Project description:Here we identify and characterize a spontaneous mutant allele that disrupts the initiation and specification of the ovule integument in Mimulus nudatus. The mutant phenotype is characterized by the early arrest of the ovule integument or alternatively, the delayed development of a carpel-like structure in place of the integument. Additionally, medusa mutant ovule primordia fail to develop female gametophytic structures. Morphological analyses indicate that a nucellar domain is specified within the ovule and a megaspore-like cell is often specified, but that cell aborts before generating the female gametophyte. We mapped the position of the mutation to a location on Chromosome1. Mapping revealed a primary candidate gene that was a member of a family of MADS-domain containing transcription factors with sequence similarity to the Arabidopsis BEL1 gene (At5G41410) and is an orthologue of the Mimulus guttatus gene MgTOL.A0789.1. Analysis of the sequence of the Mimulus nudatus homolog (referred hereafter as MnBEL1) locus, revealed the insertion of a mitochondrial DNA genomic fragment within the MnBEL1 locus that segregated with the medusa mutant phenotype. The mitochondrial insertion sequence is predicted to disrupt the coding sequence of MnBEL1. RNA expression analysis revealed dramatically reduced levels of the expression of the MnBEL1 in gynoecia from medusa mutant plants relative to wild type sisters. RNA seq analysis revealed that the medusa mutant seedpods failed to express MnBEL1 at wildtype levels. RNA sequence data comparisons between wildtype and medusa mutant seedpods also identified a set of genes, that are expressed in wildtype seedpods, but that are expressed at very low levels or that are not detected in the medusa seed pods. Homologues of many of these genes have been previously shown to be strongly expressed in female gametophyte in Arabidopsis. This RNA seq analysis further indicates a strong developmental disruption and identifies sets of genes with expected roles in female gametophyte development in Mimulus species for further investigation. Our evidence strongly suggests that MnBEL1 is a true functional orthologue of AtBEL1. To the best of our knowledge, these studies provide the first mutational analysis of a BEL1 orthologue outside of Arabidopsis and indicate the functional conservation of this gene between Mimulus and Arabidopsis. This work also presents the first identification of a mutational event in Mimulus that is linked to an insertion of a mitochondrial sequence in the nuclear genome.

Project description:Deciphering gene regulatory mechanisms through the analysis of high-throughput expression data is a challenging computational problem. Previous computational studies have used large expression datasets in order to resolve fine patterns of coexpression, producing clusters or modules of potentially coregulated genes. These methods typically examine promoter sequence information, such as DNA motifs or transcription factor occupancy data, in a separate step after clustering. We needed an alternative and more integrative approach to study the oxygen regulatory network in Saccharomyces cerevisiae using a small dataset of perturbation experiments. Mechanisms of oxygen sensing and regulation underlie many physiological and pathological processes, and only a handful of oxygen regulators have been identified in previous studies. We used a new machine learning algorithm called MEDUSA to uncover detailed information about the oxygen regulatory network using genome-wide expression changes in response to perturbations in the levels of oxygen, heme, Hap1, and Co2+. MEDUSA integrates mRNA expression, promoter sequence, and ChIP-chip occupancy data to learn a model that accurately predicts the differential expression of target genes in held-out data. We used a novel margin-based score to extract significant condition-specific regulators and assemble a global map of the oxygen sensing and regulatory network. This network includes both known oxygen and heme regulators, such as Hap1, Mga2, Hap4, and Upc2, as well as many new candidate regulators. MEDUSA also identified many DNA motifs that are consistent with previous experimentally identified transcription factor binding sites. Because MEDUSA's regulatory program associates regulators to target genes through their promoter sequences, we directly tested the predicted regulators for OLE1, a gene specifically induced under hypoxia, by experimental analysis of the activity of its promoter. In each case, deletion of the candidate regulator resulted in the predicted effect on promoter activity, confirming that several novel regulators identified by MEDUSA are indeed involved in oxygen regulation. MEDUSA can reveal important information from a small dataset and generate testable hypotheses for further experimental analysis.

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Loimia medusa

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