Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Sets of RNA ladders can be synthesized by transcription of a bacteriophage-encoded RNA polymerase using 3'-deoxynucleotides as chain terminators. These ladders can be used for sequencing of DNA. Using a nicked form of phage SP6 RNA polymerase in this study substantially enhanced yields of transcriptional sequencing ladders. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) of chain-terminated RNA ladders allowed DNA sequence determination of up to 56 nt. It is also demonstrated that A-->G and C-->T variations in heterozygous and homozygous samples can be unambiguously identified by the mass spectrometric analysis. As a step towards single-tube sequencing reactions, alpha-thiotriphosphate nucleotide analogs were used to overcome problems caused by chain terminator-independent, premature termination and by the small mass difference between natural pyrimidine nucleotides.
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PMID:DNA sequencing and genotyping by transcriptional synthesis of chain-terminated RNA ladders and MALDI-TOF mass spectrometry. 1116 Sep 13

It was known previously that overwintering larvae of the beetle Dendroides canadensis produce antifreeze proteins (DAFPs) consisting of a family of 12 similar proteins, and based on sequence variations the DAFPs may be separated into three groups. DAFPs were known to be present in hemolymph, midgut fluid and in/on epidermal cells located immediately under the cuticle. However, only DAFPs-1, 2, and 4 were known to be present in the hemolymph, leaving the location of the others unknown. In this study, matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry of hemolymph confirmed the presence of DAFPs-1, 2, and 4 (Group I), plus a protein consistent with the mass of DAFP-6 (Group I). Also, a review of older data revealed the co-purification of DAFP-6 along with DAFP-4 in hemolymph. However, none of the other DAFPs (Groups II and III) were present in hemolymph. In contrast, mass spectrometry of midgut fluid demonstrated the absence of DAFPs-1, 2, 4, or 6, however, proteins consistent with the masses of all, or a subset of, Groups II and/or III were present. Reverse transcriptase polymerase chain reaction (RT-PCR) showed that transcripts of all 12 DAFPs were present in the fat body. However, consistent with the MALDI-TOF data, only Groups II (8, 9, 10, 11) and III (3, 5, 7, 12) transcripts were found in midgut epithelia. RT-PCR of epidermal tissue identified dafps- 4, 6, 8 and 11 (and sometimes 1 and/or 2) as the major transcripts. These data suggest that various DAFPs may have evolved to function best in certain sites.
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PMID:Site-specific forms of antifreeze protein in the beetle Dendroides canadensis. 1219 17

Here we devise a new method for high-throughput comparative sequence analysis. The developed protocol comprises a homogeneous in vitro transcription/RNase cleavage system with the accuracy and data acquisition speed of matrix-assisted laser desorption/ionization coupled with time-of-flight mass spectrometry (MALDI-TOF MS). In summary, the target region is PCR amplified using primers tagged with promoter sequences of T7 or SP6 RNA polymerase. Using RNase T1, the in vitro transcripts are base-specifically cleaved at every G-position. This reaction results in a characteristic pattern of fragment masses that is indicative of the original target sequence. To enable high-throughput analysis, samples are processed with automated liquid handling devices and nanoliter amounts are dispensed onto SpectroCHIP arrays for reliable and homogeneous MALDI preparation. This system enables rapid automated comparative sequence analysis for PCR products up to 1 kb in length. We demonstrate the feasibility of the devised method for analysis of single nucleotide polymorphisms (SNPs) and pathogen identification.
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PMID:RNase T1 mediated base-specific cleavage and MALDI-TOF MS for high-throughput comparative sequence analysis. 1271 92

Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) after base-specific cleavage of PCR amplified and in vitro-transcribed 16S rRNA gene (rDNA) was used for the identification of mycobacteria. Full-length 16S rDNA reference sequences of 12 type strains of Mycobacterium spp. frequently isolated from clinical specimens were determined by PCR, cloning, and sequencing. For MALDI-TOF MS-based comparative sequence analysis, mycobacterial 16S rDNA signature sequences ( approximately 500 bp) of the 12 type strains and 24 clinical isolates were PCR amplified using RNA promoter-tagged forward primers. T7 RNA polymerase-mediated transcription of forward strands in the presence of 5-methyl ribo-CTP maximized mass differences of fragments generated by base-specific cleavage. In vitro transcripts were subsequently treated with RNase T1, resulting in G-specific cleavage. Sample analysis by MALDI-TOF MS showed a specific mass signal pattern for each of the 12 type strains, allowing unambiguous identification. All 24 clinical isolates were identified unequivocally by comparing their detected mass signal pattern to the reference sequence-derived in silico pattern of the type strains and to the in silico mass patterns of published 16S rDNA sequences. A 16S rDNA microheterogeneity of the Mycobacterium xenopi type strain (DSM 43995) was detected by MALDI-TOF MS and later confirmed by Sanger dideoxy sequencing. In conclusion, analysis of 16S rDNA amplicons by MS after base-specific cleavage of RNA transcripts allowed fast and reliable identification of the Mycobacterium tuberculosis complex and ubiquitous mycobacteria (mycobacteria other than tuberculosis). The technology delivers an open platform for high-throughput microbial identification on the basis of any specific genotypic marker region.
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PMID:Novel mass spectrometry-based tool for genotypic identification of mycobacteria. 1471 74

The general human RNA polymerase III transcription factor (TF) IIIC1 has hitherto been ill defined with respect to the polypeptides required for reconstitution of its activity. Here we identify Homo sapiens TFIIIB" (HsBdp1) as an essential component of hTFIIIC1 and hTFIIIC1-like activities. Several forms of HsBdp1 are described. The 250-kDa form of HsBdp1, also designated the "transcription factor-like nuclear regulator," strictly co-eluted with TFIIIC1 activity over multiple chromatographic purification steps as revealed by Western blot with anti-HsBdp1 antibodies and by MALDI-TOF analysis. In addition, TFIIIC1 activity could be depleted from partially purified fractions with anti-HsBdp1 antibodies but not with control antibodies. Moreover, highly purified recombinant HsBdp1 could replace TFIIIC1 activity in reconstituted transcription of the VAI gene in vitro. Furthermore, smaller proteins of approximately 90-150 kDa that were recognized by anti-HsBdp1 antibodies co-eluted with TFIIIC1-like activity. Finally, cytoplasmic extracts from differentiated mouse F9 fibroblast cells that lacked TFIIIC1 activity could be made competent for transcription of the VA1 gene by the addition of TFIIIC1, TFIIIC1-like, or recombinant HsBdp1. These results suggest that HsBdp1 proteins represent essential components of TFIIIC1 and TFIIIC1-like activities.
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PMID:Transcription factor (TF)-like nuclear regulator, the 250-kDa form of Homo sapiens TFIIIB", is an essential component of human TFIIIC1 activity. 1509 1

We present evidence that rat and mouse thymi contain mitochondrial uncoupling protein (UCP 1). Reverse transcriptase-PCR detected RNA transcripts for UCP 1 in whole thymus and in thymocytes. Furthermore, using antibodies to UCP 1 the protein was also detected in mitochondria isolated from whole thymus and thymocytes but not in thymus mitochondria from UCP 1 knock-out mice. Evidence for functional UCP 1 in thymus mitochondria was obtained by a comparative analysis with the kinetics of GDP binding in mitochondria from brown adipose tissue. Both tissues showed equivalent B(max) and K(D) values. In addition, a large component of the nonphosphorylating oxygen consumption by thymus mitochondria was inhibited by GDP and subsequently stimulated by addition of nanomolar concentrations of palmitate. UCP 1 was purified from thymus mitochondria by hydroxyapatite chromatography. The isolated protein was identified by peptide mass mapping and tandem mass spectrometry by using MALDI-TOF and LC-MS/MS, respectively. We conclude that the thymus contains a functioning UCP 1 that has the capacity to regulate metabolic flux and production of reactive oxygen-containing molecules in the thymus.
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PMID:Identification of a functioning mitochondrial uncoupling protein 1 in thymus. 1569 16

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease, and some of its forms are progressive. This study describes the profiling of hepatic gene expression and serum protein content in patients with different subtypes of NAFLD. Liver biopsy specimens from 98 bariatric surgery patients were classified as normal, steatosis alone, steatosis with nonspecific inflammation, and nonalcoholic steatohepatitis (NASH). Microarray hybridizations were performed in triplicate and the microarray expression levels of a selected group of genes were confirmed using real-time quantitative reverse-transcriptase polymerase chain reaction. Serum protein profiles of the same patients were determined by SELDI-TOF mass spectrometry. Of 98 obese patients, 91 were diagnosed with NAFLD (12 steatosis alone, 52 steatosis with nonspecific inflammation, and 27 NASH), and 7 patients without NAFLD served as obese controls. Each group of NAFLD patients was compared with the obese controls, and 22 genes with more than twofold differences in expression levels were revealed. Proteomics analyses were performed for the same group comparisons and revealed twelve significantly different protein peaks. In conclusion, this genomic/proteomic analysis suggests differential expression of several genes and protein peaks in patients within and across the forms of NAFLD. These findings may help clarify the pathogenesis of NAFLD and identify potential targets for therapeutic intervention.
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PMID:A genomic and proteomic study of the spectrum of nonalcoholic fatty liver disease. 1611 32

We report the chemical synthesis of 5'-amino- and 5'-thiol-hexaethylene glycol guanosine nucleotides and their enzymatic incorporation into RNA, followed by chemical modifications at their nucleophilic ends. By using two similar routes, the conjugates of guanosine-5'-monophosphate and hexaethylene glycol with attached reactive groups (SH or NH(2)) were synthesized using phosphoramidite chemistry, and characterized by MALDI TOF mass spectrometry. These initiator molecules were efficiently incorporated into RNA at the 5'-end by run-off transcription using T7 RNA polymerase. The potential of these RNA conjugates for a broad reaction range with electrophiles is shown here, thereby enabling their use for diverse biochemical applications.
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PMID:Universal initiator nucleotides for the enzymatic synthesis of 5'-amino- and 5'-thiol-modified RNA. 1663 8

Two-dimensional gel electrophoresis (2DE) and MALDI-TOF MS were used to obtain a global view of the cytoplasmic proteins expressed by Thermoplasma acidophilum. In addition, glycerol gradient ultracentrifugation coupled to 2DE-MALDI-TOF MS analysis was used to identify subunits of macromolecular complexes. With the 2DE proteomics approach, over 900 spots were resolved of which 271 proteins were identified. A significant number of these form macromolecular complexes, among them the ribosome, proteasome, and thermosome, which are expressed at high levels. In the glycerol gradient heavy fractions, 10 as yet uncharacterized proteins (besides the well known ribosomal subunits, translation initiation factor eIF-6-related protein, elongation factor 1, and DNA-dependent RNA polymerase) were identified that are putative building blocks of protein complexes. These proteins belong to the categories of hypothetical or conserved hypothetical proteins, and they are present in the cytosol at low concentrations. Although these proteins exhibit homology to known sequences, their structures, subunit compositions, and biological functions are not yet known.
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PMID:Proteomics analysis of Thermoplasma acidophilum with a focus on protein complexes. 1715 Oct 18

Using T-type maize cytoplasmic male sterile line (T-CMS) and maintainer line as experimental materials, we separated mitochondrial proteins from leaves at seedling, shooting, booting stages, mesocotyl, root and anther at meiosis of pollen mother cell, single-double nucleus stage of pollen grain by two-dimensional electrophoresis with immobilized pH3-10 gradients. About 150 mitochondrial protein spots in seedling leaves, 150 spots in mesocotyls, 150 spots in roots and 100 spots in meiosis anther were observed respectively in this investigation. 6 difference protein spots were identified by MALDI-TOF-MS analysis and NCBI database searching. r40c1 protein was present in mesocotyl of T-CMS and absent in maintainer line. Mature anther-specific protein, DNA-directed RNA polymerase 23kDa subunit, hexokinase II were present and glutathione S-transferase, putative polyprotein were absent in pollen aborted anther of T-CMS. Developmental changes in mitochondrial proteins were found in leaves but no differences were observed in T-CMS and its maintainer line. Obvious differences of mitochondrial proteins were found at single-double nucleus stage anther in T-CMS and maintainer line. These different proteins were considered to be associated to pollen aborted in T-CMS.
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PMID:[Different proteins in mitochondrial proteome of T-type maize cytoplasmic male-sterile line and its maintainer line]. 1819 83


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