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:2.7.7.6 (RNA polymerase)
34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The synthesis of ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBPCase) in Rhodospirillum rubrum was regulated by the CO2 concentration in the culture medium. The specific activity of RuBPCase in cells grown photolithotrophically in low concentrations of CO2 (1.5%) was five to ten times higher than that in cultures grown at high concentrations of CO2 (10%). Increased enzyme activity was reflected by an increase in both RuBPCase mRNA and RuBPCase protein. RuBPCase expression was also studied in vitro with a plasmid-borne genomic clone (pRR117) as the template in a partially defined Escherichia coli system containing either E. coli or R. rubrum RNA polymerase. With both enzymes there was excellent synthesis of RuBPCase mRNA, but no significant synthesis of RuBPCase was detected. The promoter region of the RuBPCase gene was sequenced, and mRNA start sites were mapped. A single major in vivo transcriptional start site was detected in RuBPCase mRNA extracted from R. rubrum. However, transcripts synthesized from pRR117 in vitro or from E. coli transformed with pRR117 started at upstream sites that were different from the in vivo transcription site. Two major features of the RuBPCase promoter region are three 6-base-pair direct repeats and a 31-base-pair region of dyad symmetry.
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PMID:Regulation of ribulose bisphosphate carboxylase expression in Rhodospirillum rubrum: characteristics of mRNA synthesized in vivo and in vitro. 284 1

Pyruvate decarboxylase (EC 4.1.1.1) from Zymomonas mobilis purified to homogeneity by using dye-ligand and ion-exchange chromatography. Antibodies produced against the enzyme and the amino-terminal sequence obtained for the pure enzyme were used to select and confirm the identity of a genomic clone encoding the enzyme selected from a genomic library of Z. mobilis DNA cloned into pUC9. The genomic fragment encoding the enzyme expressed high levels of pyruvate decarboxylase in Escherichia coli. Possible RNA polymerase and ribosome-binding sites have been identified in the 5'-untranslated region of the pyruvate decarboxylase gene.
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PMID:Pyruvate decarboxylase of Zymomonas mobilis: isolation, properties, and genetic expression in Escherichia coli. 354 63

Of the several hundred 7SL RNA-like sequences that are dispersed in human DNA, no more than four are likely to represent genes for 7SL RNA; the majority are 7SL pseudogenes which appear to result from the reverse flow of genetic information from 7SL RNA back into genomic DNA. We present the sequence of five 7SL pseudogenes displaying an unprecedented diversity of structures. All are truncated copies of 7SL RNA, but the site of truncation can occur at either the 5' end, the 3' end or at both ends of the RNA sequence. We suggest that such diverse 7SL pseudogenes are generated by different but related pathways. In particular, we argue that two of the loci are secondary 7SL pseudogenes which derive from RNA polymerase III transcripts of primary (preexisting) 7SL pseudogenes. We also report the isolation and characterisation of a human genomic clone carrying two linked 7SL RNA coding regions, 7L30.1 and 7L30.2. The 7L30.2 locus differs by several single base changes from the known human 7SL RNA sequences and does not appear to be expressed at a detectable level in HeLa cells. The 7L30.1 locus is an authentic 7SL RNA gene encoding one of the three sequence variants of human 7SL RNA.
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PMID:Human genes and pseudogenes for the 7SL RNA component of signal recognition particle. 608 97

Anti-Ro autoantibodies precipitate several small cytoplasmic ribonucleoproteins from mammalian cells. The RNA components of these particles, designated hY1-hY5 in human cells and mY1 and mY2 in mouse cells, are about 100 nucleotides long. We have analyzed a genomic clone that appears to contain true RNA-coding regions for two of the human Ro RNAs, hY1 and hY3. These RNAs exhibit many sequence and secondary structure homologies, both with each other and with the recently sequenced hY5 RNA. The hY2 RNA is a slightly truncated form of hY1; several shorter versions of hY3 are also detected in cell extracts and immunoprecipitates. The human hY1 and hY3 genes cross-hybridize with the mouse Ro RNAs, mY1 and mY2, respectively; we show that the mouse Ro RNAs are exclusively contained in Ro particles. The genes for hY1 and hY3 are transcribed in vitro by RNA polymerase III. In contrast with all other mammalian class III genes described, they appear to be present as single copies in the human genome.
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PMID:Genes for two small cytoplasmic Ro RNAs are adjacent and appear to be single-copy in the human genome. 618 71

The lima bean lectin recognizes terminal alpha-D-GalNAc groups and agglutinates human type A erythrocytes. We have cloned a portion of the gene encoding the alpha subunit of the lima bean lectin. The clone was obtained using the polymerase chain reaction and verified from a genomic clone encoding the mature protein of 253 amino acids. The deduced amino acid sequence has significant overall homology with other leguminous plant lectins and contains all of the known peptide sequences isolated from lima bean lectin (LBL). Southern blot analysis reveals the presence of several genes which hybridize to the cloned gene and which we propose are genes included in the lima bean lectin gene family. We report here the sequence, expression, and characterization of LBL 2, the second member of this gene family. Milligram quantities of soluble active recombinant lima bean lectin (rLBL) were obtained from Escherichia coli, using the T7 RNA polymerase expression system. SDS-polyacrylamide gel electrophoresis and Western blot analysis indicate expression of one protein band of about 27 kDa in induced E. coli cells. This protein cross-reacts with polyclonal antibodies raised against seed lectin (sLBL) and gave a reaction of identity with seed lectin by Ouchterlony double diffusion, specifically agglutinates type A blood cells, and is specifically inhibited by D-GalNAc. The isoelectric point of rLBL is 5.86, whereas those of the seed lectin subunits were determined to be 5.86, 5.58, and 5.20 (previously designated alpha, beta, alpha', respectively). rLBL binds to hydrophobic ligands independent of sugar binding, an observation similar to results obtained with sLBL. However, despite the similar activities described, several significant differences between recombinant and native lima bean lectin were found, including mobility on gel filtration, aggregation in solution, and its CD spectrum. These differences may be due to a number of factors, which will be discussed.
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PMID:The sequence of a second member of the lima bean lectin gene family and the expression and characterization of recombinant lectin in Escherichia coli. 812 94

Examination of cDNAs for the laminin-binding alpha 7 integrin subunit identified two different sequences (designated X1 and X2) coding for the variable region between the III and IV homology repeat domains near the putative ligand-binding site. Sequencing of a mouse alpha 7 genomic clone established that the X1 and X2 regions are derived by mutually exclusive alternative mRNA splicing. Reverse transcriptase-polymerase chain reaction analysis of alpha 7 mRNA indicated that the X1 and X2 isoforms were present in equal amounts in mouse skeletal myoblasts and adult heart. However, in adult skeletal muscle, the X2 variant was exclusively expressed. Amino acid sequence homologies in the III/IV segment suggest that alpha 3 and alpha 6 are also alternatively spliced at this site. We identified alternatively spliced exons in a human alpha 6 genomic clone that encode X1- and X2-like segments. Analysis of the alpha 7 cytoplasmic domain indicated that this region was also alternatively spliced and like alpha 3 and alpha 6 could exist as the A or B form. In mouse skeletal and cardiac muscle the B form of alpha 7 was strongly expressed. However, we identified alpha 7A in neonate and adult skeletal muscle but not in cardiac tissue. High levels of alpha 7A were detected in differentiating myotubes, but in proliferating myoblasts only the alpha 7B isoform was present. These results indicate that alternative splicing of alpha 7 mRNA is differentially regulated during development and generates variant integrin chains with structurally and presumably functionally unique ligand-binding and cytoplasmic domains.
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PMID:Alternative extracellular and cytoplasmic domains of the integrin alpha 7 subunit are differentially expressed during development. 825 14

We report the cloning of two full-length cDNAs coding for the human beta 1-integrin which diverge from each other for their 5'-untranslated sequences. Characterization of a genomic clone containing these two sequences showed that they are contiguous, spaced by 261 nucleotides, and both followed by donor splice sites. Analysis by primer extension and transient transfection in a human osteogenic sarcoma cell line (MG-63) demonstrated the existence of two independent promoters for transcription initiation. The two promoter regions are very G+C-rich, and lack both a TATA box and a CAAT box. Northern blot analysis showed that transcripts starting from the distal promoter (with respect to the first coding exon) are at least 20-fold more abundant than transcripts originating from the proximal one. The levels of both transcripts increase after transforming growth factor-beta 1 induction, however, mRNAs originating from the proximal promoter increase at an higher extent. Reverse transcriptase/polymerase chain reaction analysis performed on different human tissues and cell lines revealed that, while the distal promoter is ubiquitously active, the proximal promoter is not. These findings suggest a possible complex pattern for regulation of the human beta 1-integrin gene expression.
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PMID:Human beta 1-integrin gene expression is regulated by two promoter regions. 844 90

A genomic clone of the small, round-structured virus Southampton virus (SV) was constructed from a set of overlapping PCR amplicons. Sequence analysis confirmed the absence of mutations and accurate ligation of the PCR products. The SV cDNA was cloned into a vector for in vitro production of RNA and subsequent translation by rabbit reticulocyte lysate. Two polypeptides corresponding to the N-terminal and C-terminal regions of the viral polyprotein were expressed in Escherichia coli and used to produce murine antisera for detection of translation products. Three major translation products of 113, 48, and 41 kDa were identified in a coupled transcription-translation system. The large 113-kDa protein reacted with antisera raised against the C-terminal region of the polyprotein and represents a precursor of the viral RNA polymerase. The 48-kDa protein detected in vitro reacted specifically with antisera raised against the polyprotein N terminus, showing that translation was initiated in SV at the three tandem in-frame AUG codons at the 5' end of the genome. A series of nested 3' deletions of the large open reading frame encoding the viral polyprotein was used to define the translation initiation site and genomic location of the viral protease. The results are consistent with a model in which translation of the viral genome is initiated at one of the three in-frame AUG codons starting at nucleotide position 5 and in which active viral protease is produced following translation of a region located between NheI (nucleotide 3052) and SphI (nucleotide 4056), resulting in rapid cleavage of a large precursor protein. Abolition of the viral 3C-like protease activity by site-directed mutagenesis of the putative active-site cysteine (Cys-1238) resulted in production of a large protein of approximately 200 kDa which reacted with both N-terminal and C-terminal antisera. Two potential polyprotein cleavage sites containing the preferred picornaviral QG recognition site were identified on either side of the putative 2C-like helicase region of the polyprotein. Proteolysis at these positions would give rise to products with relative molecular masses identical to those of the products detected in the rabbit reticulocyte system. Site-directed mutagenesis was used to introduce a single base change which resulted in the substitution of glutamine residues with proline residues at amino acids 399 and 762. These mutations completely abolished cleavage of the polyprotein at these positions and gave rise to alternative products with molecular masses which matched the predicted sizes for a single cleavage at either Q-399 or Q-762. These data indicate that the small, round-structured virus Southampton virus produces a 3C-like protease which has two primary cleavage sites at positions 399 and 762. Proteolytic cleavage at these positions releases the putative viral 2C-like helicase.
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PMID:Polyprotein processing in Southampton virus: identification of 3C-like protease cleavage sites by in vitro mutagenesis. 864 93

A VH gene segment that could not be assigned to any of the six known VH gene families of the channel catfish was identified in a genomic clone containing VH gene segments. This gene segment (designated VH7.1) exhibited the structural features characteristic of vertebrate VH genes, specifically potential upstream regulatory sequences, a leader sequence split by an intron, a reading frame that could be readily divided into framework and complementarity determining regions, and a 3' recombination signal sequence. Two regions of nucleotide deletions coupled with degeneracy in the nonamer sequence indicate that this VH gene segment is a pseudogene. Genomic DNA restricted with different enzymes and hybridized under stringent conditions with probes derived from VH7.1 showed that 8-10 bands were present in Southern blots. Reverse transcriptase PCR approaches were used to determine if any of these related sequences were expressed. Sequence analysis of cloned PCR products indicates that different VH gene segments exhibiting > 80% similarity to germline VH7.1 are expressed. Multiple sequence alignments showed that the expressed cVH7a cDNA sequence shared less than 60% nucleotide similarity with representative cDNA sequences from the other known catfish VH gene families. These combined results thus fulfil the criteria for the definition of a new family of catfish VH gene segments. This newly defined, small VH family is designated VH7.
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PMID:Characterization of a seventh family of immunoglobulin heavy chain VH gene segments in the channel catfish, Ictalurus punctatus. 883 18

A bovine genomic clone containing a 17.4-kb DNA fragment was isolated and found to contain a solitary arginine tRNA gene with an anticodon of CCG that has a 100% identity to its cognate tRNA. This arginine tRNA gene, symbolized as TRR4, has a characteristic internal split promoter and a typical termination site for RNA polymerase III. The tRNA gene was transcribed in vitro by RNA polymerase III using a HeLa cell-free extract to yield a mature-sized tRNA product. The gene was mapped to bovine chromosome 19 using a panel of bovine-rodent somatic cell hybrid DNAs.
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PMID:Structural analysis of a bovine arginine tRNA(CCG) gene. 919 43


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