The exon͞intron architecture of genes determines whether components of the spliceosome recognize splice sites across the intron or across the exon. Using in vitro splicing assays, we demonstrate that splice-site recognition across introns ceases when intron size is between 200 and 250 nucleotides. Beyond this threshold, splice sites are recognized across the exon. Splice-site recognition across the intron is significantly more efficient than splice-site recognition across the exon, resulting in enhanced inclusion of exons with weak splice sites. Thus, intron size can profoundly influence the likelihood that an exon is constitutively or alternatively spliced. An EST-based alternative-splicing database was used to determine whether the exon͞intron architecture influences the probability of alternative splicing in the Drosophila and human genomes. Drosophila exons flanked by long introns display an up to 90-foldhigher probability of being alternatively spliced compared with exons flanked by two short introns, demonstrating that the exon͞ intron architecture in Drosophila is a major determinant in governing the frequency of alternative splicing. Exon skipping is also more likely to occur when exons are flanked by long introns in the human genome. Interestingly, experimental and computational analyses show that the length of the upstream intron is more influential in inducing alternative splicing than is the length of the downstream intron. We conclude that the size and location of the flanking introns control the mechanism of splice-site recognition and influence the frequency and the type of alternative splicing that a pre-mRNA transcript undergoes.alternative splicing ͉ bioinformatics ͉ EST database ͉ intron length P re-mRNA splicing is an essential process that accounts for many aspects of regulated gene expression. Of the Ϸ25,000 genes encoded by the human genome (1), Ͼ60% are believed to produce transcripts that are alternatively spliced. Thus, alternative splicing of pre-mRNAs can lead to the production of multiple protein isoforms from a single pre-mRNA, exponentially enriching the proteomic diversity of higher eukaryotic organisms (2, 3). Because regulation of this process can determine when and where a particular protein isoform is produced, changes in alternative-splicing patterns modulate many cellular activities.The spliceosome assembles onto the pre-mRNA in a coordinated manner by binding to sequences located at the 5Ј and 3Ј ends of introns. Spliceosome assembly is initiated by the stable associations of the U1 small nuclear ribonucleoprotein particle with the 5Ј splice site, branch-point-binding protein͞SF1 with the branch point, and U2 snRNP auxiliary factor with the pyrimidine tract (4). ATP hydrolysis then leads to the stable association of U2 snRNP at the branch-point and functional splice-site pairing (5).Intron size has been correlated with rates of evolution (6) and the regulation of genome size (7,8). The exon͞intron architecture has also been shown to influence splice-site recognition (9-11)....

The work presented here is a first step toward a long term goal of systems biology, the complete elucidation of the gene regulatory networks of a living organism. To this end, we have employed DNA microarray technology to identify genes involved in the regulatory networks that facilitate the transition of Escherichia coli cells from an aerobic to an anaerobic growth state. We also report the identification of a subset of these genes that are regulated by a global regulatory protein for anaerobic metabolism, FNR. Analysis of these data demonstrated that the expression of over one-third of the genes expressed during growth under aerobic conditions are altered when E. coli cells transition to an anaerobic growth state, and that the expression of 712 (49%) of these genes are either directly or indirectly modulated by FNR. The results presented here also suggest interactions between the FNR and the leucine-responsive regulatory protein (Lrp) regulatory networks. Because computational methods to analyze and interpret high dimensional DNA microarray data are still at an early stage, and because basic issues of data analysis are still being sorted out, much of the emphasis of this work is directed toward the development of methods to identify differentially expressed genes with a high level of confidence. In particular, we describe an approach for identifying gene expression patterns (clusters) obtained from multiple perturbation experiments based on a subset of genes that exhibit high probability for differential expression values.The enteric bacterium Escherichia coli, like many commensal and pathogenic microorganisms, thrives in the gastrointestinal tract of humans and other warm-blooded animals. In this environment, oxygen required for respiration and energy generation is in limited supply. Thus, the cell must derive energy from anaerobic respiration with alternative electron acceptors such as nitrate and fumarate or by fermentation of simple sugars. Metabolic transitions between aerobic and anaerobic growth states occur when E. coli cells enter an animal host and colonize the gastrointestinal tract, and when individual cells reposition themselves in new microenvironments inside the host. Each of these transitions is accompanied by fluctuations in oxygen tension. The cell responds to these fluctuations by modulating its central metabolic pathways for carbon and energy flow

The ArcAB two-component system of Escherichia coli regulates the aerobic/anaerobic expression of genes that encode respiratory proteins whose synthesis is coordinated during aerobic/anaerobic cell growth. A genomic study of E. coli was undertaken to identify other potential targets of oxygen and ArcA regulation. A group of 175 genes generated from this study and our previous study on oxygen regulation (Salmon, K., Hung, S. P., Mekjian, K., Baldi, P., Hatfield, G. W., and Gunsalus, R. P. (2003) J. Biol. Chem. 278, 29837-29855), called our gold standard gene set, have p values <0.00013 and a posterior probability of differential expression value of 0.99. These 175 genes clustered into eight expression patterns and represent genes involved in a large number of cell processes, including small molecule biosynthesis, macromolecular synthesis, and aerobic/anaerobic respiration and fermentation. In addition, 119 of these 175 genes were also identified in our previous study of the fnr allele. A MEME/weight matrix method was used to identify a new putative ArcA-binding site for all genes of the E. coli genome. 16 new sites were identified upstream of genes in our gold standard set. The strict statistical analyses that we have performed on our data allow us to predict that 1139 genes in the E. coli genome are regulated either directly or indirectly by the ArcA protein with a 99% confidence level.

Exercise leads to increases in circulating levels of peripheral blood mononuclear cells (PBMCs) and to a simultaneous, seemingly paradoxical increase in both pro- and anti-inflammatory mediators. Whether this is paralleled by changes in gene expression within the circulating population of PBMCs is not fully understood. Fifteen healthy men (18-30 yr old) performed 30 min of constant work rate cycle ergometry (approximately 80% peak O2 uptake). Blood samples were obtained preexercise (Pre), end-exercise (End-Ex), and 60 min into recovery (Recovery), and gene expression was measured using microarray analysis (Affymetrix GeneChips). Significant differential gene expression was defined with a posterior probability of differential expression of 0.99 and a Bayesian P value of 0.005. Significant changes were observed from Pre to End-Ex in 311 genes, from End-Ex to Recovery in 552 genes, and from Pre to Recovery in 293 genes. Pre to End-Ex upregulation of PBMC genes related to stress and inflammation [e.g., heat shock protein 70 (3.70-fold) and dual-specificity phosphatase-1 (4.45-fold)] was followed by a return of these genes to baseline by Recovery. The gene for interleukin-1 receptor antagonist (an anti-inflammatory mediator) increased between End-Ex and Recovery (1.52-fold). Chemokine genes associated with inflammatory diseases [macrophage inflammatory protein-1alpha (1.84-fold) and -1beta (2.88-fold), and regulation-on-activation, normal T cell expressed and secreted (1.34-fold)] were upregulated but returned to baseline by Recovery. Exercise also upregulated growth and repair genes such as epiregulin (3.50-fold), platelet-derived growth factor (1.55-fold), and hypoxia-inducible factor-I (2.40-fold). A single bout of heavy exercise substantially alters PBMC gene expression characterized in many cases by a brisk activation and deactivation of genes associated with stress, inflammation, and tissue repair.

The twin-domain model [Liu, L. F. & Wang, J. C. (1987) Proc. Natl. Acad. Sci. USA 84, 7024 -7027] suggests that closely spaced, divergent, superhelically sensitive promoters can affect the transcriptional activity of one another by transcriptionally induced negative DNA supercoiling generated in the divergent promoter region. This gene arrangement is observed for many LysR-type-regulated operons in bacteria. We have examined the effects of divergent transcription in the prototypic LysR-type system, the ilvYC operon of Escherichia coli. Double-reporter constructs with the lacZ gene under transcriptional control of the ilvC promoter and the galK gene under control of the divergent ilvY promoter were used to demonstrate that a down-promoter mutation in the ilvY promoter severely decreases in vivo transcription from the ilvC promoter. However, a down-promoter mutation in the ilvC promoter only slightly affects transcription from the ilvY promoter. In vitro transcription assays with DNA topoisomers showed that transcription from the ilvC promoter increases over the entire range of physiological superhelical densities, whereas transcription initiation from the ilvY promoter exhibits a broad optimum at a midphysiological superhelical density. Evidence that this promoter coupling is DNA supercoiling-dependent is provided by the observation that a novobiocin-induced decrease in global negative superhelicity results in an increase in ilvY promoter activity and a decrease in ilvC promoter activity predicted by the in vitro data. We suggest that this transcriptional coupling is important for coordinating basal level expression of the ilvYC operon with the nutritional and environmental conditions of cell growth.

SummaryHere, we review briefly the sources of experimental and biological variance that affect the interpretation of high-dimensional DNA microarray experiments. We discuss methods using a regularized t -test based on a Bayesian statistical framework that allow the identification of differentially regulated genes with a higher level of confidence than a simple t -test when only a few experimental replicates are available. We also describe a computational method for calculating the global false-positive and false-negative levels inherent in a DNA microarray data set. This method provides a probability of differential expression for each gene based on experiment-wide false-positive and -negative levels driven by experimental error and biological variance.

We describe a non-invasive approach for recovering RNA from the surface of skin via a simple tape stripping procedure that permits a direct quantitative and qualitative assessment of pathologic and physiologic biomarkers. Using semi-quantitative RT-PCR we show that tape-harvested RNA is comparable in quality and utility to RNA recovered by biopsy. It is likely that tape-harvested RNA is derived from epidermal cells residing close to the surface and includes adnexal structures and present data showing that tape and biopsy likely recover different cell populations. We report the successful amplification of tape-harvested RNA for hybridization to DNA microarrays. These experiments showed no significant gene expression level differences between replicate sites on a subject and minimal differences between a male and female subject. We also compared the array generated RNA profiles between normal and 24 h 1% SLS-occluded skin and observed that SLS treatment resulted in statistically significant changes in the expression levels of more than 1,700 genes. These data establish the utility of tape harvesting as a non-invasive method for capturing RNA from human skin and support the hypothesis that tape harvesting is an efficient method for sampling the epidermis and identifying select differentially regulated epidermal biomarkers.

A collagen-mimetic polymer that can be easily engineered with specific cell-responsive and mechanical properties would be of significant interest for fundamental cell-matrix studies and applications in regenerative medicine. However, oligonucleotide-based synthesis of full-length collagen has been encumbered by the characteristic glycine-X-Y sequence repetition, which promotes mismatched oligonucleotide hybridizations during de novo gene assembly. In this work, we report a novel, modular synthesis strategy that yields full-length human collagen III and specifically defined variants. We used a computational algorithm that applies codon degeneracy to design oligonucleotides that favor correct hybridizations while disrupting incorrect ones for gene synthesis. The resulting recombinant polymers were expressed in Saccharomyces cereVisiae engineered with prolyl-4-hydroxylase. Our modular approach enabled mixing-and-matching domains to fabricate different combinations of collagen variants that contained different secretion signals at the N-terminus and cysteine residues imbedded within the triple-helical domain at precisely defined locations. This work shows the flexibility of our strategy for designing and assembling specifically tailored biomimetic collagen polymers with re-engineered properties.