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Query: EC:3.1.27.5 (RNase)
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We have identified and characterized protein factors from mung bean (Vigna radiata) nuclear extracts that specifically bind the single-stranded G-rich telomeric DNA repeats. Nuclear extracts were prepared from three different types of plant tissue, radicle, hypocotyl, and root, in order to examine changes in the expression patterns of telomere-binding proteins during the development of mung bean. At least three types of specific complexes (A, B, and C) were detected by gel retardation assays with synthetic telomere and nuclear extract from radicle tissue, whereas the two major faster-migrating complexes (A and B) were formed with nuclear extracts from hypocotyl and root tissues. Gel retardation assays also revealed differences in relative amount of each complex forming activity in radicle, hypocotyl, and root nuclear extracts. These data suggest that the expression of telomere-binding proteins is developmentally regulated in plants, and that the factor involved in the formation of complex C may be required during the early stages of development. The binding factors have properties of proteins and are hence designated as mung bean G-rich telomere-binding proteins (MGBP). MGBPs bind DNA substrates with three or more single-stranded TTTAGGG repeats, while none of them show binding affinity to either double-stranded or single-stranded C-rich telomeric DNA. These proteins have a lower affinity to human telomeric sequences than to plant telomeric sequences and do not exhibit a significant binding activity to Tetrahymena telomeric sequence or mutated plant telomeric sequences, indicating that their binding activities are specific to plant telomere. Furthermore, RNase treatment of the nuclear extracts did not affect the complex formation activities. This result indicates that the single-stranded telomere-binding activities may be attributed to a simple protein but not a ribonucleoprotein. The ability of MGBPs to bind specifically the single-stranded TTTAGGG repeats may suggest their in vivo functions in the chromosome ends of plants.
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PMID:Characterization and developmental expression of single-stranded telomeric DNA-binding proteins from mung bean (Vigna radiata). 1080 1

The dbl oncogene is generated by substitution of the 5' portion of its normal counterpart with an unrelated human sequence. To analyze the genomic structure and transcriptional regulation of the dbl proto-oncogene, we have isolated human genomic clones containing the entire human proto-dbl gene, localized in Xq26. Restriction mapping of a 600kb YAC clone (yWXD311) placed proto-dbl about 50kb telomeric to the coagulation Factor IX gene. The genomic DNA fragment containing the 5' end of proto-dbl was subcloned into plasmid vectors and the nucleotide sequences of exon 1, the flanking intronic region and genomic DNA 5' of the first codon were determined. Sequence analysis of 85119bp from the region revealed the genomic structure of proto-dbl. It contains 25 exons coding for a 4.7kb transcript including large 5'- and 3'- (1218bp and 701bp, respectively) untranslated regions (UTRs). RNase protection and primer extension assays on RNA from medullary thyroid carcinoma (TT) cells, which normally express dbl, revealed a transcription start site 1218bp upstream of the ATG of the first exon. A 1.6kb genomic 5' of the translation start sites drives the expression of a CAT-reporter in transient transfections in the TT cell line, though lacking TATA or CAAT boxes.
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PMID:Human dbl proto-oncogene in 85 kb of xq26, and determination of the transcription initiation site. 1092 7

The ribonucleoprotein enzyme telomerase adds telomeric repeats to the ends of linear chromosomes. The Tetrahymena telomerase reverse transcriptase (TERT) protein and the telomerase RNA can be reconstituted into an active complex in vitro in rabbit reticulocyte lysates. We have probed the structure of the telomerase RNA in the reconstituted complex with RNases T1 and V1. Upon TERT binding to the RNA, sites of both protection and enhancement of cleavage were observed, suggesting potential protein-binding sites and conformational changes in the RNA. Especially prominent was a large region of RNase V1 protection in stem-loop IV. A number of loop IV mutants still bound TERT but showed drastic decreases in the level of telomerase activity and the loss of protein-dependent folding of the pseudoknot region of the telomerase RNA. The telomerase activity defect and the misfolding of the pseudoknot were partially separable, leading to the proposal of two functions for stem-loop IV: to aid in the folding of the pseudoknot and to function more directly in the active site of telomerase. Thus an RNA element far from the template makes a major contribution to Tetrahymena telomerase enzyme activity.
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PMID:A stem-loop of Tetrahymena telomerase RNA distant from the template potentiates RNA folding and telomerase activity. 1140 44

The chromosomal ends of Trypanosoma brucei, like those of most eukaryotes, contain conserved 5'-TTAGGG-3' repeated sequences and are maintained by the action of telomerase. Fractionated T. brucei cell extracts with telomerase activity were used as a source of potential regulatory factors or telomerase-associated components that might interact with T. brucei telomeres. Electrophoretic mobility shift assays and UV cross-linking were used to detect possible single-stranded telomeric protein.DNA complexes and to estimate the approximate size of the protein constituents. Three single-stranded telomeric protein.DNA complexes were observed. Complex C3 was highly specific for the G-strand telomeric repeat sequence and shares biochemical characteristics with G-rich, single-stranded telomeric binding proteins and with components of the telomerase holoenzyme described in yeast, ciliates, and humans. Susceptibility to RNase A or chemical nuclease (hydroxyl radical) pre-treatment showed that complex C3 was tightly associated with an RNA component. Matrix-assisted laser desorption/ionization-time of flight mass spectrometry was used to estimate the molecular mass of the peptides obtained by in-gel Lys-C digestion of low abundance C3-associated proteins. The molecular masses of the peptides showed no homologies with other proteins from trypanosomes or with any protein in the data bases screened.
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PMID:A Trypanosoma brucei protein complex that binds G-overhangs and co-purifies with telomerase activity. 1167 53

Increased expression of telomerase is critical in the pathogenesis of cancer. Telomerase expression is reported variably in foregut cancers, possibly as a result of telomerase inhibition or ribonucleases. We performed experiments to assess telomerase and telomerase RNA expression in foregut cancers and to quantify and characterize telomerase inhibition. Cancer specimens were obtained from 27 patients. Telomerase activity of cancers was determined by the telomeric repeat amplification protocol, the presence of telomerase RNA component (hTERC) by reverse transcription PCR, and the quantity of telomerase inhibitors in mixing experiments. Ribonuclease activity was measured by assessing degradation of labeled RNA by cancers. Telomerase was found in 8/11 adenocarcinomas of the esophagus or gastroesophageal junction and 6/16 distal gastric adenocarcinomas; hTERC was detectable in all cancers. Telomerase inhibition was more marked in distal compared to proximal adenocarcinomas (P = 0.01) and correlated with ribonuclease activity (rS = 0.65). Ribonucleases contribute significantly to telomerase inhibitory activity detectable in foregut cancer specimens. In vitro, the presence of telomerase inhibitors in some specimens did not prevent the detection of telomerase by the TRAP assay. This suggests a more complex relationship between telomerase and its inhibitors. Site-specificity of telomerase inhibitors generally and ribonuclease activity specifically suggests a putative regulatory role in vivo.
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PMID:Inhibition of telomerase by site-specific ribonucleases in gastric and esophageal adenocarcinoma. 1176 58

We have analysed an extracted RNase sensitive fraction containing telomeric repeat sequences in the telomerase negative dipteran Chironomus tentans. It shows a slow and well-defined electophoretic migration corresponding to > 20 kb and is sensitive not only to RNase, but also to DNase. It hybridizes to both strands of the telomeric repeat with about equal intensities. DNA is probably the dominant component since the fraction is only slightly heavier than genomic DNA in isopycnic gradients but considerably lighter than RNA. It can, nevertheless, be shown to incorporate tritiated uridine. The material might represent another example of extrachromosomal telomeric repeats in telomerase negative cells.
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PMID:Extrachromosomal RNA-DNA complex containing long telomeric repeats in chironomids. 1196 82

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.
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PMID:Ty1 /copia- and Ty3 /gypsy-like DNA sequences in Helianthus species. 1235 9

RNA interference (RNAi) is a widespread silencing mechanism that acts at both the posttranscriptional and transcriptional levels. Here, we describe the purification of an RNAi effector complex termed RITS (RNA-induced initiation of transcriptional gene silencing) that is required for heterochromatin assembly in fission yeast. The RITS complex contains Ago1 (the fission yeast Argonaute homolog), Chp1 (a heterochromatin-associated chromodomain protein), and Tas3 (a novel protein). In addition, the complex contains small RNAs that require the Dicer ribonuclease for their production. These small RNAs are homologous to centromeric repeats and are required for the localization of RITS to heterochromatic domains. The results suggest a mechanism for the role of the RNAi machinery and small RNAs in targeting of heterochromatin complexes and epigenetic gene silencing at specific chromosomal loci.
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PMID:RNAi-mediated targeting of heterochromatin by the RITS complex. 1470 33

Telomerase is a ribonucleoprotein enzyme that can elongate telomeric DNA, which is thought to be required for the development of cellular immortality and oncogenesis in mammals. We examined telomerase activity in tissues and primary cultured lymphoid cells of adult penaeid shrimps. Using the telomeric repeat amplification protocol (TRAP), we studied the characteristics of a putative novel telomerase in Penaeus japonicus. This telomerase could be inactivated by heating or treatment with RNase A or proteinase K. At elongation, this telomerase required dATP, dGTP, and dTTP, but not dCTP, as substrates. Sequence analysis of the TRAP product revealed that this telomerase synthesized (TTAGG)(n) repeated sequences. The activity of this telomerase was decreased but still readily detectable in 100 ng of protein extract from lymphoid tissue. The telomerase activity was detected in all examined tissues including testis, ovary, lymphoid, heart, hepatopancreas, and muscle. The highest telomerase activity was in the extract of ovarian tissues. In primary cultured lymphoid cells, the telomerase activity was retained. Thus, primary cultured lymphoid cells of Penaeus japonicus possess one of the factors necessary for cell line establishment.
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PMID:Detection of telomerase activity in tissues and primary cultured lymphoid cells of Penaeus japonicus. 1513 20

We have identified and characterized a protein factor in soybean (Glycine max) nuclear extracts that binds to plant single stranded telomeric DNA repeats. A single DNA-protein complex was detected in gel retardation assays using synthetic telomeres and nuclear extracts. The protein forming this complex was designated soy-bean (Glycine max) single stranded telomeric DNA-binding protein (Gm-STBP). Gm-STBP binds to single stranded telomeric DNA containing more than two repeats. It does not bind to Tetrahymena, human or mutated plant telomere sequences, and its binding activity is not affected by RNase treatment. Gm-STBP activity gradually decreased after suspension cultures entered stationary phase. A slower migrating band was formed with extracts of earlier and later phases of soybean suspension cultures. Our findings suggest that binding of Gm-STBP to plant single stranded telomeric DNA may play a role in the proper functioning of telomeres during development.
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PMID:Characterization of single stranded telomeric DNA-binding proteins in cultured soybean (Glycine max) cells. 1523 26


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