The plant-specific protein FEHLSTART controls male meiotic entry, initializing meiotic synchronization in Arabidopsis.

Meiosis marks the transition from the sporophyte to the gametophyte generation in the life cycle of flowering plants, and creates genetic variations through homologous recombination. In most flowering plants, meiosis is highly synchronized within each anther, which is significant for efficient fertilization. To date, little is known about the molecular mechanisms of entry into meiosis and exit from it, and only a few genes in Arabidopsis have been characterized with a role in regulating meiotic progression. In this study, we report the functional characterization of a plant-specific basic helix-loop-helix (bHLH) protein, FEHLSTART (FST), a defect in which leads to premature meiotic entry and asynchronous meiosis, and results in decreased seed yield. Investigation of the time course of meiosis showed that the onset of leptotene, the first stage of prophase I, frequently occurred earlier in fst-1 than in the wild type. Asynchronous meiosis followed, which could manifest in the disruption of regular spindle structures and symmetric cell divisions in fst-1 mutants during the meiosis I/II transition. In accordance with frequently accelerated meiotic entry, whole-transcriptome analysis of fst-1 anthers undergoing meiosis revealed that 19 circadian rhythm genes were affected and 47 pollen-related genes were prematurely expressed at a higher level. Taken together, we propose that FST is required for normal meiotic entry and the establishment of meiotic synchrony.

A transcriptomic approach to elucidate the physiological significance of human cytochrome P450 2S1 in bronchial epithelial cells.

BACKGROUND: Cytochrome P450 2S1 (CYP2S1) is an orphan P450 with an unknown biological function. Data from our laboratory and others suggest that CYP2S1 may have an important physiological role in modulating the synthesis and metabolism of bioactive lipids including prostaglandins and retinoids. CYP2S1 expression is elevated in multiple epithelial-derived cancers as well as in the chronic hyperproliferative disease psoriasis. Whether CYP2S1 expression in proliferative disease is protective, detrimental, or neutral to disease progression remains to be determined. Two human bronchial epithelial cells (BEAS-2B) were constructed to represent chronic depletion of CYP2S1 using short-hairpin RNA (shRNA) silencing directed toward the 3'UTR (759) and exon 3 (984) of the CYP2S1 gene and compared with a non-targeting shRNA control (SCRAM). Both CYP2S1 mRNA and protein were depleted by approximately 75% in stable cell lines derived from both targeted shRNA constructs (759 and 984). To elucidate the biological significance of CYP2S1, we analyzed transcriptome alterations in response to CYP2S1 depletion in human lung cells. RESULTS: RNA-sequencing (RNA-seq) analysis was performed to compare the transcriptome of the control (SCRAM) and the CYP2S1-depleted (759) BEAS-2B cell lines. Transcriptomes of the replicates from the two cell lines were found to be distinct populations as determined using Principal Component Analysis and hierarchical clustering. Approximately 1000 genes were differentially expressed in response to CYP2S1 depletion. Consistent with our previous phenotypes, DAVID analysis revealed altered regulation in key pathways implicated in cell proliferation and migration. Transcriptomic profiles were also consistent with the metabolism of proposed endogenous substrates. Pathway analysis also revealed significant expression changes within mTOR signaling, a critical pathway in cell growth. To determine whether these changes manifest as altered cell size, cell diameter and volume were calculated, revealing that CYP2S1 depletion promotes cell growth in BEAS-2B cells. CONCLUSIONS: These data suggest that pathway analysis of sequence-based gene expression is a powerful method to identify pathways and phenotypic alterations in response to changes in orphan enzyme expression. Our results suggest a novel role for CYP2S1-mediated metabolism in modulating BEAS-2B cell size. These findings warrant further studies on CYP2S1 regulated pathways to elucidate potential substrates of CYP2S1.

Transcriptome profiling of cytokinin and auxin regulation in tomato root.

Tomato is a model and economically important crop plant with little information available about gene expression in roots. Currently, there have only been a few studies that examine hormonal responses in tomato roots and none at a genome-wide level. This study examined the transcriptome atlas of tomato root regions (root tip, lateral roots, and whole roots) and the transcriptional regulation of each root region in response to the plant hormones cytokinin and auxin using Illumina RNA sequencing. More than 165 million 1×54 base pair reads were mapped onto the Solanum lycopersicum reference genome and differential expression patterns in each root region in response to each hormone were assessed. Many novel cytokinin- and auxin-induced and -repressed genes were identified as significantly differentially expressed and the expression levels of several were confirmed by qPCR. A number of these regulated genes represent tomato orthologues of cytokinin- or auxin-regulated genes identified in other species, including CKXs, type-A RRs, Aux/IAAs, and ARFs. Additionally, the data confirm some of the hormone regulation studies for recently examined genes in tomato such as SlIAAs and SlGH3s. Moreover, genes expressed abundantly in each root region were identified which provide a spatial distribution of many classes of genes, including plant defence, secondary metabolite production, and general metabolism across the root. Overall this study presents the first global expression patterns of hormone-regulated transcripts in tomato roots, which will be functionally relevant for future studies directed towards tomato root growth and development.

Characterization of a set of novel meiotically-active promoters in Arabidopsis.

BACKGROUND: Homologous recombination, together with selection, laid the foundation for traditional plant breeding. The recombination process that takes place during meiotic cell division is crucial for the creation of novel variations of highly desired traits by breeders. Gaining control over this process is important for molecular breeding to achieve more precise, large-scale and quicker plant improvement. As conventional ubiquitous promoters are neither tissue-specific nor efficient in driving gene expression in meiocytes, promoters with high meiotic activities are potential candidates for manipulating the recombination process. So far, only a few meiotically-active promoters have been reported. Recently developed techniques to profile the transcriptome landscape of isolated meiocytes provided the means to discover promoters from genes that are actively expressed in meiosis. RESULTS: In a screen for meiotically-active promoters, we examined ten promoter sequences that are associated with novel meiotic candidate genes. Each promoter was tested by expressing a GFP reporter gene in Arabidopsis. Characterization of regulatory regions revealed that these meiotically-active promoters possessed conserved motifs and motif arrangement. Some of the promoters unite optimal properties which are invaluable for meiosis-directed studies such as delivering specific gene expression in early meiosis I and/or meiosis II. Furthermore, the examination of homologs of the corresponding genes within green plants points to a great potential of applying the information from Arabidopsis to other species, especially crop plants. CONCLUSIONS: We identified ten novel meiotically-active promoters; which, along with their homologs, are prime candidates to specifically drive gene expression during meiosis in plants and can thus provide important tools for meiosis study and crop breeding.

Population-based study of genetic variation in individuals with autism spectrum disorders from Croatia.

BACKGROUND: Genome-wide studies on autism spectrum disorders (ASDs) have mostly focused on large-scale population samples, but examination of rare variations in isolated populations may provide additional insights into the disease pathogenesis. METHODS: As a first step in the genetic analysis of ASD in Croatia, we characterized genetic variation in a sample of 103 subjects with ASD and 203 control individuals, who were genotyped using the Illumina HumanHap550 BeadChip. We analyzed the genetic diversity of the Croatian population and its relationship to other populations, the degree of relatedness via Runs of Homozygosity (ROHs), and the distribution of large (>500 Kb) copy number variations. RESULTS: Combining the Croatian cohort with several previously published populations in the FastME analysis (an alternative to Neighbor Joining) revealed that Croatian subjects cluster, as expected, with Southern Europeans; in addition, individuals from the same geographic region within Europe cluster together. Whereas Croatian subjects could be separated from a sample of healthy control subjects of European origin from North America, Croatian ASD cases and controls are well mixed. A comparison of runs of homozygosity indicated that the number and the median length of regions of homozygosity are higher for ASD subjects than for controls (p = 6 × 10(-3)). Furthermore, analysis of copy number variants found a higher frequency of large chromosomal rearrangements (>2 Mb) in ASD cases (5/103) than in ethnically matched control subjects (1/197, p = 0.019). CONCLUSIONS: Our findings illustrate the remarkable utility of high-density genotype data for subjects from a limited geographic area in dissecting genetic heterogeneity with respect to population and disease related variation.

Draft Genome Sequences of Vancomycin-Susceptible Staphylococcus aureus Related to Heterogeneous Vancomycin-Intermediate S. aureus.

We report the draft genome sequences of three vancomycin-susceptible methicillin-resistant Staphylococcus aureus strains. S. aureus strain MV8 is a sequence type 8 (ST-8) staphylococcal cassette chromosome mec element type IV (SCCmec IV) derivative, while the other two strains (S. aureus MM25 and MM61) are ST-5 SCCmec II strains. MM61 is also closely related to the heterogeneous vancomycin-intermediate S. aureus strain MM66.

Draft Genomes of Heterogeneous Vancomycin-Intermediate Staphylococcus aureus Strain MM66 and MM66 Derivatives with Altered Vancomycin Resistance Levels.

The draft genomes of heterogeneous vancomycin-intermediate Staphylococcus aureus (hVISA) strain MM66 and MM66 isolates demonstrating altered vancomycin resistance levels were produced in an effort to provide information on mutations contributing to the vancomycin resistance levels observed in these strains.

Comparative genome analysis of non-toxigenic non-O1 versus toxigenic O1 Vibrio cholerae.

Pathogenic strains of Vibrio cholerae are responsible for endemic and pandemic outbreaks of the disease cholera. The complete toxigenic mechanisms underlying virulence in Vibrio strains are poorly understood. The hypothesis of this work was that virulent versus non-virulent strains of V. cholerae harbor distinctive genomic elements that encode virulence. The purpose of this study was to elucidate genomic differences between the O1 serotypes and non-O1 V. cholerae PS15, a non-toxigenic strain, in order to identify novel genes potentially responsible for virulence. In this study, we compared the whole genome of the non-O1 PS15 strain to the whole genomes of toxigenic serotypes at the phylogenetic level, and found that the PS15 genome was distantly related to those of toxigenic V. cholerae. Thus we focused on a detailed gene comparison between PS15 and the distantly related O1 V. cholerae N16961. Based on sequence alignment we tentatively assigned chromosome numbers 1 and 2 to elements within the genome of non-O1 V. cholerae PS15. Further, we found that PS15 and O1 V. cholerae N16961 shared 98% identity and 766 genes, but of the genes present in N16961 that were missing in the non-O1 V. cholerae PS15 genome, 56 were predicted to encode not only for virulence-related genes (colonization, antimicrobial resistance, and regulation of persister cells) but also genes involved in the metabolic biosynthesis of lipids, nucleosides and sulfur compounds. Additionally, we found 113 genes unique to PS15 that were predicted to encode other properties related to virulence, disease, defense, membrane transport, and DNA metabolism. Here, we identified distinctive and novel genomic elements between O1 and non-O1 V. cholerae genomes as potential virulence factors and, thus, targets for future therapeutics. Modulation of such novel targets may eventually enhance eradication efforts of endemic and pandemic disease cholera in afflicted nations.

Transcriptome analysis of cytokinin response in tomato leaves.

Tomato is one of the most economically and agriculturally important Solanaceous species and vegetable crops, serving as a model for examination of fruit biology and compound leaf development. Cytokinin is a plant hormone linked to the control of leaf development and is known to regulate a wide range of genes including many transcription factors. Currently there is little known of the leaf transcriptome in tomato and how it might be regulated by cytokinin. We employ high throughput mRNA sequencing technology and bioinformatic methodologies to robustly analyze cytokinin regulated tomato leaf transcriptomes. Leaf samples of two ages, 13d and 35d were treated with cytokinin or the solvent vehicle control dimethyl sulfoxide (DMSO) for 2 h or 24 h, after which RNA was extracted for sequencing. To confirm the accuracy of RNA sequencing results, we performed qPCR analysis of select transcripts identified as cytokinin regulated by the RNA sequencing approach. The resulting data provide the first hormone transcriptome analysis of leaves in tomato. Specifically we identified several previously untested tomato orthologs of cytokinin-related genes as well as numerous novel cytokinin-regulated transcripts in tomato leaves. Principal component analysis of the data indicates that length of cytokinin treatment and plant age are the major factors responsible for changes in transcripts observed in this study. Two hour cytokinin treatment showed a more robust transcript response indicated by both greater fold change of induced transcripts and the induction of twice as many cytokinin-related genes involved in signaling, metabolism, and transport in young vs. older leaves. This difference in transcriptome response in younger vs. older leaves was also found to a lesser extent with an extended (24 h) cytokinin treatment. Overall data presented here provides a solid foundation for future study of cytokinin and cytokinin regulated genes involved in compound leaf development or other developmental processes in tomato.

Genome Sequence of Non-O1 Vibrio cholerae PS15.

The draft genome sequence of a non-O1 Vibrio cholerae strain, PS15, organized into 3,512 open reading frames within a 3.9-Mb genome, was determined. The PS15 genome sequence will allow for the study of the evolution of virulence and environmental adaptation in V. cholerae.

Genome-wide association studies: progress and potential for drug discovery and development.

Although genetic studies have been critically important for the identification of therapeutic targets in Mendelian disorders, genetic approaches aiming to identify targets for common, complex diseases have traditionally had much more limited success. However, during the past year, a novel genetic approach - genome-wide association (GWA) - has demonstrated its potential to identify common genetic variants associated with complex diseases such as diabetes, inflammatory bowel disease and cancer. Here, we highlight some of these recent successes, and discuss the potential for GWA studies to identify novel therapeutic targets and genetic biomarkers that will be useful for drug discovery, patient selection and stratification in common diseases.

Genomic convergence analysis of schizophrenia: mRNA sequencing reveals altered synaptic vesicular transport in post-mortem cerebellum.

Schizophrenia (SCZ) is a common, disabling mental illness with high heritability but complex, poorly understood genetic etiology. As the first phase of a genomic convergence analysis of SCZ, we generated 16.7 billion nucleotides of short read, shotgun sequences of cDNA from post-mortem cerebellar cortices of 14 patients and six, matched controls. A rigorous analysis pipeline was developed for analysis of digital gene expression studies. Sequences aligned to approximately 33,200 transcripts in each sample, with average coverage of 450 reads per gene. Following adjustments for confounding clinical, sample and experimental sources of variation, 215 genes differed significantly in expression between cases and controls. Golgi apparatus, vesicular transport, membrane association, Zinc binding and regulation of transcription were over-represented among differentially expressed genes. Twenty three genes with altered expression and involvement in presynaptic vesicular transport, Golgi function and GABAergic neurotransmission define a unifying molecular hypothesis for dysfunction in cerebellar cortex in SCZ.

Genome-wide association studies: progress in identifying genetic biomarkers in common, complex diseases.

Novel, comprehensive approaches for biomarker discovery and validation are urgently needed. One particular area of methodologic need is for discovery of novel genetic biomarkers in complex diseases and traits. Here, we review recent successes in the use of genome wide association (GWA) approaches to identify genetic biomarkers in common human diseases and traits. Such studies are yielding initial insights into the allelic architecture of complex traits. In general, it appears that complex diseases are associated with many common polymorphisms, implying profound genetic heterogeneity between affected individuals.