Telomerase reverse transcriptase gene is a direct target of c-Myc but is not functionally equivalent in cellular transformation.
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The telomerase reverse transcriptase component (TERT) is not expressed in most primary somatic human cells and tissues, but is upregulated in the majority of immortalized cell lines and tumors. Here, we identify the c-Myc transcription factor as a direct mediator of telomerase activation in primary human fibroblasts through its ability to specifically induce TERT gene expression. Through the use of a hormone inducible form of c-Myc (c-Myc-ER), we demonstrate that Myc-induced activation of the hTERT promoter requires an evolutionarily conserved E-box and that c-Myc-ER-induced accumulation of hTERT mRNA takes place in the absence of de novo protein synthesis. These findings demonstrate that the TERT gene is a direct transcriptional target of c-Myc. Since telomerase activation frequently correlates with immortalization and telomerase functions to stabilize telomers in cycling cells, we tested whether Myc-induced activation of TERT gene expression represents an important mechanism through which c-Myc acts to immortalize cells. Employing the rat embryo fibroblast cooperation assay, we show that TERT is unable to substitute for c-Myc in the transformation of primary rodent fibroblasts, suggesting that the transforming activities of Myc extend beyond its ability to activate TERT gene expression and hence telomerase activity. (+info)
Evolutionary dynamics of Ty1-copia group retrotransposons in grass shown by reverse transcriptase domain analysis.
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The evolutionary dynamics of Ty1-copia group retrotransposons in grass were examined by reverse transcriptase (RT) domain analysis. Twenty-three rice RT sequences were newly determined for this report. Phylogenetic analysis of 177 RT sequences, mostly derived from wheat, rice, and, maize, showed four distinct families, which were designated G1, G2, G3, and G4. Three of these families have elements obtained from distantly related species, indicative of origins prior to the radiation of grass species. Results of Southern hybridization and detailed comparisons between the wheat and rice sequences indicated that each of the families had undergone a distinct pattern of evolution. Multiple families appear to have evolved in parallel in a host species. Analyses of synonymous and nonsynonymous substitutions suggested that there is a low percentage of elements carrying functional RT domains in the G4 family, indicating that the production of new G4 elements has been controlled by a small number of elements carrying functional RT domains. (+info)
Cellular localization and evolution of prolactin receptor mRNA in ovine endometrium during pregnancy.
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In this study, we have investigated the expression of the prolactin receptor gene in ovine endometrium during oestrus cycle and pregnancy. Using reverse transcription-PCR analysis, we provided evidence that the prolactin receptor gene is specifically transcribed in this tissue. As shown by Northern blot analysis, the level of the prolactin receptor transcripts increased dramatically during late pregnancy. In situ hybridization experiments revealed that prolactin receptor mRNA was specifically expressed in the glandular compartment and confirmed the dramatic increase of its expression that occurs at the end of pregnancy. Taken together, these findings are consistent with a putative role of prolactin and/or related molecules in the regulation of the proliferation of the glandular compartment and/or in the control of the secretory activity of the endometrium. (+info)
Two types of HTLV-1 particles are released from MT-2 cells.
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The MT-2 cell line transformed by human T-cell leukemia virus type 1 (HTLV-1) contains one complete provirus and seven defective proviruses. Four defective genomes have an identical structure (LTR-MA-deltaCA-pX-LTR) with an open reading frame that spans from MA to pX, giving rise to a 3.4-kb (24S) RNA transcript encoding a chimeric Gag-pX protein, p28. MT-2 cells release two distinct types of virions. The major "classic" type of particle has a buoyant density of 1.155-1.16 g/cm3 and contains the standard HTLV-I structural proteins and reverse transcriptase (RT). In addition, about 5% of particles are "light," approximately 1.12 g/cm3, and contain p28, RT activity, and the 3.4-kb RNA transcript. RT-PCR and in vitro translation indicate that some of the classic HTLV-1 particles package 3.4-kb RNA as well as full-length 8.5-kb RNA. In addition to matrix features, the p28 protein has a motif resembling a zinc finger at the C-terminal, pX0 region, which may play a role in the assembly of the defective light virions. (+info)
Natural selection of the Pol gene of bovine immunodeficiency virus.
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Genetic variability is a salient feature of lentiviruses, contributing to the pathogenesis of these viruses by enabling them to persist in the host and to resist anti-retroviral treatment. Bovine immunodeficiency virus (BIV), a lentivirus of unknown pathology, infects cattle in the United States and worldwide. Genetic diversity of BIV that is associated with naturally infected cattle is not well studied. We examined the genetic diversity and natural selection of a segment of the BIV pol gene amplified from the leukocyte DNA of naturally infected cattle. A portion of the reverse transcriptase domain (183 bp) of the pol region was targeted for amplification by PCR. PCR products were sequenced directly and aligned. When compared to the sequences of BIV R29-127, a molecular clone of the original BIV R29 isolate, all isolates were greater than 91% identical in nucleotide sequences and 77% identical in amino acid sequences. Pol genotypes were polymorphic at 14% of the nucleotide sites. The ratio of nonsynonymous to synonymous nucleotide substitutions (relative to the number of respective sites, Ka/Ks) was 0.16, indicating that this region of the BIV genome, like that of HIV-1, is subject to purifying selection. Based on the McDonald-Kreitman analysis, this region also was under positive Darwinian selection as HIV-1 and BIV diverged from a common progenitor. Phylogenetic analysis revealed that genotypes were geographically distinct, possibly indicating a common source of infection for animals within a herd. (+info)
Expression of mutated Paramecium telomerase RNAs in vivo leads to templating errors that resemble those made by retroviral reverse transcriptase.
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Telomeric DNA consists of short, tandemly repeated sequences at the ends of chromosomes. Telomeric DNA in the ciliate Paramecium tetraurelia is synthesized by an error-prone telomerase with an RNA template specific for GGGGTT repeats. We have previously shown that misincorporation of TTP residues at the telomerase RNA templating nucleotide C52 accounts for the 30% GGGTTT repeats randomly distributed in wild-type telomeres. To more completely characterize variable repeat synthesis in P. tetraurelia, telomerase RNA genes mutated at C52 (A, U, and G) were expressed in vivo. De novo telomeric repeats from transformants indicate that the predominant TTP misincorporation error seen in the wild-type telomerase is dependent on the presence of a C residue at template position 52. Paradoxically, the effects of various other telomerase RNA template and alignment region mutations on de novo telomeres include significant changes in fidelity, as well as the synthesis of aberrant, 5-nucleotide telomeric repeats. The occurrence of deletion errors and the altered fidelity of mutated P. tetraurelia telomerase, in conjunction with misincorporation by the wild-type enzyme, suggest that the telomerase RNA template domain may be analogous to homopolymeric mutational hot spots that lead to similar errors by the human immunodeficiency virus proofreading-deficient reverse transcriptase. (+info)
Functional analysis of mutations conferring lamivudine resistance on hepatitis B virus.
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Two patterns of mutation are commonly observed in the polymerase gene of lamivudine [(-)2'-deoxy-3'-thiacytidine]-resistant hepatitis B virus (HBV). The M539I substitution in the conserved YMDD motif occurs independently of other changes, whereas the M539V substitution is associated with an additional upstream change (L515M). These mutations were introduced into a common background and their effects on HBV DNA replication and lamivudine resistance studied. The L515M and M539V mutations provided only partial resistance while the M539I mutation conferred a high degree of lamivudine resistance. The combination of the L515M and M539V mutations gave an intermediate level of replication competence, compared with either mutation alone, and increased resistance to lamivudine. This probably accounts for these two mutations always being observed together. The M539I mutation reduced replication competence. (+info)
Properties of monoclonal antibodies directed against hepatitis B virus polymerase protein.
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Hepadnavirus polymerases are multifunctional enzymes that play critical roles during the viral life cycle but have been difficult to study due to a lack of a well-defined panel of monoclonal antibodies (MAbs). We have used recombinant human hepatitis B virus (HBV) polymerase (Pol) expressed in and purified from baculovirus-infected insect cells to generate a panel of six MAbs directed against HBV Pol protein. Such MAbs were subsequently characterized with respect to their isotypes and functions in analytical and preparative assays. Using these MAbs as probes together with various deletion mutants of Pol expressed in insect cells, we mapped the B-cell epitopes of Pol recognized by these MAbs to amino acids (aa) 8 to 20 and 20 to 30 in the terminal protein (TP) region of Pol, to aa 225 to 250 in the spacer region, and to aa 800 to 832 in the RNase H domain. Confocal microscopy and immunocytochemical studies using various Pol-specific MAbs revealed that the protein itself appears to be exclusively localized to the cytoplasm. Finally, MAbs specific for the TP domain, but not MAbs specific for the spacer or RNase H regions of Pol, appeared to inhibit Pol function in the in vitro priming assay, suggesting that antibody-mediated interference with TP may now be assessed in the context of HBV replication. (+info)