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Polycomb group (PcG) proteins are transcriptional repressors with a central role in the establishment and maintenance of gene expression patterns during development. We have investigated the role of polycomb repressive complexes (PRCs) in hematopoietic stem cells (HSCs) and progenitor populations. We show that mice with loss of function mutations in PRC2 components display enhanced HSC/progenitor population activity, whereas mutations that disrupt PRC1 or pleiohomeotic repressive complex are associated with HSC/progenitor cell defects. Because the hierarchical model of PRC action would predict synergistic effects of PRC1 and PRC2 mutation, these opposing effects suggest this model does not hold true in HSC/progenitor cells. To investigate the molecular targets of each complex in HSC/progenitor cells, we measured genome-wide expression changes associated with PRC deficiency, and identified transcriptional networks that are differentially regulated by PRC1 and PRC2. These studies provide new insights into the mechanistic interplay between distinct PRCs and have important implications for approaching PcG proteins as therapeutic targets.

Grazing occurs over a third of the earth's land surface and may potentially influence the storage of 10(9) Mg year(-1) of greenhouse gases as soil C. Displacement of native herbivores by high densities of livestock has often led to overgrazing and soil C loss. However, it remains unknown whether matching livestock densities to those of native herbivores can yield equivalent soil C sequestration. In the Trans-Himalayas we found that, despite comparable grazing intensities, watersheds converted to pastoralism had 49% lower soil C than watersheds which retain native herbivores. Experimental grazer-exclusion within each watershed type, show that this difference appears to be driven by indirect effects of livestock diet selection, leading to vegetation shifts that lower plant production and reduce likely soil C inputs from vegetation by c. 25 gC m(-2) year(-1). Our results suggest that while accounting for direct impacts (stocking density) is a major step, managing indirect impacts on vegetation composition are equally important in influencing soil C sequestration in grazing ecosystems.

Background: Down syndrome (DS) individuals suffer mental retardation with further cognitive decline and early onset Alzheimer's disease., Methodology/principal Findings: To understand how trisomy 21 causes these neurological abnormalities we investigated changes in gene expression networks combined with a systematic cell lineage analysis of adult neurogenesis using the Ts1Cje mouse model of DS. We demonstrated down regulation of a number of key genes involved in proliferation and cell cycle progression including Mcm7, Brca2, Prim1, Cenpo and Aurka in trisomic neurospheres. We found that trisomy did not affect the number of adult neural stem cells but resulted in reduced numbers of neural progenitors and neuroblasts. Analysis of differentiating adult Ts1Cje neural progenitors showed a severe reduction in numbers of neurons produced with a tendency for less elaborate neurites, whilst the numbers of astrocytes was increased., Conclusions/significance: We have shown that trisomy affects a number of elements of adult neurogenesis likely to result in a progressive pathogenesis and consequently providing the potential for the development of therapies to slow progression of, or even ameliorate the neuronal deficits suffered by DS individuals.

Background: High-throughput measurement of allele-specific expression (ASE) is a relatively new and exciting application area for array-based technologies. In this paper, we explore several data sets which make use of Illumina's GoldenGate BeadArray technology to measure ASE. This platform exploits coding SNPs to obtain relative expression measurements for alleles at approximately 1500 positions in the genome., Results: We analyze data from a mixture experiment where genomic DNA samples from pairs of individuals of known genotypes are pooled to create allelic imbalances at varying levels for the majority of SNPs on the array. We observe that GoldenGate has less sensitivity at detecting subtle allelic imbalances (around 1.3 fold) compared to extreme imbalances, and note the benefit of applying local background correction to the data. Analysis of data from a dye-swap control experiment allowed us to quantify dye-bias, which can be reduced considerably by careful normalization. The need to filter the data before carrying out further downstream analysis to remove non-responding probes, which show either weak, or non-specific signal for each allele, was also demonstrated. Throughout this paper, we find that a linear model analysis of the data from each SNP is a flexible modelling strategy that allows for testing of allelic imbalances in each sample when replicate hybridizations are available., Conclusions: Our analysis shows that local background correction carried out by Illumina's software, together with quantile normalization of the red and green channels within each array, provides optimal performance in terms of false positive rates. In addition, we strongly encourage intensity-based filtering to remove SNPs which only measure non-specific signal. We anticipate that a similar analysis strategy will prove useful when quantifying ASE on Illumina's higher density Infinium BeadChips.

Background: Imprinted genes show expression from one parental allele only and are important for development and behaviour. This extreme mode of allelic imbalance has been described for approximately 56 human genes. Imprinting status is often disrupted in cancer and dysmorphic syndromes. More subtle variation of gene expression, that is not parent-of-origin specific, termed 'allele-specific gene expression' (ASE) is more common and may give rise to milder phenotypic differences. Using two allele-specific high-throughput technologies alongside bioinformatics predictions, normal term human placenta was screened to find new imprinted genes and to ascertain the extent of ASE in this tissue., Results: Twenty-three family trios of placental cDNA, placental genomic DNA (gDNA) and gDNA from both parents were tested for 130 candidate genes with the Sequenom MassArray system. Six genes were found differentially expressed but none imprinted. The Illumina ASE BeadArray platform was then used to test 1536 SNPs in 932 genes. The array was enriched for the human orthologues of 124 mouse candidate genes from bioinformatics predictions and 10 human candidate imprinted genes from EST database mining. After quality control pruning, a total of 261 informative SNPs (214 genes) remained for analysis. Imprinting with maternal expression was demonstrated for the lymphocyte imprinted gene ZNF331 in human placenta. Two potential differentially methylated regions (DMRs) were found in the vicinity of ZNF331. None of the bioinformatically predicted candidates tested showed imprinting except for a skewed allelic expression in a parent-specific manner observed for PHACTR2, a neighbour of the imprinted PLAGL1 gene. ASE was detected for two or more individuals in 39 candidate genes (18%)., Conclusions: Both Sequenom and Illumina assays were sensitive enough to study imprinting and strong allelic bias. Previous bioinformatics approaches were not predictive of new imprinted genes in the human term placenta. ZNF331 is imprinted in human term placenta and might be a new ubiquitously imprinted gene, part of a primate-specific locus. Demonstration of partial imprinting of PHACTR2 calls for re-evaluation of the allelic pattern of expression for the PHACTR2-PLAGL1 locus. ASE was common in human term placenta.

The nature and extent of microbial biodiversity remain controversial with persistent debates over patterns of distributions (i.e. cosmopolitanism versus endemism) and the processes that structure these patterns (neutrality versus selection). We used culture-independent approaches to address these issues focusing on two groups of ciliates, the Oligotrichia (Spirotrichea) and Choreotrichia (Spirotrichea) across an environmental gradient. We assessed SSU rDNA diversity in ciliate communities at six stations in Long Island Sound spanning the frontal region that separates the fresher Connecticut River outflow plume from the open Sound. As in previous studies, we find one abundant cosmopolitan species (Strombidium biarmatum), a few moderately abundant sequences, and a long list of rare sequences. Furthermore, neither ciliate diversity nor species composition showed any clear relationship to measured environmental parameters (temperature, salinity, accessory pigment composition and chorophyll). Overall, we observed that diversity decreased moving from nearshore to offshore. We also conducted analyses to detect clustering among the sampled communities using the software Unifrac. This approach revealed three significant clusters grouping samples from nearshore, surface and deep/well mixed stations. We find no strong fit of our communities to log series, geometric or log normal distributions, though one of the 3 clusters is most consistent with a log series distribution. However, when we remove the abundant cosmopolitan species S. biarmatum, all three clusters fit to a log series distribution. These analyses suggest that, with the exception of one cosmopolitan species, the oligotrich and choreotrich communities at these stations may be distributed in a neutral manner.

Background: Extensive transcription of non-coding RNAs has been detected in eukaryotic genomes and is thought to constitute an additional layer in the regulation of gene expression. Despite this role, their transcription through the cell cycle has not been studied; genome-wide approaches have only focused on protein-coding genes. To explore the complex transcriptome architecture underlying the budding yeast cell cycle, we used 8 bp tiling arrays to generate a 5 minute-resolution, strand-specific expression atlas of the whole genome., Results: We discovered 523 antisense transcripts, of which 80 cycle or are located opposite periodically expressed mRNAs, 135 unannotated intergenic non-coding RNAs, of which 11 cycle, and 109 cell-cycle-regulated protein-coding genes that had not previously been shown to cycle. We detected periodic expression coupling of sense and antisense transcript pairs, including antisense transcripts opposite of key cell-cycle regulators, like FAR1 and TAF2., Conclusions: Our dataset presents the most comprehensive resource to date on gene expression during the budding yeast cell cycle. It reveals periodic expression of both protein-coding and non-coding RNA and profiles the expression of non-annotated RNAs throughout the cell cycle for the first time. This data enables hypothesis-driven mechanistic studies concerning the functions of non-coding RNAs.

Illumina BeadArrays are among the most popular and reliable platforms for gene expression profiling. However, little external scrutiny has been given to the design, selection and annotation of BeadArray probes, which is a fundamental issue in data quality and interpretation. Here we present a pipeline for the complete genomic and transcriptomic re-annotation of Illumina probe sequences, also applicable to other platforms, with its output available through a Web interface and incorporated into Bioconductor packages. We have identified several problems with the design of individual probes and we show the benefits of probe re-annotation on the analysis of BeadArray gene expression data sets. We discuss the importance of aspects such as probe coverage of individual transcripts, alternative messenger RNA splicing, single-nucleotide polymorphisms, repeat sequences, RNA degradation biases and probes targeting genomic regions with no known transcription. We conclude that many of the Illumina probes have unreliable original annotation and that our re-annotation allows analyses to focus on the good quality probes, which form the majority, and also to expand the scope of biological information that can be extracted.

Background: Illumina Sentrix-6 Whole-Genome Expression BeadChips are relatively new microarray platforms which have been used in many microarray studies in the past few years. These Chips have a unique design in which each Chip contains six microarrays and each microarray consists of two separate physical strips, posing special challenges for precise between-array normalization of expression values., Results: None of the normalization strategies proposed so far for this microarray platform allow for the possibility of systematic variation between the two strips comprising each array. That this variation can be substantial is illustrated by a data example. We demonstrate that normalizing at the strip-level rather than at the array-level can effectively remove this between-strip variation, improve the precision of gene expression measurements and discover more differentially expressed genes. The gain is substantial, yielding a 20% increase in statistical information and doubling the number of genes detected at a 5% false discovery rate. Functional analysis reveals that the extra genes found tend to have interesting biological meanings, dramatically strengthening the biological conclusions from the experiment. Strip-level normalization still outperforms array-level normalization when non-expressed probes are filtered out., Conclusion: Plots are proposed which demonstrate how the need for strip-level normalization relates to inconsistent intensity range variation between the strips. Strip-level normalization is recommended for the preprocessing of Illumina Sentrix-6 BeadChips whenever the intensity range is seen to be inconsistent between the strips. R code is provided to implement the recommended plots and normalization algorithms.

Unlabelled: Illumina produces a number of microarray-based technologies for human genotyping. An Infinium BeadChip is a two-color platform that types between 10(5) and 10(6) single nucleotide polymorphisms (SNPs) per sample. Despite being widely used, there is a shortage of open source software to process the raw intensities from this platform into genotype calls. To this end, we have developed the R/Bioconductor package crlmm for analyzing BeadChip data. After careful preprocessing, our software applies the CRLMM algorithm to produce genotype calls, confidence scores and other quality metrics at both the SNP and sample levels. We provide access to the raw summary-level intensity data, allowing users to develop their own methods for genotype calling or copy number analysis if they wish., Availability and Implementation: The crlmm Bioconductor package is available from http://www.bioconductor.org. Data packages and documentation are available from http://rafalab.jhsph.edu/software.html.

Tree cover is a fundamental structural characteristic and driver of ecosystem processes in terrestrial ecosystems, and trees are a major global carbon (C) sink. Fire and herbivores have been hypothesized to play dominant roles in regulating trees in African savannas, but the evidence for this is conflicting. Moving up a trophic scale, the factors that regulate fire occurrence and herbivores, such as disease and predation, are poorly understood for any given ecosystem. We used a Bayesian state-space model to show that the wildebeest population eruption that followed disease (rinderpest) eradication in the Serengeti ecosystem of East Africa led to a widespread reduction in the extent of fire and an ongoing recovery of the tree population. This supports the hypothesis that disease has played a key role in the regulation of this ecosystem. We then link our state-space model with theoretical and empirical results quantifying the effects of grazing and fire on soil carbon to predict that this cascade may have led to important shifts in the size of pools of C stored in soil and biomass. Our results suggest that the dynamics of herbivores and fire are tightly coupled at landscape scales, that fire exerts clear top-down effects on tree density, and that disease outbreaks in dominant herbivores can lead to complex trophic cascades in savanna ecosystems. We propose that the long-term status of the Serengeti and other intensely grazed savannas as sources or sinks for C may be fundamentally linked to the control of disease outbreaks and poaching., Competing Interests: The authors have declared that no competing interests exist.

Motivation: Primary data analysis methods are of critical importance in second generation DNA sequencing. Improved methods have the potential to increase yield and reduce the error rates. Openly documented analysis tools enable the user to understand the primary data, this is important for the optimization and validity of their scientific work., Results: In this article, we describe Swift, a new tool for performing primary data analysis on the Illumina Solexa Sequencing Platform. Swift is the first tool, outside of the vendors own software, which completes the full analysis process, from raw images through to base calls. As such it provides an alternative to, and independent validation of, the vendor supplied tool. Our results show that Swift is able to increase yield by 13.8%, at comparable error rate.

Summary: With their many replicates and their random layouts, Illumina BeadArrays provide greater scope fordetecting spatial artefacts than do other microarray technologies. They are also robust to artefact exclusion, yet there is a lack of tools that can perform these tasks for Illumina. We present BASH, a tool for this purpose. BASH adopts the concepts of Harshlight, but implements them in a manner that utilizes the unique characteristics of the Illumina technology. Using bead-level data, spatial artefacts of various kinds can thus be identified and excluded from further analyses., Availability: The beadarray Bioconductor package (version 1.10 onwards), www.bioconductor.org

Genome-wide associations have shown a lot of promise in dissecting the genetics of complex traits in humans with single variants, yet a large fraction of the genetic effects is still unaccounted for. Analyzing genetic interactions between variants (epistasis) is one of the potential ways forward. We investigated the abundance and functional impact of a specific type of epistasis, namely the interaction between regulatory and protein-coding variants. Using genotype and gene expression data from the 210 unrelated individuals of the original four HapMap populations, we have explored the combined effects of regulatory and protein-coding single nucleotide polymorphisms (SNPs). We predict that about 18% (1,502 out of 8,233 nsSNPs) of protein-coding variants are differentially expressed among individuals and demonstrate that regulatory variants can modify the functional effect of a coding variant in cis. Furthermore, we show that such interactions in cis can affect the expression of downstream targets of the gene containing the protein-coding SNP. In this way, a cis interaction between regulatory and protein-coding variants has a trans impact on gene expression. Given the abundance of both types of variants in human populations, we propose that joint consideration of regulatory and protein-coding variants may reveal additional genetic effects underlying complex traits and disease and may shed light on causes of differential penetrance of known disease variants., Competing Interests: The authors have declared that no competing interests exist.

Background: The RUNX1 transcription factor gene is frequently mutated in sporadic myeloid and lymphoid leukemia through translocation, point mutation or amplification. It is also responsible for a familial platelet disorder with predisposition to acute myeloid leukemia (FPD-AML). The disruption of the largely unknown biological pathways controlled by RUNX1 is likely to be responsible for the development of leukemia. We have used multiple microarray platforms and bioinformatic techniques to help identify these biological pathways to aid in the understanding of why RUNX1 mutations lead to leukemia., Results: Here we report genes regulated either directly or indirectly by RUNX1 based on the study of gene expression profiles generated from 3 different human and mouse platforms. The platforms used were global gene expression profiling of: 1) cell lines with RUNX1 mutations from FPD-AML patients, 2) over-expression of RUNX1 and CBFbeta, and 3) Runx1 knockout mouse embryos using either cDNA or Affymetrix microarrays. We observe that our datasets (lists of differentially expressed genes) significantly correlate with published microarray data from sporadic AML patients with mutations in either RUNX1 or its cofactor, CBFbeta. A number of biological processes were identified among the differentially expressed genes and functional assays suggest that heterozygous RUNX1 point mutations in patients with FPD-AML impair cell proliferation, microtubule dynamics and possibly genetic stability. In addition, analysis of the regulatory regions of the differentially expressed genes has for the first time systematically identified numerous potential novel RUNX1 target genes., Conclusion: This work is the first large-scale study attempting to identify the genetic networks regulated by RUNX1, a master regulator in the development of the hematopoietic system and leukemia. The biological pathways and target genes controlled by RUNX1 will have considerable importance in disease progression in both familial and sporadic leukemia as well as therapeutic implications.

Background: Variance-stabilizing techniques have been used for some time in the analysis of gene expression microarray data. A new adaptation, the variance-stabilizing transformation (VST), has recently been developed to take advantage of the unique features of Illumina BeadArrays. VST has been shown to perform well in comparison with the widely-used approach of taking a log2 transformation, but has not been validated on a spike-in experiment. We apply VST to the data from a recently published spike-in experiment and compare it both to a regular log2 analysis and a recently recommended analysis that can be applied if all raw data are available., Findings: VST provides more power to detect differentially expressed genes than a log2 transformation. However, the gain in power is roughly the same as utilizing the raw data from an experiment and weighting observations accordingly. VST is still advantageous when large changes in expression are anticipated, while a weighted log2 approach performs better for smaller changes., Conclusion: VST can be recommended for summarized Illumina data regardless of which Illumina pre-processing options have been used. However, using the raw data is still encouraged whenever possible.

The hypertrophic heart rat (HHR) was derived from the spontaneously hypertensive rat of the Okamoto strain and develops cardiac hypertrophy in the absence of hypertension. The genetic basis of this hypertrophy is unknown. Therefore, we compared gene expression profiles in the left ventricular myocardium of young (8-10 weeks of age) and old (38-50 weeks) HHR with rats from an age-matched control strain, the normal heart rat (NHR). cDNA microarrays (National Institute of Aging [NIA], 15,247 clones) were used to evaluate gene expression in cardiac-derived Cy3 and Cy5 labeled cDNA. M values (log2[Cy5/Cy3]) were obtained and significant differential expression was identified using an empirical Bayesian approach with specific results verified using real-time PCR. Compared with NHR, HHR cardiac weight index (heart weight/ body weight) was significantly elevated at both ages (young: 5.5 +/- 0.5 vs. 3.9 +/- 0.2; old: 4.2 +/- 0.3 vs. 3.4 +/- 0.2 mg/g; p < 0.05) with no difference in body weight or in tail-cuff blood pressure detected between the strains at either age. Differential expression was observed in 65 and 390 clones in young and old HHR, respectively, with more genes exhibiting down-regulation than up-regulation in both instances (young: down 44 vs. up 21; old: down 292 vs. up 98). Our data suggest a role for the Ras/mitogen-activated protein kinase (MAPK) signaling pathway and the tumor necrosis factor (TNF) receptor-mediated activation of nuclear factor-kappaB (NF-kappaB) in the etiology of cardiac enlargement in the HHR. These findings support the candidature of previously identified cardiotrophic agents in contributing to the cardiac enlargement in the normotensive HHR, and also identify novel genetic factors which may be involved in the genesis of primary cardiac hypertrophy.

Background: Illumina bead-based arrays are becoming increasingly popular due to their high degree of replication and reported high data quality. However, little attention has been paid to the pre-processing of Illumina data. In this paper, we present our experience of analysing the raw data from an Illumina spike-in experiment and offer guidelines for those wishing to analyse expression data or develop new methodologies for this technology., Results: We find that the local background estimated by Illumina is consistently low, and subtracting this background is beneficial for detecting differential expression (DE). Illumina's summary method performs well at removing outliers, producing estimates which are less biased and are less variable than other robust summary methods. However, quality assessment on summarised data may miss spatial artefacts present in the raw data. Also, we find that the background normalisation method used in Illumina's proprietary software (BeadStudio) can cause problems with a standard DE analysis. We demonstrate that variances calculated from the raw data can be used as inverse weights in the DE analysis to improve power. Finally, variability in both expression levels and DE statistics can be attributed to differences in probe composition. These differences are not accounted for by current analysis methods and require further investigation., Conclusion: Analysing Illumina expression data using BeadStudio is reasonable because of the conservative estimates of summary values produced by the software. Improvements can however be made by not using background normalisation. Access to the raw data allows for a more detailed quality assessment and flexible analyses. In the case of a gene expression study, data can be analysed on an appropriate scale using established tools. Similar improvements can be expected for other Illumina assays.

Impaired glucose uptake is associated with both cardiac hypertrophy and contractile dysfunction, but whether there are common underlying mechanisms linking these conditions is yet to be determined. Using a 'gene dose' Cre-Lox GLUT4-deficient murine model, we examined the effect of suppressed glucose availability on global myocardial gene expression and glycolysis substrate bypass on the function of isolated perfused hearts. Performance of hearts from 22- to 60-week-old male GLUT4 knockout (KO, >95% reduction in GLUT4), GLUT4 knockdown (KD, 85% reduction in cardiac GLUT4) and C57Bl/6 wild-type (WT) controls was measured ex vivo in Langendorff mode perfusion. DNA microarray was used to profile mRNA expression differences between GLUT4-KO and GLUT4-KD hearts. At 22 weeks, GLUT4-KO hearts exhibited cardiac hypertrophy and impaired contractile function ex vivo, characterized by a 40% decrease in developed pressure. At 60 weeks, dysfunction was accentuated in GLUT4-KO hearts and evident in GLUT4-KD hearts. Exogenous pyruvate (5 mM) restored systolic pressure to a level equivalent to WT (GLUT4-KO, 176.8+/-13.2 mmHg vs. WT, 146.4+/-9.56 mmHg) in 22-week-old GLUT4-KO hearts but not in 60-week-old GLUT4-KO hearts. In GLUT4-KO, DNA microarray analysis detected downregulation of a number of genes centrally involved in mitochondrial oxidation and upregulation of other genes indicative of a shift to cytosolic beta-oxidation of long chain fatty acids. A direct link between cardiomyocyte GLUT4 deficiency, hypertrophy and contractile dysfunction is demonstrated. These data provide mechanistic insight into the myocardial metabolic adaptations associated with short and long-term insulin resistance and indicate a window of opportunity for substrate intervention and functional 'rescue'.

Motivation: Microarray data must be background corrected to remove the effects of non-specific binding or spatial heterogeneity across the array, but this practice typically causes other problems such as negative corrected intensities and high variability of low intensity log-ratios. Different estimators of background, and various model-based processing methods, are compared in this study in search of the best option for differential expression analyses of small microarray experiments., Results: Using data where some independent truth in gene expression is known, eight different background correction alternatives are compared, in terms of precision and bias of the resulting gene expression measures, and in terms of their ability to detect differentially expressed genes as judged by two popular algorithms, SAM and limma eBayes. A new background processing method (normexp) is introduced which is based on a convolution model. The model-based correction methods are shown to be markedly superior to the usual practice of subtracting local background estimates. Methods which stabilize the variances of the log-ratios along the intensity range perform the best. The normexp+offset method is found to give the lowest false discovery rate overall, followed by morph and vsn. Like vsn, normexp is applicable to most types of two-colour microarray data., Availability: The background correction methods compared in this article are available in the R package limma (Smyth, 2005) from http://www.bioconductor.org., Supplementary Information: Supplementary data are available from http://bioinf.wehi.edu.au/resources/webReferences.html.

Fire and herbivory are important determinants of nutrient availability in savanna ecosystems. Fire and herbivory effects on the nutritive quality of savanna vegetation can occur directly, independent of changes in the plant community, or indirectly, via effects on the plant community. Indirect effects can be further subdivided into those occurring because of changes in plant species composition or plant abundance (i.e., quality versus quantity). We studied relationships between fire, herbivory, rainfall, soil fertility, and leaf nitrogen (N), phosphorus (P), and sodium (Na) at 30 sites inside and outside of Serengeti National Park. Using structural equation modeling, we asked whether fire and herbivory influences were largely direct or indirect and how their signs and strengths differed within the context of natural savanna processes. Herbivory was associated with enhanced leaf N and P through changes in plant biomass and community composition. Fire was associated with reduced leaf nutrient concentrations through changes in plant community composition. Additionally, fire had direct positive effects on Na and nonlinear direct effects on P that partially mitigated the indirect negative effects. Key mechanisms by which fire reduced plant nutritive quality were through reductions of Na-rich grasses and increased abundance of Themeda triandra, which had below-average leaf nutrients.

Unlabelled: The R/Bioconductor package beadarray allows raw data from Illumina experiments to be read and stored in convenient R classes. Users are free to choose between various methods of image processing, background correction and normalization in their analysis rather than using the defaults in Illumina's; proprietary software. The package also allows quality assessment to be carried out on the raw data. The data can then be summarized and stored in a format which can be used by other R/Bioconductor packages to perform downstream analyses. Summarized data processed by Illumina's; BeadStudio software can also be read and analysed in the same manner., Availability: The beadarray package is available from the Bioconductor web page at www.bioconductor.org. A user's guide and example data sets are provided with the package.

Mineral nitrogen (N) has been suggested as a potential factor causing declines in amphibian populations, especially in agricultural landscapes; however, there is a question as to whether it remains in the water column long enough to be toxic. We explored the hypothesis that mineral N can cause both lethal and sublethal toxic effects in amphibian embryos and larvae in a manipulative field experiment. We sampled 12 ponds, fertilizing half with ammonium nitrate fertilizer early in the spring, and measured hatching, survival, development, growth, and the incidence of deformities in native populations of wood frog (Rana sylvatica) and eastern tiger salamander (Ambystoma tigrinum tigrinum) embryos and larvae held in in situ enclosures. We found that higher ammonium concentrations negatively affect R. sylvatica more strongly than A. tigrinum. R. sylvatica tended to have lower survival as embryos and young tadpoles, slowed embryonic development, and an increased proportion of hatchlings with deformities at experimentally elevated ammonium. A. tigrinum did not experience significantly reduced survival, but their larval development was slowed in response to elevated ammonium and the abundance of large invertebrate predators. Variable species susceptibility, such as that shown by R sylvatica and A. tigrinum, could have large indirect effects on aquatic community structure through modification of competitive or predator-prey relationships. Ammonium and nitrate + nitrite concentrations were not correlated with other measures that might have affected amphibians, such as pH, pond area, depth, or vegetation. Our results highlight the potential importance of elevated ammonium on the growth, development and survival of amphibians, especially those that breed in surface waters receiving anthropogenic N inputs.

Terrestrial plant community responses to herbivory depend on resource availability, but the separate influences of different resources are difficult to study because they often correlate across natural environmental gradients. We studied the effects of excluding ungulate herbivores on plant species richness and composition, as well as available soil nitrogen (N) and phosphorus (P), across eight grassland sites in Serengeti National Park (SNP), Tanzania. These sites varied independently in rainfall and available soil N and P. Excluding herbivores decreased plant species richness at all sites and by an average of 5.4 species across all plots. Although plant species richness was a unimodal function of rainfall in both grazed and ungrazed plots, fences caused a greater decrease in plant species richness at sites of intermediate rainfall compared to sites of high or low rainfall. In terms of the relative or proportional decreases in plant species richness, excluding herbivores caused the strongest relative decreases at lower rainfall and where exclusion of herbivores increased available soil P. Herbivore exclusion increased among-plot heterogeneity in species composition but decreased coexistence of congeneric grasses. Compositional similarity between grazed and ungrazed treatments decreased with increasing rainfall due to greater forb richness in exclosures and greater sedge richness outside exclosures and was not related to effects of excluding herbivores on soil nutrients. Our results show that plant resources, especially water and P, appear to modulate the effects of herbivores on tropical grassland plant diversity and composition. We show that herbivore effects on soil P may be an important and previously unappreciated mechanism by which herbivores influence plant diversity, at least in tropical grasslands.

Background: Assessment of array quality is an essential step in the analysis of data from microarray experiments. Once detected, less reliable arrays are typically excluded or "filtered" from further analysis to avoid misleading results., Results: In this article, a graduated approach to array quality is considered based on empirical reproducibility of the gene expression measures from replicate arrays. Weights are assigned to each microarray by fitting a heteroscedastic linear model with shared array variance terms. A novel gene-by-gene update algorithm is used to efficiently estimate the array variances. The inverse variances are used as weights in the linear model analysis to identify differentially expressed genes. The method successfully assigns lower weights to less reproducible arrays from different experiments. Down-weighting the observations from suspect arrays increases the power to detect differential expression. In smaller experiments, this approach outperforms the usual method of filtering the data. The method is available in the limma software package which is implemented in the R software environment., Conclusion: This method complements existing normalisation and spot quality procedures, and allows poorer quality arrays, which would otherwise be discarded, to be included in an analysis. It is applicable to microarray data from experiments with some level of replication.

Allometric foraging theory suggests that herbivores of greatly differing size should co-exist through niche segregation, but a few studies of large-small herbivore foraging relationships have reported competitive interactions. This study addresses the potential roles of habitat productivity and large herbivore grazing intensities on large-small herbivore foraging interactions. We examined effects of different intensity simulated grazing treatments on forage abundance and quality for Utah prairie dogs (Cynomys parvidens) in a low productivity ecosystem, and consequent effects on prairie dog individual growth rates, foraging preferences, and activity budgets. We hypothesized that simulated grazing would have predominantly facilitative impacts on Utah prairie dogs, as was found for black-tailed prairie dogs in higher productivity ecosystems. To test this hypothesis, we measured the effects of simulated grazing on forage nitrogen, digestibility, and biomass. Simulated grazing increased average forage nitrogen and digestibility while decreasing forage biomass. These effects were associated with reduced individual growth rates, increased juvenile foraging time, and reduced juvenile vigilance. Results suggest that the negative effects of reduced vegetation biomass greatly outweighed positive treatment effects in this study. However, prairie dogs in the moderate intensity defoliation treatment showed some preference for "grazed" plots over "ungrazed" plots, and this preference increased with time. Our study lends support to the idea that habitat productivity and herbivore densities may mediate shifts between facilitative and competitive interactions between different-sized herbivores.

One cornerstone of ecological theory is that nutrient availability limits the number of species that can inhabit a community. However, the relationship between the spatial distribution of limiting nutrients and species diversity is not well established because there is no single scale appropriate for measuring variation in resource distribution. Instead, the correct scale for analyzing resource variation depends on the range of species sizes within the community. To quantify the relationship between nutrient distribution and plant species diversity, we measured NO(3)(-) distribution and plant species diversity in 16 paired, modified Whittaker grassland plots in Serengeti National Park, Tanzania. Semivariograms were used to quantify the spatial structure of NO(3)(-) from scales of 0.4-26 m. Plant species diversity (Shannon-Weiner diversity index; H ') was quantified in 1-m(2) plots, while plant species richness was measured at multiple spatial scales between 1 and 1000 m(2). Small-scale variation in NO(3)(-) (<0.4 m) was positively correlated with 1-m(2) H ', while 1000-m(2) species richness was a log-normal function of average NO(3)(-) patch size. Nine of the 16 grassland plots had a fractal (self-similar across scales) NO(3)(-) spatial distribution; of the nine fractal plots, five were adjacent to plots that had a non-fractal distribution of NO(3)(-). This finding offered the unique opportunity to test predictions of Ritchie and Olff (1999): when the spatial distribution of limiting resources is fractal, communities should display a left-skewed log-size distribution and a log-normal relationship between net primary production and species richness. These predictions were supported by comparisons of plant size distributions and biomass-richness relationships in paired plots, one with a fractal and one with a non-fractal distribution of NO(3)(-). In addition, fractal plots had greater large-scale richness than paired non-fractal plots (1,0-1000 m(2)), but neither species diversity ( H') nor richness was significantly different at small scales (1 m(2)). This result is most likely explained by differences in the scale of resource variation among plots: fractal and non-fractal plots had equivalent NO(3)(-) variation at small scales but differed in NO(3)(-) variation at large scales (as measured by the fractal dimension). We propose that small-scale variation in NO(3)(-) is largely due to the direct effects of plants on soil, while patterns of species richness at large scales is controlled by the patch size and fractal dimension of NO(3)(-) in the landscape. This study provides an important empirical step in understanding the relationship between the spatial distribution of resources and patterns of species diversity across multiple spatial scales.

Scaling laws that describe complex interactions between organisms and their environment as a function of body size offer exciting potential for synthesis in biology. Home range size, or the area used by individual organisms, is a critical ecological variable that integrates behaviour, physiology and population density and strongly depends on organism size. Here we present a new model of home range-body size scaling based on fractal resource distributions, in which resource encounter rates are a function of body size. The model predicts no universally constant scaling exponent for home range, but defines a possible range of values set by geometric limits to resource density and distribution. The model unifies apparently conflicting earlier results and explains differences in scaling exponents among herbivorous and carnivorous mammals and birds. We apply the model to predict that home range increases with habitat fragmentation, and that the home ranges of larger species should be much more sensitive to habitat fragmentation than those of smaller species.

Large mammalian herbivores occupy half of the earth's land surface and are important both ecologically and economically, but their diversity is threatened by human activities. We investigated how the diversity of large herbivores changes across gradients of global precipitation and soil fertility. Here we show that more plant-available moisture reduces the nutrient content of plants but increases productivity, whereas more plant-available nutrients increase both of these factors. Because larger herbivore species tolerate lower plant nutrient content but require greater plant abundance, the highest potential herbivore diversity should occur in locations with intermediate moisture and high nutrients. These areas are dry enough to yield high quality plants and support smaller herbivores, but productive enough to support larger herbivores. These predictions fit with observed patterns of body size and diversity for large mammalian herbivores in North America, Africa and Australia, and yield a global map of regions with potentially high herbivore diversity. Thus, gradients of precipitation, temperature and soil fertility might explain the global distribution of large herbivore diversity and help to identify crucial areas for conservation and restoration.

Wetlands provide many important ecosystem services to human society, which may depend on how plant diversity influences biomass production and nutrient retention. Vascular aquatic plant diversity may not necessarily enhance wetland ecosystem functioning, however, because competition among these plant species can be strong, often resulting in the local dominance of a single species. Here we have manipulated the species richness of rooted, submerged aquatic plant (macrophyte) communities in experimental wetland mesocosms. We found higher algal and total plant (algal plus macrophyte) biomass, as well as lower loss of total phosphorus, in mesocosms with a greater richness of macrophyte species. Greater plant biomass resulted from a sampling effect; that is, the increased chance in species mixtures that algal production would be facilitated by the presence of a less competitive species-in this case, crisped pondweed. Lower losses of total phosphorus resulted from the greater chance in species mixtures of a high algal biomass and the presence of sago pondweed, which physically filter particulate phosphorus from the water. These indirect and direct effects of macrophyte species richness on algal production, total plant biomass and phosphorus loss suggest that management practices that maintain macrophyte diversity may enhance the functioning and associated services of wetland ecosystems.

Coronary artery anomalies are uncommon in the general population and most are asymptomatic. We report a rare congenital coronary anomaly and discuss diagnosis and management strategies as well as review the pertinent literature.

No-reflow is an unpredictable complication following percutaneous coronary intervention. No-reflow is associated with myocardial ischemia and infarction and increased mortality. A case of refractory no-reflow is described that was rapidly and successfully treated with multiple infusions of high doses of verapamil and adenosine applied directly at the site of no-reflow through a perfusion catheter.

The incidence of thrombocytopenia with ticlopidine and clopidogrel when used in conjunction with abciximab has not been systematically addressed. We evaluated the rate of thrombocytopenia in patients undergoing intracoronary stent implantation receiving bolus plus infusion of abciximab and either ticlopidine or clopidogrel. We noted an incidence of 24% with the combination of 300-mg clopidogrel and abciximab. Other doses of ticlopidine (250 and 500 mg) and clopidogrel (75 mg) did not result in a statistically significant increase in thrombocytopenia over that of the 2.5%-5.2% reported incidence with abciximab alone. Length of hospital stay was 2.3 vs. 6.4 days in those developing thrombocytopenia (P = 0.06). Four (25%) developed thrombocytopenia requiring blood transfusion. Eight (50%) had no sequelae. The combination of 300-mg clopidogrel and abciximab results in a significant increase in the incidence of thrombocytopenia. This is an important clinical observation that merits further study.

Protein kinase C (PKC) and mitogen-activated protein (MAP) kinase activation appear important in conferring hypertrophy in vitro. However, the response of PKC and MAP kinase to stimuli known to induce hypertrophy in vivo has not been determined. We recently demonstrated that pressure-overload hypertrophy induced a transiently transfected gene driven by an hypertrophy responsive enhancer (HRE) through a marked increase in binding activity of its interacting nuclear factor (HRF). These data suggested that the HRE/HRF could serve as a target for evaluating the signal transduction events responsible for hypertrophy in vivo. Accordingly, we characterized MAP kinase and PKC isoform activation, injected HRE driven reporter gene expression, and HRF binding activity in rat hearts subjected to ascending aortic clipping or sham operation in the presence of the angiotensin-converting enzyme (ACE) inhibitor fosinopril, hydralazine, or no treatment. Analyses showed that PKC-epsilon and MAP kinase were acutely activated following ascending aortic ligature and that fosinopril significantly inhibited but did not completely abrogate PKC-epsilon and MAP kinase activation. However, fosinopril completely prevented pressure overload-mediated induction of HRE containing constructs and obviated increased HRF binding activity. These results suggest a direct relationship between ACE activity and HRE/HRF-mediated gene activation and imply that PKC-epsilon and MAP kinase may be involved in transducing this signal.

Declining biodiversity represents one of the most dramatic and irreversible aspects of anthropogenic global change, yet the ecological implications of this change are poorly understood. Recent studies have shown that biodiversity loss of basal species, such as autotrophs or plants, affects fundamental ecosystem processes such as nutrient dynamics and autotrophic production. Ecological theory predicts that changes induced by the loss of biodiversity at the base of an ecosystem should impact the entire system. Here we show that experimental reductions in grassland plant richness increase ecosystem vulnerability to invasions by plant species, enhance the spread of plant fungal diseases, and alter the richness and structure of insect communities. These results suggest that the loss of basal species may have profound effects on the integrity and functioning of ecosystems., (Blackwell Science Ltd.)

Ecologists still search for common principles that predict well-known responses of biological diversity to different factors. Such factors include the number of available niches in space, productivity, area, species' body size and habitat fragmentation. Here we show that all these patterns can arise from simple constraints on how organisms acquire resources in space. We use spatial scaling laws to describe how species of different sizes find food in patches of varying size and resource concentration. We then derive a mathematical rule for the minimum similarity in size of species that share these resources. This packing rule yields a theory of species diversity that predicts relations between diversity and productivity more effectively than previous models. Size and diversity patterns for locally coexisting East African grazing mammals and North American savanna plants strongly support these predictions. The theory also predicts relations between diversity and area and between diversity and habitat fragmentation. Thus, spatial scaling laws provide potentially unifying first principles that may explain many important patterns of species diversity.

Background: Nuclear factor-kappaB (NF-kappaB) resides inactive in the cytoplasm of lymphocytes, monocytes, endothelial cells, and smooth muscle cells, where, after stimulation, it transcriptionally activates interleukins, interferon, tumor necrosis factor-alpha, and adhesion molecules. Because acute inflammation may play a role in coronary artery plaque rupture, it was hypothesized that NF-kappaB activation correlated with coronary artery disease (CAD) activity., Methods and Results: Evidence of NF-kappaB activation in the circulation of 102 consecutive patients without an acute myocardial infarction who were undergoing cardiac catheterization was determined. Of these, 19 had unstable angina (USA) and were within 24 hours of the last episode of chest pain. The remaining 83 were being evaluated for stable angina (53), valvular heart disease (8), atypical chest pain (12), or congestive heart failure (10). Evidence of NF-kappaB activation was determined by electromobility shift assays (EMSAs) with the NF-kappaB binding-site-specific probe and nuclear proteins isolated from the buffy coat of blood obtained at the beginning of the procedure. Specificity of this DNA-protein interaction was confirmed by competition and supershift EMSAs. Analyses showed that 17 of 19 patients with USA had marked activation of NF-kappaB. Despite a significant number of patients with severe CAD (69%), only 2 of the 83 without USA showed marked NF-kappaB activation. A lack of NF-kappaB activation was not due to a lack of functional cell/protein because NF-kappaB was appropriately activated by lipopolysaccharide ex vivo in all patients. NF-kappaB activation was not a nonspecific response of all transcription factors because neither Sp1 or Oct1 was activated in patients with activated NF-kappaB. There was no relationship between drugs used, hemodynamic status, or other clinical characteristics and state of NF-kappaB activation., Conclusions: These data show that NF-kappaB is specifically and significantly activated in unstable angina pectoris and is not affected by severity of CAD or medical therapy. Furthermore, because NF-kappaB is activated before a clinical event, it may be mechanistically involved in the plaque disruption that produces acute coronary artery syndromes.

This report describes three patients in whom unsuspected large central pulmonary emboli were identified by transesophageal echocardiography. We discuss the utility and limitations of transesophageal echocardiography in diagnosing pulmonary emboli and its potential beneficial impact on the management of patients in the intensive care unit, particularly those with unexplained hypotension.

The role of herbivores in controlling plant species richness is a critical issue in the conservation and management of grassland biodiversity. Numerous field experiments in grassland plant communities show that herbivores often, but not always, increase plant diversity. Recent work suggests that the mechanisms of these effects involve alteration of local colonization of species from regional species pools or local extinction of species, and recent syntheses and models suggest that herbivore effects on plant diversity should vary across environmental gradients of soil fertility and precipitation.

Coronary artery vasospasm, the most common cause of sudden death in patients with structurally normal hearts, is not well recognized. We describe 2 survivors of sudden death with ergonovine-inducible coronary artery vasospasm successfully treated long term with calcium channel blockers. Of the reported (17 worldwide) patients with documented vasospasm-mediated sudden cardiac death treated with calcium blockers, none have had a recurrent event. This is substantially less than the 6-month sudden-death rate of 18 % of untreated vasospasm. We advocate that ergonovine provocative testing of sudden-death survivors with structurally normal hearts, followed by appropriate therapy with calcium channel blockers, be the standard approach for these highly treatable patients.

Left ventricular pseudoaneurysms are an uncommon complication of myocardial infarction and need urgent surgical repair. Though it is critical that they be accurately identified, pseudoaneurysms are occasionally misdiagnosed. We report an abnormality which may be mislabeled a pseudoaneurysm which we term a pseudo-pseudoaneurysm. The approach to accurate diagnosis of pseudoaneurysms is discussed.

Changes in gene expression occurring during skeletal muscle differentiation are exemplified by downregulation of brain creatine kinase (BCK) and induction of muscle creatine kinase (MCK). Although both are transcriptionally regulated, there appears to be no transcription factor-element overlap, suggesting that their coordinate expression results from culture medium-related influences. Basic fibroblast growth factor (bFGF) prevents myogenesis and represses MCK expression by inhibiting transcriptional activation. It was hypothesized that bFGF similarly influenced BCK by inducing its expression. Accordingly, BCK promoter constructs were transiently transfected into C2C12 cells and, after a switch to differentiation medium, were treated with bFGF, bFGF plus herbimycin, adenosine 3',5'-cyclic monophosphate (cAMP), or phorbol 12-myristate 13-acetate (PMA). Analyses demonstrated that bFGF responsiveness was contained within a 33-base pair element. Electromobility shift assays showed that bFGF induction increased the abundance of the nuclear factor binding the element. Both effects were prevented by herbimycin. Neither cAMP nor PMA specifically induced the construct containing the bFGF-responsive element. The induced factor required phosphorylation to bind, implying that bFGF-mediated increases in binding may be due to transcription factor phosphorylation.

During cardiogenesis, genes indicative of the adult phenotype are transcriptionally activated while genes characteristic of the embryonic phenotype are down-regulated. The regulation of embryonic genes such as the brain isoform of creatine kinase (BCK) during cardiac development has not been characterized. Accordingly, the transcriptional regulation of BCK in the developing heart was determined. In vitro and in vivo promoter analyses of the human BCK gene identified an element between +25 and +57 that functioned as an enhancer. Electromobility shift assays using adult and neonatal nuclear extracts identified a specific complex binding this element, the abundance of which correlated with the developmental level of endogenous cardiac BCK expression. Mutations at +47 and +53 led to a loss of activity in transfected cells and obviated binding in electromobility shift assays. These data show that a nuclear factor in cardiocytes interacts with an enhancer element (+25 and +57), via nucleotides +47 and +53, to drive BCK expression in the heart and suggest that developmental BCK expression is via abundance of this factor. The nuclear factor has not been identified but as described previously binding sites are not present in the enhancer, it is either a known factor interacting with a new recognition site or a new factor.

While the activation of muscle specific genes is well characterized, the mechanism mediating the regulation during muscle development of embryonic genes is poorly understood. To begin to investigate this area, the transcriptional regulation of the human brain creatine kinase (BCK) gene was characterized during in vitro myogenesis of C2C12 muscle cells. Initial analyses identified the first exon as important for high level expression in general and for the decrease in expression in response to C2C12 differentiation. In vivo competition confirmed the functional importance of the first exon. Electrophoretic mobility shift assays using portions of the exon showed that two separate proteins bound to sequences +1 to +27 and +25 to +57, respectively. The +1 to +27 interacting factor is present in skeletal but not cardiac muscle and thus may function as a skeletal muscle determining factor. The +25 to +57 interacting factor functions as a positive effector in myoblasts. This region also imparts the differentiation dependent decrease in expression via either modification of the myoblast factor or due to the presence of an additional factor. Site directed mutagenesis show that base pair +26 is critical for appropriate down regulation of BCK.

Platelet thromboxane receptors are acutely and reversibly upregulated after acute myocardial infarction. To determine if platelet thromboxane receptors are under transcriptional control, we isolated and characterized human genomic DNA clones containing the 5' flanking region of the thromboxane receptor gene. The exon-intron structure of the 5' portion of the thromboxane receptor gene was determined initially by comparing the nucleotide sequence of the 5' flanking genomic clone with that of a novel human uterine thromboxane receptor cDNA that extended the mRNA 141 bp further upstream than the previously identified human placental cDNA. A major transcription initiation site was located in three human tissues approximately 560 bp upstream from the translation initiation codon and 380 bp upstream from any previously identified transcription initiation site. The thromboxane receptor gene has neither a TATA nor a CAAT consensus site. Promoter function of the 5' flanking region of the thromboxane receptor gene was evaluated by transfection of thromboxane receptor gene promoter/chloramphenicol acetyltransferase (CAT) chimera plasmids into platelet-like K562 cells. Thromboxane receptor promoter activity, as assessed by CAT expression, was relatively weak but was significantly enhanced by phorbol ester treatment. Functional analysis of 5' deletion constructs in transfected K562 cells and gel mobility shift localized the major phorbol ester-responsive motifs in the thromboxane receptor gene promoter to a cluster of activator protein-2 (AP-2) binding consensus sites located approximately 1.8 kb 5' from the transcription initiation site. These studies are the first to determine the structure and organization of the 5' end of the thromboxane receptor gene and demonstrate that thromboxane receptor gene expression can be regulated by activation of protein kinase C via induction of an AP-2-like nuclear factor binding to upstream promoter elements. These findings strongly suggest that the mechanism for previously described upregulation of platelet thromboxane receptors after acute myocardial infarction is increased thromboxane receptor gene transcription in platelet-progenitor cells.

We tested the ability of consumer-resource theory to predict direct and indirect interactions among species, using an experimental system of insect herbivores and herbaceous plants. Specifically, we examined interactions among three species of grasshoppers (Melanoplus femur-rubrum, Spharagemon collare, andPhoetaliotes nebrascensis; Orthoptera, Acrididae) and herbaceous plants in experimental field cages placed over existing fertilized or unfertilized vegetation in a Minnesota old field. For the conditions inside these cages, we addressed whether (1) grasshopper diet predicted the presence of competition among grasshopper species, and (2) direct effects of grasshoppers on plants produced indirect interactions among plants, grasshoppers and soil nitrogen. Overall,M. femur-rubrum ate a greater proportion of forbs in cages, while the other two species ate primarily grasses. As expected, a pair of grasshopper species competed if they had similar diets. However, there were important exceptions that could be explained from observed indirect effects, although alternative explanations were also possible. First, all three grasshopper species significantly shifted their diets in the presence of other species, and these shifts occurred most often when competition was expected or occurred. Second, the two grassfeeding species reduced the biomass of the dominant grass (Schizachyrium scoparium) and increased available soil nitrogen and biomass of forbs. This effect may explain why the grass-feedingP. nebrascensis had a positive effect on the forb-feedingM. femur-rubrum on unfertilized plots. Overall, we show that direct effects of consumers on resources can predict competition and other important indirect interactions within a community.

Cis-elements (-933 to -641) upstream of the human M creatine kinase gene cap site contain an enhancer that confers developmental and tissue-specific expression to the chloramphenicol acetyltransferase gene in C2C12 myogenic cells transfected in culture. Division of the enhancer at -770 into a 5' fragment that includes the MyoD binding sites (-933 to -770) and a 3' fragment that includes the MEF-2 binding site (-770 to -641) resulted in two subfragments that showed minimal activity but in combination interacted in a position- and orientation-independent fashion to enhance activity of the SV40 promoter in transient transfection experiments. A 5' enhancer construct (-877 to -832) including only one (the low affinity) MyoD binding site was active when present in multiple copies. In contrast, a 3' enhancer construct (-749 to -732) including the MEF-2 binding site was inactive even when present in multiple copies. However, if the 5' construct was extended to include the high-affinity MyoD binding site (-877 to -803) the 5' and 3' constructs interacted in a position- and orientation-independent fashion to activate the SV40 promoter. Thus, the human M creatine kinase enhancer comprises multiple functional interacting domains.

We tested whether grasshoppers in experimental field environments, i.e. cages (40×40 cm) placed on existing old field vegetation, (1) were limited in density by plant abundance and/or nitrogen content, (2) exhibited interspecific competition, and (3) altered the relative abundance of different plant species. We examined interactions among a pair of early season grasshopper species (May-June; Arphia conspersa and Pardalophora apiculata) and a late season pair (July-August; Melanoplus femur-rubrum and Melanoplus bivittatus). Each grasshopper species was placed in cages by itself and with another grasshopper species. Grasshoppers generally survived at higher density in fertilized cages and they reduced plant abundance relative to empty cages, suggesting that grasshoppers may be food limited at these densities. In unfertilized plots, early season grasshoppers preferred grasses (Schizachyrium scoparium and Poa pratensis) and favored the growth of forbs (especially Solidago spp.). However, late in summer, Melanoplus spp. preferred Solidago spp. and favored the growth of grasses.The pattern of grasshopper survivorship and plant reduction within these experimental environments provide preliminary support for some of the predictions of resource competition theory. Grasshoppers exhibited interspecific competition only if they significantly reduced plant biomass. If two species competed, a grasshopper species was eliminated only if the superior competitor, when living by itself, could reduce plant biomass to a significantly lower level than the inferior competitor. Competitors persisted only if they did not differ in their ability to reduce plant biomass or reduced the abundance of different plant species.

We characterized the developmental expression of the brain creatine kinase (BCK) gene in the C2C12 myogenic cell line with the use of isoenzyme, Western blot, and Northern blot analyses. The results show that both BCK subunit protein and mRNA are upregulated early in myogenesis, and then downregulated in fully differentiated myotubes. To characterize the transcriptional regulatory mechanisms, a chimeric construct containing 1.2 kilobase pairs of 5'-flanking DNA from the human BCK gene placed upstream of the chloramphenicol acetyltransferase gene in the promoterless plasmid pSVOCAT was transiently transfected into C2C12 cells. In myoblasts and differentiating myotubes, the time course of expression of the constructs paralleled that of endogenous BCK mRNA. Additional constructs prepared by deleting 5'-flanking DNA were also transfected into C2C12 cells. All constructs were preferentially expressed in myoblasts relative to myotubes with absolute levels of expression increasing with deletion of 5'-flanking DNA. In nonmyogenic cells expression of the plasmids also increased with deletion of 5'-flanking DNA. An element from -1150 to -388 was isolated and found to be capable of suppressing expression of the BCK promoter and of heterologous promoters independent of orientation and position and hence to function as a silencer. Thus, BCK expression is mediated by sequences contained in the 5'-flanking DNA, including negative elements active in both C2C12 cells and nonmyogenic cells and elements that mediate the developmental expression of the BCK gene in C2C12 myogenic cells.