Detection of Sad genes in various species of Neurospora

Abstract only availableNeurospora crassa is a haploid fungus that reproduces asexually during vegetative growth. However, in a nitrogen deficient environment, the two mating-type cells, A and a, can fuse together and enter the sexual cycle. During this transient diploid stage, a post-transcriptional gene silencing (PTGS) mechanism silences the expression of any unpaired gene. This mechanism is called meiotic silencing by unpaired DNA (MSUD) (Shiu, et al., 2001; Cell 107: 905-916). MSUD occurs when a gene is not paired with a homologue during meiosis. The unpaired DNA segment generates a sequence-specific signal, causing any paired or unpaired copies of that gene to be silenced. Two required genes for meiotic silencing have been identified; sad-1 encodes an RNA-directed RNA polymerase while sad-2 encodes a novel protein. Interspecific crosses between N. crassa and other related species are known to be infertile. This infertility may be due rearrangements caused by inversion and/or translocation. These could disrupt the pairings of genes needed for meiosis and ascus development, causing these genes to be silenced, resulting in sterility. This hypothesis is validated by observing that interspecific crosses within the genus Neurospora become much more fertile when the N. crassa parent contains a Sad-1 mutation. If MSUD is in other Neurospora species, it may represent another mechanism for speciation. This research project focuses to determine whether sad-1 and sad-2 are conserved in related species of Neurospora. Homologues of the sad genes were amplified in several fungal isolates by PCR (Polymerase Chain Reaction), using primers designed for those sequences. We have discovered that sad-1 and sad-2 can be found in several different Neurospora species; sad-1 is present in N. sitophila, N. tetrasperma, N. dodgei, N. galapagosensis, and N. africana, while sad-2 is found in N. sitophila, N. tetrasperma, and N. intermedia. The presence of sad genes in these species suggests MSUD may contribute to their reproductive isolation.Life Sciences Fellowships/Meyerhoff Scholars Progra

Investigating the relationship between repeated DNA and gene silencing in the model organism Neurospora crassa

Abstract only availableIn order to maintain genomic integrity, organisms must possess the capability to combat unwanted elements such as retroviruses and transposons. In the filamentous fungus Neurospora crassa, the expression from repeated genetic elements is turned off, or silenced, in a post-transcriptional manner. It is thus possible to induce gene silencing in N. crassa by inserting tandem copies of a transgene into its genome. However, the number and arrangement of transgenes required to activate this process, known as quelling, is unclear. Additionally, silenced strains tend to revert to an unsilenced phenotype after several generations. With the goal of creating a genetically-defined and stably quelled strain, we are examining the effect of various tandem transgene repeats on N. crassa. We have designed plasmid vectors containing 1 to 11 ~1kb fragments of the N. crassa caratenoid pigment producing gene, albino-1 (or al-1). Depending on the number of these constructs in a transformant, the al-1 gene could remain unsilenced (orange conidia), or be partially to completely silenced (light orange to white conidia). Transformants are currently being assayed for their spore color as well as the presence or absence of the transgene repeats. Although this work is still in progress, we have identified a transformant with a putative 5-repeat al-1 transgene that displays a slight quelling phenotype (yellow-light orange conidia). These data suggest that quelling requires the introduction of at least 5 copies of a transgene.NSF-REU Program in Biological Sciences & Biochemistr

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

A CR-hydro-NEI Model of Multi-wavelength Emission from the Vela Jr. Supernova Remnant (SNR RX J0852.0-4622)

Based largely on energy budget considerations and the observed cosmic-ray (CR) ionic composition, supernova remnant (SNR) blast waves are the most likely sources of CR ions with energies at least up to the "knee" near 3 PeV. Shocks in young shell-type TeV-bright SNRs are surely producing TeV particles, but the emission could be dominated by ions producing neutral pion-decay emission or electrons producing inverse-Compton gamma-rays. Unambiguously identifying the GeV-TeV emission process in a particular SNR will not only help pin down the origin of CRs, it will add significantly to our understanding of the diffusive shock acceleration (DSA) mechanism and improve our understanding of supernovae and the impact SNRs have on the circumstellar medium. In this study, we investigate the Vela Jr. SNR, an example of TeV-bright non-thermal SNRs. We perform hydrodynamic simulations coupled with non-linear DSA and non-equilibrium ionization near the forward shock (FS) to confront currently available multi-wavelength data. We find, with an analysis similar to that used earlier for SNR RX J1713.7-3946, that self-consistently modeling the thermal X-ray line emission with the non-thermal continuum in our one-dimensional model strongly constrains the fitting parameters, and this leads convincingly to a leptonic origin for the GeV-TeV emission for Vela Jr. This conclusion is further supported by applying additional constraints from observation, including the radial brightness profiles of the SNR shell in TeV gamma-rays, and the spatial variation of the X-ray synchrotron spectral index. We will discuss implications of our models on future observations by the next-generation telescopes.Comment: 12 pages, 10 figures, to appear at the Astrophysical Journa

Map-based cloning and characterization of meiotic drive resistance

Abstract only availableRecent sequencing of the Neurospora crassa genome by The Broad Institute, in collaboration with the Neurospora research community, has given considerable insight into the understanding of Neurospora's DNA as well as its molecular structure and function. With this sequencing information, we are able to more easily characterize important loci within the N. crassa genome. One such locus, Spore killer-3 (Sk-3), is of particular interest to our laboratory. Sk-3 is considered a meiotic drive element because it segregates in excess of its Mendelian proportion during mating. In other words, instead of half the offspring receiving the Sk-3 allele, and the other half receiving the wild type allele, the Sk-3 allele is passed to nearly all of the offspring. Meiotic drive elements are often categorized as "selfish genetic elements." The reason for our interest in selfish genetic elements is that many of them cause diseases in humans. Model systems, like N. crassa, may reveal clues to their functions in higher eukaryotes. In this project, we are examining the Spore killer resistance gene (r(Sk-3)) in an attempt to gain further insight into the purpose and function of Sk-3 and ultimately of meiotic drive elements. The N. crassa genome sequence annotation has putatively placed the r(Sk-3) gene to the left of the centromere on chromosome III of the N. crassa genome. Using closely linked loci, some of which contain the hph (resistance to hygromycin) marker, we have utilized a standard three-point cross strategy to refine the known location of r(Sk-3). Mapping with additional markers is currently in progress to help narrow the r(Sk-3) locus to a ~20kb region. Genes from this region will be individually cloned and introduced into a Sk-3 susceptible host. Resistance to killing in the transformants will be used to identify the r(Sk-3) activity.Life Sciences Undergraduate Research Opportunity Progra

The Impact of Progenitor Mass Loss on the Dynamical and Spectral Evolution of Supernova Remnants

There is now substantial evidence that the progenitors of some core-collapse supernovae undergo enhanced or extreme mass loss prior to explosion. The imprint of this mass loss is observed in the spectra and dynamics of the expanding blastwave on timescales of days to years after core-collapse, and the effects on the spectral and dynamical evolution may linger long after the supernova has evolved into the remnant stage. In this paper, we present for the first time, largely self-consistent end-to-end simulations for the evolution of a massive star from the pre-main sequence, up to and through core collapse, and into the remnant phase. We present three models and compare and contrast how the progenitor mass loss history impacts the dynamics and spectral evolution of the supernovae and supernova remnants. We study a model which only includes steady mass loss, a model with enhanced mass loss over a period of $\sim$ 5000 years prior to core-collapse, and a model with extreme mass loss over a period of $\sim$ 500 years prior to core collapse. The models are not meant to address any particular supernova or supernova remnant, but rather to highlight the important role that the progenitor evolution plays in the observable qualities of supernovae and supernova remnants. Through comparisons of these three different progenitor evolution scenarios, we find that the mass loss in late stages (during and after core carbon burning) can have a profound impact on the dynamics and spectral evolution of the supernova remnant centuries after core-collapse.Comment: 18 pages, 11 figures; submitted to the Astrophysical Journa

Meiotic Silencing by Unpaired DNA

AbstractThe silencing of gene expression by segments of DNA present in excess of the normal number is called cosuppression in plants and quelling in fungi. We describe a related process, meiotic silencing by unpaired DNA (MSUD). DNA unpaired in meiosis causes silencing of all DNA homologous to it, including genes that are themselves paired. A semidominant Neurospora mutant, Sad-1, fails to perform MSUD. Sad-1 suppresses the sexual phenotypes of many ascus-dominant mutants. MSUD may provide insights into the function of genes necessary for meiosis, including genes for which ablation in vegetative life would be lethal. It may also contribute to reproductive isolation of species within the genus Neurospora. The wild-type allele, sad-1+, encodes a putative RNA-directed RNA polymerase