Browsing by Author "Becker, J.D."
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- Cyclooxygenase-2 (COX-2) expression by in vitro produced bovine embryos. Preliminary results = Expressão da ciclo-oxigenase-2 (COX-2) em embriões bovinos produzidos in vitro. Resultados preliminaresPublication . Marques, C.C.; Horta, A.E.; Vasques, M.I.; Baptista, M.C.; Becker, J.D.; Pimenta, J.; Pereira, R.M.Expression of the two isoforms of cyclooxygenase enzyme, cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), by in vitro produced bovine embryos was examined. Cumulus-oocyte complexes were recovered from ovaries of slaughtered animals and subsequently in vitro matured and in vitro fertilized. Presumptive zygotes were cultured in serum containing medium (TCM 199+10% bovine superovulated oestrus serum) on a granulosa cell monolayer for 12 days.
- Epigenetic reprogramming and small RNA silencing of transposable elements in pollenPublication . Slotkin, R.K.; Vaughn, M.; Borges, F.; Tanurdzic, M.; Becker, J.D.; Feijó, J.A.; Martienssen, R.The mutagenic activity of transposable elements (TEs) is suppressed by epigenetic silencing and small interfering RNAs (siRNAs), especially in gametes that could transmit transposed elements to the next generation. In pollen from the model plant Arabidopsis, we show that TEs are unexpectedly reactivated and transpose, but only in the pollen vegetative nucleus, which accompanies the sperm cells but does not provide DNA to the fertilized zygote. TE expression coincides with downregulation of the heterochromatin remodeler decrease in DNA methylation 1 and of many TE siRNAs. However, 21 nucleotide siRNAs from Athila retrotransposons are generated and accumulate in pollen and sperm, suggesting that siRNA from TEs activated in the vegetative nucleus can target silencing in gametes. We propose a conserved role for reprogramming in germline companion cells, such as nurse cells in insects and vegetative nuclei in plants, to reveal intact TEs in the genome and regulate their activity in gametes
- Gametophyte interaction and sexual reproduction: how plants make a zygotePublication . Boavida, L.C.; Vieira, A.M.; Becker, J.D.; Feijó, J.A.The evolutionary success of higher plants relies on a very short gametophytic phase, which underlies the sexual reproduction cycle. Sexual plant reproduction takes place in special organs of the flower: pollen, the male gametophyte, is released from the anthers and then adheres, grows and interacts along various tissues of the female organs, collectively known as the pistil. Finally, it fertilizes the female gametophyte, the embryo sac. Pollen is released as bi or tricellular, highly de-hydrated and presumably containing all the biochemical components and transcripts to germinate. Upon hydration on the female tissues, it develops a cytoplasmic extension, the pollen tube, which is one of the fastest growing cells in nature. Pollen is completely "ready-to-go", but despite this seemingly simple reaction, very complex interactions take place with the female tissues. In higher animals, genetic mechanisms for sex determination establish striking developmental differences between males and females. In contrast, most higher plant species develop both male and female structures within the same flower, allowing self-fertilization. Outcrossing is ensured by self-incompatibility mechanisms, which evolved under precise genetic control, controlling self-recognition and cell-to-cell interaction. Equally important is pollen selection along the female tissues, where interactions between different cell types with inherent signalling properties correspond to check-points to ensure fertilization. Last but not least, pollen-pistil interaction occurs in a way that enables the correct targeting of the pollen tubes to the receptive ovules. In this review, we cover the basic mechanisms underlying sexual plant reproduction, from the structural and cellular determinants, to the most recent genetic advances.
- Gene family analysis of the Arabidopsis pollen transcriptome reveals biological implications for cell growth, division control and gene expression regulationPublication . Pina, C.; Pinto, F.; Feijó, J.A.; Becker, J.D.Upon germination, pollen forms a tube that elongates dramatically through female tissues to reach and fertilize ovules. While essential for the life cycle of higher plants, the genetic basis underlying most of the process is not well understood. We previously used a combination of flow cytometry sorting of viable hydrated pollen grains and GeneChip array analysis of onethird of the Arabidopsis (Arabidopsis thaliana) genome to define a first overview of the pollen transcriptome. We now extend that study to approximately 80% of the genome of Arabidopsis by using Affymetrix Arabidopsis ATH1 arrays and perform comparative analysis of gene family and gene ontology representation in the transcriptome of pollen and vegetative tissues. Pollen grains have a smaller and overall unique transcriptome (6,587 genes expressed) with greater proportions of selectively expressed (11%) and enriched (26%) genes than any vegetative tissue. Relative gene ontology category representations in pollen and vegetative tissues reveal a functional skew of the pollen transcriptome toward signaling, vesicle transport, and the cytoskeleton, suggestive of a commitment to germination and tube growth. Cell cycle analysis reveals an accumulation of G2/Massociated factors that may play a role in the first mitotic division of the zygote. Despite the relative underrepresentation of transcription-associated transcripts, nonclassical MADS box genes emerge as a class with putative unique roles in pollen. The singularity of gene expression control in mature pollen grains is further highlighted by the apparent absence of small RNA pathway components.
- Genetic subtraction profiling identifies genes essential for Arabidopsis reproduction and reveals interaction between the female ganetophyte and the maternal sporophytePublication . Johnston, A.J.; Meier, P.; Gheyselinck, J.; Federer, M.; Wuest, A.E.J.; Schlagenhauf, E.; Becker, J.D.; Grossnikalus, U.The embryo sac contains the haploid maternal cell types necessary for double fertilization and subsequent seed development in plants. Large-scale identification of genes expressed in the embryo sac remains cumbersome because of its inherent microscopic and inaccessible nature. We used genetic subtraction and comparative profiling by microarray between the Arabidopsis thaliana wild-type and a sporophytic mutant lacking an embryo sac in order to identify embryo sac expressed genes in this model organism. The influences of the embryo sac on the surrounding sporophytic tissues were previously thought to be negligible or nonexistent; we investigated the extent of these interactions by transcriptome analysis.
- A genome-wide survey of sRNAs in the symbiotic nitrogen-fixing alpha-proteobacterium Sinorhizobium melilotiPublication . Schluter, J.P.; Reinkensmeier, J.; Daschkey, S.; Evguenieva-Hackenberg, E.; Janssen, S.; Janicke, S.; Becker, J.D.; Giegerich, R.; Becker, A.A total of 1,125 sRNA candidates that were classified as trans-encoded sRNAs (173), cis-encoded antisense sRNAs (117), mRNA leader transcripts (379), and sense sRNAs overlapping coding regions (456) were identified in a size range of 50 to 348 nucleotides. Among these were transcripts corresponding to 82 previously reported sRNA candidates. Enrichment for RNAs with primary 5'-ends prior to sequencing of cDNAs suggested transcriptional start sites corresponding to 466 predicted sRNA regions. The consensus sigma70 promoter motif CTTGAC-N17-CTATAT was found upstream of 101 sRNA candidates. Expression patterns derived from microarray hybridizations provided further information on conditions of expression of a number of sRNA candidates. Furthermore, GenBank, EMBL, DDBJ, PDB, and Rfam databases were searched for homologs of the sRNA candidates identified in this study. Searching Rfam family models with over 1,000 sRNA candidates, re-discovered only those sequences from S. meliloti already known and stored in Rfam, whereas BLAST searches suggested a number of homologs in related alpha-proteobacteria
- Genomic Expression Program Involving the Haalp-Regulation in Saccharomyces cerevisiae response to acetic acidPublication . Mira, N.P.; Becker, J.D.; Sa-Correia, I.The alterations occurring in yeast genomic expression during early response to acetic acid and the involvement of the transcription factor Haa1p in this transcriptional reprogramming are described in this study. Haa1p was found to regulate, directly or indirectly, the transcription of approximately 80% of the acetic acid-activated genes, suggesting that Haa1p is the main player in the control of yeast response to this weak acid. The genes identified in this work as being activated in response to acetic acid in a Haa1p-dependent manner include protein kinases, multidrug resistance transporters, proteins involved in lipid metabolism, in nucleic acid processing, and proteins of unknown function. Among these genes, the expression of SAP30 and HRK1 provided the strongest protective effect toward acetic acid. SAP30 encode a subunit of a histone deacetylase complex and HRK1 encode a protein kinase belonging to a family of protein kinases dedicated to the regulation of plasma membrane transporters activity. The deletion of the HRK1 gene was found to lead to the increase of the accumulation of labeled acetic acid into acid-stressed yeast cells, suggesting that the role of both HAA1 and HRK1 in providing protection against acetic acid is, at least partially, related with their involvement in the reduction of intracellular acetate concentration.
- Laser-microdissection unravels cell-type specific transcription in > arbuscular mycorrhizal roots, including CAAT-box TF gene expression correlating with fungal contact and spreadPublication . Hogekamp, C.; Arndt, D.; Pereira, P.A.; Becker, J.D.; Hohnjec, N.; Kuster, H.Arbuscular mycorrhizae (AM) are the most widespread symbioses on Earth, promoting nutrient supply of most terrestrial plant species. To unravel gene expression in defined stages of Medicago truncatula root colonization by AM fungi, we here combined genome-wide transcriptome profiling based on whole mycorrhizal roots with real-time RT-PCR experiments that relied on characteristic cell-types obtained via laser-microdissection. Our genome-wide approach delivered a core set of 512 genes significantly activated by the two mycorrhizal fungi Glomus intraradices and Glomus mossae. Focussing on 62 of these genes being related to membrane transport, signaling, and transcriptional regulation, we distinguished whether they are activated in arbuscule-containing or the neighbouring cortical cells harbouring fungal hyphae. In addition, cortical cells from non-mycorrhizal roots served as a reference for gene expression under non-colonized conditions. Our analysis identified 25 novel arbuscule-specific genes and 37 genes expressed both in the arbuscule-containing and the adjacent cortical cells colonized by fungal hyphae. Amongst the AM-induced genes specifying transcriptional regulators were two members encoding CAAT-box binding transcription factors (CBF), designated MtCbf1 and MtCbf2. Promoter analyses demonstrated that both genes were already activated by the first physical contact between the symbionts. Subsequently, and corresponding to our cell-type expression patterns, they were progressively up-regulated in those cortical areas colonized by fungal hyphae, including the arbuscule-containing cells. The encoded CBFs thus represent excellent candidates for regulators that mediate a sequential reprogramming of root tissues during the establishment of an AM symbiosis
- MicroRNA activity in the Arabidopsis male germlinePublication . Borges, F.; Pereira, P.A.; Slotkin, R.K.; Martienssen, R.A.; Becker, J.D.Most of the core proteins involved in the microRNA (miRNA) pathway in plants have been identified, and almost simultaneously hundreds of miRNA sequences processed in the Arabidopsis sporophyte have been discovered by exploiting next-generation sequencing technologies. However, there is very limited understanding about potentially distinct mechanisms of post-transcriptional regulation between different cell lineages. In this review the focus is on the Arabidopsis male gametophyte (pollen), where the germline differentiates after meiosis giving rise to the male gametes. Based on comparative analysis of miRNAs identified in sperm cells by in-depth sequencing, their possible functions during germ cell specification and beyond fertilization are discussed. In addition, 25 potentially novel miRNAs processed in sperm cells and pollen were identified, as well as enriched variations in the sequence length of known miRNAs, which might indicate subfunctionalization by association with a putative germline-specific Argonaute complex. ARGONAUTE 5 (AGO5), by close homology to AGO1 and localizing preferentially to the sperm cell cytoplasm in mature pollen, may be part of such a complex.
- Mucoid morphotype variation of Burkholderia multivorans during chronic cystic fibrosis lung infection is correlated with changes in metabolism, motility, biofilm formation and virulencePublication . Silva, I.N.; Ferreira, A.S.; Becker, J.D.; Zlosnik, J.E.A.; Speert, D.P.; He, J.; Mil-Homens, D.; Moreira, L.M.Burkholderia cepacia complex (Bcc) bacteria are opportunistic pathogens infecting hosts such as cystic fibrosis (CF) patients. Long-term Bcc infection of CF patients' airways has been associated with emergence of phenotypic variation. Here we studied two Burkholderia multivorans clonal isolates displaying different morphotypes from a chronically infected CF patient to evaluate trait development during lung infection. Expression profiling of mucoid D2095 and non-mucoid D2214 isolates revealed decreased expression of genes encoding products related to virulence-associated traits and metabolism in D2214. Furthermore, D2214 showed no exopolysaccharide production, lower motility and chemotaxis, and more biofilm formation, particularly under microaerophilic conditions, than the clonal mucoid isolate D2095. When Galleria mellonella was used as acute infection model, D2214 at a cell number of approximately 7×10(6) c.f.u. caused a higher survival rate than D2095, although 6 days post-infection most of the larvae were dead. Infection with the same number of cells by mucoid D2095 caused larval death by day 4. The decreased expression of genes involved in carbon and nitrogen metabolism may reflect lower metabolic needs of D2214 caused by lack of exopolysaccharide, but also by the attenuation of pathways not required for survival. As a result, D2214 showed higher survival than D2095 in minimal medium for 28 days under aerobic conditions. Overall, adaptation during Bcc chronic lung infections gave rise to genotypic and phenotypic variation among isolates, contributing to their fitness while maintaining their capacity for survival in this opportunistic human niche.