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)

Telomerase, a specialized cellular reverse transcriptase, compensates for chromosome shortening during the proliferation of most eucaryotic cells and contributes to cellular immortalization. The mechanism used by the single-celled protozoan malaria parasite Plasmodium falciparum to complete the replication of its linear chromosomes is currently unknown. In this study, telomerase activity has for the first time been identified in cell extracts of P. falciparum. The de novo synthesis of highly variable telomere repeats to the 3' end of DNA oligonucleotide primers by plasmodial telomerase is demonstrated. Permutated telomeric DNA primers are extended by the addition of the next correct base. In addition to elongating preexisting telomere sequences, P. falciparum telomerase can also add telomere repeats onto nontelomeric 3' ends. The sequence GGGTT was the predominant initial DNA sequence added to the nontelomeric 3' ends in vitro. Poly(C) at the 3' end of the oligonucleotide significantly alters the precision of the new telomerase added repeats. The efficiency of nontelomeric primer elongation was dependent on the presence of a G-rich cassette upstream of the 3' terminus. Oligonucleotide primers based on natural P. falciparum chromosome breakpoints are efficiently used as telomerase substrates. These results imply that P. falciparum telomerase contributes to chromosome maintenance and to de novo telomere formation on broken chromosomes. Reverse transcriptase inhibitors such as dideoxy GTP efficiently inhibit P. falciparum telomerase activity in vitro. These data point to malaria telomerase as a new target for the development of drugs that could induce parasite cell senescence.
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PMID:Plasmodium falciparum telomerase: de novo telomere addition to telomeric and nontelomeric sequences and role in chromosome healing. 944 88

MLL (ALL1, Htrx, HRX), which is located on chromosome band 11q23, frequently is rearranged in patients with therapy-related acute myeloid leukemia who previously were treated with DNA topoisomerase II inhibitors. In this study, we have identified a fusion partner of MLL in a 10-year-old female who developed therapy-related acute myeloid leukemia 17 months after treatment for Hodgkin's disease. Leukemia cells of this patient had a t(11;17)(q23;q25), which involved MLL as demonstrated by Southern blot analysis. The partner gene was cloned from cDNA of the leukemia cells by use of a combination of adapter reverse transcriptase-PCR, rapid amplification of 5' cDNA ends, and BLAST database analysis to identify expressed sequence tags. The full-length cDNA of 2.8 kb was found to be an additional member of the septin family, therefore it was named MSF (MLL septin-like fusion). Members of the septin family conserve the GTP binding domain, localize in the cytoplasm, and interact with cytoskeletal filaments. A major 4-kb transcript of MSF was expressed ubiquitously; a 1.7-kb transcript was found in most tissues. An additional 3-kb transcript was found only in hematopoietic tissues. By amplification with MLL exon 5 forward primer and reverse primers in MSF, the appropriately sized products were obtained. MSF is highly homologous to hCDCrel-1, which is a partner gene of MLL in leukemias with a t(11;22)(q23;q11.2). Further analysis of MSF may help to delineate the function of MLL partner genes in leukemia, particularly in therapy-related leukemia.
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PMID:MSF (MLL septin-like fusion), a fusion partner gene of MLL, in a therapy-related acute myeloid leukemia with a t(11;17)(q23;q25). 1033 4

In the ciliate protozoon, Tetrahymena pyriformis, mitochondrial protein-coding genes are highly divergent in sequence, and in a number of cases they lack AUG initiation codons. We asked whether RNA editing might be acting to generate protein sequences that are more conventional than those inferred from the corresponding gene sequences, and/or to create standard AUG initiation codons where these are absent. However, comparison of genomic and cDNA sequences (the latter generated by reverse transcriptase sequencing of T. pyriformis mitochondrial mRNAs) yielded no evidence of mitochondrial RNA editing in this organism. To delineate the 5' ends of mitochondrial protein-coding transcripts, primer extension experiments were conducted. In all cases, 5' termini were found to map within a few nucleotides of potential initiation codons, indicating that T. pyriformis mitochondrial mRNAs have little or no 5' untranslated leader sequence. The pattern of strong primer extension stops suggested that both standard (AUG) and non-standard (AUU, AUA, GUG, UUG) initiation codons are utilized by the Tetrahymena mitochondrial translation system. We also investigated expression of the nad1 gene, which in both T. pyriformis and Paramecium aurelia is split into two portions that are encoded by and transcribed from different DNA strands. Northern hybridization analysis showed that the corresponding transcripts are not trans-spliced, implying that separate N-terminal and C-terminal portions of Nad1 are made in this system. Finally, in a search for primary transcripts, we isolated from a T. pyriformis mitochondrial fraction several small RNAs that were reproducibly labeled by incubation in the presence of [alpha-(32)P]GTP and guanylyltransferase. Partial sequence information revealed that none of these cappable RNAs is encoded in the T. pyriformis mitochondrial genome.
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PMID:Expression of mitochondrial protein-coding genes in Tetrahymena pyriformis. 1071 8

We demonstrate that the homozygous weaver granule neurons cultured on a laminin substratum fail to express inwardly rectifying potassium currents, including a functional G-protein coupled inwardly rectifying potassium (GIRK)2 potassium channel. By contrast, both normal and weaver Purkinje cells express inwardly rectifying potassium currents, and normal granule cells exhibit inwardly rectifying potassium currents inducible with GTP-gamma-S. In protein extracts of the vermal postnatal day (P)5-9 weaver cerebellum, the GIRK2 protein could not be detected by Western analysis, although the GIRK2 protein was detectable in extracts of the normal vermis. Northern analysis indicated that during early postnatal cerebellar development, the GIRK2 mRNA is expressed at extremely low levels being detectable at P18-23 in the normal but not yet in the homozygous weaver cerebellum. Using reverse transcriptase-polymerase chain reaction (RT-PCR), the GIRK2 mRNA was detected in both normal and weaver cerebella, but quantitative PCR confirmed that the weaver cerebellum expressed the GIRK2 gene at significantly lower levels as compared to the normal cerebellum (P = 0.01, paired t-test). Sequencing indicated that the weaver GIRK2 channel gene had the point mutation proposed to be responsible for the weaver phenotype. Rescue of both survival and neurite outgrowth of the cultured vermal weaver granule neurons by verapamil (Liesi and Wright, 1996; Liesi et al., 1999) induced expression of immunocytochemically detectable levels of the GIRK2 protein. Sequencing revealed that the GIRK2 mRNA of the rescued weaver granule neurons remained the mutated variant of the GIRK2 channel gene. Our results indicate that expression of the mutated GIRK2 protein and/or mRNA in the weaver granule neurons may be an indicator of rescue rather than death of the weaver granule neurons. That the weaver granule neurons expressed no functional GIRK2 receptors during a time period of neuronal death and migration failure suggests that the point mutation in the H5 membrane spanning region of the GIRK2 gene may associate with, but not be responsible for the weaver phenotype.
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PMID:Involvement of GIRK2 in postnatal development of the weaver cerebellum. 1074 Feb 21

ADP-ribosylation factors (ARFs) constitute a family of structurally related proteins that forms a subset of the Ras superfamily of regulatory GTP-binding proteins. Like other GTPases, activation of ARFs is facilitated by specific guanine nucleotide exchange factors (GEFs). In chromaffin cells, ARF6 is associated with the membrane of secretory granules. Stimulation of intact cells or direct elevation of cytosolic calcium in permeabilized cells triggers the rapid translocation of ARF6 to the plasma membrane and the concomitant activation of phospholipase D (PLD) in the plasma membrane. Both calcium-evoked PLD activation and catecholamine secretion in permeabilized cells are strongly inhibited by a synthetic peptide corresponding to the N-terminal domain of ARF6, suggesting that the ARF6-dependent PLD activation near the exocytotic sites represents a key event in the exocytotic reaction in chromaffin cells. In the present study, we demonstrate the occurrence of a brefeldin A-insensitive ARF6-GEF activity in the plasma membrane and in the cytosol of chromaffin cells. Furthermore, reverse transcriptase-polymerase chain reaction and immunoreplica analysis indicate that ARNO, a member of the brefeldin A-insensitive ARF-GEF family, is expressed and predominantly localized in the cytosol and in the plasma membrane of chromaffin cells. Using permeabilized chromaffin cells, we found that the introduction of anti-ARNO antibodies into the cytosol inhibits, in a dose-dependent manner, both PLD activation and catecholamine secretion in calcium-stimulated cells. Furthermore, co-expression in PC12 cells of a catalytically inactive ARNO mutant with human growth hormone as a marker of secretory granules in transfected cells resulted in a 50% inhibition of growth hormone secretion evoked by depolarization with high K(+). The possibility that the plasma membrane-associated ARNO participates in the exocytotic pathway by activating ARF6 and downstream PLD is discussed.
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PMID:Identification of a plasma membrane-associated guanine nucleotide exchange factor for ARF6 in chromaffin cells. Possible role in the regulated exocytotic pathway. 1074 97

Replication of the Saccharomyces cerevisiae Ty1 retrotransposon requires a reverse transcriptase capable of synthesizing Ty1 DNA. The first description of an active form of a recombinant Ty1 enzyme with polymerase and RNase H activities is reported here. The Ty1 enzyme was expressed as a hexahistidine-tagged fusion protein in Escherichia coli to facilitate purification of the recombinant protein by metal-chelate chromatography. Catalytic activity of the recombinant protein was detected only when amino acid residues encoded by the integrase gene were added to the N-terminus of the reverse transcriptase-RNase H domain. This suggests that the integrase domain could play a role in proper folding of reverse transcriptase. Several biochemical properties of the Ty1 enzyme were analysed, including the effect of MgCl(2), NaCl, temperature and of the chain terminator dideoxy GTP on its polymerase activity. RNase H activity was examined by monitoring the cleavage of a RNA-DNA template-primer. Our results suggest that the distance between the RNase H and polymerase active sites corresponds to the length of a 14-nucleotide RNA-DNA heteroduplex. The recombinant protein produced in E. coli should be useful for further biochemical and structural analyses and for a better understanding of the role of integrase in the activation of reverse transcriptase.
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PMID:Expression of an active form of recombinant Ty1 reverse transcriptase in Escherichia coli: a fusion protein containing the C-terminal region of the Ty1 integrase linked to the reverse transcriptase-RNase H domain exhibits polymerase and RNase H activities. 1081 27

We have attempted to determine whether muscarinic stimulation induces RhoA/ROCK-mediated Ca2+ sensitization of contractions in chicken gizzard smooth muscles. rhoA is a small GTP-binding protein, and ROCK is a rhoA-associated coiled coil-forming serine/threonine kinase. The relationship between the cytosolic Ca2+ level ([Ca2+]i) and muscle force in the presence of a high K+ concentration was not different from that in the presence of carbachol. Verapamil inhibited muscle force in proportion to the decrease in [Ca2+]i in both the muscle stimulated with high K+ and that stimulated with carbachol. In addition, Y-27632 (10 microM), a ROCKs inhibitor, had no effect on the contractions. In the alpha-toxin-permeabilized muscles, Ca2+ induced a greater contraction in the presence of guanosine 5'-O-(3-thiotriphosphapte) (GTP[gamma-S]), whereas carbachol with GTP was not effective. The GTP[gamma-S]-induced Ca2+ sensitization was completely inhibited by Clostridium botulinum exoenzyme C3. Western blot analysis revealed both rhoA and ROCKII in the muscle extract. In addition, reverse transcriptase polymerase chain reaction (RT-PCR) analysis revealed the expression of both ROCKI and ROCKII mRNAs. These results suggest that Ca2+ sensitization in the chicken gizzard is elicited via a rhoA/ROCKs pathway, and that this pathway may be responsible for the augmentation of contraction by GTP[gamma-S] in the permeabilized muscles. If such a pathway does exist, however, carbachol-induced contraction may not be coupled to it, which explains the absence of Ca2+ sensitization in the intact chicken gizzard stimulated by carbachol.
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PMID:Muscarinic stimulation does not induce rhoA/ROCK-mediated Ca2+ sensitization of the contractile element in chicken gizzard smooth muscle. 1121 Nov 3

The melanosome is a unique secretory granule of the melanocyte in which melanin pigments are synthesized by tyrosinase gene family glycoproteins. Melanogenesis is a highly regulated process because of its inherent toxicity. An understanding of the various regulatory mechanisms is important in delineating the pathophysiology involved in pigmentary disorders and melanoma. We have purified and analyzed the total melanosomal proteins from B16 mouse melanoma tumors in order to identify new proteins that may be involved in the control of the melanogenesis process. Melanosomal proteins were resolved by two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis, a predominant spot (27 kDa with isoelectric point 5.8-6.4) was excised and digested with cyanogen bromide, and the fragments were sequenced. Synthetic oligonucleotide primers were synthesized corresponding to the peptide sequences, and reverse transcriptase polymerase chain reaction amplification of total RNA from B16 cells was carried out. Sequencing of one of the polymerase-chain-reaction-mediated clones demonstrated 80%-97% sequence homology of 200 bp nucleotide with GTP-binding proteins at the 3'-untranslated region. GTP-binding assay on two-dimensional gels of melanosomal proteins showed the presence of several (five to six) small GTP-binding proteins, suggesting that small GTP-binding proteins are associated with the melanosome. Among the known GTP-binding proteins with similar molecular weight and isoelectric point ranges, rab3, rab7, and rab8 were found to be present in the melanosomal fraction by immunoblotting. Confocal immunofluorescence microscopy showed that rab7 is colocalized with the tyrosinase-related protein 1 around the perinuclear area as well as, in part, in the perikaryon, thereby suggesting that rab7 might be involved in the intracellular transport of tyrosinase-related protein 1. Tyrosinase-related protein 1 transport was blocked by the treatment of B16 cells with antisense oligonucleotide to rab7. We suggest (i) that rab7 is a melanosome-associated molecule, (ii) that tyrosinase-related protein 1 is present in late-endosome delineated granules, and (iii) that rab7 is involved in the transport of tyrosinase-related protein 1 from the late-endosome delineated granule to the melanosome.
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PMID:Identification of rab7 as a melanosome-associated protein involved in the intracellular transport of tyrosinase-related protein 1. 1144 53

Nm23 has been identified as a gene family encoding different isoforms of nucleoside diphosphate kinase (NDPK). This protein is a key enzyme in nucleotide metabolism and has been shown to play important roles in various cellular functions. In the present study, we have investigated the expression of three isotypes in mouse dorsal root ganglia. In situ hybridization and reverse transcriptase-polymerase chain reaction analysis demonstrated high levels of nm23-M1, -M2, and -M3 mRNA expression in peripheral nervous tissue. Moreover, in situ hybridization also displayed a specific nuclear localization for nm23-M2 mRNA. Immunohistochemistry with light and electron microscopy on isoform-specific antibodies revealed a differential subcellular distribution of NDPK isoforms. Isoform A was mainly cytosolic, showing only partial association with organelles. In contrast, isoform B was also found in the nucleus, which is in agreement with its proposed role as a transcription factor. The results also indicate a preferential association of isoform C with endoplasmic reticulum and plasma membranes in neuronal cells. Furthermore, isoform C appeared to combine with other NDPK isoforms as demonstrated by double-labeling evidence by electron microscopy and might be responsible for binding NDPK oligomers to membranes. Thus, isoform C may be considered as a protein of importance for maintaining intracellular pools of GTP in the vicinity of membranes and, hence, for transmembrane signaling. The results indicate a high expression of NDPK isoforms, not only in the central but also in the peripheral nervous system. Their different subcellular compartmentalization suggests that they have isoform-specific roles in neuronal cell physiology.
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PMID:Differential expression of nm23 genes in adult mouse dorsal root ganglia. 1189 45

Hepatitis B viruses, or hepadnaviruses, are small DNA-containing viruses that replicate through reverse transcription. Their prototype, HBV, causes severe liver disease in humans. The hepadnaviral P protein is an unusual reverse transcriptase (RT) that initiates DNA synthesis by host-factor-dependent protein priming on a specific RNA stem-loop template, epsilon, yielding a short DNA oligonucleotide covalently attached to the RT. This priming reaction can be reconstituted with in vitro-translated duck hepatitis B virus (DHBV) P protein. No direct structural data are available for any P protein. However, P proteins share a number of conserved motifs with other polymerases. Box A contains an invariant bulky residue recently shown to be crucial for dNTP versus NTP discrimination in RTs and some DNA polymerases; its equivalent in DHBV P protein would be phenylalanine 451 (F451). Four mutants, containing glycine (F451G), alanine (F451A), valine (F451V) and aspartate (F451D), were therefore analyzed for their ability to utilize dNTPs and NTPs in in vitro priming. Priming efficiencies with dNTPs decreased with decreasing side chain size but GTP utilization increased; the wild-type enzyme was inactive with GTP. In the context of complete DHBV genomes, all mutant proteins were competent for RNA encapsidation, indicating the absence of global structural alterations. Because the function of the discriminatory residue depends on its specific spatial disposition this strongly suggests a similar architecture for the P protein dNTP-binding pocket as in other RTs.
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PMID:dNTP versus NTP discrimination by phenylalanine 451 in duck hepatitis B virus P protein indicates a common structure of the dNTP-binding pocket with other reverse transcriptases. 1191 30


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