Lauren M. McIntyre

Genomic scale profiling of nutrient and trace elements in Arabidopsis thaliana

Understanding the functional connections between genes, proteins, metabolites and mineral ions is one of biologys greatest challenges in the postgenomic era. We describe here the use of mineral nutrient and trace element profiling as a tool to determine the biological significance of connections between a plants genome and its elemental profile. Using inductively coupled plasma spectroscopy, we quantified 18 elements, including essential macro- and micronutrients and various nonessential elements, in shoots of 6,000 mutagenized M2 Arabidopsis thaliana plants. We isolated 51 mutants with altered elemental profiles. One mutant contains a deletion in FRD3, a gene known to control iron-deficiency responses in A. thaliana. Based on the frequency of elemental profile mutations, we estimate 2–4% of the A. thaliana genome is involved in regulating the plants nutrient and trace element content. These results demonstrate the utility of elemental profiling as a useful functional genomics tool.

Combining mapping and arraying: An approach to candidate gene identification

A combination of quantitative trait locus (QTL) mapping and microarray analysis was developed and used to identify 34 candidate genes for ovariole number, a quantitative trait, in Drosophila melanogaster. Ovariole number is related to evolutionary fitness, which has been extensively studied, but for which few a priori candidate genes exist. A set of recombinant inbred lines were assayed for ovariole number, and QTL analyses for this trait identified 5,286 positional candidate loci. Forty deletions spanning the QTL were employed to further refine the map position of genes contributing to variation in this trait between parental lines, with six deficiencies showing significant effects and reducing the number of positional candidates to 548. Parental lines were then assayed for expression differences by using Affymetrix microarray technology, and ANOVA was used to identify differentially expressed genes in these deletions. Thirty-four genes were identified that showed evidence for differential expression between the parental lines, one of which was significant even after a conservative Bonferroni correction. The list of potential candidates includes 5 genes for which previous annotations did not exist, and therefore would have been unlikely choices for follow-up from mapping studies alone. The use of microarray technology in this context allows an efficient, objective, quantitative evaluation of genes in the QTL and has the potential to reduce the overall effort needed in identifying genes causally associated with quantitative traits of interest.

Neuropathological and neuropsychological changes in "normal" aging: evidence for preclinical Alzheimer disease in cognitively normal individuals.

The presence of diffuse or primitive senile plaques in the neocortex of cognitively normal elderly at autopsy has been presumed to represent normal aging. Alternatively, these patients may have developed dementia and clinical Alzheimer disease (AD) if they had survived. In this setting, these patients could be subjects for cognitive or pharmacologic intervention to delay disease onset. We have thus followed a cohort of cognitively normal elderly subjects with a Clinical Dementia Rating (CDR) of 0 at autopsy. Thirty-one brains were examined at postmortem according to Consortium to Establish a Registry for Alzheimer Disease (CERAD) criteria and staged according to Braak. Ten patients were pathologically normal according to CERAD criteria (1a). Two of these patients were Braak Stage II. Seven very elderly subjects exhibited a few primitive neuritic plaques in the cortex and thus represented CERAD 1b. These individuals ranged in age from 85 to 105 years and were thus older than the CERAD la group that ranged in age from 72 to 93. Fourteen patients displayed Possible AD according to CERAD with ages ranging from 66 to 95. Three of these were Braak Stage I, 4 were Braak Stage II, and 7 were Braak Stage III. The Apolipoprotein E4 allele was over-represented in this possible AD group. Neuropsychological data were available on 12 individuals. In these 12 individuals, Possible AD at autopsy could be predicted by cognitive deficits in 1 or more areas including savings scores on memory testing and overall performance on some measures of frontal executive function.

Persistent Bacteremia Due to Methicillin-Resistant Staphylococcus aureus Infection Is Associated with agr Dysfunction and Low-Level In Vitro Resistance to Thrombin-Induced Platelet Microbicidal Protein

BACKGROUND The causes of persistent bacteremia (PB) due to methicillin-resistant Staphylococcus aureus (MRSA) are poorly understood. This investigation examined potential associations between PB with key clinical features and several in vitro bacterial genotypic and phenotypic characteristics, in isolates from 1 institution. METHODS Pulsed-field gel electrophoresis (PFGE) relatedness, thrombin-induced platelet microbicidal protein (tPMP)-susceptibility phenotype, accessory gene regulator (agr) genotype and functionality (via delta-lysin production), and autolysis phenotypes were assessed in MRSA isolates from the bloodstream of 21 prospectively identified patients with PB (blood cultures positive after > or =7 days of therapy) and of 18 patients with resolving bacteremia (RB) (sterile blood cultures within the first 2-4 days of therapy) due to MRSA. RESULTS The 2 groups had comparable baseline characteristics but differed in their clinical courses (e.g., endocarditis was more frequent in patients with PB than in those with RB [43% vs. 0%, respectively; P=.0016]); isolates from patients with PB exhibited higher rates of (1) survival in vitro after exposure to tPMP (22.4+/-14.8% vs. 11.6+/-6.5%, respectively; P=.005); (2) defective delta-lysin production (71.4% vs. 38.9%, respectively; P=.057); (3) non-agr genotype II profile (100% vs. 77.8%, respectively; P=.037); and (4) overrepresentation of a specific PFGE genotype (85.7% vs. 44.4%, respectively; P=.015). CONCLUSIONS Isolates from patients with PB differed from those in patients with RB, in several in vitro characteristics. Further studies will be necessary to define how these factors might affect clinical outcome.

Successful range-expanding plants experience less above-ground and below-ground enemy impact

Many species are currently moving to higher latitudes and altitudes. However, little is known about the factors that influence the future performance of range-expanding species in their new habitats. Here we show that range-expanding plant species from a riverine area were better defended against shoot and root enemies than were related native plant species growing in the same area. We grew fifteen plant species with and without non-coevolved polyphagous locusts and cosmopolitan, polyphagous aphids. Contrary to our expectations, the locusts performed more poorly on the range-expanding plant species than on the congeneric native plant species, whereas the aphids showed no difference. The shoot herbivores reduced the biomass of the native plants more than they did that of the congeneric range expanders. Also, the range-expanding plants developed fewer pathogenic effects in their root-zone soil than did the related native species. Current predictions forecast biodiversity loss due to limitations in the ability of species to adjust to climate warming conditions in their range. Our results strongly suggest that the plants that shift ranges towards higher latitudes and altitudes may include potential invaders, as the successful range expanders may experience less control by above-ground or below-ground enemies than the natives.

Powdery Mildew Induces Defense-Oriented Reprogramming of the Transcriptome in a Susceptible But Not in a Resistant Grapevine

Grapevines exhibit a wide spectrum of resistance to the powdery mildew fungus (PM), Erysiphe necator (Schw.) Burr., but little is known about the transcriptional basis of the defense to PM. Our microscopic observations showed that PM produced less hyphal growth and induced more brown-colored epidermal cells on leaves of PM-resistant Vitis aestivalis ‘Norton’ than on leaves of PM-susceptible Vitis vinifera ‘Cabernet sauvignon’. We found that endogenous salicylic acid levels were higher in V. aestivalis than in V. vinifera in the absence of the fungus and that salicylic acid levels increased in V. vinifera at 120 h postinoculation with PM. To test the hypothesis that gene expression differences would be apparent when V. aestivalis and V. vinifera were mounting a response to PM, we conducted a comprehensive Vitis GeneChip analysis. We examined the transcriptome at 0, 4, 8, 12, 24, and 48 h postinoculation with PM. We found only three PM-responsive transcripts in V. aestivalis and 625 in V. vinifera. There was a significant increase in the abundance of transcripts encoding ENHANCED DISEASE SUSCEPTIBILITY1, mitogen-activated protein kinase kinase, WRKY, PATHOGENESIS-RELATED1, PATHOGENESIS-RELATED10, and stilbene synthase in PM-infected V. vinifera, suggesting an induction of the basal defense response. The overall changes in the PM-responsive V. vinifera transcriptome also indicated a possible reprogramming of metabolism toward the increased synthesis of the secondary metabolites. These results suggested that resistance to PM in V. aestivalis was not associated with overall reprogramming of the transcriptome. However, PM induced defense-oriented transcriptional changes in V. vinifera.

RNA-seq: technical variability and sampling.

BackgroundRNA-seq is revolutionizing the way we study transcriptomes. mRNA can be surveyed without prior knowledge of gene transcripts. Alternative splicing of transcript isoforms and the identification of previously unknown exons are being reported. Initial reports of differences in exon usage, and splicing between samples as well as quantitative differences among samples are beginning to surface. Biological variation has been reported to be larger than technical variation. In addition, technical variation has been reported to be in line with expectations due to random sampling. However, strategies for dealing with technical variation will differ depending on the magnitude. The size of technical variance, and the role of sampling are examined in this manuscript.ResultsIn this study three independent Solexa/Illumina experiments containing technical replicates are analyzed. When coverage is low, large disagreements between technical replicates are apparent. Exon detection between technical replicates is highly variable when the coverage is less than 5 reads per nucleotide and estimates of gene expression are more likely to disagree when coverage is low. Although large disagreements in the estimates of expression are observed at all levels of coverage.ConclusionsTechnical variability is too high to ignore. Technical variability results in inconsistent detection of exons at low levels of coverage. Further, the estimate of the relative abundance of a transcript can substantially disagree, even when coverage levels are high. This may be due to the low sampling fraction and if so, it will persist as an issue needing to be addressed in experimental design even as the next wave of technology produces larger numbers of reads. We provide practical recommendations for dealing with the technical variability, without dramatic cost increases.

Differential gene expression in response to hydrogen peroxide and the putative PerR regulon of Synechocystis sp. strain PCC 6803.

We utilized a full genome cDNA microarray to identify the genes that comprise the peroxide stimulon in the cyanobacterium Synechocystis sp. strain PCC 6803. We determined that a gene (slr1738) encoding a protein similar to PerR in Bacillus subtilis was induced by peroxide. We constructed a PerR knockout strain and used it to help identify components of the PerR regulon, and we found that the regulatory properties were consistent with the hypothesis that PerR functions as a repressor. This effort was guided by finding putative PerR boxes in positions upstream of specific genes and by careful statistical analysis. PerR and sll1621 (ahpC), which codes for a peroxiredoxin, share a divergent promoter that is regulated by PerR. We found that isiA, encoding a Chl protein that is induced under low-iron conditions, was strongly induced by a short-term peroxide stress. Other genes that were strongly induced by peroxide included sigD, sigB, and genes encoding peroxiredoxins and Dsb-like proteins that have not been studied yet in this strain. A gene (slr1894) that encoded a protein similar to MrgA in B. subtilis was upregulated by peroxide, and a strain containing an mrgA knockout mutation was highly sensitive to peroxide. A number of genes were downregulated, including key genes in the chlorophyll biosynthesis pathway and numerous regulatory genes, including those encoding histidine kinases. We used PerR mutants and a thioredoxin mutant (TrxA1) to study differential expression in response to peroxide and determined that neither PerR nor TrxA1 is essential for the peroxide protective response.

Microarray Analysis of the Genome-Wide Response to Iron Deficiency and Iron Reconstitution in the Cyanobacterium Synechocystis sp. PCC 6803

A full-genome microarray of the (oxy)photosynthetic cyanobacterium Synechocystis sp. PCC 6803 was used to identify genes that were transcriptionally regulated by growth in iron (Fe)-deficient versus Fe-sufficient media. Transcript accumulation for 3,165 genes in the genome was analyzed using an analysis of variance model that accounted for slide and replicate (random) effects and dye (a fixed) effect in testing for differences in the four time periods. We determined that 85 genes showed statistically significant changes in the level of transcription (P ≤ 0.05/3,165 = 0.0000158) across the four time points examined, whereas 781 genes were characterized as interesting (P ≤ 0.05 but greater than 0.0000158; 731 of these had a fold change >1.25×). The genes identified included those known previously to be Fe regulated, such as isiA that encodes a novel chlorophyll-binding protein responsible for the pigment characteristics of low-Fe (LoFe) cells. ATP synthetase and phycobilisome genes were down-regulated in LoFe, and there were interesting changes in the transcription of genes involved in chlorophyll biosynthesis, in photosystem I and II assembly, and in energy metabolism. Hierarchical clustering demonstrated that photosynthesis genes, as a class, were repressed in LoFe and induced upon the re-addition of Fe. Specific regulatory genes were transcriptionally active in LoFe, including two genes that show homology to plant phytochromes (cph1 and cph2). These observations established the existence of a complex network of regulatory interactions and coordination in response to Fe availability.

The chemical interactions underlying tomato flavor preferences.

Although human perception of food flavors involves integration of multiple sensory inputs, the most salient sensations are taste and olfaction. Ortho- and retronasal olfaction are particularly crucial to flavor because they provide the qualitative diversity so important to identify safe versus dangerous foods. Historically, flavor research has prioritized aroma volatiles present at levels exceeding the orthonasally measured odor threshold, ignoring the variation in the rate at which odor intensities grow above threshold. Furthermore, the chemical composition of a food in itself tells us very little about whether or not that food will be liked. Clearly, alternative approaches are needed to elucidate flavor chemistry. Here we use targeted metabolomics and natural variation in flavor-associated sugars, acids, and aroma volatiles to evaluate the chemistry of tomato fruits, creating a predictive and testable model of liking. This nontraditional approach provides novel insights into flavor chemistry, the interactions between taste and retronasal olfaction, and a paradigm for enhancing liking of natural products. Some of the most abundant volatiles do not contribute to consumer liking, whereas other less abundant ones do. Aroma volatiles make contributions to perceived sweetness independent of sugar concentration, suggesting a novel way to increase perception of sweetness without adding sugar.