Southern-by-Sequencing: A Robust Screening Approach for Molecular Characterization of Genetically Modified Crops.

Molecular characterization of events is an integral part of the advancement process during genetically modified (GM) crop product development. Assessment of these events is traditionally accomplished by polymerase chain reaction (PCR) and Southern blot analyses. Southern blot analysis can be time-consuming and comparatively expensive and does not provide sequence-level detail. We have developed a sequence-based application, Southern-by-Sequencing (SbS), utilizing sequence capture coupled with next-generation sequencing (NGS) technology to replace Southern blot analysis for event selection in a high-throughput molecular characterization environment. SbS is accomplished by hybridizing indexed and pooled whole-genome DNA libraries from GM plants to biotinylated probes designed to target the sequence of transformation plasmids used to generate events within the pool. This sequence capture process enriches the sequence data obtained for targeted regions of interest (transformation plasmid DNA). Taking advantage of the DNA adjacent to the targeted bases (referred to as next-to-target sequence) that accompanies the targeted transformation plasmid sequence, the data analysis detects plasmid-to-genome and plasmid-to-plasmid junctions introduced during insertion into the plant genome. Analysis of these junction sequences provides sequence-level information as to the following: the number of insertion loci including detection of unlinked, independently segregating, small DNA fragments; copy number; rearrangements, truncations, or deletions of the intended insertion DNA; and the presence of transformation plasmid backbone sequences. This molecular evidence from SbS analysis is used to characterize and select GM plants meeting optimal molecular characterization criteria. SbS technology has proven to be a robust event screening tool for use in a high-throughput molecular characterization environment.

Maize global transcriptomics reveals pervasive leaf diurnal rhythms but rhythms in developing ears are largely limited to the core oscillator.

BACKGROUND: Plant diurnal rhythms are vital environmental adaptations to coordinate internal physiological responses to alternating day-night cycles. A comprehensive view of diurnal biology has been lacking for maize (Zea mays), a major world crop. METHODOLOGY: A photosynthetic tissue, the leaf, and a non-photosynthetic tissue, the developing ear, were sampled under natural field conditions. Genome-wide transcript profiling was conducted on a high-density 105 K Agilent microarray to investigate diurnal rhythms. CONCLUSIONS: In both leaves and ears, the core oscillators were intact and diurnally cycling. Maize core oscillator genes are found to be largely conserved with their Arabidopsis counterparts. Diurnal gene regulation occurs in leaves, with some 23% of expressed transcripts exhibiting a diurnal cycling pattern. These transcripts can be assigned to over 1700 gene ontology functional terms, underscoring the pervasive impact of diurnal rhythms on plant biology. Considering the peak expression time for each diurnally regulated gene, and its corresponding functional assignment, most gene functions display temporal enrichment in the day, often with distinct patterns, such as dawn or midday preferred, indicating that there is a staged procession of biological events undulating with the diurnal cycle. Notably, many gene functions display a bimodal enrichment flanking the midday photosynthetic maximum, with an initial peak in mid-morning followed by another peak during the afternoon/evening. In contrast to leaves, in developing ears as few as 47 gene transcripts are diurnally regulated, and this set of transcripts includes primarily the core oscillators. In developing ears, which are largely shielded from light, the core oscillator therefore is intact with little outward effect on transcription.

Target lesion revascularization after bare-metal or drug-eluting stents: clinical presentations and outcomes.

OBJECTIVE: We sought to examine the clinical presentations and subsequent clinical outcomes of patients undergoing target lesion revascularization (TLR) after either bare-metal stent (BMS) or drug-eluting stent (DES) placement. BACKGROUND: The widely held notion that BMS TLR is benign has recently been challenged. While DES substantially reduce TLR, little is known about the clinical syndromes accompanying DES TLR and the long-term clinical outcomes after TLR. METHODS: The clinical syndrome at the time of hospitalization when TLR was performed and subsequent clinical outcomes after TLR were assessed in 1,147 BMS patients and 1,246 DES patients who were followed for 3 years. Patients were considered to have TLR when repeat target lesion PCI was required including those with myocardial infarction (MI) and stent thrombosis. RESULTS: At 3 years, the overall incidence of TLR was higher after BMS compared to DES 98/1,147 (9.2%) vs. 56/1,246 (4.5%); p < 0.001. The clinical presentations at the time of TLR were not always benign with non-STelevation myocardial infarction (N-STEMI) or STEMI in 25% of BMS vs. 34% DES; p = 0.217. The risk of non-fatal MI or death outcomes over 3 years were significantly worse in those with TLR compared to those without TLR; hazard ratio (HR) 2.65 (2.00-3.52), independent of stent type. CONCLUSIONS: The clinical presentation at the time of TLR is not always a benign clinical event and identifies a subgroup of stent-treated patients at high risk for non-fatal MI or death in the 3 years following the index percutaneous coronary intervention, independent of stent type.

Harmonics of circadian gene transcription in mammals.

The circadian clock is a molecular and cellular oscillator found in most mammalian tissues that regulates rhythmic physiology and behavior. Numerous investigations have addressed the contribution of circadian rhythmicity to cellular, organ, and organismal physiology. We recently developed a method to look at transcriptional oscillations with unprecedented precision and accuracy using high-density time sampling. Here, we report a comparison of oscillating transcription from mouse liver, NIH3T3, and U2OS cells. Several surprising observations resulted from this study, including a 100-fold difference in the number of cycling transcripts in autonomous cellular models of the oscillator versus tissues harvested from intact mice. Strikingly, we found two clusters of genes that cycle at the second and third harmonic of circadian rhythmicity in liver, but not cultured cells. Validation experiments show that 12-hour oscillatory transcripts occur in several other peripheral tissues as well including heart, kidney, and lungs. These harmonics are lost ex vivo, as well as under restricted feeding conditions. Taken in sum, these studies illustrate the importance of time sampling with respect to multiple testing, suggest caution in use of autonomous cellular models to study clock output, and demonstrate the existence of harmonics of circadian gene expression in the mouse.

Expression quantitative trait loci mapping identifies new genetic models of glutathione S-transferase variation.

Expression quantitative trait loci (eQTL) mapping can be used to identify the genetic variations that underlie inherited differences in gene transcription. We performed eQTL mapping by combining whole genome transcriptional data from the hypothalami of 33 strains of inbred mice with a detailed haplotype map of those same strains, revealing 10,655 trans associations and 31 cis eQTLs. One of the cis associations was found to be driven by strain-specific variation in the expression of Glutathione S-transferase, mu 5 (Gstm5). Gstm5 is one of seven members of the glutathione S-transferase, Mu family of genes. The glutathione S-transferases are phase II metabolic enzymes and are key regulators of drug and toxin clearance. In mouse, all seven family members are tightly clustered on mouse chromosome 3. Investigation of the Gstm5 cis association in multiple tissues types revealed that an 84-kilobase region on MMU3 acts as a haplotype-specific locus control region for the glutathione S-transferase, Mu cluster. In the strains that share the minor haplotype, drastic reductions in mRNA levels in multiple members of the Gst Mu family were observed. The strain-specific differences in Gst Mu transcription characterized here accurately model the human population, in which extreme variations in expression of GST Mu family members have been observed. Furthermore, the reduction in Gst Mu levels has important relevance for pharmacology and toxicology studies conducted in these strains. For instance, the reduced levels of Gst Mu in general and Gstm5 in particular have implications in models of dopamine metabolism, Parkinson's disease, and chemical neurotoxicity.

Hepatic transcriptional networks induced by exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin.

The environmental contaminant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) serves as a prototype for a range of environmental toxicants and as a pharmacologic probe to study signal transduction by the aryl hydrocarbon receptor (AHR). Despite a detailed understanding of how TCDD exposure leads to the transcriptional up-regulation of cytochrome P450-dependent monooxygenases, we know little about how compounds like TCDD lead to a variety of AHR-dependent toxic end points such as liver pathology, terata, thymic involution, and cancer. Using an acute exposure protocol and the toxic response of the mouse liver as a model system, we have begun a detailed microarray analysis to describe the transcriptional changes that occur after various TCDD doses and treatment times. Through correlation analysis of time- and dose-dependent toxicological end points, we are able to identify coordinately responsive transcriptional events that can be defined as primary transcriptional events and downstream events that may represent mechanistically linked sequelae or that have potential as biomarkers of toxicity.

Circadian and CLOCK-controlled regulation of the mouse transcriptome and cell proliferation.

Circadian rhythms of cell and organismal physiology are controlled by an autoregulatory transcription-translation feedback loop that regulates the expression of rhythmic genes in a tissue-specific manner. Recent studies have suggested that components of the circadian pacemaker, such as the Clock and Per2 gene products, regulate a wide variety of processes, including obesity, sensitization to cocaine, cancer susceptibility, and morbidity to chemotherapeutic agents. To identify a more complete cohort of genes that are transcriptionally regulated by CLOCK and/or circadian rhythms, we used a DNA array interrogating the mouse protein-encoding transcriptome to measure gene expression in liver and skeletal muscle from WT and Clock mutant mice. In WT tissue, we found that a large percentage of expressed genes were transcription factors that were rhythmic in either muscle or liver, but not in both, suggesting that tissue-specific output of the pacemaker is regulated in part by a transcriptional cascade. In comparing tissues from WT and Clock mutant mice, we found that the Clock mutation affects the expression of many genes that are rhythmic in WT tissue, but also profoundly affects many nonrhythmic genes. In both liver and skeletal muscle, a significant number of CLOCK-regulated genes were associated with the cell cycle and cell proliferation. To determine whether the observed patterns in cell-cycle gene expression in Clock mutants resulted in functional dysregulation, we compared proliferation rates of fibroblasts derived from WT or Clock mutant embryos and found that the Clock mutation significantly inhibits cell growth and proliferation.

Comparative genomics as a tool in the understanding of eukaryotic transcriptional regulation.

Comparative genomics approaches are having a remarkable impact on the study of transcriptional regulation in eukaryotes. Many eukaryotic genome sequences are being explored by new computational methods and high-throughput experimental tools such as DNA arrays and genome-wide location analyses. These tools are enabling efficient panning for common regulatory cassettes underlying fundamental biological processes, extending the use of existing techniques for the discovery of response elements to mammals, deciphering the transcriptional regulatory code in eukaryotes and providing the first global insights into a recently described post-transcriptional regulatory mechanism. Collectively, these approaches are rapidly expanding both our knowledge and our definition of transcriptional regulation.

Circadian clocks are seeing the systems biology light.

Circadian rhythms are those biological rhythms that have a periodicity of around 24 hours. Recently, the generation of a circadian transcriptional network -- compiled from RNA-expression and promoter-element analysis and phase information -- has led to a better understanding of the gene-expression patterns that regulate the precise 24-hour clock.

EDGE: a centralized resource for the comparison, analysis, and distribution of toxicogenomic information.

Transcriptional profiling via microarrays holds great promise for toxicant classification and hazard prediction. Unfortunately, the use of different microarray platforms, protocols, and informatics often hinders the meaningful comparison of transcriptional profiling data across laboratories. One solution to this problem is to provide a low-cost and centralized resource that enables researchers to share toxicogenomic data that has been generated on a common platform. In an effort to create such a resource, we developed a standardized set of microarray reagents and reproducible protocols to simplify the analysis of liver gene expression in the mouse model. This resource, referred to as EDGE, was then used to generate a training set of 117 publicly accessible transcriptional profiles that can be accessed at http://edge.oncology.wisc.edu/. The Web-accessible database was also linked to an informatics suite that allows on-line clustering and K-means analyses as well as Boolean and sequence-based searches of the data. We propose that EDGE can serve as a prototype resource for the sharing of toxicogenomics information and be used to develop algorithms for efficient chemical classification and hazard prediction.

Application of genomics to toxicology research.

Traditional models of toxicity have relied on dissecting chemical action into pharmacokinetic and pharmacodynamic processes. However, the integration of genomic information with toxicology will enhance our basic understanding of these processes and significantly change the way we apply toxicological information to risk assessment and regulatory problems. In this article, we summarize the application of gene expression information and polymorphism discovery to four areas in toxicology: toxicity testing, cross-species extrapolation, understanding mechanism of action, and susceptibility.