Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rat cells produce two different transcripts of DNA polymerase beta (beta-Pol). The low-molecular-weight transcript (1.4 kb) was already sequenced. We report here the cloning and sequencing of the full-length cDNA, corresponding to the high-molecular-weight (HMW) transcript (4.0 kb) of beta-Pol. Sequence data strongly suggest that both transcripts are produced from a single gene by alternative polyadenylation. The HMW transcript contains the entire 1.4 kb transcript sequence and additional 2.2 kb on the 3' end. The 3' UTR of the HMW transcript contains some regulatory sequences which are not present in the 1.4-kb transcript. The A + U-rich fragment and (GU)21 sequence are believed to influence the stability of the mRNA. The functional significance of the A-rich region locally destabilizing double-stranded secondary structure remains unknown.
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PMID:Alternative polyadenylation of the gene transcripts encoding a rat DNA polymerase beta. 891 52

The catalytic subunit (UL54) and accessory protein (UL44) of human cytomegalovirus (HCMV) DNA polymerase have been cloned and expressed in an in vitro-coupled transcription/translation reticulocyte lysate system. The influence of the 5'-untranslated region (5'-UTR) on the efficiency of expression from the circular plasmids has been investigated. For expression of both UL54 and UL44, a truncated form of the alfalfa mosaic virus (AMV) RNA4 5'-UTR was found to be superior to the full-length AMV 5'-UTR or the original HCMV 5'-UTRs of different lengths. Protein products with Mr approximately 140 and 55 kDa were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis in the UL54 and UL44 in vitro expression reactions, respectively. The properties of the expressed enzyme were compared with those of native HCMV DNA polymerase purified from HCMV-infected cells. DNA polymerase and 3'-5' exonuclease activities of the expressed UL54/UL44 complex were found to be dependent on salt concentration in the same manner as the activities of the native enzyme. The in vitro-expressed enzyme resembles the purified HCMV DNA polymerase in its affinity for deoxynucleoside triphosphates as well as in its sensitivity to known inhibitors (cidofovir diphosphate, ganciclovir triphosphate, and foscarnet). This straightforward method for protein expression may also be applicable to other enzymes where reproducible generation of fully functional products is desirable.
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PMID:Expression of the catalytic subunit (UL54) and the accessory protein (UL44) of human cytomegalovirus DNA polymerase in a coupled in vitro transcription/translation system. 936 18

Differential mRNA display (DD-PCR) amplifies short cDNAs (average size 100-350 bp), representing mainly the 3' untranslated regions (3' UTR) of transcripts. Sequencing of these cDNAs is predominantly uninformative for prediction of function and selection of clones for further analysis. Differential display of longer amplicons (0.5-2.0 kb) could enable isolation of cDNAs that encompass both 3' UTR and at least part of the 3' end of the coding region. The coding sequence information could facilitate selection of candidate clones for further analysis without the necessity of screening cDNA libraries. By combining DD-PCR protocols with long-distance PCR and using hot-start PCR with rTth DNA polymerase we have successfully amplified and comparatively displayed cDNAs ranging in size from 150 bp to 2 kb. Long-distance DD-PCR (LDD-PCR) has generated highly reproducible primer-specific patterns of cDNA fragments, as well as reproducible duplicate fingerprints, obtained from different RNA and cDNA samples. Sequencing and expression analyses of LDD-PCR clones have shown that LDD-PCR (a) enables nonredundant clone sampling, (b) generates many clones that encompass part of the coding region, and (c) samples both abundant and rare transcripts, approximately 60% of which are differentially expressed as confirmed by Northern analysis. Coupled with high-throughput cDNA sequencing and multiplex hybridization of cDNA microarrays for confirmation of differential expression, LDD-PCR could prove to be useful for simultaneous scanning of transcripts from multiple cDNA samples and faster selection of differentially expressed transcripts of interest.
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PMID:Identification and cloning of differentially expressed genes by long-distance differential display. 961 2

The variability of the 5' untranslated genomic region (5'UTR) of bovine viral diarrhoea virus (BVDV) RNA obtained from a single individual was analysed. Lung, kidney and spleen tissues from a naturally infected foetus were used as the source of viral RNA. A fragment of 288 bases of the internal ribosome entry site from the BVDV 5'UTR was amplified by RT-PCR using a proofreading DNA polymerase. PCR products were cloned into pGem and, subsequently, transformed into Escherichia coli. The single-strand conformational polymorphisms of 158 lung-derived clones were analysed; a total of 11 banding patterns was observed. DNAs corresponding to all patterns were sequenced. Of the randomly selected clones, 11 and 10 clones derived from the kidney and spleen, respectively, were also sequenced. All sequences presented differences ranging from 1 to 6 nt substitutions. Analysis of the secondary structure of the variant sequences and comparisons to variant nucleotide sites from the 5'UTR of several BVDV isolates showed that the observed changes were almost free of randomness. Clustering and phylogenetic analyses suggested the existence of low-kinetic variants. BVDV quasispecies may be involved in establishing persistent infections by means of eluding maternal antibodies. The methods described here may be adapted easily both to analyse large numbers of samples from other genomic regions and for the study of BVDV quasispecies evolution in other systems.
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PMID:Quasispecies in the 5' untranslated genomic region of bovine viral diarrhoea virus from a single individual. 1218 69

The majority of expressed sequence tag (EST) sequences available today have been derived from the 5' ends of cDNA clones. Obtaining high-quality DNA sequences from the 3' ends of oligo(dT)-primed cDNA on a large scale has been difficult because of slippage of the DNA polymerase enzyme used in direct PCR and cycle sequencing. With the completion of whole genome sequencing for more and more organisms, mRNA 3'-UTR sequences can be particularly useful for clustering large numbers of ESTs for the effective discrimination of individual genes and gene families. We have identified a flaw in the widely used oligo(dT) primers for cDNA synthesis, and here we describe an improved priming approach to effectively synthesize cDNA devoid of homopolymeric nucleotide stretches from mRNA poly(A) tails to enable highly efficient and reliable DNA sequence determination from 3' mRNA ends. Using this method, we produced a rat lung cDNA library and successfully sequenced the 3' ends of 98% of all attempted clones.
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PMID:Construction of cDNA libraries for highly efficient DNA sequencing from the 3' end of expressed genes. 1270 1

Aberrant expression of DNA polymerase beta, a key enzyme involved in base excision repair, leads to genetic instability and carcinogenesis. Pol beta expression has been previously shown to be regulated at the level of transcription, but there is also evidence of post-transcriptional regulation, since rat transcripts undergo alternative polyadenylation, and the resulting 3'UTR contain at least one regulatory element. Data presented here indicate that RNA of the short 3'UTR folds to form a strong secondary structure (hairpin). Its regulatory role was established utilizing a luciferase-based reporter system. Further studies led to the identification of a protein factor, which binds to this element-the anti-apoptotic, cytoskeleton-related protein Hax-1. The results of in vitro binding analysis indicate that the formation of the RNA-protein complex is significantly impaired by disruption of the hairpin motif. We demonstrate that Hax-1 binds to Pol beta mRNA exclusively in the form of a dimer. Biochemical analysis revealed the presence of Hax-1 in mitochondria, but also in the nuclear matrix, which, along with its transcript-binding properties, suggests that Hax-1 plays a role in post-transcriptional regulation of expression of Pol beta.
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PMID:Hairpin structure within the 3'UTR of DNA polymerase beta mRNA acts as a post-transcriptional regulatory element and interacts with Hax-1. 1770 38

MicroRNAs (miRNAs) have been implicated in sequence-specific cleavage, translational repression or deadenylation of specific target mRNAs resulting in post-transcriptional gene silencing. Epstein-Barr virus (EBV) encodes 23 miRNAs of unknown function. Here we show that the EBV-encoded miRNA miR-BART2 down-regulates the viral DNA polymerase BALF5. MiR-BART2 guides cleavage within the 3'-untranslated region (3'UTR) of BALF5 by virtue of its complete complementarity to its target. Induction of the lytic viral replication cycle results in a reduction of the level of miR-BART2 with a strong concomitant decrease of cleavage of the BALF5 3'UTR. Expression of miR-BART2 down-regulates the activity of a luciferase reporter gene containing the BALF5 3'UTR. Forced expression of miR-BART2 during lytic replication resulted in a 40-50% reduction of the level of BALF5 protein and a 20% reduction of the amount of virus released from EBV-infected cells. Our results are compatible with the notion that EBV-miR-BART2 inhibits transition from latent to lytic viral replication.
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PMID:Epstein-Barr virus-encoded microRNA miR-BART2 down-regulates the viral DNA polymerase BALF5. 1807 97

Small nucleolar RNAs (snoRNAs) are localized within the nucleolus, a sub-nuclear compartment, in which they guide ribosomal or spliceosomal RNA modifications, respectively. Up until now, snoRNAs have only been identified in eukaryal and archaeal genomes, but are notably absent in bacteria. By screening B lymphocytes for expression of non-coding RNAs (ncRNAs) induced by the Epstein-Barr virus (EBV), we here report, for the first time, the identification of a snoRNA gene within a viral genome, designated as v-snoRNA1. This genetic element displays all hallmark sequence motifs of a canonical C/D box snoRNA, namely C/C'- as well as D/D'-boxes. The nucleolar localization of v-snoRNA1 was verified by in situ hybridisation of EBV-infected cells. We also confirmed binding of the three canonical snoRNA proteins, fibrillarin, Nop56 and Nop58, to v-snoRNA1. The C-box motif of v-snoRNA1 was shown to be crucial for the stability of the viral snoRNA; its selective deletion in the viral genome led to a complete down-regulation of v-snoRNA1 expression levels within EBV-infected B cells. We further provide evidence that v-snoRNA1 might serve as a miRNA-like precursor, which is processed into 24 nt sized RNA species, designated as v-snoRNA1(24pp). A potential target site of v-snoRNA1(24pp) was identified within the 3'-UTR of BALF5 mRNA which encodes the viral DNA polymerase. V-snoRNA1 was found to be expressed in all investigated EBV-positive cell lines, including lymphoblastoid cell lines (LCL). Interestingly, induction of the lytic cycle markedly up-regulated expression levels of v-snoRNA1 up to 30-fold. By a computational approach, we identified a v-snoRNA1 homolog in the rhesus lymphocryptovirus genome. This evolutionary conservation suggests an important role of v-snoRNA1 during gamma-herpesvirus infection.
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PMID:Expression and processing of a small nucleolar RNA from the Epstein-Barr virus genome. 1968 May 35

The addition of a poly(A)-tail to the 3' termini of RNA molecules influences stability, nuclear export, and efficiency of translation. In the cytoplasm, dynamic changes in the length of the poly(A)-tail have long been recognized as reflective of the switch between translational silence and activation. Thus, measurement of the poly(A)-tail associated with any given mRNA at steady-state can serve as a surrogate readout of its translation-state. Here, we describe a simple new method to 3'-tag adenylated RNA in total RNA samples using the intrinsic property of Escherichia coli DNA polymerase I to extend an RNA primer using a DNA template. This tag can serve as an anchor for cDNA synthesis and subsequent gene-specific PCR to assess poly(A)-tail length. We call this method extension Poly(A) Test (ePAT). The ePAT approach is as efficient as traditional Ligation-Mediated Poly(A) Test (LM-PAT) assays, avoids problems of internal priming associated with oligo-dT-based methods, and allows for the accurate analysis of both the poly(A)-tail length and alternate 3' UTR usage in 3' RACE applications.
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PMID:ePAT: a simple method to tag adenylated RNA to measure poly(A)-tail length and other 3' RACE applications. 2254 66

The human fragile X mental retardation 1 (FMR1) gene contains a (CGG)(n) trinucleotide repeat in its 5' untranslated region (5'UTR). Expansions of this repeat result in a number of clinical disorders with distinct molecular pathologies, including fragile X syndrome (FXS; full mutation range, greater than 200 CGG repeats) and fragile X-associated tremor/ataxia syndrome (FXTAS; premutation range, 55-200 repeats). Study of these diseases has been limited by an inability to sequence expanded CGG repeats, particularly in the full mutation range, with existing DNA sequencing technologies. Single-molecule, real-time (SMRT) sequencing provides an approach to sequencing that is fundamentally different from other "next-generation" sequencing platforms, and is well suited for long, repetitive DNA sequences. We report the first sequence data for expanded CGG-repeat FMR1 alleles in the full mutation range that reveal the confounding effects of CGG-repeat tracts on both cloning and PCR. A unique feature of SMRT sequencing is its ability to yield real-time information on the rates of nucleoside addition by the tethered DNA polymerase; for the CGG-repeat alleles, we find a strand-specific effect of CGG-repeat DNA on the interpulse distance. This kinetic signature reveals a novel aspect of the repeat element; namely, that the particular G bias within the CGG/CCG-repeat element influences polymerase activity in a manner that extends beyond simple nearest-neighbor effects. These observations provide a baseline for future kinetic studies of repeat elements, as well as for studies of epigenetic and other chemical modifications thereof.
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PMID:Sequencing the unsequenceable: expanded CGG-repeat alleles of the fragile X gene. 2306 52


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