Complement component C8gamma is expressed in human fetal and adult kidney independent of C8alpha.
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Human complement component C8gamma is an unusual complement factor since it shows no homology to other complement proteins but is a member of the lipocalin superfamily. So far, it has been found exclusively in plasma, covalently linked to C8alpha by disulfide bridging. We have used dot blot and Northern blot analyses of a large number of different human tissues to survey systematically the expression pattern of C8gamma. Our experiments clearly showed that besides in liver, this gene is also expressed in fetal and adult kidney. Renal expression of C8gamma is not dependent on C8alpha expression, since we could not detect C8alpha expression in kidney. Thus its physiological function is not restricted to a specific action in association with complement components. As a prerequisite for further characterization of the structure and binding activities of the uncomplexed C8gamma, we have expressed the encoding cDNA in Escherichia coli. To increase the probability for proper folding of the characteristic intramolecular disulfide bridge the recombinant protein was produced by secretion to the periplasm. (+info)
Expression and characterization of a novel structural protein of human cytomegalovirus, pUL25.
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Human cytomegalovirus (HCMV) UL25 has recently been found to encode a new structural protein that is present in both virion and defective viral particles (C. J. Baldick and T. Shenk, J. Virol. 70:6097-6105, 1996). In the present work a polyclonal antibody was raised against a prokaryotic pUL25 fusion protein in order to investigate the biosynthesis and localization of the UL25 product (pUL25) during HCMV replication in human fibroblasts. Furthermore, pUL25 was transiently expressed in its native form and fused to the FLAG epitope, in COS7 and U373MG cells, in order to compare the properties of the isolated protein and that produced during infection. Immunoblotting analysis revealed a group of polypeptides, ranging from 80 to 100 kDa, in both transfected and infected cells; in vivo labeling experiments with infected cells demonstrated they are posttranslationally modified by phosphorylation. The transcriptional analysis of the UL25 open reading frame combined with the study of pUL25 biosynthesis showed true late kinetics for this protein in infected human fibroblasts. By indirect immunofluorescence both recombinant and viral pUL25 were detected exclusively in the cytoplasm of transfected or infected cells. Interestingly, pUL25 was shown to localize in typical condensed structures in the perinuclear region as already observed for other HCMV tegument proteins. Colocalization of ppUL99 in the same vacuoles suggests that these structure are endosomal cisternae, which are proposed to be a preferential site of viral particle envelopment. Our data suggest that pUL25 is most likely a novel tegument protein and possibly plays a key role in the process of envelopment. (+info)
Expression of prokaryotic 1-deoxy-D-xylulose-5-phosphatases in Escherichia coli increases carotenoid and ubiquinone biosynthesis.
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Isopentenyl diphosphate (IPP) acts as the common, five-carbon building block in the biosynthesis of all isoprenoids. The first reaction of IPP biosynthesis in Escherichia coli is the formation of 1-deoxy-D-xylulose-5-phosphate, catalysed by 1-deoxy-D-xylulose-5-phosphate synthase (DXPS). E. coli engineered to produce lycopene, was transformed with dxps genes cloned from Bacillus subtilis and Synechocystis sp. 6803. Increases in lycopene levels were observed in strains expressing exogenous DXPS compared to controls. The recombinant strains also exhibited elevated levels of ubiquinone-8. These increases corresponded with enhanced DXP synthase activity in the recombinant E. coli strains. (+info)
The Escherichia coli homologue of yeast RER2, a key enzyme of dolichol synthesis, is essential for carrier lipid formation in bacterial cell wall synthesis.
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We found in the Escherichia coli genome sequence a homologue of RER2, a Saccharomyces cerevisiae gene required for proper localization of an endoplasmic reticulum protein, and designated it rth (RER2 homologue). The disruption of this gene was lethal for E. coli. To reveal its biological function, we isolated temperature-sensitive mutants of the rth gene. The mutant cells became swollen and burst at the nonpermissive temperature, indicating that their cell wall integrity was defective. Further analysis showed that the mutant cells were deficient in the activity of cis-prenyltransferase, namely, undecaprenyl diphosphate synthase, a key enzyme of the carrier lipid formation of peptidoglycan synthesis. The cellular level of undecaprenyl phosphate was in fact markedly decreased in the mutants. These results are consistent with the fact that the Rer2 homologue of Micrococcus luteus shows undecaprenyl diphosphate synthase activity (N. Shimizu, T. Koyama, and K. Ogura, J. Biol. Chem. 273:19476-19481, 1998) and demonstrate that E. coli Rth is indeed responsible for the maintenance of cell wall rigidity. Our work on the yeast rer2 mutants shows that they are defective in the activity of cis-prenyltransferase, namely, dehydrodolichyl diphosphate synthase, a key enzyme of dolichol synthesis. Taking these data together, we conclude that the RER2 gene family encodes cis-prenyltransferase, which plays an essential role in cell wall biosynthesis in bacteria and in dolichol synthesis in eukaryotic cells and has been well conserved during evolution. (+info)
How does replication-associated mutational pressure influence amino acid composition of proteins?
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We have performed detrended DNA walks on whole prokaryotic genomes, on noncoding sequences and, separately, on each position in codons of coding sequences. Our method enables us to distinguish between the mutational pressure associated with replication and the mutational pressure associated with transcription and other mechanisms that introduce asymmetry into prokaryotic chromosomes. In many prokaryotic genomes, each component of mutational pressure affects coding sequences not only in silent positions but also in positions in which changes cause amino acid substitutions in coded proteins. Asymmetry in the silent positions of codons differentiates the rate of translation of mRNA produced from leading and lagging strands. Asymmetry in the amino acid composition of proteins resulting from replication-associated mutational pressure also corresponds to leading and lagging roles of DNA strands, whereas asymmetry connected with transcription and coding function corresponds to the distance of genes from the origin or terminus of chromosome replication. (+info)
Two distinct SECIS structures capable of directing selenocysteine incorporation in eukaryotes.
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Translation of UGA as selenocysteine requires specific RNA secondary structures in the mRNAs of selenoproteins. These elements differ in sequence, structure, and location in the mRNA, that is, coding versus 3' untranslated region, in prokaryotes, eukaryotes, and archaea. Analyses of eukaryotic selenocysteine insertion sequence (SECIS) elements via computer folding programs, mutagenesis studies, and chemical and enzymatic probing has led to the derivation of a predicted consensus structural model for these elements. This model consists of a stem-loop or hairpin, with conserved nucleotides in the loop and in a non-Watson-Crick motif at the base of the stem. However, the sequences of a number of SECIS elements predict that they would diverge from the consensus structure in the loop region. Using site-directed mutagenesis to introduce mutations predicted to either disrupt or restore structure, or to manipulate loop size or stem length, we show that eukaryotic SECIS elements fall into two distinct classes, termed forms 1 and 2. Form 2 elements have additional secondary structures not present in form 1 elements. By either insertion or deletion of the sequences and structures distinguishing the two classes of elements while maintaining appropriate loop size, conversion of a form 1 element to a functional form 2-like element and of a form 2 to a functional form 1-like element was achieved. These results suggest commonality of function of the two classes. The information obtained regarding the existence of two classes of SECIS elements and the tolerances for manipulations of stem length and loop size should facilitate designing RNA molecules for obtaining high-resolution structural information about these elements. (+info)
DNA bending and wrapping around RNA polymerase: a "revolutionary" model describing transcriptional mechanisms.
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A model is proposed in which bending and wrapping of DNA around RNA polymerase causes untwisting of the DNA helix at the RNA polymerase catalytic center to stimulate strand separation prior to initiation. During elongation, DNA bending through the RNA polymerase active site is proposed to lower the energetic barrier to the advance of the transcription bubble. Recent experiments with mammalian RNA polymerase II along with accumulating evidence from studies of Escherichia coli RNA polymerase indicate the importance of DNA bending and wrapping in transcriptional mechanisms. The DNA-wrapping model describes specific roles for general RNA polymerase II transcription factors (TATA-binding protein [TBP], TFIIB, TFIIF, TFIIE, and TFIIH), provides a plausible explanation for preinitiation complex isomerization, suggests mechanisms underlying the synergy between transcriptional activators, and suggests an unforseen role for TBP-associating factors in transcription. (+info)
PAS domains: internal sensors of oxygen, redox potential, and light.
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PAS domains are newly recognized signaling domains that are widely distributed in proteins from members of the Archaea and Bacteria and from fungi, plants, insects, and vertebrates. They function as input modules in proteins that sense oxygen, redox potential, light, and some other stimuli. Specificity in sensing arises, in part, from different cofactors that may be associated with the PAS fold. Transduction of redox signals may be a common mechanistic theme in many different PAS domains. PAS proteins are always located intracellularly but may monitor the external as well as the internal environment. One way in which prokaryotic PAS proteins sense the environment is by detecting changes in the electron transport system. This serves as an early warning system for any reduction in cellular energy levels. Human PAS proteins include hypoxia-inducible factors and voltage-sensitive ion channels; other PAS proteins are integral components of circadian clocks. Although PAS domains were only recently identified, the signaling functions with which they are associated have long been recognized as fundamental properties of living cells. (+info)