Reprogrammed Transcriptome in Rhesus-Bovine Interspecies Somatic Cell Nuclear Transfer Embryos

Global activation of the embryonic genome (EGA), one of the most critical steps in early mammalian embryo development, is recognized as the time when interspecies somatic cell nuclear transfer (iSCNT) embryos fail to thrive.In this study, we analyzed the EGA-related transcriptome of rhesus-bovine iSCNT 8- to 16-cell embryos and dissected the reprogramming process in terms of embryonic gene activation, somatic gene silencing, and maternal RNA degradation. Compared with fibroblast donor cells, two thousand and seven genes were activated in iSCNT embryos, one quarter of them reaching expression levels comparable to those found in in vitro fertilized (IVF) rhesus embryos. This suggested that EGA in iSCNT embryos had partially recapitulated rhesus embryonic development. Eight hundred and sixty somatic genes were not silenced properly and continued to be expressed in iSCNT embryos, which indicated incomplete nuclear reprogramming. We compared maternal RNA degradation in bovine oocytes between bovine-bovine SCNT and iSCNT embryos. While maternal RNA degradation occurred in both SCNT and iSCNT embryos, we saw more limited overall degradation of maternal RNA in iSCNT embryos than in SCNT embryos. Several important maternal RNAs, like GPF9, were not properly processed in SCNT embryos.Our data suggested that iSCNT embryos are capable of triggering EGA, while a portion of somatic cell-associated genes maintain their expression. Maternal RNA degradation seems to be impaired in iSCNT embryos. Further understanding of the biological roles of these genes, networks, and pathways revealed by iSCNT may expand our knowledge about cell reprogramming, pluripotency, and differentiation

Septation of Infectious Hyphae Is Critical for Appressoria Formation and Virulence in the Smut Fungus Ustilago Maydis

Differentiation of hyphae into specialized infection structures, known as appressoria, is a common feature of plant pathogenic fungi that penetrate the plant cuticle. Appressorium formation in U. maydis is triggered by environmental signals but the molecular mechanism of this hyphal differentiation is largely unknown. Infectious hyphae grow on the leaf surface by inserting regularly spaced retraction septa at the distal end of the tip cell leaving empty sections of collapsed hyphae behind. Here we show that formation of retraction septa is critical for appressorium formation and virulence in U. maydis. We demonstrate that the diaphanous-related formin Drf1 is necessary for actomyosin ring formation during septation of infectious hyphae. Drf1 acts as an effector of a Cdc42 GTPase signaling module, which also consists of the Cdc42-specific guanine nucleotide exchange factor Don1 and the Ste20-like kinase Don3. Deletion of drf1, don1 or don3 abolished formation of retraction septa resulting in reduced virulence. Appressorium formation in these mutants was not completely blocked but infection structures were found only at the tip of short filaments indicating that retraction septa are necessary for appressorium formation in extended infectious hyphae. In addition, appressoria of drf1 mutants penetrated the plant tissue less frequently

A tripartite paternally methylated region within the Gpr1-Zdbf2 imprinted domain on mouse chromosome 1 identified by meDIP-on-chip

The parent-of-origin specific expression of imprinted genes relies on DNA methylation of CpG-dinucleotides at differentially methylated regions (DMRs) during gametogenesis. To date, four paternally methylated DMRs have been identified in screens based on conventional approaches. These DMRs are linked to the imprinted genes H19, Gtl2 (IG-DMR), Rasgrf1 and, most recently, Zdbf2 which encodes zinc finger, DBF-type containing 2. In this study, we applied a novel methylated-DNA immunoprecipitation-on-chip (meDIP-on-chip) method to genomic DNA from mouse parthenogenetic- and androgenetic-derived stem cells and sperm and identified 458 putative DMRs. This included the majority of known DMRs. We further characterized the paternally methylated Zdbf2/ZDBF2 DMR. In mice, this extensive germ line DMR spanned 16 kb and possessed an unusual tripartite structure. Methylation was dependent on DNA methyltransferase 3a (Dnmt3a), similar to H19 DMR and IG-DMR. In both humans and mice, the adjacent gene, Gpr1/GPR1, which encodes a G-protein-coupled receptor 1 protein with transmembrane domain, was also imprinted and paternally expressed. The Gpr1-Zdbf2 domain was most similar to the Rasgrf1 domain as both DNA methylation and the actively expressed allele were in cis on the paternal chromosome. This work demonstrates the effectiveness of meDIP-on-chip as a technique for identifying DMRs

Transcriptome-Wide Identification of Novel Imprinted Genes in Neonatal Mouse Brain

Imprinted genes display differential allelic expression in a manner that depends on the sex of the transmitting parent. The degree of imprinting is often tissue-specific and/or developmental stage-specific, and may be altered in some diseases including cancer. Here we applied Illumina/Solexa sequencing of the transcriptomes of reciprocal F1 mouse neonatal brains and identified 26 genes with parent-of-origin dependent differential allelic expression. Allele-specific Pyrosequencing verified 17 of them, including three novel imprinted genes. The known and novel imprinted genes all are found in proximity to previously reported differentially methylated regions (DMRs). Ten genes known to be imprinted in placenta had sufficient expression levels to attain a read depth that provided statistical power to detect imprinting, and yet all were consistent with non-imprinting in our transcript count data for neonatal brain. Three closely linked and reciprocally imprinted gene pairs were also discovered, and their pattern of expression suggests transcriptional interference. Despite the coverage of more than 5000 genes, this scan only identified three novel imprinted refseq genes in neonatal brain, suggesting that this tissue is nearly exhaustively characterized. This approach has the potential to yield an complete catalog of imprinted genes after application to multiple tissues and developmental stages, shedding light on the mechanism, bioinformatic prediction, and evolution of imprinted genes and diseases associated with genomic imprinting

How doctors generate diagnostic hypotheses: a study of radiological diagnosis with functional magnetic resonance imaging.

In medical practice, diagnostic hypotheses are often made by physicians in the first moments of contact with patients; sometimes even before they report their symptoms. We propose that generation of diagnostic hypotheses in this context is the result of cognitive processes subserved by brain mechanisms that are similar to those involved in naming objects or concepts in everyday life

Efeito da luz e temperatura na germinação e crescimento inicial de Vochysia tucanorum Mart., Vochysiaceae, em solo de cerrado e mata sob diferentes níveis de radiação

Vochysia tucanorum Mart. (Vochysiaceae) is a very widely distributed species in the cerrado and forests in Brazil, from the State of Bahia to Paraná. Its seed germination was analysed under eight treatments using different conditions of light and temperature. Up to 73 days, at least ca. 93% of the seeds germinated in all treatments, except under alternating temperatures of 35°/10°C (62%). Light enhanced germination, except under constant temperature of 25°C when seeds are not photoblastic. At this temperature, the highest percentage of germination was found. Initial vegetative growth was analysed by comparing the effects of shading, by means of different percentages of full daylight (100%, 45%, 10.6%, 4.8%), and soils from cerrado and forest. During 123 days, no mortality was recorded and cotyledons persisted on the seedlings of all treatments. In general, the seedlings of V. tucanorum were found to be more or less indifferent to the soil from cerrado or forest and shading seems to promote height increment. However, total dry mass was larger under a radiation of 45% full daylight and smaller under extreme conditions (100% or 4.8% full daylight).Vochysia tucanorum Mart. (Vochysiaceae) é uma espécie amplamente distribuída em cerrado e florestas do Brasil, do estado da Bahia até o Paraná. Sua germinação foi analisada sob oito tratamentos usando diferentes condições de luz e temperatura. Até o 73 dia, pelo menos cerca de 93% das sementes germinaram em todos os tratamentos, exceto em alternância de temperaturas de 35°/10°C (62%). A luz favoreceu a germinação, exceto sob temperatura constante de 25°C, na qual as sementes não se mostraram fotoblásticas. Nesta temperatura, foi encontrada a maior porcentagem de germinação. O crescimento inicial foi analisado comparando-se os efeitos de diferentes porcentagens da luz do dia (100%, 45%, 10,6%, 4,8%), e do solo de cerrado e mata. Durante 123 dias não houve mortalidade e os cotilédones persistiram em plântulas de todos os tratamentos. Em geral, as plântulas de V. tucanorum foram indiferentes ao tipo de solo, sendo que o sombreamento parece ter promovido incremento na altura das plantas. Entretanto, a massa seca total foi maior sob a radiação de 45% da luz solar e menor sob as condições extremas utilizadas (100% ou 4,8% de pleno sol).275280Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES