Sequences important for heterokaryon incompatibility function in MAT A-1 of Neurospora crassa

Using chimeric constructs between the Neurospora crassa mat A-1 gene and the Podospora anserina FMR1 gene, we identified the amino acids important for the heterokaryon incompatibility function in the mating-type protein MAT A-1. Strains of Neurospora crassa exist as two alternative mating type forms, A and a; differences in mating type are required for the initiation of the sexual cycle (Shiu and Glass, 2000). The mating-type (mat) locus also acts as a heterokaryon incompatibility (het) locus, such that hyphal fusion between A and a strains results in a heterokaryon that shows extremely inhibited growth, absence of conidiation, and hyphal compartmentation and death (Glass et al., 2000). The A and a mating type sequences occupy the same locus in A and a strains, but are highly dissimilar in sequence. The mat a-1 gene, which encodes a putative HMG (high mobility group) type of transcriptional regulator, provides all the functions for the a mating type, including mating, ascospore formation, and heterokaryon incompatibility (Chang and Staben, 1994). The mat A locus encodes three proteins. MAT A-2 and MAT A-3 are responsible for ascospore formation (Ferreira et al., 1998); MAT A-3 is a putative HMG type of transcriptional regulator. MAT A-1 is predicted to be a á-domain type of transcriptional regulator and is both necessary and sufficient to confer A mating specificity and trigger heterokaryon incompatibility with a strains (Glass et al., 1990). Mutations in an unlinked locus, tol, suppress mating-type incompatibility such that tol A and tol a strains are capable of forming a vigorous heterokaryon (Newmeyer, 1970; Shiu and Glass, 1999). To determine the region required for the heterokaryon incompatibility function in MAT A-1 (293 amino acids), a series o

SAD-3, a Putative Helicase Required for Meiotic Silencing by Unpaired DNA, Interacts with Other Components of the Silencing Machinery

In Neurospora crassa, genes lacking a pairing partner during meiosis are suppressed by a process known as meiotic silencing by unpaired DNA (MSUD). To identify novel MSUD components, we have developed a high-throughput reverse-genetic screen for use with the N. crassa knockout library. Here we describe the screening method and the characterization of a gene (sad-3) subsequently discovered. SAD-3 is a putative helicase required for MSUD and sexual spore production. It exists in a complex with other known MSUD proteins in the perinuclear region, a center for meiotic silencing activity. Orthologs of SAD-3 include Schizosaccharomyces pombe Hrr1, a helicase required for RNAi-induced heterochromatin formation. Both SAD-3 and Hrr1 interact with an RNA-directed RNA polymerase and an Argonaute, suggesting that certain aspects of silencing complex formation may be conserved between the two fungal species

The Non-Coding RNA Journal Club: Highlights on Recent Papers—11

We are delighted to share with you our eleventh Journal Club and highlight some of the most interesting papers published recently [...

Identification and Functional Analysis of Light-Responsive Unique Genes and Gene Family Members in Rice

Functional redundancy limits detailed analysis of genes in many organisms. Here, we report a method to efficiently overcome this obstacle by combining gene expression data with analysis of gene-indexed mutants. Using a rice NSF45K oligo-microarray to compare 2-week-old light- and dark-grown rice leaf tissue, we identified 365 genes that showed significant 8-fold or greater induction in the light relative to dark conditions. We then screened collections of rice T-DNA insertional mutants to identify rice lines with mutations in the strongly light-induced genes. From this analysis, we identified 74 different lines comprising two independent mutant lines for each of 37 light-induced genes. This list was further refined by mining gene expression data to exclude genes that had potential functional redundancy due to co-expressed family members (12 genes) and genes that had inconsistent light responses across other publicly available microarray datasets (five genes). We next characterized the phenotypes of rice lines carrying mutations in ten of the remaining candidate genes and then carried out co-expression analysis associated with these genes. This analysis effectively provided candidate functions for two genes of previously unknown function and for one gene not directly linked to the tested biochemical pathways. These data demonstrate the efficiency of combining gene family-based expression profiles with analyses of insertional mutants to identify novel genes and their functions, even among members of multi-gene families

Refinement of Light-Responsive Transcript Lists Using Rice Oligonucleotide Arrays: Evaluation of Gene-Redundancy

Studies of gene function are often hampered by gene-redundancy, especially in organisms with large genomes such as rice (Oryza sativa). We present an approach for using transcriptomics data to focus functional studies and address redundancy. To this end, we have constructed and validated an inexpensive and publicly available rice oligonucleotide near-whole genome array, called the rice NSF45K array. We generated expression profiles for light- vs. dark-grown rice leaf tissue and validated the biological significance of the data by analyzing sources of variation and confirming expression trends with reverse transcription polymerase chain reaction. We examined trends in the data by evaluating enrichment of gene ontology terms at multiple false discovery rate thresholds. To compare data generated with the NSF45K array with published results, we developed publicly available, web-based tools (www.ricearray.org). The Oligo and EST Anatomy Viewer enables visualization of EST-based expression profiling data for all genes on the array. The Rice Multi-platform Microarray Search Tool facilitates comparison of gene expression profiles across multiple rice microarray platforms. Finally, we incorporated gene expression and biochemical pathway data to reduce the number of candidate gene products putatively participating in the eight steps of the photorespiration pathway from 52 to 10, based on expression levels of putatively functionally redundant genes. We confirmed the efficacy of this method to cope with redundancy by correctly predicting participation in photorespiration of a gene with five paralogs. Applying these methods will accelerate rice functional genomics

CMS physics technical design report : Addendum on high density QCD with heavy ions

Peer reviewe

Riociguat treatment in patients with chronic thromboembolic pulmonary hypertension: Final safety data from the EXPERT registry

Objective: The soluble guanylate cyclase stimulator riociguat is approved for the treatment of adult patients with pulmonary arterial hypertension (PAH) and inoperable or persistent/recurrent chronic thromboembolic pulmonary hypertension (CTEPH) following Phase