Selection of AU-rich transiently expressed sequences: reversal of cDNA abundance.
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Study of early and transient response gene expression is important for understanding the mechanisms of response to growth stimuli and exogenous agents such as microbes, stress, and radiation. Many of the cytokines, proto-oncogenes, and other transiently expressed gene products are encoded by mRNAs that contain AU-rich elements (AREs) in their 3' untranslated regions (UTRs). In this article, we describe an approach to selectively synthesize ARE-containing cDNA (ARE-cDNA) using an innovative combination of culture treatment, thermostabilization of reverse transcriptase (RT) by the disaccharide trehalose, and use of optimized ARE-specific oligomers. The monocytic cell line, THP-1, was treated with cycloheximide and endotoxin to enrich for ARE-mediated gene expression followed by the RT procedure. Selection of ARE-cDNA with simultaneous suppression of abundant cDNA was made possible using the procedure as monitored by the preferential expression of IL-8, an ARE-cDNA molecule, over the abundant housekeeping cDNA, beta-actin. The use of trehalose dramatically reversed cDNA abundance, resulting in almost complete suppression of housekeeping cDNA. Finally, construction of specialized ARE-cDNA libraries confirmed the selectivity of ARE-cDNAs and the presence of rare genes. The ability to reverse the abundance of housekeeping and other highly expressed genes toward ARE genes facilitates the discovery and study of rare early response and transiently expressed genes. (+info)
Comprehensive analysis of the base composition around the transcription start site in Metazoa.
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BACKGROUND: The transcription start site of a metazoan gene remains poorly understood, mostly because there is no clear signal present in all genes. Now that several sequenced metazoan genomes have been annotated, we have been able to compare the base composition around the transcription start site for all annotated genes across multiple genomes. RESULTS: The most prominent feature in the base compositions is a significant local variation in G+C content over a large region around the transcription start site. The change is present in all animal phyla but the extent of variation is different between distinct classes of vertebrates, and the shape of the variation is completely different between vertebrates and arthropods. Furthermore, the height of the variation correlates with CpG frequencies in vertebrates but not in invertebrates and it also correlates with gene expression, especially in mammals. We also detect GC and AT skews in all clades (where %G is not equal to %C or %A is not equal to %T respectively) but these occur in a more confined region around the transcription start site and in the coding region. CONCLUSIONS: The dramatic changes in nucleotide composition in humans are a consequence of CpG nucleotide frequencies and of gene expression, the changes in Fugu could point to primordial CpG islands, and the changes in the fly are of a totally different kind and unrelated to dinucleotide frequencies. (+info)
Bacterial genomes as new gene homes: the genealogy of ORFans in E. coli.
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Differences in gene repertoire among bacterial genomes are usually ascribed to gene loss or to lateral gene transfer from unrelated cellular organisms. However, most bacteria contain large numbers of ORFans, that is, annotated genes that are restricted to a particular genome and that possess no known homologs. The uniqueness of ORFans within a genome has precluded the use of a comparative approach to examine their function and evolution. However, by identifying sequences unique to monophyletic groups at increasing phylogenetic depths, we can make direct comparisons of the characteristics of ORFans of different ages in the Escherichia coli genome, and establish their functional status and evolutionary rates. Relative to the genes ancestral to gamma-Proteobacteria and to those genes distributed sporadically in other prokaryotic species, ORFans in the E. coli lineage are short, A+T rich, and evolve quickly. Moreover, most encode functional proteins. Based on these features, ORFans are not attributable to errors in gene annotation, limitations of current databases, or to failure of methods for detecting homology. Rather, ORFans in the genomes of free-living microorganisms apparently derive from bacteriophage and occasionally become established by assuming roles in key cellular functions. (+info)
Transcriptional Repressor CopR: use of SELEX to study the copR operator indicates that evolution was directed at maximal binding affinity.
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CopR is one of the two copy number control elements of the streptococcal plasmid pIP501. It represses transcription of the repR mRNA encoding the essential replication initiator protein about 10- to 20-fold by binding to its operator region upstream of the repR promoter pII. CopR binds at two consecutive sites in the major groove of the DNA that share the consensus motif 5'-CGTG. Previously, the minimal operator was narrowed down to 17 bp, and equilibrium dissociation constants for DNA binding and dimerization were determined to be 0.4 nM and 1.4 microM, respectively. In this work, we used a SELEX procedure to study copR operator sequences of different lengths in combination with electrophoretic mobility shift assays of mutated copR operators as well as copy number determinations to assess the sequence requirements for CopR binding. The results suggest that in vivo evolution was directed at maximal binding affinity. Three simultaneous nucleotide exchanges outside the bases directly contacted by CopR only slightly affected CopR binding in vitro or copy numbers in vivo. Furthermore, the optimal spacer sequence was found to comprise 7 bp, to be AT rich, and to need an A/T and a T at the 3' positions, whereas broad variations in the sequences flanking the minimal 17-bp operator were well tolerated. (+info)
Sequence-dependence of the energetics of opening of at basepairs in DNA.
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Proton exchange and nuclear magnetic resonance spectroscopy are being used to characterize the energetics of opening of AT/TA basepairs in the DNA dodecamer 5'-d(GCTATAAAAGGG)-3'/5'-d(CCCTTTTATAGC)-3'. The dodecamer contains the TATA box of the adenovirus major late promoter. The equilibrium constants for opening of each basepair are measured from the dependence of the exchange rates of imino protons on ammonia concentration. The enthalpy, entropy, and free energy changes in the opening reaction of each basepair are determined from the temperature dependence of the exchange rates. The results reveal that the opening enthalpy changes encompass a wide range of values, namely, from 17 to 29 kcal/mol. The largest values are observed for the AT basepairs in 7th and 8th positions. These values, and the exchange rates of the corresponding imino protons, suggest that these two basepairs open in a single concerted reaction. The enthalpy changes for opening of the central six basepairs are correlated to the opening entropy changes. This enthalpy-entropy compensation minimizes the variations in the opening free energies among these central basepairs. Deviations from the enthalpy-entropy compensation pattern are observed for basepairs located close to the ends of the duplex structure, suggesting a different mode of opening for these basepairs. (+info)
Genomic specification and epigenetic regulation of eukaryotic DNA replication origins.
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Identification of DNA replication origins (ORIs) at a genome-wide level in eukaryotes has proved to be difficult due to the high degree of degeneracy of their sequences. Recent structural and functional approaches, however, have circumvented this limitation and have provided reliable predictions of their genomic distribution in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, and they have also significantly increased the number of characterized ORIs in animals. This article reviews recent evidence on how ORIs are specified and maintained in these systems and on their regulation and sensitivity to epigenetic signals. It also discusses the possible additional involvement of ORIs in processes other than DNA replication. (+info)
Fragile X-related protein FXR1P regulates proinflammatory cytokine tumor necrosis factor expression at the post-transcriptional level.
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Tumor necrosis factor (TNF) is regulated post-transcriptionally by the AU-rich element (ARE) within the 3'-untranslated region of its mRNA. This regulation modulates translational efficacy and mRNA stability. By using a cRNA probe containing the TNF ARE sequence, we screened a macrophage protein expression library and identified FXR1P. Macrophages that we generated from FXR1 knock-out mice had enhanced TNF protein production compared with wild type macrophages following activation. Expression of several other proteins that are regulated by ARE sequences was also affected by FXR1P deficiency. A GFP-ARE reporter that has green fluorescent protein (GFP) expression under control of the 3'-untranslated region of TNF mRNA had enhanced expression in transfected macrophages deficient in FXR1P. Finally, we found that the ablation of FXR1P led to a dramatically enhanced association of the TNF mRNA with polyribosomes demonstrating the important role of FXR1P in the post-transcriptional regulation of TNF expression. Our data suggest that release of this repression by FXR1P occurs during lipopolysaccharide-induced macrophage activation. Finally, complementation of the knock-out macrophages with recombinant FXR1P resulted in decreased TNF protein production, supporting our findings that FXR1P operates as a repressor of TNF translation. (+info)
Homopolymer tract length dependent enrichments in functional regions of 27 eukaryotes and their novel dependence on the organism DNA (G+C)% composition.
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BACKGROUND: DNA homopolymer tracts, poly(dA).poly(dT) and poly(dG).poly(dC), are the simplest of simple sequence repeats. Homopolymer tracts have been systematically examined in the coding, intron and flanking regions of a limited number of eukaryotes. As the number of DNA sequences publicly available increases, the representation (over and under) of homopolymer tracts of different lengths in these regions of different genomes can be compared. RESULTS: We carried out a survey of the extent of homopolymer tract over-representation (enrichment) and over-proportional length distribution (above expected length) primarily in the single gene documents, but including some whole chromosomes of 27 eukaryotics across the (G+C)% composition range from 20 - 60%. A total of 5.2 x 10(7) bases from 15,560 cleaned (redundancy removed) sequence documents were analyzed. Calculated frequencies of non-overlapping long homopolymer tracts were found over-represented in non-coding sequences of eukaryotes. Long poly(dA).poly(dT) tracts demonstrated an exponential increase with tract length compared to predicted frequencies. A novel negative slope was observed for all eukaryotes between their (G+C)% composition and the threshold length N where poly(dA).poly(dT) tracts exhibited over-representation and a corresponding positive slope was observed for poly(dG).poly(dC) tracts. Tract size thresholds where over-representation of tracts in different eukaryotes began to occur was between 4 - 11 bp depending upon the organism (G+C)% composition. The higher the GC%, the lower the threshold N value was for poly(dA).poly(dT) tracts, meaning that the over-representation happens at relatively lower tract length in more GC-rich surrounding sequence. We also observed a novel relationship between the highest over-representations, as well as lengths of homopolymer tracts in excess of their random occurrence expected maximum lengths. CONCLUSIONS: We discuss how our novel tract over-representation observations can be accounted for by a few models. A likely model for poly(dA).poly(dT) tract over-representation involves the known insertion into genomes of DNA synthesized from retroviral mRNAs containing 3' polyA tails. A proposed model that can account for a number of our observed results, concerns the origin of the isochore nature of eukaryotic genomes via a non-equilibrium GC% dependent mutation rate mechanism. Our data also suggest that tract lengthening via slip strand replication is not governed by a simple thermodynamic loop energy model. (+info)