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)

OprE is a channel-forming outer membrane protein of Pseudomonas aeruginosa, the expression of which is induced under anaerobic conditions. We constructed various mutants and observed the effects on oprE expression. Deficiency in RpoN, an alternative sigma factor for RNA polymerase, abolished oprE expression under aerobic conditions, but did not affect the expression under anaerobic conditions. One mutation on the putative RpoN recognition site also caused reduction of oprE expression. The region 500 nucleotides upstream of the mRNA start site was required for optimal oprE transcription, which contains an AT-rich region including a putative integration host factor binding site. These results indicate that OprE production is directly or indirectly controlled by RpoN but also require some other regulatory proteins bound to the upstream region.
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PMID:Involvement of the RpoN protein in the transcription of the oprE gene in Pseudomonas aeruginosa. 959 61

The outer-membrane protein ShlB of Serratia marcescens activates and secretes hemolytic ShlA into the culture medium. Without ShlB, inactive ShlA (termed ShlA*) remains in the periplasm. Since Proteus mirabilis L-form cells lack an outer membrane and a periplasm, it was of interest to determine in which compartment recombinant ShlA* and ShlB are localized and whether ShlB activates ShlA*. The cloned shlB and shlA genes were transcribed in P. mirabilis stable L-form cells by the temperature-inducible phage T7 RNA polymerase. Radiolabeling, Western blotting, and complementation with C-terminally truncated ShlA (ShlA255) identified inactive ShlA* in the culture supernatant. ShlB remained cell-bound and did not activate ShlA without integration in an outer membrane. Although hemolytic ShlA added to L-form cells had access to the cytoplasmic membrane, it did not affect L-form cells. Synthesis of the large ShlA protein (165 kDa) in P. mirabilis L-form cells under phage T7 promoter control demonstrates that L-form cells are suitable for the synthesis and secretion of large recombinant proteins. This property and the easy isolation of released proteins make L-form cells suitable for the biotechnological production of proteins.
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PMID:The Serratia marcescens hemolysin is secreted but not activated by stable protoplast-type L-forms of Proteus mirabilis. 973 37

Escherichia coli responds to external acidification (pH 4.0 to 5.0) by synthesizing a newly identified, approximately 450-nucleotide RNA component. At maximal levels of induction it is one of the most abundant small RNAs in the cell and is relatively stable bacterial RNA. The acid-inducible RNA was purified, and the gene encoding it, designated asr (for acid shock RNA), mapped at 35.98 min on the E. coli chromosome. Analysis of the asr DNA sequence revealed an open reading frame coding for a 111-amino-acid polypeptide with a deduced molecular mass of approximately 11.6 kDa. According to computer-assisted analysis, the predicted polypeptide contains a typical signal sequence of 30 amino acids and might represent either a periplasmic or an outer membrane protein. The asr gene cloned downstream from a T7 promoter was translated in vivo after transcription using a T7 RNA polymerase transcription system. Expression of a plasmid-encoded asr::lacZ fusion under a native asr promoter was reduced approximately 15-fold in a complex medium, such as Luria-Bertani medium, versus the minimal medium. Transcription of the chromosomal asr was abolished in the presence of a phoB-phoR (a two-component regulatory system, controlling the pho regulon inducible by phosphate starvation) deletion mutant. Acid-mediated induction of the asr gene in the Delta(phoB-phoR) mutant strain was restored by introduction of the plasmid with cloned phoB-phoR genes. Primer extension analysis of the asr transcript revealed a region similar to the Pho box (the consensus sequence found in promoters transcriptionally activated by the PhoB protein) upstream from the determined transcription start. The asr promoter DNA region was demonstrated to bind PhoB protein in vitro. We discuss our results in terms of how bacteria might employ the phoB-phoR regulatory system to sense an external acidity and regulate transcription of the asr gene.
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PMID:The acid-inducible asr gene in Escherichia coli: transcriptional control by the phoBR operon. 1009 85

Endoglin/CD105 is a membrane protein involved in the TGF-beta receptor signalling pathway. Endoglin expression has been reported to be selective for a few cell types, in particular endothelial cells, although a number of conflicting reports have been published. In this study, we performed a detailed analysis of endoglin expression in human lung tumors and different tumor and endothelial cell lines, employing reverse-transcriptase-polymerase-chain reaction as well as immunoblotting and immunohistochemistry using verified antibodies to endoglin. Our data show a clearly preferential expression of both endoglin mRNA and protein in endothelial cells. In tumors, endoglin expression was strongly elevated in the angiogenic endothelium at the tumor edges. In agreement with this observation, we find a clear correlation between endoglin expression and markers of proliferation, such as cyclin A and Ki-67, suggesting that endoglin expression is linked to cell-cycle regulation. These findings not only resolve some of the discrepancies in the literature, but also provide the basis for further applications making use of its selective localization and expression in the tumor vasculature.
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PMID:Elevated expression of endoglin, a component of the TGF-beta-receptor complex, correlates with proliferation of tumor endothelial cells. 1022 46

Western blots of two-dimensional electrophoretic maps of proteins from Chlamydia trachomatis were probed with sera from 17 seropositive patients with genital inflammatory disease. Immunoblot patterns (comprising 28 to 2 spots, average 14.8) were different for each patient; however, antibodies against a spot-cluster due to the chlamydia-specific antigen outer membrane protein-2 (OMP2) were observed in all sera. The next most frequent group of antibodies (15/17; 88%) recognized the hsp60 GroEL-like protein, described as immunopathogenic in chlamydial infections. Reactivity to the major surface-exposed and variable antigen major outer membrane protein (MOMP) was observed at a relatively lower frequency (13/17; 76%). The hsp70 DnaK-like protein was also frequently recognized (11/17; 64.7%) in this patient group. Besides the above confirmatory findings, the study detected several new immunoreactive proteins, with frequencies ranging from 11/17 to 1/17. Some were characterized also by N-terminal amino acid sequencing and homology searches. Amongst these were a novel outer membrane protein (OmpB) and, interestingly, five conserved bacterial proteins: four (23%) sera reacted with the RNA polymerase alpha-subunit, five (29%) recognized the ribosomal protein S1, eight (47%) the protein elongation factor EF-Tu, seven (41%) a putative stress-induced protease of the HtrA family, and seven sera (41%) the ribosomal protein L7/L12. Homologs of the last two proteins were shown to confer protective immunity in other bacterial infections. The data show that immunological sensitization processes commonly thought to play a role in chlamydial pathogenicity may be sustained not only by the hsp60 GroEl-like protein, but also by other conserved bacterial antigens, some of which may be also considered as potential vaccine candidates.
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PMID:Identification of immunoreactive proteins of Chlamydia trachomatis by Western blot analysis of a two-dimensional electrophoresis map with patient sera. 1049 31

The conversion to mucoid phenotype in Pseudomonas aeruginosa during chronic infections in cystic fibrosis (CF) is due to mutations in the algU mucABCD gene cluster. This cluster encodes an extreme stress response system conserved in Gram-negative bacteria. The system includes an ECF sigma factor, AlgU (sigmaE), an inner membrane protein, MucA, which inhibits AlgU activity, and MucB, a periplasmic protein that negatively controls AlgU. In this work, we investigated whether and how these factor interact to transduce signals between different cellular compartments. The mutation mucADeltaG440, which renders a large fraction of P. aeruginosa CF isolates mucoid, did not abrogate AlgU-MucA interactions, although it eliminated MucA-MucB interactions in the yeast two-hybrid system. The mucADeltaG440 truncation of the periplasmic C-terminal tail of MucA destabilized the molecule resulting in low or undetectable steady-state levels in P. aeruginosa. Somewhat reduced levels of MucA were also seen in cells with inactivated mucB or with the mucACF53 allele carrying the missense P184S mutation, which mildly affected interactions with MucB. The events downstream from MucA destabilization were also investigated. AlgU was found to associate with inner membranes in mucA+ cells. In mutants destabilizing MucA, a limited redistribution of AlgU from the membrane to the cytosol was observed. The redistribution was spontaneous in mucADeltaG440 cells, while in mucB and mucACF53 mutants it required additional signals. Despite a large reduction in MucA levels in mucADeltaG440 cells, only a small fraction of AlgU was redistributed to the cytosol and a significant portion of this sigma factor remained membrane bound and behaved as a peripheral inner membrane protein. The fraction of AlgU that depended on MucA for association with the membrane also brought RNA polymerase into this compartment. These results are consistent with a model in which MucB-MucA-AlgU-RNA polymerase interactions at the membrane allow transduction of potentially lethal stress signals with both rapid reaction times of the preassembled complexes and efficient resupply at the membrane from the prebound components.
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PMID:Membrane-to-cytosol redistribution of ECF sigma factor AlgU and conversion to mucoidy in Pseudomonas aeruginosa isolates from cystic fibrosis patients. 1079 19

LALP70 is a novel lysosomal membrane protein belonging to the apyrase protein family. The apyrase protein family comprises enzymes capable of cleaving nucleotide tri- and diphosphates in a calcium- or magnesium-dependent manner, not being altered by P-type, F-type, or V-type NTPase inhibitors. In this study we have cloned and sequenced the human LALP70 gene to determine the genomic structure. The gene is organized in 11 introns and 12 exons covering a genomic region of approximately 16 kilobase pairs. By fluorescence in situ hybridization analysis, the hLALP70 gene was mapped to the human chromosome 8p21.1-p21.3. We further show that there is at least one alternatively spliced variant, hLALP70v, which can be generated via an alternative splice side at the 3'-end of exon 7, leading to a protein variant differing in 8 amino acids (VSFASSQQ). This is the first splice variant that has been described in the apyrase protein family. Reverse transcriptase polymerase chain reaction analysis showed an ubiquitous expression of both variants, with different relative mRNA expression levels in different tissues. Comparison of the enzymatic properties of the splice variants revealed a broader substrate specificity for hLALP70v with CTP, UDP, CDP, GTP, and GDP as preferred substrates, while hLALP70 utilized UTP and TTP preferentially. Furthermore, enzyme activity of hLALP70v was equally dependent on Ca(2+) and Mg(2+), being saturated already at 1 mm concentration. In contrast, hLALP70 enzymatic activity were unsaturated up to 10 mm Ca(2+), while Mg(2+) showed a saturation at already 1 mm concentration with 2-3-fold lower enzymatic activity as observed with Ca(2+). Our data suggest that the presence or absence of the 8-amino acid motif VSFASSQQ provoke differences in substrate specificity and divalent cation dependence of hLALP70/hLALP70v.
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PMID:First apyrase splice variants have different enzymatic properties. 1085 52

A 130-kDa glycoprotein (p130) has been found to be associated with surrogate light chain on pro- and pre-B I cells. Using peptide sequences obtained from purified p130 we have cloned its gene. The gene encodes a typical cadherin type 1 membrane protein with six extracellular cadherin domains (one pseudo domain) but lacking the catenin-binding site in its cytoplasmic part. Even without this catenin-binding site, p130 mediates Ca(2+)-dependent homotypic adhesion of cells. The interaction of p130 with surrogate light chain is confirmed by co-transfection and co-immunoprecipitation experiments. The expression of p130 is biphasic during the B cell development. Reverse transcriptase-polymerase chain reaction and flow cytometric analyses revealed that it is expressed on B220(+)c-Kit(+) pro-B and pre-B-I cells as well as on B220(+)CD25(-)IgM(+) immature and mature B cells but not on B220(+)CD25(+) pre-B-II cells. It is also expressed in fetal liver, at low levels in myeloid cells, and strongly in intestinal epithelial cells. In the spleen, p130-expressing cells are mainly localized in the marginal zone. We call this B lineage-, intestine-, liver- and leukocyte-expressed gene BILL-cadherin. The possible functions of BILL-cadherin in B cell development are discussed.
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PMID:The identification of a nonclassical cadherin expressed during B cell development and its interaction with surrogate light chain. 1090 47

The bone marrow (BM) in myelodysplastic syndromes (MDS) undergoes pathobiological changes that mimic an inflammatory process, and hence, an infectious etiology was suspected in these disorders. In the present report, we examined the bone marrow mononuclear cells (BMMNC) of 19 MDS patients and seven normal donors for the expression of one latency-related (Latency membrane protein 1 (LMP-1) and immediate early protein (IEP)) and one activation-related (BZLF and DNA-Pol) m-RNA each for two herpes viruses, Epstein-Barr virus (EBV) and cytomegalovirus (CMV), respectively. Reverse transcriptase polymerase chain reaction was used for this purpose. The latency-related transcripts (EBV-LMP-1 and CMV-IEP) were present in all the MDS and normal specimens. Intriguingly, 10/19 MDS specimens ( approximately 53%) and 2/7 normal donors ( approximately 28%) were positive for active EBV-BZLF (P=0.0067), while 2/19 MDS specimens ( approximately 11%) with 1/7 normal ( approximately 14%) showed active CMV-DNA-Pol (P=0.1588). Later, from another set of MDS patients (n=7) and normal donors (n=4), BM stromal cultures were established, which, at a 75% confluency, were overlaid with cord blood mononuclear cells (CBMNC). IEP was detectable in the CBMNC before and after co-incubation with MDS, as well as normal stroma. So, it was also present both in MDS and normal stromal cells. The other three were absent both in MDS and normal stromal layers. In CBMNC though, active EBV-BZLF and CMV-DNA-Pol m-RNA were detectable in one of seven MDS co-cultures each, albeit from different patients. None of the normal co-cultures showed active virus, either in stroma or CBMNC. Thus, the present report demonstrates, for the first time, the presence of active herpes viruses in the BMMNC of MDS patients and reveals the ability of the MDS stroma to support the viral activation.
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PMID:Presence of activation-related m-RNA for EBV and CMV in the bone marrow of patients with myelodysplastic syndromes. 1117 35

The overlapping and opposing promoter elements for the Escherichia coli fepDGC operon and the ybdA gene (encoding a 43-kDa cytoplasmic membrane protein) within the enterobactin gene cluster were investigated by measuring the effects of site-specific mutations on transcript levels and on expression of reporter genes in a bidirectional transcriptional fusion vector. Primary promoter structures for the opposing transcripts overlapped extensively such that their -10 sequences were almost directly opposed on the two strands of the DNA helix and their +1 transcription start sites were only 23 bp apart. Relative to the E. coli consensus sequence, both promoters were poorly conserved at the -35 position and mutations which strengthened the -35 element of either promoter significantly enhanced its transcription, decreased that of the opposing promoter, and dramatically altered iron-mediated regulation of expression. Both the fepD and ybdA primary promoters were shown to require a 5'-TGn-3' upstream extension of their -10 elements for optimal activities. Secondary promoters were identified for both fepD and ybdA, and their contributions to the overall expression levels were evaluated in these dual expression vector constructs. The data provided strong evidence that the architecture of the regulatory elements within the overlapping fepD and ybdA promoters is configured such that there is a direct competition for binding RNA polymerase and that the expression levels at these promoters are influenced not only by the activity of the opposing promoters but also by additional promoter sequence elements and perhaps accessory regulatory factors. Iron-mediated regulation of these promoters through the repressor protein Fur is a consequence of the relative promoter strengths and the position of an operator site that consists of two overlapping Fur-binding sequences in this compact regulatory region.
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PMID:Regulatory architecture of the iron-regulated fepD-ybdA bidirectional promoter region in Escherichia coli. 1122 6


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