Search

Graphene, due to its unique electronic structure favoring high carrier mobility, is considered a promising material for use in high-speed electronic devices in the post-silicon electronic era. For this reason, experimental research on graphene-based field-effect transistors (GFETs) has rapidly increased in the last years. However, despite the continuous progress in the optimization of such devices many critical issues remain to be solved such as their reproducibility and performance uniformity against possible variations originated by the manufacturing processes or the operating conditions. In the present work, changes of the I D - V DS characteristics of a Graphene Field-Effect Transistors, caused by a tolerance of 10% in the active channel (i.e. its length and width) and in the top oxide thickness are numerically investigated in order to assess the reliability of such devices. Design of Experiments (DoE) is adopted with the aim to identify the most influential factors on the electrical performance of the device, so that the fabrication process may be suitably optimized. [ABSTRACT FROM AUTHOR]

Insulators are a new class of genetic elements that attenuate enhancer function directionally. Previously, we characterized in sea urchin a 265-bp-long insulator, termed sns. To test insulator activity following stable integration in human cells, we placed sns between the CMV enhancer and a tk promoter upstream of a GFP transgene of plasmid or retroviral vectors. In contrast to controls, cells transfected or transduced with insulated constructs displayed a barely detectable fluorescence. Southern blot and PCR ruled out vector rearrangement following integration into host DNA; RNase protection confirmed the enhancer blocking activity. Finally, we demonstrate that two cis-acting sequences, previously characterized in sea urchin, are also specific binding sites for human proteins. We conclude that sns interferes with enhancer promoter interaction also in a human chromatin context. The relatively small size, evolutionary conservation and apparent lack of enhancer specificity might result useful in gene transfer experiments in human cells.

Insulator elements can be functionally identified by their ability to shield promoters from regulators in a position-dependent manner or their ability to protect adjacent transgenes from position effects. We have previously reported the identification of a 265bp snsDNA fragment at the 3′ end of the sea urchin H2A early histone gene that blocked expression of a reporter gene in transgenic embryos when placed between the enhancer and the promoter. Here we show that snsinterferes with enhancer-promoter interaction in a directional manner. When snsis placed between the H2A modulator and the inducible tetoperator, the modulator is barred from interaction with the basal promoter. However, the tetactivator (tTA) can still activate the promoter, even in the presence of sns,demonstrating that snsdoes not interfere with activity of a downstream enhancer. In addition, the H2A modulator can still drive expression of a divergently oriented transcription unit, suggesting that snsdoes not inhibit binding of transcription factor(s) to the enhancer. To identify cis-acting sequence elements within snswhich are responsible for insulator activity, we have performed in vitroDNase I footprinting and EMSA analysis, and in vivofunctional assays by microinjection into sea urchin embryos. We have identified three binding sites for protein complexes: a palindrome, a direct repeat, and a C+T sequence that corresponds to seven GAGA motifs on the transcribed strand. Insulator function requires all three cis-acting elements. Based on these results, we conclude that snsdisplays properties similar to the best characterized insulators and suggest that directional blocking of enhancer-activated transcription by snsdepends on the assembly of distinct DNA-protein complexes.

Localization of constitutive hsp70 in eggs and early embryos of sea urchin Paracentrotus lividusis shown by means of in situimmunostaining. An accumulation of this protein is shown in the mitotic structures (asters, spindles and centrosomes). Microinjection of anti-hsp70 antibodies into eggs causes impairment of formation of mitotic structures and of cell division. This impairment goes from a complete mitotic block, to irregular mitotic apparatus formation with irregular cleavage, depending upon the antibody concentration. The localization of hsp70 after antibody microinjection is also described. Blockage of mitotic apparatus formation by nocodazole also blocks the concentration of hsp70 molecules observed in nontreated eggs. That the constitutive hsp70 plays a role in sea urchin mitosis is indicated.

Several homeobox genes are expressed in the sea urchin embryo but their roles in development have yet to be elucidated. Of particular interest are homologues of homeobox genes that in mouse and Drosophila are involved in patterning the developing central nervous system (CNS). Here, we report the cloning of an orthopedia (Otp)-related gene from Paracentrotus lividus, PlOtp. Otp is a single copy zygotic gene that presents a unique and highly restricted expression pattern. Transcripts were first detected at the mid-gastrula stage in two pairs of oral ectoderm cells located in a ventrolateral position, overlying primary mesenchyme cell (PMC) clusters. Increases in both transcript abundance and the number of Otp-expressing cells were observed at prism and pluteus stages. Otp transcripts are symmetrically distributed in a few ectodermal cells of the oral field. Labelled cells were observed close to sites of active skeletal rod growth (tips of the budding oral and anal arms), and at the juxtaposition of stomodeum and foregut. Chemicals known to perturb PMC patterning along animal-vegetal and oral-aboral axes altered the pattern of Otp expression. Vegetalization by LiCl caused a shift in Otp-expressing cells toward the animal pole, adjacent to shifted PMC aggregates. Nickel treatment induced expression of the Otp gene in an increased number of ectodermal cells, which adopted a radialized pattern. Finally, ectopic expression of Otp mRNA affected patterning along the oral-aboral axis and caused skeletal abnormalities that resembled those exhibited by nickel-treated embryos. From these results, we conclude that the Otp homeodomain gene is involved in short-range cell signalling within the oral ectoderm for patterning the endoskeleton of the larva through epithelial- mesenchymal interactions.

Evidence is provided for the presence at the physiological temperature of 20°C of a heat shock transcriptor factor, HSF, in the nuclei ofP.lividusembryos. This HSF is able to specifically bindin vitrothe heat shock element, HSE, of the promoter of the hsp70 gene IV, as suggested by DNA–protein binding reactions and DNAse I protection assays. Upon heat-shock, at the temperature of 31°C, its ability to bind the HSE units becomes much higher. The HSF activated by heat-shock drivesin vivothe transcription of the β-galactosidase reporter gene in transgenic sea urchin gastrulae. An ATF-like transcription factor, widely described in other organisms but not at all in sea urchins, is also present in the nuclear extracts and is able to bind the consensus individuated in the hsp70 IV gene promoter.

Transcriptional regulators are thought to play a key role in cell fate determination and territorial specification in sea urchin. Our goals are to clone transcription factors for studying embryonic development. One approach has been to use promoter binding and gene transfer technology to investigate the mechanisms of transcriptional activation and repression of the early H2A histone gene. By this analysis we identified a transcriptional activator, the MBF-1, that binds to the modulator element of the H2A gene and enhances the activity of the H2A promoter. However, the enhancer activity of the modulator and its interaction with MBF-1 also occurs at the gastrula stage when the early histone genes are shut off. Therefore, the silencing of the early H2A histone gene at late stages of development requires the inactivation of the modulator function. To search for antimodulator sequence elements, we took advantage of our previous work showing the presence of phased nucleosomes specifically positioned on the 3'-spacer and in the modulator of the repressed H2A gene. Evidence is described indicating that a 3'-spacer DNA fragment cloned between the modulator and the basal promoter behaves as an antimodulator element. However, this element does not confer temporal capability to the modulator in its function, suggesting that other elements have to be involved in the regulation of the early H2A expression. The second approach relied on the cloning of genes controlling development using probes of regulators known to be involved in regional specification, such as the homeobox genes, with the aim to understand their possible role through the study of their temporal and territorial expression and through the analysis of the mechanism of regulation of their expression. We present evidence that PIHbox 12, a homeodomain encoding gene, is transiently expressed during the early/mid cleavage stages. The abundance of the transcripts reach their maximum in embryos at the 64/128-cell stage concomitantly with the segregation of the specified embryonic territories. Expression of PIHbox l2 is drastically reduced in the absence of cell contacts and/or Ca ions, suggesting that this gene is transcriptionally activated by signal transduction mechanisms. Whole mount in situ hybridization showed that PIHbox 12 transcripts are asymmetrically distributed along the A-V axis, being spatially localized in the blastomeres of the ectodermal lineage. We suggest that PIHbox l2 might be involved in the initial events that lead to the specification of the ectodermal territories.

Two homeobox-containing genes that belong to different homeodomain classes have been isolated from a sea urchin geonomic library. One, PlHbox11, is the sea urchin homologue of the human and mouse Hox B3 gene, the other, PlHbox12, shows about 55% identity with paired class genes. Expression profile analysis of the two sea urchin Hbox genes suggests that they play different roles during embryogenesis. In fact, PlHbox11 transcripts are rare and are detected only in the pluteus larva and in the Aristotle's lantern and intestine of the adult. The PlHbox12 gene is, on the contrary, transiently expressed in the very early embryo already at the four cell stage; it accumulates at the 64 cell stage and disappears at later stages of development.In situ hybridization experiments to 16 and 32 cell stage whole mount embryos showed localization of the PlHbox12 mRNA to part of the mesomere-macromere region of the early cleavage embryo. These observations suggest a possible role of this gene in early events of cell specification.

Retroviral vectors are currently the most suitable vehicles for therapeutic gene transfer in hematopoietic stem cells. However, these vectors are known to integrate rather randomly throughout the genome, suffering the so called chromosomal position effects (PE). Such a critical occurrence most probably depends upon the ability of heterochromatin to spread in the inserted vector sequences. Moreover, the use of transgenes imply genotoxicity effects, since the cis-regulatory sequences harbored by the vector can disturb the proper transcription of the resident genes neighboring the integration site, potentially leading to malignant transformation.

No relevant conflicts of interest to declare.

The nucleosomal organization of active and repressed α subtype histone genes has been investigated by micrococcal nuclease digestion of P. lividus sperm, 32-64 cell embryo and mesenchyme blastula nuclei, followed by hybridization with 32 P-labeled specific DNA probes. In sperms, fully repressed histone genes are regularly folded in nucleosomes, and exhibit a greater resistance to micrococcal nuclease cleavage than bulk chromation. In contrast, both coding and spacer α subtype histone DNA sequences acquire an altered conformation in nuclei from early cleavage stage embryos, i.e., when these genes are maximally expressed. Switching off of the α subtype histone genes, in mesenchyme blastulae, restores the typical nucleosomal organization on this chromatin region. As probed by hybridization to D.melanogaster actin cDNA actin genes retain a regular nucleosomal structure in all the investigated stages.

The SV40 enhancer microinjected into the nuclei of X. laevis oocytes is able to activate transcription about 100-fold when cloned upstream of the SV40 early promoter region or about 10-fold regardless of its orientation when located at a long distance from a test gene (bacterial chloramphenicol acetyl transferase). This effect is qualitatively and quantitatively similar to that observed when analogous constructions were transfected into mammalian cells. Making use of a direct labelling technique for the analysis of the chromatin structure we could show that the SV40 enhancer region in the microinjected plasmids is particularly accessible to cleavage with MNase or DNAse I. Single sites in the 72 bp repeats have been mapped at the nucleotide level.