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:3.1.27.5 (RNase)
17,967 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The est1 mutant was previously identified because it is defective in telomere maintenance and displays a senescent phenotype. To see if Est1 might be a component of yeast telomerase, we examined immunoprecipitated Est1. The yeast telomerase RNA Tlc1 specifically coprecipitated with Est1. Furthermore, the Est1 immunoprecipitates contained a telomerase-like activity. As expected for yeast telomerase, the activity elongated telomeric primers, it required dGTP and dTTP but not dATP or dCTP, and it was sensitive to RNase A. Further evidence suggesting that the activity was telomerase was obtained from experiments using a TLC1-1 mutant strain, which has a mutant telomerase template containing dG residues. The activity immunoprecipitated from TLC1-1 mutant strains incorporated 32P-labeled dCTP, while activity from TLC1 strains did not. Use of different telomeric primer substrates revealed two distinguishable telomerase-like activities: one was dependent on TLC1, and one was not. The TLC1-independent activity may be due to a second yeast telomerase RNA, or it may be some other kind of activity.
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PMID:Association of the Est1 protein with telomerase activity in yeast. 861 Jan 24

Telomerase is a ribonucleoprotein enzyme which elongates the G-rich strand of telomeric DNA to compensate for the progressive reduction in its length due to incomplete replication of chromosome ends, which in human somatic cells leads to cell cycle arrest upon shortening of telomeres to a critical length. To examine the possible involvement of telomerase in metabolism of plant genetic material, we used cells of Nicotiana tabacum strain TBY-2, a stable long-term culture which has kept a constant pattern of restriction fragments from chromosome termini during its 6 month period of cultivation in our laboratory. In a direct assay for telomerase, a 5' end-labeled plant telomeric oligonucleotide 5' (TTTAGGG)(3')6 was elongated in a TBY-2 cell extract, showing a pausing pattern which is a characteristic feature of telomerases from other organisms. The elongation was inhibited by RNase A pretreatment of the extract. We conclude that plant cells possess telomerase which is used for maintenance of their telomeres.
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PMID:Telomerase activity in plant cells. 876 95

Using a synthetic telomere DNA template and whole cell extracts, we have identified proteins capable of synthesizing the telomere complementary strand. Synthesis of the complementary strand required a DNA template consisting of 10 repeats of the human telomeric sequence d(TTAGGG) and deoxy- and ribonucleosidetriphosphates and was inhibited by neutralizing antibodies to DNA polymerase alpha. No evidence for RNA-independent synthesis of the lagging strand was observed, suggesting that a stable DNA secondary structure capable of priming the lagging strand is unlikely. Purified DNA polymerase alpha/primase was capable of catalyzing synthesis of the lagging strand with the same requirements as those observed in crude cell extracts. A ladder of products was observed with an interval of six bases, suggesting a unique RNA priming site and site-specific pausing or dissociation of polymerase alpha on the d(TTAGGG)10 template. Removal of the RNA primers was observed upon the addition of purified RNase HI. By varying the input rNTP, the RNA priming site was determined to be opposite the 3' thymidine nucleotide generating a five-base RNA primer with the sequence 5'-AACCC. The addition of UTP did not increase the efficiency of priming and extension, suggesting that the five-base RNA primer is sufficient for extension with dNTPs by DNA polymerase alpha. This represents the first experimental evidence for RNA priming and DNA extension as the mechanism of mammalian telomeric lagging strand replication.
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PMID:Synthesis of the mammalian telomere lagging strand in vitro. 911 15

A telomerase-like primer extension activity has been detected in chromatographic fractions derived from Schizosaccharomyces pombe extracts. This primer extension activity acts preferentially on dG-rich oligodeoxynucleotides, is sensitive to RNase A pretreatment and requires all four deoxynucleotides for optimal polymerization. The extension products are also truncated by the inclusion of any one of the four dideoxynucleotides, consistent with the presence of all four bases in the S.pombe telomeric repeats. The intensity distribution of the extension products and the dideoxynucleotide termination pattern suggest that nucleotide addition is template directed, and that telomere-like sequences are added to the primers. In particular, the sequence d(CGGTTA), a variant of the S.pombe telomeric repeat, can be added directly by the in vitro activity. Partially purified S.pombe telomerase sediments as a 35S particle, suggesting that it exists in vivo as part of a large multi-protein complex.
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PMID:Identification and characterization of a telomerase activity from Schizosaccharomyces pombe. 933 65

In this work, we have identified and characterized proteins in rice nuclear extracts that specifically bind the single-stranded G-rich telomere sequence. Three types of specific DNA-protein complexes (I, II, and III) were identified by gel retardation assays using synthetic telomere substrates consisting of two or more single-stranded TTTAGGG repeats and rice nuclear extracts. Since each complex has a unique biochemical property and differs in electrophoretic mobility, at least three different proteins interact with the G-rich telomere sequences. These proteins are called rice G-rich telomere binding protein (RGBP) and none of them show binding affinity to double-stranded telomere repeats or single-stranded C-rich sequence. Changing one or two G's to C's in the TTTAGGG repeats abolishes binding activity. RGBPs have a greatly reduced affinity for human and Tetrahymena telomeric sequence and do not efficiently bind the cognate G-rich telomere RNA sequence UUUAGGG. Like other telomere binding proteins, RGBPs are resistant to high salt concentrations. RNase sensitivity of the DNA-protein interaction. In this assay, we observed a novel complex (complex III) in gel retardation assays which did not alter the mobilities or the band intensities of the two pre-existing complexes (I and II). The complex III, in addition to binding to telomeric sequences, has a binding affinity to rice nuclear RNA, whereas two other complexes have a binding affinity to only single-stranded G-rich telomere DNA. Taken together, these studies suggest that RGBPs are new types of telomere-binding proteins that bind in vitro to single-stranded G-rich telomere DNA in the angiosperms.
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PMID:Rice proteins that bind single-stranded G-rich telomere DNA. 952 98

HIC-1 (hypermethylated in cancer) is a candidate tumor suppressor gene which is located at 17p13.3, a region which frequently undergoes allelic loss in breast and other human cancers. HIC-1 is proposed to be commonly inactivated in human cancers by hypermethylation of a normally unmethylated dense CpG island which encompasses the entire gene. To study whether HIC-1 inactivation may be important to the development of breast cancer, we first measured methylation of the HIC-1 gene in normal breast ductal tissues from microdissected frozen breast tissues and from epithelial cells purified from mammoplasty specimens. Surprisingly, in all normal breast ductal tissues we found approximately equal amounts of densely methylated HIC-1 and completely unmethylated HIC-1. This is in contrast to most normal tissues, in which all copies of HIC-1 are completely unmethylated. We then evaluated 39 primary breast cancer tissues and found virtually complete methylation of the HIC-1 gene in 26 (67%) of the cases. We also found loss of heterozygosity at the telomeric portion of chromosomal arm 17p in 22 of the 26 cases with strongly methylated HIC-1, suggesting that loss of an unmethylated HIC-1 allele may contribute to the inactivation of HIC-1 in cells with a pre-existing methylated allele. Finally, by RNase protection analysis, HIC-1 was found to be expressed in microdissected normal breast ductal tissues and unmethylated tumors but not in tumors with hypermethylation of the HIC-1 gene. These results indicate that hypermethylation of HIC-1 and associated loss of HIC-1 expression is common in primary breast cancer. Furthermore, the HIC-1 gene is densely methylated in approximately one-half of the alleles in normal breast epithelium, which may predispose this tissue to inactivation of this gene by loss of heterozygosity.
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PMID:Methylation of the HIC-1 candidate tumor suppressor gene in human breast cancer. 957 97

Telomerase, a ribonucleoprotein complex that includes the telomerase RNA component (hTR) and the telomerase catalytic subunit gene (hTERT) product, has been shown to be activated in the majority of cancer tissues and immortalized cells. To study telomerase activation during the progression of cervical cancer, the expression of hTR and hTERT RNAs in tissues of various stages of cervical cancer was analyzed using the in situ hybridization method and compared with proliferative activity as estimated by Ki-67 immunostaining. To test whether expression of these components is reflected in enzyme activity, we determined the levels of the RNAs in cervical cancer and normal tissues and in primary and immortal keratinocytes by reverse transcription-polymerase chain reaction and RNase protection assays and compared the results to telomerase activities as detected by telomeric repeat amplification protocol assay. In situ hybridization signals of hTR and hTERT were present not only in carcinoma tissues but also in normal epidermal layers. In many adenocarcinoma and fewer squamous cell carcinoma tissues, both signals were focally increased where high proliferative activity was present at the stages of dysplasia/metaplasia, in situ carcinoma, and invasive carcinoma. The level of bTERT, as quantitated by RNase protection assay, was not different between cancer and control tissues or immortal and a subset of primary keratinocytes and did not correlate with telomerase activity. These results indicate that expression of hTR and bTERT is up-regulated in at least a subset of neoplastic cells at an early stage of carcinogenesis and that unidentified factors, such as the modulation or coordination of its protein level with other products, may contribute to the activation of telomerase in cervical cancer.
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PMID:Telomerase activity and expression of telomerase RNA component and telomerase catalytic subunit gene in cervical cancer. 973 34

In Euplotes crassus, telomerase is responsible for telomere maintenance during vegetative growth and de novo telomere synthesis during macronuclear development. Here we show that telomerase in the vegetative stage of the life cycle exists as a 280-kD complex that can add telomeric repeats only onto telomeric DNA primers. Following the initiation of macronuclear development, telomerase assembles into larger complexes of 550 kD, 1600 kD, and 5 MD. In the 1600-kDa and 5-MDa complexes, telomerase is more processive than in the two smaller complexes and can add telomeres de novo onto nontelomeric 3' ends. Assembly of higher order telomerase complexes is accompanied by an extended region of RNase V1 and RNase T1 protection in the telomerase RNA subunit that is not observed with telomerase from vegetatively growing cells. The protected residues encompass a highly conserved region previously proposed to serve as a platform for formation of higher order structures. These findings provide the first direct demonstration of developmentally regulated higher order telomerase complexes with unique biochemical and structural properties.
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PMID:Developmentally programmed assembly of higher order telomerase complexes with distinct biochemical and structural properties. 974 68

Telomerase activity has been examined extensively in a variety of human cancerous and noncancerous tissues. However, it was sometimes difficult to measure telomerase activity quantitatively with the methods used and in the tissues examined. We examined telomerase activity quantitatively in gastrointestinal tissues by using the hybridization protection assay combined with the telomeric repeat amplification protocol (TRAP) to assess the diagnostic utility of measuring telomerase activity and to determine the relationship between telomerase activity and human telomerase reverse transcriptase (hTERT) expression. We report here that (i) polymerase chain reaction (PCR) inhibitors in the tissue extracts used for the telomerase assay were practically nullified by using tissue extract at 0.1 microg of protein/assay; (ii) RNase activity in tissue extracts should be blocked with 0.5 U of RNase inhibitor/microg tissue protein for the quantitative telomerase assay; (iii) no inhibitors of telomerase were found in tissue extracts other than RNase and PCR inhibitors (iv) higher telomerase activity in cancerous tissue than in noncancerous tissue from the same patients was observed in both gastric and colorectal tissues, but the telomerase activity varied from low to high levels in cancerous tissues, and it was not practical to set a general cut-off level for cancer diagnosis; (v) hTERT was expressed in both cancerous and noncancerous tissues, and (vi) the telomerase activity levels were generally lower than expected from the hTERT expression levels, suggesting posttranscriptional regulation of expression of telomerase activity.
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PMID:Quantitative reevaluation of telomerase activity in cancerous and noncancerous gastrointestinal tissues. 1056 8

Smooth muscle cells (SMC) express a battery of lineage-restricted genes whose encoded proteins impart the unique contractile phenotype that characterizes this muscle type. While the encoded function of many SMC-restricted genes has been extensively analyzed, less is known about their position within the genome and the regulatory factors governing their transcription. In this report, we define the gene structure, 5' promoter analysis, and chromosomal mapping of the rat smooth muscle calponin (CnnI) gene. The rat CnnI gene is comprised of seven exons spanning approximately 8 kb of genomic sequence. The intron-exon boundaries of the rat CnnI gene match precisely those in human and mouse. Primer extension and RNase protection assays indicate two major transcription start positions (tsp). Comparative sequence analysis of the 5' promoter region reveals several conserved cis regulatory elements, including a TA-rich element within 30 nt of the tsp that could be a recognition site for TATA-binding protein and two CCAAT boxes. Transient and stable transfection studies support the hypothesis that distal regulatory elements confer SMC-restricted expression of CnnI. Finally, using an F2 intercross, we have mapped the rat CnnI gene to the telomeric end of Chromosome (Chr) 8. These studies provide additional information relating to the control of CnnI gene expression and provide a platform to begin assessing the potential linkage of CnnI to spontaneous and experimental disease phenotypes in rats.
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PMID:Gene structure and chromosomal mapping of the rat smooth muscle calponin gene. 1065 25


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