treatment of the mRNA with HCHO was required to expose the initiator region. The initiation complex reacted quantitatively with puromycin to form a puromycin peptide, whose electrophoretic properties were identical to methionyl-puromycin formed in response to poly(A,G,U) or the initiator codon AUG. The complex was relatively stable and specific for [(35)S]Met-tRNA(F); rat liver [(35)S]Met-tRNA(M) was unreactive unless the supernatant factors EF T(1) and EF T(2) were also present. However, the addition of fusidic acid, at a concentration that did not affect complex formation with [(35)S]Met-tRNA(F), completely inhibited Met-tRNA(M) utilization. Exogenous ribosomal factors and GTP were not required unless the separated 40S and 60S subunits were further treated with 1 M KCl. The data suggest that reovirus mRNA contains AUG initiator codons that form a complex with Met-tRNA(F) at a puromycin-reactive site on ribosomes.
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PMID:Formation of a mammalian initiation complex with reovirus messenger RNA, methionyl-tRNA F , and ribosomal subunits. 450 35

The (-) enantiomers of 2',3'-dideoxy-5-fluoro-3'-thiacytidine [(-)-FTC] and 2',3'-dideoxy-3'-thiacytidine [(-)-BCH-189] were recently shown to inhibit selectively human immunodeficiency viruses (HIV) and hepatitis B virus in vitro. In the current study, the potential for HIV type 1 (HIV-1) resistance to these compounds was evaluated by serial passage of the virus in human peripheral blood mononuclear cells and MT-2 cells in the presence of increasing drug concentrations. Highly drug-resistant HIV-1 variants dominated the replicating virus population after two or more cycles of infection. The resistant variants were cross-resistant to (-)-FTC, (-)-BCH-189, and their (+) congeners but remained susceptible to 2',3'-dideoxycytidine, 3'-azido-3'-deoxythymidine, 3'-fluoro-3'-deoxythymidine, 2',3'-dideoxyinosine, phosphonoformate, the TIBO compound R82150, and the bis(heteroaryl)piperazine derivative U-87201E. Reverse transcriptase derived from drug-resistant viral particles was 15- to 50-fold less susceptible to the 5'-triphosphates of FTC and BCH-189 compared with enzyme from parental drug-susceptible virus. DNA sequence analysis of the reverse transcriptase gene amplified from resistant viruses consistently identified mutations at codon 184 from Met (ATG) to Val (GTG or GTA) or Ile (ATA). Sequence analysis of amplified reverse transcriptase from a patient who had received (-)-BCH-189 therapy for 4 months demonstrated a mixture of the Met-184-to-Val (GTG) mutation and the parental genotype, indicating that the Met-184 mutation can occur in vivo.
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PMID:Characterization of human immunodeficiency viruses resistant to oxathiolane-cytosine nucleosides. 768 16

The sequence of the entire genome of citrus tristeza virus (CTV), Florida isolate T36, was completed. The 19,296-nt CTV genome encodes 12 open reading frames (ORFs) potentially coding for at least 17 protein products. The 5'-proximal ORF 1a starts at nucleotide 108 and encodes a large polyprotein with calculated MW of 349 kDa containing domains characteristic of (from 5' to 3') two papain-like proteases (P-PRO), a methyltransferase (MT), and a helicase (HEL). Alignment of the putative P-PRO sequences of CTV with the related proteases of beet yellows closterovirus (BYV) and potyviruses allowed the prediction of catalytic cysteine and histidine residues as well as two cleavage sites, namely Val-Gly/Gly for the 5' proximal P-PRO domain and Met-Gly/Gly for the 5' distal P-PRO domain. The autoproteolytic cleavage of the polyprotein at these sites would release two N-terminal leader proteins of 54 and 55 kDa, respectively, and a 240-kDa C-terminal fragment containing MT and HEL domains. The apparent duplication of the leader domain distinguishes CTV from BYV and accounts for most of the size increase in the ORF 1a product of CTV. The downstream ORF 1b encodes a 57-kDa putative RNA-dependent RNA polymerase (RdRp), which is probably expressed via a +1 ribosomal frameshift. Sequence analysis of the frameshift region suggests that this +1 frameshift probably occurs at a rare arginine codon CGG and that elements of the RNA secondary structure are unlikely to be involved in this process. The complete polyprotein resulting from this frameshift event has a calculated MW of 401 kDa and after cleavage of the two N-terminal leaders would yield a 292-kDa protein containing the MT, HEL, and RdRp domains. Phylogenetic analysis of the three replication-associated domains, MT, HEL, and RdRp, indicates that CTV and BYV form a separate closterovirus lineage within the alpha-like supergroup of positive-strand RNA viruses. Two gene blocks or modules can be easily identified in the CTV genome. The first includes the replicative MT, HEL, and RdRp genes and is conserved throughout the entire alpha-like superfamily. The second block consists of five ORFs, 3 to 7, conserved among closteroviruses, including genes for the CTV homolog of HSP70 proteins and a duplicate of the coat protein gene. The 3'-terminal ORFs 8 to 11 encode a putative RNA-binding protein (ORF 11), and three proteins with unknown functions; this gene array is poorly conserved among closteroviruses.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Complete sequence of the citrus tristeza virus RNA genome. 774 24

Recent studies have identified a new family of inwardly rectifying K+ channels, members of which are known by the acronyms ROMK1, IRK1, and GIRK1. We have isolated cDNAs encoding the human homologue of ROMK1 from an adult kidney cDNA library. The sequences of the human kidney ROMK1 cDNA clones indicated that they were derived from at least two types of mRNAs, human ROMK1A and human ROMK1B, differing in sequence at their 5' ends. The isolation of the human ROMK1 gene, localized to chromosome band 11q24 by fluorescence in situ hybridization, indicated that the different ROMK1 transcripts were generated by alternative splicing. Human ROMK1A mRNA was predicted to encode a protein of 389 amino acids, having 93% identity with the 391-residue rat ROMK1 protein, and expression studies in Xenopus oocytes indicated that it encoded a Ba(2+)-sensitive inwardly rectifying K+ channel with properties similar to those reported for cloned rat ROMK1. Human ROMK1B mRNA was predicted to encode a protein of 372 amino acids whose sequence was truncated at the amino terminus but otherwise identical to that of the human ROMK1A protein. Translation of human ROMK1B mRNA was predicted to initiate at a codon corresponding to Met-18 of human ROMK1A mRNA. Reverse transcriptase-polymerase chain reaction amplification of human kidney mRNA revealed human ROMK1A and -B transcripts as well as a third type of transcript, human ROMK1C mRNA, which was predicted to encode a protein identical to human ROMK1B. Human ROMK1A, -B, and -C transcripts were identified in kidney, whereas only human ROMK1A mRNA could be detected in pancreatic islets and other tissues in which human ROMK1 was expressed at low levels. Thus, tissue-specific alternative splicing of human ROMK1 mRNA may result in the expression of a family of ROMK1 proteins.
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PMID:Alternative splicing of human inwardly rectifying K+ channel ROMK1 mRNA. 819 Jan 2

Mouse neuroblastoma Neuro-2a cells were examined for the expression of pro-enkephalin mRNA, protein, and Met-enkephalin ([Met]-Enk) peptide. Reverse transcriptase/polymerase chain reaction (RT/PCR) and in situ hybridization demonstrated the presence of pro-enkephalin mRNA in these cells. Immunocytochemistry using an antibody which recognizes pro-enkephalin and high pressure liquid chromatography (HPLC) followed by radioimmunoassay indicated that pro-enkephalin was synthesized in these cells and processed to yield the bioactive pentapeptide, [Met]-Enk. Furthermore, release studies showed that the [Met]-Enk was secreted from these cells with high K+ stimulation. Using double labeling, in situ hybridization combined with immunocytochemistry, we demonstrated that prohormone convertase 2 (PC2) mRNA is colocalized with pro-enkephalin in the same Neuro-2a cells, suggesting that this enzyme may be responsible for processing this precursor. we also showed the presence of vasopressin mRNA and arginine-vasopressin peptide in these cells using in situ hybridization and immunocytochemistry, respectively. Thus, the Neuro-2a cells are a multiple neuropeptide-producing cell line and an excellent model for studying the mechanisms involved in the synthesis, intracellular targeting and processing of endogenous pro-enkephalin and pro-vasopressin, as well as other transfected neuropeptide precursors.
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PMID:The Neuro-2a neuroblastoma cell line expresses [Met]-enkephalin and vasopressin mRNA and peptide. 867 23

The innermost core of rotavirus is composed of VP2, which forms a protein layer that surrounds the two minor proteins VP1 and VP3, and the genome of 11 segments of double-stranded RNA. This inner core layer surrounded by VP6, the major capsid protein, constitutes double-layered particles that are transcriptionally active. Each gene encoding a structural protein of double-layered particles has been cloned into baculovirus recombinants and expressed in insect cells. Previously, we showed that coexpression of different combinations of the structural proteins of rotavirus double-layered particles results in the formation of virus-like particles (VLPs), and each VLP containing VP1, the presumed RNA-dependent RNA polymerase, possesses replicase activity as assayed in an in vitro template-dependent assay system (C. Q.-Y. Zeng, M. J. Wentz, J. Cohen, M. E. Estes, and R. F. Ramig, J. Virol. 70:2736-2742, 1996). This work reports construction and characterization of VLPs containing a truncated VP2 (VPdelta2, containing amino acids [aa] Met-93 to 880). Expression of VPdelta2 alone resulted in the formation of single-layered delta2-VLPs. Coexpression of VPdelta2 with VP6 produced double-layered delta2/6-VLPs. VLPs formed by coexpression of VPdelta2 and VP1 or VP3, or both VP1 and VP3, resulted in the formation of VLPs lacking both VP1 and VP3. The presence of VP6 with VPdelta2 did not result in encapsidation of VP1 and VP3. To determine the domain of VP2 required for binding VP1, far-Western blot analyses using a series of truncated VP2 constructs were performed to test their ability to bind VP1. These analyses showed that (i) full-length VP2 (aa 1 to 880) binds to VP1, (ii) any N-terminal truncation lacking aa 1 to 25 fails to bind VP1, and (iii) a C-terminal 296-aa truncated VP2 construct (aa 1 to 583) maintains the ability to bind VP1. These analyses indicate that the N terminus of rotavirus VP2 is necessary for the encapsidation of VP1 and VP3.
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PMID:The N terminus of rotavirus VP2 is necessary for encapsidation of VP1 and VP3. 942 Feb 16

In order to clone candidate tumor suppressor genes whose loss contributes to the pathogenesis of neuroblastoma (NB), we performed polymerase chain reaction (PCR) screening using a high-density sequence tagged site-content map within a commonly deleted region (chromosome band 1p36) in 24 NB cell lines. We found a approximately 480 kb homozygously deleted region at chromosome band 1p36.2 in one of the 24 NB cell lines, NB-1, and cloned the human homologue (KIF1B-beta) of the mouseKif1B-beta gene in this region. The KIF1B-beta gene had at least 47 exons, all of which had a classic exon-intron boundary structure. Mouse Kif1B is a microtubule-based putative anterograde motor protein for the transport of mitochondria in neural cells. We performed mutational analysis of the KIF1B-beta gene in 23 cell lines using 46 sets of primers and also an allelic imbalance (AI) analysis of KIF1B-beta in 50 fresh NB samples. A missense mutation at codon 1554, GTG (Gly) to ATG (Met), silent mutations at codon 409 (ACG to ACA) and codon 1721 (ACC to ACT), and polymorphisms at codon 170, GAT (Asp) to GAA (Glu), and at codon 1087, TAT (Tyr), to TGT (Cys), were all identified, although their functional significances remain to be determined. The AI for KIF1B-beta was slightly higher (38%) than those for the other two markers (D1S244, D1S1350) (35 and 32%) within the commonly deleted region (1p36). Reverse transcriptase-PCR analysis of the KIF1B-beta gene revealed obvious expression in all NB cell lines except NB-1, although decreased expression of the KIF1B-beta gene was found in a subset of early- and advanced-stage NBs. These results suggest that the KIF1B-beta gene may not be a candidate for tumor suppressor gene of NB.
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PMID:Genomic structure and mutational analysis of the human KIF1B gene which is homozygously deleted in neuroblastoma at chromosome 1p36.2. 1152 94


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