http://http://dspace.unitus.it:80 The DSpace digital repository system captures, stores, indexes, preserves, and distributes digital research material. 2013-05-23T03:07:07Z http://hdl.handle.net/2067/1615 Title: Epigenetic Marks For Chromosome Imprinting During Spermatogenesis In Coccids Authors: Bongiorni, Silvia; Pugnali, Margherita; Volpi, Silvia; Bizzaro, Davide; Singh, Prim B.; Prantera, Giorgio Abstract: The establishment of sex-specific epigenetic marks during gametogenesis is one of the key feature of genomic imprinting. By immunocytological analysis, we thoroughly characterized the chromatin remodeling events that take place during gametogenesis in the mealybug Planococcus citri, in which an entire haploid set of (imprinted) chromosomes undergoes facultative heterochromatinization in male embryos. Building on our previous work, we have investigated the interplay of several epigenetic marks in the regulation of this genome-wide phenomenon. We characterized the germline patterns of histone modifications, Me(3)K9H3, Me(2)K9H3, and Me (3)K20H4, and of heterochromatic proteins, PCHET2 (HP1-like) and HP2-like during male and female gametogenesis. We found that at all stages in oogenesis chromatin is devoid of any detectable epigenetic marks. On the other hand, spermatogenesis is accompanied by a complex pattern of redistribution of epigenetic marks from euchromatin to heterochromatin, and vice versa. At the end of spermatogenesis, sperm heads are decorated by all the molecules we tested, except for PCHET2. However, only Me(3)K9H3 and Me(2)K9H3 are still detectable in the male pronucleus. We suggest that the histone H3 lysine 9 methylation is the signal used to establish the malespecific imprinting on the paternal genome, thus allowing it to be distinguished from the maternal genome in the developing embryo. Description: L'articolo è disponibile sul sito dell'editore: http://www.springerlink.com 2008-12-31T23:00:00Z http://hdl.handle.net/2067/1643 Title: HP2-like protein: a new piece of the facultative heterochromatin puzzle Authors: Volpi, Silvia; Bongiorni, Silvia; Prantera, Giorgio Abstract: In Drosophila melanogaster, the two chromosomal proteins HP1 and HP2 colocalize on heterochromatic and euchromatic sites in polytene chromosomes. Mutations in the HP2 gene act as dominant suppressors of position effect variegation, demonstrating a role for HP2 in the formation or maintenance of heterochromatin. In this paper, we investigated whether a putative homolog of the D. melanogaster HP2 is involved in the facultative heterochromatinization process in mealybugs. Using an antibody raised against the Drosophila HP2, we identified in the mealybug Planococcus citri a cross-reactive epitope, which we refer to as HP2-like. We investigated the HP2-like pattern during the male embryo development where the entire paternal haploid chromosome set becomes heterochromatic. The HP2 antibody heavily decorates the chromocenters, where it localizes with HP1, and marks the chromatin before it acquires the full cytological characteristics of the male-specific heterochromatin. In euchromatic chromosomes, HP2-like is mainly concentrated at telomeric sites. The interplay between HP2-like and HP1-like was studied by dsRNA interference experiments. Extinguishing HP1-like expression by RNAi does not prevent the association of HP2-like with facultative heterochromatin, implying that HP2-like binds to chromatin in a HP1- independent manner. Our results confirm and extend the structural and functional conservation of proteins involved in heterochromatin assembly. Description: L'articolo è disponibile sul sito dell'editore: http://www.springerlink.com 2006-12-31T23:00:00Z http://hdl.handle.net/2067/1893 Title: Transcriptomic analysis of two sheep breeds during lactation, using a new custom microarray platform Authors: Bongiorni, Silvia; Chillemi, Giovanni; Prosperini, Gianluca; Bueno, Susana; Signorelli, Federica; Moioli, Bianca; Pariset, Lorraine Abstract: We aim at understanding the genomic influence on milk quality and synthesis by comparing two sheep breeds using sheep-specific microarray technology. From sheep ESTs deposited at NCBI we generated a chip carrying about 22,000 non-redundant features in quadruplicate, achieving very good technical outcomes. Oligos were in situ generated on chip using the Combimatrix equipment. We analysed the mammary transcriptome in individuals of two sheep breeds at two lactation stages, to identify genes controlling milk production and metabolic pathways in which these genes are involved. With |FC|>1.4, and p-value≤0.05, 142 and 14 genes resulted differentially expressed in stages 01 and 02, respectively. 2011-04-07T22:00:00Z http://hdl.handle.net/2067/1888 Title: Microarrays and high throughput transcriptomic analysis for species with limited knowledge of genomic sequences F transcriptomic analysis for species with OO limited knowledge of genomic sequences Authors: Pariset, Lorraine; Chillemi, Giovanni; Bongiorni, Silvia; Spica, Vincenzo Romano; Valentini, Alessio Abstract: Microarrays produce a measurement of gene expression based on the relative measures of dye intensities that correspond to the amount of target RNA. This technology is fast developing and its application is expanding from Homo sapiens to a wide number of species, where enough information on sequences and annotations exist. Anyway, the number of species for which a dedicated platform exists is not many. The use of heterologous array hybridization, screening for gene expression in one species using an array developed for another species, is still quite frequent, even though cross-species microarray hybridization has raised many arguments. Some methods which are high throughput and do not rely on knowledge of the DNA/RNA sequence exist, namely serial analysis of gene expression (SAGE), Massively Parallel Signature Sequencing (MPSS) and deep sequencing of full transcriptome. Although very powerful methods, particularly the last one, they are still quite costly and cumbersome. In some species where genome sequences are largely unknown, several anonymous sequences are deposited in gene banks as a result of Expressed Sequence Tags (ESTs) sequencing projects. The ESTs databases represent a valuable knowledge that can be exploited with some bioinformatic effort to build species-specific microarrays. We present here a method of high-density in situ synthesized microarrays starting from available EST sequences in, Ovis aries. Our data indicate that the method is very efficient and can be easily extended to other species of which genetic sequences are present in public databases, but neglected so far with advanced devices like microarrays. As a perspective, the approach can be applied also to species of which no sequences are available to date, thanks to high-throughput deep sequencing methods. 2011-04-05T22:00:00Z http://hdl.handle.net/2067/1878 Title: Not working on mice or humans Authors: Pariset, Lorraine; Bueno, Susana; Bongiorni, Silvia; Valentini, Alessio Abstract: Microarrays technology is fast devel- oping and its application is expanding from Homo sapiens to a wide number of species where enough information on sequences and annotations are available. The number of species for which a dedicated platform exists is not high. The Expressed Sequence Tags (ESTs) databases represent a collection of anonymous sequences that can be used to build species spe- cific microarrays for species whose genome sequences are largely un- known. 2011-04-05T22:00:00Z http://hdl.handle.net/2067/1880 Title: Comparison of Milk Fat Globule Membrane (MFGM) Proteins of Chianina and Holstein Cattle Breed Milk Samples Through Proteomics Methods Authors: Murgiano, Leonardo; Timperio, Anna Maria; Zolla, Lello; Bongiorni, Silvia; Valentini, Alessio; Pariset, Lorraine Abstract: Identification of proteins involved in milk production is important to understand the biology of lactation. Many studies have advanced the understanding of mammary function and milk secretion, but the critical molecular mechanisms implicated in milk fat secretion is still incomplete. Milk Fat Globules are secreted from the apical surface of the mammary cells, surrounded by a thin membrane bilayer, the Milk Fat Globule Membrane (MFGM), formed by proteins which have been suggested to be holesterolemia-lowering factors, inhibitors of cancer cell growth, vitamin binders, bactericidal, suppressors of multiple sclerosis. Using a proteomic approach, we compared MFGM from milk samples of individuals belonging to two different cattle breeds, Chianina and Holstein,representative of selection for milk and meat traits, respectively. We were able to isolate some of the major MFGM proteins in the examined samples and to identify differences between the protein fractions of the two breeds. We detected differences in the amount of proteins linked to mammary gland development and lipid droplets formation, as well as host defence mechanisms. We have shown that proteomics is a suitable, unbiased method for the study of milk fractions proteins and a powerful tool in nutritional genomics. 2011-04-05T22:00:00Z http://hdl.handle.net/2067/1889 Title: A Tool for Sheep Product Quality: Custom Microarrays from Public Databases Authors: Bongiorni, Silvia; Chillemi, Giovanni; Prosperini, Gianluca; Bueno, Susana; Valentini, Alessio; Pariset, Lorraine Abstract: Milk and dairy products are an essential food and an economic resource in many countries. Milk component synthesis and secretion by the mammary gland involve expression of a large number of genes whose nutritional regulation remains poorly defined. The purpose of this study was to gain an understanding of the genomic influence on milk quality and synthesis by comparing two sheep breeds with different milking attitude (Sarda and Gentile di Puglia) using sheep-specific microarray technology. From sheep ESTs deposited at NCBI, we have generated the first annotated microarray developed for sheep with a coverage of most of the genome. 2011-04-05T22:00:00Z http://hdl.handle.net/2067/1642 Title: Epigenetic regulation of facultative heterochromatinisation in Planococcus citri via the Me(3)K9H3/HP1/Me(3)K20H4 pathway Authors: Bongiorni, Silvia; Pasqualini, Barbara; Taranta, Monia; Singh, Prim B.; Prantera, Giorgio Abstract: Using RNA interference (RNAi) we have conducted a functional analysis of the HP1-like chromobox gene pchet2 during embryogenesis of the mealybug Planococcus citri. Knocking down pchet2 expression results in decondensation of the male-specific chromocenter that normally arises from the developmentally-regulated facultative heterochromatinisation of the paternal chromosome complement. Together with the disappearance of the chromocenter the staining levels of two associated histone modifications, tri-methylated lysine 9 of histone H3 [Me(3)K9H3] and tri-methylated lysine 20 of histone H4 [Me(3)K20H4], are reduced to undetectable levels. Embryos treated with double-stranded RNA (dsRNA) targeting pchet2 also exhibit chromosome abnormalities, such as aberrant chromosome condensation, and also the presence of metaphases that contain ‘lagging’ chromosomes. We conclude that PCHET2 regulates chromosome behavior during metaphase and is a crucial component of a Me(3)K9H3-HP1-Me(3)K20H4 pathway involved in the facultative heterochromatinisation of the (imprinted) paternal chromosome set. Description: L'articolo è disponibile sul sito dell'editore: http://jcs.biologists.org. 2006-12-31T23:00:00Z