H. Sofia Pereira

Bisphenol A at concentrations found in human serum induces aneugenic effects in endothelial cells.

Bisphenol A (BPA) is an endocrine disrupting chemical to which humans are exposed. Continuous environmental exposure to BPA leads to its detection in the majority of individuals from developed countries, with serum concentrations ranging from 0.5 to 10ng/ml in the general population and much higher when associated with occupational exposure. In this work, human umbilical vascular endothelial cells (HUVEC) and human colon adenocarcinona (HT29) cell lines were used to represent endothelial and digestive-tract tissues, which are in direct contact to BPA in vivo. Our results demonstrate that BPA has cell-type differential effects. Notably, BPA concentrations commonly found in humans induce micronucleus formation and interfere with cell-division processes in endothelial cells, resulting in mitotic abnormalities. We also found that BPA induces up-regulation of two genes encoding proteins associated with chromosome segregation, namely CDCA8 (borealin/cell division cycle A8) and SGOL2 (shugoshin-like2). Taken together, the aneugenic effects observed in endothelial cells (HUVECs) substantiate increasing concerns about BPA exposure at levels currently detected in humans.

Polyploidization as a retraction force in plant genome evolution: sequence rearrangements in triticale.

Background Polyploidization is a major evolutionary process in plants where hybridization and chromosome doubling induce enormous genomic stress and can generate genetic and epigenetic modifications. However, proper evaluation of DNA sequence restructuring events and the precise characterization of sequences involved are still sparse. Methodology/Principal Findings Inter Retrotransposons Amplified Polymorphism (IRAP), Retrotransposons Microsatellite Amplified Polymorphism (REMAP) and Inter Simple Sequence Repeat (ISSR) largely confirmed the absence of any intraspecific variation in wheat, rye and triticale. The comparative analysis of banding profiles between wheat and rye inbred lines revealed 34% of monomorphic (common to both parental species) bands for the ten different primer combinations used. The analysis of triticale plants uncovered nearly 51% of rearranged bands in the polyploid, being the majority of these modifications, due to the loss of rye bands (83%). Sequence analysis of rye fragments absent in triticale revealed for instance homology with hydroxyproline-rich glycoproteins (HRGP), a protein that belongs to a major family of inducible defence response proteins. Conversely, a wheat-specific band absent in triticale comprises a nested structure of copia-like retrotransposons elements, namely Claudia and Barbara. Sequencing of a polyploid-specific band (absent in both parents) revealed a microsatellite related sequence. Cytological studies using Fluorescent In Situ Hybridization (FISH) with REMAP products revealed a widespread distribution of retrotransposon and/or microsatellite flanking sequences on rye chromosomes, with a preferential accumulation in heterochromatic sub-telomeric domains. Conclusions/Significance Here, we used PCR-based molecular marker techniques involving retrotransposons and microsatellites to uncover polyploidization induced genetic restructuring in triticale. Sequence analysis of rearranged genomic fragments either from rye or wheat origin showed these to be retrotransposon-related as well as coding sequences. Further FISH analysis revealed possible chromosome hotspots for sequence rearrangements. The role of chromatin condensation on the origin of genomic rearrangements mediated by polyploidization in triticale is also discussed.

Genomic analysis of Grapevine Retrotransposon 1 (Gret1) in Vitis vinifera

The complete sequence of the first retrotransposon isolated in Vitis vinifera, Gret1, was used to design primers that permitted its analysis in the genome of grapevine cultivars. This retroelement was found to be dispersed throughout the genome with sites of repeated insertions. Fluorescent in situ hybridization indicated multiple Gret1loci distributed throughout euchromatic portions of chromosomes. REMAP and IRAP proved to be useful as molecular markers in grapevine. Both of these techniques showed polymorphisms between cultivars but not between clones of the same cultivar, indicating differences in Gret1 distribution between cultivars. The combined cytological and molecular results suggest that Gret1 may have a role in gene regulation and in explaining the enormous phenotypic variability that exists between cultivars.

Interplay of ribosomal DNA Loci in nucleolar dominance: dominant NORs are up-regulated by chromatin dynamics in the wheat-rye system

Background Chromatin organizational and topological plasticity, and its functions in gene expression regulation, have been strongly revealed by the analysis of nucleolar dominance in hybrids and polyploids where one parental set of ribosomal RNA (rDNA) genes that are clustered in nucleolar organizing regions (NORs), is rendered silent by epigenetic pathways and heterochromatization. However, information on the behaviour of dominant NORs is very sparse and needed for an integrative knowledge of differential gene transcription levels and chromatin specific domain interactions. Methodology/Principal Findings Using molecular and cytological approaches in a wheat-rye addition line (wheat genome plus the rye nucleolar chromosome pair 1R), we investigated transcriptional activity and chromatin topology of the wheat dominant NORs in a nucleolar dominance situation. Herein we report dominant NORs up-regulation in the addition line through quantitative real-time PCR and silver-staining technique. Accompanying this modification in wheat rDNA trascription level, we also disclose that perinucleolar knobs of ribosomal chromatin are almost transcriptionally silent due to the residual detection of BrUTP incorporation in these domains, contrary to the marked labelling of intranucleolar condensed rDNA. Further, by comparative confocal analysis of nuclei probed to wheat and rye NORs, we found that in the wheat-rye addition line there is a significant decrease in the number of wheat-origin perinucleolar rDNA knobs, corresponding to a diminution of the rDNA heterochromatic fraction of the dominant (wheat) NORs. Conclusions/Significance We demonstrate that inter-specific interactions leading to wheat-origin NOR dominance results not only on the silencing of rye origin NOR loci, but dominant NORs are also modified in their transcriptional activity and interphase organization. The results show a cross-talk between wheat and rye NORs, mediated by ribosomal chromatin dynamics, revealing a conceptual shift from differential amphiplasty to ‘mutual amphiplasty’ in the nucleolar dominance process.

Rye Bs Disclose Ancestral Sequences in Cereal Genomes with a Potential Role in Gametophyte Chromatid Segregation

Two sequence families, E3900 and D1100, are amplified on the subtelomeric domain of the long arm of rye B chromosomes, the region that controls its drive mechanism. In this work, polymerase chain reaction (PCR) with a number of primers spanning E3900 shows that the organization and nucleotide sequence of E3900-related portions are present and highly conserved on rye A chromosomes as well as in other cereals. Quantitative Real-Time PCR estimates two E3900 sequences to be represented in 100-150 copies on Bs and at least as single copies on As. A novel E3900-related sequence, with a deletion that results in a frameshift and subsequently an open reading frame with putative DNA binding motifs, is identified. Expression analysis of E3900 indicates identical transcription levels in leaves from plants with and without Bs, showing that the expression of these sequences must be silenced on Bs and tightly regulated on As in leaves. In contrast, E3900 transcription is upregulated during meiosis exclusively in plants with Bs, maintaining a high level of transcription in the gametophyte. Interestingly, Bs not only influence their own chromatid segregation but also that of the regular chromosome complement of both rye and wheat. There is a drastic increase in frequency of disrupted metaphase and anaphase cells in the first mitosis of pollen grains carrying Bs, which appears to be due to anomalous adherences between sister chromatids. Taken together, this work provides insight into how E3900 sequences are potentially associated with important evolutionary mechanisms involved in basic cellular processes.

Rye (Secale cereale) supernumerary (B) chromosomes associated with heat tolerance during early stages of male sporogenesis

Background and aims Rye supernumerary (B) chromosomes have an accumulation mechanism involving the B subtelomeric domain highly enriched in D1100- and E3900-related sequences. In this work, the effects of heat stress during the early stages of male meiosis in 0B and +B plants were studied. Methods In-depth cytological analyses of chromatin structure and behaviour were performed on staged rye meiocytes utilizing DAPI, fluorescence in situ hybridization and 5-methylcytosine immune labelling. Quantitative real-time PCR was used to measure heat effects on the expression of the Hsp101 gene as well as the 3·9- and 2·7-kb E3900 forms in various tissues and meiotic stages. Key Results and Conclusions Quantitative real-time PCR established that heat induced equal up-regulation of the Hsp101 gene in 0B and 2B plants, with a marked peak in anthers with meiocytes staged at pachytene. Heat also resulted in significant up-regulation of E3900-related transcripts, especially at pachytene and for the truncated 2·7-kb form of E3900. Cytological heat-induced anomalies in prophase I, measured as the frequency of anomalous meiocytes, were significantly greater in 0B plants. Whereas telomeric sequences were widely distributed in a manner close to normal in the majority of 2B pachytene cells, most 0B meiocytes displayed abnormally clustered telomeres after chromosome pairing had occurred. Relevantly, bioinformatic analysis revealed a significantly high-density heat responsive cis regulatory sequence on E3900, clearly supporting stress-induced response of transcription for the truncated variant. Taken together, these results are the first indication that rye B chromosomes have implications on heat tolerance and may protect meiocytes against heat stress-induced damage.

Bisphenol A alters transcript levels of biomarker genes for Major Depressive Disorder in vascular endothelial cells and colon cancer cells

Bisphenol A (BPA) is capable of mimicking endogenous hormones with potential consequences for human health and BPA exposure has been associated with several human diseases including neuropsychiatric disorders. Here, quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) results show that BPA at low concentrations (10 ng/mL and 1 μg/mL) induces differential transcript levels of four biomarker genes for Major Depressive Disorder (MDD) in HT29 human colon adenocarcinona cell line and Human Umbilical Vein Endothelial Cells (HUVEC). These results substantiate increasing concerns of BPA exposure in levels currently detected in humans.