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:2.7.7.7 (DNA polymerase)
17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A mini-Tn10 insertion in the polA cistron (polA2099) was isolated in a search for mutations that affect patterned Mudlac replication in colonies. The polA2099 mutation had a dramatic effect on cell morphogenesis during the first few hours of microcolony development. Abnormal microcolonies containing filamentous cells were produced as a result of SOS induction. Despite gross abnormalities in early microcolonies, mature polA2099 colonies after 2 to 4 days were morphologically indistinguishable from Pol+ colonies, and 44-h polA2099 colonies displayed a cell size distribution very similar to that of Pol+ colonies. These results suggested the involvement of a protective factor produced during colony growth that compensated for the polA deficiency. The action of a diffusible substance that stimulates growth of polA2099 microcolonies was shown by spotting dilute polA2099 cultures next to established colonies. Differential transcription of polA during colony development was visualized by growing colonies containing polA-lacZ fusions on beta-galactosidase indicator agar. When polA-lacZ colonies were inoculated next to established colonies, a diffusible factor was seen to inhibit polA transcription during the earliest stages of colony development. These results show that a basic housekeeping function, DNA polymerase I, is subject to multicellular control by the changing conditions which the bacteria create as they proliferate on agar.
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PMID:Differential action and differential expression of DNA polymerase I during Escherichia coli colony development. 133 Oct 25

The gene encoding DNA ligase I, the major DNA ligase activity in proliferating mammalian cells, maps to human chromosome 19q13.2-13.3. We have determined the complete structure of the gene, which is composed of 28 exons spanning 53kb on this chromosome. The first exon is untranslated, and utilises a GC dinucleotide instead of the canonical GT splice donor. The 5' flanking region lacks a TATA box and is highly GC-rich, as is characteristic of a 'housekeeping' gene. In common with the promoters of genes encoding other DNA replication enzymes, such as DNA polymerase alpha, the 5' flanking region of the DNA ligase I gene contains recognition elements for several transcription factors which may mediate increased expression in quiescent cells in response to growth factors.
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PMID:Structure of the human DNA ligase I gene. 150 69

DNA polymerase beta (beta-polymerase) is a housekeeping enzyme involved in DNA repair in vertebrate cells. We used a cDNA probe to study abundance of beta-polymerase mRNA in cultured human cells. The mRNA level in synchronized HeLa cells, representing different stages of the cell-cycle, varied only slightly. Contact inhibited fibroblasts AG-1522 contained the same level of mRNA as growing cells. The steady-state level of mRNA in fibroblasts is equivalent to 6 molecules per cell. The results indicate that the beta-polymerase transcript is "low abundance" and is neither cell-cycle nor growth phase responsive.
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PMID:Characterization of DNA polymerase beta mRNA: cell-cycle and growth response in cultured human cells. 246 Aug 24

DNA polymerase beta (beta-pol) is a housekeeping enzyme considered to be involved in DNA repair in vertebrate cells. We cloned a fragment of genomic DNA spanning the first two exons of the human beta-pol gene and approximately 11 kilobases of the flanking region. The segment just 5' of the transcription start site can direct expression of the bacterial chloramphenicol acetyltransferase (CAT) gene in HeLa cells. A sequence containing only 113 base pairs of flanking DNA has promoter activity, and various constructs containing up to 4.8 kilobases of flanking sequence are expressed at a similar level, indicating that with this assay the important regulatory elements are located within or proximal to the approximately 100-bp core promoter. S1 nuclease mapping was used to show that transcription of the transfected genes is initiated at the same position as the endogenous beta-pol gene. The region upstream of the transcription start site is G + C rich and contains neither CAAT nor TATA boxes, but does have three decanucleotide elements matching high affinity binding sites for the RNA polymerase II transcription factor Sp1. Extending 5' from position -39 and surrounded by Sp1 consensus binding elements, there is a 10-nucleotide sequence with perfect dyad symmetry, GTGACGTCAC. Similar sequences are found in a number of cellular and viral promoters, including several adenovirus promoters. Experiments to test whether the core beta-pol promoter is activated by the adenovirus early region products showed that cotransfection with an adenovirus expression plasmid strongly activates expression of the beta-pol promoter.
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PMID:Human beta-polymerase gene. Structure of the 5'-flanking region and active promoter. 318 28

The in situ-reverse transcription-polymerase chain reaction (IS-RT-PCR) is a method that allows the direct localisation of gene expression. The method utilises the dual buffer mediated activity of the enzyme rTth DNA polymerase enabling both reverse transcription and DNA amplification. Labelled nucleoside triphosphates allow the site of expression to be labelled, rather than the PCR primers themselves, giving a more accurate localisation of transcript expression and decreased background than standard in situ hybridisation (ISH) assays. The MDA-MB-231 human breast carcinoma (HBC) cell line was assayed via the IS-RT-PCR technique, using primers encoding MT-MMP (membrane-type matrix metalloproteinase) and human beta-actin. Our results clearly indicate baseline expression of MT-MMP in the relatively invasive MDA-MB-231 cell line at a signal intensity similar to the housekeeping gene beta-actin, and results following induction with Concanavalin A (Con A) are consistent with our previous results obtained via Northern blotting.
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PMID:IS-RT-PCR assay detection of MT-MMP in a human breast cancer cell line. 882 7

Recent epidemiological and immunohistochemical studies have indicated a possible link between measles virus and inflammatory bowel disease (IBD). The aim of this study was to use a sensitive and robust method for the detection of measles virus RNA in IBD and control clinical samples. Peripheral blood mononuclear cells and intestinal resection tissue from IBD and control patients were studied. Two methods were used to determine the presence of measles virus RNA: hybrid capture, using measles virus-specific oligonucleotides linked to paramagnetic solid-phase supports, was carried out on total cellular RNA to enrich for measles virus RNA sequences. Reverse transcription followed by the polymerase chain reaction (RT-PCR) using rTth DNA polymerase was employed for amplification of measles virus N-gene sequences amongst the enriched species. Total RNA was also used for RT-PCR of a housekeeping mRNA species to assess RNA quality. RT-PCR for another region of the measles genome (the haemagglutinin (H) gene) was also undertaken in order to confirm the results obtained using N-gene primers for analysis of these samples. None of the samples were positive for measles N- or H-gene RNA using RT-PCR. Positive control samples confirmed the sensitivity of the methods employed. These results show that either measles virus RNA was not present in the samples, or was present below the sensitivity limits known to have been achieved.
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PMID:Measles virus RNA is not detected in inflammatory bowel disease using hybrid capture and reverse transcription followed by the polymerase chain reaction. 966 40

We have developed real-time PCR systems to quantitate feline cytokine gene expression. The method is based on the cleavage of fluorescent dye-labelled probes by the 5'-3' exonuclease activity of the Taq DNA polymerase during PCR and measurement of fluorescence intensity by a Sequence Detection System. The feline-specific TaqMan probes were designed to encompass an intron, thus allowing differentiation of complementary DNA versus genomic DNA amplification products. Quantitative analysis of cytokine cDNA concentrations was performed in comparison to feline GAPDH. Messenger RNA (mRNA) from the universally expressed housekeeping gene GAPDH proved to be useful as an amplification control and allowed for correction of variations in the efficiencies of RNA extraction and reverse transcription. GAPDH mRNAs were readily detectable in cDNAs prepared from unstimulated feline peripheral blood mononuclear cells (PBMCs) and from frozen cell pellets, while cytokines (Interleukin (IL)-4, IL-10, IL-12 p35, IL-12 p40, IFNgamma, IL-16) were expressed at variable amounts. IFNgamma transcription was found to be upregulated in stimulated PBMCs and feline cell lines. The synthesis of cDNA and the performance of the PCR in separate tubes proved to be of superior sensitivity compared to a single-tube based system. The assays described are highly reproducible, require no post-PCR manipulation of the amplicons and permit the analysis of several hundred PCR reactions per day. With this method it is possible to detect and quantify cytokine mRNA expression reliably in small amounts of cells even after storage of samples for at least 5 years.
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PMID:Quantitative real-time PCR for the measurement of feline cytokine mRNA. 1058 8

We have sequenced a genomic clone of the gene encoding the mouse mitochondrial DNA polymerase. The gene consists of 23 exons, which span approximately 13.2 kb, with exons ranging in size from 53 to 768 bp. All intron-exon boundaries conform to the GT-AG rule. By comparison with the human genomic sequence, we found remarkable conservation of the gene structure; the intron-exon borders are in almost identical locations for the 22 introns. The 5' upstream region contains approximately 300 bp of homology between the mouse and human sequences that presumably contain the promoter element. This region lacks any obvious TATA domain and is relatively GC rich, consistent with the housekeeping function of the mitochondrial DNA polymerase. Finally, within the 5' flanking region, both mouse and human genes have a region of 73 bp with high homology to the tRNA-Arg gene.
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PMID:Genomic structure of murine mitochondrial DNA polymerase-gamma. 1105 62

Here we present a novel methodology to quantitate bovine cytokines and growth factors contributing to immunity against bacterial infections of the mammary gland in cattle. Real-time TaqMan PCR systems were developed to overcome limitations of conventional quantitative PCR methods. The TaqMan method is based on the cleavage of fluorescent dye-labeled probes by the 5'-3' exonuclease activity of the Taq DNA polymerase during PCR and measurement of fluorescence intensity by an automated spectrophotometer integrated in a sequence detection system (Applied Biosystems, Foster City, CA). The bovine-specific TaqMan probes were designed to encompass an intron, thus allowing differentiation between complementary DNA (cDNA) and genomic DNA (gDNA) amplification products. Quantitative analysis of cytokine cDNA was performed in comparison to bovine glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Messenger RNA (mRNA) from the universally expressed housekeeping gene GAPDH proved to be useful as an amplification control and allowed for correction of variations in different numbers of cells in the starting material, in the efficiencies of RNA extraction and reverse transcription. With this method, high-throughput analysis of large numbers of samples was possible within a short time. In addition, decreasing the numbers of working steps shortened the time for analysis and increased accuracy. Profiles of cytokines (interleukin (IL)-2, IL-6, IL-8, IL-12 p40, TNF-alpha, IFN-gamma) and granulocyte-macrophage colony stimulating factor (GM-CSF) were established in normal lactating cattle. Differences of cytokine profiles obtained with the real-time TaqMan PCR system and conventional methods are discussed.
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PMID:Quantitation of bovine cytokine mRNA in milk cells of healthy cattle by real-time TaqMan polymerase chain reaction. 1113 25

To establish an easy, fast and reliable RT-PCR for the analysis of human cytokine expression, we made use of the recently developed technique of TaqMan PCR. This technique is based on the cleavage of fluorochrome-labelled internal oligodeoxynucleotide probes by the 5'-->3' nuclease activity of Taq DNA polymerase. Measurement of fluorescence intensity during each cycle of the PCR reaction with a Sequence Detection System allows the determination of a threshold cycle at which an increase in fluorescence intensity is first detectable. From these values, a starting amount of template DNA can be calculated. Here, we established specific primers and corresponding internal, fluorogenic probes for the human cytokines tumour necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma), and for the constant region of the T-cell receptor beta chain (TCRbeta) and the housekeeping gene, glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) for normalization of mRNA expression levels. Titrations of the cDNA input showed a strict inverse correlation between the threshold cycles obtained and the starting amount of template. This in turn allowed the generation of a standard curve, and thus quantification of mRNA abundance in cDNA samples. Evaluation of the method using cDNAs from peripheral blood mononuclear cells (PBMC) stimulated with lipopolysaccharide (LPS) or phytohae-magglutinin (PHA) showed basal expression of TNF-alpha and IL-1beta in untreated PBMC while IFN-y was not detectable or only weakly expressed. After stimulation with LPS, a strong induction of IL-1beta and TNF-alpha was measured, while IFN-gamma was induced to a lesser extent. PHA treatment, in contrast, led to an induction of all three cytokines with IFN-gamma being the most prominent. The method has a large dynamic range, requires no post-PCR processing and gives reliable results.
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PMID:Semiquantitative determination of human cytokine mRna expression using TaqMan RT-PCR. 1765 27


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