Giovanna Benvenuto

The Photomorphogenesis Regulator DET1 Binds the Amino-Terminal Tail of Histone H2B in a Nucleosome Context

Light provides a major source of information from the environment during plant growth and development. Recent results suggest that the key events controlling light-regulated gene expression in plants are translocation of the phytochrome photoreceptors into the nucleus, followed by their binding to transcription factors such as PIF3. Coupled with this, the degradation of positively acting intermediates such as the transcription factor HY5 by COP1 and the COP9 signalosome appears to be an important process whereby photomorphogenesis is repressed in darkness (e.g., ). Genetic analyses in Arabidopsis and tomato have revealed that the nuclear protein DET1 also plays a key role in the repression of photomorphogenesis. However, the function of this protein has remained a mystery. In a series of in vitro experiments, we provide persuasive evidence that DET1 binds to nonacetylated amino-terminal tails of the core histone H2B in the context of the nucleosome. Furthermore, we have utilized FRET (fluorescence resonance energy transfer) imaging with GFP variants to demonstrate this interaction within the nucleus of living plant cells. Given the dramatic photomorphogenic phenotypes of det1 mutants, we propose that chromatin remodeling plays a heretofore unsuspected role in regulating gene expression during photomorphogenesis.

Galectin-1 and galectin-3 expression in human bladder transitional-cell carcinomas

Galectin‐1 and galectin‐3 are galactoside‐binding proteins involved in different steps of tumor progression and potential targets for therapy. We have investigated the expression of these galectins in 38 human bladder transitional‐cell carcinomas of different histological grade and clinical stage and in 5 normal urothelium samples. Galectin‐1 mRNA levels were highly increased in most high‐grade tumors compared with normal bladder or low‐grade tumors. Western blot and immuno‐histochemical analysis of normal and neoplastic tissues revealed a higher content of galectin‐1 in tumors. Galectin‐3 mRNA levels were also increased in most tumors compared with normal urothelium, but levels were comparable among tumors of different histological grade. Int. J. Cancer (Pred. Oncol.) 84:39–43, 1999.

High mobility group I (Y) proteins bind HIPK2, a serine-threonine kinase protein which inhibits cell growth.

The HMGI proteins (HMGI, HMGY and HMGI-C) have an important role in the chromatin organization and interact with different transcriptional factors. The HMGI genes are expressed at very low levels in normal adult tissues, whereas they are very abundant during embryonic development and in several experimental and human tumours. In order to isolate proteins interacting with the HMGI(Y) proteins, a yeast two-hybrid screening was performed using the HMGI(Y) protein as bait. This analysis led to the isolation of homeodomain-interacting protein kinase-2 (HIPK2), a serine/threonine nuclear kinase. HIPK2 co-immunoprecipitates with the HMGI(Y) protein in 293T cells. The interaction between HIPK2 and HMGI(Y) occurs through the PEST domain of HIPK2 and it is direct because in vitro translated HIPK2 binds HMGI(Y). We also show that HIPK2 is able to phosphorylate the HMGI(Y) protein by an in vitro kinase assay. In order to understand a possible role of HIPK2 gene in cell growth we performed a colony assay which showed an impressive HIPK2 inhibitory effect on normal thyroid cells. Flow cytometric analysis would indicate the block of cell growth at the G2/M phase of the cell cycle. Since normal thyroid cells do not express detectable HMGI(Y) protein levels, we assume that the HIPK2 inhibitory effect is independent from the interaction with the HMGI(Y) protein.

A modified Gateway cloning strategy for overexpressing tagged proteins in plants

BackgroundRecent developments, including the sequencing of a number of plant genomes, have greatly increased the amount of data available to scientists and has enabled high throughput investigations where many genes are investigated simultaneously. To perform these studies, recombinational cloning methods such as the Gateway system have been adapted to plant transformation vectors to facilitate the creation of overexpression, tagging and silencing vectors on a large scale.ResultsHere we present a hybrid cloning strategy which combines advantages of both recombinational and traditional cloning and which is particularly amenable to low-to-medium throughput investigations of protein function using techniques of molecular biochemistry and cell biology. The system consists of a series of twelve Gateway Entry cassettes into which a gene of interest can be inserted using traditional cloning methods to generate either N- or C-terminal fusions to epitope tags and fluorescent proteins. The resulting gene-tag fusions can then be recombined into Gateway-based Destination vectors, thus providing a wide choice of resistance marker, promoter and expression system. The advantage of this modified Gateway cloning strategy is that the entire open reading frame encoding the tagged protein of interest is contained within the Entry vectors so that after recombination no additional linker sequences are added between the tag and the protein that could interfere with protein function and expression. We demonstrate the utility of this system for both transient and stable Agrobacterium-mediated plant transformations.ConclusionThis modified Gateway cloning strategy is complementary to more conventional Gateway-based systems because it expands the choice of tags and higher orders of combinations, and permits more control over the linker sequence lying between a protein of interest and an epitope tag, which can be particularly important for studies of protein function.

The conserved factor DE‐ETIOLATED 1 cooperates with CUL4–DDB1DDB2 to maintain genome integrity upon UV stress

Plants and many other eukaryotes can make use of two major pathways to cope with mutagenic effects of light, photoreactivation and nucleotide excision repair (NER). While photoreactivation allows direct repair by photolyase enzymes using light energy, NER requires a stepwise mechanism with several protein complexes acting at the levels of lesion detection, DNA incision and resynthesis. Here we investigated the involvement in NER of DE‐ETIOLATED 1 (DET1), an evolutionarily conserved factor that associates with components of the ubiquitylation machinery in plants and mammals and acts as a negative repressor of light‐driven photomorphogenic development in Arabidopsis. Evidence is provided that plant DET1 acts with CULLIN4‐based ubiquitin E3 ligase, and that appropriate dosage of DET1 protein is necessary for efficient removal of UV photoproducts through the NER pathway. Moreover, DET1 is required for CULLIN4‐dependent targeted degradation of the UV‐lesion recognition factor DDB2. Finally, DET1 protein is degraded concomitantly with DDB2 upon UV irradiation in a CUL4‐dependent mechanism. Altogether, these data suggest that DET1 and DDB2 cooperate during the excision repair process.

Control of galectin gene expression.

In this review we summarize the available information on the expression of mammalian galectins in normal and transformed cells. From all these studies it is apparent that each cell might express most of galectins; yet, during development or in various differentiation stages or under different physiological or pathological conditions, one or more galectins are preferentially expressed in each cell type. This implies a fine control of gene expression and suggests that such control should be coordinated. Nevertheless, to date very few studies have been performed on the mechanisms responsible for the regulation of galectin genes. We review the current knowledge on galectin promoter function. We believe that this area of galectin research will expand rapidly in the near future.

Sequential Modification of Serines 621 and 624 in the Raf-1 Carboxyl Terminus Produces Alterations in Its Electrophoretic Mobility

The Raf-1 serine/threonine protein kinase plays a central role in many of the mitogenic signaling pathways regulating cell growth and differentiation. The regulation of Raf-1 is complex, and involves protein-protein interactions as well as changes in the phosphorylation state of Raf-1 that are accompanied by alterations in its electrophoretic mobility. We have previously shown that a 33-kDa COOH-terminal, kinase-inactive fragment of Raf-1 underwent a mobility shift in response to the stimulation of cells with serum or phorbol esters. Here we demonstrate that treatment of NIH 3T3 cells or Sf9 cells with hydrogen peroxide (H2O2) also induces the mobility shift of the kinase-inactive Raf-1 fragment. A series of deletion mutants of the Raf-1 COOH terminus were analyzed, and the region required for the mobility shift was localized to a 78-amino acid fragment (residues 566-643). Metabolic labeling revealed that the slower migrating forms of the 33-kDa and of the smaller fragment contained phosphorus. Mutation of a previously characterized phosphorylation site, serine 621, to alanine prevented the mobility shift as well as phosphate incorporation or Src and Ras-dependent kinase activation in Sf9 cells when this mutation was engineered into the full-length Raf-1. Mutation of 621 to aspartate yielded a protein that existed in both the shifted and unshifted forms, demonstrating that a negative charge at 621 was necessary, but not sufficient, for the mobility shift to occur; however, its full-length form was still resistant to activation in the Sf9 system. Additional mutation of nearby serine 624 to alanine blocked the shift, implicating this residue as the site of the second of a two-step modification process leading to the slower migrating form. Co-expression of the 33-kDa fragment with an activated form of mitogen-activated protein kinase kinase in NIH 3T3 led to the appearance of the shifted form in a serum-independent manner. These results demonstrate that a mitogen-activated protein kinase kinase-induced event involving modification of serines 621 and 624 leads to the mobility shift of Raf-1.

High resolution methylation analysis of the galectin-1 gene promoter region in expressing and nonexpressing tissues

We conducted by bisulfite genomic sequencing a high resolution study of the methylation of the galectin‐1 gene in expressing and nonexpressing tissues. We show that: (i) hypomethylation of galectin‐1 promoter correlates with expression; (ii) differences in methylation occur in a small region, which include a CpG cluster; (iii) the density of methyl‐CpGs rather than site‐specific methylation distinguishes the nonexpressing from the expressing alleles; (iv) the modification profiles in nonexpressing tissues are highly heterogeneous; (v) a single CpG within 1300 bp is always methylated both in expressing and nonexpressing tissues; (vi) these features are conserved in rat and mouse.

Characterization and functional dissection of the galactin-1 gene promoter

The galectin‐1 gene encodes a β‐galactoside‐binding protein whose overexpression is associated with neoplastic transformation and loss of differentiation. Transient transfection assays of a series of deletions constructs (pGAT) showed that the galectin‐1 promoter is highly active in cells both expressing and non‐expressing the endogenous gene, and that the basal activity is determined by sequences encompassing the transcription start site ( ). Both an upstream ( ) and a downstream position‐dependent ( ) cis‐elements are necessary for efficient transcriptional activity and are able to bind nuclear proteins.

Ultrastructural features of the benthic dinoflagellate Ostreopsis cf. ovata (Dinophyceae).

The toxic benthic dinoflagellate Ostreopsis cf. ovata has considerably expanded its distribution range in the last decade, posing risks to human health. Several aspects of this species are still poorly known. We studied ultrastructural features of cultivated and natural populations of Ostreopsis cf. ovata from the Gulf of Naples (Mediterranean Sea) using confocal laser scanning, and scanning and transmission electron microscopy. New information on the morphology and location of several sulcal plates was gained and a new plate designation is suggested that better fits the one applied to other Gonyaulacales. The microtubular component of the cytoskeleton, revealed using an anti-β-tubulin antibody, consisted of a cortical layer of microtubules arranged asymmetrically in the episome and in the hyposome, complemented by a complex inner microtubular system running from the sulcal area towards the internal part of the cell. The conspicuous canal was delimited by two thick, burin-shaped lobes ending in a tubular ventral opening. The canal was surrounded by mucocysts discharging their content into it. A similar structure has been reported in other benthic and planktonic dinoflagellates and may be interpreted as an example of convergent evolution in species producing large amounts of mucus.