Depending on their genetic background (p53(wt) versus p53(null)), carcinoma cells are more or less sensitive to drug-induced cell cycle arrest and/or apoptosis. Among the members of the p53 family, p63 is characterized by two N-terminal isoforms, TAp63 and ΔNp63. TAp63 isoform has p53-like functions, while ΔNp63 acts as a dominant negative inhibitor of p53. We have previously published that TAp63 is involved in poly(ADP-ribose)polymerase-1 (PARP-1) signaling of DNA damage deriving from DNA topoisomerase I (TOP I) inhibition in carcinoma cells. In the present study, we treated MCF7 breast carcinoma cells (p53(+)/ΔNp63(-)) or SCC022 (p53(-)/ΔNp63(+)) squamous carcinoma cells with the TOP I inhibitor topotecan (TPT) and the PJ34 PARP inhibitor, to compare their effects in the two different cell contexts. In MCF7 cells, we found that PJ34 addition reverts TPT-dependent PARP-1 auto-modification and triggers caspase-dependent PARP-1 proteolysis. Moreover, TPT as single agent stimulates p53(ser15) phosphorylation, p53 PARylation and occupancy of the p21WAF promoter by p53 resulting in an increase of p21WAF expression. Interestingly, PJ34 in combination with TPT enhances p53 occupancy at the BAX promoter and is associated with increased BAX protein level. In SCC022 cells, instead, TPT+PJ34 combined treatment reduces the level of the anti-apoptotic ΔNp63α protein without inducing apoptosis. Remarkably, in such cells, either exogenous p53 or TAp63 can rescue the apoptotic program in response to the treatment. All together our results suggest that in cancer cells PARP inhibitor(s) can operate in the choice between growth arrest and apoptosis by modulating p53 family-dependent signal.

Vitamin D and folate are essential vitamins that pl y important roles in human physiological developme nt and are activated and degraded by sunlight, respect ively. We aimed to detect signals of positive sele ction in gene sets involved in metabolism, regulation and ac tion of these two vitamins using an existing algori thm developed by our group to universally test for evol uti nary adaption in any chosen gene set. We made use of the low coverage re-sequencing data from worldwi de populations generated by the 1000 Genomes Projec t and compared frequency spectrum based summary stati stics between vitamin D and folate pathway gene sets and matched control genes. Genes interacting with Vitamin D receptor (VDR) were selected in all populations. Within this group genes that directly interact with VDR and retinoid –X-receptor (RXR) w ere selected in Africans and Europeans, respectively. Genes involved in folate uptake were selected in Europeans only and those associated with methylatio n were selected in both Africans and Europeans. Som e genes ( ARID1A and BAZ1B) were selected in all populations while others suc h as FGR, NCOA1, RXRA and NROB2 were selected in non-Africans. The selected genes identified by this method are shared between the folate and vitamin D pathways, enriched in the nucl e s and significantly associated with chromosome organization (p-value 1.609 X 10 ) and cellular protein modification process (p-valu e 1.529 X 10). This study provides evidence for convergent evolution op erating these pathways and highlights the power of using such an approach to understand how modern hum ans have genetically adapted to environmental changes during their recent evolutionary history. 2.2. StreamingTrim 1.0: a Java software for dynamic trimming of 16SrRNA sequence data from metagenetic studies Giovanni Bacci , Marco Bazzicalupo , Anna Benedetti , Alessio Mengoni 1* 1 Department of Biology, University of Florence, via M donna del Piano 6, I-50019 Firenze, Italy. 2 Agricultural Research Council /Research Centre for the Soil-Plant System (CRA-RPS), Via della Navicella 2/4, I-00184 Roma, Italy Molecular microbial ecology is massively using next generation sequencing technologies to describe taxonomic composition and infer the functionality of microbial communities, by using metagenetic applications based on PCR amplicon library sequencing. One of the problems related to the utilization of amplicon libraries data from next generation sequencing technologies is to analyze the quality of every reads present in a sequence file and to be able to trim the low quality segment without lose too much information for the following taxonomic analyses. Here, we present StreamingTrim, a DNA reads trimming software, written in Java, capable of analyzing the quality of a DNA sequence file and to search for low quality zones in a very conservative way, by using a dynamic trimming algorithm. This software has been developed aiming to provide a tool which allow to trim amplicon library data, retaining as much as taxonomic information as possible. The software is provided with a Graphical User Interface (GUI) for a user-friendly use. From the computational point of view, StreamingTrim reads and analyzes sequences one by one from the input file, without keeping anything in memory, allowing the computation to be run on a normal desktop PC or laptop. Trimmed sequences are saved in an output file and a statistics summary, containing the mean and the standard deviation of length and quality of the whole sequence file, is also displayed. Compiled software, manual and example data sets are available under the BSD-2-Clauses License at GitHub repository at https://github.com/GiBacci/StreamingTrim/. 2.3. Genomic tools for fishery and conservation of European hake and common sole Alessia Cariani1, FishPopTrace Consortium2 and Fausto Tinti1 11 Department of Biological, Geological and Environmental Sciences, University of Bologna, via Selmi 3, 40126 Bologna, Italy 2 https://fishpoptrace.jrc.ec.europa.eu/about/consortium Next-generation sequencing (NGS) technologies offer the opportunity to move from population genetics to population genomics, even in non-model species. In the present study NGS platforms were used to discover Single Nucleotide Polymorphisms (SNPs) in the muscle transcriptome of common sole (Solea solea) and European hake (Merluccius merluccius), two of the most important demersal fisheries in Europe. Transcriptome sequencing and de novo assembly into unique contigs, in silico SNP detection, and validation by high-throughput genotyping yielded hundreds of polymorphic SNP loci for both species. Population samples from several Atlantic and Mediterranean locations were screened at the developed EST-derived loci, in order to find candidate genes for environmental adaptation at different spatial scales and to integrate information on neutral and adaptive evolutionary patterns. With the putative neutral markers we confirmed the major genetic breaks already described for the target species, while analyzing candidate outliers SNPs higher resolution of population differentiation both between and within basins was obtained. Our results suggest local adaptation as a potential driver of population structure in marine fish species. Although caution should be taken when drawing indirect inferences about adaptive processes in the wild, precautionary management strategies should contemplate the possible presence of locally adapted populations. 2.4. Analysis of Clock Genes Variation in worldwide populations Irene Dall’Ara1, Silvia Ghirotto1, Selene Ingusci1, Guido Barbujani1 and Cristiano Bertolucci1. 1University of Ferrara, Department of Life Sciences and Biotechnologies Life on Earth is marked by many biological rhythms, evolved as consequence of environmental periodic changes, such as day-light cycles or the fluctuation of seasons. The circadian rhythm, a period of ~24h, gives rise to oscillations in behaviour and physiological functions, in order to anticipate upcoming daily change and to maximally benefit from the limited natural resources. Comparisons of genotypic and phenotypic information highlight links between clock genes’ polymorphisms and several human diseases. In this project, we analyse the global diversity of clock genes and their associations with sleep disorders. To do this we used whole genome sequences from the 1000Genomes data set (1092 individuals from 14 worldwide populations). In the initial phase, we considered 18 SNPs known to be linked with human diseases, describing their levels and patterns of population diversity, and investigating the association between allele frequencies and factors such as latitude and photoperiod. Correlations between genetic and geographical distances were insignificant, contrary to what observed at most neutral loci, and thus suggesting some effects of selective pressures upon the populations considered. However, the global population structure did not seem to reflect differences in photoperiod, whereas differences were observed in the distribution of SNPs at different loci. 2.5. Molecular identification of Diptera, used in f orensic investigations, by means of variable lenght of intronic sequences Emanuela De Domenico, Debora Lombardo, Concetta Fed erico, Claudia Leotta, Alessandro Marletta, Francesco Lombardo, Salvatore Saccone Dept. Biological, Geological, and Environmental Sci ences. Section of Animal Biology “M. La Greca”. University of Catania. Via Androne 81, 95124 Catani a. E-mail: saccosal@unict.it The identification of Diptera at the larval stage i s important in forensic investigations as it helps in establishing the correct PMI (Post Mortem Interval) . In the present work we describe a procedure that allows the molecular identification of three species of Di ptera, useful in the forensic investigations, more quickly and easily respect to the Barcoding system. To achi eve this goal we taken into consideration a number of gene sequences, one of which has allowed to obtain informations to distinguish the three considered sp ecies of scavenger Diptera: Lucilia sericata (Meigen,1826), Calliphora vicina Robinseau-Desvoid, 1830 and Sarcophaga carnaria (Linnaeus, 1758). The system developed involves a PCR amplification o f a DNA segment within the wingless gene. The amplified segment presents a conserved sequence in th 3’ and 5’ ends, in order to use the same primer s in different species, and a variable length among the three species, due to the amplified sequence that c ontains an intron of wingless whose size is variable. In th is way it is possible to assign larvae of the three Diptera species simply analysing the band size on an electr ophoretic gel. This procedure could help, in the fo rensic investigations, to obtain easily and rapidly the es timation of PMI of a body colonized by these larvae . 2.6. Population structure and adaptive variation to climate in natural populations of Norway spruce (Picea abies Karst) Erica A. Di Pierro1, Elena Mosca1, Duccio Rocchini1, Giorgio Binelli2, David B. Neale13 and Nicola La Porta1 1Research and Innovation Centre, Fondazione Edmund M ach (FEM), S. Michele all’Adige, Italy ; Dipartimento di Scienze Teoriche e Applicate, Unive rsità dell’Insubria, Varese, Italy ; 3Department of Plant Sciences, University of California, Davis, USA Forest trees dominate many Alpine landscapes and are currently exposed to changing climate. Norway spruce is one of the most important conifer species of the Italian Alps due to its economic and ecological value. This study seeks to determine and quantify patterns of genetic diversity for natural populations of this species toward understanding adaptive responses to changing climate. A wide array of potential candidate genes was tested for correlation with climatic parameters characterizing sampled populations. Across the Italian species range, 24 natural stands were sampled; trees were genotyped for 384 selected Single N

Poly(ADP-ribose)polymerase 1 (PARP-1) inhibitors are thought as breakthrough for cancer treatment in solid tumors such as breast cancer through their effects on PARP's enzymatic activity. Our previous findings showed that the hydrophilic PARP inhibitor PJ34 enhances the sensitivity of p53 proficient MCF7 breast carcinoma cells to topotecan, a DNA Topoisomerase I (TOP 1) inhibitor. In the present study, we combine the classical TOP 1 poison camptothecin or its water-soluble derivative topotecan with PJ34 to investigate the potentiation of chemotherapeutic efficiency in MCF7 (p53(WT)), MDA-MB231 (p53(mut)) breast carcinoma cells and SCC022 (p53(null)) squamous carcinoma cells. We show that, following TPT-PJ34 combined treatment, MCF7 cells exhibit apoptotic death while MDA-MB231 and SCC022 cells are more resistant to these agents. Specifically, in MCF7, (i) PJ34 in combination with TPT causes a G2/M cell cycle arrest followed by massive apoptosis; (ii) PJ34 addition reverts TPT-dependent PARP-1 automodification and triggers caspase-dependent PARP-1 proteolysis; (iii) TPT, used as a single agent, stimulates p53 expression while in combination with PJ34 increases p53, TAp63α and TAp63γ protein levels with a concomitant reduction of MDM2 protein. The identification of p63 proteins as new players involved in the cancer cell response to TPT-PJ34 is relevant for a better understanding of the PARP1-dependent signaling of DNA damage. Furthermore, our data indicate that, in response to TPT-PJ34 combined chemotherapy, a functional cooperation between p53 and TAp63 proteins may occur and be essential to trigger apoptotic cell death.

A molecular approach to enhance the antitumour activity of topoisomerase 1 (TOM) inhibitors relies on the use of chemical inhibitors of poly(ADP-ribose)polymerases (PARP). Poly(ADP-ribosyl)ation is involved in the regulation of many cellular processes such as DNA repair, cell cycle progression and cell death. Recent findings showed that poly(ADP-ribosyl)ated PARP-1 and PARP-2 counteract camptothecin action facilitating resealing of DNA strand breaks. Moreover, repair of DNA strand breaks induced by poisoned TOP1 is slower in the presence of PARP inhibitors, leading to increased toxicity.In the present study we compared the effects of the camptothecin derivative topotecan (TPT), and the PARP inhibitor PJ34, in breast (MCF7) and cervix (HeLa) carcinoma cells either PARP-1 proficient or silenced, both BRCA1/2(+)/(+) and p53(+)/(+).HeLa and MCF7 cell lines gave similar results: (i) TPT-dependent cell growth inhibition and cell cycle perturbation were incremented by the presence of PJ34 and a 2 fold increase in toxicity was observed in PARP-1 stably silenced HeLa cells; (ii) higher levels of DNA strand breaks were found in cells subjected to TFT + PJ34 combined treatment; (iii) PARP-1 and -2 modification was evident in TPT-treated cells and was reduced by TPT + PJ34 combined treatment: (iv) concomitantly, a reduction of soluble/active TOP1 was observed. Furthermore, TPT-dependent induction of p53, p21 and apoptosis were found 24-72 h after treatment and were increased by PJ34 both in PARP-1 proficient and silenced cells. The characterization of such signaling network can be relevant to a strategy aimed at overcoming acquired chemoresistance to TOP1 inhibitors. (C) 2010 Elsevier Inc. All rights reserved.

Using computer stress‐induced duplex destabilization (SIDD) analysis and binding experiments, we identified a S/MAR element (−599/−200 bp) (Hp‐S/MAR) adjacent to the cis‐element (−165/−56 bp) in the rat haptoglobin gene. We examined its functional interactions with the lamins and lamin‐associated proteins in the basal state and during acute‐phase (AP) response‐induced increased transcription. Colocalization, electrophoretic mobility shift assay (EMSA), and re‐electrophoresis of nucleoprotein complexes, South‐Western and Western blot analysis and coimunoprecipitation experiments revealed that the lamins, PARP‐1, C/EBPβ, and Hp‐S/MAR assembled higher order complexes through direct lamin‐Hp‐S/MAR and probably PARP‐1–Hp‐S/MAR interactions although C/EBPβ did not bind to the Hp‐S/MAR but established direct interaction with PARP‐1. The transition from constitutive to increased haptoglobin gene transcription during the AP response was associated with quantitative and qualitative changes in Hp‐S/MAR–protein interactions, respectively, observed as increased association of the lamin(s) with the Hp‐S/MAR and as the appearance of a 90 kDa Hp‐S/MAR‐binding protein. Also, during the AP response the contact between C/EBPβ and PARP‐1 established in the basal state was lost. DNA chromatography with the haptoglobin cis‐element and Western blot analysis suggests that PARP‐1 was a coactivator during constitutive and elevated transcription. The results show that the lamin components of the nuclear matrix form a network of functional, dynamic protein–protein and protein–Hp‐S/MAR associations with multiple partners, and underline the involvement of PARP‐1 in the regulation of haptoglobin gene transcription. We concluded that the interplay of these interactions fine tunes haptoglobin gene expression to meet the changing requirements of liver cells. J. Cell. Biochem. 107: 1205–1221, 2009. © 2009 Wiley‐Liss, Inc.

Poly(ADPR)polymerases’ (PARPs) inhibitors potentiate the cytotoxic effects of chemotherapeutic agents like alkylating compounds and TOPO I poisons, while their action in combination with cisplatin still needs investigation. In fact, one of the earliest responses to DNA single- or double-strand breaks is the synthesis of poly(ADP-ribose) (PAR) by PARPs; these enzymes are components of DNA repair machineries and substrates of caspases. Cisplatin (cDDP) yields intra- and inter-strand DNA cross-links and several proteins that recognise cDDP-induced DNA damage, such as p53, are also targets of poly(ADP-ribosyl)ation. We compared the effects of treatments with cDDP and the PARPs inhibitor PJ34 in p53 mutated carcinoma cell lines (HeLa, KB, HT29) that exhibited differential sensitivities to the drugs, in terms of cell growth inhibition and onset of apoptosis. In cDDP-resistant HT29 cells we determined: (i) PJ34 potentiation of cDDP-induced cell growth inhibition; (ii) an increment of PARP-1 automodification following cDDP treatment. In cDDP-sensitive HeLa cells, we found that the drug induced apoptotic cell death associated with caspase-dependent PARP-1 proteolysis.

Glioblastomas are widely characterised by the mutation of the p53 gene and p53 disruption sensitizes glioblastoma cells to DNA topoisomerase I (TOPO I) inhibitor-mediated apoptosis.We investigated the effects of combined treatments with the DNA topoisomerase I inhibitor Topotecan and the poly(ADPR)polymerase-1 inhibitor NU1025 in D54p53wt and U251p53mut glioblastoma cell lines. Analysis of cell growth and cell cycle kinetics showed a synergistic anti-proliferative effect of 10nM TPT and 10μM NU1025 and a G2/M block of the cell cycle.We also evaluated, the influence of TPT+/−NU1025 treatment on PARP-1 and p53 activity. We got evidences of a TPT-dependent increase of PARP-1 auto-modification level in both the cells. Moreover, in the D54p53wt cells we found that in co-treatments NU1025 incremented the TPT-dependent stimulation of p53 transcriptional activity and increased the p21 nuclear amount. Conversely, in U251p53mut cells we found that NU1025 incremented the TPT-dependent apoptosis characterised by PARP-1 proteolysis.Our findings suggest that the modulation of PARP-1 can be considered a strategy in the potentiation of the chemotherapeutic action of TOPO I poisons in glioblastoma cells apart from their p53 status.

Poly(ADP-ribose)polymerase (PARP-1), a nuclear enzyme activated by DNA strand breaks, is involved in DNA repair, aging, inflammation, and neoplastic transformation. In diabetes, reactive oxygen and nitrogen species occurring in response to hyperglycemia cause DNA damages and PARP-1 activation. Because circulating mononuclear cells (MNCs) are involved in inflammation mechanisms, these cells were chosen as the experimental model to evaluate PARP-1 levels and activity in patients with type 2 diabetes. MNCs were isolated from 25 diabetic patients (118 M, 7 F, age, 63.5 +/- 10.2 years, disease duration 17.7 +/- 8.2 years) and 11 age and sex matched healthy controls. PARP-1 expression and activity were analyzed by semi-quantitative PCR, Western and activity blot, and immunofluorescence microscopy. PARP-I-mRNA expression was increased in MNCs from all diabetic patients versus controls (P < 0.01), whereas PARP-1 content and activity were significantly lower in diabetic patients (P < 0.0001). To verify whether low PARP-1 levels and activity were due to a proteolytic effect of caspase-3 like, the latter activation was measured by a fluorimetric assay. Caspase-3 activity in MNCs was significantly higher in diabetic patients versus control subjects (P < 0.0001). The different PARP-1 behavior in MNCs from patients with type 2 diabetes could therefore be responsible for the abnormal inflammation and infection responses in diabetes.

The distribution of poly(ADP‐ribose) polymerase‐1 (PARP‐1) over different nuclear compartments was studied by nuclear fractionation procedures and Western analysis revealing a prominent role of the nuclear matrix. This structure is operationally defined by the solubility properties of the A‐ and B‐type lamins under defined experimental conditions. We consistently observed that most of the nuclear matrix‐associated PARP‐1 partitioned, in an active form, with the insoluble, lamin‐enriched protein fractions that were prepared by a variety of established biochemical procedures. These PARP‐1–protein interactions resisted salt extraction, disulfide reduction, RNase and DNase digestion. An inherent ability of PARP‐1 to reassemble with the lamins became evident after a cycle of solubilization/dialysis using either urea or Triton X‐100 and disulfide reduction, indicating that these interactions were dominated by hydrophobic forces. Together with in vivo crosslinking and co‐immunoprecipitation experiments our results show that the lamins are prominent PARP‐1‐binding partners which could contribute to the functional sequestration of the enzyme on the nuclear matrix. © 2004 Wiley‐Liss, Inc.

Poly( ADP-ribose) polymerase (PARP-1) and poly( ADP-ribose) glycohydrolase (PARG) are responsible for the transient poly( ADP-ribosyl) ation of proteins in eukaryotic cells. This biochemical reaction plays an active role in DNA replication and repair, transcription, cell differentiation and death. The aim of this study was to investigate the levels and the sub-cellular distribution of such enzymes in rat germinal cells at different stages of differentiation, i.e. in primary spermatocytes and round spermatids, representing meiotic and post-meiotic cells, respectively. The determination of the level of PARP-1 mRNA and protein revealed its higher expression in primary spermatocytes, thus implying that PARP-1 is one of the meiotic genes whose expression is requested at the pachytene phase of the meiosis. We also demonstrated that rat germinal cells contain both the forms of PARG (i.e. of 110 and 60 kDa) so far described in somatic cells. In our experimental system, the large PARG was present and active mainly in the nuclear fraction of primary spermatocytes, whereas round spermatids showed a higher level of the 60 kDa PARG in the post-nuclear fraction. Collectively, our data show a different expression level of PARP-1 and a different endocellular distribution of PARG and suggest a role for the poly( ADP-ribose) turnover in distinct pathways in meiotic and post-meiotic germinal cells.

Poly (ADPribosyl) polymerase (PARP) is a 113 kD nuclear protein. Following activation by DNA strand breaks, PARP hydrolyzes NAD+ and catalyzes the formation of poly(ADP-ribose) onto itself (automodification) and other nuclear proteins (heteromodification) with the release of nicotinamide. Poly(ADPribosyl)ation is an event of major importance. It participates in fundamental processes such as DNA repair, replication, cell cycle regulation and apoptosis (d 'A m 0 u r s etai.1999). PARP also participates in the regulation of transcription. It appears to exert a dual function in this process: stimulation and inhibition. In the absence of catalytic activity, PARP has been shown to be a component of the pre-initiation transcription complex. On the other hand, protein modification with poly (ADPribose) is an efficient means of reversibly silencing polymerase Il-dependent transcription (0 e i et al. 1998a). Consequently, the activating or repressing influence of PARP on transcription depends on its catalytic activity. Poly (ADPribosyl)ation occurs only if it is initiated before the acceptor protein binds to DNA and thereby prevents the formation of active transcription complexes. Otherwise, the binding of the acceptor protein to DNA prevents its modification. Recently, it was shown that the catalytically active PARP moiety can cause silencing of transcription. It modifies transcription factorsY'Yl (0 e i et al. 2001b), p53 (We s i e r s k a Gad e k et ai. 19(6), TBP (0 e i et ai. 1998c) and FOS (A m s tad et al. 1992) by poly (ADPribosyl) ation, thereby interfering with their DNA-binding affinity. PARP exists in a form that is associated with the nuclear matrix (d' E r m e 1990). The nuclear matrix, the main structural framework of the nucleus, is a nonhistone protein scaffold that supports the attachment points of DNA loops (B ere z n e y and Wei 1998; La e m m I i et ai. 1992). It is now widely accepted that fundamental processes of the cell nucleus are actively coordinated by the nuclear matrix. DNA replication, transcription, mRNA processing and DNA repair occur on the nuclear matrix (T u b 0 and B ere z n e y 1987). Some of the protein components of the nuclear matrix appear to be poly(ADPribosyl)ated (B err i 0 s et ai. 1985), whereas others are tightly associated with PARP, suggesting a modulatory role of the nuclear matrix-associated PARP in nuclear functions. In light of the potential role of PARP in certain nuclear matrix-mediated functions, we examined the role of the nuclear matrix-associated poly (ADPribosyl) ation system in transcriptional regulation. The experimental model system of

It is known that mammalian spermatogenesis is a synchronous process of cellular differentiation during which morphological changes occur, concomitantly with alterations in the complement of constituent proteins, that reflect differences in the mRNA populations coding for stage-specif ic proteins. Moreover, the most dramatic changes in chromatin structure observed in eukaryotes, take place during spermiogenesis, and the main nuclear processes occur in well-defined cell stages. Rat test is has been used as experimental model in a research project carried out at various levels , represented by rat germinal cel ls (primary and secondary spermatocytes, round spermatids), chromatin fractions (transcriptionally active chromatin, nuclear matrix, MARs) and purified nuclear proteins (histone and non-histone proteins). Specific experiments have been carried out in order to determine the poly(ADPR)polymerase content at different stages of germ-cell differentiation, the poly(ADP-ribose) amount, length and complexity inside the nucleus, and the poly(ADP-ribose) acceptors among tissueand stage-specific nuclear proteins. The results indicate that regulation of the poly(ADPribosyl)ation system accompanies the earlier phases of the germinal cell differentiation. Indeed, poly(ADPribose)polymerase is particularly active in primary spermatocytes, being possibly implicated in the recombination events that characterize the pachytene phase of the meiot ic divis ion. Different c lasses of poly(ADPribose) modify different chromatin fractions (DNase I-sensitive, DNase I-resistant chromatin, and nuclear matrix) implicated in DNA replication, repair and transcription. Moreover, the H1 variant H1t, specifically expressed in pachytene spermatocytes, represents the main poly(ADPribose) acceptor, together with poly(ADPR)polymerase itself , in rat germ-cells . Its modification can amplify the role of histone H1 variants as modulators of chromatin structure.

The poly(ADP-ribosyl)ation system, associated with different nuclear fractions of rat testis, has been analyzed for both pADPR and pADPR acceptor proteins. The DNase I sensitive and resistant chromatin contain 35% and 40%, respectively, of the total pADPR synthesized in intact nuclei incubated with [32P]NAD. Moreover, the residual 25% were estimated to be associated with the nuclear matrix.Three different classes of pADPR are present in the nuclei. The longest and branched ADPribose polymers modify proteins present in the DNase I resistant (2 M NaCl extractable) chromatin and in the nuclear matrix, whereas polymers of > 20 residues interact with the components of the DNase I sensitive chromatin and oligomers of 6 ADPribose residues are bound specifically to the acid-soluble chromosomal proteins, present in isolated nuclear matrix. The main pADPR acceptor protein in all the nuclear fractions is represented by the PARP itself (auto-modification reaction). The hetero-modification reaction occurs mostly on histone H1 and core histones, that have been found associated to DNase I sensitive and resistant chromatin, respectively. Moreover, an oligo(ADP-ribosyl)ation occurs on core histones tightly-bound to the matrix associated regions (MARs) of chromatin loops.

The post-translational modification of proteins by ADPribose is widely distributed among living organisms (1). In prokaryotes, a single ADPR unit is transferred to acceptor proteins by mono-ADPR transferases (1). Many bacterial toxins are mono-ADPR transferases that modify host-cell proteins (1). Only three bacterial ADPribosylating systems, in Rodospirillum rubrum, Escherichia coli and Pseudomonas maltophilia, have been described, characterized by endogenous enzyme and substrate proteins (1, 2).

Culture and differentiation parameters of a human thyroid transformed cell line (HuT) were analyzed. Treatment with high concentrations of chemical agents namely dimethyl sulphoxide and retinoic acid, exerted a dramatic cytotoxic effect. The exposure of these cells to the lowest doses of retinoic acid as well as to 8 mM-16 mM 3-aminobenzamide a potent inhibitor of poly(ADPribose)polymerase, resulted in a delay of cell proliferation. Poly(ADPribose)polymerase activity was differently affected by retinoic acid (stimulation) and 3-aminobenzamide (inhibition).