Andrew Y. Gracey

Cold-Induced Expression of Δ9-Desaturase in Carp by Transcriptional and Posttranslational Mechanisms

Poikilothermic animals respond to chronic cold by increasing phosphoglyceride unsaturation to restore the fluidity of cold-rigidified membranes. Despite the importance of this compensatory response, the enzymes involved have not been clearly identified, and the mechanisms that control their activity are unknown. In carp liver, cold induces an 8- to 10-fold increase in specific activity of the microsomal stearoyl coenzyme A desaturase. Cold-induced up-regulation of gene transcription resulted in a 10-fold increase in desaturase transcript amounts after 48 to 60 hours. However, this increase was preceded by the activation of latent desaturase, probably by a posttranslational mechanism. These two mechanisms may act sequentially to match desaturase expression to the demands imposed by a progressive decrease in temperature.

The cellular response to heat stress in the goby Gillichthys mirabilis: a cDNA microarray and protein-level analysis.

SUMMARY The cellular response to stress relies on the rapid induction of genes encoding proteins involved in preventing and repairing macromolecular damage incurred as a consequence of environmental insult. To increase our understanding of the scope of this response, a cDNA microarray, consisting of 9207 cDNA clones, was used to monitor gene expression changes in the gill and white muscle tissues of a eurythermic fish, Gillichthys mirabilis (Gobiidae) exposed to ecologically relevant heat stress. In each tissue, the induction or repression of over 200 genes was observed. These genes are associated with numerous biological processes, including the maintenance of protein homeostasis, cell cycle control, cytoskeletal reorganization, metabolic regulation and signal transduction, among many others. In both tissues, the molecular chaperones, certain transcription factors and a set of additional genes with various functions were induced in a similar manner; however, the majority of genes displayed tissue-specific responses. In gill, thermal stress induced the expression of the major structural components of the cytoskeleton, whereas these same genes did not respond to heat in muscle. In muscle, many genes involved in promoting cell growth and proliferation were repressed, perhaps to conserve energy for repair and replacement of damaged macromolecules, but a similar repression was not observed in the gill. Many of the observed changes in gene expression were similar to those described in model species whereas many others were unexpected. Measurements of the concentrations of the protein products of selected genes revealed that in each case an induction in mRNA synthesis correlated with an increase in protein production, though the timing and magnitude of the increase in protein was not consistently predicted by mRNA concentration, an important consideration in assessing the condition of the stressed cell using transcriptomic analysis.

Rhythms of Gene Expression in a Fluctuating Intertidal Environment

The physiological strategies that enable organisms to thrive in habitats where environmental factors vary dramatically on a daily basis are poorly understood. One of the most variable and unpredictable habitats on earth is the marine rocky intertidal zone located at the boundary between the terrestrial and marine environments. Mussels dominate rocky intertidal habitats throughout the world and, being sessile, endure wide variations in temperature, salinity, oxygen, and food availability due to diurnal, tidal, and climatic cycles. Analysis of gene-expression changes in the California ribbed mussel (Mytilus californianus) at different phases in the tidal cycle reveals that intertidal mussels exist in at least four distinct physiological states, corresponding to a metabolism and respiration phase, a cell-division phase, and two stress-response signatures linked to moderate and severe heat-stress events. The metabolism and cell-division phases appear to be functionally linked and are anticorrelated in time. The magnitudes and timings of these states varied by vertical position on the shore and appear to be driven by microhabitat conditions. The results provide new insights into the strategies that allow life to flourish in fluctuating environments and demonstrate the importance of time course data collected from field animals in situ in understanding organism-environment interactions.

Interpreting physiological responses to environmental change through gene expression profiling.

SUMMARY Identification of differentially expressed genes in response to environmental change offers insights into the roles of the transcriptome in the regulation of physiological responses. A variety of methods are now available to implement large-scale gene expression screens, and each method has specific advantages and disadvantages. Construction of custom cDNA microarrays remains the most popular route to implement expression screens in the non-model organisms favored by comparative physiologists, and we highlight some factors that should be considered when embarking along this path. Using a carp cDNA microarray, we have undertaken a broad, system-wide gene expression screen to investigate the physiological mechanisms underlying cold and hypoxia acclimation. This dataset provides a starting point from which to explore a range of specific mechanistic hypotheses at all levels of organization, from individual biochemical pathways to the level of the whole organism. We demonstrate the utility of two data analysis methods, Gene Ontology profiling and rank-based statistical methods, to summarize the probable physiological function of acclimation-induced gene expression changes, and to prioritize specific genes as candidates for further study.

Post-genomic approaches to understanding the mechanisms of environmentally induced phenotypic plasticity

SUMMARY Post-genomic techniques offer new and detailed insights into the mechanisms underpinning all biological processes, including phenotypic plasticity and environmentally relevant phenotypes. Although they require access to genomic resources it is now possible to create these for species of comparative or environmental interest even within a modest research project. Here we describe an open transcript screen for genes responding to environmental cold that might account for the acquired cold-specific phenotype in all its complex manifestations. Construction of a cDNA microarray led to a survey of transcript expression levels in seven tissues of carp, as a function of time, and three different extents of cooling. The resulting data delineated a common stress response found in all tissues that comprises genes involved in cellular homeostasis, including energy charge, ATP turnover, protein turnover and stress protein production. These genes respond to kinds of perturbation other than cold and probably form part of a more general stress response common to other species. We also defined tissue-specific response patterns of transcript regulation whose main characteristics were investigated by a profiling technique based on categorisation of gene function. These genes underpin the highly tissue-specific pattern of physiological adaptations observed in the cold-acclimated fish. As a result we have identified a large number of candidate gene targets with which to investigate adaptive responses to environmental challenge.

An explicit test of the phospholipid saturation hypothesis of acquired cold tolerance in Caenorhabditis elegans

Protection of poikilothermic animals from seasonal cold is widely regarded as being causally linked to changes in the unsaturation of membrane phospholipids, yet in animals this proposition remains formally untested. We have now achieved this by the genetic manipulation of lipid biosynthesis of Caenorhabditis elegans independent of temperature. Worms transferred from 25°C to 10°C develop over several days a much-increased tolerance of lethal cold (0°C) and also an increased phospholipid unsaturation, as in higher animal models. Of the three C. elegans Δ9-desaturases, transcript levels of fat-7 only were up-regulated by cold transfer. RNAi suppression of fat-7 caused the induction of fat-5 desaturase, so to control desaturase expression we combined RNAi of fat-7 with a fat-5 knockout. These fat-5/fat-7 manipulated worms displayed the expected negative linear relationship between lipid saturation and cold tolerance at 0°C, an outcome confirmed by dietary rescue. However, this change in lipid saturation explains just 16% of the observed difference between cold tolerance of animals held at 25°C and 10°C. Thus, although the manipulated lipid saturation affects the tolerable thermal window, and altered Δ9-desaturase expression accounts for cold-induced lipid adjustments, the effect is relatively small and none of the lipid manipulations were sufficient to convert worms between fully cold-sensitive and fully cold-tolerant states. Critically, transfer of 10°C-acclimated worms back to 25°C led to them restoring the usual cold-sensitive phenotype within 24 h despite retaining a lipid profile characteristic of 10°C worms. Other nonlipid mechanisms of acquired cold protection clearly dominate inducible cold tolerance.

Elevated expression of protein biosynthesis genes in liver and muscle of hibernating black bears (Ursus americanus)

We conducted a large-scale gene expression screen using the 3,200 cDNA probe microarray developed specifically for Ursus americanus to detect expression differences in liver and skeletal muscle that occur during winter hibernation compared with animals sampled during summer. The expression of 12 genes, including RNA binding protein motif 3 (Rbm3), that are mostly involved in protein biosynthesis, was induced during hibernation in both liver and muscle. The Gene Ontology and Gene Set Enrichment analysis consistently showed a highly significant enrichment of the protein biosynthesis category by overexpressed genes in both liver and skeletal muscle during hibernation. Coordinated induction in transcriptional level of genes involved in protein biosynthesis is a distinctive feature of the transcriptome in hibernating black bears. This finding implies induction of translation and suggests an adaptive mechanism that contributes to a unique ability to reduce muscle atrophy over prolonged periods of immobility during hibernation. Comparing expression profiles in bears to small mammalian hibernators shows a general trend during hibernation of transcriptional changes that include induction of genes involved in lipid metabolism and carbohydrate synthesis as well as depression of genes involved in the urea cycle and detoxification function in liver.

Circadian cycles are the dominant transcriptional rhythm in the intertidal mussel Mytilus californianus

Residents in the marine intertidal, the zone where terrestrial and marine habitats converge, inhabit an environment that is subject to both the 24-h day and night daily rhythm of the terrestrial earth and also the 12.4-h ebb and flow of the tidal cycle. Here, we investigate the relative contribution of the daily and tidal cycle on the physiology of intertidal mussels, Mytilus californianus, by monitoring rhythms of gene expression in both simulated and natural tidal environments. We report that >40% of the transcriptome exhibits rhythmic gene expression, and that depending on the specific tidal conditions, between 80% and 90% of the rhythmic transcripts follow a circadian expression pattern with a period of 24 to 26 h. Consistent with the dominant effect of the circadian cycle we show that the expression of clock genes oscillates with a 24-h period. Our data indicate that the circadian 24-h cycle is the dominant driver of rhythmic gene expression in this intertidal inhabitant despite the profound environmental and physiological changes associated with aerial exposure during tidal emergence.

Mass mortality in Pacific oysters is associated with a specific gene expression signature

Mass mortality events occur in natural and cultured communities of bivalve molluscs. The Pacific oyster, Crassostrea gigas, is a dominant species in many intertidal locations as well as an important aquacultured bivalve species, and for the last 50 years, adult oysters have suffered frequent and extreme mass mortality events during summer months. To investigate the molecular changes that precede these mortality events, we employed a novel nonlethal sampling approach to collect haemolymph samples from individual oysters during the period that preceded a mortality event. Microarray‐based gene expression screening of the collected haemolymph was used to identify a mortality gene expression signature that distinguished oysters that survived the mortality event from those individuals that died during the event. The signature was cross‐validated by comparing two separate episodes of mortality. Here, we report that near‐mortality oysters can be distinguished from longer‐lived oysters by the elevated expression of genes associated with cell death, lysosomal proteolysis, and cellular assembly and organization. These results show the potential utility of nonlethal sampling approaches for investigating the environmental causes of mortality in natural populations in the field, and for predicting when such events could occur and which individuals will be affected.

Robust regression for periodicity detection in non-uniformly sampled time-course gene expression data

BackgroundIn practice many biological time series measurements, including gene microarrays, are conducted at time points that seem to be interesting in the biologists opinion and not necessarily at fixed time intervals. In many circumstances we are interested in finding targets that are expressed periodically. To tackle the problems of uneven sampling and unknown type of noise in periodicity detection, we propose to use robust regression.MethodsThe aim of this paper is to develop a general framework for robust periodicity detection and review and rank different approaches by means of simulations. We also show the results for some real measurement data.ResultsThe simulation results clearly show that when the sampling of time series gets more and more uneven, the methods that assume even sampling become unusable. We find that M-estimation provides a good compromise between robustness and computational efficiency.ConclusionSince uneven sampling occurs often in biological measurements, the robust methods developed in this paper are expected to have many uses. The regression based formulation of the periodicity detection problem easily adapts to non-uniform sampling. Using robust regression helps to reject inconsistently behaving data points.AvailabilityThe implementations are currently available for Matlab and will be made available for the users of R as well. More information can be found in the web-supplement [1].